Human ZNT8 Antibody

Abstract

The present invention relates to the fields of immunology and autoimmunity. More specifically, the present invention provides methods and compositions for the production and use of antibodies related to the pancreatic zinc transporter ZnT8. The present invention also provides an isolated antibody or antigen-binding fragment thereof that specifically binds to ZnT8, the isolated antibody or antigen-binding fragment thereof comprising a VH comprising one of the amino acid sequences shown in SEQ ID NOs: 2, 12, 22, 32, 42 and 52. In an alternative embodiment, the isolated antibody or antigen-binding fragment thereof that specifically binds to ZnT8 comprises a VL comprising one of the amino acid sequences shown in SEQ ID NOs: 7, 17, 27, 37, 47 and 57.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 719,016, filed on August 16, 2018, the contents of which are incorporated herein by reference in their entirety.

[0003] Government Interest Statement

[0004] This invention was made with government support under Grant No. GM065137 awarded by the National Institutes of Health. The government has certain rights in this invention. Technical Field

[0005] The present invention relates to the fields of immunology and autoimmunity. More specifically, the present invention provides methods and compositions for the production and use of antibodies directed to the pancreatic zinc transporter ZnT8.

[0006] Incorporation by Reference of Electronically Submitted Materials

[0007] This application contains a sequence listing. The sequence listing has been submitted electronically by EFS-Web as an ASCII text file named "P15404-02_ST25.txt". The size of the sequence listing is 69547 bytes and was created on August 15, 2019. The sequence listing is incorporated herein by reference in its entirety. Background Art

[0008] The human genome encodes a large number of zinc transporters that are involved in regulating cellular zinc homeostasis and signaling (1). Among all zinc transporters, zinc transporter 8 (ZnT8) is unique in its tissue-specific expression (2). Microarray analysis of mouse tissues (3) and single-cell transcriptome analysis of human islets (4) showed that SCL30A8 transcription is primarily limited to endocrine cells of the islets. The transcriptional profile of SLC30A8 has a similar degree of islet specificity as well-known β-cell markers (e.g., PDX1 and NKX6.1) (4). Single-cell RNAseq showed that these β-cell markers also have "spill-over" transcription to non-β-endocrine cells, but the "spill-over" does not extend to exocrine cells or ductal cells (4). In pancreatic β-cells, ZnT8 is primarily expressed in insulin secretory granules (2, 5), where ZnT8 is required for granular zinc enrichment and zinc insulin crystal encapsulation in the form of hexameric insulin (6, 7). The zinc content within insulin granules exceeds 10 mM (8). High zinc levels are associated with insulin synthesis, storage, regulation of insulin secretion, and hepatic insulin clearance after glucose-stimulated insulin secretion (GSIS) (9-11).

[0009] Pancreatic β cells are the only source of insulin in the human body. Insufficient insulin production is the main pathogenic factor for type 2 diabetes (T2D) (12). Common T2D is a complex polygenic disease associated with more than 150 T2D risk genes (13). To date, SCL30A8 is the only gene known to have a protective loss-of-function (LOF) mutation (14). Genotyping of ~150,000 individuals across multiple population cohorts showed that LOF mutation carriers had a 65% reduced risk of T2D (15). The unique position of SLC30A8 in the genetic context of T2D susceptibility makes ZnT8 an attractive therapeutic target (16). Notably, a missense single nucleotide polymorphism in SCL30A8 (rs13266634) is associated with increased susceptibility to T2D (17-19). Compared with the lower-risk W325 variant, the zinc transport activity of the higher-risk R325 variant is hyperactive (20), which corresponds to higher zinc levels in human islets from donors carrying the R325 variant (21). Furthermore, although the GSIS phenotype of ZnT8 KO mouse models is diverse (6, 7, 23, 24), transgenic mice overexpressing the R325 variant have increased islet zinc levels and impaired glucose tolerance after a high-fat diet (22). The combination of emerging evidence from ZnT8 biochemistry, animal models, and human genetics appears to show a causal relationship linking the gain-of-function R235 variant to increased risk of T2D, supporting the case for ZnT8 inhibition as a potential antidiabetic strategy (16, 25).

[0010] Two alternative approaches can be used to downregulate ZnT8 activity in β cells: inhibiting zinc transport or reducing ZnT8 expression. The mechanisms driving zinc transport are conserved from bacteria to humans (26, 27). In the bacterial homolog YiiP (28), zinc transport is susceptible to allosteric regulation through zinc binding to the cytoplasmic C-terminal domain (CTD) (29). Similarly, allosteric targeting can be used for ZnT8 inhibition using mAbs to hold ZnT8 in a fixed conformation, but proof-of-principle allosteric inhibitors are still lacking. In addition, due to the lack of ZnT8-specific reagents for tracking ZnT8 cellular levels in a heterologous background, the functional regulation of ZnT8 expression in β cells has not been studied. The discovery of mAbs with specificity and high affinity will allow testing of the allosteric inhibition hypothesis while developing assays to quantify ZnT8 cellular levels. Summary of the invention

[0011] The present invention is based at least in part on the development of monoclonal antibodies that specifically bind to the pancreatic zinc transporter ZnT8. The gene SLC30A8 encoding ZnT8 is a susceptibility gene for type 2 diabetes. Reducing ZnT8 transport activity or downregulating its cellular expression is considered to be an anti-diabetic strategy that mimics the protective effect of SLC30A8 haploinsufficiency on humans. However, there are no research tools for inhibiting ZnT8 and measuring ZnT8 cellular levels. As described herein, the present invention has developed a monoclonal ZnT8 antibody suitable for addressing these unmet needs. mAbs exhibit sub-nanomolar affinity for human ZnT8 and exhibit selectivity and epitope recognition for homologous zinc transporters with different interspecies reactivity. Antigen binding fragments (Fabs) protect ZnT8 from unfolding and inhibit ZnT8-mediated zinc transport in proteoliposomes. Negative staining electron microscopy shows a ternary binding complex of a ZnT8 monomer with a stoichiometric ratio of 1:1:1 of two different Fabs. Co-binding of two mAbs to a single ZnT8 protein multiplies the individual anti-ZnT8 specificity, enabling quantification of ZnT8 cellular levels by homogeneous time-resolved fluorescence (HTRF).The mAbs described herein can be used as allosteric inhibitors and highly specific biosensors for human ZnT8.

[0012] In a specific embodiment, the present invention provides an isolated antibody or antibody binding fragment thereof that specifically binds to ZnT8, wherein the antibody or antibody binding fragment comprises heavy chain complementarity determining regions (CDRs) 1, 2, and 3, wherein the heavy chain CDR1 comprises an amino acid sequence as shown in one of SEQ ID NOs: 3, 13, 23, 33, 43, and 53, or an amino acid sequence as shown in one of SEQ ID NOs: 3, 13, 23, 33, 43, and 53 with substitutions at two or fewer amino acid positions, the heavy chain CDR2 comprises an amino acid as shown in one of SEQ ID NOs: 4, 14, 24, 34, 44, and 54, or an amino acid as shown in one of SEQ ID NOs: 4, 14, 24, 34, 44, and 54 with substitutions at two or more amino acid positions, and the heavy chain CDR3 comprises an amino acid sequence as shown in one of SEQ ID NOs: 5, 15, 25, 35, 45, and 55, or an amino acid sequence as shown in one of SEQ ID NOs: 5, 15, 25, 35, 45, and 55 with substitutions at two or fewer amino acid positions. The amino acid sequence shown in one of NO: 5, 15, 25, 35, 45 and 55.

[0013] In other embodiments, the isolated antibody or antigen-binding fragment further comprises a light chain CDR1, CDR2 and CDR3, wherein the light chain CDR1 comprises an amino acid sequence as shown in one of SEQ ID NOs: 8, 18, 28, 38, 48 and 58, or an amino acid sequence as shown in one of SEQ ID NOs: 8, 18, 28, 38, 48 and 58 with a substitution at two or less amino acid positions, the light chain CDR2 comprises an amino acid sequence as shown in one of SEQ ID NOs: 9, 19, 29, 39, 49 and 59, or an amino acid sequence as shown in one of SEQ ID NOs: 9, 19, 29, 39, 49 and 59 with a substitution at two or less amino acid positions, and the light chain CDR3 comprises an amino acid sequence as shown in one of SEQ ID NOs: 10, 20, 30, 40, 50 and 60, or an amino acid sequence as shown in one of SEQ ID NOs: 10, 20, 30, 40, 50 and 60 with a substitution at two or less amino acid positions.

[0014] The present invention also provides an isolated antibody or antigen-binding fragment thereof that specifically binds to ZnT8, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH), the heavy chain variable region comprises CDR1, CDR2 and CDR3, and the CDR1, CDR2 and CDR3 are composed of the amino acid sequences shown below: (a) SEQ ID NOs: 3-5, respectively; (b) SEQ ID NOs: 13-15, respectively; (c) SEQ ID NOs: 23-25, respectively; (d) SEQ ID NOs: 33-35, respectively; (e) SEQ ID NOs: 43-45, respectively; and (f) SEQ ID NOs: 53-55, respectively.

[0015] In an alternative embodiment, the isolated antibody or antigen-binding fragment thereof that specifically binds to ZnT8 comprises a light chain variable region (VL), which comprises CDR1, CDR2 and CDR3, and the CDR1, CDR2 and CDR3 are composed of the amino acid sequences shown below: (a) SEQ ID NO: 8-10, respectively; (b) SEQ ID NO: 18-20, respectively; (c) SEQ ID NO: 28-30, respectively; (d) SEQ ID NO: 38-40, respectively; (e) SEQ ID NO: 48-50, respectively; and (f) SEQ ID NO: 58-60, respectively.

[0016] In a specific embodiment, the isolated antibody or antigen-binding fragment thereof that specifically binds to ZnT8 comprises (a) a VH and (b) a VL, wherein the VH comprises a CDR1, a CDR2 and a CDR, wherein the CDR1, CDR2 and CDR are composed of the amino acid sequences shown below: (i) SEQ ID NOs: 3-5, respectively; (ii) SEQ ID NOs: 13-15, respectively; (iii) SEQ ID NOs: 23-25, respectively; (iv) SEQ ID NOs: 33-35, respectively; (v) SEQ ID NOs: 43-45, respectively; or (vi) SEQ ID NOs: 53-55, respectively, and the VL comprises a CDR1, a CDR2 and a CDR3, wherein the CDR1, CDR2 and CDR3 are composed of the amino acid sequences shown below: (vii) SEQ ID NOs: 8-10, respectively; (viii) SEQ ID NOs: 18-20, respectively; (ix) SEQ ID NOs: 28-30, respectively; (x) SEQ ID NOs: 53-55, respectively. NOs: 38-40; (xi) SEQ ID NOs: 48-50, respectively; or (xii) SEQ ID NOs: 58-60, respectively.

[0017] In certain embodiments, the isolated antibody or antigen binding fragment is an antagonist of ZnT8 activity. The present invention also provides an isolated nucleic acid molecule encoding an anti-ZnT8 antibody or its antigen binding fragment as described herein. The present invention also provides a vector comprising the nucleic acid molecule as described herein. In a specific embodiment, a host cell comprises a vector as described herein. The host cell can be a prokaryotic or eukaryotic cell.

[0018] In another aspect, the present invention provides a method for producing an anti-ZnT8 antibody or an antigen-binding fragment thereof. In one embodiment, the method for producing an anti-ZnT8 antibody or an antigen-binding fragment thereof comprises the following steps: (a) culturing a host cell as described herein under conditions suitable for the host cell to express the ZnT8 antibody or an antigen-binding fragment thereof; (b) recovering the ZnT8 antibody or an antigen-binding fragment thereof. The host cell may be a prokaryotic or eukaryotic cell.

[0019] In another aspect, the present invention provides a composition comprising an anti-ZnT8 antibody or antigen-binding fragment thereof as described herein and a suitable pharmaceutical carrier. The composition can be formulated for intravenous administration, intramuscular administration, oral administration, subcutaneous administration, intraperitoneal administration, intrathecal administration or intramuscular administration.

[0020] In another aspect, the present invention provides a method for treating a disease or condition mediated by ZnT8, including but not limited to diabetes. In a specific embodiment, the method for treating diabetes in a mammal comprises the following steps: administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds to ZnT8 as described herein to the mammal.

[0021] In a specific embodiment, an isolated antibody or antigen-binding fragment thereof that specifically binds to ZnT8, wherein the antibody or antigen-binding fragment thereof comprises: (a) a VH comprising CDR1, CDR2 and CDR3 having amino acid sequences shown in SEQ ID NOs: 3-5, respectively; and a VH comprising CDR1, CDR2 and CDR3 having amino acid sequences shown in SEQ ID NOs: 8-10, respectively; or (b) a VH comprising CDR1, CDR2 and CDR3 having amino acid sequences shown in SEQ ID NOs: 13-15, respectively; and a VH comprising CDR1, CDR2 and CDR3 having amino acid sequences shown in SEQ ID NOs: 18-20, respectively; or (c) a VH comprising CDR1, CDR2 and CDR3 having amino acid sequences shown in SEQ ID NOs: 23-25, respectively; and a VH comprising CDR1, CDR2 and CDR3 having amino acid sequences shown in SEQ ID NOs: 24-25, respectively. or (d) comprising a VH having an amino acid sequence as shown in SEQ ID NOs: 33-35, respectively, CDR1, CDR2 and CDR3; and a VH comprising an amino acid sequence as shown in SEQ ID NOs: 38-40, respectively; or (e) comprising a VH having an amino acid sequence as shown in SEQ ID NOs: 43-45, respectively, CDR1, CDR2 and CDR3; and a VH comprising an amino acid sequence as shown in SEQ ID NOs: 48-50, respectively, CDR1, CDR2 and CDR3; or (f) comprising a VH having an amino acid sequence as shown in SEQ ID NOs: 53-55, respectively, CDR1, CDR2 and CDR3; and a VH comprising an amino acid sequence as shown in SEQ ID NOs: 54-55, respectively, CDR2 and CDR3. VH of CDR1, CDR2 and CDR3 of the amino acid sequence shown in NOs: 58 to 60. In certain embodiments, the antigen-binding fragment is selected from the group consisting of scFv, sc(Fv)2, Fab, F(ab)2 and diabody.

[0022] The present invention also provides an isolated antibody or antigen-binding fragment thereof that specifically binds to ZnT8 comprising a VH comprising one of the amino acid sequences shown in SEQ ID NOs: 2, 12, 22, 32, 42 and 52. In an alternative embodiment, the isolated antibody or antigen-binding fragment thereof that specifically binds to ZnT8 comprises a VL comprising one of the amino acid sequences shown in SEQ ID NOs: 7, 17, 27, 37, 47 and 57.

[0023] In other embodiments, the isolated antibody or antigen-binding fragment thereof that specifically binds to ZnT8 includes: (a) a VH comprising the amino acid sequence shown in SEQ ID NO: 2, and a VL comprising the amino acid sequence shown in SEQ ID NO: 7; (b) a VH comprising the amino acid sequence shown in SEQ ID NO: 12, and a VL comprising the amino acid sequence shown in SEQ ID NO: 17; (c) a VH comprising the amino acid sequence shown in SEQ ID NO: 22, and a VL comprising the amino acid sequence shown in SEQ ID NO: 27; (d) a VH comprising the amino acid sequence shown in SEQ ID NO: 32, and a VL comprising the amino acid sequence shown in SEQ ID NO: 37; (e) a VH comprising the amino acid sequence shown in SEQ ID NO: 42, and a VL comprising the amino acid sequence shown in SEQ ID NO: 47; or (f) a VH comprising the amino acid sequence shown in SEQ ID NO: 52, and a VL comprising the amino acid sequence shown in SEQ ID NO: 57. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1A-1B Mapping of mAb binding sites for ZnT8 (TMD+CTD) immobilized in proteoliposomes by passive encapsulation ( Figure 1A )ELISA. Bound antibodies were detected by HRP-conjugated secondary antibodies. The assay was calibrated with CTDA and NTDA as positive controls and MSA as negative control, with a Z' factor > 0.5. Error bars represent standard deviations. ( Figure 1B ) ELISA of monomeric CTD antigen immobilized on Ni-NTA plates via a C-terminal poly-histidine tag. CTDA and NTDA were used as positive and negative controls, respectively (Z>0.5);

[0025] Figure 2.Size exclusion HPLC chromatograms of ZnT8-antibody complexes obtained by complexing ZnT8 with mAb20, Fab20 and / or mAb39 in various combinations and binding stoichiometries. Eight different peaks were resolved, labeled 1 to 8. The corresponding complex molecular weights and predicted complex compositions are summarized in Table 1. mAb20 / Fab20 and mAb39 were fluorescently labeled and detected in the green (shown as blue trace) or red (red trace) channel of a multichannel fluorescence detector. The gray trace is unbound ZnT8 detected by UV absorption;

[0026] Figure 3A-Figure 3F .EM analysis of ZnT8-Fab complex. ( Figure 3A ) Representative primary particles of negatively stained binary complex ZnT8-(Fab20)2. Scale bar is ( Figure 3B ) Representative reference-free 2D class averages for this binary complex. Figure 3C ) Representative primary particles of the ternary complex (Fab20)2-ZnT8-(Fab39)2. Figure 3D ) Representative reference-free 2D class averages for this ternary complex. Figure 3E ) 3D image of ZnT8-(Fab20)2 in two views related by a 90° rotation about the two-fold axis perpendicular to the membrane plane (black dashed line). Atomic models of Fab20 (PDB ID 1M71) and the ZnT8 homologue (PDB ID 3H90) were fitted into the low-resolution envelope and colored in cyan and yellow, respectively. ( Figure 3F ) The 3D model of (Fab20)2-ZnT8-(Fab39)2 in two views is docked with four Fab structures, which represent two Fab20 (cyan) and two Fab39 (magenta). The ZnT8 dimer model is shown in yellow. The blue dotted curve represents the boundary between the two Fab39 molecules on the top of the adjacent;

[0027] Figure 4A-4C mAb binding stabilizes ZnT8 folding ( Figure 4A ), mAb binding to folded or denatured ZnT8. ZnT8-His in lotion micelles was immobilized on Ni-NTA plates in replicates without SDS denaturation. ELISAs of mAb20 and mAb39 against denatured (triangles) and undenatured ZnT8 (circles) were compared as shown. CTDA and MSA were used as positive and negative controls, respectively. Error bars are standard deviations. ( Figure 4B) mAb binding to folded ZnT8-GFP in lotion micelles. mAb20 or mAb39 was mixed with ZnT8-GFP and the binding mixture was analyzed by size exclusion HPLC and GFP fluorescence detection. Note that mAb20 shifted the unbound ZnT8-GFP peak (black) to the left to the mAb20-ZnT8-GFP position (red), while mAb39 did not cause a peak shift (magenta). ( Figure 4C ) Protection of ZnT8 unfolding. Aliquots of ZnT8 in lotion micelles were incubated with Fab20 (red), Fab39 (magenta), or Fab20+Fab39 (brown). Free ZnT8 was used as a control (black). After the indicated thermal denaturation times, protein aggregates were removed by ultracentrifugation, and the residual amount of ZnT8 in the supernatant was detected by mAb20 immunoblotting (above). Error bars are the standard errors of 6 independent densitometry measurements of a representative experiment performed using ImageJ;

[0028] Figure 5A-Figure 5B Inhibition of zinc transport by Fab binding in response to rapid mixing of ZnT8 proteoliposomes with extracellular zinc buffer ( Figure 5A ) Fluozin-3 fluorescence trace. As indicated by the arrow, the free zinc concentration in the buffer increased from 1.6 μM to 116 μM. ( Figure 5B ) Steady-state kinetics of zinc transport mediated by ZnT8 or ZnT8 bound to Fab20, Fab39, or Fab20+Fab39. The solid line is the Michaelis–Menten equation fitted by the least squares method. Table 2 summarizes the Km values ​​and Vmax values;

[0029] Figure 6A-6C .mAb binding affinity. ( Fig. 6A ) Representative confocal microscopy images of immunofluorescence staining of fixed and saponin-permeabilized HEK293 cells stained with mAb20 or mAb39 as indicated. ZnT8(+) or ZnT8(-) indicates HEK293 cells without ZnT8 overexpression. Red: mAb20 or mAb39 staining, blue: DAPI, scale bar: 5 μm. ( Figure 6B ) Histogram of flow cytometric count events. Arrows indicate the left shift of the histogram due to serial dilution of mAb. Figure 6C ) As shown, mAbs bind to HEK293 cells without ZnT8 overexpression. Figure 6B ) obtained immunofluorescence intensity of serially diluted mAb20 or mAb39 markers. The solid line is the least squares fit of the binding data to the single component binding process;

[0030] Figure 7A-7D .mAb binding specificity ( Fig. 7A ), mAb bound to ZnT8-His immobilized on Ni-NTA plate. Bound mAb20 (red) or mAb39 (magenta) was detected by HRP-conjugated secondary antibody. Solid lines are the least squares fit of the binding data points to the single component binding process. Error bars are standard errors (n=8). ( Figure 7B ) SDS-denatured mouse ZnT8-His (labeled M) and human ZnT8-His (labeled H) on immunoblots were detected by mAb20 or mAb39 as indicated. Replicate immunoblots were probed with anti-His-tag antibody, showing that approximately the same amount of HEK293 crude lysate was loaded in each lane. ( Figure 7C ) Endogenous human ZnT8 in EndoC-βH cells was detected by immunoblotting with mAb20 or mAb38. Equal amounts of EndoC-βH lysate (5 μg total protein) were loaded into each lane. ( Fig.7D ) Immunoblots of HEK lysates with ZnT homologs or ZnT homolog overexpression as indicated. Approximately equal amounts of total lysate protein (5 μg) were placed in each lane and probed with mAb20, mAb39, or anti-FALG antibody as indicated;

[0031] Figure 8 .HTRF responses to human ZnT8, mouse ZnT8, and human ZnT3. DETAILED DESCRIPTION

[0032] It should be understood that the present invention is not limited to the specific methods and components described herein, etc., as they may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the present invention. It must be noted that the singular forms "a", "an", and "the" as used herein and in the appended claims include plural forms unless the context clearly indicates 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.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, specific methods, devices, and materials are described.

[0034] All publications cited herein are incorporated herein by reference, including all journal articles, books, manuals, published patent applications, and issued patents. In addition, the meanings of certain terms and phrases used in the specification, examples, and appended claims are provided. The definitions are not meant to be limiting in nature, but are intended to provide a clearer understanding of certain aspects of the invention.

[0035] I. Definitions

[0036] The term "antibody" means an immunoglobulin molecule that recognizes and specifically binds to a target (e.g., a protein (e.g., ZNT8, its subunits or receptor complexes), a polypeptide, a peptide, a carbohydrate, a polynucleotide, a lipid, or a combination thereof) through at least one antigen recognition site in the variable region of the immunoglobulin molecule. A typical antibody comprises at least two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds. Each heavy chain consists 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 consists of three domains, CH1, CH2, and CH3. Each light chain consists 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 consists of one domain, CI. The VH and VL regions can be further subdivided into highly variable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL region consists of three CDRs and four FRs, arranged in the following order from amino to carboxyl terminus: FRI, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. As used herein, the term "antibody" encompasses: intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, Fd, Facb, and Fv fragments), single-chain Fv (scFv), miniantibodies (e.g., sc(Fv)2, diabodies), multispecific antibodies (e.g., bispecific antibodies produced from at least two intact antibodies), chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising antigenic determinants of antibodies, and any other modified immunoglobulin molecules comprising antigen recognition sites, as long as the antibody exhibits the desired biological activity. Therefore, the term "antibody" includes whole antibodies and any antigen-binding fragments or single chains thereof. Antibodies may be naked or conjugated to other molecules (e.g., toxins, radioisotopes, small molecule drugs, polypeptides, etc.).

[0037] The term "isolated antibody" refers to an antibody that has been identified, separated and / or recovered from a component of its natural environment. Contaminating components of its natural environment are substances that 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 purified (1) to greater than 95 wt % of the antibody, e.g., as determined by the Lowry method, and including greater than 99 wt %, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequencer, or (3) to a homogeneity obtained by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or silver staining. Isolated antibodies include antibodies in situ within recombinant cells because at least one component of the antibody's natural environment will not be present. However, typically isolated antibodies will be prepared by at least one purification step.

[0038] The term "humanized" immunoglobulin refers to an immunoglobulin comprising a human framework region and one or more CDRs from a non-human (usually mouse or rat) immunoglobulin. The non-human immunoglobulin providing the CDR is called a "donor", and the human immunoglobulin providing the framework is called an "acceptor". The constant region does not have to be present, but if it is present, they must be substantially identical to the human immunoglobulin constant region, i.e., at least about 85%-90%, preferably about 95% or higher. Therefore, all parts of the humanized immunoglobulin (which may not include CDR) are substantially identical to the corresponding parts of the natural human immunoglobulin sequence. A "humanized antibody" is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. For example, a humanized antibody will not encompass a typical chimeric antibody as defined above, for example, because the entire variable region of the chimeric antibody is non-human.

[0039] The term "antigen-binding fragment" refers to a portion of a complete antibody, and refers to the antigen-determining variable region of a complete antibody. It is known in the art that the antigen-binding function of an antibody can be performed by a fragment 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 by antibody fragments. In some cases, antibody fragments can be prepared by proteolytic digestion of a complete antibody or a whole antibody. For example, antibody fragments can be obtained by treating a whole antibody with an enzyme (e.g., papain, pepsin, or plasmin). Papain digestion of the whole antibody produces F(ab)2 or Fab fragments; pepsin digestion of the whole antibody produces F(ab')2 or Fab'; plasmin digestion of the whole antibody produces Facb fragments.

[0040] The term "Fab" refers to an antibody fragment substantially equivalent to an antibody obtained by digesting an immunoglobulin (usually IgG) with papain. The heavy chain segment of the Fab fragment is the Fd segment. Such a fragment can be: enzymatically produced or chemically produced by fragmenting a complete antibody, produced by recombination of a gene encoding a partial antibody sequence, or it can be synthesized in whole or in part. The term "F(ab')2" refers to an antibody fragment substantially equivalent to a fragment obtained by digesting an immunoglobulin (usually IgG) with pepsin at pH 4.0-4.5. Such a fragment can be: enzymatically produced or chemically produced by fragmenting a complete antibody, produced by recombination of a gene encoding a partial antibody sequence, or it can be synthesized in whole or in part. The term "Fv" refers to an antibody fragment consisting of an NH and an N domain held together by non-covalent interactions.

[0041] The terms "ZNT8 antibody", "anti-ZNT8 antibody", "anti-ZNT8", "antibody that binds to ZNT8" and any grammatical variations thereof refer to an antibody that can specifically bind to ZNT8 with sufficient affinity so that the antibody can be used as a therapeutic or diagnostic agent targeting ZNT8. TM The extent of binding of the anti-ZNT8 antibodies disclosed herein to unrelated, non-ZNT8 proteins is less than about 10% of the binding of the antibodies to ZNT8 as determined by (using recombinant ZNT8 as an analyte and the antibody as a ligand, or vice versa) or other binding assays known in the art. In certain embodiments, the dissociation constant (KD) of the antibody binding to ZNT8 is <1 μM, <100 nM, <50 nM, <10 nM, or <1 nM.

[0042] The term "% identity" between two polypeptide (or polynucleotide) sequences refers to the number of identical matching positions shared by the sequences in the comparison window, taking into account the additions or deletions (i.e., gaps) that must be introduced in order to optimally align the two sequences. A matching position is any position where the same nucleotide or amino acid is present in both the target sequence and the reference sequence. Since a gap is not a nucleotide or amino acid, the gaps that occur in the target sequence are not counted. Similarly, since the nucleotides or amino acids of the target sequence are counted instead of the nucleotides or amino acids of the reference sequence, the gaps in the reference sequence are not counted. The percentage of sequence identity is calculated by determining the number of positions where the same amino acid residue or nucleic acid base occurs in the two sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and then multiplying the result by 100 to obtain the percentage of sequence identity. The alignment of the sequences and the determination of the percentage sequence identity between the two sequences can be accomplished using software that is easy to use both online and for download. Suitable software programs are available from a variety of sources and are used for alignment of protein and nucleotide sequences. A suitable program for determining percentage sequence identity is bl2seq, which is part of the program BLAST suite, which can be obtained from the BLAST website of the National Center for Biotechnology Information of the U.S. government. Bl2seq uses BLASTN or BLASTP algorithms to compare between two sequences. 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 can also be obtained from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa. In some embodiments, the percent identity "X" of the first amino acid sequence and the second sequence amino acid is calculated as 100 × (Y / Z), wherein Y is the number of amino acid residues scored as identical matches in the comparison of the first sequence and the second sequence (as compared by visual inspection or a specific sequence comparison program), and Z is the total number of residues in the second sequence. If the length of the first sequence is greater than the length of 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. It will be appreciated by those skilled in the art that the generation of sequence alignments for calculating percent sequence identity is not limited to binary sequence-sequence alignments driven by primary sequence data. Sequence alignments can be derived from multiple sequence alignments. A 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 optionally available from, for example, the European Bioinformatics Institute (EBI).

[0043] The term "therapeutic agent" refers to any biological or chemical agent used in the treatment of a disease or condition. Therapeutic agents include any suitable biologically active compounds, biologically derived components (e.g., cells, peptides, antibodies, and polynucleotides), and radiochemical therapeutic agents (e.g., radioisotopes). In some embodiments, the therapeutic agent includes a chemotherapeutic agent or an analgesic agent.

[0044] As used herein, the terms "treatment of", "treating of", "treat", and the like refer to obtaining a desired pharmacological and / or physiological effect. The term is also used for administering a "therapeutically effective amount" of an agent, such as an anti-ZnT8 antibody. The effect may be prophylactic in terms of complete or partial prevention of a particular outcome: a disease or its symptoms, and / or therapeutic in terms of partial or complete cure of a disease and / or adverse effects attributable to the disease. As used herein, "treatment of" encompasses any treatment of a disease in a subject, particularly a human, and includes: (a) preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., arresting its development; and (c) alleviating the disease, e.g., causing regression of the disease, e.g., to completely or partially eliminate the symptoms of the disease. In a specific embodiment, the term is used to prevent or treat any condition of a ZnT8-mediated disease, including diabetes.

[0045] II. Anti-ZnT8 Antibodies

[0046] The antibodies or antigen-binding fragments thereof disclosed herein specifically bind to ZNT8. In a specific embodiment, these antibodies or antigen-binding fragments specifically bind to human ZNT8. As used herein, "specific binding" means that the antibody or antigen-binding fragment preferentially binds to ZNT8 (e.g., human ZNT8, mouse ZNT8) over other proteins. In some cases, the anti-ZNT8 antibodies disclosed herein have a higher affinity for ZNT8 than for other proteins. The binding affinity of the anti-ZNT8 antibodies that specifically bind to ZNT8 for human ZNT8 may be less than or equal to 1×10 -7 M, less than or equal to 2×10 -7 M, less than or equal to 3×10 -7 M, less than or equal to 4×10 -7 M, less than or equal to 5×10 -7 M, less than or equal to 6×10 -7 M, less than or equal to 7×10 -7M, less than or equal to 8×10 -7 M, less than or equal to 9×10 -7 M, less than or equal to 1×10 -8 M, less than or equal to 2×10 -8 M, less than or equal to 3×10 -8 M, less than or equal to 4×10 -8 M, less than or equal to 5×10 -8 M, less than or equal to 6×10 -8 M, less than or equal to 7×10 -8 M, less than or equal to 8×10 -8 M, less than or equal to 9×10 -8 M, less than or equal to 1×10 -9 M, less than or equal to 2×10 -9 M, less than or equal to 3×10 -9 M, less than or equal to 4×10 -9 M, less than or equal to 5×10 -9 M, less than or equal to 6×10 -9 M, less than or equal to 7×10 -9 M, less than or equal to 8×10 -9 M, less than or equal to 9×10 -9 M, less than or equal to 1×10 -10 M, less than or equal to 2×10 -10 M, less than or equal to 3×10 -10 M, less than or equal to 4×10 -10 M, less than or equal to 5×10 -10 M, less than or equal to 6×10 -10 M, less than or equal to 7×10 -10 M, less than or equal to 8×10 -10 M, less than or equal to 9×10 -10 M, less than or equal to 1×10 -11 M, less than or equal to 2×10 -11 M, less than or equal to 3×10 -11 M, less than or equal to 4×10 -11 M, less than or equal to 5×10 -11 M, less than or equal to 6×10 -11 M, less than or equal to 7×10 -11 M, less than or equal to 8×10 -11 M, less than or equal to 9×10 -11 M, less than or equal to 1×10 -12 M, less than or equal to 2×10 -12 M, less than or equal to 3×10 -12 M, less than or equal to 4×10-12 M, less than or equal to 5×10 -12 M, less than or equal to 6×10 -12 M, less than or equal to 7×10 -12 M, less than or equal to 8×10 -12 M or less than or equal to 9×10 -12M. Methods for measuring antibody binding affinity are well known in the art and include surface plasmon resonance (SPR) (Morton and Myszka "Kinetic analysis of macromolecular interactions using surface plasmon resonance biosensors" Methods in Enzymology (1998) 295, 268-294), bio-layer interferometry (Bio-Layer Interferometry) (Abdiche et al "Determining Kinetics and Affinities of Protein Interactions Using a Parallel Real-time Label-free Biosensor, the Octet" Analytical Biochemistry (2008) 377, 209-217), kinetic exclusion assay (KinExA) (Darling and Brault "Kinetic exclusion assay technology: characterization of molecular interactions" Assay and Drug Dev Tech (2004) 2, 647-657), isothermal calorimetry (Pierce et al "Kinetic Exclusion Assay Technology: Characterization of Molecular Interactions" Assay and Drug Dev Tech (2004) 2, 647-657), and isothermal calorimetry (Pierce et al "Kinetic Exclusion Assay Technology: Characterization of Molecular Interactions" Assay and Drug Dev Tech (2004) 2, 647-657). et al "IsothermalTitration Calorimetry of Protein-Protein Interactions" Methods (1999) 19, 213-221) and analytical ultracentrifugation (Lebowitz et al "Modemanalytical ultracentrifugation in protein science: A tutorial review" ProteinScience (2002), 11:2067-2079).

[0047] A. Antibody fragments

[0048] The present disclosure encompasses antibody fragments or domains described herein that retain the ability to specifically bind to ZNT8 (e.g., human ZNT8). Antibody fragments include, for example, Fab, Fab', F(ab')2, Facb, and Fv. These fragments can be humanized or fully human. Antibody fragments can be prepared by proteolytic digestion of intact antibodies. For example, antibody fragments can be obtained by treating the whole antibody with an enzyme (e.g., papain, pepsin, or plasmin). Papain digestion of the whole antibody produces F(ab)2 or Fab fragments; pepsin digestion of the whole antibody produces F(ab')2 or Fab'; plasmin digestion of the whole antibody produces Facb fragments.

[0049] Alternatively, antibody fragments can be produced recombinantly. For example, a nucleic acid encoding an antibody fragment of interest can be constructed, introduced into an expression vector, and expressed in a suitable host cell. See, for example, 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 can be expressed in E. coli and secreted from E. coli, thus making it easy to produce large quantities of these fragments. 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 method, F(ab')2 fragments can be directly isolated from recombinant host cell cultures. Fab and F(ab')22 fragments with increased in vivo half-life containing salvage receptor binding epitope residues are described in U.S. Patent No. 5,869,046.

[0050] B.Minibody

[0051] Also contemplated 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).

[0052] "Diabodies" are bivalent miniantibodies constructed by gene fusion (see, e.g., Holliger, P. et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993); EP 404,097; WO 93 / 11161). Diabodies are dimers consisting of two polypeptide chains. The VL and VH domains of each polypeptide chain of the diabody are bound by a linker. The number of amino acid residues constituting the linker can be between 2 and 12 residues (e.g., 3-10 residues or 5 or about 5 residues). The linker of the polypeptides in the diabody is usually too short so that VL and VH cannot bind to each other. Therefore, the VL and VH encoded in the same polypeptide chain cannot form a single-chain variable region fragment, but form a dimer with different single-chain variable region fragments. As a result, the diabody has two antigen binding sites.

[0053] scFv is a single-chain polypeptide antibody obtained by connecting 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, Ed Resenburg and Moore, Springer Verlag, New York, pp. 269-315, (1994). Each variable domain (or a portion thereof) is derived from the same or different antibody. The single-chain Fv molecule preferably comprises an scFv linker inserted between the VH domain and the VL domain. Exemplary scFv molecules are known in the art and are described, for example, in: U.S. Pat. No. 5,892,019; Ho et al, Gene, 77:51 (1989); Bird et al., Science, 242:423 (1988); Pantoliano et al., Gene, 77:51 (1989); al, Biochemistry, 30: 10117 (1991); Milenic et al, Cancer Research, 51: 6363 (1991); Takkinen et al, Protein Engineering, 4: 837 (1991).

[0054] As used herein, the term "scFv linker" refers to the portion inserted between the VL and VH domains of a scFv. The scFv linker preferably maintains the scFv molecule in an antigen binding conformation. In one embodiment, the scFv linker comprises or consists of a scFv linker peptide. In certain embodiments, the scFv linker peptide comprises or consists of a Gly-Ser linker peptide. In other embodiments, the scFv linker comprises a disulfide bond.

[0055] The order in which VH and VL are to be linked is not particularly limited, and they can 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 can be derived from any anti-ZNT8 antibody or antigen-binding fragment thereof described herein.

[0056] sc(Fv)2 is a mini antibody in which two VH and two VL are connected by a linker to form a single chain (Hudson, et al., J Immunol. Methods, (1999) 231: 177-189 (1999)). For example, sc(Fv)2 can be prepared by connecting scFv to a linker. The sc(Fv)2 of the present invention includes an antibody, preferably in which two VH and two VL are arranged in the following order: starting from the N-terminus of the single-chain polypeptide, VH, VL, VH and VL ([VH] linker [VL] linker [VH] linker [VL]); however, the order of the two VH and the two VL is not limited to the above arrangement, and they can be arranged in any order. The following lists the arrangement examples:

[0057] [VL] Connector [VH] Connector [VH] Connector [VL]

[0058] [VH]Connector [VL]Connector [VL]Connector [VH]

[0059] [VH]Connector [VH]Connector [VL]Connector [VL]

[0060] [VL] Connector [VL] Connector [VH] Connector [VH]

[0061] [VL] Connector [VH] Connector [VL] Connector [VH]

[0062] Typically, when four antibody variable regions are connected, three linkers are required; the linkers used can be the same or different. The linker connecting the VH and VL regions of the miniantibody is not particularly limited. In some embodiments, the linker is a peptide linker. Any single-chain peptide containing about 3 to 25 residues (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18) can be used as a linker.

[0063] In other embodiments, the linker is a synthetic compound linker (chemical cross-linker). Examples of commercially available cross-linkers include: N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS3), dithiobis(succinimidyl propionate) (DSP), dithiobis(sulfosuccinimidyl propionate) (DTSSP), ethylene glycol bis(succinimidyl succinate) (EGS), ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimidyloxycarbonyloxy)ethyl]sulfone (BSOCOES) and bis[2-(sulfosuccinimidyloxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES).

[0064] The amino acid sequence of VH or VL in an antibody fragment or miniantibody may include modifications, such as replacements, deletions, additions and / or insertions. For example, the modification may be in one or more CDRs of an anti-ZNT8 antibody as described herein. In certain embodiments, the modification involves 1, 2 or 3 amino acid replacements in 1, 2 or 3 CDRs of the VH and / or 1, 2 or 3 CDRs of the VL domain of an anti-ZNT8 miniantibody. Such replacements are performed to improve the binding activity and / or functional activity of the anti-ZNT8 miniantibody. In other embodiments, 1, 2 or 3 amino acids of one or more of the six CDRs of an anti-ZNT8 antibody or its antigen-binding fragment may be deleted or added, as long as there is ZNT8 binding and / or functional activity when VH and VL are associated.

[0065] C.VHH

[0066] VHHs, also known as nanobodies, are derived from the antigen-binding variable heavy chain region (VHH) of heavy chain antibodies (lacking light chains) in camels and llamas. The present disclosure encompasses VHHs that specifically bind to ZNT8.

[0067] D. Variable Domain of New Antigen Receptor (VNAR)

[0068] VNAR is the variable domain of new antigen receptor (IgNAR). IgNARs are present in the serum of sharks in the form of covalently linked heavy chain homodimers. It exists in soluble and receptor-bound forms and is composed of variable domains (VNAR) and different numbers of constant domains. VNAR is composed of CDR1 and CDR3, and replaces CDR2 with HV2 and HV4 domains (see, e.g., Barelle and Porter, Antibodies, 4: 240-258 (2015)). The present disclosure covers VNARs that specifically bind to ZNT8.

[0069] E. Constant region

[0070] The antibodies disclosed herein can be whole antibodies or single-chain Fc (scFc), and can include any constant region known in the art. The light chain constant region can be, for example, a κ-type or λ-type light chain constant region, such as a human κ or human λ light chain constant region. The heavy chain constant region can be, for example, an α-type, δ-type, ε-type, γ-type or μ-type heavy chain constant region, such as a human α-type, a human δ-type, a human ε-type, a human γ-type or a human μ-type heavy chain constant region. In some cases, 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.

[0071] In one embodiment, the light chain or heavy chain constant region is a fragment, derivative, variant or mutant protein of a naturally occurring constant region. In some embodiments, the variable heavy chain of the anti-ZNT8 antibody described herein is connected to a heavy chain constant region comprising a CH1 domain and a hinge region. In some embodiments, the variable heavy chain is connected to a heavy chain constant region comprising a CH2 domain. In some embodiments, the variable heavy chain is connected to a heavy chain constant region comprising a CH3 domain. In some embodiments, the variable heavy chain is connected to a heavy chain constant region comprising a CH2 and CH3 domain. In some embodiments, the variable heavy chain is connected to a heavy chain constant region comprising a hinge region, a CH2 and a CH3 domain. CH1, a hinge region, CH2 and / or CH3 can be from an IgG antibody (e.g., IgG1, IgG4). In certain embodiments, the variable heavy chain of the anti-ZNT8 antibody described herein is connected to a heavy chain constant region, and the heavy chain constant region comprises a CHI domain, a hinge region and a CH2 domain from IgG4 and a CH3 domain from IgG1. In certain embodiments, such chimeric antibodies may comprise one or more additional mutations in the heavy chain constant region that increase the stability of the chimeric antibody.In certain embodiments, the heavy chain constant region comprises substitutions that modify the properties of the antibody.

[0072] In certain embodiments, the anti-ZNT8 antibodies of the present disclosure are IgG isotype antibodies. In some embodiments, the antibody is IgG1. In another embodiment, the antibody is IgG2. In yet another embodiment, the antibody is IgG4. In some cases, the IgG4 antibody has one or more mutations that reduce or prevent it from adopting a functional monovalent form. For example, the hinge region of IgG4 can be mutated to make its amino acid sequence identical to the hinge region of human IgG1 (serine in the human IgG4 hinge is mutated to proline). In some embodiments, the antibody has a chimeric heavy chain constant region (e.g., having the CH1 region, hinge region, and CH2 region of IgG4 and the CH3 region of IgG1).

[0073] F. Bispecific Antibodies

[0074] In certain embodiments, the anti-ZNT8 antibodies of the present disclosure are bispecific antibodies. Bispecific antibodies are antibodies that have binding specificity to at least two different epitopes. Exemplary bispecific antibodies can bind to two different epitopes of the ZNT8 protein. Other such antibodies can bind the ZNT8 binding site to the binding site of another protein. Bispecific antibodies can be prepared as full-length antibodies or low molecular weight forms thereof (e.g., F(ab')2 bispecific antibodies, sc(Fv)2 bispecific antibodies, double antibody bispecific antibodies).

[0075] Traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, in which the two chains have different specificities (Millstein et al., Nature, 305: 537-539 (1983)). In a different approach, the antibody variable domains with the desired binding specificity are fused to the immunoglobulin constant domain sequence. The DNA encoding the immunoglobulin heavy chain fusion and (if necessary) the immunoglobulin light chain are inserted into separate expression vectors and co-transfected into suitable host cells. This provides greater flexibility in adjusting the ratio of the three polypeptide fragments. However, when at least two polypeptide chains are expressed at an equal ratio resulting in high yields, the coding sequences of two or all three polypeptide chains can be inserted into a single expression vector.

[0076] According to another method described in U.S. Patent No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture.Preferred interfaces include at least a portion of CH3 domains.In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan).By replacing large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine), a compensation "cavity (cavity)" of the same or similar size as large side chains is produced on the interface of the second antibody molecule.This provides for improving the yield of heterodimers relative to other unwanted final products (e.g., homodimers) provides a mechanism.

[0077] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one antibody in the heteroconjugate can be coupled to avidin and the other can be coupled to biotin. Heteroconjugate antibodies can be prepared using any convenient cross-linking method.

[0078] "Diabody" technology provides an alternative mechanism for preparing bispecific antibody fragments. The fragments contain VH connected to VL by a linker that is too short to allow pairing between the two domains on the same chain. Therefore, the VH domain and VL domain of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites.

[0079] G. Conjugated Antibodies

[0080] The antibodies or antigen-binding fragments disclosed herein can be conjugated to various molecules, including: macromolecular substances, such as polymers (e.g., polyethylene glycol (PEG), polyethyleneimine modified with PEG (PEI-PEG), polyglutamic acid (PGA) (N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer); human serum albumin or fragments thereof; radioactive substances (e.g., 90Y, 131I); fluorescent substances; luminescent substances; haptens; enzymes; metal chelates; and drugs.

[0081] In certain embodiments, portions of the anti-ZNT8 antibody or antigen-binding fragment thereof are modified, which improves its stability and / or retention in the circulation (e.g., in blood, serum, or other tissues), for example, by at least 1.5 times, 2 times, 5 times, 10 times, 15 times, 20 times, 25 times, 30 times, 40 times, or 50 times. For example, the anti-ZNT8 antibody or antigen-binding fragment thereof can be associated with (e.g., conjugated to) a polymer, such as a substantially non-antigenic polymer, such as a polyalkylene oxide or polyethylene oxide. Suitable polymers will vary greatly in weight. A polymer having a number average molecular weight of about 200 Daltons to about 35,000 Daltons (or about 1,000 Daltons to about 15,000 Daltons, and 2,000 Daltons to about 12,500 Daltons) 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 polyethylene polymer, such as polyvinyl alcohol or polyvinyl pyrrolidone). Examples of such polymers include: polyalkylene oxide homopolymers (e.g., polyethylene glycol (PEG) or polypropylene glycol), polyoxyethylated polyols, copolymers thereof, and block copolymers thereof, provided that the water solubility of the block copolymers is maintained. Other useful polymers include: polyoxyalkylenes, such as polyethylene oxide, polypropylene oxide, and block copolymers of polyethylene oxide and polypropylene oxide; polymethacrylates; carbomers; and branched or unbranched polysaccharides.

[0082] The conjugated antibodies or fragments described above can be prepared by chemically modifying the antibodies or their lower molecular weight forms as described herein. Methods for modifying antibodies are well known in the art.

[0083] II. Characterization of Antibodies

[0084] 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: Surface plasmon resonance (SPR), BIACORE TM Analysis, Enzyme Linked Immunosorbent Assay (ELISA), EIA (Enzyme Immunoassay), RIA (Radioimmunoassay) and Fluorescence Resonance Energy Transfer (FRET).

[0085] The binding interaction between the target protein (anti-ZNT8 antibody or its functional fragment) and the target (e.g., ZNT8) can be determined using In this method, one of several variations of an instrument made by ForteBio (e.g., QKe and QK) to determine protein interactions, binding specificity, and epitope localization. The system provides an easy way to monitor real-time binding by measuring changes in polarized light that travels down the custom tip and back to the sensor.

[0086] Surface plasmon resonance (SPR) can be used to analyze the binding interaction of the target protein (anti-ZNT8 antibody or its functional fragment) with the target (e.g., ZNT8). SPR or biomolecular interaction analysis (BIA) detects biospecific interactions in real time without labeling any interactants.

[0087] Changes in the mass of the binding surface of the BIA chip (indicative of a binding event) result in changes in the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)). The change in refractive index produces 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 on-line resources provided by BIAcore International AB (Uppsala, Sweden). Information from SPR can be used to provide accurate and quantitative measurements of the equilibrium dissociation constant (Kd) and kinetic parameters (including Kon and Koff) of biomolecule binding to a target.

[0088] Epitopes can also be directly mapped using BIACORE chromatography technology by evaluating the ability of different anti-ZNT8 antibodies or functional fragments thereof to compete with each other for binding to human ZNT8 (Pharmacia BIAtechnology Handbook, "Epitope Mapping", Section 6.3.2, (May 1994); see also Johne et al. (1993) J. Immunol. Methods, 160: 191-198).

[0089] When using enzyme immunoassay, a sample containing the antibody, e.g., culture supernatant of antibody-producing cells or purified antibody, is added to an antigen-coated plate. A secondary antibody labeled with an enzyme (e.g., alkaline phosphatase) is added, the plate is incubated, washed, an enzyme substrate (e.g., p-nitrophenyl phosphate) is added, and the absorbance is measured to assess antigen binding activity.

[0090] Other general guidelines for evaluating antibodies, such as Western blots and immunoprecipitation, can be found in Antibodies: A Laboratory Manual, ed. by Harlow and Lane, Cold Spring Harbor press (1988).

[0091] III. Affinity Maturation

[0092] In one embodiment, an anti-ZNT8 antibody or an 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, a display library technique is used to select or screen a modified antibody library. Then, for example, by using higher stringency or more competitive binding and washing conditions, antibodies with higher affinity are identified from the second library. Other screening techniques may also be used. Methods for affecting affinity maturation include random mutagenesis (e.g., Fukuda et al., Nucleic Acids Res., 34: el27 (2006); directed mutagenesis (e.g., Rajpal et al., Proc. Natl. Acad. Sci. USA, 102: 8466-71 (2005); shuffling approach (e.g., Jermutus et al., Proc. Natl. Acad. Sci. USA, 98: 75-80 (2001); and in silica approach (e.g., Lippow et al., Nat. Biotechnol., 25: 1171-6 (2005).

[0093] In some embodiments, mutagenesis targets a known region or regions that may be at the binding interface. If, for example, the identified binding protein is an antibody, mutagenesis can be directed to the CDR regions of the heavy or light chains described herein. In addition, mutagenesis can be directed to framework regions near or adjacent to CDRs, for example, framework regions within 10, 5, or 3 amino acids that are connected to the CDRs. In the case of antibodies, mutagenesis can also be limited to one or several CDRs, for example, to make stepwise improvements.

[0094] In one embodiment, mutagenesis is used to make an antibody more similar to one or more germline sequences. An exemplary germline method may include: identifying one or more germline sequences that are similar to the sequence of the separated antibody (e.g., most similar in a particular database). Then, mutations (at the amino acid level) can be made in the separated antibody, either incremental mutations, combinatorial mutations, or both. For example, a nucleic acid library containing sequences encoding some or all possible germline mutations is prepared. The mutated antibodies are then evaluated, for example, to identify antibodies that have one or more additional germline residues relative to the separated antibody and are still useful (e.g., functionally active). In one embodiment, as many germline residues as possible are introduced into the separated antibody.

[0095] In one embodiment, mutagenesis is used to replace one or more germline residues or insert them into a CDR region. For example, the germline CDR residues can be from a germline sequence that is similar (e.g., most similar) to the variable region being modified. After mutagenesis, the activity (e.g., binding or other functional activity) of the antibody can be evaluated to determine whether the germline residue or residues are tolerated. Similar mutagenesis can be performed in the framework region.

[0096] Germline sequences can be selected in different ways. For example, if the germline sequence meets a predetermined standard of selectivity or similarity, for example, at least a certain percentage of identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97%, 98%, 99% or 99.5% identity relative to the donor non-human antibody, then the germline sequence can be selected. At least 2, 3, 5 or 10 germline sequences can be used for selection. In the case of CDR1 and CDR2, identifying similar germline sequences can include selecting such a sequence. In the case of CDR3, identifying similar germline sequences can include selecting such a sequence, but can include using two germline sequences that contribute to the amino terminal portion and the carboxyl terminal portion, respectively. In other embodiments, more than one or two germline sequences are used, for example to form a consensus sequence.

[0097] The calculation of "sequence identity" between two sequences is as follows. For the purpose of optimal comparison, the sequences are compared (for example, for the purpose of optimal comparison, a gap can be introduced in one or both of the first and second amino acid or nucleic acid sequences, and for the purpose of comparison, non-homologous sequences can be ignored). The GAP program in the GCG software package is used to determine the best comparison according to the best score, wherein the gap penalty of the Blossum 62 scoring matrix is ​​12, the gap extension penalty is 4, and the frameshift gap penalty is 5. The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When the position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at this position. The percentage identity between the two sequences is a function of the number of identical positions shared by the sequences.

[0098] In other embodiments, the antibody can be modified to have an altered glycosylation pattern (i.e., changed from the original or native glycosylation pattern). As used herein, "altered" means the deletion of one or more carbohydrate moieties, and / or the addition of one or more glycosylation sites to the original antibody. Adding glycosylation sites to the currently disclosed antibodies can be achieved by changing the amino acid sequence to include a glycosylation site consensus sequence; such techniques are well known in the art. Another method of increasing the number of carbohydrate moieties on an antibody is by chemical or enzymatic coupling of glycosides to antibody amino acid residues. These methods are described, for example, in WO 87 / 05330 and Aplin and Wriston (1981) CRC Crit. Rev. Biochem., 22: 259-306. Removal of any 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. Pat. No. 5,869,046 for modifications that increase in vivo half-life by providing salvage receptor binding epitopes.

[0099] In one embodiment, the anti-ZNT8 antibody has one or more CDR sequences different from those described herein (e.g., Chothia, enhanced Chothia or Kabat CDRs). In one embodiment, the anti-ZNT8 antibody has one or more CDR sequences comprising amino acid changes, such as if the length of the CDR is 5-7 amino acids, 1, 2, 3 or 4 amino acids are replaced, or if the length of the CDR is 8 amino acids or more amino acids, 1, 2, 3, 4 or 5 amino acids are replaced in the CDR sequence. The replaced amino acids can have similar charge, hydrophobicity or stereochemical characteristics. In some embodiments, amino acid replacement is a conservative replacement. "Conservative amino acid replacement" is an amino acid replacement in which an amino acid residue is replaced by an amino acid residue having a side chain with a similar charge. Families of amino acid residues with side chains with similar charges have been 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), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In other embodiments, the amino acid substitution is a non-conservative substitution. Antibodies or antibody fragments thereof containing the substituted CDRs can be screened to identify the antibody of interest.

[0100] Unlike CDR, more substantial changes can be made to the structural framework region (FR) without adversely affecting the binding properties of the antibody. Changes to the FR include, but are not limited to, humanizing a non-human derived framework or engineering certain framework residues that are important for antigen contact or for stabilizing the binding site, for example, changing the class or subclass of the constant region, changing specific amino acid residues that may change effector function (e.g., Fc receptor binding) (Lund et al., J Immun., 147: 26S7-62 (1991); Morgan et al., Immunology, 86: 319-24 (199S)), or changing the species from which the constant region is derived.

[0101] IV. Methods for Producing Anti-ZNT8 Antibodies

[0102] The anti-ZNT8 antibodies (or antigen-binding domains of antibodies or functional fragments thereof) disclosed herein can be produced in bacteria or eukaryotic cells. In order to produce a target polypeptide, 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 into host cells, select transformants, culture host cells, and recover antibodies.

[0103] If the antibody is to be expressed in bacterial cells (e.g., Escherichia coli), the expression vector should have the feature of allowing the vector to be amplified in bacterial cells. In addition, when using Escherichia coli (e.g., JM109, DH5a, HB101 or XLI-Blue) as a host, the vector must have a promoter that can allow effective expression in Escherichia coli, for example, lacZ promoter (Ward et al., 341: 544-546 (1989)), araB promoter (Better et al., Science, 240: 1041-1043 (1988)) or T7 promoter. The example of this vector includes, for example, M13 series vectors, pUC series vectors, pBR322, pBluescript, pCR-Script, pGEX-5X-1 (Pharmacia), "QIAexpress system" (QIAGEN), pEGFP and pET (when using the expression vector, the host is preferably BL21 expressing T7 RNA polymerase). The expression vector may include a signal sequence for antibody secretion. For production in the periplasm of E. coli, the pelB signal sequence (Lei et al., J. Bacteriol., 169:4379 (1987)) can be used as a signal sequence for antibody secretion. For bacterial expression, the expression vector can be introduced into bacterial cells using the calcium chloride method or electroporation method.

[0104] If the antibody is to be expressed in animal cells such as CHO, COS, 293, 293T and NIH3T3 cells, the expression vector includes a promoter necessary for expression in these cells, for example, SV40 promoter (Mulligan et al., Nature, 277: 108 (1979)), MMLV-LTR promoter, EF 1a promoter (Mizushima et al., Nucleic Acids Res., 18: 5322 (1990)) or CMV promoter. In addition to the nucleic acid sequence encoding the immunoglobulin or its domain, the recombinant expression vector may also carry other sequences, such as sequences (e.g., replication origins) and selection marker genes that regulate the replication of the vector in the host cell. The selection marker gene helps to select the host cell into which the vector has been introduced (see, e.g., U.S. Patents 4,399,216, 4,634,665 and 5,179,017). For example, typically, the selectable marker gene confers resistance to drugs (e.g., G418, hygromycin or methotrexate) on the host cell into which the vector has been introduced. Examples of vectors with selectable markers include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV and pOP13.

[0105] In one embodiment, the antibody is produced in a mammalian cell. Exemplary mammalian host cells for expressing polypeptides include: Chinese Hamster Ovary (CHO cells) (including dhfr-CHO cells, as described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220, used with a DHFR selection marker, e.g., as described in Kaufman and Sharp (1982) Mol. Biol. 159: 601 621), human embryonic kidney 293 cells (e.g., 293, 293E, 293T), COS cells, NIH3T3 cells, lymphocyte 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 mammary epithelial cell.

[0106] The antibodies of the present disclosure can be separated from the inside or outside (e.g., culture medium) of the host cell and purified into substantially pure and homogeneous antibodies. The method for separation and purification of polypeptides can generally be used for the separation and purification of antibodies described herein, and is not limited to any particular method. Antibodies can be separated and purified by appropriately selecting and merging (e.g., column chromatography, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, and recrystallization). Chromatography includes, for example, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). Liquid chromatography (e.g., HPLC and FPLC) can be used for chromatographic analysis. The column used for affinity chromatography includes protein A column and protein G column. Examples of columns using a protein A column include Hyper D, POROS, and Sepharose FF (GE Healthcare Biosciences). The present disclosure also includes antibodies that are highly purified using these purification methods.

[0107] The present disclosure also provides nucleic acid molecules or groups of nucleic acid molecules encoding anti-ZNT8 antibodies or antigen binding molecules disclosed herein. In one embodiment, the present invention includes nucleic acid molecules encoding polypeptide chains, which nucleic acid molecules include light chains of anti-ZNT8 antibodies or antigen binding molecules thereof as described herein. In one embodiment, the present invention includes nucleic acid molecules encoding polypeptide chains, which nucleic acid molecules include heavy chains of anti-ZNT8 antibodies or antigen binding molecules thereof as described herein.

[0108] Also provided are vectors or sets of vectors comprising such nucleic acid molecules or sets of nucleic acid molecules or their complement, and host cells comprising the vectors.

[0109] The present disclosure also provides a method for producing ZNT8 or an antigen-binding molecule or chimeric molecule thereof disclosed herein, the method comprising culturing the host cell disclosed herein and recovering the antibody, antigen-binding molecule or chimeric molecule thereof from the culture medium.

[0110] There are a variety of methods for recombinantly producing the ZNT8 antibodies or antigen binding molecules thereof disclosed herein or the chimeric molecules disclosed herein. It should be understood that due to the degeneracy of the code, a variety of nucleic acid sequences will encode the amino acid sequence of the polypeptide. The desired polynucleotides can be produced by de novo solid phase DNA synthesis or by PCR mutagenesis of an earlier prepared polynucleotide.

[0111] For recombinant production, a polynucleotide sequence encoding a polypeptide (e.g., a ZNT8 antibody or antigen-binding molecule thereof disclosed herein, or any chimeric molecule disclosed herein) is inserted into an appropriate expression vector, i.e., a vector that contains the necessary elements for transcription and translation of the inserted coding sequence, or in the case of an RNA viral vector, a vector that contains the necessary elements for replication and translation.

[0112] The nucleic acid encoding the polypeptide (e.g., ZNT8 antibody or its antigen binding molecule disclosed herein, or any chimeric molecule disclosed herein) is inserted into the vector in an appropriate reading frame. The expression vector is then transfected into a suitable target cell, which will express the polypeptide. Transfection techniques known in the art include, but are not limited to, calcium phosphate precipitation (Wigler et al. 1978, Cell 14: 725) and electroporation (Neumann et al. 1982, EMBO J. 1: 841). A variety of host expression vector systems can be used to express the polypeptides described herein (e.g., ZNT8 antibody or its antigen binding molecule disclosed herein, or any chimeric molecule disclosed herein) in eukaryotic cells. In one embodiment, the eukaryotic cell is an animal cell, including mammalian cells (e.g., 293 cells, PerC6, CHO, BHK, Cos, HeLa cells). When the polypeptide is expressed in a eukaryotic cell, the DNA encoding the polypeptide (e.g., ZNT8 antibody or its antigen binding molecule disclosed herein, or any chimeric molecule disclosed herein) can also encode a signal sequence that will allow the polypeptide to be secreted. Those skilled in the art will appreciate that when the polypeptide is translated, the signal sequence is cleaved by the cell to form a mature chimeric molecule. Various signal sequences are known in the art and are familiar to the skilled person. Alternatively, in the absence of a signal sequence, the polypeptide (e.g., a ZNT8 antibody or antigen binding molecule disclosed herein, or any chimeric molecule disclosed herein) can be recovered by lysing the cell.

[0113] V. Pharmaceutical Compositions

[0114] The present disclosure also provides a pharmaceutical composition comprising one or more of the following: (i) a ZNT8 antibody or antigen-binding molecule thereof disclosed herein; (ii) a nucleic acid molecule or a group of nucleic acid molecules encoding a ZNT8 antibody or antigen-binding molecule as disclosed herein; or (iii) a vector or a group of vectors disclosed herein, and a pharmaceutically acceptable carrier.

[0115] The anti-ZNT8 antibodies or fragments thereof described herein can be formulated into pharmaceutical compositions for administration to a subject (e.g., for treating a condition described herein). Typically, the pharmaceutical composition comprises a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents that are physiologically compatible. The composition may comprise a pharmaceutically acceptable salt, e.g., an acid addition salt or a base addition salt (see, e.g., Berge, SM, et al. (1977) J. Pharm. Sci. 66: 1-19).

[0116] Pharmaceutical formulation is an established art and is further described in, for example, 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).

[0117] Pharmaceutical compositions can be in a variety of forms. These include, for example, liquid, semisolid and solid dosage forms, for example, liquid solutions (e.g., injectable and infusible solutions), dispersants or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form may depend on the intended mode of administration and therapeutic application. Typically, the composition of the agents described herein is in the form of an injectable or infusible solution.

[0118] In one embodiment, the antibodies described herein are formulated with excipient materials such as sodium citrate, disodium hydrogen phosphate heptahydrate, sodium dihydrogen phosphate, -80 and a stabilizer. It can be provided, for example, in a buffer solution of appropriate concentration and can be stored at 2° C.-8° C. In some other embodiments, the pH of the composition is about 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, 7.2, 7.3, 7.4 and 7.5).

[0119] The pharmaceutical composition may also be included in the reagent that reduces antibody aggregation when formulated. The example of aggregation reducing agent includes one or more amino acids selected from the group consisting of: methionine, arginine, lysine, aspartic acid, glycine and glutamic acid. These amino acids can be added to the preparation to a concentration of about 0.5mM to about 145mM (e.g., 0.5mM, 1mM, 2mM, 5mM, 10mM, 25mM, 50mM, 100mM). The pharmaceutical composition may also include sugar (e.g., sucrose, trehalose, mannitol, sorbitol or xylitol) and / or tension modifier (e.g., sodium chloride, mannitol or sorbitol) and / or surfactant (e.g., polysorbate 20 or polysorbate 80).

[0120] The composition can be formulated into a solution, microemulsion, dispersion, liposome or other ordered structure suitable for stable storage at high concentrations. As required, a sterile injectable solution can be prepared by incorporating the required amount of an agent described herein into a suitable solvent with a combination of one or more of the ingredients listed, followed by filtration sterilization.

[0121] Typically, dispersions are prepared by incorporating the agents described herein into a sterile solvent containing a basic dispersion medium and the other ingredients listed above as required. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze drying, which obtain powders of the agents described herein and any other desired ingredients from their previous sterile filtered solutions. For example, by using a coating such as a phospholipid, by maintaining the desired particle size (in the case of dispersion) and by using a surfactant, the appropriate fluidity of the solution can be maintained. Extended absorption of the injectable composition can be achieved by including an agent that delays absorption in the composition, such as monostearate and gelatin.

[0122] In certain embodiments, the antibody can be prepared with a carrier that will protect the compound from rapid release (e.g., a controlled release formulation, 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 have been patented or are generally known. See, for example, Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York (1978).

[0123] In one embodiment, the pharmaceutical formulation comprises a concentration of about 0.005 mg / mL to 500 mg / mL (e.g., 0.005 mg / mL, 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 5 mg / mL, 10 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL) formulated with a pharmaceutically acceptable carrier. In some embodiments, the antibody is prepared in sterile distilled water or phosphate buffered saline. The pH of the pharmaceutical formulation can be between 5.5 and 7.5 (e.g., 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2 6.3, 6.4 6.5, 6.6 6.7, 6.8, 6.9 7.0, 7.1, 7.3, 7.4, 7.5).

[0124] A pharmaceutical composition may contain a "therapeutically effective amount" of an agent described herein. Such an effective amount may be determined based on the effect of the agent administered or, if more than one agent is used, the combined effect of the agents. 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 and the ability of the compound to elicit a desired response in the individual, such as improving at least one parameter of the condition or improving at least one symptom of the condition. A therapeutically effective amount is also an amount in which the therapeutically beneficial effects outweigh any toxic or deleterious effects of the composition.

[0125] A. Drug administration

[0126] The antibodies or antigen-binding fragments thereof of the present disclosure or nucleic acids encoding the antibodies or antigen-binding fragments thereof of the present disclosure can be administered to a subject, such as a subject in need, such as a human or animal subject, by a variety of methods. For many applications, the route of administration is one of the following: intravenous injection or parenteral, infusion (IV), subcutaneous injection (SC), intraperitoneal (IP) or intramuscular injection, intratumoral (IT). Other parenteral administration methods can also be used. Examples of such methods include: intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, and epidural and intrasternal injections.

[0127] In one embodiment, 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 vaginal administration. The intravenous form of parenteral administration is 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, suitable pharmaceutical compositions for injection may include a buffer (e.g., acetate, phosphate or citrate buffer), a surfactant (e.g., polysorbate), optionally a stabilizer (e.g., human albumin), etc. However, in other methods compatible with the teachings herein, the polypeptide can be delivered directly to the site of the bad cell population, thereby increasing the exposure of the diseased tissue to the therapeutic agent.

[0128] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (e.g. olive oil) and injectable organic esters (e.g. ethyl oleate). Aqueous vehicles include water, alcohol / aqueous solutions, emulsions or suspensions, including saline and buffered media.

[0129] Pharmaceutically acceptable carriers include, but are not limited to, phosphate buffer or 0.8% saline of 0.01M-0.1M and preferably 0.05M. Other common parenteral vehicles include sodium phosphate solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient supplements, electrolyte supplements (e.g., supplements based on Ringer's dextrose), etc. Preservatives and other additives may also be present, for example, antimicrobial agents, antioxidants, chelating agents, and inert gases, etc.

[0130] More particularly, the pharmaceutical composition suitable for injection includes sterile aqueous solution (in the case of water-soluble) or dispersion and the sterile powder for the temporary preparation of sterile injectable solution or dispersion. In this case, the composition must be sterile and should flow to the extent that easy injection exists. It should be stable under manufacturing and storage conditions, and will preferably be preserved, resisting the contamination of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium, which includes, for example, water, ethanol, polyols (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. For example, by using a coating such as phospholipids, by maintaining the required particle size (in the case of dispersion) and by using a surfactant, appropriate fluidity can be maintained.

[0131] Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols (e.g., mannitol, sorbitol), or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0132] In any case, sterile injectable solutions can be prepared by incorporating the active compound (eg, the polypeptide itself or in combination with other active agents) in the required amount in an appropriate solvent with one or more of the ingredients enumerated above, as required, followed by filtered sterilization.

[0133] Typically, dispersions are prepared by incorporating the active compound into a sterile solvent containing a basic dispersion medium and other required ingredients listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze drying, which obtain powders of the active ingredient and any other desired ingredients from their previously sterile filtered solutions. The preparations for injection 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, these preparations can be packaged and sold in the form of kits. Such products will preferably have a label or package insert indicating that the relevant composition can be used to treat subjects suffering from or susceptible to coagulation disorders.

[0134] The effective dose of the disclosed compositions for treating a condition varies depending on many different factors, including: the mode of administration, the target site, the physiological state of the patient, whether the patient is human or animal, other drugs administered, and whether the treatment is preventive or therapeutic. Typically, the patient is a human, but non-human mammals, including transgenic mammals, can also be treated. Conventional methods known to those skilled in the art can be used to titrate the therapeutic dose to optimize safety and efficacy.

[0135] The route and / or mode of administration of the anti-ZNT8 antibody or fragment thereof can also be tailored to the individual situation, for example, by monitoring the subject.

[0136] Antibodies or fragments thereof can be administered in fixed doses or in mg / kg dosages. Dosages can also be selected to reduce or avoid producing antibodies against anti-ZNT8 antibodies or fragments thereof. Dosage regimens are adjusted to provide desired responses, such as therapeutic responses or combined therapeutic effects. Typically, a dose of an antibody or fragment thereof (and optionally a second agent) can be used to provide a bioavailable amount of agent to an object. For example, the dosage range that can be administered is 0.1mg / kg-100mg / kg, 0.5mg / kg-100mg / kg, 1mg / kg-100mg / kg, 0.5mg / kg-20mg / kg, 0.1mg / kg-10mg / kg or 1mg / kg-10mg / kg. Other dosages can also be used. In certain embodiments, an antibody or fragment thereof of a dosage of about 1mg / kg to about 30mg / kg is administered to an object that needs to be treated with an antibody or fragment thereof. In some embodiments, a dose of 1 mg / kg, 2 mg / kg, 4 mg / kg, 5 mg / kg, 7 mg / kg, 10 mg / kg, 12 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 28 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg or 50 mg / kg of the antibody or fragment thereof is administered to a subject in need of treatment with an anti-ZNT8 antibody or fragment thereof. In a specific embodiment, the antibody or fragment thereof is administered subcutaneously at a dose of 1 mg / kg to 3 mg / kg. In another embodiment, the antibody or fragment thereof is administered intravenously at a dose of 4 mg / kg to 30 mg / kg.

[0137] The composition can comprise about 1 mg / mL to 100 mg / mL, or about 10 mg / mL to 100 mg / mL, or about 50 mg / mL to 250 mg / mL, or about 100 mg / mL to 150 mg / mL, or about 100 to 250 mg / mL of the antibody or fragment thereof.

[0138] As used herein, unit dosage form or "fixed dose" refers to physically discrete units suitable as unit dosages for the subject to be treated; each unit contains a predetermined amount of the antibody or fragment thereof calculated to produce the desired therapeutic effect in combination with the required pharmaceutical carrier and optionally other agents. A single dose or multiple doses may be administered. Alternatively or additionally, the antibody or fragment thereof may be administered by continuous infusion.

[0139] The dosage of the antibody or fragment thereof can be, for example, sufficient to cover: at least 2 doses, 3 doses, 5 doses, 10 doses or more, for example once or twice a day, or about one to four times a week, or preferably weekly, biweekly (every two weeks), every three weeks, monthly, for example, about 1 to 12 weeks, preferably 2 to 8 weeks, more preferably about 3 to 7 weeks, even more preferably about 4 weeks, 5 weeks or 6 weeks of time period (a course of treatment) administered at periodic intervals. Factors that may affect the dosage and time required to effectively treat a subject include, for example, the stage or severity of the disease or condition, formulation, delivery route, previous treatment, the overall health and / or age of the subject, and the presence of other diseases. In addition, treatment of a subject with a therapeutically effective amount of a compound can include a single treatment, or preferably, can include a series of treatments.

[0140] If a subject is at risk of developing a condition described herein, the antibody or fragment thereof may be administered prior to the full onset of the condition, e.g., as a preventive measure. The duration of such preventive treatment may be a single dose of the antibody or fragment thereof, or the treatment may continue (e.g., multiple doses). For example, a subject at risk of or susceptible to the condition may be treated with the antibody or fragment thereof for days, weeks, months, or even years to prevent the onset or outbreak of the condition.

[0141] In certain embodiments, the dosage form is about 1 mg / mL to about 500 mg / mL (e.g., 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, In one embodiment, 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 one embodiment, the antibody or fragment thereof is administered intravenously at a concentration of 50 mg / mL.

[0142] Intermediate doses within the above ranges are also intended to be within the scope of the invention. Such doses may be administered to a subject daily, every other day, weekly, or according to any other schedule determined by empirical analysis. An exemplary method of treatment requires administration of multiple doses over an extended period of, for example, at least six months. In some methods, two or more polypeptides may be administered simultaneously, in which case the dose of each polypeptide administered is within a specified range.

[0143] The polypeptides of the present invention can be administered at multiple times. The intervals between single doses can be daily, weekly, monthly or annual. Intervals can also be irregular as indicated by measuring the blood levels of the modified polypeptide or antigen in the patient. Alternatively, the polypeptide can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency depend on the half-life of the polypeptide in the patient.

[0144] The dosage and frequency of administration can vary depending on whether the treatment is preventive or therapeutic. In preventive applications, a composition containing a polypeptide of the present invention or a mixture thereof is administered to a patient who is not yet in a disease state to enhance the patient's resistance or minimize the effects of the disease. This amount is defined as a "preventive effective dose". Over a long period of time, a relatively low dose is administered at relatively infrequent intervals. Some patients continue to receive treatment throughout their lives.

[0145] VI. Devices and Kits for Treatment

[0146] Anti-ZNT8 antibodies or fragments thereof may be provided in a kit. In one embodiment, the kit includes: (a) a container comprising a composition comprising an anti-ZNT8 antibody or fragment thereof as described herein; and optionally (b) informational material. The informational material may be descriptive, instructional, marketing or other material related to the use of the methods and / or agents described herein for therapeutic benefit.

[0147] In one embodiment, the kit further comprises a second agent for treating a condition described herein. For example, the kit comprises: a first container comprising a composition comprising an anti-ZNT8 antibody or fragment thereof; and a second container comprising a second agent.

[0148] The information material of the kit is not limited to its form. In one embodiment, the information material may include information about the production of the compound, the molecular weight, concentration, expiration date, batch or production site information of the compound, etc. In one embodiment, the information material relates to, for example, a method of administering an anti-ZNT8 antibody or fragment thereof in a suitable dose, dosage form or administration (e.g., a dose, dosage form or administration as described herein) to treat an object that has suffered from a disease described herein or is at risk of suffering from a disease described herein. Information can be provided in a variety of formats, including printed text, computer-readable material, video recording or audio recording, or information providing a link or address of substantial material such as on the Internet.

[0149] In addition to anti-ZNT8 antibodies or fragments thereof, the composition in the kit may also include other ingredients, such as solvents or buffers, stabilizers or preservatives. Anti-ZNT8 antibodies or fragments thereof may be provided in any form, such as liquid, dry or lyophilized form, preferably substantially pure and / or sterile. When the reagent is provided in a liquid solution, the liquid solution is preferably an aqueous solution. In some embodiments, the concentration of anti-ZNT8 antibodies or fragments thereof in the liquid solution is about 25mg / mL to about 250mg / mL (e.g., 40mg / mL, 50mg / mL, 60mg / mL, 75mg / mL, 85mg / mL, 100mg / mL, 125mg / mL, 150mg / mL and 200mg / mL). When anti-ZNT8 antibodies or fragments thereof are provided as lyophilized products, anti-ZNT8 antibodies or fragments thereof are about 75mg / vial to about 200mg / vial (e.g., 100mg / vial, 108.5mg / vial, 125mg / vial, 150mg / vial). Lyophilized powders are typically reconstituted by adding a suitable solvent. A solvent, such as sterile water or a buffer (eg, PBS) may optionally be provided in the kit.

[0150] The kit may include one or more containers for one or more compositions containing reagents. In some embodiments, the kit includes separate containers, dividers or compartments for compositions and informational materials. 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 other embodiments, the separate elements of the kit are contained in a single, undivided container. For example, the composition is contained in a bottle, vial or syringe with informational material in the form of a label. In some embodiments, the kit includes a plurality of (e.g., a pack) of separate containers, each container containing one or more unit dosage forms of a reagent (e.g., a dosage form described herein). The container may include a combined unit dose, such as a unit containing an anti-ZNT8 antibody or a fragment thereof and a second reagent, such as in a desired ratio. For example, the kit includes a plurality of syringes, ampoules, foil bags, blister packs or medical devices, for example, each containing a single combined unit dose. The container of the kit may be airtight, waterproof (e.g., impermeable to changes in moisture or evaporation) and / or opaque.

[0151] The kit may optionally include a device suitable for administering the composition, for example, a syringe or other suitable delivery device. The device may be pre-filled with one or both reagents, or may be empty but suitable for loading.

[0152] 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 following examples are merely illustrative, and are not intended to limit the remainder of the disclosure in any way whatsoever.

[0153] Example

[0154] The following examples are presented to provide a person of ordinary skill in the art with a complete disclosure and description of how to prepare and evaluate the compounds, compositions, articles, devices and / or methods described and claimed herein, and the examples are exemplary only and are not intended to limit the scope of the invention that the inventors consider to be their inventions. Efforts have been made to ensure the accuracy of numbers (e.g., quantity, temperature, etc.), but some errors and deviations should be considered herein. Unless otherwise stated, parts are parts by weight, temperatures are degrees Celsius or at ambient temperature, and pressures are atmospheric pressure or near atmospheric pressure. Reaction conditions have a variety of variations and combinations, such as component concentrations, desired solvents, solvent mixtures, temperature, pressure, and other reaction ranges and conditions, which can be used to optimize the product purity and yield obtained from the method. Only reasonable and conventional experiments will be required to optimize such process conditions.

[0155] As described below, the inventors identified two mAbs (mAb20, mAb39) by multilayer screening for the purified human ZnT8 of native form. Detailed mAb characterization shows unique binding properties and specificity, and it has the common ability of stabilizing ZnT8 structural folding, and therefore inhibits its transport activity by slowing down the turnover rate of zinc. These findings prove the practicality of mAb as the allosteric inhibitor of human ZnT8. The inventors also identified the stable ternary complex of ZnT8 monomer and two different Fab. The adjacent relationship of two mAbs mediated by ZnT8 allows the FRET signal between a pair of donors and the covalently attached acceptor fluorophore combined with mAb to carry out ZnT8 quantitatively. In this assay, we used a caged lanthanide donor (terbium cryptate), which is characterized by a long fluorescence emission half-life. After the short background fluorescence decays, the FRET signal derived from the ternary complex can be obtained using delayed fluorescence measurement. Therefore, this assay can be carried out in a homogeneous form without separating the binding complex from uncombined mAb (30). This simple add-and-read HTRF assay highlights another utility of the mAb as a ZnT8-specific biosensor.

[0156] Materials and methods

[0157] Generation of hybridomas. Four pairs of male / female homozygous Slc30a8- / - mice were immunized by weekly intraperitoneal injection of human ZnT8 antigen (R325 variant) in reconstituted proteoliposomes. All experiments performed on mice were approved by the Institutional Animal Care and Use Committee of Johns Hopkins University School of Medicine. Serum antibody titration and validation of anti-ZnT8 specificity by CRISPR / Cas9 ZnT8 knockout have been described previously (36). After the final immune boost, spleens were isolated. Splenocytes were fused with the immortalized myeloma cell line Sp2 / 0-Ag14 (CRL-1581; ATCC) by adding 1 ml of 50% PEG1500 (w / v; Roche) dropwise to the cell mixture over 1 minute, with a spleen cell to myeloma ratio of 4:1 (37). The cells were allowed to rest in the PEG solution for one minute under gentle mixing, and then 20 ml of serum-free DMEM was added stepwise over 4-5 minutes. The cells were peeled off and then resuspended in 15 ml of DMEM, to which 10% FBS and hybridoma fusion and cloning supplement (HFCS) and hypoxanthine, aminopterin and thymidine (HAT) were added. After fusion for eight days, dead Sp2 / 0 cells were removed, and hybridoma cells were seeded in 96-well plates at a density of 5 cells per well and kept under HAT selection.

[0158] Screening hybridomas. A multiplex ELISA was developed to evaluate hybridoma supernatants. The first screening was performed by passive coating (200ng ZnT8 / well) using native human ZnT8 antigen in reconstituted proteoliposomes. The coated plate was pre-blocked with 5% BSA in assay buffer (20mM HEPES, 100mM NaCl, pH7.0), and 20μl of hybridoma supernatant was added to each well, incubated at RT for 2hr, and then washed 3 times with assay buffer plus 1% BSA. The bound antibodies were detected by 1:3000 dilution of HRP-conjugated goat anti-mouse secondary antibody (Invitrogen, catalog number 62-6500) in assay buffer, followed by ABTS colorimetric reaction (Life Technologies) and 415nm signal reading on a microplate reader (Molecular Device). The second screening was performed by affinity binding to Ni-NTA plates (100ng CTD / well) using native CTD-His monomers. The ELISA procedure was the same as above. The third screening used native ZnT8 in lotion micelles. Antigen binding was between two layers of antibodies: the capture antibody was mAb20 fixed on a protein A / G plate (800ng / well). Only detection antibodies that bind to non-overlapping epitopes gave positive ELISA readings. Purified MSA and commercially available NTDA (Proteintech, catalog number 16169-I-AP) and CTDA (Amgen) were used as negative and positive controls to calibrate ELISA dynamic range and sensitivity.

[0159] Validation of mAbs . Monoclonal hybridoma cell lines were established by single-cell sorting by flow cytometry to ensure clonal purity. After clonal expansion, secreted mAbs were purified by protein A / G affinity chromatography (Thermo Scientific, catalog number 20423), isoformed (Thermo Scientific, catalog number 37503), and Fab fragments were prepared by papain digestion (ThermoScientific, catalog number 44985). Purified mAbs or Fabs were tested for: (i) binding complex formation with purified ZnT8 or ZnT8-GFP on size exclusion HPLC; (ii) immunofluorescence staining of HEK293 cells (without ZnT8 expression) followed by confocal microscopy imaging; (iii) immunofluorescence staining of large cell populations followed by flow cytometric quantification; and (iv) formation of Fab-ZnT8 complexes by EM negative staining and single particle analysis.

[0160] Fluorescence size exclusion HPLC. Purified mAb20 / Fab20 or mAb39 was mixed with Alexa Fluor 488 or AlexaFluor555 carboxylic acid succinimidyl ester in PBS buffer at a molar ratio of 1:20 at RT for 60 min, and the labeling reaction was terminated by quenching with 100 mM Tris-HCL. The fluorescently labeled mAb or Fab was HPLC purified to remove traces of free fluorescent dye and concentrated to 1 mg / ml for storage in PBS buffer at 4°C. The detergent micelles of purified human ZnT8 were incubated with mAb20 / Fab20 or mAb30 for 2 hours, centrifuged at 258000×g for 30 min to precipitate cross-linked ZnT8 aggregates, and then injected into a TSKgel G3000SWXL size exclusion HPLC column (Tosoh Biosciences) pre-equilibrated with buffer (supplemented with 0.02% DDM). Alternatively, ZnT8-GFP in the wash crude extract of INS-1E cells stably expressing human ZnT8 is purified by GFP-fluorescence sizing-HPLC. Next, the peak fraction of ZnT8-GFP is incubated with mAb20 or mAb39 at 4 ° C for 3 hr, and the cross-linked aggregates are removed by centrifugation, and then injected into TSK columns as described above. The elution graph is monitored using a multi-channel fluorescence detector with the following excitation / emission pairs: AlexaFluor 488, Alexa Fluor 555 and GFP fluorescence are respectively monitored by 480 / 520nm, 485 / 555nm, 480 / 520nm. The individual ZnT8 peak is monitored by a UV detector connected to a fluorescence detector.

[0161] Negative stain electron microscopy . Human ZnT8 was purified as described previously (20). Fab20 or Fab20+Fab39 was added to ZnT8 at a molar ratio of 10:1. The binding complex was purified again by size exclusion HPLC and concentrated to 1 mg / ml-2 mg / ml. The protein sample was diluted to 20 μg / ml, and a 3 μl aliquot of the diluted sample was applied to a glow-discharged EM grid covered with a continuous thin carbon film and stained with 2% aqueous uranyl acetate for 1.5 min. The grid was loaded onto a Tecnai Spirit electron microscope operating at a high voltage of 120 kV. A Gatan Orius CCD camera was used in low-dose mode Raw images were collected with a camera with a defocus value range of 1 μm to 2.5 μm and a magnification of 30,000, corresponding to specimen-level / pixel. We recorded 221 and 71 micrographs of the binary (Fab20)2-ZnT8 complex and the ternary (Fab20)2-ZnT8-(Fab39)2 complex, respectively. The contrast transfer function parameters of each image were determined by CTFFIND4.1.5 (38). 76,274 binary particles and 6,107 ternary particles were selected from the micrographs. After 2D classification in RELION2.1.0 (39) and 3D classification in cryoSPARC (40), 15,745 binary particles and 2,369 ternary particles were retained for 3D reconstruction. 3D refinement was performed using cryoSPARC. The estimated resolution of both binary and ternary complex maps was 3 nm. The 3D map was docked in UCSF Chimera (42) using the crystal structures of Fabs targeting Shigella flexneri Y lipopolysaccharide (PDB ID 1M71) (41) and the Escherichia coli zinc transporter YiiP (PDB ID 3H90) (29).

[0162] Immunofluorescence staining and cell imaging . Flp-In, T-REx-HEK293 cells stably expressing human ZnT8 variants were grown and induced on coverslips to a confluence of 50% as described previously (20). Cells were fixed (R&D Systems, catalog number FC004) and permeabilized (R&D Systems, catalog number FC005). HEK293 cells were stained with mAb20 or mAb39 at a dilution of 1:3000 from a 1 mg / ml mAb stock, while EndoC-βH cells were stained with mAb20 and mAb39 from the same batch at a dilution of 1:1000. Following the same procedure, parental Flp-In, T-REx-HEK293 cells without ZnT8 expression were used as negative controls. Fluorescence imaging was performed on a Zeiss LSM 700 inverted confocal microscope with a 63× oil objective using goat anti-mouse secondary antibodies conjugated to Alexa Fluor-594 (Thermo Scientific, catalog number A11005, dilution 1:1000) and DAPI as described above ( 20 ).

[0163] Flow cytometric analysis HEK293 cells without ZnT8 overexpression were fixed, permeabilized, and cultured at 1 × 10 6The density of 100 μ l of cell suspension was resuspended in PBS plus 5% BSA at 1:1000. The aliquots of 100 μ l cell suspension were stained with 3-fold serial dilutions of mAb20 or mAb39 for 2 hours at RT, and then stained with 5% BSA and Alexa fluor 647-conjugated donkey anti-mouse secondary antibody (Thermo Fisher Scientific, catalog number A31571) diluted 1:1000. The labeled cells were analyzed immediately by flow cytometry. Cells gated in forward scatter and side scatter showed a separate group, which was represented by a histogram of ~3000 cell counting events. The average immunofluorescence intensity and standard error were obtained using flow cytometer analysis software Summit 4.3 (Beckman Coulter). EndoC-βH cells and HEK293 cells as ZnT8-KO substitutes were fixed and permeabilized as described above, and stained with mAb20 and mAb39 at 1:1000 dilutions, and then stained with secondary antibodies as described above. The same flow cytometry protocol was used to analyze labeled EndoC-βH cells and HEK293 cells.

[0164] Immunoblotting SDS-lysed cell lysates of EndoC-βH cells or HEK293 cells overexpressing ZnT8 were subjected to SDS-PAGE and probed with mAb20 or mAb39 at 1:3000, followed by anti-mouse HRP-conjugated secondary antibody (GE Healthcare, catalog number NA931V) at 1:3000. Coomassie staining was performed on gel replicas showing equal protein loading per lane (100 μg). To compare the reactivity of mAb20 or mAb39 with different ZnT homologs, we used commercial HEK293 lysates lentivirally overexpressing ZnT1, ZnT2, ZnT3, ZnT4, ZnT7, ZnT8, ZnT10, each tagged with a Flag tag at the C-terminus (Origene, catalog number LY500001, LC412033, LC423751, LC401170, LY415673, LC428544, LC406320, and LC412886). Approximately equal amounts of total protein (5 μg) were loaded into each lane and then probed with mAb20, mAb39, or anti-FLAG antibody (Origene, catalog number TA50011-100) as described above. The expression levels of ZnT homologs varied with ZnT1 being the most abundantly expressed.

[0165] Stopped-flow dynamicsHuman ZnT8 (R325 variant) was purified and reconstituted into proteoliposomes (wt / wt) with a defined lipid composition of DOPC:DOPG:DOPE (2:1:1) at a protein to lipid ratio of 1:50 as described previously (20). Preformed proteoliposomes were mixed with 200 μM Fluozin-3 (without 100 μg Fab) in 0.2 ml assay buffer and subjected to three cycles of freeze-thaw followed by 10 s sonication to complete encapsulation of dye and Fab. Extravesicular Fluozin-3 was removed by washing the vesicles with 3 × 25 ml assay buffer, followed by the addition of an additional 100 μg Fab to ensure saturation of Fab binding. The estimated molar ratio of Fab to ZnT8 was 2.8:1. ZnT8-mediated zinc transport into the proteoliposomes was triggered by rapid mixing of the proteoliposomes and assay buffer on a SFM-3000 stopped-flow apparatus (Bio-logic) as described previously (20). According to maxchelator (maxchelator.stanford.edu), increasing amounts of ZnSO4 were added to the assay buffer of 1 mM sodium citrate, which buffered free zinc concentrations from 6 μM to 116 μM. All kinetic traces are cumulative averages of 9 consecutive recordings. The liposome trace was collected as a baseline and subtracted from the proteoliposome trace to obtain the net fluorescence change ΔF. ΔF / ΔF was obtained by normalizing ΔF to the maximal liposome response induced by the assay buffer containing 2 mM ZnSO4 and 2% β-OG. max The initial rate of zinc influx was obtained by linear regression of the data points in the quasi-linear phase of the initial fluorescence rise (t < 10 s). max M / (M+K m The concentration dependence data were analyzed by least squares fitting of the initial transport rate of the hyperbola defined by , where M represents the free zinc ion concentration, V max is the maximum transport rate when the transport rate approaches the quasi-steady state, and K m is the Michaelis-Menten constant. The experimental data were fitted using the data analysis software SIGMAPLOT (SPSS Inc.).

[0166] HTRF assay. Purified mAb20 and mAb39 were labeled with terbium cryptates and d2, respectively, according to the manufacturer's protocol (Cisbio Bioassays, Bedford, MA). The molecular ratio between the molecule and the dye in the conjugate was xx for mAb20 and xx for mAb39. The labeled mAbs were HPLC purified in PBS (?), concentrated to 1 mg / ml, and stored at 4°C. 293-F cells transiently expressing human ZnT8, human ZnT3, or mouse ZnT8 in suspension culture were harvested. The cell pellet was solubilized with 1% DDM in 20mM HEPES, 100mM NaCl (pH 7.0) and a protease mixture, and centrifuged at 16000g for 15 minutes to remove debris to prepare a wash crude extract. The supernatant was collected for ZnT8 detection. The precipitate was dissolved with a 2% DDM solution in PBS, and the crude extract was cleared of debris for ZnT8 detection. HTRF analysis was performed in 384-well plates (Greiner Bio-one, catalog number 784075) by adding labeled mAb20, mAb39 in a 1:1 ratio and wash extracts of 293-F cells or EndoC-βH cells at appropriate dilutions to a total volume of 20 μl. After 2 hours of binding to mAb at room temperature, fluorescence emission was measured using a microplate reader (Molecular Devices) equipped with a flash lamp for fluorophore excitation with a 40 μs delay at 340 nm, a 620 nm filter for terbium cryptate fluorescence readings, and a 665 nm filter for d2 fluorescence detection. Data were dropped according to the ratio of fluorescence measured at 665 nm (d2) and 620 nm (terbium cryptate), using the 620 nm signal as an internal standard. Ratio measurements were corrected for well-to-well differences and signal quenching from potential interfering components in the binding mixture. The background signal was measured and used to calculate the relative ZnT8 reading ΔF for inter-batch comparison: ΔF = (ratio 样品 -ratio 背景 ) / ratio 背景 The actual ZnT8 concentration in the assay was estimated based on the ZnT8 production transiently expressed by 293-F cells and analyzed by linear regression (r 2 =0.99) were correlated to the HTRF readings to generate a standard curve. LoD and LoQ values ​​were obtained by interpolation of the measured data points on the standard curve. Z' factor values ​​for HTRF assays were calculated by comparing multiple replicates of HTRF readings of crude wash extracts collected from EndoC-βH, HEK293, and INS-1E cells in a single experiment as described previously (44).

[0167] result

[0168] Identification of different anti-ZnT8 mAbsHuman ZnT8 (type B) is a homodimer of two 35.1 kDa promoters, each consisting of a 23.8 kDa transmembrane domain (TMD) followed by an 11.3 kDa CTD. The ZnT8 homodimer is hereinafter referred to as ZnT8. Immunization of ZnT8-KO mice with purified human ZnT8 in proteoliposomes produced serum antibodies that exhibited an antibody titer that was ~100-fold higher than that of two commercially available antibodies directed against linear peptide sequences of the N-terminal domain and the C-terminal domain (NTD and CTD), respectively (31). Splenocytes from immunized mice were isolated and fused with myeloma cells to establish immortalized hybridomas. Preliminary screening by enzyme-linked immunosorbent assay (ELISA) identified anti-ZnT8 antibodies that showed different reactivity to the full-length ZnT8 antigen and the monomeric CTD antigen (aa 275-369) in proteoliposomes. These hybridoma cells were single-cell sorted and clonal expanded, and then the two secreted monoclonal antibodies (mAb20 and mAb39) were purified for further characterization. Commercially available anti-NTD antibodies and anti-CTD antibodies (NTDA and CTDA) or purified mouse serum antibodies (MSA) from non-immunized ZnT8-KO mice were used as positive or negative controls. mAb20, mAb39, CTDA, and NTDA all produced positive readouts against the ZnT8 proteoliposome antigen ( Figure 1A In contrast, only mAb20 and the positive control CTDA recognized the monomeric CTD antigen. mAb39 and the negative control NTDA gave negative results in the ELISA with CTD probing ( Figure 1B ), indicating that the epitope recognized by mAb20 is different from that recognized by mAb39.

[0169] Stable Antibody-ZnT8 Complex The formation of stable mAb-ZnT8 complexes or Fab-ZnT8 complexes was examined by analytical size exclusion HPLC. mAb in excess of ZnT8 tended to form cross-linked protein aggregates. By adjusting the stoichiometry of mAb:ZnT8, we detected Figure 2 A series of eight different monodisperse peaks marked by 1-8 in . mAb20 and Fab20 are labeled with green fluorophores, while mAb39 is labeled with red fluorophores. mAb20 / Fab20 and mAb39 compounded with ZnT8 are monitored in separate channels of the fluorescence detector ( Figure 2). The apparent molecular weight (MW) of each peak was calculated, and the predicted complex compositions consistent with the MW are summarized in Table 1. For binary binding, Fab20 formed a single (Fab20)2-ZnT8 complex (peak 3), and mAb20 mainly formed a mAb20-ZnT8 complex (peak 2). In contrast, mAb39 mainly formed a (mAb39)2-ZnT8 complex (peak 1) and a mAb39-ZnT8 minor complex aligned with peak 2. For ternary binding between ZnT8, mAb20 / Fab20 and mAb39, similar (mAb39)2-ZnT8-mAb20 complexes (peaks 5 and 7) and mAb39-ZnT8-(Fab20)2 complexes (peaks 6 and 8) were recorded in both the green and red channels, indicating that mAb20 / Fab20 and mAb39 independently bound to a single ZnT8 (peak 4).

[0170] Table 1. Summary of binary and ternary complex peaks

[0171]

[0172] Single particle electron microscopy (EM) analysis To further characterize antibody binding, we purified the binary (Fab20)2-ZnT8 and ternary (Fab20)2-ZnT8-(Fab39)2 complexes and directly visualized Fab binding by negative stain EM. Raw image of binary complex ( Figure 3A ) and the corresponding 2D reference-free average show two Fabs in an intact complex, each with a clearly visible characteristic Fab groove between the variable and constant regions ( Figure 3B ). Therefore, 2-fold symmetry was applied to the single-particle 3D reconstruction, resulting in a low-resolution (~3 nm) 3D map. The 3D envelope was fitted to the crystal structure of two Fab molecules (PDB ID 1M71) and a ZnT8 homolog YiiP (PDB ID 3H90). The ZnT8 envelope is larger than the crystal structure, which may be due to the presence of detergent micelles around the transmembrane region ( Figure 3E ). For the ternary complex, the original image shows the binding of multiple Fab molecules to a single ZnT8 dimer ( Figure 3C Representative 2D averages also show multiple Fab binding ( Figure 3D ). The 3D reconstruction shows three discrete protrusions on the surface of the CTD ( Figure 3F As observed in ZnT8-(Fab20)2, the two external protrusions are smaller and in similar but slightly lower positions, thus belonging to a pair of Fab20 molecules ( Figure 3FThe middle protrusion is larger and can be assembled by two Fab molecules spanning the two-fold axis of the ZnT8 dimer interface, and is therefore attributed to two Fab39 molecules ( Figure 3F ). Although the atlas resolution was insufficient to distinguish different types of Fab in the ternary complex, docking of the 3D model with homologous structures indicated that CTD dimerization is required for mAb39 binding. This finding was supported by negative mAb39 readouts against monomeric CTD antigen in CTD-ELISA ( Figure 1B By comparison, each Fab20 binds to a single CTD monomer in the 3D model ( Figure 3E-Figure 3F ), which is consistent with the positive mAb20 readout in the CTD-ELISA.

[0173] Collapse Dependencies To compare the differences in mAb binding to native and denatured ZnT8, we unfolded immobilized ZnT8-His on Ni-NTA plates using 2% SDS and then compared the denatured ELISA readings with undenatured replicas. The native readout for mAb20 was approximately twice that of the denatured readout ( Figure 4A ), indicating moderate selectivity for the folded conformation compared to the unfolded ZnT8. As a control, CTDA was reacted indiscriminately with native ZnT8 and denatured ZnT8. Unexpectedly, although mAb39 bound to the dimer interface and was anti-CTD negative ( Figure 1B ), but for denatured ZnT8 immobilized on Ni-NTA plate, it was ELISA positive ( Figure 4A ). One possibility is that the mAb39 epitope is close to the C-terminus, where a pair of His tags from the ZnT8 dimer may stabilize local dimer interactions via Ni-NTA binding. To test this hypothesis, we added a larger green fluorescence protein (GFP) to the C-terminus and examined its potential steric hindrance to mAb binding. Size exclusion HPLC showed that mAb20 shifted the ZnT8-GFP peak to the left, as predicted for the mAb20-ZnT8-GFP binary complex ( Figure 4B ). In contrast, C-terminal GFP completely blocked the formation of the mAb39-ZnT8-GFP binary complex. The steric clash between GFP and mAb39 binding localized the mAb39 binding site to the C-terminus. This result further confirmed that mAb39 docks to the distal CTD surface across the dimer interface ( Figure 3F ).

[0174] Enhance the stability of ZnT8. Binding of mAbs to conformational epitopes is expected to stabilize protein folding. Unfolded ZnT8 can be precipitated as protein aggregates by ultracentrifugation. To evaluate the effect of antibody binding on ZnT8 stability, we incubated detergent-solubilized ZnT8 with or without Fab, then exposed protein samples to heat denaturation at 37°C and monitored folded ZnT8 in the supernatant after ultracentrifugation. Quantitative immunoblotting showed that ZnT8 was unstable and began to precipitate after one hour of heat exposure ( Figure 4C ). Fab20 or Fab39 binding delayed the onset of ZnT8 precipitation to 2 hours, and this protective effect was further enhanced by simultaneous binding of Fab20 and Fab39. At 3 hours, only 12% of the initial ZnT8 remained in the supernatant, while more than 70% of (Fab20)2-ZnT8-(Fab39)2 was still soluble.

[0175] Inhibits zinc transport The stabilization effect of Fab20 and Fab39 suggests that Fab binding maintains ZnT8 in a more stable conformation. We further examined the effect of conformational restriction on the kinetics of ZnT8-mediated zinc transport. Purified human ZnT8 was reconstituted in proteoliposomes encapsulated with the zinc fluorescent indicator Fluozin-3 and then exposed to a range of extracellular zinc concentrations ( Figure 5A ). Fixed 1 mM citrate was added to the assay buffer containing increasing zinc concentrations, resulting in free zinc concentrations of 1.6 μM to 116 μM. This ensured a continuous supply of free zinc to meet steady-state conditions. Based on the differences in mAb immunofluorescence staining between sealed and lotion-perforated proteoliposomes, it was estimated that the transmembrane orientation of the reconstituted ZnT8 exposed 50%-75% of the CTD outside the proteoliposome capsule. Fab20 or Fab39 was loaded onto the proteoliposomes by brief sonication to balance the intracapsular Fab and extracapsular Fab. Size HPLC of the re-dissolved, Fab-treated proteoliposomes confirmed complete occupancy of Fab binding. Rapid mixing of the proteoliposomes with the zinc buffer triggered a quasi-linear increase in Fluozin-3 fluorescence within the first 10 seconds ( Figure 5A ). Calculate the rate of fluorescence rise and fit it with the Michaelis-Menten equation ( Figure 5B The steady-state kinetics of V are summarized in Table 2. max Value and K m The values ​​show that Fab20 or Fab39 increased V max The inhibitory effects of individual Fab binding were additive, and when ZnT8 was dually bound to Fab20 and Fab39, V maxThe magnitude of the Fab inhibition effect was similar to that of the low-risk W325 variant and was ∼50% of that of the high-risk R325 variant (20). No K m The significant difference in the values ​​indicated that Fab inhibited ZnT8 by reducing its transport rate.

[0176] Table 2. Steady-state zinc transport kinetic parameters

[0177]

[0178] Binding affinity We examined the binding of the mAbs to HEK293 cells stably expressing human ZnT8. Confocal microscopy imaging of immunofluorescence staining using mAb20 and mAb39 showed strong intracellular signals ( Fig. 6A ). No mAb staining was detected in parental HEK293 cells without ZnT8 overexpression. Next, we extended the imaging analysis to large cell populations using flow cytometry to analyze serial dilutions of stably transfected cells or parental cells labeled with mAb20 or mAb39. For each mAb concentration, approximately 3000 cell counting events were recorded to form a fluorescence intensity histogram ( Figure 6B ). mAb dilution gradually shifted the peak to the left, yielding a quantitative measure of concentration-dependent mAb labeling. mAb20 and mAb39 showed similar binding curves with affinities of 0.8 nM ± 0.1 nM and 0.7 nM ± 0.1 nM, respectively ( Figure 6C ).

[0179] Binding specificity We first examined cross-species specificity by comparing mAb binding to human and mouse ZnT8, both overexpressed in HEK293 cells, then solubilized with detergent (DDM) and immobilized on Ni-NTA plates via a C-terminal polyhistidine tag. mAb titration of mAb20 showed a strong difference between human and mouse, shifting the mouse binding curve to the right with a 566.8-fold difference in apparent binding affinity (2.5 nM ± 0.2 nM vs. 1422.0 nM ± 72.6 nM) ( Fig. 7A In contrast, the binding of mAb39 to human ZnT8 was similar to that to mouse ZnT8, with only a negligible 2.0-fold difference (17.6 nM ± 1.7 nM vs. 35.8 nM ± 3.0 nM) ( Fig. 7A). These results indicate that mAb20 is specific for human ZnT8, while mAb39 recognizes both human and mouse ZnT8. Notably, epitope masking by ZnT8 immobilization attenuated the apparent binding affinity of both mAbs compared to the affinity determined by flow cytometry, and this attenuation was more pronounced for mAb39, likely due to the closer proximity of the mAb39 epitope to the C-terminal His tag. Next, we compared the immunoreactivity of the mAbs to denatured ZnT8. Immunoblotting lysates of HEK293 transfectants showed that denaturation of ZnT8 on SDS-PAGE significantly reduced the immunoreactivity of mAb39 to both human and mouse ZnT8. On the other hand, mAb20 only detected human ZnT8 but not mouse ZnT8 ( Figure 7B Similar levels of human ZnT8 and mouse ZnT8 were detected by anti-His tag antibody on duplicate blots, confirming similar protein loading ( Figure 7B The reduction in mAb39 immunoreactivity was attributed to the loss of ZnT8 dimer association on SDS-PAGE. Although mAb39 was much weaker than mAb20 for denatured ZnT8 detection, the ZnT8 reactivity of mAb39 appeared comparable to that of a commercial antibody against His-tagged ZnT8 on the blot ( Figure 7B Next, we used mAbs to probe endogenous ZnT8 expression in EndoC-βH, an insulinoma cell line derived from human pancreatic β cells (32). Immunoblotting of EndoC-βH lysates with mAb20 revealed a single ZnT8 protein band above negligible background. mAb39 detected neither ZnT8 nor nonspecific endogenous proteins ( Figure 7C ). Finally, we examined the specificity of mAb20 and mAb39 for ZnT homologs using lysates of HEK293 cells overexpressing ZnT1, ZnT2, ZnT3, ZnT4, ZnT7, ZnT8, or ZnT10 (each tagged with a C-terminal FLAG octapeptide). With similar sample loading in each lane, both mAbs detected only ZnT8, demonstrating that both mAbs have excellent selectivity for human ZnT8 relative to other ZnT homologs.

[0180] Quantification of cellular ZnT8 levels. Co-binding of the mAb20-mAb39 pair to a single ZnT8 protein allows quantification of ZnT8 levels by measuring the FRET signal derived from the closely-proximally labeled fluorescent donor and acceptor. mAb20 was labeled with a long-emitting terbium cryptate as an energy donor, while mAb39 was labeled with d2 as a near-infrared energy acceptor. Fixed amounts of donor and acceptor were added in a 1:1 ratio to lysates of HEK293 cells overexpressing human ZnT8, mouse ZnT8, or human ZnT3. HTRF calibration was performed on a series of lysate dilutions, showing a linear HTRF response up to a human ZnT8 concentration of 50 ng / mL, after which the concentration level bent due to binding saturation ( Figure 8 ). Even at a high ZnT concentration of 500 ng / ml, mouse ZnT8 or human ZnT3 (the closest homolog to human ZnT8) showed negligible or no HTRF response. Therefore, the mouse ZnT8 and human ZnT3 readings were taken as HTRF background readings. The limit of detection (LoD) of human ZnT8 was 0.51 ng / ml at 3× SD above background, while the limit of quantification (LoQ) was 1.71 ng / ml at 10× SD above background ( Figure 8 , inset). The coefficient of variance (CV) for LoQ was 9.8%.

[0181] discuss

[0182] The distinctive biochemical properties of ZnT8 present a significant challenge for the development of ZnT8-specific reagents. To date, all anti-ZnT8 antibodies have been generated against linear epitopes derived from the ZnT8 protein sequence and, therefore, lack sufficient specificity for the immunodetection of endogenous ZnT8 in pancreatic β-cells. In this work, we characterized two anti-ZnT8 mAbs against full-length human ZnT8 in reconstituted proteoliposomes. Both mAbs recognized folded ZnT8 with subnanomolar binding affinity and were able to protect ZnT8 from unfolding and partially inhibit ZnT8 transport activity. Although the reactivity of mAb39 was greatly reduced, the folding dependency did not prevent the mAbs from recognizing denatured ZnT8 on SDS-PAGE. Both mAbs were highly specific for human ZnT8, showing no detectable reactivity to a panel of human ZnT homologs on immunoblots. Specific immunodetection of human ZnT8 was further enhanced by simultaneous binding of two ZnT8-specific mAbs to a single ZnT8 protein. Because mAb20 does not recognize mouse ZnT8, the mAb20-mAb39 pair did not produce detectable FRET readout for mouse ZnT8 ( Figure 7BBinding of mAb39 alone to mouse ZnT8 ( Fig. 7A ) failed to elicit a FRET response above the assayed LoD, indicating that the specificity of the HTRF assay is the product of the multiplication of the two individual mAb specificities.

[0183] The inhibition of Fab binding reflects the effect of the polymorphic R to W substitution at position 325 of human ZnT8. Early comparison of steady-state kinetics between the R325 and W325 variants showed that W325 converts V max decreased by 50%, while K m No significant effect (20). Based on homology modeling of human ZnT8, the R325W polymorphic variant is located at the distal dimer interface near the C-terminus (20). Coincidentally, Fab39 appears to bind to the same region by our EM analysis. Functional characterization of the polymorphic CTD variants showed that CTD-W325 has a higher dimerization affinity than CTD-R325 (33), while removal of zinc binding to the CTD was shown to trigger a hinge-like movement, resulting in two open CTDs (29, 34). Both the W325 variant and Fab39 binding lead to stabilization of the dimer interaction, resulting in partial inhibition of ZnT8 transport activity. Therefore, Fab39 binding can mimic the inhibitory effect of the W325 variant by acting on the same allosteric pathway. Our results demonstrate the utility of the mAb as an allosteric inhibitor.

[0184] Emerging data suggest that human ZnT8 activity in pancreatic β cells may be inversely associated with the risk of developing T2D (15, 20, 22). Currently, the molecular mechanisms underlying the causal relationship between LOF mutations in human ZnT8 and a lower risk of T2D are unclear. A mechanistic understanding of this natural protective effect could facilitate translation into novel antidiabetic therapies. The frequency of the risk R325 allele is approximately 72% in the general population (35), but its expression levels in human islets have not been quantified and correlated with T2D risk. The HTRF assay is highly specific for human ZnT8, accurate, and easy to perform in a clinical research setting. The assay could be adapted to measure the abundance of human ZnT8 in healthy and diseased islets to correlate islet ZnT8 expression profiles with clinical markers associated with T2D. Alterations in R325 expression are expected to exacerbate or attenuate hyperactive zinc transport. The application of the mAb as a specific ZnT8 biosensor in clinical research could yield new insights into the pathophysiology of ZnT8.

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[0230] Table 3. Description of sequence listing

[0231]

[0232] Sequence Listing <110> Johns Hopkins University <120> Human ZNT8 Antibody <130> P15820-02 <150> 62 / 719,016 <151> 2018-08-16 <160> 62 <170> PatentIn version 3.5 <210> 1 <211> 1404 <212> DNA <213> Artificial Sequence <220> <223> mAB12 heavy chain <400> 1 atggacatga gggtgcccgc tcagctcctg gggctcctgc tgctgtggct gagaggtgcg 60 cgctgtcaga tccagttggt gcagtctgga cctgagctga agaagcctgg agagacagtc 120 aagatctcct gcaaggcttc tgggtatacc ttcacaaact atccaatgca ctgggtgaag 180 cagactccag gaaagggttt aaagtggatg ggctggataa acacctactc tggagtgcca 240 acatatggag atgacttcaa gggacggttt gccttctctt tggaaacctc tgccagtact 300 gcatatttgc agatcaacaa cctcaaaaat gaagacatgg ctacatattt ctgtgcaaga 360 tcgaacccct atgattactt gtatgctatg gactcctggg gtcaaggaac ctcagtcacc 420 gtctctagtg ccaaaacgac acccccatct gtctacccac tggcccctgg atctgctgcc 480 caaactaact ccatggtgac cctgggatgc ctggtcaagg gctatttccc tgagccagtg 540 acagtgacct ggaactctgg atccctgtcc agcggtgtgc acaccttccc agctgtcctg 600 cagtctgacc tctacactct gagcagctca gtgactgtcc cctccagcac ctggcccagc 660 gagaccgtca cctgcaacgt tgcccacccg gccagcagca ccaaggtgga caagaaaatt 720 gtgcccaggg attgtggttg taagccttgc atatgtacag tcccagaagt atcatctgtc 780 ttcatcttcc ccccaaagcc caaggatgtg ctcaccatta ctctgactcc taaggtcacg 840 tgtgttgtgg tagacatcag caaggatgat cccgaggtcc agttcagctg gtttgtagat 900 gatgtggagg tgcacacagc tcagacgcaa ccccgggagg agcagttcaa cagcactttc 960 cgctcagtca gtgaacttcc catcatgcac caggactggc tcaatggcaa ggagttcaaa 1020 tgcagggtca acagtgcagc tttccctgcc cccatcgaga aaaccatctc caaaaccaaa 1080 ggcagaccga aggctccaca ggtgtacacc attccacctc ccaaggagca gatggccaag 1140 gataaagtca gtctgacctg catgataaca gacttcttcc ctgaagacat tactgtggag 1200 tggcagtgga atgggcagcc agcggagaac tacaagaaca ctcagcccat catggacaca 1260 gatggctctt acttcgtcta cagcaagctc aatgtgcaga agagcaactg ggaggcagga 1320 aatactttca cctgctctgt gttacatgag ggcctgcaca accaccatac tgagaagagc 1380 ctctcccact ctcctggtaa atga 1404 <210> 2 <211> 467 <212> PRT <213> Artificial Sequence <220> <223> mAB12 heavy chain <400> 2 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Gln Ile Gln Leu Val Gln Ser Gly Pro Glu 20 25 30 Leu Lys Lys Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly 35 40 45 Tyr Thr Phe Thr Asn Tyr Pro Met His Trp Val Lys Gln Thr Pro Gly 50 55 60 Lys Gly Leu Lys Trp Met Gly Trp Ile Asn Thr Tyr Ser Gly Val Pro 65 70 75 80 Thr Tyr Gly Asp Asp Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr 85 90 95 Ser Ala Ser Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp 100 105 110 Met Ala Thr Tyr Phe Cys Ala Arg Ser Asn Pro Tyr Asp Tyr Leu Tyr 115 120 125 Ala Met Asp Ser Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala 130 135 140 Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala 145 150 155 160 Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe 165 170 175 Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly 180 185 190 Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser 195 200 205 Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr 210 215 220 Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile 225 230 235 240 Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu 245 250 255 Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr 260 265 270 Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys 275 280 285 Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val 290 295 300 His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe 305 310 315 320 Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly 325 330 335 Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile 340 345 350 Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val 355 360 365 Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser 370 375 380 Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu 385 390 395 400 Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro 405 410 415 Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val 420 425 430 Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu 435 440 445 His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His Ser 450 455 460 Pro Gly Lys 465 <210> 3 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> mAb12 heavy chain CDR1 <400> 3 Gly Tyr Thr Phe Thr Asn Tyr 1 5 <210> 4 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> mAb12 heavy chain CDR2 <400> 4 Thr Tyr Ser Gly Val 1 5 <210> 5 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> mAB heavy chain CDR3 <400> 5 Ser Asn Pro Tyr Asp Tyr Leu Tyr Ala Met Asp Ser 1 5 10 <210> 6 <211> 726 <212> DNA <213> Artificial Sequence <220> <223> mAb12 light chain <400> 6 atggacatga gggtgcccgc tcagctcctg gggctcctgc tgctgtggct gagaggtgcg 60 cgctgtgatg ttgtgatgac ccaaactcca ctctccctgc ctgtcagtct tggagatcaa 120 gcctccatct cctgcagatc tagtcagagc cttgtacaca tcaatggaaa cacctatata 180 cattggtacc tgcagaagcc aggccagtct ccaaagctcc tgatctacaa agtttccaac 240 cgattttctg gggtcccaga caggttcagt ggcagtggat cagggacaga ttttacactc 300 aagatcagaa gagtggaggc tgaggatctg ggagtttatt tctgctctca aaatacacat 360 gttccattca cattcggctc ggggacaaag ttggaaataa aacgtgcaga tgctgcgcca 420 actgtatcca tcttcccacc atctagcgag cagttaacat ctggaggtgc ctcagtcgtg 480 tgcttcttga acaacttcta ccccaaagac atcaatgtca agtggaagat tgatggcagt 540 gaacgacaaa atggcgtcct gaacagttgg actgatcagg acagcaaaga cagcacctac 600 agcatgagca gcaccctcac gttgaccaag gacgagtatg aacgacataa cagctatacc 660 tgtgaggcca ctcacaagac atcaacttca cccattgtca agagcttcaa caggaatgag 720 tgttag 726 <210> 7 <211> 241 <212> PRT <213> Artificial Sequence <220> <223> mAb12 light chain <400> 7 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Asp Val Val Met Thr Gln Thr Pro Leu Ser 20 25 30 Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser 35 40 45 Gln Ser Leu Val His Ile Asn Gly Asn Thr Tyr Ile His Trp Tyr Leu 50 55 60 Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn 65 70 75 80 Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr 85 90 95 Asp Phe Thr Leu Lys Ile Arg Arg Val Glu Ala Glu Asp Leu Gly Val 100 105 110 Tyr Phe Cys Ser Gln Asn Thr His Val Pro Phe Thr Phe Gly Ser Gly 115 120 125 Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile 130 135 140 Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val 145 150 155 160 Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys 165 170 175 Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp 180 185 190 Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu 195 200 205 Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr 210 215 220 His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu 225 230 235 240 Cys <210> 8 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> mAb12 light chain CDR1 <400> 8 Arg Ser Ser Gln Ser Leu Val His Ile Asn Gly Asn Thr Tyr Ile His 1 5 10 15 <210> 9 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> mAb12 light chain CDR2 <400> 9 Lys Val Ser Asn Arg Phe Ser 1 5 <210> 10 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> mAb12 light chain CDR3 <400> 10 Ser Gln Asn Thr His Val Pro Phe Thr 1 5 <210> 11 <211> 1401 <212> DNA <213> Artificial Sequence <220> <223> mAb16 heavy chain <400> 11 atggacatga gggtgcccgc tcagctcctg gggctcctgc tgctgtggct gagaggtgcg 60 cgctgtcaga tccagttggt gcagtctgga cctgagctga agaagcctgg agagacagtc 120 acgatctcct gcaaggcttc tggatatacc ttcacacact atccagtgca ctgggtgaag 180 caggctccag gaaagggttt acagtggatg ggctggataa acacctactc tggagtgcca 240 acatatgcag atgccttcaa gaaacgtttt gccttctctt tggaaacctc tgccagcact 300 gcatatttgc agatcaacaa cctcaaaagt gaggacatgg ctacatattt ctgtgcaaga 360 tcgagggtct atgatgggta ctattttgac tactggggcc aaggcaccac tctcaccgtc 420 tctagtgcca aaacgacacc cccatctgtc tacccactgg cccctggatc tgctgcccaa 480 actaactcca tggtgaccct gggatgcctg gtcaagggct atttccctga gccagtgaca 540 gtgacctgga actctggatc cctgtccagc ggtgtgcaca ccttcccagc tgtcctgcag 600 tctgacctct acactctgag cagctcagtg actgtcccct ccagcacctg gcccagcgag 660 accgtcacct gcaacgttgc ccacccggcc agcagcacca aggtggacaa gaaaattgtg 720 cccagggatt gtggttgtaa gccttgcata tgtacagtcc cagaagtatc atctgtcttc 780 atcttccccc caaagcccaa ggatgtgctc accattactc tgactcctaa ggtcacgtgt 840 gttgtggtag acatcagcaa ggatgatccc gaggtccagt tcagctggtt tgtagatgat 900 gtggaggtgc acacagctca gacgcaaccc cgggaggagc agttcaacag cactttccgc 960 tcagtcagtg aacttcccat catgcaccag gactggctca atggcaagga gttcaaatgc 1020 agggtcaaca gtgcagcttt ccctgccccc atcgagaaaa ccatctccaa aaccaaaggc 1080 agaccgaagg ctccacaggt gtacaccatt ccacctccca aggagcagat ggccaaggat 1140 aaagtcagtc tgacctgcat gataacagac ttcttccctg aagacattac tgtggagtgg 1200 cagtggaatg ggcagccagc ggagaactac aagaacactc agcccatcat ggacacagat 1260 ggctcttact tcgtctacag caagctcaat gtgcagaaga gcaactggga ggcaggaaat 1320 actttcacct gctctgtgtt acatgagggc ctgcacaacc accatactga gaagagcctc 1380 tcccactctc ctggtaaatg a 1401 <210> 12 <211> 466 <212> PRT <213> Artificial Sequence <220> <223> mAb16 heavy chain <400> 12 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Gln Ile Gln Leu Val Gln Ser Gly Pro Glu 20 25 30 Leu Lys Lys Pro Gly Glu Thr Val Thr Ile Ser Cys Lys Ala Ser Gly 35 40 45 Tyr Thr Phe Thr His Tyr Pro Val His Trp Val Lys Gln Ala Pro Gly 50 55 60 Lys Gly Leu Gln Trp Met Gly Trp Ile Asn Thr Tyr Ser Gly Val Pro 65 70 75 80 Thr Tyr Ala Asp Ala Phe Lys Lys Arg Phe Ala Phe Ser Leu Glu Thr 85 90 95 Ser Ala Ser Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Ser Glu Asp 100 105 110 Met Ala Thr Tyr Phe Cys Ala Arg Ser Arg Val Tyr Asp Gly Tyr Tyr 115 120 125 Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Lys 130 135 140 Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln 145 150 155 160 Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro 165 170 175 Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val 180 185 190 His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser 195 200 205 Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys 210 215 220 Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val 225 230 235 240 Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val 245 250 255 Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile 260 265 270 Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp 275 280 285 Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His 290 295 300 Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg 305 310 315 320 Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys 325 330 335 Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu 340 345 350 Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr 355 360 365 Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu 370 375 380 Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp 385 390 395 400 Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile 405 410 415 Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln 420 425 430 Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His 435 440 445 Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro 450 455 460 Gly Lys 465 <210> 13 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> mAb16 heavy chain CDR1 <400> 13 Gly Tyr Thr Phe Thr His Tyr 1 5 <210> 14 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> mAb16 heavy chain CDR2 <400> 14 Asn Thr Tyr Ser Gly Val 1 5 <210> 15 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> mAb16 heavy chain CDR3 <400> 15 Ser Arg Val Tyr Asp Gly Tyr Tyr Phe Asp Tyr 1 5 10 <210> 16 <211> 726 <212> DNA <213> Artificial Sequence <220> <223> mAb16 light chain <400> 16 atggacatga gggtgcccgc tcagctcctg gggctcctgc tgctgtggct gagaggtgcg 60 cgctgtgatg ttgtgatgac ccaaactcca ctctccctgc ctgtcagtct tggagatcaa 120 gcctccatct cttgcagatc tagtcagagc cttgtacaca gtaatggaaa gacctattta 180 cattggtacc tgcagaagcc aggccagtct ccaaacctcc tgatctacaa agtttccaac 240 cgattttctg gggtcccaga caggttcagt ggcagtggat cagggacaga tttcacactc 300 aagatcagca gagtggaggc tgaggatctg ggagtttatt tctgctctca acttacacat 360 gttccgtgga cgttcggtgg aggcaccaag ctggaaatca aacgtgcaga tgctgcgcca 420 actgtatcca tcttcccacc atctagcgag cagttaacat ctggaggtgc ctcagtcgtg 480 tgcttcttga acaacttcta ccccaaagac atcaatgtca agtggaagat tgatggcagt 540 gaacgacaaa atggcgtcct gaacagttgg actgatcagg acagcaaaga cagcacctac 600 agcatgagca gcaccctcac gttgaccaag gacgagtatg aacgacataa cagctatacc 660 tgtgaggcca ctcacaagac atcaacttca cccattgtca agagcttcaa caggaatgag 720 tgttag 726 <210> 17 <211> 241 <212> PRT <213> Artificial Sequence <220> <223> mAb16 light chain <400> 17 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Asp Val Val Met Thr Gln Thr Pro Leu Ser 20 25 30 Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser 35 40 45 Gln Ser Leu Val His Ser Asn Gly Lys Thr Tyr Leu His Trp Tyr Leu 50 55 60 Gln Lys Pro Gly Gln Ser Pro Asn Leu Leu Ile Tyr Lys Val Ser Asn 65 70 75 80 Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr 85 90 95 Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val 100 105 110 Tyr Phe Cys Ser Gln Leu Thr His Val Pro Trp Thr Phe Gly Gly Gly 115 120 125 Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile 130 135 140 Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val 145 150 155 160 Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys 165 170 175 Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp 180 185 190 Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu 195 200 205 Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr 210 215 220 His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu 225 230 235 240 Cys <210> 18 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> mAb16 light chain CDR1 <400> 18 Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Lys Thr Tyr Leu His 1 5 10 15 <210> 19 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> mAb16 light chain CDR2 <400> 19 Lys Val Ser Asn Arg Phe Ser 1 5 <210> 20 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> mAb16 light chain CDR3 <400> 20 Ser Gln Leu Thr His Val Pro Trp 1 5 <210> 21 <211> 1407 <212> DNA <213> Artificial Sequence <220> <223> mAb17 heavy chain <400> 21 atggacatga gggtgcccgc tcagctcctg gggctcctgc tgctgtggct gagaggtgcg 60 cgctgtcaga tccagttggt gcagtctgga cctgagctga agaagcctgg agagacagtc 120 aagatctcct gcaaggcttc tgggtatacc ttcacaaact atccaatgca ctggttgaag 180 caggctccag gaaagggttt aaagtggatg ggctggataa acacctactc tggagtgcca 240 acatatgcag atgacttcaa gggacggttt gccttctctt tggaaacctc tgccagcact 300 gcatatttgc agatcaacaa cctcaaaaat gaggacatgg ctacatattt ctgtacaaaa 360 tcgcgcatta ctacgatggg gggttatgct atggactgct ggggtcaagg aacctcagtc 420 accgtctcta gtgccaaaac gacaccccca tctgtctacc cactggcccc tggatctgct 480 gcccaaacta actccatggt gaccctggga tgcctggtca agggctattt ccctgagcca 540 gtgacagtga cctggaactc tggatccctg tccagcggtg tgcacacctt cccagctgtc 600 ctgcagtctg acctctacac tctgagcagc tcagtgactg tcccctccag cacctggccc 660 agcgagaccg tcacctgcaa cgttgcccac ccggccagca gcaccaaggt ggacaagaaa 720 attgtgccca gggattgtgg ttgtaagcct tgcatatgta cagtcccaga agtatcatct 780 gtcttcatct tccccccaaa gcccaaggat gtgctcacca ttactctgac tcctaaggtc 840 acgtgtgttg tggtagacat cagcaaggat gatcccgagg tccagttcag ctggtttgta 900 gatgatgtgg aggtgcacac agctcagacg caaccccggg aggagcagtt caacagcact 960 ttccgctcag tcagtgaact tcccatcatg caccaggact ggctcaatgg caaggagttc 1020 aaatgcaggg tcaacagtgc agctttccct gcccccatcg agaaaaccat ctccaaaacc 1080 aaaggcagac cgaaggctcc acaggtgtac accattccac ctcccaagga gcagatggcc 1140 aaggataaag tcagtctgac ctgcatgata acagacttct tcctgaaga cattactgtg 1200 gagtggcagt ggaatgggca gccagcggag aactacaaga acactcagcc catcatggac 1260 acagatggct cttacttcgt ctacagcaag ctcaatgtgc agaagagcaa ctgggaggca 1320 ggaaatactt tcacctgctc tgtgttacat gagggcctgc acaaccacca tactgagaag 1380 agcctctccc actctcctgg taaatga 1407 <210> 22 <211> 468 <212> PRT <213> Artificial Sequence <220> <223> mAb17 heavy chain <400> 22 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Gln Ile Gln Leu Val Gln Ser Gly Pro Glu 20 25 30 Leu Lys Lys Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly 35 40 45 Tyr Thr Phe Thr Asn Tyr Pro Met His Trp Leu Lys Gln Ala Pro Gly 50 55 60 Lys Gly Leu Lys Trp Met Gly Trp Ile Asn Thr Tyr Ser Gly Val Pro 65 70 75 80 Thr Tyr Ala Asp Asp Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr 85 90 95 Ser Ala Ser Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp 100 105 110 Met Ala Thr Tyr Phe Cys Thr Lys Ser Arg Ile Thr Thr Met Gly Gly 115 120 125 Tyr Ala Met Asp Cys Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 130 135 140 Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala 145 150 155 160 Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr 165 170 175 Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser 180 185 190 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu 195 200 205 Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val 210 215 220 Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys 225 230 235 240 Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro 245 250 255 Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu 260 265 270 Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser 275 280 285 Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu 290 295 300 Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr 305 310 315 320 Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn 325 330 335 Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro 340 345 350 Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln 355 360 365 Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val 370 375 380 Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val 385 390 395 400 Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln 405 410 415 Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn 420 425 430 Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val 435 440 445 Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His 450 455 460 Ser Pro Gly Lys 465 <210> 23 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> mAb17 heavy chain CDR1 <400> 23 Gly Tyr Thr Phe Thr Asn Tyr 1 5 <210> 24 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> mAb17 heavy chain CDR2 <400> 24 Asn Thr Tyr Ser Gly Val 1 5 <210> 25 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> mAb17 heavy chain CDR3 <400> 25 Ser Arg Ile Thr Thr Met Gly Gly Tyr Ala Met Asp Cys 1 5 10 <210> 26 <211> 726 <212> DNA <213> Artificial Sequence <220> <223> mAb17 light chain <400> 26 atggacatga gggtgcccgc tcagctcctg gggctcctgc tgctgtggct gagaggtgcg 60 cgctgtgaag ttgtgatgac ccaaactcca ctctccctgc ctgtcagtct tggagatcaa 120 gcctccatct cttgcagatc tagtcagagc cttctacaca gtaatggaaa cacctattta 180 cattggtacc tgcagaggcc aggccagtct ccaaacctcc tgatctccaa agtttccaac 240 cgatttctg gggtcccaga caggttcagt ggcagtggat cagggacaga tttcacactc 300 aagatcagca gagtggaggc tgaggatctg ggagtttatt tctgctctca aaatacacat 360 gttccgtgga cgttcggtgg aggcaccaag ctggaaatca aacgtgcaga tgctgcgcca 420 actgtatcca tcttcccacc atctagcgag cagttaacat ctggaggtgc ctcagtcgtg 480 tgcttcttga acaacttcta ccccaaagac atcaatgtca agtggaagat tgatggcagt 540 gaacgacaaa atggcgtcct gaacagttgg actgatcagg acagcaaaga cagcacctac 600 agcatgagca gcaccctcac gttgaccaag gacgagtatg aacgacataa cagctatacc 660 tgtgaggcca ctcacaagac atcaacttca cccattgtca agagcttcaa caggaatgag 720 tgtag 726 <210> 27 <211> 241 <212> PRT <213> Artificial Sequence <220> <223> mAb17 light chain <400> 27 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Glu Val Val Met Thr Gln Thr Pro Leu Ser 20 25 30 Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser 35 40 45 Gln Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu 50 55 60 Gln Arg Pro Gly Gln Ser Pro Asn Leu Leu Ile Ser Lys Val Ser Asn 65 70 75 80 Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr 85 90 95 Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val 100 105 110 Tyr Phe Cys Ser Gln Asn Thr His Val Pro Trp Thr Phe Gly Gly Gly 115 120 125 Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile 130 135 140 Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val 145 150 155 160 Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys 165 170 175 Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp 180 185 190 Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu 195 200 205 Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr 210 215 220 His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu 225 230 235 240 Cys <210> 28 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> mAb17 light chain CDR1 <400> 28 Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu His 1 5 10 15 <210> 29 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> mAb17 light chain CDR2 <400> 29 Lys Val Ser Asn Arg Phe Ser 1 5 <210> 30 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> mAb17 light chain CDR3 <400> 30 Ser Gln Asn Thr His Val Pro Trp Thr 1 5 <210> 31 <211> 1407 <212> DNA <213> Artificial Sequence <220> <223> mAb20 heavy chain <400> 31 atggacatga gggtgcccgc tcagctcctg gggctcctgc tgctgtggct gagaggtgcg 60 cgctgtcaga tccagttggt gcagtctgga cctgagctga agaagcctgg agagacagtc 120 aagatctcct gcaaggcttc tgggtatatc ttcacaaact atccaatgca ctgggtgaag 180 caggctccag gaaagggttt aaagtggatg ggctggataa acacctactc tggagtgcca 240 acacatgcag atgacttcaa gggacggttt gccttctctt tggaaacctc tgccaacagt 300 gcatttttgc agatcaacaa cctcaaaaat gaggacacgg ctacatattt ctgtacaaga 360 tcgcgcatta ctccgacggg gggctatgct atggactact ggggtcaagg aacctcagtc 420 accgtctcta gtgccaaaac cakaccccca tctgtctacc cactggcccc tggatctgct 480 gcccaaacta actccatggt gaccctggga tgcctggtca agggctatt ccctgagcca 540 gtgacagtga cctggaacctc tggatccctg tccagcggtg tgcacacctt cccagctgtc 600 ctgcagtctg acctctacac tctgagcagc tcagtgactg tccctccag cacctgccc 660 agcgagaccg tcacctgcaa cgttgcccac ccggccagca gcaccaaggt ggacaagaaa 720 attgtgccca gggattgtgg ttgtaagcct tgcatatgta cagtcccaga agtatcatct 780 gtcttcatct tccccaaa gcccaggat gtgctcacca ttactctgac tcctaggtc 840 acgtgtgttg tggtagacat cagcaggat gatcccgagg tccagttcag ctggtttgta 900 gatgatgtgg aggtgcacac agctcacacg caccccggg aggagcagtt cacacact 960 ttccgctcag tcagtgact tcccatcatg caccaggact ggctcaatgg tcagtg 1020 aaatgcagggg tcacagtgc agctttccct gccccatcg agaaaccact ctccaaacc 1080 aaaggcagac cgaggctcc acaggtgtac accattccac ctcccagga gcagatggcc 1140 aaggataaag tcagtctgac ctgcatgata acagacttct tccctgaaga cattactgtg 1200 gagtggcagt ggaatgggca gccagcggag aactacaaga acaccagcc catcatggac 1260 acgatggct cttacttcgt ctacagcaag ctcaatgtgc agagagcaa ctgggaggca 1320 ggaaatactt tcacctgctc tgtgttacat gagggcctgc acacca tactgagaag 1380 agccctctccc actctccctgg call 1407 <210> 32 <211> 468 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain mAb20 <400> 32 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Gln Ile Gln Leu Val Gln Ser Gly Pro Glu 20 25 30 Leu Lys Lys Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly 35 40 45 Tyr Ile Phe Thr Asn Tyr Pro Met His Trp Val Lys Gln Ala Pro Gly 50 55 60 Lys Gly Leu Lys Trp Met Gly Trp Ile Asn Thr Tyr Ser Gly Val Pro 65 70 75 80 Thr His Ala Asp Asp Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr 85 90 95 Ser Ala Asn Ser Ala Phe Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp 100 105 110 Thr Ala Thr Tyr Phe Cys Thr Arg Ser Arg Ile Thr Pro Thr Gly Gly 115 120 125 Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 130 135 140 Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala 145 150 155 160 Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr 165 170 175 Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser 180 185 190 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu 195 200 205 Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val 210 215 220 Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys 225 230 235 240 Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro 245 250 255 Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu 260 265 270 Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser 275 280 285 Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu 290 295 300 Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr 305 310 315 320 Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn 325 330 335 Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro 340 345 350 Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln 355 360 365 Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val 370 375 380 Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val 385 390 395 400 Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln 405 410 415 Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn 420 425 430 Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val 435 440 445 Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His 450 455 460 Ser Pro Gly Lys 465 <210> 33 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> mAb20 heavy chain CDR1 <400> 33 Gly Tyr Ile Phe Thr Asn Tyr 1 5 <210> 34 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> mAb20 heavy chain CDR2 <400> 34 Asn Thr Tyr Ser Gly Val 1 5 <210> 35 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> mAb20 heavy chain CDR3 <400> 35 Ser Arg Ile Thr Pro Thr Gly Gly Tyr Ala Met Asp Tyr 1 5 10 <210> 36 <211> 726 <212> DNA <213> Artificial Sequence <220> <223> mAb20 light chain <400> 36 atggacatga gggtgcccgc tcagctcctg gggctcctgc tgctgtggct gagaggtgcg 60 cgctgtgatg ttgtgatgac ccaaactcca ctctccctgc ctgtcagtct tggagatcaa 120 gcctccatct cttgcagatc tagtcagagc cttgtacaca gtaatggaaa cacctattta 180 cattggtacc tgcagaagcc aggccagtct ccaaacctcc tgatctccaa agtttccaac 240 cgattttctg gggtcccaga aaggttcagt ggcagtggat cagggacaga tttcacactc 300 aagatcagca gagtggaggc tgaggatctg ggagtttatt tctgctctca aaatacacat 360 <h2 style=";text-align:left;direction:ltr">gttccgtgga cgttcggtgg aggcaccaag ctggaaatca aacgtgcaga tgctgcgcca 420<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> actgtatcca tcttcccacc atctagcgag cagttaacat ctggaggtgc ctcagtcgtg 480<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tgcttcttga acaacttcta ccccaaagac atcaatgtca agtggaagat tgatggcagt 540<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gaacgacaaa atggcgtcct gaacagttgg actgatcagg acagcaaaga cagcacctac 600<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> agcatgagca gcaccctcac gttgaccaag gacgagtatg aacgacataa cagctatacc 660<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tgtgaggcca ctcacaagac atcaacttca cccattgtca agagcttcaa caggaatgag 720<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tgttag 726<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <210> 37<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <211> 241<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <212> PRT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <213> Artificial Sequence<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <220><h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <223> mAb20 light chain<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 37<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1 5 10 15<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Leu Arg Gly Ala Arg Cys Asp Val Val Met Thr Gln Thr Pro Leu Ser<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 20 25 30<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 35 40 45<h2 style=";text-align:left;direction:ltr"> Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu 50 55 60 Gln Lys Pro Gly Gln Ser Pro Asn Leu Leu Ile Ser Lys Val Ser Asn 65 70 75 80 Arg Phe Ser Gly Val Pro Glu Arg Phe Ser Gly Ser Gly Ser Gly Thr 85 90 95 Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val 100 105 110 Tyr Phe Cys Ser Gln Asn Thr His Val Pro Trp Thr Phe Gly Gly Gly 115 120 125 Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile 130 135 140 Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val 145 150 155 160 Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys 165 170 175 Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp 180 185 190 Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu 195 200 205 Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr 210 215 220 His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu 225 230 235 240 Cys <210> 38 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> mAb20 light chain CDR1 <400> 38 Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His 1 5 10 15 <210> 39 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> mAb20 light chain CDR2 <400> 39 Lys Val Ser Asn Arg Phe Ser 1 5 <210> 40 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> mAb40 light chain CDR3 <400> 40 Ser Gln Asn Thr His Val Pro Trp Thr 1 5 <210> 41 <211> 1407 <212> DNA <213> Artificial Sequence <220> <223> mAb28 heavy chain <400> 41 atggacatga gggtgcccgc tcagctcctg gggctcctgc tgctgtggct gagaggtgcg 60 cgctgtcaga tccagttggt gcagtctgga cctgagctga agaagcctgg agagacagtc 120 aagatctcct gcaaggcttc tgggtatacc ttcacaaact atccaatgca ctgggtaaag 180 caggctccag gaaaggattt aaagtggatg ggctggataa acacctactc tggaatgtca 240 acatatgcag atgacttcaa gggacggttt gccttctctt tggaaacctc tgccagcact 300 gcgtatttgc agatcaacaa cctcaaaaat gaggacatgg ctacatattt ctgtgcaaga 360 tcgcgcatta caacgatggg gggctatgct atggactact ggggtcaagg agcctcagtc 420 accgtctcta gtgccaaaac gacaccccca tctgtctacc cactggcccc tggatctgct 480 gcccaaacta actccatggt gaccctggga tgcctggtca agggctattt ccctgagcca 540 gtgacagtga cctggaactc tggatccctg tccagcggtg tgcacacctt cccagctgtc 600 ctgcagtctg acctctacac tctgagcagc tcagtgactg tccctccag cacctggccc 660 agcgagaccg tcacctgcaa cgttgcccac ccggccagca gcaccaaggt ggacaagaaa 720 attgtgccca gggattgtgg ttgtaagcct tgcatatgta cagtcccaga agtatcatct 780 gtcttcatct tccccccaaa gcccaaggat gtgctcacca ttactctgac tcctaaggtc 840 acgtgtgttg tggtagacat cagcaaggat gatcccgagg tccagttcag ctggtttgta 900 gatgatgtgg aggtgcacac agctcagacg caaccccggg aggagcagtt caacagcact 960 ttccgctcag tcagtgaact tcccatcatg caccaggact ggctcaatgg caaggagttc 1020 aaatgcaggg tcaacagtgc agctttccct gcccccatcg agaaaaccat ctccaaaacc 1080 aaaggcagac cgaaggctcc acaggtgtac accattccac ctcccaagga gcagatggcc 1140 aaggataaag tcagtctgac ctgcatgata acagacttct tcctgaaga cattactgtg 1200 gagtggcagt ggaatgggca gccagcggag aactacaaga acactcagcc catcatggac 1260 acagatggct cttacttcgt ctacagcaag ctcaatgtgc agaagagcaa ctgggaggca 1320 ggaaatactt tcacctgctc tgtgttacat gagggcctgc acacca tactgagaag 1380 agccctctccc actctccctgg call 1407 <210> 42 <211> 468 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain mAb28 <400> 42 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Gln Ile Gln Leu Val Gln Ser Gly Pro Glu 20 25 30 Leu Lys Lys Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly 35 40 45 Tyr Thr Phe Thr Asn Tyr Pro Met His Trp Val Lys Gln Ala Pro Gly 50 55 60 Lys Asp Leu Lys Trp Met Gly Trp Ile Asn Thr Tyr Ser Gly Met Ser 65 70 75 80 Thr Tyr Ala Asp Asp Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr 85 90 95 Ser Ala Ser Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp 100 105 110 Met Ala Thr Tyr Phe Cys Ala Arg Ser Arg Ile Thr Thr Met Gly Gly 115 120 125 Tyr Ala Met Asp Tyr Trp Gly Gln Gly Ala Ser Val Thr Val Ser Ser 130 135 140 Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala 145 150 155 160 Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr 165 170 175 Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser 180 185 190 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu 195 200 205 Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val 210 215 220 Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys 225 230 235 240 Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro 245 250 255 Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu 260 265 270 Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser 275 280 285 Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu 290 295 300 Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr 305 310 315 320 Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn 325 330 335 Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro 340 345 350 Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln 355 360 365 Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val 370 375 380 Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val 385 390 395 400 Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln 405 410 415 Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn 420 425 430 Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val 435 440 445 Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His 450 455 460 Ser Pro Gly Lys 465 <210> 43 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> mAb28 heavy chain CDR1 <400> 43 Gly Tyr Thr Phe Thr Asn Tyr 1 5 <210> 44 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> mAb28 heavy chain CDR2 <400> 44 Asn Thr Tyr Ser Gly Met 1 5 <210> 45 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> mAb28 heavy chain CDR3 <400> 45 Ser Arg Ile Thr Thr Met Gly Gly Tyr Ala Met Asp Tyr 1 5 10 <210> 46 <211> 726 <212> DNA <213> Artificial Sequence <220> <223> mAb28 light chain <400> 46 atggacatga gggtgcccgc tcagctcctg gggctcctgc tgctgtggct gagaggtgcg 60 cgctgtgaag ttgtgatgac ccaaactcca ctctccctgc ctgtcagtct tggagatcaa 120 gcctccatct cttgcagatc tagtcagagc cttgtacaca gtaatggaaa cacctattta 180 cattggtacc tgcagaagcc aggccagtct ccaaagctcc tgatctccaa agtttccaac 240 cgattttctg gggtcccaga caggttcagt ggcagtggat cagggacaga tttcacactc 300 aagatcatca gagtggaggc tgaggatctg ggagtttatt tctgctctca aaatacacat 360 gttccgtgga cgttcggtgg aggcaccaag ctggaaatca aacgtgcaga tgctgcgcca 420 actgtatcca tcttcccacc atctagcgag cagttaacat ctggaggtgc ctcagtcgtg 480 tgcttcttga acaacttcta ccccaaagac atcaatgtca agtggaagat tgatggcagt 540 gaacgacaaa atggcgtcct gaacagttgg actgatcagg acagcaaaga cagcacctac 600 agcatgagca gcaccctcac gttgaccaag gacgagtatg aacgacataa cagctatacc 660 tgtgaggcca ctcacaagac atcaacttca cccattgtca agagcttcaa caggaatgag 720 tgtag 726 <210> 47 <211> 241 <212> PRT <213> Artificial Sequence <220> <223> mAb28 light chain <400> 47 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Arg Gly Ala Arg Cys Glu Val Val Met Thr Gln Thr Pro Leu Ser 20 25 30 Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser 35 40 45 Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu 50 55 60 Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Ser Lys Val Ser Asn 65 70 75 80 Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr 85 90 95 Asp Phe Thr Leu Lys Ile Ile Arg Val Glu Ala Glu Asp Leu Gly Val 100 105 110 Tyr Phe Cys Ser Gln Asn Thr His Val Pro Trp Thr Phe Gly Gly Gly 115 120 125 Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile 130 135 140 Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val 145 150 155 160 Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys 165 170 175 Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp 180 185 190 Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu 195 200 205 Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr 210 215 220 His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu 225 230 235 240 Cys <210> 48 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> mAb28 light chain CDR1 <400> 48 Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His 1 5 10 15 <210> 49 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> mAb28 light chain CDR2 <400> 49 Lys Val Ser Asn Arg Phe Ser 1 5 <210> 50 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> mAb28 light chain CDR3 <400> 50 Ser Gln Asn Thr His Val Pro Trp Thr 1 5 <210> 51 <211> 1389 <212> DNA <213> Artificial Sequence <220> <223> mAb39 heavy chain <400> 51 atgggatgga gctgtatcat gttctttttg gtagcaacag ctacagatgt ccactcccag 60 gtccaactgc agcagcctgg ggctgaactg gtgaagcctg gggcttcagt gaagctgtcc 180. tgcaaggctt ctggctactc cttcaccacc tactggatgc actgggtgaa gcagaggcct ggacaaggcc taggtgggt tggagatatt aatcctagga acggtcgtac taactacaat 300. 300. 300. 300. 300. 300. 300. 300. 300. 300. 300. 300 caagtcagca gcctgacatc tgaggactct gcggtctatt actgtgcaat atggtcgggt gctatggact actggggtcc aggaacctca gtcaccgtct cctcagccaa aacaacagcc ccatcggtct atccactggc ccctgtgtgt ggagatacaa ctggctcctc ggtgactcta 480 ggatgcctgg tcaagggtta tttccctgag ccagtgacct tgacctgga ctctggatcc 540 ctgtccagtg atgtgcacac cttcccagct ctcctgcagt ctggcctcta caccctcagc 600 agctcagtga ctgtaaccac ctggcccagc cagaccatca cctgcaatgt ggcccacccg gcaagcagca ccaaagtgga caagaaatt gagcccagag ggtccccaac acataaaccc tgtcctccat gcccagctcc taacctcttg ggtggaccat ccgtcttcat cttccctcca 780 aagatcaagg atgtactcat gatctccctg agcccatgg tcacgtgtgt ggtggtggat 840 gtgagcgagg atgacccaga tgtccatgtc agctggttcg tgaacaacgt ggaagtacac 900 acagctcaga cacaaaccca tagagagat tacaacagta ctatccgggt ggtcagtgcc 960 ctccccatcc agcaccagga ctggatgagt ggcaaggagt tcaaatgcaa ggtcaacaac 1020 aaagccctcc cagcgcccat cgagagaacc atctcaaaac ccaaagggcc agtaagagct 1080 ccacaggtat atgtcttgcc tccaccagaa gaagagatga ctaagaaaca ggtcactctg 1140 acctgcatga tcacagactt catgcctgaa gacatttacg tggagtggac caacaacggg 1200 caaacagagc taaactacaa gaacactgaa ccagtcctgg actctgatgg ttcttacttc 1260 atgtacagca agctgagagt ggaaaagaag aactgggtgg aaagaaatag ctactcctgt 1320 tcagtggtcc acgagggtct gcacaatcac cacacgacta agagcttctc ccggtctccg 1380 ggtaaatga 1389 <210> 52 <211> 462 <212> PRT <213> Artificial Sequence <220> <223> mAb39 heavy chain <400> 52 Met Gly Trp Ser Cys Ile Met Phe Phe Leu Val Ala Thr Ala Thr Asp 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys 20 25 30 Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe 35 40 45 Thr Thr Tyr Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu 50 55 60 Glu Trp Val Gly Asp Ile Asn Pro Arg Asn Gly Arg Thr Asn Tyr Asn 65 70 75 80 Glu Lys Ser Lys Ser Lys Ala Thr Leu Thr Val Asp Ile Ser Ser Ser 85 90 95 Thr Val Tyr Met Gln Val Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110 Tyr Tyr Cys Ala Ile Trp Ser Gly Ala Met Asp Tyr Trp Gly Pro Gly 115 120 125 Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr 130 135 140 Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu 145 150 155 160 Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp 165 170 175 Asn Ser Gly Ser Leu Ser Ser Asp Val His Thr Phe Pro Ala Leu Leu 180 185 190 Gln Ser Gly Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Thr Trp 195 200 205 Pro Ser Gln Thr Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr 210 215 220 Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Ser Pro Thr His Lys Pro 225 230 235 240 Cys Pro Pro Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe 245 250 255 Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro 260 265 270 Met Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val 275 280 285 His Val Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr 290 295 300 Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Ile Arg Val Val Ser Ala 305 310 315 320 Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys 325 330 335 Lys Val Asn Asn Lys Ala Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser 340 345 350 Lys Pro Lys Gly Pro Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro 355 360 365 Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Ile 370 375 380 Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly 385 390 395 400 Gln Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp 405 410 415 Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp 420 425 430 Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His 435 440 445 Asn His His Thr Thr Lys Ser Phe Ser Arg Ser Pro Gly Lys 450 455 460 <210> 53 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> mAb39 heavy chain CDR1 <400> 53 Gly Tyr Ser Phe Thr Thr Tyr 1 5 <210> 54 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> mAb39 heavy chain CDR2 <400> 54 Asn Pro Arg Asn Gly Arg 1 5 <210> 55 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> mAb39 heavy chain CDR3 <400> 55 Trp Dear Gly Here Put Asp Tyr 1 5 <210> 56 <211> 717 <212> DNA <213> Artificial Sequence <220> <223> mAb39 light chain <400> 56 atgaagttgc ctgttaggct gttggtgctg atgttctgga ttcctgcttc cagcagtgat 60 gttgtgatga cccaaactcc actctccctg cctgtcagtc ttggagatca accctccatc 120 tcttgcaaat ctagtcagag ccttgtacac aataatggaa acacctattt acattggtac 180 ctgcagaagc caggccagtc tccaaagctc ctgatctaca aagtttccaa ccgattttct 240 ggggtcccag acaggttcag tggcagtgga tcagggacag atttcacact caagatcagc 300 agagtggagg ctgaggatct gggagtttat ttctgctctc aaactacaca tgttcctccg 360 acgttcggtg gaggcaccaa gctggaaatc aaacgggctg atgctgcacc aactgtatcc 420 atcttcccac catccagtga gcagttaaca tctggaggtg cctcagtcgt gtgcttcttg 480 aacaacttct accccaaaga catcaatgtc aagtggaaga ttgatggcag tgaacgacaa 540 aatggcgtcc tgaacagttg gactgatcag gacagcaaag acagcaccta cagcatgagc 600 agcaccctca cgttgaccaa ggacgagtat gaacgacata acagctatac ctgtgaggcc 660 actcacaaga catcaacttc acccattgtc aagagcttca acaggggaga gtgttga 717 <210> 57 <211> 238 <212> PRT <213> Artificial Sequence <220> <223> mAb39 light chain <400> 57 Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Ala 1 5 10 15 Ser Ser Ser Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val 20 25 30 Ser Leu Gly Asp Gln Pro Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu 35 40 45 Val His Asn Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro 50 55 60 Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser 65 70 75 80 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 85 90 95 Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys 100 105 110 Ser Gln Thr Thr His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu 115 120 125 Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro 130 135 140 Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu 145 150 155 160 Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly 165 170 175 Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser 180 185 190 Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp 195 200 205 Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr 210 215 220 Ser Thr Ser Pro Ile Val Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 58 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> mAb39 light chain CDR1 <400> 58 Lys Ser Ser Gln Ser Leu Val His Asn Asn Gly Asn Thr Tyr Leu His 1 5 10 15 <210> 59 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> mAb39 light chain CDR2 <400> 59 Lys Val Ser Asn Arg Phe Ser 1 5 <210> 60 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> mAb39 light chain CDR3 <400> 60 Ser Gln Thr Thr His Val Pro Pro Thr 1 5 <210> 61 <211> 1389 <212> DNA <213> Artificial Sequence <220> <223> mAb39 heavy chain v2 (t1375a-->S459T) <400> 61 atgggatgga gctgtatcat gttctttttg gtagcaacag ctacagatgt ccactcccag gtccaactgc agcagcctgg ggctgaactg gtgaagcctg gggcttcagt gaagctgtcc 180. tgcaaggctt ctggctactc cttcaccacc tactggatgc actgggtgaa gcagaggcct ggacaaggcc taggtgggt tggagatatt aatcctagga acggtcgtac taactacaat 300. 300. 300. 300. 300. 300. 300. 300. 300. 300. 300. 300 caagtcagca gcctgacatc tgaggactct gcggtctatt actgtgcaat atggtcgggt gctatggact actggggtcc aggaacctca gtcaccgtct cctcagccaa aacaacagcc 420 ccatcggtct atccactggc ccctgtgtgt ggagatacaa ctggctcctc ggtgactcta 480 ggatgcctgg tcaagggtta tttccctgag ccagtgacct tgacctggaa ctctggatcc 540 ctgtccagtg atgtgcacac cttcccagct ctcctgcagt ctggcctcta caccctcagc 600 agctcagtga ctgtaaccac ctggcccagc cagaccatca cctgcaatgt ggcccacccg 660 ccaagcagca ccaaagtgga caagaaaatt gagcccagag ggtccccaac acataaaccc 720 tgtcctccat gcccagctcc taacctcttg ggtggaccat ccgtcttcat cttccctcca 780 aagatcaagg atgtactcat gatctccctg agcccatgg tcacgtgtgt ggtggtggat 840 gtgagcgagg atgacccaga tgtccatgtc agctggttcg tgaacaacgt ggagtacac 900 960 ctcccccatcc agcaccagga ctggatgagt ggcaaggagt tcaaatgcaa ggtcaacaac 1020 aaagccctcc cagcgcccat cgagagaacc atctcaaaac ccaaaggcc agtaagagct 1080 ccacaggtat atgtcttgcc tccaccagaa gaagagatga ctaagaaca ggtcactctg 1140 acctgcatga tcacagactt catgcctgaa gatattacg tggagtggac cacaacggg 1200 caacagagc taaactacaa gaacactgaa ccagtcctgg actctgatgg ttcttactc 1260 atgtacagca agctgagagt ggaaagaag aactgggtgg aaagaatag ctactcctgt 1320 tcagtggtcc acgaggttct gcacaatcac cacacgacta agagctctc ccggactccg 1380 ggTAATGA 1389 <210> 62 <211> 462 <212> PRT <213> Artificial Sequence <220> <223> mAb39 heavy chain (S459T) <400> 62 Met Gly Trp Ser Cys Ile Met Phe Phe Leu Val Ala Thr Ala Thr Asp 1 5 10 15 Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys 20 25 30 Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe 35 40 45 Three Thr Tyrr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu 50 55 60 Glu Trp Val Gly Asp Ile Asn Pro Arg Asn Gly Arg Thr Asn Tyr Asn 65 70 75 80 Glu Lys Ser Lys Ser Lys Ala Thr Leu Thr Val Asp Ile Ser Ser Ser 85 90 95 Thr Val Tyr Met Gln Val Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110 Tyr Tyr Cys Ala Ile Trp Ser Gly Ala Met Asp Tyr Trp Gly Pro Gly 115 120 125 Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr 130 135 140 Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu 145 150 155 160 Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp 165 170 175 Asn Ser Gly Ser Leu Ser Ser Asp Val His Thr Phe Pro Ala Leu Leu 180 185 190 Gln Ser Gly Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Thr Trp 195 200 205 Pro Ser Gln Thr Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr 210 215 220 Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Ser Pro Thr His Lys Pro 225 230 235 240 Cys Pro Pro Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe 245 250 255 Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro 260 265 270 Met Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val 275 280 285 His Val Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr 290 295 300 Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Ile Arg Val Val Ser Ala 305 310 315 320 Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys 325 330 335 Lys Val Asn Asn Lys Ala Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser 340 345 350 Lys Pro Lys Gly Pro Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro 355 360 365 Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Ile 370 375 380 Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly 385 390 395 400 Gln Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp 405 410 415 Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp 420 425 430 Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His 435 440 445 Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys 450 455 460

Claims

1. An isolated antibody or antigen-binding fragment thereof that specifically binds to ZnT8, wherein the antibody or antigen-binding fragment thereof comprises heavy chain complementary determining regions (CDRs) 1, 2, and 3 and light chain CDR1, CDR2, and CDR3, wherein: the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO:33, the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO:34, the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO:35, the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO:38, the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO:39, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO:40; or The amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO:53, the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO:54, the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO:55, the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO:58, the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO:59, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO:

60.

2. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof is an antagonist of ZnT8 activity.

3. The antibody or antigen-binding fragment according to claim 1, wherein The antigen binding fragment is selected from the group consisting of scFv, sc(Fv)2, Fab, and F(ab)2. 4 . The antibody or antigen-binding fragment thereof according to claim 1 , wherein the antibody or antigen-binding fragment thereof comprises a VH comprising one of the amino acid sequences shown in SEQ ID NO: 32 or 52. 5 . The antibody or antigen-binding fragment thereof according to claim 1 , wherein the antibody or antigen-binding fragment thereof comprises a VL comprising one of the amino acid sequences shown in SEQ ID NO: 37 or 57.

6. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises: (a) a VH comprising the amino acid sequence of SEQ ID NO: 32, and a VL comprising the amino acid sequence of SEQ ID NO: 37; or (b) VH comprising the amino acid sequence shown in SEQ ID NO:52, and VL comprising the amino acid sequence shown in SEQ ID NO:

57.

7. An isolated nucleic acid molecule encoding the anti-ZnT8 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

8. A vector comprising the nucleic acid molecule of claim 7.

9. A host cell comprising the vector of claim 8.

10. The host cell according to claim 9, wherein The host cell is a prokaryotic or eukaryotic cell.

11. A method for producing an anti-ZnT8 antibody or an antigen-binding fragment thereof, the method comprising the steps of: (a) culturing the host cell of claim 9 under conditions suitable for the host cell to express the ZnT8 antibody or the antigen-binding fragment thereof; and (b) recovering the ZnT8 antibody or the antigen-binding fragment thereof.

12. The method according to claim 11, wherein: The host cell is a prokaryotic or eukaryotic cell.

13. A composition comprising the anti-ZnT8 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 and a suitable pharmaceutical carrier.

14. The composition according to claim 13, wherein The composition is formulated for intravenous, oral, subcutaneous, intraperitoneal, intrathecal, or intramuscular administration.

15. Use of the isolated antibody or antigen-binding fragment thereof that specifically binds to ZnT8 according to any one of claims 1 to 6 in the preparation of a reagent for detecting ZnT8.

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