WO2010096930A1 - Single chain antibodies for targeting pancreatic alpha and beta cells - Google Patents

Abstract

Single chain antibodies binding specifically to cell types of the islets of Langerhans are provided herein, as well as diagnostic, prognostic, theranostic and therapeutic methods using the antibodies to identify cell types of the islets of Langerhans and for identifying a change in β cell mass via in vivo imaging, particularly for diseases related to the pancreas such as diabetes

Description

SINGLE CHAIN ANTIBODIES FOR TARGETING PANCREATIC ALPHA AND

BETA CELLS

FIELD OF THE INVENTION

[0001] Visualization and quantification of the native β-cell mass in vivo in humans are important in the study of the natural course of diabetes, and in ongoing trials aimed at preserving β-cell mass in patients with diabetes. There is a great need for development of a non-invasive method for visualization and quantification of the native β-cell mass.

[0002] The disclosure relates to human single chain antibodies (SCA) and analogs, homologs, fragments and variants thereof for non-invasive in vivo measurement of pancreatic α and β cell mass in rodents and humans. BACKGROUND

[0003] Diabetes mellitus affects over 100 million individuals worldwide.

In the U.S., the estimated healthcare costs of those affected by diabetes is approximately 136 billion dollars annually. Diabetes mellitus is a disorder of the metabolism that is characterized by the inability of the pancreas to secrete sufficient amounts of insulin, which results in large fluctuations in blood glucose levels and can have both short- and long-term physiological consequences. Long-term complications arising from elevated blood glucose levels (hyperglycemia) in patients with Type I diabetes (insulin-dependent diabetes mellitus, or IDDM) include retinopathy, neuropathy, nephropathy and other vascular complications. Low glucose levels (hypoglycemia) can lead to diabetic coma, seizures, accidents, anoxia, brain damage, decreased cognitive function, and death.

[0004] Type Il diabetes, also known as non-insulin dependent diabetes mellitus or NIDDM, is a progressive disease characterized by impaired glucose metabolism resulting in elevated blood glucose levels. Patients with type Il diabetes exhibit impaired pancreatic beta-cell function resulting in failure of the pancreatic beta-cells to secrete an appropriate amount of insulin in response to a hyperglycemic signal, and resistance to the action of insulin at its target tissues (insulin resistance). Docket no. 05001770-409PCT - 2 -

[0005] Current treatments of type Il diabetes aim to reverse insulin resistance, control intestinal glucose absorption, normalise hepatic glucose production, and improve beta-cell glucose sensing and insulin secretion. Because of the shortcomings of current treatments for diabetes, new treatments for type I and type Il diabetes, as well as new diagnostic and prognostic methods, are highly desirable.

[0006] Pancreatic β-cell mass is markedly reduced in patients with type

I diabetes as a result of selective autoimmune destruction of β-cells (Weir, G. C. et al., Diabetes 39, 401-415 (1990)). In patients with type Il diabetes, a reduction in β-cell mass can also be observed, possibly because of an increased apoptosis rate (Butler, A.E. et a/., Diabetes 52, 102-110 (2003)). Due to these pathophysiologic conditions, it would be advantageous to measure β-cell mass non-invasively in vivo. In addition, restoration of β-cell mass provides a potential cure for diabetic patients, and a number of therapeutic strategies have been proposed to modulate β-cell mass. Indeed, data from animal studies has suggested that several antidiabetic drugs (such as glucagon-like peptide-1 (GLP-1) analogues and dipeptidyl peptidase-4 inhibitors) might not only improve the control of plasma glucose, but also increase β-cell mass (Staffers, D.A. et al., Diabetes 49, 741-748 (2000)). [0007] As yet the majority of studies focusing on changes in β-cell mass have been carried out in rodents, where β-cell mass can readily be determined in the harvested pancreas after sacrificing the animals. Currently, no method exists that could either accurately or non-invasively determine the beta-cell mass in humans. Longitudinal studies aiming to restore β-cell mass in humans have been hampered by the lack of reliable imaging methods to directly quantify β-cell mass in vivo. Therefore, most studies have applied functional tests of insulin secretion, e.g. in response to intravenous arginine-, glucagon- or glucose-administration as surrogate markers of β-cell mass. Although some correlation between β-cell mass and such functional indices as insulin secretion exists (Larson et al., 2006), the validity of these markers Docket no. 05001770-409PCT - 3 - in humans is not yet proven. In addition, human insulin secretory capacity is subject to large day-to-day variability.

[0008] The quantification of beta-cells would allow a better understanding of the pathophysiology of both type I and Il diabetes, such as the relationship between β-cell mass, β-cell function and glucose homeostasis; identification of pre-diabetic patients; and monitoring of therapeutic treatments, such as cellular therapies (e.g. islet-transplantations).

However, so far no method exists that allows the imaging of a single islet or a single β-cell in pancreas for the accurate and non-invasive assessment of the native β-cell mass.

[0009] There is a need therefore for precise tools for the accurate and non-invasive assessment of pancreatic α and β cell mass in vivo.

SUMMARY OF THE INVENTION [0010] Accordingly, the present invention provides single chain antibodies which specifically bind to cell types of the islets of Langerhans and are suitable for use for non-invasive imaging of pancreatic cells in vivo. In particular, single chain antibodies (SCAs) binding specifically to pancreatic α- or β-cells, analogs, homologs, fragments and variants of the SCAs, and pharmaceutical compositions thereof, are provided herein. Diagnostic, prognostic, theranostic and therapeutic methods using the antibodies of the invention to identify cell types of the islets of Langerhans via in vivo imaging, particularly for diseases related to the pancreas such as type I and type Il diabetes, are also provided. [0011] In one embodiment, the present invention relates to a single chain antibody specifically binding to a cell type of the islets of Langerhans, wherein said single chain antibody comprises a heavy chain CDR1 amino acid sequence, CDR2 amino acid sequence, and CDR3 amino acid sequence, respectively, of amino acid residues 34-38, 53-69, 102-108 of SEQ Docket no. 05001770-409PCT - A -

ID NO:2; and a light chain CDR1 amino acid sequence, CDR2 amino acid sequence, and CDR3 amino acid sequence, respectively of amino acid residues 159-169, 185-191 , 224-232 of SEQ ID NO:2; or an analog, homolog, fragment or variant thereof. In one aspect, an analog, homolog, fragment or variant retains the binding specificity of the SCA.

[0012] In another embodiment, there is provided herein a single chain antibody or an analog, homolog, fragment or variant thereof, comprising heavy and light chain CDR1 , CDR2 and CDR3 amino acid sequences 34-38, 53-69, 102-108, 159-169, 185-191 , and 224-232, respectively, of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10, and binding specifically to β-cells of the pancreas. In one embodiment, the β-cell is identified in a subject. In an aspect, the subject is a mammal, including but not limited to a rodent, canine, pig, primate or preferably a human.

[0013] In yet another embodiment, the single chain antibodies of the invention may be labeled with a radioactive isotope. For example, the antibodies may be labeled with ¹¹C, ¹⁸F, ¹⁸O, ¹³N, ⁷⁶Br and ¹²⁴I for use as a radioligand in PET imaging, or with ⁸⁶Y, ^99mTc, ¹¹¹In, ¹²³I, and ²⁰¹TI for use as a radioligand in SPECT imaging.

[0014] The single chain antibodies of the invention may also be used for diagnosing a change in β cell mass. For example, an increase in β cell mass may be diagnostic of an insulinoma, a nesidioblastoma, an endocrine tumor, or onset of obesity, or may confirm the use of an effective amount of a drug or medicament for the treatment of type I or type Il diabetes, or may confirm the success of a β cell transplantation. Alternatively, a decrease in β cell mass may be diagnostic of type I or type Il diabetes, or may confirm the use of an effective amount of a drug for the treatment of obesity, or may support the surveillance of a resected insulinoma, nesidioblastoma, or endocrine tumor. In certain embodiments, the β cell is in a subject, such as a rodent, a canine, a pig, a primate or a human. Docket no. 05001770-409PCT

- 5 -

[0015] Also provided herein are nucleic acid molecules comprising a nucleic acid sequence encoding the antibodies provided herein. In an embodiment, the nucleic acid molecules have the sequence shown in SEQ ID NO:1 , 3, 5, 7 or 9. In another embodiment, the nucleic acid molecules are operably linked to an expression control sequence to form an expression vector, which may then be propagated in a suitable cell.

[0016] The invention further provides the use of an effective amount of a single chain antibody of the invention or an analog, homolog, fragment or variant thereof, which specifically binds to a cell type (e.g. α- or β-cell) of the islets of Langerhans, in the identification of the cell type by in vivo imaging. The antibody may be labeled with a radioactive isotope such as ¹¹C, ¹⁸F, ¹⁸O, ¹³N, ⁷⁶Br or ¹²⁴I for use as a radioligand in PET imaging, or such as ⁸⁶Y, ^99mTc, ¹¹¹In, ¹²³I, and ²⁰¹TI for use as a radioligand in SPECT imaging.

[0017] In one embodiment, the antibody may be used to detect a change in β cell mass in vivo. For example, in certain embodiments an increase of β cell mass is diagnostic of an insulinoma, a nesidioblastoma, an endocrine tumor or onset of obesity, or confirms the use of an effective amount of a drug for the treatment of type I or type Il diabetes, or confirms a successful β cell transplantation. In other embodiments, a decrease of β cell mass is diagnostic of type I or type Il diabetes, or confirms the use of an effective amount of a drug for the treatment of obesity, or supports the surveillance of a resected insulinoma, nesidioblastoma, or endocrine tumor. In some embodiments the β cell being detected is in a subject, for example a rodent, a canine, a pig, a primate or a human. [0018] A method for identifying a cell type of the islets of Langerhans in a subject in vivo is also provided herein, wherein a single chain antibody of the invention or an analog, homolog, fragment or variant thereof is detectable labeled and then imaged in the subject. For example, the antibody may be labeled with a radioactive isotope, such as ¹¹C, ¹⁸F, ¹⁸O, ¹³N, ⁷⁶Br and ¹²⁴I for PET imaging or ⁸⁶Y, ^99mTc, ¹¹¹In, ¹²⁴I, and ²⁰¹TI for SPECT imaging. In Docket no. 05001770-409PCT - 6 - another embodiment, the antibody is used as a superparamagnetic contrast agent and visualized using magnetic-resonance imaging (MRI). Other imaging labels and methods may also be used, including without limitation bioluminescence and sonography. [0019] Further provided herein is a method for identifying a change in β cell mass in a subject in vivo, wherein β cells are identified in the subject using the methods provided herein; an increase in β cell mass is diagnostic of an insulinoma, a nesidioblastoma, an endocrine tumor or onset of obesity, or confirms the use of an effective amount of a drug for the treatment of type I or type Il diabetes, or confirms a successful β cell transplantation, or indicates increase in size of a resected insulinoma, nesidioblastoma, or endocrine tumor; and/or a decrease of β cell mass is diagnostic of type I or type Il diabetes, or confirms the use of an effective amount of a drug for the treatment of obesity, or indicates reduction in size of a resected insulinoma, nesidioblastoma, or endocrine tumor.

[0020] In one embodiment, the single chain antibody or analog, homolog, fragment or variant thereof is humanized. In another embodiment, the single chain antibody or analog, homolog, fragment or variant thereof is administered by injection, orally, intravenously, intraperitoneal^, intramuscularly or subcutaneously.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Particular embodiments of the present invention will now be explained by way of example and with reference to the accompanying drawings, in which:

[0022] Figure 1 shows the efficiency of phage-display screening for human single chain antibodies (SCAs) binding to rat pancreatic islets. Successive rounds of panning of phage transducing units (TU) per rat islet Docket no. 05001770-409PCT

- 7 - recovered with the in vivo enrichment (a) are compared to the in vitro enrichment approach (b).

[0023] Figure 2 shows the amino acid sequences of the β-cell specific

SCA B1 to B5 (SEQ ID NO: 2, 4, 6, 8 and 10) and the α-cell specific SCA A1 and A2 (SEQ ID NO: 12, 14). Amino acids are shown in one letter codes. Positions which are diverse in the repertoire are marked in bold. Boxes indicate the complementarity determining region (CDR) determined according to Kabat et al., 2000. Heavy and light chain regions are underlined (heav^ chain, light. chain). [0024] Figure 3 shows immunofluorescence analyses of the ISPC/SCA binding affinity to control tissue after intravenous administration of ISPC or SCA in a rat. Staining in green colour for ISPC 1 (a), ISPC 3 (b), SCA B1 (c) or SCA A1 (d). Nuclei were stained with DAPI in blue colour. Neither the ISPC nor the SCA revealed any binding activity to the tested control organs. All images were acquired at 40 x magnification.

[0025] Figure 4 shows immunofluorescence analyses of the ISPC/SCA binding affinity to pancreatic islets after intravenous administration of ISPCs, a control ISPC or SCAs in a rat. Double staining of ISPC (a-f, green) and insulin- (a,c,e, red) or glucagon (b,d,f, red) and nucleus using DAPI (blue). ISPC 1 (a,b) co-localized exclusively with insulin, whereas ISPC 3 (c,d) co- localized selectively with glucagon. In contrast, an insertless control ISPC (e,f) was not detectable in the islets. Moreover, double staining of SCA (g-j, green) and insulin- (g,i, red) or glucagon (h,j, red) and nucleus using DAPI (blue) was performed. This confirmed the highly selective uptake of SCA 1 in β-cells (g,h) and of SCA 3 in α-cells (i,j). All images were acquired at 40 x magnification.

[0026] Figure 5 shows ultrastructural analyses of exact intracellular localization of the SCAs. Transmission electron microscopy detected the β- cell specific SCA 1 (a,b) and the α-cell specific SCA 3 (c,d) in the Docket no. 05001770-409PCT - 8 - endoplasmatic reticulum (a,c) and at the secretory granule membrane (b,d) of the respective target cells exclusively. Scale bars, 90 nm.

[0027] Figure 6 shows analyses of the binding process of selected

[¹²⁵l]-labelled SCAs to different endocrine and exocrine cell lines in vitro, and determination of their pharmacokinetic profiles in vivo. Binding specificity of SCA B1 to INS-1 cells (a,^*P = 0.0016 vs. α-TC1 or AR42J) and SCA A1 to α- TC1 cells (b,^*P < 0.0001 vs. INS-1 or AR42J). Time-course of binding of SCA B1 to INS-1 cells (c, t1/2 = 8.0 min) or SCA A1 to α-TC1 cells (d, t_1/2 = 5.3 min). Competition assay of SCA B1 (e,*P < 0.0001 vs. preincubation (Pl) with SCA B1) or SCA A1 (f,*P <0.0001 vs. Pl with SCA A1) with unlabelled SCAs. Dose response of SCA B1 binding to INS-1 cells (g, r² = 0.96) or SCA A1 to α-TC1 cells (h, r² = 0.96). Time-course of elimination of SCA B1 (i, t_1/2 = 22.7 min, r² = 0.87) or SCA A1 (j, t_1/2 = 19.2 min, r² = 0.97) from the vascular system. Error bars represent SEM. [0028] Figure 7 shows immunofluorescent staining for binding of the

SCA to human islets in a representative pancreas from a non-diabetic subject. SCA, green; insulin or glucagon, red; DAPI (nucleus), blue. The SCA B1 co- localized selectively with insulin- (a), but not glucagon-staining (b). The SCA A1 co-localized with glucagon-(c), but not insulin-expressing cells (d). All images were acquired at 40 x magnification.

[0029] Figure 8 shows effects of SCAs on islet function in vivo and cell viability in vitro. Time-course of (a) plasma glucose, (b) insulin- and (c) glucagon-levels in rats during an IPGTT, 7 days after intravenous injection of β-cell specific SCA B1 (o, n = 5), α-cell specific SCA A1 (T, n = 5) or in vehicle-treated rats (•, n = 5]. (d) Viability of INS-1 and α-TC1-cells (SCA1 is SCA B1 and SCA3 is SCA A1) and (e) flow cytometric analysis of cells stained for the apoptosis marker FITC-Annexin-V after overnight exposure to SCA in vitro compared to non-treated controls (n = 6 experiments) (SCA1 is SCA B1 and SCA3 is SCA A1). Error bars represent SEM. Docket no 05001770-409PCT - 9 -

[0030] Figure 9 shows the quantification of β-cell mass with the radio- labelled SCA B1 in rats. Linear regression analyses show a close correlation between pancreatic uptake of SCA B1 and β-cell mass in normal (▼), low- (o) and high-dose (•) STZ animals (a, r² = 0.937). Moreover, a strong non-linear association between the probe accumulation in the pancreas and the AUC for glucose during an IPGTT in the respective animals was depicted (b, r² = 0.876). Each data point represents an animal.

[0031] Figure 10 shows a dynamic in vivo PET scan showing the selective accumulation of [I¹²⁴]-labeled SCA B1 in the pancreatic gland 30 minutes (a) and 24 hours (b) after injection. Neither heart (H) nor lungs (L) nor kidneys (K) demonstrate any accumulation of SCA Bl Non-bound tracer molecules temporarily accumulate in the bladder (B) as they are eliminated through the urinary tract (a).

[0032] Figure 11 shows anatomical in vivo images of the mouse abdomen using MRI at ultra high fields (16.4T) and the SCA B1 antibody, as follows: a) Axial FLASH, 80x80x300 μm, TR/TE=250/5.2ms, α=45°, NA=8; b)

Axial FLASH, 100x100x200 μm, TR/TE=15/2ms, α=20°, 12 segments, NA=4; c) Coronal FLASH, 100x100x200 μm, TR/TE= 14/1.65ms, α=15°, 8 segments,

NA=4; d) Coronal FLASH, 80x80x375 μm, TR/TE=9.63/4.25ms, α=90°, NA= 1 ; e) Coronal FLASH, 100x100x375 μm, TR/TE=500/4.21 ms, α=60°,

NA=10; K: Kidney, L: Liver, M: Skeletal Muscle, S: Spleen, ST: Stomach; pancreatic structures are circled or marked with an arrow.

[0033] Figure 12 shows images of a dissected pancreas ex vivo using

MRI and the SCA B1 antibody, as follows: a) FLASH, 50x50x317 μm, TR/TE=55/4.21 ms, α=40°, NA=10; b) FLASH, 50x50x200 μm, TR/TE=55/4.21 ms, α=40°, NA=10; labelled islets are marked with an arrow.

[0034] Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating particular embodiments of the invention are given by way of Docket no. 05001770-409PCT - 10 - illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

DETAILED DESCRIPTION OF THE INVENTION

[0035] The present invention provides single chain antibodies (SCAs) and analogs, homologs, fragments and variants thereof which are highly specific for either β- or α-cells of the pancreas, and diagnostic, prognostic, theranostic and therapeutic methods of use thereof. In particular, the SCAs of the invention may be used to measure β- or α-cell mass non-invasively in vivo.

[0036] As used herein, "β-cells" refer to the fully differentiated insulin- producing β-cells of the islets of Langerhans in the pancreas. Pancreatic β- cells are characterized by their secretion of insulin and typically by their cell surface expression of the islet amyloid polypeptide (IAPP). "α-cells" refer to cells of the islets of Langerhans in the pancreas which make and release glucagon.

[0037] The selective detection of islet cells, such as islet β-cells, within the pancreas requires contrast agents exhibiting high specificity for islet cells and preferentially having a high affinity for binding sites expressed at high levels on the islet cells. In addition, such agents should be excreted rapidly from the vascular system, and should exert no in vivo toxicity on the islet cells or other tissues. Theoretical requirements for the imaging of the β-cell mass are reviewed by Schneider, S., Obesity and Metabolism 10, 1-10 (2008). [0038] The development of such tools has been limited due to various hurdles. First, the total β-cell mass constitutes only -1-2 % of the pancreas, with ~1 million individual islets being scattered throughout the organ. This mass is even smaller in patients with diabetes. Second, the differences in density and echogenicity between the islets and the exocrine cells of the Docket no 05001770-409PCT

- 11 - pancreas are relatively small, thereby limiting the application of conventional ultrasonographic or scintigraphic methods (Meier, J.J. Diabetologia 51 , 703- 713 (2008)). Third, the general knowledge about potential targets that are exclusively expressed on the α- or β-cell surface is poor. [0039] In order to overcome such limitations, we have used phage- display technology to identify single-chain antibodies which bind specifically to β- or α-cells of the pancreas. Phage display is a powerful approach for isolating peptides or antibodies that bind to mammalian cell surface receptors. Repeated phage-panning has been used to isolate human SCAs specific for epitopes important in the medical field for imaging and therapy (Riechmann L. et al. Nature. 332, 323-7 (1988)).

[0040] A SCA contains an antigen binding domain in one polypeptide instead of two as is the case for a complete antibody. The antigen binding domain of an antibody comprises two separate regions: a heavy chain variable domain (VH) and a light chain variable domain (V_L: which can be either V_κ or V_λ). The antigen binding site itself is formed by six polypeptide loops: three from V_H domain (H 1 , H2 and H3) and three from VL domain (L1 , L2 and L3).

[0041] A diverse primary repertoire of V genes that encode the V_H and V_L domains is produced by the combinatorial rearrangement of gene segments. The V_H gene is produced by the recombination of three gene segments, V_H, D and J_H. In humans, there are approximately 51 functional V_H segments, 25 functional D segments and 6 functional J_H segments, depending on the haplotype. The V_H segment encodes the region of the polypeptide chain which forms the first and second antigen binding loops of the V_H domain

(H 1 and H2), whilst the V_H, D and J_H segments combine to form the third antigen binding loop of the VH domain (H3). The V_L gene is produced by the recombination of only two gene segments, V_L and J_L. In humans, there are approximately 40 functional V_κ segments, 31 functional V_λ segments , 5 functional J_κ segments and 4 functional J_λ segments, depending on the Docket no. 05001770-409PCT - 12 - haplotype (Williams S.C. et al., J MoI Biol. 264, 220-32 (1996); Corbett S.J. et al., J MoI Biol. 270, 587-97 (1997)). The V_L segment encodes the region of the polypeptide chain which forms the first and second antigen binding loops of the V_L domain (L1 and L2), whilst the V_L and J_L segments combine to form the third antigen binding loop of the V_L domain (L3).

[0042] Antibodies selected from this primary repertoire are believed to be sufficiently diverse to bind almost all antigens with at least moderate affinity. High affinity antibodies are produced by "affinity maturation" of the rearranged genes, in which point mutations are generated and selected by the immune system on the basis of improved binding.

[0043] Analysis of the structures and sequences of antibodies has shown that five of the six antigen binding loops (H 1 , H2, L1 , L2, L3) possess a limited number of main-chain conformations or canonical structures. The main-chain conformations are determined by (i) the length of the antigen binding loop, and (ii) particular residues, or types of residue, at certain key positions in the antigen binding loop and the antibody framework. Analysis of the loop lengths and key residues has allowed the prediction of the main- chain conformations of H1 , H2, L1 , L2 and L3 encoded by the majority of human antibody sequences (Chothia C. et al., J MoI Biol. 227, 799-817 (1992)). Although the H3 region is much more diverse in terms of sequence, length and structure due to the use of D segments, it also forms a limited number of main-chain conformations for short loop lengths which depend on the length and the presence of particular residues, or types of residue, at key positions in the loop and the antibody framework (Shirai H. et al., FEBS Lett. 399, 1-8 (1996)).

[0044] Using phage-display technology, we identified a single chain antibody which binds to a β-cell specific epitope and a single chain antibody which binds to an α-cell specific epitope. These antibodies and their analogs, homologs, fragments and variants can be used for in vivo imaging of β- or α- cell mass. Such in vivo imaging of islet cell mass has a variety of uses, for Docket no. 05001770-409PCT

- 13 - example to diagnose diseases of the pancreas such as type I or type Il diabetes, to monitor disease progression or to evaluate efficacy of therapeutic regimens.

[0045] In one aspect, the present invention provides a single chain antibody (SCA) that binds to an epitope on the islets of Langerhans of the pancreas, comprising a heavy chain and a light chain variable region amino acid sequence, each variable region amino acid sequence comprising a contiguous amino acid sequence from within an FR1 sequence through an FR3 sequence that comprises at least one of the amino acid substitutions in the HCDR2 sequences, the HCDR3 sequences, the LCDR2 sequences, and the LCDR3 sequences shown in Figure 2. In one embodiment, the SCAs bind to an epitope on glucagon secreting α-cells. In another embodiment, the SCAs bind to an epitope on insulin secreting β-cells. In yet another embodiment, the SCAs have complementarity determining regions. In a further embodiment, the β-cell specific SCA has the amino acid sequence shown in SEQ ID NO: 2 or an analog thereof, including but not limited to the analogs shown in SEQ ID NO: 4, 6, 8, and 10 or homologs thereof. In another embodiment, the α-cell specific SCAs have the amino acid sequence shown in SEQ ID NO: 12 or an analog thereof, including but not limited to the analog shown in SEQ ID NO: 14 or a homolog thereof.

[0046] In another aspect, the present invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding the SCA. In one embodiment, the nucleic acid molecule encoding the conjugate protein has the nucleotide sequence shown in SEQ ID NO:1 or an analog thereof, including but not limited to the analogs shown in SEQ ID NO: 3, 5, 7, and 9 or homologs thereof. In a further embodiment, the invention provides an expression vector comprising the nucleic acid operably linked to an expression control sequence. In yet another embodiment, the present invention provides a cell comprising the expression vector or progeny of such a cell, wherein the cell expresses the conjugate protein. Docket no. 05001770-409PCT - 14 -

[0047] In a further aspect, the present invention provides a method to identify the α- or β-cell mass of the endocrine pancreas by non-invasive visualization and quantitative measurement using an imaging method, and this method provides support for new experimental therapies, as well as monitoring and management of α- or β-cell associated disorders.

[0048] In another aspect, the disclosure provides a method for determining the α- or β-cell mass in the pancreas of a subject in need thereof by administering to the subject or to an organ isolated from the subject an effective amount of a SCA in order to diagnose a disease (e.g. type I or type Il diabetes), to determine the therapeutic effect of a medicament, or to monitor the transplantation of β-cell mass from a xenogenic or allogeneic source.

[0049] In one embodiment, the subject can be a rodent, a canine, a pig, a primate or a human. Although methods of the present invention can be used to determine the α- or β-cell mass in any mammal, the subject is preferably a human.

[0050] In another embodiment, the therapeutic effect of a medicament or drug can be determined by measuring a decline of β-cell mass in a tumor (including but not limited to insulinoma or nesidioblastosis) or an increase in β-cell mass in the treatment of diabetes type I or type II. In a further embodiment, the increase of β-cells from xenogeneic or allogeneic sources can be determined as successful β-cell transplantation. In yet another embodiment, the increase of β-cells can be determined during onset of obesity.

[0051] In another embodiment, the therapeutic effect of a medicament or drug can be determined as a decline of α-cell mass in a tumor (including but not limited to glucagonoma). In another embodiment a steady α-cell mass can serve as a control for a varying β-cell mass of the same islet of Langerhans. Docket no. 05001770-409PCT - 15 -

[0052] The present invention also provides methods for diagnosing a metabolic or neuroendocrine disorder in a subject by administering to the subject an effective amount of a SCA; obtaining one or more computerized image(s) of at least a portion of the pancreas of the subject; quantitatively analyzing the computerized image(s) in order to determine the α- or β-cell mass in the pancreas of the subject; and comparing the α- or β-cell mass with a baseline measure of α- or β-cell mass, where decreased α- or β-cell mass or increased α- or β-cell mass versus the baseline measure is associated with the presence of a metabolic or neuroendocrine disorder. In another embodiment of the present invention, the computerized image is obtained using PET, SPECT, bioluminescence, magnetic-resonance imaging (MRI), or sonography.

[0053] In one embodiment of the invention, SCA B1 or analogs, homologs, fragments or variants thereof are labeled with radioactive isotopes to form a radioligand used to generate the computerized image obtained using PET and SPECT. In another embodiment of the invention, the metabolic disorder is a β-cell associated disorder, including but not necessarily limited to, an insulinoma, nesidioblastosis or another neuroendocrine tumor. In another embodiment, the β-cell associated disorder is diabetes. In a particular embodiment of the invention, the metabolic disorder is type I diabetes. In another embodiment, the metabolic disorder is type Il diabetes. In a further embodiment, the metabolic disorder is preclinical type I diabetes.

[0054] In one embodiment of the invention, SCA A1 or analogs, homologs, fragments or variants thereof are labeled with radioactive isotopes to form a radioligand used to generate the computerized image obtained using PET and SPECT. In another embodiment of the invention, the metabolic disorder is an α-cell associated disorder, including but not necessarily limited to, a glucagonoma.

[0055] The present invention additionally provides methods for assessing the prognosis of a subject at risk for developing diabetes by Docket no. 05001770-409PCT - 16 - periodically administering to the subject an effective amount of a SCA B1 or its analog or homolog radioligands; obtaining one or more computerized image(s) of at least a portion of the pancreas of the subject; quantitatively analyzing the computerized image(s) in order to determine the β-cell mass in the pancreas of the subject; and comparing the periodically determined β-cell mass with a baseline measure of β-cell mass, where decreased β-cell mass versus the baseline measure is associated with the progression from a prediabetic condition to a diabetic condition. In an embodiment of the invention, the radioligand is SCA B1 or analogs, homologs, fragments or variants thereof and the computerized image is obtained using PET. In another embodiment of the present invention, the subject is at risk for developing type I or type Il diabetes.

[0056] The present invention also provides methods for determining the efficacy of therapy of a metabolic disorder by periodically administering to the subject an effective amount of SCA B1 or analogs, homologs, fragments or variants thereof.for example as a radioligand; obtaining one or more computerized image(s) of at least a portion of the pancreas of the subject; quantitatively analyzing the computerized image(s) in order to determine the β-cell mass in the pancreas of the subject; and comparing the periodically determined β-cell mass with a baseline measure of β-cell mass, where a β-cell mass generally equivalent to the baseline measure is indicative of successful therapy to treat the metabolic disorder. In an embodiment of the invention, the radioligand is SCA B1 or analogs, homologs, fragments or variants thereof and the computerized image is obtained using PET. In a further embodiment of the invention, the metabolic disorder is a pancreatic β-cell associated disorder. In a specific embodiment, the β-cell disorder is an insulinoma, nesidioblastosis or a neuroendocrine tumor. In yet another specific embodiment, the β-cell disorder is diabetes. In still other embodiments of the invention, the β-cell disorder is type I diabetes, type Il diabetes or preclinical type I diabetes. Docket no. 05001770-409PCT - 17 -

[0057] The invention also provides methods for managing the treatment or prevention of diabetes by periodically administering to the subject an effective amount of a radioligand; obtaining one or more computerized image(s) of at least a portion of the pancreas of the subject; quantitatively analyzing the computerized image(s) in order to determine the β-cell mass in the pancreas of the subject; and comparing the periodically determined β-cell mass with a baseline measure of β-cell mass, where decreased β-cell mass versus the baseline measure is associated with the need for further therapy. In an embodiment of the invention, the radioligand is SCA B1 and its analogs or homologs and the computerized image is obtained using PET. In one embodiment, the diabetes is type Il diabetes. In another embodiment, the diabetes is type I diabetes.

[0058] The present invention also provides methods for determining the success of an islet cell transplantation, e.g. a β-cell transplantation, by periodically administering to the subject an effective amount of a SCA B1 or analogs, homologs, fragments or variants thereof, for example as a radioligand. Increase of β-cell mass after transplantation indicates success of a β-cell transplantation, whereas decrease in β-cell mass after transplantation indicates lack of success of a β-cell transplantation. [0059] Thus, the present invention provides antibodies and analogs, homologs, fragments and variants thereof for the detection of a β-cell specific epitope. Also provided herein are antibodies and analogs, homologs, fragments and variants thereof for the detection of an α-cell specific epitope. Such antibodies can be used for in vivo imaging of cells of the pancreas, for example to diagnose disease of the pancreas, to monitor disease progression or to assess efficacy of a therapeutic treatment. Accordingly, the present inventions provides SCAs and analogs, homologs, fragments or variants thereof which are specific for the identification of a- or β-cell mass in the pancreas. Docket no. 05001770-409PCT - 18 -

[0060] The term "ISPC" as used herein refers to islet specific phage clones which have been isolated from a phage library consisting of nucleic acid molecules comprising a first DNA sequence encoding the signal peptide of a bacteriophage protein linked at its 3' end to a second DNA sequence encoding a tag, wherein the second DNA sequence is linked at its 3' end to a third DNA sequence encoding an immunoglobulin variable region polypeptide. Such ISPCs are known in the art and have been described previously, for example in published US patent application number US/2004/0202995. In one embodiment, the ISPC nucleic acid sequence is human and has the nucleotide sequence shown in SEQ ID NO:1 , or an analog or homolog thereof. In another embodiment, the nucleic acid sequence is human and has the nucleotide sequence shown in SEQ ID NO: 3, 5, 7, 9 or 11 , or an analog or homolog thereof.

[0061] The term "single chain antibody" or "SCA" as used herein refers to a single chain antibody that has been derived from an ISPC. Single chain antibodies are known in the art.

[0062] In one embodiment, there is provided herein a single chain antibody (SCA) called SCA B1 which comprises a human CDR and frame region protein sequences and has the amino acid sequence shown in SEQ ID NO:2 (Fig. 2). In another embodiment, single chain antibodies specific for β- cells are provided herein. Such antibodies are for example selected from a group of analogs including but not limited to SCA B1 , SCA B2, SCA B3, SCA B4, and SCA B5.

[0063] In one embodiment, there is provided herein a single chain antibody SCA A1 which comprises a human CDR and frame region protein sequences and has the amino acid sequence shown in SEQ ID NO: 12 (Fig. 2). In another embodiment, single chain antibodies specific for a α-cells are provided herein. Such antibodies are for example selected from a group of analogs including but not limited to SCA A1 and SCA A2. Docket no 05001770-409PCT - 19 -

[0064] The term "homolog" is used to mean those amino acid or nucleic acid sequences which have slight or inconsequential sequence variations from the sequences in SEQ ID NOs: 2, 4, 6, 8, 10, 12, and 14, i.e., the sequences function in substantially the same manner. The sequence variations may be attributable to local mutations or structural modifications. Sequences having substantial homology include nucleic acid sequences having at least 65%, more preferably at least 85%, and most preferably 90- 95% identity with the sequences shown in SEQ ID NOs: 1 , 3, 5, 7, 9, 11 , and 13. Sequence identity can be calculated according to methods known in the art. Nucleic acid sequence identity is most preferably assessed by the algorithm of BLAST version 2.1 advanced search. A series of programs is available at http://www.ncbi.nlm.nih.gov/BLAST. The advanced blast search (http://www.ncbi.nlm.nih.gov/blast/blast.cgi7Jform =1) may be set to default parameters, (ie Matrix BLOSUM62; Gap existence cost 11 ; Per residue gap cost 1 ; Lambda ratio 0.85 default). Examples of references to BLAST searches are: Altschul, S. F. et al., (1990), J. MoI. Biol. 215:403410; Gish, W. & States, DJ. (1993), Nature Genet. 3:266272; Madden, T.L. et al., (1996), Meth. Enzymol. 266:131-141 ; Altschul, S.F. et al., (1997), Nucleic Acids Res. 25:33893402; and Zhang, J. & Madden, T.L. (1997), Genome Res. 7:649656. [0065] The term "analog" is used to mean an amino acid or nucleic acid sequence which has been modified as compared to the sequence of SEQ ID NOs: 1-14 wherein the modification does not alter the utility of the sequence (e.g. the binding specificity) as described herein. The modified sequence or analog may have improved properties over the sequences shown in SEQ ID NOs: 1-14. One example of a nucleic acid modification to prepare an analog is to replace one of the naturally occurring bases (i.e. adenine, guanine, cytosine or thymidine) of the sequence with a modified base such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines, 8- Docket no. 05001770-409PCT - 20 - halo guanines, 8 amino guanine, 8-thiol guanine, 8-thiolalkyl guanines, 8- hydroxyl guanine and other 8-substituted guanines, other aza and deaza uracils, thymidines, cytosines, adenines, or guanines, 5-trifluoromethyl uracil and 5-trifluoro cytosine. [0066] Another example of a modification is to include modified phosphorous or oxygen heteroatoms in the phosphate backbone, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages in the nucleic acid molecules shown in SEQ ID NO:1 or 13. For example, the nucleic acid sequences may contain phosphorothioates, phosphotriesters, methyl phosphonates, and phosphorodithioates.

[0067] A further example of an analog of a nucleic acid molecule of the disclosure is a peptide nucleic acid (PNA) wherein the deoxyribose (or ribose) phosphate backbone in the DNA (or RNA), is replaced with a polyamide backbone which is similar to that found in peptides (P. E. Nielsen, et al Science 1991 , 254, 1497). PNA analogs have been shown to be resistant to degradation by enzymes and to have extended lives in vivo and in vitro. PNAs also bind more strongly to a complementary DNA sequence due to the lack of charge repulsion between the PNA strand and the DNA strand. Other nucleic acid analogs may contain nucleotides containing polymer backbones, cyclic backbones, or acyclic backbones. For example, the nucleotides may have morpholino backbone structures (as described in U.S. Pat. No. 5,034,506). The analogs may also contain groups such as reporter groups, or groups for improving the pharmacokinetic or pharmacodynamic properties of the nucleic acid sequence.

[0068] Also encompassed are sequences that hybridize to the sequences shown in SEQ ID NO:1 or 13 or a fragment thereof, and maintain the property of binding a β- or an α-cell. The term "sequence that hybridizes" means a nucleic acid sequence that can hybridize to a sequence of SEQ ID NO:1 or 13 under stringent hybridization conditions. Appropriate "stringent Docket no 05001770-409PCT - 21 - hybridization conditions" which promote DNA hybridization are known to those skilled in the art, and may be found for example in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. The term "stringent hybridization conditions" as used herein means that conditions are selected which promote selective hybridization between two complementary nucleic acid molecules in solution. Hybridization may occur to all or a portion of a nucleic acid sequence molecule. The hybridizing portion is at least 50% the length with respect to one of the polynucleotide sequences encoding a polypeptide. In this regard, the stability of a nucleic acid duplex, or hybrids, is determined by the Tm, which in sodium containing buffers is a function of the sodium ion concentration, G/C content of labeled nucleic acid, length of nucleic acid probe (I), and temperature (Tm = 81.5°C - 16.6 (Log10 [Na+]) + 0.41 (%(G+C) - 600/I). Accordingly, the parameters in the wash conditions that determine hybrid stability are sodium ion concentration and temperature. In order to identify molecules that are similar, but not identical, to a known nucleic acid molecule a 1 % mismatch may be assumed to result in about a 1⁰C decrease in Tm, for example if nucleic acid molecules are sought that have a greater than 95% identity, the final wash will be reduced by 5°C. Based on these considerations, in one embodiment stringent hybridization conditions are defined as: hybridization at 5 x sodium chloride/sodium citrate (SSC)/5 x Denhardt's solution/1.0% SDS at Tm (based on the above equation) - 5°C, followed by a wash of 0.2 x SSC/0.1 % SDS at 6O⁰C.

[0069] The SCA may be modified to contain amino acid substitutions, insertions and/or deletions that do not alter the binding properties of the SCA protein. Conservative amino acid substitutions involve replacing one or more amino acids of the SCA with amino acids of similar charge, size, and/or hydrophobicity characteristics. When only conservative substitutions are made, it is expected that the resulting analog would be functionally equivalent to the unsubstituted protein. Non-conservative substitutions involve replacing one or more amino acids of the SCA with one or more amino acids which possess dissimilar charge, size, and/or hydrophobicity characteristics. Docket no. 05001770-409PCT - 22 -

[0070] The SCA may be modified to make it more therapeutically effective or suitable. For example, the SCA of the present invention may be converted into a pharmaceutically-acceptable salt by reacting with inorganic acids such as, for example, hydrochloric acid, sulphuric acid, hydrobromic acid, phosphoric acid, etc., or organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid, benzenesulphonic acid, and tolunesulphonic acids, for example. Pharmaceutically-acceptable salts are well-known in the art and pharmaceutically-acceptable salts of the antibodies and analogs, homologs, fragments and variants thereof are encompassed herein.

[0071] The present invention also provides expression vectors comprising a nucleic acid sequence encoding a SCA of the invention or a fragment or analog thereof. [0072] Possible expression vectors include, but are not limited to, cosmids, plasmids, artificial chromosomes, viral vectors or modified viruses (e.g. replication defective retroviruses, adenoviruses and adeno-associated viruses), so long as the vector is compatible with the host cell used. The expression vectors are "suitable for transformation of a host cell", which means that the expression vectors contain a nucleic acid molecule of the invention and regulatory sequences selected on the basis of the host cells to be used for expression, operatively linked to the nucleic acid molecule of the invention. Operatively linked" is intended to mean that the nucleic acid is linked to regulatory sequences in a manner which allows expression of the nucleic acid.

[0073] There is provided herein a recombinant expression vector containing a nucleic acid molecule of the invention, or a fragment or analog thereof, and the necessary regulatory sequences for the transcription and translation of the inserted protein-encoding sequence. Docket no. 05001770-409PCT

- 23 -

[0074] Suitable regulatory sequences may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes (for example, see the regulatory sequences described in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Selection of appropriate regulatory sequences is dependent on the host cell, and may be readily accomplished by one of ordinary skill in the art. Examples of such regulatory sequences include: a transcriptional promoter and enhancer or RNA polymerase binding sequence, a ribosomal binding sequence, including a translation initiation signal. Additionally, depending on the host cell chosen and the vector employed, other sequences, such as an origin of replication, additional DNA restriction sites, enhancers, and sequences conferring inducibility of transcription may be incorporated into the expression vector. It will also be appreciated that the necessary regulatory sequences may be supplied by the SCA and/or their flanking regions.

[0075] The recombinant expression vectors of the invention may also contain a selectable marker gene which facilitates the selection of host cells transformed or transfected with a recombinant molecule of the disclosure. Examples of selectable marker genes are genes encoding a protein such as G418 and hygromycin which confer resistance to certain drugs, β- galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or an immunoglobulin or portion thereof such as the Fc portion of an immunoglobulin, such as IgG. Transcription of the selectable marker gene is monitored by changes in the concentration of the selectable marker protein such as β-galactosidase, chloramphenicol acetyltransferase, or firefly luciferase. If the selectable marker gene encodes a protein conferring antibiotic resistance such as neomycin resistance, transformant cells can be selected with G418. Cells that have incorporated the selectable marker gene will survive, while the other cells die. This makes it possible to visualize and assay for expression of recombinant expression vectors of the disclosure and in particular to determine the effect of a mutation on expression and Docket no. 05001770-409PCT

- 24 - phenotype. It will be appreciated that selectable markers can be introduced on a separate vector from the nucleic acid of interest.

[0076] The recombinant expression vectors provided herein may also contain genes which encode a moiety which provides increased expression of the recombinant protein; increased solubility of the recombinant protein; and/or aid in the purification of the target recombinant protein by acting as a ligand in affinity purification. For example, a proteolytic cleavage site may be added to the target recombinant protein to allow separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Typical fusion expression vectors include pGEX (Amrad Corp., Melbourne, Australia), pMal (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the recombinant protein. [0077] Recombinant expression vectors can be introduced into host cells to produce a transformed host cell. The term "transformed host cell" is intended to include cells that are capable of being transformed or transfected with a recombinant expression vector of the invention. The terms "transduced", "transformed with", "transfected with", "transformation" and "transfection" are intended to encompass introduction of nucleic acid (e.g. a vector or naked RNA or DNA) into a cell by one of many possible techniques known in the art. Prokaryotic cells can be transformed with nucleic acid by, for example, electroporation or calcium-chloride mediated transformation. For example, nucleic acid can be introduced into mammalian cells via conventional techniques such as calcium phosphate or calcium chloride co- precipitation, DEAE-dextran mediated transfection, lipofectin, electroporation, microinjection, RNA transfer, DNA transfer, artificial chromosomes, viral vectors and any emerging gene transfer technologies. Suitable methods for transforming and transfecting host cells can be found in Sambrook et al. Docket no. 05001770-409PCT

- 25 -

(Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory textbooks.

[0078] Suitable host cells include a wide variety of eukaryotic host cells and prokaryotic cells. For example, the proteins of the disclosure may be expressed in yeast cells or mammalian cells. Other suitable host cells can be found in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1991). In addition, the proteins of the disclosure may be expressed in prokaryotic cells, such as Escherichia coli (Zhang et al., Science 303(5656): 371-3 (2004)). [0079] Mammalian cells suitable for use in the methods described herein include, among others: COS (e.g., ATCC No. CRL 1650 or 1651), BHK (e.g. ATCC No. CRL 6281), CHO (ATCC No. CCL 61), and HeLa (e.g., ATCC No. CCL 2) and 3T3 mouse fibroblasts (e.g. ATCC No. CCL92).

[0080] Suitable expression vectors for directing expression in mammalian cells generally include a promoter (e.g., derived from viral material such as polyoma, Adenovirus 2, cytomegalovirus and Simian Virus

40), as well as other transcriptional and translational control sequences.

Examples of mammalian expression vectors include without limitation pCDMδ

(Seed, B., Nature 329:840 (1987)), pMT2PC (Kaufman et al., EMBO J. 6:187- 195 (1987)) and pCMV (Clontech, California, U.S.A.).

[0081] Alternatively, the SCAs of the invention may also be expressed in non-human transgenic animals, such as rats, mice, rabbits, sheep and pigs (Hammer et al. Nature 315:680-683 (1985); Palmiter et al. Science 222:809- 814 (1983); Brinster et al. Proc. Natl. Acad. Sci. USA 82:4438-4442 (1985); Palmiter and Brinster Cell 41 :343-345 (1985) and U.S. Patent No. 4,736,866). The present invention also encompasses tissues and cells derived or isolated from such animals.

[0082] In addition to the analogs and homologs described above, in certain embodiments, the antibodies of the invention may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or Docket no. 05001770-409PCT

- 26 - chemically conjugated (including covalent and non-covalent conjugations) to polypeptides or other compositions. For example, antibodies may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 396,387. Any type of molecule may be covalently attached to the antibodies of the invention as long as it does not prevent the antibody from binding to α-cells or β-cells or alter the binding specificity of the antibody. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids. The heterologous polypeptide to which the antibody is fused may be useful for example to increase the in vivo half life of the antibody, or for use in immunoassays using methods known in the art, or for use in diagnostic medical imaging methods as known in the art. Antibodies of the invention can be fused to marker sequences, such as a peptide to facilitate their purification or detection. In general, it should be understood that antibodies of the present invention may be used in non- conjugated form or may be conjugated to at least one of a variety of molecules, e.g., to improve the therapeutic properties of the molecule, to facilitate target detection, or for imaging in the patient.

[0083] In certain embodiments, an antibody of the inventions includes an amino acid sequence or one or more moieties not normally associated with an antibody. Exemplary modifications are described in more detail below. For example, the single chain antibodies of the invention may comprise a flexible Docket no. 05001770-409PCT - 27 - linker sequence, or may be modified to add a functional moiety (e.g., PEG, a drug, a toxin, an imaging agent or a label).

[0084] Furthermore, nucleotide or amino acid substitutions, deletions, or insertions leading to conservative substitutions or changes at "non- essential" amino acid regions may be made. For example, a polypeptide or amino acid sequence derived from a designated protein may be identical to the starting sequence except for one or more individual amino acid substitutions, insertions, or deletions, e.g., one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty or more individual amino acid substitutions, insertions, or deletions, a polypeptide or amino acid sequence derived from a designated protein may be identical to the starting sequence except for one or more individual amino acid substitutions, insertions, or deletions, e.g., one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty or more individual amino acid substitutions, insertions, or deletions. In other embodiments, a polypeptide or amino acid sequence derived from a designated protein may be identical to the starting sequence except for two or fewer, three or fewer, four or fewer, five or fewer, six or fewer, seven or fewer, eight or fewer, nine or fewer, ten or fewer, fifteen or fewer, or twenty or fewer individual amino acid substitutions, insertions, or deletions. In certain embodiments, a polypeptide or amino acid sequence derived from a designated protein has one to five, one to ten, one to fifteen, or one to twenty individual amino acid substitutions, insertions, or deletions relative to the starting sequence.

[0085] Also encompassed in the present invention are fragments, derivatives, modifications, or variants of the antibodies described herein, as well as the analogs and homologs described above, and any combination thereof. The terms "fragment," "variant," "derivative", "modification",

"homolog" and "analog" when referring to antibodies of the present invention include any polypeptides which retain at least some of the antigen-binding properties (e.g. antigen-binding specificity) of the corresponding native Docket no. 05001770-409PCT

- 28 - antibody. The terms "variant," "derivative" and "modification" are used interchangeably herein. Fragments of antibodies of the present invention include proteolytic fragments or deletion fragments. Variants of the antibodies of the present invention include fragments, polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions as described herein, and modifications as described herein. Various derivatives which have been altered so as to exhibit additional features not found on the native polypeptide are also described herein and include, for example, fusion proteins and antibodies conjugated to imaging agents. Variants may occur naturally or be non-naturally occurring (e.g. produced using art-known mutagenesis techniques. Variant polypeptides may comprise conservative or non-conservative amino acid substitutions, deletions or additions as described herein. Derivatives may also have one or more residues chemically derivatized by reaction of a functional side group. Also included as "derivatives" are those peptides which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For example, 4- hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine.

[0086] Thus in one embodiment, analogs, homologs, fragments or variants of the single chain antibodies disclosed herein are encompassed by the present invention. In an embodiment, the analogs, homologs, fragments or variants retain the antigen-binding specificity and/or properties of the single chain antibodies disclosed herein.

[0087] By "specifically binds," it is generally meant that an antibody binds to an epitope via its antigen binding domain, and that the binding entails some complementarity between the antigen binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope when it binds to that epitope, via its antigen binding domain more Docket no. 05001770-409PCT

- 29 - readily than it would bind to a random, unrelated epitope. The term "specificity" is used herein to qualify the relative affinity by which a certain antibody binds to a certain epitope. For example, antibody "A" may be deemed to have a higher specificity for a given epitope than antibody "B," or antibody "A" may be said to bind to epitope "C" with a higher specificity than it has for related epitope "D."

[0088] Alternatively, in another embodiment, mutations may be introduced randomly along all or part of the immunoglobulin coding sequence, such as by saturation mutagenesis, and the resultant mutants can be incorporated into antibodies for use in the diagnostic and treatment methods disclosed herein and screened for their ability to bind to the desired antigen, e.g., α-cells or β-cells. It will also be understood by one of ordinary skill in the art that antibodies of the invention may be modified such that they vary in amino acid sequence from the naturally occurring binding polypeptide from which they were derived and may have a certain percent identity to the starting sequence, e.g., it may be 60%, 70%, 75%, 80%, 85%, 90%, or 95% identical to the starting sequence.

[0089] The present invention is further directed to isolated polypeptides which make up the antibodies of the invention, and polynucleotides encoding such polypeptides. A polypeptide or amino acid sequence "derived from" a designated protein refers to the origin of the polypeptide having a certain amino acid sequence. In certain cases, the polypeptide or amino acid sequence which is derived from a particular starting polypeptide or amino acid sequence has an amino acid sequence that is essentially identical to that of the starting sequence, or a portion thereof, wherein the portion consists of at least 10-20 amino acids, at least 20-30 amino acids, at least 30-50 amino acids, or which is otherwise identifiable to one of ordinary skill in the art as having its origin in the starting sequence.

[0090] In one embodiment, the invention encompasses antibodies, or antigen-binding fragments, variants, analogs, homologs, modifications or Docket no. 05001770-409PCT

- 30 - derivatives thereof of the invention conjugated to a diagnostic agent. The antibodies can be used diagnostically to, for example, monitor the development or progression of diabetes in a subject as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment and/or prevention regimen. Detection can be facilitated by coupling the antibody of the invention to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions; such substances are well-known in the art. See, for example, U.S. Pat. No. 4,741 ,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, .beta.-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include 1251, .sup.1311, .sup.H Hn or .sup.99Tc.

[0091] Other suitable imaging agents for use with the antibodies of the present invention include, without limitation, ¹¹C, ¹⁸F, ¹⁸O, ¹³N, ⁷⁶Br and ¹²⁴I as radioligands in PET imaging Or ⁸⁶Y, ^99mTc, ¹¹¹In, ¹²³I, and ²⁰¹TI as radioligands in SPECT imaging.

[0092] The person skilled in the art will appreciate that many imaging methods are known and can be used with the antibodies of the present invention. For example, presence of the labeled molecule can be detected in the patient using methods known in the art for in vivo scanning. These methods depend upon the type of label used. Skilled artisans will be able to Docket no. 05001770-409PCT - 31 - determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods of the invention include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), SPECT, bioluminescence, fluorescence and sonography. The person skilled in the art will be able to choose appropriate ligands, agents and methods using routine procedures. It will also be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. [0093] In a specific embodiment, the antibody is labeled with a radioisotope and is detected in the patient using a radiation responsive surgical instrument (see e.g. Thurston et al., U.S. Pat. No. 5,441 ,050). In another embodiment, the antibody is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive scanning instrument. In another embodiment, the antibody is labeled with a positron emitting metal and is detected in the patent using positron emission- tomography. In yet another embodiment, the antibody is labeled with a paramagnetic label and is detected in a patient using magnetic resonance imaging (MRI). [0094] Techniques for conjugating various moieties to an antibody of the invention are well known, see, e.g., Arnon et al., "Monoclonal Antibodies For lmmunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), Marcel Dekker, Inc., pp. 623-53 (1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Docket no. 05001770-409PCT

- 32 -

Baldwin et al. (eds.), Academic Press pp. 303-16 (1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev. 62:119-58 (1982).

[0095] With respect to the use of radiolabeled conjugates in conjunction with the antibodies of the present invention, it should be understood that the antibodies may be directly labeled (such as through iodination) or may be labeled indirectly through the use of a chelating agent. As used herein, the phrases "indirect labeling" and "indirect labeling approach" both mean that a chelating agent is covalently attached to a binding molecule and at least one radionuclide is associated with the chelating agent. Such chelating agents are typically referred to as bifunctional chelating agents as they bind both the polypeptide and the radioisotope. Particularly preferred chelating agents comprise 1-isothiocycmatobenzyl-3-methyldiothelene triaminepentaacetic acid ("MX-DTPA") and cyclohexyl diethylenetriamine pentaacetic acid ("CHX- DTPA") derivatives. Other chelating agents comprise P-DOTA and EDTA derivatives. Radionuclides for indirect labeling include .sup.H Hn and .sup.9OY.

[0096] As used herein, the phrases "direct labeling" and "direct labeling approach" both mean that a radionuclide is covalently attached directly to a polypeptide (typically via an amino acid residue). More specifically, these linking technologies include random labeling and site-directed labeling. In the latter case, the labeling is directed at specific sites on the polypeptide, such as the N-linked sugar residues present only on the Fc portion of the conjugates. Further, various direct labeling techniques and protocols are compatible with the instant invention. For example, Technetium-99 labeled polypeptides may be prepared by ligand exchange processes, by reducing pertechnate (TcO. sub.4. sup.-) with stannous ion solution, chelating the reduced technetium onto a Sephadex column and applying the binding polypeptides to this column, or by batch labeling techniques, e.g. by incubating pertechnate, a reducing agent such as SnCI. sub.2, a buffer Docket no. 05001770-409PCT

- 33 - solution such as a sodium-potassium phthalate-solution, and the antibodies. Preferred radionuclides for directly labeling antibodies are well known in the art.

[0097] It will be appreciated that, in accordance with the teachings herein, the antibodies may be conjugated to many different radiolabels for diagnostic and imaging purposes. A variety of radionuclides are applicable to the present invention and those skilled in the can readily determine which radionuclide is most appropriate under various circumstances. Antibodies can also be detectably labeled using fluorescence emitting metals such as 152Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0098] It is known in the art that after phage selection, as described herein, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria. For example, techniques to recombinantly produce Fab, Fab' and F(ab').sub.2 fragments can be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al., Science 240:1041-1043 (1988) (said references incorporated by reference in their entireties). Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040 (1988).

[0099] For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. A chimeric antibody is a molecule in which Docket no. 05001770-409PCT - 34 - different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., J. Immunol. Methods 125:191-202 (1989); U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporated herein by reference in their entireties. Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entireties.) Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101 ; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91 :969- 973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332).

[00100] Completely human antibodies are particularly desirable for therapeutic treatment or diagnosis of human patients. Human antibodies can be made by a variety of methods known in the art including phage display methods described herein using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and Docket no. 05001770-409PCT

- 35 -

4,716,111 ; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741 ; each of which is incorporated herein by reference in its entirety.

[00101] In an embodiment, the single chain antibodies or analogs, homologs, fragments or variants thereof provided herein are humanized or fully human.

[00102] In another embodiment, any antigen-binding polypeptide which binds specifically to the same α-cell or β-cell specific epitope as the SCAs provided herein, is encompassed. Pharmaceutical Compositions and Methods of Administration

[00103] Pharmaceutical compositions encompassing the antibodies of the invention are also encompassed herein. The antibodies of the present invention can be administered to a subject in a conventional dosage form prepared by combining the antibody of the invention with a conventional pharmaceutically acceptable carrier or diluent according to known techniques. It will be recognized by one of skill in the art that the form and character of the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables. [00104] Methods of preparing and administering SCAs or antigen- specific analogs, homologs, fragments or variants thereof to a subject are well-known in the art or are readily determined by those skilled in the art. The route of administration of the antibodies of the invention may be, for example, oral, parenteral, by inhalation or topical. The term parenteral as used herein includes, e.g., intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration.

[00105] Usually, a suitable pharmaceutical composition for injection may comprise a buffer (e.g. acetate, phosphate or citrate buffer), a surfactant (e.g. polysorbate), optionally a stabilizer agent (e.g. human albumin), etc. Docket no 05001770-409PCT

- 36 -

Preparations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. In the subject invention, pharmaceutically acceptable carriers include, but are not limited to, 0.01 -0.1 M and preferably 0.05M phosphate buffer or 0.8% saline. Other common parenteral vehicles include sodium phosphate solutions, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present such as for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like. [00106] More particularly, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In such cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and will preferably be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Suitable formulations for use in the therapeutic methods disclosed herein are described in Remington's Pharmaceutical Sciences, Mack Publishing Co., 16th ed. (1980). Docket no. 05001770-409PCT

- 37 -

[00107] Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[00108] In any case, sterile injectable solutions can be prepared by incorporating an antibody of the invention (by itself or in combination with other active agents) in the required amount in an appropriate solvent with one or a combination of ingredients, as required and easily determined by a person of skill in the art, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yields a powder of an active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The preparations for injections are processed, filled into containers such as ampoules, bags, bottles, syringes or vials, and sealed under aseptic conditions according to methods known in the art.

[00109] Further, the preparations may be packaged and sold in the form of a kit. Such articles of manufacture will preferably have labels or package inserts providing instructions for use and may have additional components required for the use of the preparations.

[00110] Those skilled in the art will appreciate that effective doses of the antibodies and compositions of the present invention, e.g. for in vivo imaging of α- or β- cells of the pancreas as described herein, vary depending upon many different factors, including means of administration, characteristics or Docket no. 05001770-409PCT

- 38 - physiological state of the subject (such as state of health), other medications being administered, whether the treatment is diagnostic, prognostic, prophylactic or therapeutic, and so on. The dosage may be determined using routine methods known to those of skill in the art in order to optimize safety and efficacy.

[00111] It should also be understood that antibodies of the invention can optionally be administered in combination with other agents that are effective in treating the disorder or condition in need of treatment. In keeping with the scope of the present disclosure, α- or β- cell specific antibodies of the invention may be used as α- or β- cell targeting agents to deliver other therapeutic or prophylactic agents directly to the α- or β- cells. Examples of such therapeutic or prophylactic agents include, without limitation, anti- apoptotic substances such as the Nemo-Binding Domain and compounds that induce proliferation such as CDK-6, CDK-4 and Cyclin D1.

EXAMPLES

[00112] The present invention will be more readily understood by referring to the following examples, which are provided to illustrate the invention and are not to be construed as limiting the scope thereof in any manner.

Generation of SCAs binding selectively to β- or α-cells

[00113] For the purpose of identifying and generating agents specifically binding to pancreatic islets in vivo, a phage library was screened for SCAs on rat islets using two different approaches: 1) Islets were isolated from rats after intravenous injection of the library for a circulation time of 5 minutes; and 2) Rat islets were isolated and the library was panned in the isolated islets in vitro. Five rounds of selection were carried out and demonstrated a marked increase in the phage transducing units (TU) per islet over successive rounds of panning, representing a 700- and 500-fold enrichment within the first and Docket no. 05001770-409PCT

- 39 - the second approach, respectively (Fig. 1). Subsequently, the DNA encoding the corresponding phage-displayed SCAs was sequenced. The first approach supplied five islet-specific phage clones (ISPCs), ISPC1 to 4 and 7. The second approach generated two ISPCs, ISPC5 and 6 (Fig. 2). [00114] The selective binding of the ISPCs to rat islets in vivo was determined by the harvest of the pancreas and control organs after intravenous administration of the ISPCs for a circulation time of 2 hours. In rats that received ISPC1 , phage immunostaining was clearly identifiable in the cytoplasm of the islets and the signal overlapped with insulin expression, whereas no co-staining with glucagon was detectable, suggesting that the ISPC1 was exclusively taken up by β-cells (Fig. 4 a,b). The same staining patterns were observed for ISPC 2, 5, 6 and 7 (data not shown). In contrast, in animals that received ISPC3 (Fig. 4 c,d) or 4 (data not show), phage immunostaining was clearly identifiable and overlapped exclusively with glucagon expression, suggesting that ISPC3 and 4 were taken up by α-cells. Importantly, a control ISPC without insert was undetectable in the islet (Fig. 4 e,f). Moreover, none of the β-cell specific ISPC 1 , 2, 5 to 7 (Fig. 3 a) and the a-cell specific ISPC 3 and 4 (Fig. 3 b) exhibited any binding to control organs, such as liver, kidney, spleen, heart, lung and exocrine cells (Fig. 4 a-d). [00115] Next, all ISPCs were produced as soluble SCAs containing a c- Myc tag and a HiS₆ tag in small scale cultures, purified by metal affinity chromatography and then intravenously applied to rats. Highly selective uptake into the cytoplasm of β-cells was confirmed for SCA B1 (Fig. 4 g,h), 2, 5, 6 and 7 (data not shown), whereas SCA A1 (Fig. 4 i,j) and 4 (data not shown) were accumulated selectively in the α-cells of the islet. In addition, none of SCA B1 to B5 (Fig. 3 c) and SCA A1 and 2 (Fig. 3 d) exhibited any binding to control organs, such as liver, kidney, spleen, heart, lung and exocrine cells (Fig. 4 a-d). Finally, electron microscopy imaging confirmed selective intracellular localization for the β-cell specific SCA1 (SCA B1) (Fig. 5 a,b) as well as for the α-cell specific SCA3 (SCA A1) (Fig. 5 c,d) and linked Docket no. 05001770-409PCT

- 40 - them to the endoplasmatic reticulum (ER) and the secretory granule of their target cells.

Determination of binding properties of radio-labeled SCAs in vitro and pharmacokinetic profile in vivo

[00116] A systematic in vitro analysis was performed in order to assess the binding properties of selected [¹²⁵l]-labeled SCA to the β-cell line INS-1 , the α-cell line α-TC1 and the exocrine cell line AR42J and to compare them quantitatively to previously evaluated agents. Binding of the β-cell specific SCA B1 was 514 times higher to INS-1 cells relative to α-TC1- and AR42J- cells (p = 0.0016; Fig. 6 a), whereas binding of the α-cell specific SCA A1 was 210-fold higher to α-TC1- relative to INS-1- and AR42J-cells (p < 0.0001 ; Fig. 6 b). This pattern correlated well with the in vivo data, showing selective binding to either β- or α-cells (Fig. 4 a-d). Furthermore, the assay revealed rapid binding of the β-cell specific SCA B1 to INS-1 as well as of the α-cell specific SCA A1 to α-TC1 cells (ti_/2 = 8.0 min and 5.3 min for SCA B1 and SCA A1 , respectively; Fig. 6 c,d).

[00117] The results of the competition assay suggest that binding and/or internalization of β-cell specific SCA B1 is mediated by a different membrane protein or receptor than α-cell specific SCA A1 , since binding of radio-labeled SCA B1 was not inhibited when cells were preincubated with unlabelled SCA A1 and vice versa (Fig. 6 e,f). However, the process is saturable in the case of both SCAs, since full inhibition was observed by preincubation with corresponding unlabelled SCAs (Fig. 6 g,h). Finally, calculations from the dose-response curves indicated that a high number of SCA molecules coulde be internalized by the cells (approximately 650,307 SCA B1-molecules/β-cell and 669,945 SCA A1-molecules/α-cell, could be internalized maximally) (Fig. 6 g,h).

[00118] Since unbound label circulating in the vasculature will interfere with the imaging signal coming from label bound to either the β- or α-cells, we next determined the plasma clearance kinetics of selected SCA (Fig. 6 i,j). Docket no. 05001770-409PCT - 41 -

Preparations of radio-labeled SCA were administered intravenously to rats and the radioactivity was measured in the plasma over time. We found that the tested β- cell- and α-cell-specific SCA were eliminated quickly from the blood (tι_/2 = 22.7 min and 19.2 min for SCA B1 and SCA A1 , respectively). Taken together, the results indicate that the SCA are promising candidates for specific delivery of imaging agents to β- or α-cells in vivo.

Specific binding to human islets in situ and exclusion of toxicity

[00119] We next asked whether the generated SCA detect a protein or receptor that is expressed selectively in human pancreatic β- or α-cells. The selectivity of the SCAs was demonstrated by exposure to human pancreatic tissue slides, where SCAs were detected using a monoclonal anti-cMyc antibody, and co-staining with specific anti-insulin- and anti-glucagon- antibodies was used to identify the cell type. Staining for β-cell specific SCA B1 was clearly identified and restricted to the islets and co-localized with insulin expression, whereas none was detectable in α- or exocrine cells (Fig. 7 a,b). In contrast, the α-cell specific SCA A1 overlapped with glucagon staining, but not with insulin expressing- or exocrine-cells (Fig. 7 c,d). These data strongly suggest that the SCA bind to a protein/receptor that is selectively expressed in human islets. [00120] Next, we determined that β- or α-cells labeled with a SCA retain their ability to secrete insulin and glucagon, in order to ensure that labeling does not affect islet function. An intraperitoneal glucose tolerance test (IPGTT) was performed during seven days after the SCA were injected intravenously in rats. We found that the course of plasma glucose or insulin- and glucagon secretion during an IPGTT was unchanged compared to sham treated animals (Fig. 8 a-c). Moreover, after overnight exposure to increasing amounts of SCA in vitro, viability (Fig. 8 d,e) as well as apoptosis rate (Fig. 8 f) for INS-1- or α-TC1 -cells remained unchanged when compared to non- treated controls. Quantification of β-cell mass with radio-labeled SCA1 Docket no. 05001770-409PCT - 42 -

[00121] The applicability of the SCA to serve as probes for the quantification of β-cell mass was explored by intravenous administration of the β-cell-specific [¹²⁵l]-labeled SCA B1 in rats for a circulation time of 2 hours. The accumulation of the [¹²⁵l]-labeled SCA B1 was measured in the explanted pancreas and plotted against the β-cell mass of the given pancreas and the area under the curve (AUC) for glucose during an IPGTT. In non-diabetic rats, β-cell mass was higher than in low- and high-dose STZ-diabetic rats, as was the accumulation of the probe (Fig. 9 a). A strong correlation was detectable between probe accumulation and β-cell mass within the three subgroups (r² = 0.937). The overall probe accumulation has a clear tendency to decrease with reduction of β-cell mass. Moreover, we detected a strong correlation between probe accumulation and AUC for glucose during an IPGTT (r² = 0.876, Fig. 9 b). Taken together, the results indicate that even after radio-labeling SCA B1 is highly specific for β-cells and allows quantification of β-cell mass, and that accumulation of radio-labeled SCA B1 correlates strongly with islet function.

Dynamic in vivo PET-scan after intravenous injection of β cell specific P⁴I]- labeled SCA B1 in non diabetic rats

[00122] In an experiment similar to the one described above, [I¹²⁴]- labeled SCA B1 was tested in vivo by intravenous administration. Within 30 min, [I¹²⁴]-labeled SCA B1 rapidly and selectively accumulated in the pancreatic gland, without any binding to non-targeted tissue. Any non-bound tracer molecules were eliminated through the urinary tract (Fig. 10 a). Twenty- four hours after administration the tracer was still clearly visible in the pancreas while completely eliminated from the rest of the body (Fig. 10 b). Internal calibration of α or β cell mass by the opposite cell type

[00123] Increases of β-cell mass occur in insulinoma, nesidioblastosis, and islet cell carcinoma, while decreases in β-cell mass are linked to type I and type Il diabetes. In contrast, increases in α-cell mass are linked to glucagonoma. While the treatment of insulinoma, nesidioblastosis and islet cell carcinoma leads to a decrease in β-cell mass, the α-cell mass is Docket no. 05001770-409PCT - 43 - unaffected. The demonstration of the functionality of SCA B1 and SCA A1 to detect β-cells and α-cells, respectively, makes these antibodies and their analogs of use for analyzing β-cells and α-cells independently in the same animal or human subject (i.e., they form a reciprocal control pair). In vivo visualization of pancreatic β -cells

[00124] We used in vivo MRI of the mouse abdomen and pancreas at ultra high fields (16.4T) in order to visualize pancreatic islets using a superparamagnetic contrast agent based on the beta-cell specific SCA B1 antibody. [00125] C57BL/6J-mice were anaesthetized and a constant breathing rate was maintained. MR-images were recorded on a 16.4T horizontal animal system. The contrast agent was injected intravenously into the tail vein. Fig. 11 shows anatomical in vivo images of the mouse abdomen. The measurements were triggered on breathing and acquired using volume (Fig. 11a) or surface coils (Figs. 11 b-e). All organs can be easily identified. Figures 11d and 11e show enhanced sections of the pancreas. The vasculature providing blood flow to the pancreas and the spleen can be seen (also visible on Fig. 11b next to the spleen, marked with an asterisk in Figs. 11d and 11e). By finding these structures first, the pancreas can be localized. Similar characteristic structures are also visible in images of a dissected pancreas (Fig. 12a). Results using the targeted superparamagnetic contrast agent directed against beta-cells (SCA B1) demonstrated punctuate loss of signal intensity in an excised pancreas 24h after intravenous injection (Fig. 12b). The sizes of the areas with signal loss matched the sizes of islets of Langerhans which are ~100μm in diameter. Although this signal-loss was not yet detectable in vivo, binding of the prospective contrast agent to beta-cells in the islets of the excised pancreas was verified by immunofluorescence (data not shown).

[00126] These results represent the first demonstration of the feasibility of in vivo MRI of the mouse abdomen at the ultra high field of 16.4T. The Docket no. 05001770-409PCT

- 44 - sensitivity of the MR-microscopy allows identification for structures <100μm and anatomical details (e.g. the vasculature) of the pancreas. The molecular imaging at 16.4 T also demonstrates that the antibodies of the invention can function as targeted contrast agents for identification of alpha-cells or beta-cell containing islets of Langerhans.

Summary of Experimental Results

[00127] Thus, we used repeated phage-panning in rodents to identify and generate SCA with highly specific binding to pancreatic β-cells or α-cells in vivo, and which can be used as agents for non-invasive quantification of β- cell or α-cell mass. We isolated five highly selective SCA homologs and demonstrated that the SCA homologs were internalized into the insulin producing β-cells of rat pancreas in vivo, bound to human β-cells in situ. The SCA homologs were linked to the endoplasmatic reticulum and the insulin secretory granule membrane by transmission electron microscopy. Similarly, we identified two SCA homologs that were selectively taken up by the glucagon-producing α-cells of rats in vivo, exhibited highly specific binding to human α-cells in situ and were linked as well to the endoplasmatic reticulum and the glucagon secretory granule. Importantly, no binding to exocrine cells or other tested tissues was detected for any of the generated SCAs in vivo. [00128] These results, together with the unexpected specificity ratios for target- vs. non-target-cells determined on cell lines in vitro, indicate strongly that the selectivity of the generated SCA to either β- (> 500:1) or α-cells (>200:1) is well-above the specificity ratio required for imaging purposes (at least >100:1 is estimated to be required for imaging purposes), in order to overcome the signal-to-background ratio in vivo.

[00129] The signal-to-background ratio measured for the SCAs is far superior to the low specificity of 70:1 achieved by the ¹¹C-labeled dihydrotetrabenazine, which was used previously to image β-cell mass with positron emission tomography in a rodent model of spontaneous type I Docket no. 05001770-409PCT - 45 - diabetes, but failed as a tracer in human subjects (Souza, F. et al., J CHn Invest 116:, 1506-1513 (2006); Liu, E. H. et al., Diabetes 56, A83-A83 (2007)).

[00130] Without wishing to be bound by theory, it is likely that the mechanism of binding and cellular uptake within the short time frame of minutes and at the observed high quantities of more than 650,000 SCA per cell, to either β- or α-cells, is a membrane protein/receptor driven process. The results also indicate strongly that the cellular target differs for the β- and α-cell specific SCAs, as indicated by the competition assay. However, SCAs not specific for either β- or α-cells were eliminated from the circulation in rats within minutes. These results are consistent with recent studies showing the rapid elimination of SCA mainly via the kidneys due to their small molecular weight of approximately 31 kD.

[00131] Moreover, our findings that the gradual loss of β-cell mass of approximately 50 to 100% in STZ-diabetic rats was accompanied by a corresponding decline in pancreatic uptake of the radio-labeled β-cell specific SCA B1 (r² = 0.937) and was strongly correlated with the AUC for glucose during an IPGTT (r² = 0.876), and that exposure to SCA B1 and its homologs affected neither islet function in rats in vivo nor the viability of cell lines in vitro. Thus a direct correlation between pancreatic uptake of radio-labelled SCAs and β-cell mass was observed in normal and diabetic animals. These findings are consistent with the use of SCA B1 and its homologs as agents for the quantification of β-cell mass.

[00132] In summary, the results presented herein confirm SCA B1 and its homologs and SCA A1 and its homologs as highly selective tools to identify β- or α-cells in vivo for non-invasive imaging by for example PET, SPECT, or magnetic resonance imaging.

EXPERIMENTAL METHODS

Animal models, human tissue samples, phage library, cell lines Docket no. 05001770-409PCT

- 46 -

[00133] Female 6-week old CD-rats were purchased from Charles River Laboratories. Mild or severe diabetic animals were established by intraperitoneal injection with 30 or 60 mg/kg STZ two weeks prior to the experiments. Animals with plasma glucose levels of more than 350 mg/dl in four consecutive measurements were used in the experiments approved by the Landesamt fur Naturschutz (No. 50.10.32.08.037). The use of human pancreatic tissue samples, obtained from non-diabetic patients undergoing partial pancreatectomy, was approved by the ethics committee of the Ruhr- University Bochum (No. 2528, amendment 3). [00134] The recombinant phage-library Tomlinson I is constructed in the plT2 vector (derived from pHEN1), consists of about 1.4 x 10⁸ different human single chain variable fragments and was provided by MRC Genservice (Cambridge, UK). The library is based on a single human framework for VH (V3-23/DP-47 and JH4b) and V (O12/O2/DPK9 and J 1) with diversified (DVT) side chains incorporated in complementary determining region 2 and complementary determining region 3 at positions in the antigen binding site that make contacts to antigen in known structures and are highly diverse in the mature repertoire.

[00135] Cell lines used in the studies were rat β-cell line INS-1 , the murine α-cell line α-TC1 and the rat exocrine cell line AR42J.

Phage library screening

[00136] For the in vivo approach a rat was injected through the jugular vein with 10¹² phage TU for a circulation time of 5 minutes, followed by islet isolation using collagenase digestion. Subsequently islet purification was achieved using a discontinuous three-phase Ficoll density gradient. In the in vitro approach rat islets were directly isolated, and incubated with 10¹² TU for 1 h at 37⁰C with gentle shaking.

[00137] Following both screening approaches, the islets were washed twice in 1 ml HBSS, once in 1-ml wash with 0.1 N HCI-Glycine (pH 2.2). Subsequently, the cells were lysed by the addition of 1 ml hypotonic solution Docket no. 05001770-409PCT - 47 -

(30 mmol/l Tris-HCI, pH 8.0), followed by a single freeze-thaw cycle. Phages from the output fraction were amplified according to standard protocols for use in the next round of panning. Aliquots of the initial input and output samples were titered, and the output-to-input phage ratio based on colony-forming units was determined to monitor the progress of each round of library panning.

Generation and purification of SCA

[00138] HB2151 -bacteria (ODgQO = °-⁴) ^were infected with the ISPC of interest and grown overnight at 37⁰C on 2TY plates containing 100 μg/ml ampicillin and 1% glucose. On the following day individual colonies were picked and grown for 12 h in 10 ml 2TY containing 100 μg/ml ampicillin and 1% glucose (shaking at 220 rpm at 37⁰C). Subsequently, the 10 ml culture was added to 2 L of 2TY medium (0.1 % glucose and 100 μg/ml ampicillin) and grown at 37⁰C until an OD₆oo = 0.6 was obtained, lsopropyl β-D- thiogalactoside (IPTG; AppliChem GmbH, Darmstadt, Germany) (final concentration 1mM) was added to the culture to induce SCA expression. The culture was grown for 4 h at 3O⁰C (shaking at 220 rpm), followed by centrifugation at 6000 g for 15 min at 4°C. The pellet was re-suspended in 50 ml PBS and 1mM Phenylmethylsulfonylfluoride (PMSF; Roche Applied Science, Mannheim, Germany) and incubated for 1 h on ice, vortexed intermittently, followed by centrifugation for 1 h at 11600 g at 4⁰C. The supernatant containing the SCA was purified by immobilized metal affinity chromatography on a nickel column (Nunc ProPur^®, Nunc GmbH, Germany) according to the manufacturer's protocol, except for more intensive washing (3-4 times). Imidazole (80 mM) was used to elute the SCA from the column. The purified sample was then dialyzed overnight in PBS and purity was checked by SDS gel electrophoresis. The SCA concentration was determined using the BCA protein assay kit (Pierce, Rockford, USA). Cell culture [00139] INS-1 cells (kind gift from C. Wollheim, Geneva) were grown in RPMI 1640 (Gibco BRL, Grand Island, NY) supplemented with 10% (v/v) fetal bovine serum (FBS), 2 mM glutamine (all from Gibco BRL), 1 mM pyruvate Docket no 05001770-409PCT

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and 50 μM β-mercaptoethanol (Sigma Chemical, St. Louis, MO). AR42J (ATCC, Manassas, VA) cells were grown in Dulbecco's modified Eagle's medium (DMEM) media (Gibco BRL) containing 10% FBS. Cells were detached with trypsin (0.05% trypsin, 0.53 mM EDTA-4Na, Gibco BRL) 2 hours prior to conducting the screening assays as follows. Culture media bathing the cells was aspirated and the cells were washed with phosphate- buffered saline (PBS) to remove residual media/serum. Trypsin/EDTA (2.5 mL) was added to each T-75 and the flasks were incubated for 3 minutes after placing in a CO₂ incubator maintained at 37°C. Cell culture media (RPMI with 10%FBS) was added and the cell suspension was transferred to a 50 mL conical centrifuge tube, and centrifuged at 1000Xg for 3 minutes. The supernatant was aspirated; the cells were resuspended in culture media to achieve approximately 1 million cells/ml, and then stored in a CO₂ incubator at 37⁰C until the binding studies were conducted. lmmunohistochemistry

[00140] Staining of formalin-fixed, paraffin-embedded rat and human tissue sections (5 μm) were performed as follows: Sections were deparaffinized using Xylol twice for 10 min and followed by EtOH three times for 5 min and Aqua dest. for another 5 min. Afterwards, the sections were permeabilized by heating in the microwave in antigen unmasking solution pH 6 and cooling down for 45 min. Blocking was done for 1 h at 24°C with PBS containing 2% BSA. Primary and secondary antibodies were diluted in PBS with 2% BSA. Primary antibodies were incubated at 4°C overnight, except for insulin and glucagon for which the incubation period was 1 h at 37°C. Secondary antibodies were incubated for 30 min at 24°C and the same holds true for the Cy2- and Cy3-conjugated streptavidin reagents. The following primary antibodies and dilutions were used: SCA B1 and SCA A1 , 1 :200; monoclonal mouse anti-c Myc antibody, 1 :200 (Cell Signaling,#2276); polyclonal guinea pig anti-swine-insulin antibody, 1 :400 (Dako,#A0564); and monoclonal mouse anti-glucagon antibody, 1 :200 (Affinity BioReagents,#MA1 -20210). Secondary antibodies were monoclonal mouse anti-c Myc antibody, 1 :200 (Cell Signaling,#2276); biotinylated anti-rabbit IgG Docket no. 05001770-409PCT - 49 - and biotinylated anti-mouse IgG, 1 :200 (Linaris,#BA-1000,#BA-2001); Cy3- conjugated goat anti-mouse IgG, 1 :200 (Jackson ImmunoResearch Laboratories,#115-165-044); Cy3-conjugated goat anti-guinea pig IgG, 1 :800 (Jackson ImmunoResearch Laboratories, #106-165-003). Third reagents were Cy2-conjugated streptavidin, 1 :200 (Jackson ImmunoResearch Laboratories,#016-220-084). Tissue slides were analyzed using a Zeiss Axioplan microscope.

Electron microscopy

[00141] Rats were transcardially perfused with PBS and 2.5% glutaraldehyde to fix the pancreas before extraction, postfixed in 2.5% glutaraldehyde, rinsed with PBS, postfixed in 1 % osmiumtetroxide and dehydrated in ascending concentrations of ethanol and propylenoxide and embedded in durcupan. Ultrathin sections were stained with ultrostain 1 and 2 using the ultrostain. Residual aldehyde groups were inactivated with 0.05M lysine in PBS buffer. Block Step: 5% BSA and 0.1 % CWSFs gelatin supplemented with 5% serum, followed by incubation for 1 h with monoclonal mouse anti-c Myc antibody (1 :200; Cell Signaling, #2276) and a biotinylated

IgG (1 :200, Vector Laboratories). For gold labelling ultra small gold (Aurion,

Wageningen, Netherlands) and silver enhancement (Aurion, Wageningen, Netherlands) was used. Sections were analyzed on a ZEISS 109 transmission electron microscope.

Radio-labeling

[00142] The SCAs (0.1 mg) were labeled with ¹²⁵I or ¹²⁴I using the Chloramine-T method. Briefly, the pH of the SCAs dissolved in PBS was adjusted to 7.4 by the addition of 1/10 volume of 0.2 M sodium phosphate buffer. Subsequently, Na124l (100 mCi/ml) (IBA, Belgium) was added at a 1 :1 ratio (mg protein to mCi) and the reaction was allowed to start by adding 1/10 volume of Chloramine-T (1 mg/ml). The reaction proceeds for 1 min at 24°C before quenching with 1/10 volume of sodium metabisulfite (1 mg/ml). The mixture was then passed by gravity flow over a G-25 Sephadex columnn Docket no. 05001770-409PCT - 50 -

(NAP-10, GE Healthcare, Buckinghamshire, UK) to collect the flow- through in fifteen 250 μl fractions in PBS. Fractions were measured for radioactivity in a gamma-counter and a BCA test was performed to measure the protein content, followed by combining the fractions of interest. In vitro screening assay

[00143] Polypropylene test tubes (12 x 75 mm) containing cells (0.25 x 10⁶) in 100 μl of RPM11640 (prepared as described above) were placed in an incubator (5% CO2/37°C) for 30 min. The incubator door was opened, and the addition of the radiolabeled SCA was accomplished using an Eppendorf Repeater pipet and subsequently the cells were further incubated for 30 min (or as noted). Accumulation of radiolabel was determined by separating the cell-associated radioactivity (CAR) from the free radioactivity by transferring the cell suspension to a 0.4-ml centrifuge tube (USA Scientific, Ocala, FL) containing a layer of oil consisting of 1 :90.8 volume to volume ratio of n- dodecane: bromo-dodecane (yields 1.017 g/ml; Sigma-Aldrich), and spinning for 8 seconds in a Beckman E centrifuge (maximum speed = 12,535 g Beckman Coulter Inc., Fullerton, CA). The tubes were placed briefly in liquid nitrogen, cut through the radioactive-free oil layer with a razor blade, and the bottom portion of the tube containing the cell pellet was placed into a 12 x 75 mm polypropylene test tube and counted in a gamma counter. Retention was determined similarly except that after incubating the cells for 30 min in the presence of the radiolabel, cells were washed twice with RPM11640 and further incubated in radiolabel-free media prior to spinning the cells through the oil layer. In order to account for the cell type specific cell volume the CAR (cpm/cell) was normalized with the average cell volume (fl), determined in each experiment with a CASY^®. To evaluate specificity of binding, SCAs were preincubated with selected unlabelled SCAs (20 μg) for competition assays.

Pharmacokinetic analysis

[00144] Rats injected with radio-labeled SCA were sacrificed at indicated time points, and blood samples obtained, followed by sedimentation of cellular Docket no. 05001770-409PCT - 51 - material and precipitation of supernatant with trichloroacetic-acid. Radioactivity associated with pellets and supernatant was measured. Blood content of radiolabel was expressed as a percentage of injected dose per gram of blood (%ID/g). Intraperitoneal glucose tolerance test (IPGTT)

[00145] IPGTT tests were performed in non-diabetic rats 7 days after SCA injection and in another set in diabetic and non-diabetic rats just before injection of radio-labeled SCA. Rats were fasted for 12 h. After baseline blood sampling, animals received an intraperitoneal injection of glucose (2g per kg body weight) with glucose-, insulin- and glucagon-levels measured at 30, 60 and 120 min later. Blood samples were taken from tail vein, and glucose determined by a clinical analyzer (Nova Biomedical), insulin with an ELISA (Mercodia) and glucagon using an EIA (Alpco).

Cell viability and apoptosis [00146] INS-1- or α-TC1-cells (10⁶) were exposed overnight to SCA (5 μg or 20 μg). Apoptosis was assessed by a standard test with FITC-Annexin- V/propidium iodide (Pharmigen) and analysis with a FACSscan flowcytometer. Viability was determined by staining the cells with calcein-AM (Calbiochem) and propidium iodine (Molecular Probes), photographed with a fluorescence microscope connected to a digital camera and images were digitally processed using Cell P software (Olympus) and Photoshop 6.0 software (Adobe). Values were compared to non-treated controls.

Biodistribution and estimation of β-cell mass

[00147] Radio-labeled SCA B1 (0.5 μCi, 100 μg) was injected in diabetic and non-diabetic rats, and pancreases were removed 2 hours later, weighed and assayed in a gamma counter for radioactivity .Accumulation of SCA B1 was expressed as a percentage of injected dose per gram of tissue (%ID/g) corrected for background, and estimated in the glandula parotis. β-cell mass (mg) of the corresponding pancreases was estimated by morphometry Docket no. 05001770-409PCT

- 52 - according to the following formula: BCF (%): Beta-Cell Fraction = Insulin- positive area/total pancreatic area; Beta-Cell Mass per pancreas (mg) = BCF x Pancreatic weight (mg).

PET protocol [00148] Rats were anaesthetized with isoflurane (inhalation). The [¹²⁴I]- labeled SCA were dissolved in isotonic saline containing 10% ethanol and then injected in the jugular vein (15-25 MBq). Simultaneously with the tracer injection, a dynamic PET scan using three-dimensional (3D) acquisition mode was started with the following frames: 5 min x 10 frames and subsequently 7 min x 20 frames. Moreover, due to the long half life of ¹²⁴I, it was possible to perform on the following day (24h later) another set of dynamic scans. After the abdomen scan, a static whole body scan (skull base-proxima femora) was performed in 3D acquisition mode in order to image whole body tracer distribution. Statistical Analysis

[00149] Curve fits were modelled in the form of nonlinear regression. For wash-out experiments, data were adapted to an exponential decay function with y = a + b^*exp(-c^*x), where x denotes the time axis and y the measured count rate. For saturation experiments results were adapted to an exponential rise function as y = a + b^*(1 - exp(-c^*x)). Decision criteria were squared regression coefficients (r*r). Parametric comparisons of continuous data were calculated with Student's t-test for unpaired data with unequal variance. Main null-hypothesis was equal distribution of measured counts in both investigated cell types or with and without preincubation, respectively. AUC for glucose, insulin and glucagon was calculated using the trapezoidal method. All calculations have been performed with KaleidaGraph 4.0.3 for Macintosh Computers (Synergy Software, Reading, PA. USA). Docket no. 05001770-409PCT

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[00150] While the disclosure has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the disclosure following, in general, the principles of the disclosure and including such departures from the present disclosures as come within known or customary practice within the art to which the disclosure pertains and as may be applied to the essential features herein before set forth, and as follows in the scope of the appended claims.

[00151] Unless defined otherwise or the context clearly dictates otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It should be understood that any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention. [00152] The contents of all documents and references cited herein are hereby incorporated by reference in their entirety.

Docket no.05001770-409PCT -54-

TABLE OF SEQUENCES: Islet Cell Specific Phage Clone 1 -SCA B1

DNA Sequence (SEQ ID NO:1)

GCCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAG CCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTA GCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGG AGTGGGTCTCATCTATTACTGCTGAGGGTACGCATACATGGTACGCAGA CTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACG CTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATT ACTGTGCGAAAACGTCTTATCGGTTTGACTACTGGGGCCAGGGAACCCT GGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCG GCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCC TGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCA GAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCC CCTAAGCTCCTGATCTATAAGGCATCCCGTTTGCAAAGTGGGGTCCCAT CAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAG CAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAAGTGGG ATCCTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGGG CGGCCGCACATCATCATCAC

Amino-acid Sequence (SEQ ID NO:2) A M A E V Q L L E S G G G L V Q P G G S L R L S C A A S G F T F S S Y A M S W V R Q A P G K G L E W V S S I T A E G T H T W Y A D S V K G R F T I S R D N S K N T L Y L Q M N S L R A E D T A V Y Y C A K T S Y R F D Y W G Q G T L V T V S S G G G G S G G G G S G G G G S T D I Q M T Q S P S S L S A S V G D R V T I T C R A S Q S I S S Y L N W Y Q Q K P G K A P K L L I Y K A S R L Q S G V P S R F S G S G S G T D F T L T I S S L Q P E D F A T Y Y C Q Q K W D P P R T F G Q G T K V E I K R A A A H H H H H H G A A E Q K L I S E E D L N

Islet Cell Specific Phage Clone 2 -SCA B2

DNA Sequence (SEQ ID NO:3)

GCCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAG CCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTA GCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGG AGTGGGTCTCACGGATTAAGATTTTTGGTTCGAAGACAAAGTTCGCAGAC TCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGC Docket no.05001770-409PCT -55-

TGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTA CTGTGCGAAACATTCTACGCATTTTGACTACTGGGGCCAGGGAACCCTG GTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGG CGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCT GTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAG AGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCC CTAAGCTCCTGATCTATAGGGCATCCAGTTTGCAAAGTGGGGTCCCATC AAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGC AGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGCTTCAGAG TACTCCTAGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGGGC GGCCGCACATCATCATCATCACCATCACGGGGCCGCAGAACAAAAACTC ATCTCAGGAGAGGATCTGAAT

Amino-acid Sequence (SEQ ID NO:4)

A M A E V Q L L E S G G G L V Q P G G S L R L S C A A S G F T F S S Y A M S W V R Q A P G K G L E W V S R I K I F G S K T K F A D S V K G R F T I S R D N S K N T L Y L Q M N S L R A E D T A V Y Y C A K H S T H F D Y W G Q G T L V T V S S G G G G S G G G G S G G G G S T D I Q M T Q S P S S L S A S V G D R V T I T C R A S Q S I S S Y L N W Y Q Q K P G K A P K L L I Y R A S S L Q S G V P S R F S G S G S G T D F T L T I S S L Q P E D F A T Y Y C Q Q L Q S T P R T F G Q G T K V E I K R A A A H H H H H H G A A E Q K L I S E E D L N

Islet Cell Specific Phage Clone 5 -SCA B3 DNA Sequence (SEQ ID NO:5)

GCCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAG CCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTA GCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGG AGTGGGTCTCATCGATTCATCCTAAGGGTTACCCTACACGGTACGCAGA CTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACG CTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATT ACTGTGCGAAATCGACGACTCCTTTTGACTACTGGGGCCAGGGAACCCT GGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCG GCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCC TGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCA GAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCC CCTAAGCTCCTGATCTATGCTGCATCCTCTTTGCAAAGTGGGGTCCCATC AAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGC AGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGATGGGGAG GGATCCTAGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGGGC Docket no.05001770-409PCT -56-

GGCCGCACATCATCATCATCACCATCACGGGGCCGCAGAACAAAAACTC ATCTCAGGAGAGGATCTGAAT

Amino-acid Sequence (SEQ ID NO:6)

AM A E V Q L L E S G G G L V Q P G G S L R L S C A A S G F T F S S Y A M S W V R Q A P G K G L E W V S S I H P K G Y P T R Y A D S V K G R F T I S R D N S K N T L Y L Q M N S L R A E D T A V Y Y C A K S T T P F D Y W G Q G T L V T V S S G G G G S G G G G S G G G G S T D I Q M T Q S P S S L S A S V G D R V T I T C R A S Q S I S S Y L N W Y Q Q K P G K A P K L L I Y A A S S L Q S G V P S R F S G S G S G T D F T L T I S S L Q P E D F A T Y Y C Q Q M G R D P R T F G Q G T K V E I K R A A A H H H H H H G A A E Q K L I S E E D L N

Islet Cell Specific Phage Clone 6 -SCA B4 DNA Sequence (SEQ ID NO:7)

GCCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAG CCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTA GCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGG AGTGGGTCTCAAGGATTCAGTTTTTTGGTTCGCATACATACTTCGCAGAC TCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGC TGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTA CTGTGCGAAACATTCGACGCATTTTGATTACTGGGGCCAGGGAACCCTG GTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGG CGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCT GTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAG AGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCC CTAAGCTCCTGATCTATAGGGCATCCATTTTGCAAAGTGGGGTCCCATCA AGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCA GTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAATAGGAGA ATTCCTAGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGGGCG GCCGCACATCATCATCATCACCATCACGGGGCCGCAGAACAAAAACTCA TCTCAGGAGAGGATCTGAAT Amino-acid Sequence (SEQ ID NO:8)

A M A E V Q L L E S G G G L V Q P G G S L R L S C A A S G F T F S S YA M S W V R Q A P G K G L E W V S R I Q F F G S H T Y F A D S V K G R F T I S R D N S K N T L Y L Q M N S L R A E D T A V Y Y C A K H S T H F D Y W G Q G T L V T V S S G G G G S G G G G S G G G G S T D I Q Docket no 05001770-409PCT -57-

M T Q S P S S L S A S V G D R V T I T C R A S Q S I S S Y L N W Y Q Q K P G K A P K L L I Y R A S I L Q S G V P S R F S G S G S G T D F T L T I S S L Q P E D F A T Y Y C Q Q N R R I P R T F G Q G T K V E I K R A A A H H H H H H G A A E Q K L I S E E D L N

Islet Cell Specific Phage Clone 7 -SCA B5

DNA Sequence (SEQ ID NO:9)

GCCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAG CCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTA GCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGG AGTGGGTCTCATCTATTAGTTCTACTGGTGATTCTACAAGTTACGCAGAC TCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGC TGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTA CTGTGCGAAAGCTGCTGATAGTTTTGACTACTGGGGCCAGGGAACCCTG GTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGG CGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCT GTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAG AGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCC CTAAGCTCCTGATCTATGGTGCATCCTCTTTGCAAAGTGGGGTCCCATCA AGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCA GTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGACTAATGGT GCTCCTACTACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGGGCG GCCGCACATCATCATCACCATCACGGGGCCGCAGAACAAAAACTCATCT CAGGAGAGGATCTGAAT

Amino-acid Sequence (SEQ ID NO:10)

A M A E V Q L L E S G G G L V Q P G G S L R L S C A A S G F T F S S YA M S W V R Q A P G K G L E W V S S I S S T G D S T S Y A D S V K G R F T I S R D N S K N T L Y L Q M N S L R A E D T A V Y Y C A K A A D S F D Y W G Q G T L V T V S S G G G G S G G G G S G G G G S T D I Q M T Q S P S S L S A S V G D R V T I T C R A S Q S I S S Y L N W Y Q Q K P G K A P K L L I Y G A S S L Q S G V P S R F S G S G S G T D FT L T I S S L Q P E D F A T Y Y C Q Q T N G A P T T F G Q G T K V E I K R A A A H H H H H H G A A E Q K L I S E E D L N

Islet Cell Specific Phage Clone 3 -SCA A1 Docket no.05001770-409PCT

-58- DNA Sequence (SEQ ID NO:11)

ATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCT GGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCA GCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGT GGGTCTCACGGATTAGTGTGGCTGGTCGGCGGACAGCTTACGCAGACT CCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCT GTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTAC TGTGCGAAAAAGCGGCCTCCGTTTGACTACTGGGGCCAGGGAACCCTG GTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGG CGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCT GTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAG AGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCC CTAAGCTCCTGATCTATGCTGCATCCTCTTTGCAAAGTGGGGTCCCATCA AGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCA GTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGATGGGGAG GGATCCTAGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGGGC GGCCGCACATCATCATCACCAT

Amino-acid Sequence (SEQ ID NO: 12)

A M A E V Q L L E S G G G L V Q P G G S L R L S C A A S G F T F S S Y A M S W V R Q A P G K G L E W V S R I S V A G R R T A Y A D S V K G R F T I S R D N S K N T L Y L Q M N S L R A E D T A V Y Y C A K K R P P F D Y W G Q G T L V T V S S G G G G S G G G G S G G G G S T D I Q M T Q S P S S L S A S V G D R V T I T C R A S Q S I S S Y L N W Y Q Q K P G K A P K L L I Y A A S S L Q S G V P S R F S G S G S G T D F T L T I S S L Q P E D F A T Y Y C Q Q M G R D P R T F G Q G T K V E I K R A A A H H H H H H G A A E Q K L I S E E D L N

Islet Cell Specific Phage Clone 4 -SCA A2 DNA Sequence (SEQ ID NO:13)

GCCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAG CCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTA GCAGCTATGCCATGAGCTGGGTCCGCCAGGCtCCAGGGAAGGGGCTGG AGTGGGTCTCACCTATTGCGTCGCGGGGTGCTCGGACAAATTACGCAGA CTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACG CTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATT Docket no 05001770-409PCT

-59-

ACTGTGCGAAAAAGCCTAGTAGTTTTGACTACTGGGGCCAGGGAACCCT GGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCG GCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCC TGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCA GAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCC CCTAAGCTCCTGATCTATAAGGCATCCCCTTTGCAAAGTGGGGTCCCAT CAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAG CAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGTCGATGC AGGTTCCTTCTACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGGGC GGCCGCACATCATCATCATCACCATCACGGGGCCGCAGAACAAAAACTC ATCTCAGGAGAGGATCTGAAT

Amino-acid Sequence (SEQ ID NO:14)

A M A E V Q L L E S G G G L V Q P G G S L R L S C A A S G F T F S S YA M S W V R Q A P G K G L E W V S P I A S R G A R T N Y A D S V K G R F T I S R D N S K N T L Y L Q M N S L R A E D T A V Y Y C A K K P S S F D Y W G Q G T L V T V S S G G G G S G G G G S G G G G S T D I Q M T Q S P S S L S A S V G D R V T I T C R A S Q S I S S Y L N W Y Q Q K P G K A P K L L I Y K A S P L Q S G V P S R F S G S G S G T D F T L T I S S L Q P E D F A T Y Y C Q Q S M Q V P S T F GQ G T K V E I K R A A A H H H H H H G A A E Q K L I S E E D L N

Claims

Docket no. 05001770-409PCT - 60 -CLAIMS What is claimed is:

1. A single chain antibody specifically binding to a cell type of the islets of Langerhans, wherein said single chain antibody comprises:

i) a heavy chain CDR1 amino acid sequence, CDR2 amino acid sequence, and CDR3 amino acid sequence, respectively, of amino acid residues 34-38, 53-69, 102-108 of SEQ ID NO:2; and ii) a light chain CDR1 amino acid sequence, CDR2 amino acid sequence, and CDR3 amino acid sequence, respectively of amino acid residues 159-169, 185-191 , 224-232 of SEQ ID NO:2.

2. The single chain antibody as in claim 1 or an analog, homolog, fragment or variant thereof comprising heavy and light chain CDR1 , CDR2 and CDR3 amino acid sequences 34-38, 53-69, 102-108, 159- 169, 185-191 , and 224-232, respectively, of SEQ ID NO:4, SEQ ID

NO:6, SEQ ID NO:8, or SEQ ID NO:10, and specifically binding to a β cell.

3. The β cell of claim 2, wherein said β cell is identified in a subject, and said subject is a rodent, canine, pig, primate or human.

4. The single chain antibody of claim 1 or 2 labeled with a radioactive isotope selected from the group consisting of ¹¹C, ¹⁸F, ¹⁸O, ¹³N, ⁷⁶Br and ¹²⁴I for use as a radioligand in PET imaging or selected from the group consisting of ⁸⁶Y, ^99mTc, ¹¹¹In, ¹²³I, and ²⁰¹TI for use as a radioligand in SPECT imaging.

5. The single chain antibody of claim 4 for diagnosing a change in β cell mass, wherein i) an increase of β cell mass is diagnostic of an insulinoma, a nesidioblastoma, an endocrine tumor, or onset of obesity, or Docket no. 05001770-409PCT - 61 - confirms the use of an effective amount of a drug for the treatment of type I or type Il diabetes, or confirms a successful β cell transplantation; and ii) a decrease of β cell mass is diagnostic of type I or type Il diabetes, or confirms the use of an effective amount of a drug for the treatment of obesity, or supports the surveillance of a resected insulinoma, nesidioblastoma, or endocrine tumor; and iii) said β cell is in a subject, wherein the subject is a rodent, a canine, a pig, a primate or a human.

6. A nucleic acid molecule comprising a nucleic acid sequence encoding the antibody of claim 1 or 2 and having the sequence shown in SEQ ID NO:1 , 3, 5, 7 or 9.

7. The nucleic acid molecule of claim 6 operably linked to an expression control sequence to form an expression vector, wherein said expression vector is propagated in a suitable cell.

8. Use of an effective amount of a single chain antibody or an analog, homolog, fragment or variant thereof specifically binding to a cell type of the islets of Langerhans to identify said cell type by in vivo imaging.

9. The use according to claim 8 wherein the single chain antibody comprises: i) a heavy chain CDR1 amino acid sequence, CDR2 amino acid sequence, and CDR3 amino acid sequence, respectively, of amino acid residues 34-38, 53-69, and 102-108 of SEQ ID NO:2; and ii) a light chain CDR1 amino acid sequence, CDR2 amino acid sequence, and CDR3 amino acid sequence, respectively of amino acid residues 159-169, 185-191 , and 224-232 of SEQ ID NO:2; or an analog, homolog, fragment or variant thereof. Docket no. 05001770-409PCT

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10. The use according to claim 9, wherein the single chain antibody comprises heavy and light chain CDR1 , CDR2 and CDR3 amino acid sequences selected from the group consisting of amino acids 34-38, 53-69, 102-108, 159-169, 185-191 , and 224-232 of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 or an analog homolog, fragment or variant thereof, and the cell type identified is a β cell.

11. The use according to any one of claims 8 to 10, wherein said single chain antibody is labeled with a radioactive isotope selected from the group consisting of ¹¹C, ¹⁸F, ¹⁸O, ¹³N, ⁷⁶Br and ¹²⁴I for use as a radioligand in PET imaging or selected from the group consisting of

⁸⁶Y, ^99mTc, ¹¹¹In, ¹²³I, and ²⁰¹TI for use as a radioligand in SPECT imaging.

12. The use according to claim 11 , wherein a change in β cell mass is detected in vivo, wherein i) an increase of β cell mass is diagnostic of an insulinoma, a nesidioblastoma, an endocrine tumor or onset of obesity, or confirms the use of an effective amount of a drug for the treatment of type I or type Il diabetes, or confirms a successful β cell transplantation; and ii) a decrease of β cell mass is diagnostic of type I or type Il diabetes, or confirms the use of an effective amount of a drug for the treatment of obesity, or supports the surveillance of a resected insulinoma, nesidioblastoma, or endocrine tumor; and iii) said β cell is in a subject, wherein the subject is a rodent, a canine, a pig, a primate or a human.

13. A method for identifying a cell type of the islets of Langerhans in a subject in vivo comprising labeling a single chain antibody or an analog, homolog, fragment or variant thereof and imaging said labeled Docket no 05001770-409PCT

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14. The method of claim 13 wherein said single chain antibody comprises: i) a heavy chain CDR1 amino acid sequence, CDR2 amino acid sequence, and CDR3 amino acid sequence, respectively, of amino acid residues 34-38, 53-69, and 102-108 of SEQ ID NO:2; and ii) a light chain CDR1 amino acid sequence, CDR2 amino acid sequence, and CDR3 amino acid sequence, respectively of amino acid residues 159-169, 185-191 , and 224-232 of SEQ ID NO:2; or an analog, homolog, fragment or variant thereof.

15. The method of claim 14 wherein the single chain antibody comprises heavy and light chain CDR1 , CDR2 and CDR3 amino acid sequences selected from the group consisting of 34-38, 53-69, 102-108, 159-169, 185-191 , and 224-232 of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO: 10, or an analog, homolog, fragment or variant thereof, and the cell type identified is a β cell.

16. The method of any one of claims 13 to 15, wherein said single chain antibody is labeled with a radioactive isotope selected from the group consisting of ¹¹C, ¹⁸F, ¹⁸O, ¹³N, ⁷⁶Br and ¹²⁴I for use as a radioligand in PET imaging or selected from the group consisting of ⁸⁶Y, ^99mTc, ¹¹¹In,

¹²⁴I, and ²⁰¹TI for use as a radioligand in SPECT imaging.

17. A method for identifying a change in β cell mass in a subject in vivo, wherein i) β cells are identified in the subject using the method of any one of claims 13 to 16; ii) an increase of β cell mass is diagnostic of an insulinoma, a nesidioblastoma, an endocrine tumor or onset of obesity, or confirms the use of an effective amount of a drug for the treatment Docket no 05001770-409PCT - 64 - of type I or type Il diabetes, or confirms a successful β cell transplantation, or indicates increase in size of a resected insulinoma, nesidioblastoma, or endocrine tumor; and iii) a decrease of β cell mass is diagnostic of type I or type Il diabetes, or confirms the use of an effective amount of a drug for the treatment of obesity, or indicates reduction in size of a resected insulinoma, nesidioblastoma, or endocrine tumor; wherein the subject is a rodent, a canine, a pig, a primate or a human.

18. An analog, homolog, fragment or variant thereof of the single chain antibody of any one of the preceding claims, wherein the analog, homolog, fragment or variant retains the binding specificity of the single chain antibody.

19. The method of any one of the preceding claims, wherein said single chain antibody or analog, homolog, fragment or variant thereof is used as a superparamagnetic contrast agent and visualized using MRI.

20. The method of any one of the preceding claims, wherein said single chain antibody or analog, homolog, fragment or variant thereof is administered by injection, orally, intravenously, intraperitoneal^, intramuscularly or subcutaneously.

21. The method of antibody of any one of the preceding claims, wherein said single chain antibody or analog, homolog, fragment or variant thereof is humanized.