WO2009105548A2 - Methods for treating diabetes - Google Patents

Abstract

The present invention provides, among other things, methods for preventing or delaying the onset of diabetes, restoring normoglycemia, or establishing long-term tolerance towards autoantigens by administering an effective amount of anti-CD22 antibody, or a fragment thereof. The present invention further provides methods for identifying genes or proteins differentially expressed in re-emerging B cells that may be used as biomarkers or therapeutic targets for the treatment of diabetes and related diseases, disorders or conditions.

Description

METHODS FOR TREATING DIABETES

RELATED APPLICATIONS

[0001] This application claims priority to U.S. Application No. 61/066,377 filed on

February 19, 2008, the contents of which is hereby incorporated by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to methods for treating autoimmune diabetes. In particular, this invention relates to methods for treating or delaying onset of type I diabetes by administering antibodies against B-cell antigens.

BACKGROUND OF THE INVENTION

[0003] Diabetes is a lifelong disease for which there is not yet a cure. There are several forms of diabetes. Type 1 diabetes (TlD) is a disorder involving insulin deficiency resulting from the autoimmune destruction of β cells (Atkinson MA, et al. Lancet. 2001; 358:221-229). Indeed, most individuals affected by TlD exhibit multiple features associated with autoimmune disease, including autoantibodies against a variety of islet cell antigens (Todd JA, et al. Immunity. 2001; 15:387-395; Larsson K, et al. Diabetes Metab Res Rev. 2004; 20:429-437). Different monoclonal antibodies directed against various lymphocyte subsets (e.g., anti-CD3) or their products (e.g., IL-2 receptor, CD154, IFN-γ, etc.) have been investigated in nonobese diabetic (NOD) mice (Harlan DM, et al. Nat Med. 2005; 11:716- 718; Graca L, et al. Curr Opin Immunol. 2003; 15:499-506). However, few treatments have been able to restore normoglycemia in NOD mice after the onset of hyperglycemia (Chatenoud L, et al. J Immunol. 1997; 158:2947-2954; Belghith M, et al. Nat Med. 2003; 9: 1202-1208; Kodama S, et al. Science. 2003; 302: 1223-1227; Ogawa N, et al. Diabetes. 2004; 53: 1700- 1705; Chong AS, e? α/. Science. 2006; 311: 1774-1775; Tarbell KV, et al. J Exp Med. 2007; 204:191-201). For example, anti-CD3 treatment in NOD mice given before hyperglycemic onset failed to prevent TlD or was not long-lasting (Chatenoud L, et al. J Immunol. 1997; 158:2947-2954), supporting the notion that agents should be tested throughout the natural history of diabetes to undercover therapeutic windows of opportunity. In addition, anti-CD3 used in the clinic also causes many adverse events (Herold KC, et al. N Engl J Med. 2002; 346:1692-1698). One of the great challenges with any immunological treatment for TlD is to develop safer tolerogenic protocols to allow their use in new-onset diabetic children.

SUMMARY OF THE INVENTION

[0004] The present invention provides a new therapeutic approach for autoimmune diabetes, in particular, type 1 diabetes. Specifically, the present invention provides methods for treating type 1 diabetes based on B-cell depletion strategies. In particular, the present invention provides methods for preventing or delaying diabetes onset, restoring normoglycemia, or establishing long-term tolerance towards autoantigens.

[0005] Thus, in one aspect, the present invention provides a method for preventing or delaying the onset of diabetes in a subject at risk for developing diabetes. In one embodiment, the method includes administering to the subject an effective amount of anti-CD22 antibody, or a fragment thereof. In some embodiments, the diabetes is type 1 diabetes. In some embodiments, the anti-CD22 antibody, or a fragment thereof, is selected from the group consisting of intact IgG, F(ab')2, F(ab)2, Fab', Fab, scFvs, diabodies, triabodies or tetrabodies. In certain embodiments, the anti-CD22 antibody is a monoclonal antibody. In particular embodiments, the anti-CD22 antibody is a mouse monoclonal antibody. In other embodiments, the anti-CD22 antibody is a humanized monoclonal antibody.

[0006] In some embodiments, the anti-CD22 antibody, or a fragment thereof, is conjugated to a cytotoxic agent. In certain embodiments, the cytotoxic agent is calicheamicin or a derivative thereof. In particular embodiments, the calicheamicin is N-acetyl-calicheamicin dimethyl acid.

[0007] In certain embodiments, the ratio of the amount of N-acetyl-calicheamicin dimethyl acid to the amount of the anti-CD22 antibody ranges from about 0.1 to about 15.0 moles calicheamicin/mol antibody. In certain embodiments, the ratio of the amount of N-acetyl- calicheamicin dimethyl acid to the amount of the anti-CD22 antibody ranges from about 1.0 to about 4.0 calicheamicin/mol antibody. In certain embodiments, the ratio of the amount of N- acetyl-calicheamicin dimethyl acid to the amount of the anti-CD22 antibody ranges from about 1.2 to about 2.6 calicheamicin/mol antibody. In certain embodiments, the ratio of the amount of N-acetyl-calicheamicin dimethyl acid to the amount of the anti-CD22 antibody ranges from about 0.1 to about 5.0 calicheamicin/mol antibody. In certain embodiments, the ratio of the amount of N-acetyl-calicheamicin dimethyl acid to the amount of the anti-CD22 antibody ranges from about 1.0 to about 10.0 calicheamicin/mol antibody. In certain embodiments, the ratio of the amount of N-acetyl-calicheamicin dimethyl acid to the amount of the anti-CD22 antibody ranges from about 5.0 to about 10.0 calicheamicin/mol antibody. In certain embodiments, the ratio of the amount of N-acetyl-calicheamicin dimethyl acid to the amount of the anti-CD22 antibody ranges from about 5.0 to about 15.0 calicheamicin/mol antibody.

[0008] In some embodiments, the effective amount of the anti-CD22 antibody, or a fragment thereof, ranges from 20 milligrams to 2 grams protein per dose. In some embodiments, the effective amount of the anti-CD22 antibody, or a fragment thereof, ranges from 20 to 1000 milligrams protein per dose. In some embodiments, the effective amount of the anti-CD22 antibody, or a fragment thereof, ranges from 20 to 500 milligrams protein per dose. In some embodiments, the effective amount of the anti-CD22 antibody, or a fragment thereof, ranges from 20 to 150 milligrams protein per dose. In certain embodiments, the effective amount of the anti-CD22 antibody, or a fragment thereof, ranges from 20 to 100 milligrams protein per dose.

[0009] In some embodiments, the anti-CD22 antibody, or a fragment thereof, is administered bimonthly, monthly, triweekly, biweekly, weekly, twice a week, daily, or at variable intervals.

[0010] In another aspect, the present invention provides a method for restoring normoglycemia in a subject suffering from type 1 diabetes. In particular, the method includes administering to the subject an effective amount of anti-CD22 antibody, or a fragment thereof.

[0011] In some embodiments, the anti-CD22 antibody, or a fragment thereof, is selected from the group consisting of intact IgG, F(ab')2, F(ab)2, Fab', Fab, scFvs, diabodies, triabodies or tetrabodies. In certain embodiments, the anti-CD22 antibody is a monoclonal antibody. In certain embodiments, the anti-CD22 antibody is a mouse monoclonal antibody. In certain embodiments, the anti-CD22 antibody is a humanized monoclonal antibody.

[0012] In some embodiments, the anti-CD22 antibody, or a fragment thereof, is conjugated to a cytotoxic agent. In certain embodiments, the cytotoxic agent is calicheamicin or a derivative thereof. In certain embodiments, the calicheamicin is N-acetyl-calicheamicin dimethyl acid. [0013] In certain embodiments, the ratio of the amount of N-acetyl-calicheamicin dimethyl acid to the amount of the anti-CD22 antibody ranges from about 0.1 to about 15.0 moles calicheamicin/mol antibody. In certain embodiments, the ratio of the amount of N-acetyl- calicheamicin dimethyl acid to the amount of the anti-CD22 antibody ranges from about 1.0 to about 4.0 calicheamicin/mol antibody. In certain embodiments, the ratio of the amount of N- acetyl-calicheamicin dimethyl acid to the amount of the anti-CD22 antibody ranges from about 1.2 to about 2.6 calicheamicin/mol antibody. In certain embodiments, the ratio of the amount of N-acetyl-calicheamicin dimethyl acid to the amount of the anti-CD22 antibody ranges from about 0.1 to about 5.0 calicheamicin/mol antibody. In certain embodiments, the ratio of the amount of N-acetyl-calicheamicin dimethyl acid to the amount of the anti-CD22 antibody ranges from about 1.0 to about 10.0 calicheamicin/mol antibody. In certain embodiments, the ratio of the amount of N-acetyl-calicheamicin dimethyl acid to the amount of the anti-CD22 antibody ranges from about 5.0 to about 10.0 calicheamicin/mol antibody. In certain embodiments, the ratio of the amount of N-acetyl-calicheamicin dimethyl acid to the amount of the anti-CD22 antibody ranges from about 5.0 to about 15.0 calicheamicin/mol antibody.

[0014] In some embodiments, the effective amount of the anti-CD22 antibody, or a fragment thereof, ranges from 20 milligrams to 2 grams protein per dose. In some embodiments, the effective amount of the anti-CD22 antibody, or a fragment thereof, ranges from 20 to 1000 milligrams protein per dose. In some embodiments, the effective amount of the anti-CD22 antibody, or a fragment thereof, ranges from 20 to 500 milligrams protein per dose. In some embodiments, the effective amount of the anti-CD22 antibody, or a fragment thereof, ranges from 20 to 150 milligrams protein per dose. In certain embodiments, the effective amount of the anti-CD22 antibody, or a fragment thereof, ranges from 20 to 100 milligrams protein per dose.

[0015] In some embodiments, the anti-CD22 antibody, or a fragment thereof, is administered bimonthly, monthly, triweekly, biweekly, weekly, twice a week, daily, or at variable intervals.

[0016] In some embodiments, the method of this aspect of the invention further includes administering a T-cell targeting agent.

[0017] In yet another aspect, the present invention provides a method for inducing T-cell tolerance in a subject suffering from type 1 diabetes. In particular, the method includes administering to the subject an effective amount of anti-CD22 antibody, or a fragment thereof. [0018] In some embodiments, the anti-CD22 antibody, or a fragment thereof, is selected from the group consisting of intact IgG, F(ab')2, F(ab)2, Fab', Fab, scFvs, diabodies, triabodies or tetrabodies. In certain embodiments, the anti-CD22 antibody is a monoclonal antibody. In certain embodiments, the anti-CD22 antibody is a mouse monoclonal antibody. In certain embodiments, the anti-CD22 antibody is a humanized monoclonal antibody.

[0019] In some embodiments, the anti-CD22 antibody, or a fragment thereof, is conjugated to a cytotoxic agent. In certain embodiments, the cytotoxic agent is calicheamicin or a derivative thereof. In certain embodiments, the calicheamicin is N-acetyl-calicheamicin dimethyl acid.

[0020] In certain embodiments, the ratio of the amount of N-acetyl-calicheamicin dimethyl acid to the amount of the anti-CD22 antibody ranges from about 0.1 to about 15.0 moles calicheamicin/mol antibody. In certain embodiments, the ratio of the amount of N-acetyl- calicheamicin dimethyl acid to the amount of the anti-CD22 antibody ranges from about 1.0 to about 4.0 calicheamicin/mol antibody. In certain embodiments, the ratio of the amount of N- acetyl-calicheamicin dimethyl acid to the amount of the anti-CD22 antibody ranges from about 1.2 to about 2.6 calicheamicin/mol antibody. In certain embodiments, the ratio of the amount of N-acetyl-calicheamicin dimethyl acid to the amount of the anti-CD22 antibody ranges from about 0.1 to about 5.0 calicheamicin/mol antibody. In certain embodiments, the ratio of the amount of N-acetyl-calicheamicin dimethyl acid to the amount of the anti-CD22 antibody ranges from about 1.0 to about 10.0 calicheamicin/mol antibody. In certain embodiments, the ratio of the amount of N-acetyl-calicheamicin dimethyl acid to the amount of the anti-CD22 antibody ranges from about 5.0 to about 10.0 calicheamicin/mol antibody. In certain embodiments, the ratio of the amount of N-acetyl-calicheamicin dimethyl acid to the amount of the anti-CD22 antibody ranges from about 5.0 to about 15.0 calicheamicin/mol antibody.

[0021] In some embodiments, the effective amount of the anti-CD22 antibody, or a fragment thereof, ranges from 20 milligrams to 2 grams protein per dose. In some embodiments, the effective amount of the anti-CD22 antibody, or a fragment thereof, ranges from 20 to 1000 milligrams protein per dose. In some embodiments, the effective amount of the anti-CD22 antibody, or a fragment thereof, ranges from 20 to 500 milligrams protein per dose. In some embodiments, the effective amount of the anti-CD22 antibody, or a fragment thereof, ranges from 20 to 150 milligrams protein per dose. In certain embodiments, the effective amount of the anti-CD22 antibody, or a fragment thereof, ranges from 20 to 100 milligrams protein per dose. [0022] In some embodiments, the anti-CD22 antibody, or a fragment thereof, is administered bimonthly, monthly, triweekly, biweekly, weekly, twice a week, daily, or at variable intervals.

[0023] In still another aspect, the present invention provides a method for inhibiting immune-mediated destruction of pancreatic islets in a subject. In particular, the method includes administering to the subject an effective amount of anti-CD22 antibody, or a fragment thereof. In some embodiments, the subject is suffering from type 1 diabetes. In other embodiments, the subject has received an islet transplantation.

[0024] In some embodiments, the anti-CD22 antibody, or a fragment thereof, is selected from the group consisting of intact IgG, F(ab')2, F(ab)2, Fab', Fab, scFvs, diabodies, triabodies or tetrabodies. In certain embodiments, the anti-CD22 antibody is a monoclonal antibody. In certain embodiments, the anti-CD22 antibody is a mouse monoclonal antibody. In certain embodiments, the anti-CD22 antibody is a humanized monoclonal antibody.

[0025] In some embodiments, the anti-CD22 antibody, or a fragment thereof, is conjugated to a cytotoxic agent. In certain embodiments, the cytotoxic agent is calicheamicin or a derivative thereof. In certain embodiments, the calicheamicin is N-acetyl-calicheamicin dimethyl acid.

[0026] In certain embodiments, the ratio of the amount of N-acetyl-calicheamicin dimethyl acid to the amount of the anti-CD22 antibody ranges from about 0.1 to about 15.0 moles calicheamicin/mol antibody. In certain embodiments, the ratio of the amount of N-acetyl- calicheamicin dimethyl acid to the amount of the anti-CD22 antibody ranges from about 1.0 to about 4.0 calicheamicin/mol antibody. In certain embodiments, the ratio of the amount of N- acetyl-calicheamicin dimethyl acid to the amount of the anti-CD22 antibody ranges from about 1.2 to about 2.6 calicheamicin/mol antibody. In certain embodiments, the ratio of the amount of N-acetyl-calicheamicin dimethyl acid to the amount of the anti-CD22 antibody ranges from about 0.1 to about 5.0 calicheamicin/mol antibody. In certain embodiments, the ratio of the amount of N-acetyl-calicheamicin dimethyl acid to the amount of the anti-CD22 antibody ranges from about 1.0 to about 10.0 calicheamicin/mol antibody. In certain embodiments, the ratio of the amount of N-acetyl-calicheamicin dimethyl acid to the amount of the anti-CD22 antibody ranges from about 5.0 to about 10.0 calicheamicin/mol antibody. In certain embodiments, the ratio of the amount of N-acetyl-calicheamicin dimethyl acid to the amount of the anti-CD22 antibody ranges from about 5.0 to about 15.0 calicheamicin/mol antibody. [0027] In some embodiments, the effective amount of the anti-CD22 antibody, or a fragment thereof, ranges from 20 milligrams to 2 grams protein per dose. In some embodiments, the effective amount of the anti-CD22 antibody, or a fragment thereof, ranges from 20 to 1000 milligrams protein per dose. In some embodiments, the effective amount of the anti-CD22 antibody, or a fragment thereof, ranges from 20 to 500 milligrams protein per dose. In some embodiments, the effective amount of the anti-CD22 antibody, or a fragment thereof, ranges from 20 to 150 milligrams protein per dose. In certain embodiments, the effective amount of the anti-CD22 antibody, or a fragment thereof, ranges from 20 to 100 milligrams protein per dose.

[0028] In some embodiments, the anti-CD22 antibody, or a fragment thereof, is administered bimonthly, monthly, triweekly, biweekly, weekly, twice a week, daily, or at variable intervals.

[0029] In another aspect, the present invention provides methods of identifying genes or proteins associated with re-programming B-cells, in particular, following a treatment or therapy for diabetes. In some embodiments, a method of the invention comprises includes one or more steps of (a) providing a biological sample comprising reemerging B cells after an anti-diabetic treatment; (b) generating an expression profile of genes or proteins of the reemerging B-cells; (c) identifying one or more differentially expressed genes or proteins in the reemerging B-cells as compared to a reference expression profile, thereby identifying genes or proteins associated with re-programming B-cells. In some embodiments, the anti-diabetic treatment is a treatment with a B-cell depleting agent. In some embodiments, a B-cell depleting agent suitable for the invention is an anti-CD22 antibody, or a fragment thereof. In some embodiments, a reference expression profile suitable for the invention is an expression profile of corresponding untreated B-cells. In some embodiments, a reference expression profile suitable for the invention is an expression profile of naϊve B-cells from an untreated subject suffering from diabetes. In some embodiments, a method of the invention further comprises a step of determining if any of the differentially expressed genes or proteins are indicative or regulatory of diabetes.

[0030] In yet another aspect, the present invention provides methods of diagnosis or prognosis of diabetes based on the differentially expressed genes and/or proteins identified herein. In some embodiments, a method according to the invention includes one or more steps of (a) providing a biological sample comprising B-cells obtained from a subject suffering from or at risk of diabetes; (b) determining an expression or activity of a differentially expressed gene or protein identified according to methods described herein (e.g., one or more genes or proteins identified in Table 1 or Figure 6) in the biological sample; and (c) providing a diagnosis or prognosis based on the expression or activity of the differentially expressed gene or protein as determined at step (b). In some embodiments, a method of the invention further comprises a step of providing a treatment of diabetes (e.g., use of particular drugs, doses or dosing schedules) based on the diagnosis or prognosis described herein.

[0031] In this application, the use of "or" means "and/or" unless stated otherwise. As used in this application, the term "comprise" and variations of the term, such as "comprising" and "comprises," are not intended to exclude other additives, components, integers or steps. As used in this application, the terms "about" and "approximately" are used as equivalents. Any numerals used in this application with or without about/approximately are meant to cover any normal fluctuations appreciated by one of ordinary skill in the relevant art.

[0032] Other features, objects, and advantages of the present invention are apparent in the detailed description, drawings and claims that follow. It should be understood, however, that the detailed description, the drawings, and the claims, while indicating embodiments of the present invention, are given by way of illustration only, not limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings are for illustration purposes only, not for limitation.

[0033] Figure 1 illustrates exemplary depletional studies. Splenocytes were extracted from normoglycemic 10-week-old NOD mice (n=5 mice) and were analyzed at flow cytometry for CD 19 and CD22 expression on B220⁺ cells and CD138⁺ cells (plasmacells). CD 19 and CD22 were similarly expressed on B220⁺ cells (Panels A and B). CD138⁺ cells expressed both CD 19 and CD22 (Panel C). We then analyzed CD22 expression on CD138⁺ cells (plasmacells) and examined by flow cytometry the infiltrating cells in the pancreata of 4, 8, 12-week-old and hyperglycaemic NOD mice (> 14-weeks-old) (n=5 mice per group). Most of the infiltrate is constituted by CD45⁺CD19⁺ cells (B cells), (Panel D). B cells pancreatic infiltration in the NOD mice peaks around 8 to 10 weeks (p<0.05; Panel D). while CD45 CD3⁺ cells (T cells) remained stable over time (Panel E). Two injections (160 μg i.p. 5 days apart, day 0 and day 5) of anti- CD22/cal mAb elicited a quick and profound depletion of B cells in the peripheral blood of 10- week-old NOD mice (n=6 mice per group) by week one and lasted for 6-7 weeks (Panels F and H). In control NOD mice, B cells appeared to be not-depleted (Panels F and G), while in the group treated with unconjugated anti-CD22, a transient and partial B cells depletion was observed (Panels F and I). After 8-10 weeks after depletion, B cells recovered almost completely (Panels F and H).

[0034] Figure 2 illustrates exemplary diabetes prevention studies. We observed a significant delay in diabetes onset in anti-CD22/cal mAb treated female 10-week-old NOD mice (n=20 mice) compared to controls (n=30 mice, p<0.01), (Panel A). The calicheamicin alone treated group developed diabetes similarly to untreated control (n=10 mice, p<0.01 vs. anti- CD22/cal mAb treated NOD mice). Unconjugated anti-CD22 treatment slightly delayed diabetes onset (n=10 mice, p=0.06 vs. untreated control). At 25 weeks post- injections, an increase in the percentage of CD4⁺CD25⁺FoxP3⁺ cells was evident in the pancreatic lymph nodes (PLn) of anti- CD22/cal mAb treated NOD mice (n=4 mice) compared to 10-week-old untreated control NOD (n=4 mice, p=0.02) and compared to hyperglycemic more than 14 weeks-old NOD mice (n=4 mice, p=0.009), (Panel B). CD4⁺ cells extracted from splenocytes of anti-CD22/cal mAb treated NOD mice at 25 weeks after depletion produced less IFN-γ when challenged with the BDC2.5 peptide compared to untreated age-matched control hyperglycemic NOD mice (p=0.001) and 10- week-old NOD mice (p=0.04), (n=4 mice each group), (Panel C).

[0035] Isolated BDC2.5 TCR Tg⁺ CD4⁺ cells were CFSE-labeled and transferred into two groups of NOD. SCID mice reconstituted with NOD splenocytes with and without anti- CD22/cal mAb treatment, respectively. A higher proliferation of BDC2.5 TCR Tg⁺ CD4⁺ cells was evident in the control NOD. SCID host (Panels E and F. lower quadrant) with fewer cells recovered (Panel D) compared to the anti-CD22/cal mAb treated NOD. SCID host (Panels E and F, upper quadrant). Insulitis score analysis revealed better preserved islets in the anti-CD22/cal mAb treated NOD mice at 5 and 25 weeks after injections (Panel G).

[0036] Figure 3 illustrates exemplary histology of prevention studies. At baseline, NOD mouse showed some mild perinsulitis (Panel Al) with many B220⁺ cells (Panel A2) and some CD3⁺ cells (Panel A3), but still with well preserved insulin and glucagon staining (Panels A5 and A6). Fopx3⁺ cells were merely present at baseline (Panel A4). Interestingly, after 5 weeks from injections, treated NOD mice showed reduced infiltrate compared to control NOD mouse (Panels Bl and Cl) with no B220⁺ cells (Panel B2) and fewer CD3⁺ cells (Panel B3), while in the control, B220⁺ and CD3⁺ cells were abundantly represented (Panel C2 and C3). After 25 weeks from the treatment, the treated group showed cleaner pancreatic compared to the untreated control hyperglycemic NOD mice (Panels Dl and El). B220⁺ and CD3⁺ cells were not infiltrating the islets in the treated group (Panels D2 and D3), while in the controls, islets were massively infiltrated by B220⁺ and CD3⁺ cells (Panels E2 and E3). Islets morphology was well preserved in the treated group at 5 and 25 weeks after injections (Panels B5. B6 and D5. D6). but not in the control group (Panels C5. C6 and E5. E6). FoxP3 staining of pancreatic islets at 5 and 25 weeks after treatment revealed a persistently reduced FoxP3 expression, particularly if compared with the massive presence of T cells, in the controls compared to treated NOD mice (Panels B4. D4 and C4. E4).

[0037] Figure 4 illustrates exemplary hyperglycemia reversal studies. A rapid reversal of hyperglycemia (within 2 days) was observed in all of the treated hyperglycemia NOD mice (10 out of 10) (Panel A). Six out of 10 remained normoglycemic in the long term. Three mice remained normoglycemic for 20-40 days and then again became hyperglycemic and one returned to hyperglycemia around 60 days (Panel A). None of the untreated newly hyperglycemic control NOD mice reverted from hyperglycemia (Panel B). After 5 days from hyperglycemia onset (n=6 mice), anti-CD22/cal mAb treatment was not able to restore normoglycemia in the long-term (Panel B). Either calicheamicin alone (GG5/cal) (n=5 mice) or unconjugated anti-CD22 treatment failed to restore normoglycemia in the long-term (Panel B). Serum samples were obtained in the reversal studies at baseline, day 10 and day 100 after the treatment from anti- CD22/cal mAb treated and control NOD mice. Pro-inflammatory cytokines including IL- 17, TNF-α were reduced 10 days after the treatment (Panel C). IFN-γ was slightly reduced as well (Panel C). The percentage of CD4⁺CD25 FoxP3⁺ cells increases in long-term tolerant mice treated with anti-CD22/cal mAb compared to that in control NOD mice, for example, in the PLn (anti-CD22/cal mAb treated vs. normoglycemic 10-week-old, p=0.007 and vs. hyperglycemic, p=0.03, Panel D) and in the spleen (anti-CD22/cal mAb treated vs. normoglycemic 10-week- old, p=0.02 and vs. hyperglycemic, p=0.01, Panel E). Insulitis score confirmed that anti- CD22/cal mAb treated NOD mice showed better preserved and less infiltrated islets compared to untreated control NOD mice both at baseline and 10 days after hyperglycemia onset (Panel F).

[0038] Figure 5 illustrates exemplary histology of hyperglycemia reversal studies.

Untreated, hyperglycemic mice, at baseline, showed islets heavily infiltrated by lymphocytes (Panels Al). predominantly B220⁺ B-cells (Panels A2). and to a lesser extent by CD3⁺ T cells (Panels A3). Rare insulin positive cells and more glucagon positive cells can be detected (Panels A5 and A6). The infiltrate consists of only a small number of FoxP3⁺ Tregs (Panels A4). Ten days following treatment with anti-CD22/cal mAb, islets appeared rarely infiltrated compared to the untreated controls (Panels Bl and Cl), with few B220⁺ and CD3⁺ cells (Panels B2, B3 and C2, C3), but with an increase in FoxP3⁺ cells (Panels B4 and C4). In treated animals, but not in the controls, islets showed abundant stainable insulin (Panels B5 and C5) and glucagon (Panels

B6 and C6). One hundred days post-treatment, 2 histologic patterns were seen in the treated group. Many of the islets showed essentially no lymphoid infiltrating at all (Panels D1-D3). few cells stain for insulin while more for glucagon (Panels D5 and D6). A smaller subset of islets showed an abundant B220⁺ lymphoid infiltrates (Panel E2) but also frequent CD3⁺ T cells (Panel E3) similar to untreated hyperglycemic mice. However in contrast to hyperglycemic mice, the infiltrates remained largely confined to the periphery of the islet (compare Panels El and Al and Bl), and a larger percentage of FoxP3⁺ Tregs (Panel E4). Glucagon was easily detected (Panel E6), but insulin staining was rare (Panel E5).

[0039] Figure 6 illustrates exemplary transcriptome analysis of re-emerging B cells. B cells were extracted (using CD 19 magnetic beads) from 10-week-old NOD mice, from hyperglycemic NOD mice as well as from the re-emerging B cell pool from age-matched B cell- depleted NOD mice in which the B cell repertoire is recovered. A gene array analysis was performed to evaluate gene expression of more than 40,000 genes. Genes which are differentially expressed in naϊve B cells extracted from normoglycemic 10-week-old or hyperglycemic NOD mice and re-emerging B cells are shown in the heat map (Panels A-C). Blue represents lesser expression and red higher expression. 200 genes are downregulated in the re-emerging B cells compared to naϊve B cells from 10-week-old NOD mice (Panel A). 38 genes are downregulated in the re-emerging B cells compared to naϊve B cells from hyperglycemic NOD mice (Panel B). 21 genes are downregulated similarly in the re-emerging B cells compared to naϊve B cells from 10-week-old and hyperglycemic NOD mice (Panel C).

[0040] Figure 7 illustrates exemplary characterization of re-emerging B cells. FACS analysis of CD80, CD86, CD40, Class II and IgM did not reveal any differences between re- emerging and naϊve B cells extracted from splenocytes (the latter from either normo- or hyperglycemic NOD mice) (representative of 5 mice, Panel A). Interestingly, we observed by FACS analysis a higher percentage of anergic B cells (B220⁺CD93⁺CD23⁺IgM^lc cells) in the re- emerging B cell pool compared to naϊve B cells from hyperglycemic age-matched untreated NOD mice (representative of 5 mice, Panel B, with anergic B cells circled). We customized an in vitro assay in which B cells are used as APCs and autoreactive BDC2.5 TCR Tg⁺ CD4⁺ cells are used as responders in the presence of the BDC2.5 peptide. When re-emerging B cells were APCs, a lower IFN-γ production by BDC2.5 TCR Tg⁺ CD4⁺ cells was evident compared to when naϊve B cells were used (Panel C). Supernatant were collected from the experiment described above and cytokine profile was assessed with a Luminex assay. Interestingly, when re-emerging B cells, but not naϊve B cells, were used as APCs, BDC2.5 autoreactive CD4⁺ cells downregulated the production of pro-inflammatory cytokines (IL-2, IL- 17, TNF-α and IFN-γ), (Panels

M). [0041] We then co-adoptively transferred CD19⁺ cells (obtained from re-emerging or from naive B cell pool) into NOD. SCID recipients with diabetogenic CD4⁺ cells obtained from hyperglycemic NOD mice. When re-emerging B cells were transferred, but not naϊve B cells, they completely abrogated the onset of diabetes mediated by the transfer of diabetogenic CD4⁺ cells (Panel D), (n=5 mice/group). We also analyzed the percentage of CD4⁺CD25⁺FoxP3⁺ cells in the spleen of NOD. SCID recipients of diabetogenic CD4 T cells and re-emerging B cells or controls (B cells from hyperglycemic animals or no cells) at day 30 post-adoptive transfer. Panel E shows that there is no difference between the groups (n=5 mice/group).

DETAILED DESCRIPTION OF THE INVENTION

[0042] The present invention provides a new therapeutic approach for autoimmune diabetes, for example, type 1 diabetes. Specifically, the present invention provides methods for treating type 1 diabetes based on B-cell depletion strategies. In particular, the present invention provides methods for preventing or delaying diabetes onset, restoring normoglycemia, or establishing long-term tolerance towards autoantigens by administering a B-cell depleting agent, such as, an anti-CD22 antibody or an immunoconjugate thereof.

[0043] Various aspects of the invention are described in detail in the following sections.

The use of sections is not meant to limit the invention. Each section can apply to any aspect of the invention. In this application, the use of "or" means "and/or" unless stated otherwise.

Type 1 diabetes

[0044] Type 1 diabetes is a form of diabetes mellitus. Type 1 diabetes (TlD) is also known as diabetes mellitus type 1, insulin-dependent diabetes mellitus (IDDM), childhood diabetes, juvenile diabetes, or insulin-dependent diabetes. Type 1 diabetes is an autoimmune disease that results in the permanent destruction of insulin producing beta cells of the pancreas. As a result, cells of the pancreas produce little or no insulin, the hormone that allows glucose to enter body cells. Consequently, glucose builds up in the bloodstream instead of going into the cells causing high blood glucose levels, known as hyperglycemia. Chronic hyperglycemia is the defining characteristic of diabetes mellitus, including type 1 diabetes. Typically, symptoms of hyperglycemia include, but are not limited to, polyphagia (frequently hungry), polyuria (frequently urinating), polydipsia (frequently thirsty), blurred vision, fatigue, weight loss, poor wound healing (cuts, scrapes, etc.), dry mouth, dry or itchy skin, male impotence, recurrent infections. Acute episodes of hyperglycemia without an obvious cause may indicate developing diabetes or a predisposition to the disorder. Type 1 diabetes can occur at any age. Some patients are diagnosed as kids. Many patients, however, are diagnosed after age 20.

[0045] Typically, type 1 diabetes develops due to an autoimmune disorder. As used herein, an "autoimmune disorder," "autoimmune disease" or "autoimmune problem," or grammatical equivalents, refers to a disorder when the body's immune system attacks one of it's own tissues as foreign. In the case of type 1 diabetes, the body's immune system attacks the islet cells (e.g., β cells) of the pancreas that produce insulin. Typically, in patients suffering from type 1 diabetes, the body produces antibodies to fight the islet cells of the pancreas and destroys the islet cells ability to produce insulin. Indeed, most individuals affected by TlD exhibit multiple features associated with autoimmune disease, including autoantibodies against a variety of islet cell antigens (Todd JA, et al. Immunity. 2001; 15:387-395; Larsson K, et al. Diabetes Metab Res Rev. 2004; 20:429-437). Type 1 diabetes can be caused by genetic defects, viruses, and other auto-immune problems. For example, type 1 diabetes develops following a viral infection such as mumps, rubella, cytomegalovirus, measles, influenza, encephalitis, polio or Epstein-Barr virus. Type 1 diabetes can also be caused by injury to the pancreas from toxins, trauma, or after the surgical removal of the majority (or all) of the pancreas.

[0046] The methods of the present invention are equally effective in treating individuals affected by infantile-, juvenile- or adult-onset type 1 diabetes. The methods of the present invention are equally effective in treating type 1 diabetes caused by genetic defects, various viruses, injury to the pancreas and other auto-immune problems.

Treatment of type 1 diabetes based on B-cell depletion strategies

[0047] B cells normally produce antibodies against foreign antigens. B cells bear autoantibody Ig-receptors are present in normal individuals. Autoimmunity results when these B-cells become overactive, and mature to plasma cells that secrete autoantibody. In accordance with the present invention, B-cell depletion can effectively treat type 1 diabetes.

[0048] As used herein, the term "B-cell depletion" refers to selectively reducing B cell circulating levels or functions. The term "reducing" as used herein, indicate values that are relative to a baseline measurement, such as a measurement in the same individual prior to initiation of the B-cell depletion treatment described herein, or a measurement in a control individual (or multiple control individuals) in the absence of the B-cell depletion treatment described herein. A "control individual" is an individual afflicted with the same form of type 1 diabetes (either infantile, juvenile or adult-onset) as the individual being treated, who is about the same age as the individual being treated (to ensure that the stages of the disease in the treated individual and the control individual(s) are comparable). For example, B-cell depletion includes killing mature B cells, inhibiting B-cell differentiation or blocking one or more B cell functions. B-cell depletion includes both incomplete or partial B-cell depletion and complete B-cell depletion. B-cell depletion strategies suitable for the invention can be achieved by administering B-cell depleting agents described below.

[0049] As discussed below in the Examples section, the present invention showed for the very first time that B-cell depletion can effectively cure type 1 diabetes based on the studies done in nonobese diabetic (NOD) mice, an animal model for human autoimmune type 1 diabetes. In particular, the present invention showed that B-cell depletion is capable of preventing or delaying diabetes onset, restoring normoglycemia, and establishing long-term tolerance towards autoantigens.

[0050] In particular, as described in the Examples section, the present invention showed that 100% of newly diabetic mice were successfully treated with a B-cell-depleting agent, and 70% of the treated animal maintained long-term sustained normoglycemia, with the appearance of tolerance toward autoantigens. Interestingly, the B-cell-depleted mice resemble normal mice in histology and blood glucose levels, suggesting considerable restoration of B-cell function. Importantly, after the complete recovery of B cells, NOD mice remained protected in the diabetes prevention studies and remained normoglycemic in the reversal study.

[0051] Moreover, the present studies showed for the very first time that re-emerging B cells in NOD mice displayed a more anergic phenotype compared to naϊve B cells and are less capable of activating T cells in the presence of autoantigen compared to naϊve B cells. Without wishing to be bound by any theories, it is contemplated that the B-cell depletion strategies according to the present invention not only deplete B cells but also change their immunological properties. As a result, it is contemplated that reconstitution of the B-cell compartment following B-cell depletion according to the present invention can purge the pool of autoreactive B cells.

[0052] As discussed in the Examples section, it was observed that B-cell-depleted NOD mice that became tolerant towards autoantigen showed an increased percentage of CD4⁺CD25⁺FoxP3⁺ cells, which resume the phenotype of regulatory T cells. This was detected both in the prevention studies (in the pancreatic lymph nodes (PLn)) and in the reversal of hyperglycemia (in PLn and spleen). The present invention showed that the absence of B cells reduces autoreactive T cell proliferation. Without wishing to be bound by any theories, it was contemplated that a persistent interaction of B cells and autoreactive T cells may be important in maintaining the autoimmune response. For example, B cells may sustain autoreactive T cells through the release of soluble factor (e.g., lymphotoxin) or through cell-to-cell interaction.

[0053] The present invention also showed that the re-emerging B cells display a regulatory phenotype. The adoptive transfer studies described below showed that re-emerging B cells can change the phenotype of autoreactive T cells by reducing the production of proinflammatory cytokines and can abrogate the transfer of diabetes in NOD. SCID by diabetogenic CD4⁺ cells. This regulatory role from re-emerging B cells was also confirmed by histological studies, which showed the absence of any T cells islets infiltrate in absence of islets, and even when B cells recovered, B and T cells remained confined to the islet border without infiltrating insulin-producing cells. Regarding the reversal studies, the present invention discovered surprisingly that B-cell depletion cleans islets of infiltrates faster than other therapies. Even the anti-CD3 strategy appeared to be slower than B-cell depletion of the present invention in restoring normoglycemia. For example, 20% of anti-CD3 treated animals did not revert from hyperglycemia. In the present studies, 100% of hyperglycemic NOD mice reverted to normoglycemia in 2-3 days.

[0054] In general, it is understood by scientists that a cure for type 1 diabetes requires the reestablishment of immunological tolerance. Prior to the present invention, most of the scientists believed that the ideal targets were T cells. The present invention showed that targeting B cells may provide a sufficient signal to instruct either B or T cells toward tolerance. In addition, based on the animal studies, the present invention showed that the diabetic patients may retain sufficient β cell mass in the short term, which can be rejuvenated or regenerated to reverse disease upon dampening of autoimmunity.

[0055] Thus, the present invention showed for the very first time that B-cell depletion can effectively cure diabetes by restoring normoglycemia in new-onset hyperglycemic patients and that a B-cell depletion strategy according to the present invention (such as an anti-CD22 therapy) can not only deplete B cells but modify the immunological properties of B- and T-cells. The present strategy provides an ultimate tolerogenic protocol for diabetes that applies to both healthy individuals at high risk for developing Tl D or patients with overt Tl D. [0056] In some embodiments, the present invention provides a method for treating patients with new-onset type 1 diabetes based on B-cell depletion strategies. In some embodiments, the method of the invention is used to treat patients with new-onset type 1 diabetes that retain about 10% of their normal β cell mass at diagnosis.

[0057] In some embodiments, a B-cell depletion strategy contemplated herein can be used in combination with immunosuppressive drugs. In particular, the B-cell depletion strategy can be used with the transient use of immunosuppressive drugs, especially, in already diabetic patients. In some embodiments, a B-cell-depletion strategy may be used in combination with an agent that targets T cells, such as Rapamycin or cytotoxic T-lymphocyte-associated antigen 4 immunoglobulin (CTLA4-Ig), or an islet-regenerating agent (e.g. , exendin-4), to treat patients whose diabetes onset is not recent. Additional T-cell targeting or islet-regenerating agents suitable for the invention are well-known in the art.

[0058] The present invention also provides a therapeutic strategy for individuals at risk for developing diabetes (such as those individuals identified by genetic markers or the presence of high-affinity islet autoantibodies). For example, the B-cell depletion strategies according to the invention can be used to prevent or delay the onset of type 1 diabetes in individuals at risk for developing diabetes. In some embodiments, partial or incomplete B-cell depletion strategies are used to prevent or delay the onset of type 1 diabetes in individuals at risk for developing diabetes. In some embodiments, the B-cell depletion strategies according to the invention can be used to prevent or delay the onset of type 1 diabetes in individuals with high risk for developing diabetes.

[0059] In some embodiments, the present invention provides methods for inducing T-cell tolerance and/or halting immune- mediated destruction of pancreatic islets in type 1 diabetes patients based on B-cell depletion strategies. In some embodiments, the present invention provides methods for inducing T-cell tolerance in patients who have received islet transplantation. Transplanted islets face either allo- or autoimmune response in the host. B-cell depletion strategies according to the invention can prolong islet allograft survival. In some embodiments, the B-cell depletion strategies of the invention can be used in combination with T- cell targeting agents.

[0060] Without wishing to be bound by any theories, it is contemplated that the use of B cells, rather than T cells, depleting strategy in diabetes can be potentially advantageous because of the virtual absence of the cytokine release syndrome associated with virtually any T-cell depleting strategy, which leads to a dramatic release of inflammatory cytokines such as IL-2, TNF-α, and IFN-γ secondary to lympholysis and T-cell activation (Herold KC, et al. N Engl J Med. 2002; 346: 1692-1698).

B-cell depleting agents

[0061] B-cell depletion can be achieved by administering B-cell depleting agents. As used herein, the term "B-cell depleting agent" refers to any agent that reduces B cell circulating levels in an organism or that reduces or interferes with the activity of B cells in an organism. For example, suitable B cell depleting agents include, but are not limited to, antibodies, cytotoxins, peptides, small molecules, nucleic acids, and other B-cell antagonists. In particular, B cell depleting agents include antibodies that selectively binds to a B-cell antigen including, but not limited to, the CD22, CD20, CD 19, and CD74 or HLA-DR antigen.

[0062] As used herein, the term "antibody" is intended to include immunoglobulins and fragments thereof which are specifically reactive to the designated protein or peptide, or fragments thereof. An antibody can include human antibodies, primatized antibodies, chimeric antibodies, bispecific antibodies, humanized antibodies, conjugated antibodies (i.e., antibodies conjugated or fused to other proteins, radiolabels, cytotoxins), and antibody fragments. As used herein, the term "antibody" also includes intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.

[0063] As used herein, an "antibody fragment" includes a portion of an intact antibody, such as, for example, the antigen-binding or variable region of an antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; triabodies; tetrabodies; linear antibodies; single-chain antibody molecules; and multi specific antibodies formed from antibody fragments. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex. For example, antibody fragments include isolated fragments, "Fv" fragments, consisting of the variable regions of the heavy and light chains, recombinant single chain polypeptide molecules in which light and heavy chain variable regions are connected by a peptide linker ("sFv proteins"), and minimal recognition units consisting of the amino acid residues that mimic the hypervariable region.

[0064] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins), as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al. Proc. Natl. Acad. ScL USA 1984; 81:6851-6855; Oi et al. Biotechnologies 1986: 4G):214-221; and Liu et al. Proc. Natl. Acad ScL USA 1987: 84:3439-43).

[0065] As used herein, "humanized" or "CDR grafted" forms of non-human (e.g., murine) antibodies are human immunoglobulins (recipient antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are also replaced by corresponding non-human residues (so called "back mutations"). Furthermore, humanized antibodies may be modified to comprise residues which are not found in the recipient antibody or in the donor antibody, in order to further improve antibody properties, such as affinity. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al. Nature 1986; 321:522-525; and Reichmann et al. Nature 1988; 332:323-329.

[0066] As used herein, "single-chain Fv" or "sFv" antibody fragments comprise the V_H and V_L domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the V_H and V_L domains which enables the sFv to form the desired structure for antigen binding. See, Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer- Verlag, New York, pp. 269-315 (1994).

[0067] As used herein, the term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (V_H) connected to a light chain variable domain (V_L) in the same polypeptide chain (V_H - V_L). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al. Proc. Natl. Acad. ScL USA 1993; 90:6444-6448.

[0068] As used herein, the term "triabodies" refers to the combination of three single chain antibodies. Triabodies is also known as "trivalent trimers." Triabodies are constructed with the amino acid terminus of a V_L or V_H domain, i.e., without any linker sequence. A triabody has three Fv heads with the polypeptides arranged in a cyclic, head-to-tail fashion. A possible conformation of the triabody is planar with the three binding sites located in a plane at an angle of 120 degrees from one another. Triabodies can be monospecific, bispecific or trispecific.

[0069] As used herein, the term "tetrabodies" refers to a complex including four antigen- binding domains, where the four antigen-binding domains may be directed towards the same or different epitopes. Tetrabodies are constructed with the amino acid terminus of a V_L or V_H domain, i.e., without any linker sequence. A tetrabody can be combination of three single chain antibodies.

[0070] As used herein, the term "linear antibodies" refers to these antibodies including a pair of tandem Fv segments (V_H-C_HI- V_H-C_HI) which form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific. Details are described in Zapata et al. Protein Eng. 1995; 8(10): 1057-1062.

[0071] Anti-CD20, anti-CD22, anti-CD19, anti-CD74, anti-HLA-DR and other antibodies against B cells are known generally to those of skill in the art. See, for example, Ghetie et al. Cancer Res. 1988; 48:2610; Hekman et al. Cancer Immunol. Immunother. 1991; 32:364; Kaminski ^ α/. N. Engl. J. Med. 1993; 329:459; Press et al. N. Engl. J. Med. 1993; 329: 1219; Maloney et al. Blood 1994; 84:2457; Press et al. Lancet 1995; 346:336; Longo, Curr. Opin. Oncol. 1996; 8:353. More particularly, monoclonal antibodies to CD22, CD20, CD 19, or CD74 antigens can be obtained by methods known to those skilled in the art. See generally, for example, Kohler et al. Nature 1975; 256:495, and Coligan et al. (eds.), CURRENT PROTOCOLS IN IMMUNOLOGY. VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons 1991) ["Coligan"].

Anti-CD22 antibodies [0072] In particular embodiments, anti-CD22 antibodies are used for the method of the present invention. CD22 is an inhibitory co-receptor that down-modulates BCR signaling and functions as a molecular switch, determining whether antigen-stimulated B cells undergo apoptosis or proliferation (Nitschke L, et al Trends Immunol. 2004; 25:543-550). CD22 regulates B cell homeostasis and survival, the promotion of BCR- induced cell cycle progression, and is a potent regulator of CD40 signaling. Unlike other B-cell markers, CD22 membrane expression is limited to the late differentiation stages of mature B cells. Anti-CD22 treatment may be particularly useful to deplete mature B cells and to modify immunoproperties of B-cells. Thus, anti-CD22 strategies have the potential advantage of blocking B-cell function without inducing complete B-cell depletion. This is particularly important for delaying or preventing diabetes onset in subjects at risk of developing Tl D, in whom blocking B-cell depletion without complete depleting is desirable.

[0073] Anti-CD22 antibodies can be generated using standard methods known in the art.

For example, suitable amounts of well-characterized antigen for production of antibodies can be obtained using standard techniques. As an example, CD22 can be immunoprecipitated from B- lymphocyte protein using the deposited antibodies described by Tedder et al, U.S. Pat. No. 5,484,892 (1996). Alternatively, CD22 antigen proteins can be obtained from transfected cultured cells that overproduce the antigen of interest. Expression vectors that comprise DNA molecules encoding CD22 can be constructed using published nucleotide sequences. See, for example, Wilson et al J. Exp. Med. 1991; 173: 137; Wilson et al J. Immunol. 1993; 150:5013). As an illustration, DNA molecules encoding CD22 can be obtained by synthesizing DNA molecules using mutually priming long oligonucleotides. See, for example, Ausubel et al, (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, pages 8.2.8 to 8.2.13 (1990) ["Ausubel"]. Also, see Wosnick et al Gene 1987; 60: 115; and Ausubel et al (eds.), SHORT PROTOCOLS IN MOLECULAR BIOLOGY, 3rd Edition, pages 8-8 to 8-9 (John Wiley & Sons, Inc. 1995). DNA molecules encoding CD22 can also be obtained using the polymerase chain reaction (Adang et al Plant Molec. Biol. 1993 ; 21 : 1131 ; Bambot et al PCR Methods and Applications 1993; 2:266; Dillon et al METHODS IN MOLECULAR BIOLOGY. Vol. 15: PCR PROTOCOLS: CURRENT METHODS AND APPLICATIONS, White (ed.), pages 263- 268, (Humana Press, Inc. 1993)).

[0074] Monoclonal antibodies can be generated using various methods well known in the art. For example, monoclonal antibody can be obtained by fusing myeloma cells with spleen cells from mice immunized with a murine pre-B cell line stably transfected with cDNA which encodes a CD22 antigen of interest. See Tedder et al, U.S. Pat. No. 5,484,892 (1996). [0075] Suitable anti-CD22 antibodies include any antibodies that bind to one or more epitopes of CD22. For example, suitable anti-CD22 antibodies include monoclonal antibodies that bind CD22 epitopes such as epitope A, epitope B, epitope C, epitope D or epitope E. See, for example, Schwartz-Albiez et al., "The Carbohydrate Moiety of the CD22 Antigen Can Be Modulated by Inhibitors of the Glycosylation Pathway," in LEUKOCYTE TYPING IV. WHITE CELL DIFFERENTIATION ANTIGENS, Knapp et al. (eds.), P- 65 (Oxford University Press 1989). One example of a suitable murine anti-CD22 monoclonal antibody is the LL2 (formerly EPB-2) monoclonal antibody, which was produced against human Raji cells derived from a Burkitt lymphoma. Pawlak-Byczkowska et al. Cancer Res. 1989: 49:4568. The LL2 antibody binds with epitope B (Stein et al. Cancer Immunol. Immunother. 1993; 37:293).

[0076] Additional exemplary anti-CD22 antibodies suitable for the invention include, but are not limited to, anti-CD22 monoclonal antibodies designated HB22-7, HB22-23, HB22-33, HB22-5, HB22-13, and HB22-196. These antibodies are disclosed in U.S. Pat. No. 5,484,892, Tuscano et al. Eur. J. Immunol. 1996; 26: 1246, and Tuscano et al. Blood 1999; 94(4), 1382- 1392, the teachings of which are hereby incorporated by reference. In addition, HB22-7 and HB22-23 are available from the American Type Culture Collection (ATCC), 12302 Parklawn Drive, Rockville, Md. 20852, under Accession Nos. HB22347 and HBl 1349, respectively.

[0077] Anti-CD22 antibodies suitable for the present invention include human and non- human anti-CD22 antibodies. Non-human anti-CD22 antibodies include, but are not limited to, anti-CD22 antibodies obtained from mouse, rat, rabbit, pig, monkey, horse, dog, cat. For example, anti-mouse CD22 monoclonal antibody purified from Cy34.1 hybridoma (American Type Culture Collection [ATCC], Rockville, MD) (hereinafter, monoclonal antibody Cy34.1) may be used for the present invention.

[0078] In some embodiments, an anti-CD22 antibody suitable for the present invention is a chimeric antibody in which the variable regions of a human antibody have been replaced by the variable regions of a non-human (e.g., a rodent) anti-CD22 antibody. The advantages of chimeric antibodies include decreased immunogenicity and increased in vivo stability.

[0079] Techniques for constructing chimeric antibodies are well known to those of skill in the art. As an example, Leung et al. Hvbridoma 1994; 13:469, describe how they produced an LL2 chimera by combining DNA sequences encoding the V_kappa and V_H domains of LL2 monoclonal antibody with respective human .kappa, and IgGl constant region domains. This publication also provides the nucleotide sequences of the LL2 light and heavy chain variable regions, V_ka_PPa and V_H, respectively. Similar techniques can be used to construct a Cy34.1 chimera.

[0080] In another embodiment, an anti-CD22 antibody suitable for the present invention is a subhuman primate antibody. General techniques for raising therapeutically useful antibodies in baboons may be found, for example, in Goldenberg et al. , international patent publication No. WO 91/11465 (1991), and in Losman et al Int. J. Cancer 1990; 46: 310.

[0081] In yet another embodiment, an anti-CD22 antibody suitable for the present invention is a "humanized" monoclonal antibody. That is, mouse complementarity determining regions are transferred from heavy and light variable chains of the mouse immunoglobulin into a human variable domain, followed by the replacement of some human residues in the framework regions of their murine counterparts. General techniques for cloning murine immunoglobulin variable domains are described, for example, by the publication of Orlandi et al Proc. Nat'l Acad. ScL USA 1989; 86: 3833. Techniques for producing humanized monoclonal antibodies are described, for example, by Jones et al. Nature 1986; 321 :522, Riechmann et al. Nature 1988; 332:323, Verhoeyen ef α/. Science 1988: 239: 1534. Carter et al Proc. Nat'l Acad. ScL USA 1992; 89:4285, Sandhu, Crit. Rev. Biotech. 1992; 12:437, and Singer et al. J. Immun. 1993; 150:2844. The publication of Leung et al. MoI. Immunol. 1995; 32: 1413, describes the construction of humanized LL2 antibody. Similar techniques can be used to construct humanized Cy34.1.

[0082] In another embodiment, an anti-CD22 antibody suitable for the present invention is a human monoclonal antibody. Such antibodies are obtained from transgenic mice that have been "engineered" to produce specific human antibodies in response to antigenic challenge. In this technique, elements of the human heavy and light chain locus are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy chain and light chain loci. The transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody-secreting hybridomas. Methods for obtaining human antibodies from transgenic mice are described by Green et al. Nature Genet. 1994; 7: 13, Lonberg et al. Nature 1994; 368:856, and Taylor et al. Int. Immun. 1994; 6:579.

[0083] As discussed above, anti-CD22 antibodies also encompass antibody fragments.

The antibody fragments are antigen binding portions of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, and the like. The antibody fragments bind to the same antigen that is recognized by the intact antibody. For example, an anti-CD22 monoclonal antibody fragment binds to an epitope of CD22.

Cytotoxic agents

[0084] In some embodiments, a B-cell depleting agent, such as an antibody against the

CD22, CD20, CD 19, CD74 or HLA-DR antigen as described above, is conjugated or fused to a cytotoxic agent. Suitable cytotoxic agents include, but are not limited to, drugs, toxins, enzymes, hormones, cytokines, immunomodulators, boron compounds and therapeutic radioisotopes. These conjugates and fusion proteins may be used alone, or in combination with naked B-cell antibodies. Alternatively, an antibody used can include an arm that is specific for a low- molecular weight hapten to which a therapeutic agent is conjugated or fused. In this case, the antibody pretargets the B-cells, and the low-molecular weight hapten with the attached therapeutic agent is administered after the antibody has bound to the B-cell targets. Examples of recognizable haptens include, but are not limited to, chelators, such as DTPA, fluorescein isothiocyanate, vitamin B- 12 and other moieties to which specific antibodies can be raised.

[0085] Drugs which are known to act on B-cells, plasma cells and/or T-cells are particularly useful in accordance with the present invention, whether conjugated to a B-cell antibody, or administered as a separate component in combination with a naked or conjugated B- cell antibody. These include, but are not limited to, methotrexate, phenyl butyrate, bryostatin, cyclophosphamide, etoposide, bleomycin, doxorubicin, carmustine, vincristine, procarbazine, dexamethasone, leucovorin, prednisone, maytansinoids such as DMl, calicheamicin, rapamycin, leflunomide, FK506, immuran, fludarabine, azathiopine, mycophenolate, cyclosporine, thiotepa, taxanes, vincristine, daunorubicin, doxorubicin, epirubicin, actinomycin, authramycin, azaserines, bleomycins, tamoxifen, idarubicin, dolastatins/auristatins, hemiasterlins, maytansinoids, esperamicins, FK506 and derivatives thereof. For example, B-cell depleting agents can be conjugated with calicheamicins or calicheamicin derivatives. Exemplary calicheamicin derivatives include, but are not limited to, gamma calicheamicin, N-acetyl calicheamicin, a disulfide analog of calicheamicin, N-acetyl gamma calicheamicin dimethyl hydrazide (N-acetyl calicheamicin DMH), or N-acetyl-calicheamicin dimethyl acid, a member of the enedyne antitumor antibiotic family (Dunussi-Joannopoulos K, et al. Blood. 2005; 106:2235-2243).

[0086] In particular, an anti-mouse CD22 monoclonal Ig and N-acetyl-calicheamicin dimethyl acid (referred to as "CD22/cal mAb") conjugate is used for the present invention. Upon binding to CD22- expressing murine B cells, the conjugate is internalized and exhibits potent dose-dependent cytotoxicity due to DNA damage caused by calicheamicin (Damle NK, et al. Curr Opin Pharmacol. 2003; 3:386-390). The immunoconjugate dramatically increases the direct cytotoxic effect of anti-CD22 treatment.

[0087] Exemplary toxins suitable for the present invention include, but are not limited to, ricin, abrin, ribonuclease, DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtherin toxin, Pseudomonas exotoxin, Pseudomonas endotoxin and RNAses, such as onconase. See, for example, Pastan et al. Cell 1986; 47:641, and Goldenberg, CA. Cancer Journal for Clinicians 1994; 44:43.

[0088] Other suitable cytotoxic agents are known to those of skill in the art.

[0089] Methods for making conjugated antibodies are well known in the art. For example, exemplary methods for making calicheamicin conjugates, such as anti-CD 22/calicheamicin conjugates, are described in U.S. publication nos. 20040192900, 20060002942, and 20070213511, the teachings of all of which are hereby incorporated by reference.

[0090] For example, a B-cell depleting agent (e.g., an anti-CD22 antibody) can be covalently attached to any number of calicheamicin molecules. The number of calicheamicin moieties covalently attached to an antibody is also referred to as drug loading. For example, the average loading suitable for the present invention can be anywhere from about 0.1 to about 10 or about 15 calicheamicin moieties per antibody. In particular, the average loading can be about 0.1 to about 5, about 1.0 to about 10, about 5 to about 10, about 5 to about 15, about 1.0 to about 4.0, or about 1.2 to about 2.6 calicheamicin per antibody. A given population of conjugates (e.g., in a composition or formulation) can be either heterogeous or homogenous in terms of drug loading. In a heterogeneous population, since average loading represents the average number of drug molecules (or moles) conjugated to an antibody, the actual number of drug moieties per antibody can vary substantially.

Administration of B-cell depleting agents

[0091] In the methods of the invention, B-cell depleting agents are typically administered to the individual alone, or in compositions or medicaments comprising the B-cell depleting agents as described herein. The compositions can be formulated with a physiologically acceptable carrier or excipient to prepare a pharmaceutical composition. A component is considered to be a "pharmaceutically acceptable carrier" if its administration can be tolerated by a recipient patient. Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions (e.g., NaCl), saline, buffered saline (e.g., phosphate-buffered saline), alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, sugars such as mannitol, sucrose, or others, dextrose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrolidone, etc., as well as combinations thereof. The pharmaceutical preparations can, if desired, be mixed with auxiliary agents (e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like) which do not deleteriously react with the active compounds or interference with their activity. In a preferred embodiment, a water-soluble carrier suitable for intravenous administration is used. Other suitable carriers are well-known to those in the art. See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed. (1995). The carrier and composition can be sterile. The formulation should suit the mode of administration.

[0092] The composition or medicament, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. The composition can also be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrollidone, sodium saccharine, cellulose, magnesium carbonate, etc.

[0093] The antibodies, alone or conjugated to liposomes, can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the therapeutic proteins are combined in a mixture with a pharmaceutically acceptable carrier.

[0094] The composition or medicament can be formulated in accordance with the routine procedures as a pharmaceutical composition adapted for administration to human beings. For example, in a preferred embodiment, a composition for intravenous administration typically is a solution in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose/water. Where the composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[0095] The B-cell depleting agents can be formulated as neutral or salt forms.

Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

[0096] Control release preparations can be prepared through the use of polymers to complex or adsorb the antibody. For example, biocompatible polymers include matrices of poly(ethylene-co-vinyl acetate) and matrices of a polyanhydride copolymer of a stearic acid dimer and sebacic acid. Sherwood et al. Bio/Technology 1992; 10: 1446. The rate of release of an antibody from such a matrix depends upon the molecular weight of the protein, the amount of antibody within the matrix, and the size of dispersed particles. Saltzman et al. Biophys. J. 1989; 55: 163; Sherwood et al, supra. Other solid dosage forms are described in REMINGTON'S PHARMACEUTICAL SCIENCES, 19th ed. (1995).

[0097] B-cell depleting agents (or a composition or medicament containing B-cell depleting agents) is administered by any appropriate route. For example, administration of B- cell depleting agents (e.g. , antibodies) to a patient can be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, by perfusion through a regional catheter, or by direct intralesional injection. When administering therapeutic proteins by injection, the administration may be by continuous infusion or by single or multiple boluses. Intravenous injection provides a useful mode of administration due to the thoroughness of the circulation in rapidly distributing antibodies. More than one route can be used concurrently, if desired.

[0098] B-cell depleting agents (or composition or medicament containing B-cell depleting agents) can be administered alone, or in conjunction with other therapeutic agents. For example, B-cell depleting agents may be administered in conjunction with therapeutics that are targeted against T-cells, plasma cells or macrophages, such as antibodies directed against T-cell epitopes, more particularly against the CD4 and CD5 epitopes. Gamma globulins also may be co-administered. In some cases, it may be desirable to co-administer immunosupproessive drugs such as corticosteroids and possibly also cytotoxic drugs. In this case, lower doses of the corticosteroids and cytotoxic drugs can be used as compared to the doses used in conventional therapies, thereby reducing the negative side effects of these therapeutics. The supplemental therapeutic compositions can be administered before, concurrently or after administration of the B-cell depleting agents.

[0099] Other useful secondary therapeutics useful in conjunction with B-cell depleting agents are cytokines, such as IL-2, GM-CSF, tumor necrosis factor alpha (TNF _α) and interleukin- 1 (IL-I), cytokine agonists and antagonists, such as anti-TNF_α reagents (e.g., Infiximab and Etanercept (Embrel)) and anti-IL-1 reagents.

[0100] The term, "in conjunction with," indicates that the agent is administered prior to, at about the same time as, or following the B-cell depleting agents (or composition containing B- cell depleting agents). For example, the agent can be mixed into a composition containing B-cell depleting agents, and thereby administered contemporaneously with the B-cell depleting agents; alternatively, the agent can be administered contemporaneously, without mixing (e.g., by "piggybacking" delivery of the agent on the intravenous line by which the B-cell depleting agents is also administered, or vice versa). In another example, the agent can be administered separately (e.g., not admixed), but within a short time frame (e.g., within 24 hours) of administration of the B-cell depleting agents.

[0101] For purposes of therapy, B-cell depleting agents (e.g., antibodies) are administered to a patient in a therapeutically effective amount. As used herein, a "therapeutically effective amount" is a dosage amount that, when administered at regular intervals, is sufficient to reduce the B-cell levels in circulation by inactivating or killing B-cells, or sufficient to treat type 1 diabetes. As used herein, the term "treat" or "treatment" refers to ameliorating one or more symptoms associated with the disease, for example, reducing blood glucose levels or ameliorating one or more symptoms associated with hyperglycemia, in some cases, restoring normoglycemia, preventing or delaying the onset of type 1 diabetes, or establishing tolerance towards autoantigens.

[0102] The terms, "improve," "increase" or "reduce," as used herein, indicate values that are relative to a baseline measurement, such as a measurement in the same individual prior to initiation of the treatment described herein, or a measurement in a control individual (or multiple control individuals) in the absence of the treatment described herein. A "control individual" is an individual afflicted with the same form of type 1 diabetes (either infantile, juvenile or adult- onset) as the individual being treated, who is about the same age as the individual being treated (to ensure that the stages of the disease in the treated individual and the control individual(s) are comparable).

[0103] The individual (also referred to as "patient" or "subject") being treated is an individual (fetus, infant, child, adolescent, or adult human) having type 1 diabetes or at risk for developing type 1 diabetes.

[0104] In general, the dosage of administered antibodies will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition and previous medical history. Typically, it is desirable to provide the recipient with a dosage of antibody component, immunoconjugate or fusion protein which is in the range of from about 1 pg/kg to 10 mg/kg (amount of agent/body weight of patient), although a lower or higher dosage also may be administered as circumstances dictate.

[0105] The dose which will be therapeutically effective for the treatment of the disease will also depend on the nature and extent of the disease's effects, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges, such as those exemplified below. The precise dose to be employed will also depend on the route of administration, and the seriousness of the disease, and should be decided according to the judgment of a practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. The therapeutically effective dosage amount can be, for example, 20 milligrams to 2 grams protein per dose. In particular, the therapeutically effective dosage amount of conjugated anti-CD22 antibodies can range from about 20 to about 1000 milligrams protein per dose, or from about 20 to about 500 milligrams protein per dose, or from 20 to about 100 milligrams protein per dose.

[0106] The effective dose for a particular individual can be varied (e.g., increased or decreased) over time, depending on the needs of the individual.

[0107] The therapeutically effective amount of B-cell depleting agents (or composition or medicament containing B-cell depleting agents) is administered at regular intervals, depending on the nature and extent of the disease's effects, and on an ongoing basis. Administration at an "interval," as used herein, indicates that the therapeutically effective amount is administered periodically (as distinguished from a one-time dose). The interval can be determined by standard clinical techniques. In preferred embodiments, B-cell depleting agents is administered bimonthly, monthly, twice monthly, triweekly, biweekly, weekly, twice weekly, thrice weekly, or daily. The administration interval for a single individual need not be a fixed interval, but can be varied over time, depending on the needs of the individual.

[0108] As used herein, the term "bimonthly" means administration once per two months

(i.e., once every two months); the term "monthly" means administration once per month; the term "triweekly" means administration once per three weeks (i.e., once every three weeks); the term "biweekly" means administration once per two weeks (i.e., once every two weeks); the term "weekly" means administration once per week; and the term "daily" means administration once per day.

[0109] The invention additionally pertains to a pharmaceutical composition comprising

B-cell depleting agents, as described herein, in a container (e.g., a vial, bottle, bag for intravenous administration, syringe, etc.) with a label containing instructions for administration of the composition for treatment of type 1 diabetes, such as by the methods described herein.

Identification of differentially expressed genes or proteins in re-emerging B cells

[0110] The present invention also encompasses the finding that differential gene expression analysis, such as transcriptome analysis, of re-emerging B cells showed significant changes of expression of certain genes, in particular, proinflammatory genes. Differentially expressed genes or proteins are likely to be involved in re-programming B cells. Thus, differentially expressed genes or proteins in re-emerging B cells can be used as biomarkers for diagnosis and/or prognosis of diabetes and related diseases, disorders or conditions. In some embodiments, certain differentially expressed genes or proteins can be used as biomarkers to indicate the risk of developing diabetes and other related diseases, disorders and conditions. In some embodiments, certain differentially expressed genes or proteins can be used as biomarkers to indicate patients' responses to a therapy or treatment of diabetes and other related diseases, disorders and conditions. In some embodiments, certain differentially expressed genes or proteins can be used to predict likely outcomes of a treatment or therapy. In some embodiments, certain differentially expressed genes or proteins can also be used as biomarkers to test the effectiveness of a particular therapy or treatment (e.g., a new drug, dose, and/or dosing schedule). In some embodiments, a new therapy can be provided or recommended based on the expression or activity of certain differentially expressed genes or proteins. Additionally or alternatively, differentially expressed genes in re-emerging B cells can be used as therapeutic targets for the treatment of diabetes and related diseases, disorders or conditions. [0111] Differentially expressed genes can be identified by comparing the expression profiles between treated and untreated B cells. For example, the gene expression profile of re- emerging B cells (e.g., obtained from a normoglycemic diabetic patient treated in accordance with the present invention) can be compared with that of B cells before the treatment (e.g., naive normoglycemic B cells before the onset of diabetes) and/or with that of hyperglycemic untreated control B cells (e.g., B cells obtained from a patient or subject suffering from diabetes). Treated and untreated B cells can be obtained from humans and other animal models such as mice, rats, guinea pigs, etc. Methods of preparing B cells are well known in the art and exemplary methods are described in the Examples section.

[0112] Methods suitable for differential gene expression profiling analysis are well known in the art. Exemplary methods include, but are not limited to, transcriptome analysis (also referred to as high-coverage gene expression profiling, HiCEP), DNA microarrays, quantitative RT-PCR and northern hybridization. Exemplary transcriptome analysis is described in the Examples section.

[0113] Methods used to detect the hybridization profile of target nucleic acids with oligonucleotide probes are well known in the art. In particular, means of detecting and recording fluorescence of target nucleic acid-oligonucleotide probe hybrid have been well established and are well known in the art, for example, as described in U.S. Pat. No. 5,631,734, U.S. Publication No. 20060010513, incorporated herein in their entirety by reference. In some embodiments, a confocal microscope can be controlled by a computer to automatically detect the hybridization profile of the entire array.

[0114] It will be appreciated by one of skill in the art that evaluation of the hybridization profile is dependent on the composition of the array, i.e., which oligonucleotide probes were included for analysis. For example, where the array includes oligonucleotide probes to consensus sequences only, or consensus sequences and transgene sequences only, (i.e., the array does not include control probes to normalize for variation between experiments, samples, stringency requirements, and preparations of target nucleic acids), the hybridization profile is evaluated by measuring the absolute signal intensity of each location on the array. Alternatively, the mean, trimmed mean (i.e., the mean signal intensity of all probes after 2-5% of the probe sets with the lowest and highest signal intensities are removed), or median signal intensity of the array may be scaled to a preset target value to generate a scaling factor, which will subsequently be applied to each probeset on the array to generate a normalized expression value for each gene (see, e.g., Affymetrix (2000) Expression Analysis Technical Manual, pp. A5-14). Conversely, where the array further comprises control oligonucleotide probes, the resulting hybridization profile is evaluated by normalizing the absolute signal intensity of each location occupied by a test oligonucleotide probe by means of mathematical manipulations with the absolute signal intensity of each location occupied by a control oligonucleotide probe. Typical normalization strategies are well known in the art, and are included, for example, in U.S. Pat. No. 6,040,138 and Hill et al. Genome Biol. 2001; 2(12):research0055.1-0055.13.

[0115] Signals gathered from oligonucleotide arrays can be analyzed using commercially available software, such as those provide by Affymetrix or Agilent Technologies. Controls, such as for scan sensitivity, probe labeling and cDNA or cRNA quantitation, may be included in the hybridization experiments. The array hybridization signals can be scaled or normalized before being subjected to further analysis. For instance, the hybridization signal for each probe can be normalized to take into account variations in hybridization intensities when more than one array is used under similar test conditions. Signals for individual target nucleic acids hybridized with complementary probes can also be normalized using the intensities derived from internal normalization controls contained on each array. In addition, genes with relatively consistent expression levels across the samples can be used to normalize the expression levels of other genes.

[0116] The present invention also provides methods for identifying differentially expressed proteins by protein expression profiling analysis. Protein expression profiles can be generated by any method permitting the resolution and detection of proteins from a sample from a subject. Methods with higher resolving power are generally preferred, as increased resolution can permit the analysis of greater numbers of individual proteins, increasing the power and usefulness of the profile. A sample can be pre-treated to remove abundant proteins from a sample, such as by immunodepletion, prior to protein resolution and detection, as the presence of an abundant protein may mask more subtle changes in expression of other proteins, particularly for low-abundance proteins. A sample can also be subjected to one or more procedures to reduce the complexity of the sample. For example, chromatography can be used to fractionate a sample; each fraction would have a reduced complexity, facilitating the analysis of the proteins within the fractions.

[0117] Useful methods for simultaneously resolving and detecting several proteins include, but are not limited to, array-based methods; mass-spectrometry based methods; and two- dimensional gel electrophoresis based methods. [0118] Protein arrays generally involve a significant number of different protein capture reagents, such as antibodies or antibody variable regions, each immobilized at a different location on a solid support. Such arrays are available, for example, from Sigma-Aldrich as part of their Panorama™ line of arrays. The array is exposed to a protein sample and the capture reagents selectively capture the specific protein targets. The captured proteins are detected by detection of a label. For example, the proteins can be labeled before exposure to the array; detection of a label at a particular location on the array indicates the detection of the corresponding protein. If the array is not saturated, the amount of label detected may correlate with the concentration or amount of the protein in the sample. Captured proteins can also be detected by subsequent exposure to a second capture reagent, which can itself be labeled or otherwise detected, as in a sandwich immunoassay format.

[0119] Mass spectrometry-based methods include, for example, matrix-assisted laser desorption/ionization (MALDI), Liquid Chromatography/Mass Spectrometry/Mass Spectrometry (LC -MS/MS) and surface enhanced laser desorption/ ionization (SELDI) techniques. For example, a protein profile can be generated using electrospray ionization and MALDI. SELDI, as described, for example, in U.S. Patent No. 6,225,047, incorporates a retention surface on a mass spectrometry chip. A subset of proteins in a protein sample are retained on the surface, reducing the complexity of the mixture. Subsequent time-of-flight mass spectrometry generates a "fingerprint" of the retained proteins.

[0120] In methods involving two-dimensional gel electrophoresis, proteins in a sample are generally separated in a first dimension by isoelectric point and in a second dimension by molecular weight during SDS-PAGE. By virtue of the two dimensions of resolution, hundreds or thousands of proteins can be simultaneously resolved and analyzed. The proteins are detected by application of a stain, such as a silver stain, or by the presence of a label on the proteins, such as a Cy2, Cy3, or Cy5 dye. To identify a protein, a gel spot can be cut out and in-gel tryptic digestion performed. The tryptic digest can be analyzed by mass spectrometry, such as MALDI. The resulting mass spectrum of peptides, the peptide mass fingerprint or PMF, is searched against a sequence database. The PMF is compared to the masses of all theoretical tryptic peptides generated in silico by the search program. Programs such as Prospector, Sequest, and MasCot (Matrix Science, Ltd., London, UK) can be used for the database searching. For example, MasCot produces a statistically -based Mowse score indicates if any matches are significant or not. MS/MS can be used to increase the likelihood of getting a database match. CID-MS/MS (collision induced dissociation of tandem MS) of peptides can be used to give a spectrum of fragment ions that contain information about the amino acid sequence. Adding this information to a peptide mass fingerprint allows Mascot to increase the statistical significance of a match. It is also possible in some cases to identify a protein by submitting only a raw MS/MS spectrum of a single peptide.

[0121] Exemplary expression profiling analysis is described in details in the Examples section. Exemplary genes or proteins differentially expressed in re-emerging B cells are shown in Figure 6 and Table 1. One of skill in the art would readily appreciate that nucleotide and/or amino acid sequences associated with the genes or proteins shown in Figure 6 and Table 1 may be retrieved from public sequence data bases (e.g., Genbank) using appropriate gene names identified herein. The nucleotide and/or amino acid sequences associated with each of the genes and/or proteins listed in Figure 6 and Table 1 available in Genbank as of the filing date of the present application are hereby incorporated by reference in their entireties.

[0122] The present invention further contemplates methods and compositions that may be used to detect the expression and/or the activity of the genes or proteins corresponding to differentially expressed genes or proteins identified herein. For example, the expression and/or activity of differentially expressed genes or proteins can be detected using nucleic acid hybridization or antibody staining (e.g., Western blotting or immunohistochemistry) using various methods known in the art. The expression and/or activity of differentially expressed genes or proteins can also be detected using relevant biological activity assays known in the art.

[0123] The present invention further contemplates methods and compositions that may be used to alter (i.e., regulate (e.g., enhance, reduce, or modify)) the expression and/or the activity of the genes or proteins corresponding to the genes or proteins differentially expressed in B-cells identified herein. Altered expression in a cell or organism may be achieved through down-regulating or up-regulating of a gene or protein of interest. For example, a differentially expressed B cell sequence may be down-regulated by the use of various inhibitory polynucleotides, such as antisense polynucleotides, ribozymes that bind and/or cleave the mRNA transcribed from the gene of interest, triplex-forming oligonucleotides that target regulatory regions of the target gene, and short interfering RNA that causes sequence-specific degradation of target mRNA (e.g., Galderisi et al. J. Cell. Physiol. 1999; 181:251-57; Sioud, Curr. MoI. Med. 2001; 1:575-88; Knauert ef α/. Hum. MoI. Genet. 2001; 10:2243-51; Bass. Nature 2001; 411 :428-29). As another example, a differentially expressed B cell sequence may be down- regulated by the use of various inhibitory antibodies, other exogenous agents such as small molecules, pharmaceutical compounds, or other factors that may be directly or indirectly modulating the activity of the gene or protein of interest. [0124] The present invention, thus generally described, will be understood more readily by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention.

EXAMPLES

Example 1. Animal model for autoimmune type 1 diabetes

[0125] Nonobese diabetic (NOD) mice, an animal model for autoimmune type 1 diabetes were used in the experiments described herein. Female NOD and NOD. SCID mice of various ages were obtained from The Jackson laboratory and cared for in accordance with institutional guidelines. BDC2.5 TCR transgenic mice on the NOD genetic background were purchased from the Jackson Laboratory and bred under specific pathogen-free conditions. Mice were used according to appropriate guidelines. Protocols were approved by the Institutional Animal Care and Use Committee.

Example 2. Anti-CD22/calicheamicin immunoconjugate

[0126] The immunoconjugate (referred here as anti-CD22/cal mAb) is a conjugate of an anti-mouse CD22 mAb and N-acetyl-calicheamicin dimethyl acid, a member of the enediyne antitumor antibiotics. Anti-mouse CD22 is a mouse IgGl mAb purified from Cy34.1 hybridoma (American Type Culture Collection [ATCC], Rockville, MD). Anti-CD22/cal mAb has an average loading of 17 to 30 g calicheamicin/mg antibody protein (i.e., 1.2-2.6 moles calicheamicin/mol antibody). On binding to CD22-expressing mouse B cells, the conjugate is internalized and exhibits potent dose-dependent cytotoxicity due to DNA damage caused by calicheamicin (Dunussi-Joannopoulos K, et al. Blood. 2005; 106:2235-2243). A mouse IgGl anti-rat very late antigen 4 (VLA-4) mAb (does not bind on mouse cells) conjugated to calicheamicin (GG5/cal) was used as a control in the in vitro and in vivo cytotoxicity studies.

Example 3. Monitoring for Diabetes

[0127] Clinical diabetes was defined as blood glucose levels >250 mg/dL for three consecutive days. Blood glucose was measured by Accu-Chek Advantage glucometers (Roche Diagnostics). After any treatments, mice were monitored daily by measuring blood glucose for 3 weeks, followed by three times a week until the mice were killed.

Example 4. Flow cytometry (FACS) [0128] Rat anti-mouse CD 19 PE, CD20 PE, CD22 PE, B220 PE, B220 PerCP, CD80

(B7-1) PE, CD86 (B7-2) PE, H-2^d FITC, CD4 FITC, CD23 FITC, CD25 PE, CD44 PE, CD45 FITC, CD62L APC, CD93 PE (CIqRp) and IgM PAPC, were purchased from BD Pharmingen and eBiosciences (San Diego, CA). FoxP3 APC was purchased from eBiosciences (San Diego, CA). Cells recovered from spleens and peripheral lymphoid tissues were subjected to FACS analysis and were run on a FACSCalibur™ (Becton Dickinson, San Jose, CA). Data were analyzed using FIoJo software version 6.3.2 (Treestar, Ashland, OR). The level of maturation of B cells was determined by FAGS analysis by examining the expression of CD80 and 86. B-cell anergic phenotype was determined at FACS using quadruple staining (for B220 CD93 CD23^" IgM¹⁰ cells), as recently shown (Merrell KT BR, et al. Immunity. 2006;25:953-962) FoxP3 analysis was performed following overnight permeabilization of cells extracted from spleens and peripheral lymphoid tissue using commercially available antibodies and gating on CD4⁺CD25⁺ cells.

Example 5. Pancreata digestion and lymphocytes extraction

[0129] The pancreata were collected in cold HBSS medium supplemented with 10%

FBS, cut into small pieces, and pressed through a 70-pm cell strainer. After washing, mononuclear leukocytes were separated from the other cells by density-gradient centrifugation using Histopaque 1077 (Sigma, St Louis, MO). Following centrifugation for 30 min at 2500 rpm, the interphase containing the mononuclear cells was removed and the cells were washed twice with Ca²⁺/Mg²⁺-free PBS.

Example 6. Islet pathology and immunohistochemistry

[0130] Immunohistochemistry was performed using 5 -micron-thick formalin-fixed, paraffin-embedded tissue sections. Briefly, slides were soaked in xylene, passed through graded alcohols, and put in distilled water. Slides were then pre-treated with 10-mM citrate, pH 6.0 (Zymed, South San Francisco, CA) or with 1 mM EDTA (pH 8.0) in a steam pressure cooker (Decloaking Chamber, BioCare Medical, Walnut Creeek, CA) as per manufacturer's instructions, followed by washing in distilled water for antigen retrieval. All further steps were performed at room temperature in a hydrated chamber. Slides were pretreated with Peroxidase Block (DAKO USA, Carpinteria, CA) for 5 minutes to quench endogenous peroxidase activity. The following primary antibodies were used (including company, clone/reference, dilution, and retrieval method, respectively): anti-CD3 (Cell Marque, CMC363, 1: 1500, EDTA), anti-CD45/ 8220 (BD Pharmingen, #550286, 1:200, citrate), anti-FoxP3 (eBioscience, #14-5773, 1:25, citrate), anti-insulin (DAKO, N 1542, undilute, EDTA), and anti-glucagon (Abeam, Ab 18461, 1 :50, EDTA). All primary antibodies were applied to slides in DAKO diluent for 1 hour. Slides were then washed in 50-mM Tris-CI, pH 7.4, and the appropriate horseradish peroxidase- coniugated secondary antibody (Envision detection kits, DAKO) was applied for 30 minutes. After further washing, immunoperoxidase staining was developed using a DAB chromogen kit (DAKO) per the manufacturer and counterstained with hematoxylin. Photomicrographs were taken on an Olympus BX41 microscope (Center Valley, PA) at indicated magnifications using an Olympus Q-color5 digital camera and analyzed with Adobe Photoshop Elements 2.0 (San Jose, CA).

Example 7. Insulitis score

[0131] Insulitis scoring was performed on hematoxylin- and eosin-stained pancreatic sections. A score from 0 to 4 was assigned based to islet infiltration as previously described⁴⁰. The sections were stained for Hematoxylin and Eosin; at least 30 islets per group were analyzed, pooled from different mice. Insulitis score was graded as follows: grade 0, normal islets; grade 1, mild mononuclear infiltration (<25%) at the periphery; grade 2, 25-50% of the islet infiltrated; grade 3, more than 50% of the islet infiltrated; grade 4, islet completely infiltrated with no residual parenchyma left.

Example 8. In vitro generation of bone marrow (BM)-derived dendritic cells (DC)

[0132] DC were isolated by culturing murine BM cells from NOD or BALB/c mice.

Briefly, the femurs and tibiae of mice were flushed, and the cells were seeded in Petri dishes at a density of 2 x 10⁶/ml of RPMI- 1640 medium supplemented with 20 ng/ml of recombinant murine granulocyte-monocyte colony stimulating factor (rmGM-CSF, R&D Systems, Minneapolis, MN). At days 3, 6, and 8, additional medium and rmGM-CSF were added. On day 9, non-adherent cells were either harvested for analysis or allowed to mature with the addition of lipopolysaccharide (LPS) to the culture medium.

Example 9. In vitro autoreactive T cells functional assay

[0133] In vitro functional assays were used to challenge CD4⁺ cells extracted from B- cell-depleted and control NOD mice with BDC2.5 peptide in an ELISpot assay to determine IFN-γ production. This assay allowed us to evaluate the immune response of CD4⁺ cells compartment toward autoantigen and the eventual positive effects of different treatments. CD4⁺ were extracted from B-cell-depleted and control NOD mice with magnetic microbeads (Miltenji Inc., Upsala) and then plated in 64 wells with irradiated BM-derived DC from NOD mice and BDC2.5 peptide (an islet peptide) in an ELISpot assay. Purity of more than 95% was assessed by FACS analysis (data not shown). A purity of 99% was obtained for gene expression profile using multiple columns and isolations with the magnetic device (data not shown).

Example 10. ELISpot

[0134] To measure IFN-γ production by CD4⁺ cells extracted from B-cell-depleted and control NOD mice, we used our ELISpot assay as previously described (Makhlouf L, et al. Diabetes. 2002; 51:3202-3210). Briefly, Millipore immunospot plates (Millipore Corporation, Bedford, MA) were coated with capture antibodies conjugated to murine IFN-γ (BD Biosciences). Plates are blocked with 1% bovine serum albumin (BSA) to prevent non-specific binding, and stimulators and responders are added to plates. BM-derived DC from NOD were used as stimulators, while NOD CD4⁺ cells extracted from B-celldepleted and control mice were used as responders. Along with BDC2.5 peptide, 5xlO⁵ cells each of stimulators and responders were added to each well. All stimulators were irradiated with 3000 rads to render them unable to proliferate. The capacity of syngeneic DC to stimulate responders is also evaluated to exclude autoproliferation, and 10 μg/ml con A is used to stimulate cells as a positive control. The capacity of peptide to stimulate CD4⁺ cell proliferation was previously tested in a titration assay, and the optimal concentration identified in our lab (data not shown). After 24 (for IFN-γ) of incubation at 37° C and 5% CO₂, plates were washed, and biotinylated antibodies specific for each cytokine were added to wells and incubated at 4° C for 24 hours. Plates were then washed and incubated at room temperature with streptavidin-horseradish peroxidase (HRP) for 2 hours and developed using aminoethyl carbazole (AEC, Sigma Aldrich) diluted in N,N- dimethylformamide. Spots were counted on an immunospot analyzer (Cellular Technology Ltd., Cleveland, OH). In another ELISpot experiment, IxIO⁵ NOD CD19⁺ cells from treated or untreated NOD mice were used as stimulators of 1x10⁵ CD4 cells from BDC2.5 mice in the presence of 15 ng/ml BDC2.5 peptide for 24h. The supernatant of each culture was used for Luminex analysis as well.

Example 11. Rt-PCR

[0135] RNA extraction was performed using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol; RNA was then reverse-transcribed to synthesize 60 μl of cDNA and 250 ng of cDNA; 10 μl of SYBR Green master mix (Applied Biosystems, Foster City, CA) and 250 nmol of sense and anti-sense primer were used in a 20 μl QPCR reaction. Primers were designed with Primer Express software (sequences shown below). QPCR reaction conditions were as follows: 50⁰C for 2 minutes, 95°C for 10 minutes, then 40 cycles of 95°C for 15 seconds, and 60⁰C for 1 minute. For each reaction, emitted fluorescence was measured during the annealing/extension phase. The calculated number of copies was divided by the number of copies of the housekeeping gene GAPDH.

Example 12. Adoptive cell transfer

[0136] Two settings of adoptive transfer have been set up: (i) adoptive transfer of re- emerging or naive B cells in combination with diabetogenic T cells to evaluate B-cell regulatory function. For adoptive transfer studies, recipients were adult 6- to 8-week-old NOD-SCID mice, injected intravenously with the required type of cells (splenocytes or CD4⁺/CD19⁺ cells), diabetes onset was monitored in the following weeks.

[0137] Diabetogenic CD4⁺ cells extracted from hyperglycemic NOD mice were adoptively transferred alone or in combination with re-emerging or naive CD19⁺ cells (B cells) into NOD. SCID hosts. Naϊve B cells were extracted from both normoglycemic and hyperglycemic NOD mice, while re-emerging B cells were extracted from B-cell-depleted NOD mice after the recovery of the B-cell pool.

[0138] Mice receiving 1OxIO⁶ splenocytes from control mice with recent onset Tl D served as methodological controls. For the CD4⁺ and CD19⁺ cell adoptive transfer, 5xlO⁶ diabetogenic CD4⁺ cells were co-adoptively transferred with the same amount of CD19⁺ cells into NOD. SCID mice. While CD4⁺ cells were extracted from hyperglycemic female NOD mice, CD19⁺ cells were extracted from normoglycemic 10-week-old female NOD mice, from hyperglycemic >14- week-old female NOD mice, and from B-cell-depleted NOD mice once B cells were recovered. Diabetes onset was monitored in the following weeks. We also analyzed the percentage of CD4⁺CD25⁺FoxP3⁺ cells in the spleen of NOD.SCID recipients of diabetogenic CD4⁺ T cells and re-emerging B cells or controls (B cells from hyperglycemic animals or no cells) at day 30 post-adoptive transfer.

Example 13. In vivo tracking of autoreactive T cells

[0139] For in vivo tracking of autoreactive T cells, we used CD4⁺ cells from the BDC2.5 mice, in which autoreactive CD4⁺ cells can be easily tracked using the anti-ideotypic antibody against Vβ4 chain of the TCR receptor. CD4⁺ cells were adoptively transferred in a NOD.SCID host with splenocytes extracted from NOD mice freshly depleted of B cells. Then after 72 hours cells were recovered, autoreactive T cells were counted, and apoptosis or proliferation of CD4⁺Vβ4⁺ autoreactive T cells was evaluated.

Example 14. Preparation of CFSE-labeled cells

[0140] For in vivo tracking, cells were labeled with CFSE before adoptive transfer. To label cells with the CFSE dye, cells were washed with PBS, pelleted, and resuspended at Ix 10⁶ cells/ml in PBS containing 5% FCS. The CFSE dye (Sigma) [1OmM stock] was added to the cell suspension to a final concentration of 5 μM. The cells were incubated for 6 minutes at 37°C to allow labeling of the cells and washed and resuspended in PBS/5% FCS or re-plated in culture media. CFSE dilution was considered a marker of cell proliferation.

Example 15. Apoptosis detection

[0141] Apoptotic cells were quantified using dual Annexin V-FITC/7AAD staining with

FACS analysis. After extraction, cells were stained with Annexin V-PE (BD Pharmingen) and 7AAD-Perpc (BD Pharmingen) according to the manufacturer's directions and analyzed by flow cytometry. Cells stained with Annexin V-FITC and negative for PI were considered apoptotic.

Example 16. Mixed lymphocyte reaction (MLR)

[0142] To compare immunocompetence of B-cell-depleted and control NOD mice, an

MLR assay was performed. Cultured BM-derived DC from BALB/c were used after irradiation with 3000 rads to stimulate CD4⁺ NOD cells isolated from splenocytes obtained from B-cell- depleted and control NOD mice by magnetic bead separation (Miltenyi Biotec, Auburn, CA) at a ratio of 1 : 1 DC-splenocytes. Proliferation was measured at day 3 of incubation at 37° C and 5% CO₂ following pulsing with ^[3H]TdR (Perkin Elmer, Wellesley, MA) using a liquid scintillation counter. Splenocytes were also stimulated with con A as a positive control.

Example 17. Luminex serum cytokine determination

[0143] B-cell depleted and control age-matched NOD mice were bled via tail perforation at selected times (0, 2, 4, 6, 8, and 10 weeks) after injection in the prevention studies (every other week up to 10 weeks after injection) or at 0, 10, and 100 days after injection in the reversal studies, and serum samples were collected. Serum from in vitro studies (ELISpot assay) was also collected to analyze cytokine profile. Samples were subjected to automated calculation using a MASCOT Hemavet 850 CBC Analyzer (Drew Scientific, Dallas, TX). The resulting sera were subjected to cytokine analysis with IL-lα, IL-I β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IL- 10, IL-12p70, IL- 15, IL- 17, IFN-γ, and TNF-α using the Lincloplex platform (Linco, St. Louis, MO), as previously described (Goudy KS, et al. J Immunol. 2003; 171:2270-2278). Briefly, supernatant samples were incubated overnight at room temperature with beads conjugated to the aforementioned cytokines; matched biotinylated reporters were added and incubated for 1.5 hours, and streptavidin-phycoerythrin solution was incubated with samples for 30 minutes. Following addition of stop solution, sample cytokine levels were calculated from a standard curve using a LuminexlOO reader from Luminex Corporation (Austin, TX).

Example 18. RNA amplification and hybridization on Illumina beadchip mouseόyl. l

[0144] The integrity of RNA was evaluated using an Experion™ (Bio-RAD). 100 ng total RNA was used to generate cRNA by the use of Illumina TotalPrep RNA Amplification Kit (Ambion, Austin TX). Reverse transcription was performed with the T7 oligo (dT) primer to obtain first-strand cDNA, which then underwent second-strand synthesis and RNA degradation by DNA Polymerase and RNase H followed by clean up. In vitro transcription (IVT) technology along with biotin UTP was employed to generate multiple copies of biotinylated cRNA. The labeled cRNA was purified via Filter Cartridge and quantified by NanoDrop and RiboGreen^® (Molecular Probes Inc. Ugene, OR).

[0145] The labeled cRNA target (1.5 μg each) was used for hybridization to each array according to Illumina Sentrix beadchip array mouse-6vl.l protocol. A maximum of 10 μl cRNA was mixed with 20 μl GEX-HYB hybridization solution. The preheated 30 μl assay sample was dispensed onto the large sample port of each array and was incubated for 18 hours at 58 ⁰C at a rocker speed of 5. Following hybridization, the samples were washed according to the protocol and scanned with a BeadArray Reader (Illumina, San Diego, CA).

Example 19. Beadarray data analysis

[0146] The bead array data was analyzed by Bead Studio Version 2 (Illumina). The differential expression of genes between the treated group and control group and between the treated group and hyper group was obtained after rank invariant normalization. The criteria for this analysis is differing score less than -13 or more than 13, which is corresponding to P value less than 0.05. The heat map of differential expression genes was generated by Multiple Array Viewer software after data standardization.

Example 20. Autoantibody evaluation (IAA assay) [0147] Autoantibodies have been shown to be associated with diabetes onset; we evaluated serum antibodies in NOD mice during treatment in collaboration with the Barbara Davis Center in Colorado. Briefly, IAA (IA2) were measured with a standard IAA radioassay⁴²'⁴³ utilizing competition with unlabeled insulin with 600 μl of sera per determination (150 μl duplicates with and without unlabeled insulin). After 7-day incubation at 4°C, antibody- antigen complexes were precipitated with polyethylene glycol 8000, and the results were calculated as the difference between the tube without cold insulin and the tube with cold insulin and were expressed as nU/ml.

Example 21. Statistical Analyses

[0148] Data are expressed as mean±standard error. Kaplan-Meier analysis was used for survival analysis. ANOVA (for parametric data) and Kruskal-Wallis test (for non-parametric data) were used. Chi-square test for categorical variables was used when necessary. When the 2 groups were compared cross-sectionally, two-sided unpaired Student t-test (for parametric data) or Mann-Whitney test (for non-parametric data) were used according to distribution. A P value of less than 0.05 (by two-tailed testing) was considered an indicator of statistical significance. Analysis of data was done using an SPSS statistical package for Windows (SPSS Inc., Chicago, Illinois). Prism software was used for drawing graphs (GraphPad Software, Inc., San Diego, CA).

Example 22. CD22 is widely expressed on mature B cells in NOD mice

[0149] This experiment is designed to exam if CD22 is expressed by the B-cell compartment in NOD mice. Splenocytes from 10-week-old female NOD mice were extracted and analyzed by flow cytometry the expression of CD 19 and CD22 on B220⁺ cells. No differences were observed in terms of CD 19 and CD22 expression on B220⁺ cells, suggesting that CD22 is widely expressed by B cells and that CD22 is a good target for a B-cell-depleting approach (B220⁺CD19⁺ cells=82.0±2.5 vs. B220⁺CD22⁺ cells=83.1±2.7 %, ns), (Figure Ia-Ib). CD22 appeared to be expressed on CD138⁺ cells (namely plasmacells).

Example 23. B cells represent the majority of infiltrating cells in the pancreata of NOD mice

[0150] This experiment was designed to examine the presence of infiltrating cells in the pancreata of 4-, 8-, and 12-week-old female NOD mice, and hyperglycemic female NOD mice (>14 weeks) by flow cytometry. BALB/c mice age-matched were used as further control. Pancreata from NOD or BALB/c mice at different ages were extracted as described above. A collagenase digestion of the pancreata with a final lymphocyte Percoll extraction were performed. Lymphocytes were then stained for different T- and B-cell markers. An abundant infiltration of CD45⁺CD19⁺ cells (B cells) were observed in the pancreata of 10-week-old NOD mice as compared to BALB/c age-matched mice (average CD19⁺ cells out of CD45⁺ cells: NOD=55.1±12.3 vs. BALB/c=15.9±8.9% p<0.05) (Figure Ic and data not shown). Few cells can be recovered after pancreas digestion in the BALB/c mice, due to the absence of infiltrating cells. However, most of them appeared to be CD45⁺CD3⁺ cells (T cells) (average CD3⁺ cells out of CD45⁺ cells NOD=45.4±5.7 vs. BALB/c=76.3±14.9% p<0.05) (Figure Id and data not shown), and very few were B cells (CD45 CD19⁺ cells). Interestingly, the kinetics of CD45⁺CD19⁺ (B cells) infiltration in the pancreas of NOD mice showed an increase around 8 to 10 weeks, when the mice began to exhibit islet peri-infiltration (from 22.8±7.8 at 4 weeks up to 65.1±5.0% at 8 weeks of age, p<0.01), (Figure Ic). We did not detect any further increase of B cells when the NOD mice became hyperglycemic (Figure Ic). This increase was not observed for CD45⁺CD3⁺ cells (T cells) (Figure Ie).

Example 24. Anti-CD22/cal mAb produces a profound depletion of B cells in NOD mice

[0151] There is no information in the literature on whether NOD mice can be B-cell- depleted. NOD mice were treated with anti-CD22/cal mAb to evaluate whether the antibody can successfully deplete B cells in NOD mice. Two injections (160 μg i.p. 5 days apart, D0-D5) of anti-CD22/cal mAb elicited a quick and profound depletion of B cells in the peripheral blood of 10-week-old NOD mice. The effect took one week and lasted for 5 to 7 weeks (Figures If and Ih). Control NOD mice did not appear to be depleted of B cells (Figures If and Ig), while the group treated with unconjugated anti-CD22 showed transient and partial B-cell depletion (Figures If and Ii). At 8-10 weeks after depletion, B cells had recovered almost completely (Figures If and Ih). Interestingly, the depletion appeared to be general and consistent in both lymphoid organs and in pancreas (data not shown). Finally, other cellular subsets such as T-cell compartment (CD3, CD4, and CD8 percentages) were comparable among controls and those treated with either anti-CD22/cal mAb or anti-CD22 unconjugated (data not shown). No B-cell depletion was evident by using immunotoxin-calicheamicin alone (data not shown).

Example 25. B-cell depletion slightly affected autoantibody production

[0152] Autoantibodies (e.g., IA2) can be associated with diabetes onset and has been associated with shortened islet allograft survival in human patients⁴⁴. However, their role has not been totally clarified. This experiment evaluated the effect of B cell depletion on autoantibody levels (IA2) in the serum of NOD mice during follow-up in prevention studies. In the control group, 4 of 8 NOD mice showed detectable IA2 levels at baseline (10 weeks of age), as did 2 of 8 in the B-cell-depleted group. Both NOD mice positive for autoantibodies in the B-cell- depleted group showed a reduction in IA2 levels, with disappearance of B-cell depletion after 10 weeks (data not shown), while only one NOD mouse out of 4 positive for autoantibodies in the control group had significantly reduced IA2 levels (data not shown).

Example 26. B-cell depletion with the anti-CD22/cal mAb prevents diabetes onset in prediabetic NOD mice

[0153] The effect of anti-CD22/cal mAb treatment on diabetes onset was evaluated in female 10-week-old NOD mice. The primary goal of the treatment was maintenance of normoglycemia in the long-term (up to 25 weeks after injection), with the achievement of a state of stable tolerance towards autoantigens. NOD mice were treated with 2 injections of 160 μg of anti-CD22/cal mAb 5 days apart and monitored for diabetes development (defined as blood glucose persistently higher than 250 mg/dl for 3 consecutive days). As shown in Figure 2a, we observed a significant delay in diabetes onset in B-cell-depleted mice (n=20 mice) compared to untreated controls (n=30 mice, p<0.01) and the group treated with calicheamicin alone (n=10 mice, p<0.01). Fifty percent of female 10-week-old NOD mice whose B cells had been depleted with anti-CD22/cal mAb appeared to be protected in the long term (25 weeks after injection) and became tolerant towards autoantigen, compared to fewer than 10% of controls (Figure 2a). Interestingly, no mice became hyperglycemic during the period in which B cells were completely depleted (Figure 2a). Administration of calicheamicin alone (n=10 mice) was ineffective in delaying or protecting from diabetes, and diabetes progression in the calicheamicin group was similar to that in untreated controls (Figure 2a).

[0154] 10-week-old female NOD mice were also treated with the unconjugated anti-

CD22 mAb that only partially depletes B cells (n=20 mice) (Figures If). Mice were treated with 2 injections 5 days apart to mimic the same protocol used for the anti-CD22/cal mAb. Diabetes onset was slightly delayed compared to controls (p=0.06, Figure 2a); however long-term protection was achieved in fewer than 20% of mice compared to 10% of control mice.

Example 27. B-cell depletion in prediabetic mice is associated with an increase in CD4 CD25 FoxP3⁺ cells percentage in the pancreatic lymph nodes (PLn)

[0155] This experiment was designed to examine the effect of B-cell depletion in NOD mice from a mechanistic point of view. In particular, two time points were considered: 5 weeks after the injection, when B cells are still depleted, and 25 weeks after the depletion, when B cells have recovered completely. Flow cytometry analysis were performed to compare T cell phenotype between B-cell-depleted vs. control NOD mice. CD4/CD8 effector cells and CD4/CD8 regulatory T cells were analyzed in spleens and PLn. No major differences were observed in terms of CD4 or CD8 effector T cells; particularly, CD4⁺CD44⁺CD62L^lc and CD8⁺CD44⁺CD62L^lc percentages were similar.

[0156] Five weeks after the injection, effector T cells were similar in the PLn (for example, CD4 effectors: B-cell-depleted =4.0±1.4 vs. control=7.0±1.7%, ns; CD8 effectors: B- cell-depleted =3.2±1.3 vs. control=3.1±0.1%, ns) and in the spleen (CD4 effectors: B-cell- depleted =15.3±0.5 vs. control=16.9±1.9%, ns; CD8 effectors: B-cell-depleted =9.1±1.1 vs. control=8.8±2.1%, ns) between B-cell-depleted and untreated control NOD mice (data not shown).

[0157] Twenty-five weeks after injection, an increase in CD4/CD8 effector T cells was evident in the spleen but not in the PLn (data not shown) of B-cell-depleted compared to untreated controls NOD mice. Five weeks after the depletion, CD4⁺CD25⁺FoxP3⁺ cells appeared to be slightly reduced in the spleen (B-cell-depleted =5.0±0.2 vs. control=6.1±0.1%, p=0.02), while in PLn, CD4⁺CD25⁺FoxP3⁺ cells appeared similar in B-cell-depleted and control NOD mice (B-cell-depleted =4.9±0.1 vs. control=4.2±0.1%, p=0.05 data not shown). At twenty- five weeks after treatment, no differences were evident for CD4⁺CD25⁺FoxP3⁺ cells in the spleen (data not shown). However, in the PLn, a significant increase in the percentage of CD4 CD25 FoxP3⁺ cells (Tregs) is evident compared to either hyperglycemic and normoglycemic control NOD mice (B-cell-depleted=20.3±3.1 vs. Normoglycemic control=8.1±0.6, p=0.02 and vs. Hyperglycemic control=7.7±3.1 %, p=0.009, Figure 2b).

Example 28. Hvporesponsiveness of CD4⁺ cells towards autoantigen in B-cell-depleted NOD mice

[0158] NOD CD4⁺ T cells have been shown to produce IFN-γ when stimulated with

BDC2.5 peptide and syngeneic DC. IFN-γ production can be determined by ELISpot and can be considered an index of the T cell anti-islet response. This experiment was designed to determine whether B cell depletion can modify BDC2.5 peptide-driven IFN-γ production of T cells and thus whether it can modify the anti-islet response in NOD mice. CD4⁺ cells extracted from B- cell-depleted and control NOD mice in the prevention studies were isolated 5 and 25 weeks after depletion and were challenged with the BDC2.5 peptide in an ELISpot assay to evaluate IFN-γ production. Only after 25 weeks, but not 5 weeks after depletion, CD4⁺ cells extracted from splenocytes of B-cell-depleted mice produced less significant IFN-γ when challenged with the BDC2.5 peptide in an ELISpot assay compared to age-matched controls (Figure 2c).

Example 29. Lack of B cells reduces proliferation of autoreactive T cells

[0159] This experiment was designed to track the effect of B-cell depletion on survival/apoptosis and proliferation of autoreactive CD4⁺ T cells by using a fluorescently-labeled tetramer of the Ag⁷/2.5mi complex. 2.5 mi is an MHC-mimetic peptide with high agonistic affinity for the diabetogenic clone, BDC2.5. Ag⁷ is the lone MHC class II molecule of NOD mice. This tetramer labels a BDC2.5 TCR-specific autoreactive population in the islets and in the periphery of the NOD mouse, allowing us to determine the effect of B-cell depletion on the behavior of such cells (i.e. their expansion, activation, and apoptosis) (Katz JD, et al. Cell. 1993 ;74: 1089-1100; Masteller EL, et al. J Immunol. 2005; 175:3053-3059; Tang Q, et al. Nat Immunol. 2006; 7:83-92).

[0160] NOD. SCID mice were reconstituted with the same number of splenocytes from normoglycemic NOD 10-week-old mice. After 7 days (allowing reconstitution of the immune system), mice were either treated with anti-CD22/cal mAb or not treated. After another 7 days, in order to allow ample time for B cell depletion, isolated BDC2.5 TCR Tg⁺ CD4⁺ cells were labeled with CFSE and transferred into B-cell-depleted or untreated NOD. SCID mice. After 72 hours, mice were euthanized and examined for autoreactive CD4⁺ cells (easily tracked using the anti-ideotypic antibody against Vγ4 chain of the TCR receptor) survival/apoptosis and proliferation (based on CFSE dilution) (Tang Q, et al. Nat Immunol. 2006; 7:83-92).

[0161] Interestingly, when B cells are absent, fewer BDC2.5 TCR Tg⁺ CD4⁺ cells can be recovered from the host (-50%) (Figure 2d). This parallels an increase of autoreactive T-cell apoptosis (data not shown) and is associated with a higher proliferation of BDC2.5 TCR Tg⁺ CD4⁺ cells in the control NOD. SCID host (Figures 2e and 2f, lower quadrant) compared to the B-cell-depleted NOD mice (Figures 2e and 2f, upper quadrant). This experiment indicates a crucial role of B cells in autoreactive T-cell survival and proliferation and suggests that B-cell depletion can reduce autoreactive T cells proliferation in vivo.

Example 30. CD4⁺ cells from B cell-depleted mice are immunocompetent in the long-term

[0162] To evaluate the immunocompetence of B-cell-depleted NOD mice and to exclude the possibility that the observed effects are related to a state of long-term immunosuppression rather than tolerance toward autoantigens, CD4⁺ cells extracted from B-cell-depleted and untreated control NOD mice were challenged with BALB/c BM-derived DC in an MLR assay. CD4⁺ cells extracted with magnetic beads from splenocytes obtained from a B-cell-depleted NOD mice were capable of mounting an immune response to alloantigen similar to that found in the control NOD mice at both 5 and 25 weeks after the depletion (data not shown), suggesting that CD4⁺ cells are immunocompetent.

Example 31. Five weeks after B-cell depletion: islets appeared free from infiltrates and had well-preserved morphology

[0163] Sections of pancreatic islets from 10-week-old normoglycemic NOD mice and

15-week-old normoglycemic treated and untreated control NOD mice were fixed in formalin and analyzed. Hematoxylin & eosin showed that at baseline (10 weeks of age) NOD mice started to show some mild perinsulitis (Figure 3al) with many B220⁺ cells (Figure 3a2) and some CD3⁺ cells (Figure 3a3) on the islets' borders, but still with well-preserved insulin (Figure 3a5) and glucagon (Figure 3a6) staining. Fopx3⁺ cells can be barely detected at 10 weeks of age in the pre-diabetic NOD mice (Figure 3a4). Interestingly, 5 weeks after injections in the B-cell- depleted NOD mice, infiltrate was reduced compared to untreated control NOD mice (Figures 3bl and 3cl). B220⁺ cells were completely absent in the B-cell-depleted NOD mice (Figure 3b2), while in the controls, B220⁺ cells were abundant inside the islets (Figure 3c2). Surprisingly, very few CD3⁺ cells can be detected in the B-cell-depleted NOD mice and they were confined to islet borders (Figure 3b3). On the contrary, in the untreated control NOD mice, islets appeared to be greatly infiltrated by clusters of CD3⁺ cells (Figure 3c3). This is despite the fact that B-cell-depleted animals are not depleted of CD3⁺ cells. Insulin (Figures 3b5 and 3c5) and glucagon (Figures 3b6 and 3c6) staining were performed to evaluate islet structure. In both groups (B-cell-depleted and untreated control NOD mice) islets appeared to be preserved (particularly in the B-cell-depleted group), with some signs of insulin loss in the untreated control group (Figure 3c5). Five weeks after injection in the B-cell-depleted NOD mice, an increase in FoxP3⁺ cells was evident within the islets compared to untreated control NOD mice (Figures 3b4 and 3c4). If we consider the ratio between CD3⁺ and Foxp3⁺ cells, a great imbalance towards a reduction in Foxp3⁺ cells is evident in the untreated controls but not in the B-cell-depleted group.

Example 32. After 25 weeks from B-cell depletion: islets remained free of infiltrate despite the complete recovery of the B-cell pool [0164] Twenty-five weeks after treatment (35 weeks of age), despite the complete recovery of the B-cell pool in the originally B-cell-depleted group, pancreatic islets appeared to contain much less infiltrate than untreated control hyperglycemic NOD mice (Figures 3dl and 3el). Again, neither the B220⁺ nor the CD3⁺ cells were infiltrating the islets but remain at their border in the B-cell-depleted group (Figures 3d2 and 3d3). In the B-cell-depleted group, T cells appeared to be incapable of infiltrating the islets and remained confined to the borders, threatening but not injuring islet cells (Figure 3d3). Conversely, in the hyperglycemic age- matched untreated control NOD mice, islets are massively infiltrated by B220⁺ and CD3⁺ cells (Figures 3e2 and 3e3). Islet morphology, as evaluated by insulin and glucagon staining, confirmed the presence of many well-preserved islets in the B-cell-depleted group (Figures 3d5 and 3d6). Again, the control group showed degenerated islets with virtually no insulin or glucagon staining (Figures 3e5 and 3e6). Foxp3 staining of pancreatic islets revealed reduced Foxp3 expression, particularly if compared with the massive presence of T cells in the untreated control NOD mice (Figure 3d4-3e4). Finally, insulitis score in B-cell-depleted and untreated control NOD mice at different points after injection were calculated (Figure 2g). More well- preserved islets (0-50% of infiltration) were evident in the B-cell-depleted compared to untreated control NOD mice 5 and 25 weeks after injections (Figure 2g).

Example 33. B-cell depletion restores normoglycemia in newly hyperglycemic NOD mice

[0165] Newly hyperglycemic NOD mice (defined on the basis of glucose levels higher than 250 mg/dl for 3 consecutive days) were treated with a protocol identical to that outlined above using the anti-CD22/cal mAb. A rapid reversal of hyperglycemia (within 2 days) was observed in all the B-cell-depleted NOD mice (10 out of 10; Figure 4a). Six out of 10 remained normoglycemic in the long term (for more than 100 days). Three mice remained normoglycemic for 20-40 days and then reverted to hyperglycemia (Figure 4a). One mouse remained normoglycemic for more than 50 days and then reverted to hyperglycemia (Figure 4a). None of the control NOD mice (n=10 mice) ever reverted from hyperglycemia to normoglycemia spontaneously after 3 consecutive days of hyperglycemia (Figure 4b).

[0166] When hyperglycemic NOD mice were treated with anti-CD22/cal mAb after 5 days (n=6 mice) from hyperglycemia onset a transient return to normoglycemia was evident in five out of six NOD mice, and it lasted for 20 days; then all the mice reverted to hyperglycemia. This suggests that it is desirable to treat the mice between 3 and 5 days after diabetes onset (Figure 4b). Finally, newly hyperglycemic NOD mice were treated with calicheamicin alone (n=5 mice), and no effect was observed in 3 out of 5 mice treated, while in 2 NOD mice treated ,some glycemic oscillations were observed before the return to stable hyperglycemia (Figure 4b).

[0167] B-cell depletion appeared to be important for the restoration of normoglycemia.

In fact, when B cells made up more than 3-5% of the blood, normoglycemia was not restored. Only 2 out of 10 partially depleted (with unconjugated CD22 treatment) hyperglycemic NOD mice showed a transient return to normoglycemia with a quick reappearance of hyperglycemia (Figure 4b).

Example 34. B-cell depletion reduces pro-inflammatory peripheral cytokine levels in hyperglycemic NOD mice

[0168] In the reversal studies, serum samples were obtained at baseline, on day 10, and on day 100 after treatment of the B-cell-depleted and in the untreated control NOD mice. Remarkably, a change in peripheral cytokine levels was observed during the restoration of normoglycemia (Figure 4c). Most of the pro-inflammatory cytokines were reduced 10 days after the treatment, when normoglycemia was restored (Figure 4c). Particularly IL- 17 and TNF-α, and to some extent IFN-γ, peripheral levels were reduced in B-cell-depleted mice. Interestingly, in the long-term tolerant mice, IL- 17 and TNF-α peripheral levels were similar to those in newly hyperglycemic NOD mice (Figure 4c).

Example 35. B-cell depletion in the reversal study is associated with changes in the percentage of CD4⁺CD25⁺FoxP3⁺ cells

[0169] The percentage of CD4⁺CD25⁺FoxP3⁺ cells in spleens and PLn of B-cell-depleted and untreated control NOD mice were calculated with FACS analysis. CD4⁺CD25⁺FoxP3⁺ cell percentage is increased in B-cell-depleted long-term tolerant compared to control NOD mice, in both the PLn (B-cell-depleted=14.3+1.9 vs. normoglycemic 10-week-old=6.4+0.7, p=0.007 and vs. hyperglycemic=8.7+1.1%, p=0.03, Figure 4d) and in the spleen (B-cell-depleted=12.8+1.7 vs. normoglycemic 10-week-old=7.9+0.4, p=0.02 and vs. hyperglycemic=8.4+0.2%, p=0.01, Figure 4e). It was evaluated whether the percentage of CD4⁺CD44⁺CD62L^lc and CD8⁺CD44⁺CD62L^lc cells (Teffs) differed between the B-cell-depleted and the untreated control NOD mice in any of the usual tissues (spleens and PLn). No differences were observed between the two groups at day 10 (data not shown). Only in the long-term reverted NOD mice, there was an increase in Teffs evident in the spleens of B-cell-depleted compared to untreated control mice (data not shown). Example 36. B-cell depletion in new onset hyperglycemic mice clear islet infiltrate and halt islets destruction

[0170] The histological evaluation of pancreata from B-cell-depleted and control NOD mice in the reversal studies revealed that after 3 days of hyperglycemia the islets are discretely infiltrated (Figure 5al) with disrupted structure and reduction in insulin and glucagon staining (Figures 5a5 and 5a6). The infiltrate contains both B220⁺ and CD3⁺ cells (Figures 5a2 and 5a3). After 10 days pancreas histology and immunohistochemistry in untreated control NOD mice showed massive islet infiltration (Figure 5b 1) with both B220⁺ and CD3⁺ spreading throughout the islets (Figures 5b2 and 5b3) and absence of any islet structure (as shown by insulin and glucagon staining), (Figures 5b5 and 5b6). Surprisingly, in the B-cell-depleted NOD mice 10 days after treatment, islets showed no infiltrate (or very mild infiltrate confined to the borders of beta cells) (Figure 5c 1) with the total absence of B220⁺ and CD3⁺ (Figures 5c2 and 5c3). Islet structure appeared well-maintained and preserved with obvious insulin and glucagon staining (Figures 5c5 and 5c6). One hundred days after the treatment, in the B-cell-depleted NOD mice, islets still appeared completely free of infiltrate (Figure 5dl) with few B220⁺ and CD3⁺ cells inside the islets (Figures 5d2 and 5d3). Many small but well-preserved islets were present in the pancreas, with scant insulin staining (Figure 5d5) and strong glucagon staining (Figure 5d6). Finally, Foxp3 staining of pancreatic islets revealed the presence of some Foxp3⁺ cells at baseline and 10 days later in untreated control NOD mice, within many CD3⁺ cells (Figures 5a4 and 5b4). Conversely, in the B-cell-depleted NOD mice, after 10 days most of the infiltrating cells appeared to be Foxp3⁺ cells (Figure 5c4). One hundred days after treatment in the B-cell- depleted group, Foxp3⁺ cells also appeared to be greatly diminished in the B-cell-depleted NOD mice (Figure 5d4). Insulitis score confirmed that B-cell-depleted NOD mice showed better- preserved and less-infiltrated islets compared to untreated control NOD mice both at baseline and 10 days after hyperglycemia onset (Figure 4f). Surprisingly, more than 100 days after reversal of hyperglycemia, islets appeared to be well-preserved with a low Insulitis core compared to hyperglycemic baseline NOD mice (Figure 4f).

Example 37. Transcriptome analysis revealed a reprogramming of re-emerging B cells compared to naϊve B cells

[0171] We examined the gene expression profile of re-emerging B cells (obtained from normoglycemic NOD mice treated with anti-CD22/cal mAb 100 days after B cell depletion) and compared it to that of B cells obtained from naϊve normoglycemic 10-week-old or hyperglycemic untreated control NOD mice. CD19⁺ cells were extracted from splenocytes with microbeads. Interestingly, a significant downregulation of inducible gene transcription was observed within the re-emerging B cell pool. Almost 200 genes were downregulated in re- emerging B cells compared to B cells extracted from normoglycemic 10-week-old NOD mice (Figure 6A), and 38 genes were downregulated in re-emerging B cells compared to B cells extracted from hyperglycemic NOD mice (Figure 6B).

[0172] When all 3 groups of B cells were compared (naϊve normoglycemic 10-week-old

NOD mice, naϊve hyperglycemic NOD mice and re-emerging), 21 genes appeared to be downregulated in the re-emerging B cell population (Table 1 and Figure 6C). Without wishing to be bound by any theories, it was contemplated that the downregulation of many extracellular lytic enzyme products (elastase 1 and 2, lysozime, chymotrypsinogen Bl, amylase) may be associated with directed islet damage or a sustained pro-inflammatory effect (Table 1 and Figure 6C). Genes of the complement cascade (Fcna, Clqb) and pro-inflammatory (Hebpl, Pilrbl, PPARγ and Hmox-1) genes are downregulated in re-emerging B cells as well (see, Table 1 and Figure 6C).

Table 1. Exemplary downregulated genes in re-emerging B cells compared to B cells extracted from naϊve normoglycemic or hyperglycemic NOD mice.

Example 38. B-cell-depleted mice are immunocompetent in the long-term

[0173] CD4⁺ cells extracted from B-cell-depleted and control NOD mice were challenged with BALB/c bone marrow-derived dendritic cells (DC) in an MLR assay to evaluate the immunocompetence of T cells 10 and 100 days after B cell depletion. Both B-cell-depleted and control NOD mice were capable of mounting a comparable immune response to alloantigen at baseline and 10 and 100 days after the treatment (data not shown).

Example 39. Re-emerging B cells displayed a more anergic phenotvpe compared to naive B cells

[0174] The phenotypes of re-emerging B cells and naive B cells were evaluated. FACS analysis of the most common co-stimulatory molecules (CD80, CD86, and CD40) as well as of Class II and IgM, revealed no major differences between re-emerging and naϊve B cells (the latter from either normo- or hyperglycemic NOD mice) (Figure 7a). The proportion of different B cell subpopulations before depletion and after reconstitution were then analyzed by FACS with respect to immaturity/maturity and the presence of anergic B cells, which can be identified within the B220⁺CD93⁺CD23TgM^lc cells, as previously shown (Merrell KT BR, et al. Immunity. 2006; 25:953-962). A higher percentage of B220⁺CD93⁺CD23TgM^l0 cells were detected in the re-emerging B-cell pool compared to naϊve B cells from either normo- or hyperglycemic NOD mice (Figure 7b).

[0175] Then, the ability of re-emerging and naϊve B cells to present autoantigen were evaluated. An in vitro assay in which B cells are used as APC and CD4⁺ cells from NOD mice are used as responders in the presence of BDC2.5 peptide was designed. Interestingly, B cells from NOD mice are capable of presenting autoantigen and stimulating IFN-γ production by CD4⁺ cells (Figure 7c). When re-emerging B cells were used, IFN-γ production by CD4⁺ cells was lower than when naϊve B cells were used (p=0.008 compared to normoglycemic untreated control NOD mice, Figure 7c), suggesting a reduced ability to present autoantigen in vitro.

Example 40. Re-emerging B cells are regulatory in vitro and down-regulate pro-inflammatory cytokine production in BDC2.5 CD4⁺ T cells

[0176] The ability of re-emerging B cells to modulate cytokine profile of autoreactive T cells were evaluated. We generated an assay in which naϊve or re-emerging B cells extracted with magnetic beads from NOD mice were used as stimulatory APC, and autoreactive CD4⁺ cells extracted from BDC2.5 mice with the use of anti-idiotype antibody VB4 were used as responders in the presence of BDC2.5 peptide. Cells were incubated for 72 hours in 86-well plates and serum was collected to assess cytokine profile with a Luminex assay. Interestingly, when re-emerging B cells, but not naϊve B cells, were used, BDC2.5 autoreactive CD4⁺ cells down-regulated the production of pro-inflammatory cytokine (IL-2, IL- 17, IFN-γ, TNF-α), (Figures 7f-i). Particularly, when re-emerging B cells were used, autoreactive T cells produced less TNF-α compared to naϊve B cells extracted from normoglycemic and hyperglycemic untreated control NOD mice (p<0.05 vs. both); less IL- 17 compared to hyperglycemic untreated control NOD mice (p=0.004) and less IFN-γ compared to hyperglycemic untreated control NOD mice (p=0.02), (Figure 7f-i).

Example 41. Re-emerging B cells are regulatory in vivo and halt the transfer of diabetes from diabetogenic CD4⁺ T cells

[0177] To compare the regulatory function of re-emerging and naϊve B cells, CD4⁺ cells extracted with magnetic beads from splenocytes obtained from hyperglycemic NOD mice were adoptively transferred into NOD. SCID hosts. CD19⁺ cells extracted with magnetic beads from splenocytes obtained from re-emerging B cells or from naϊve B cells were then co-adoptively transferred. Interestingly, when naϊve B cells were used, NOD. SCID developed diabetes as expected (particularly when naive B cells were extracted from hyperglycemic NOD mice), (Figure 6d). Conversely, when re-emerging B cells were used, they completely abrogated the onset of diabetes mediated by the transfer of CD4⁺ cells from hyperglycemic NOD mice (Figure 7d). This experiment suggested the existence of an active regulatory mechanism associated with re-emerging B cells. In order to determine whether this protection is related to induction/expansion of Tregs in vivo we analyzed the percentage of CD4⁺CD25 FoxP3⁺ cells (Tregs) in spleen of the NOD. SCID recipients of the diabetogenic CD4⁺ T cells and re-emerging B cells or controls (B cells from hyperglycemic animals or no cells) at day 30 post-adoptive transfer. As seen in Figure 7e, no significant differences were detected among the three groups.

EQUIVALENTS

[0178] The foregoing has been a description of certain non-limiting embodiments of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.

[0179] In the claims articles such as "a,", "an" and "the" may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the claims or from relevant portions of the description is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a composition, it is to be understood that methods of using the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known in the art are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. In addition, the invention encompasses compositions made according to any of the methods for preparing compositions disclosed herein.

[0180] Where elements are presented as lists, e.g., in Markush group format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It is also noted that the term "comprising" is intended to be open and permits the inclusion of additional elements or steps. It should be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, steps, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, steps, etc. For purposes of simplicity those embodiments have not been specifically set forth in haec verba herein. Thus for each embodiment of the invention that comprises one or more elements, features, steps, etc., the invention also provides embodiments that consist or consist essentially of those elements, features, steps, etc.

[0181] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

[0182] In addition, it is to be understood that any particular embodiment of the present invention may be explicitly excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and/or methods of the invention can be excluded from any one or more claims. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects is excluded are not set forth explicitly herein.

INCORPORATION OF REFERENCES

[0183] All publications and patent documents cited in this application are incorporated by reference in their entirety to the same extent as if the contents of each individual publication or patent document were incorporated herein.

[0184] What is claimed is:

Claims

1. A method for preventing or delaying the onset of diabetes in a subject at risk for developing diabetes, the method comprising administering to the subject an effective amount of anti-CD22 antibody, or a fragment thereof.

2. A method for restoring normoglycemia in a subject suffering from diabetes, the method comprising administering to the subject an effective amount of anti-CD22 antibody, or a fragment thereof.

3. A method for inducing T-cell tolerance in a subject suffering from diabetes, the method comprising administering to the subject an effective amount of anti-CD22 antibody, or a fragment thereof.

4. The method of any one of claims 1-3, wherein the diabetes is type 1 diabetes.

5. A method for inhibiting immune-mediated destruction of pancreatic islets in a subject, the method comprising administering to the subject an effective amount of anti-CD22 antibody, or a fragment thereof.

6. The method of claim 5, wherein the subject is suffering from type 1 diabetes.

7. The method of claim 5, wherein the subject has received an islet transplantation.

8. The method of any of the preceding claims, wherein the anti-CD22 antibody, or a fragment thereof, is selected from the group consisting of intact IgG, F(ab')2, F(ab)2, Fab', Fab, scFvs, diabodies, triabodies or tetrabodies.

9. The method of any one of claims 1-5,, wherein the anti-CD22 antibody is a monoclonal antibody.

10. The method of claim 9, wherein the anti-CD22 antibody is a mouse monoclonal antibody.

11. The method of claim 9, wherein the anti-CD22 antibody is a humanized monoclonal antibody.

12. The method of any one of the preceding claims, wherein the anti-CD22 antibody, or a fragment thereof, is conjugated to a cytotoxic agent.

13. The method of claim 12, wherein the cytotoxic agent is calicheamicin or a derivative thereof.

14. The method of claim 13, wherein the calicheamicin is N-acetyl-calicheamicin dimethyl acid.

15. The method of claim 14, wherein the ratio of the amount of N-acetyl-calicheamicin dimethyl acid to the amount of the anti-CD22 antibody ranges from about 0.1 to about 15.0 moles calicheamicin/mol antibody.

16. The method of claim 15, wherein the ratio of the amount of N-acetyl-calicheamicin dimethyl acid to the amount of the anti-CD22 antibody ranges from about 1.2 to about 2.6 calicheamicin/mol antibody.

17. The method of any one of the preceding claims, wherein the effective amount of the anti- CD22 antibody, or a fragment thereof, ranges from 20 to 600 milligrams protein per dose.

18. The method of claim 17, wherein the effective amount of the anti-CD22 antibody, or a fragment thereof, ranges from 20 to 150 milligrams protein per dose.

19. The method of claim 18, wherein the effective amount of the anti-CD22 antibody, or a fragment thereof, ranges from 20 to 100 milligrams protein per dose.

20. The method of any one of the preceding claims, wherein the anti-CD22 antibody, or a fragment thereof, is administered bimonthly, monthly, triweekly, biweekly, weekly, twice a week, daily, or at variable intervals.

21. The method of any one of the preceding claims, wherein the method further comprises administering a T-cell targeting agent.

22. A method of identifying genes or proteins associated with re-programming B cells, the method comprising: providing a biological sample comprising re-emerging B cells after an anti-diabetic treatment; generating an expression profile of genes or proteins of the re-emerging B cells; identifying one or more differentially expressed genes or proteins in the re-emerging B cells as compared to a reference expression profile, thereby identifying genes or proteins associated with re-programming B cells.

23. The method of claim 22, wherein the anti-diabetic treatment is a treatment with a B cell depleting agent.

24. The method of claim 23, wherein the B cell depleting agent is an anti-CD22 antibody, or a fragment thereof.

25. The method of any one of claims 22-24, wherein the reference expression profile is an expression profile of corresponding untreated B cells.

26. The method of any one of claims 22-24, wherein the reference expression profile is an expression profile of naϊve B cells from an untreated subject suffering from diabetes.

27. The method of any one of claims 22-26, wherein the method further comprises a step of determining if any of the differentially expressed genes or proteins are indicative or regulatory of diabetes.

28. A method of diagnosis or prognosis of diabetes, the method comprising the steps of:

(a) providing a biological sample comprising B cells obtained from a subject suffering from or at risk of diabetes;

(b) determining an expression or activity of a differentially expressed gene or protein identified according to claim 22 in the biological sample; and

(c) providing a diagnosis or prognosis based on the expression or activity of the differentially expressed gene or protein as determined at step (b).

29. A method of diagnosis or prognosis of diabetes, the method comprising the steps of:

(a) providing a biological sample comprising B cells obtained from a subject suffering from or at risk of diabetes;

(b) determining an expression or activity of one or more genes or proteins identified in Table 1 or Figure 6 in the biological sample; and

(c) providing a diagnosis or prognosis based on the expression or activity of the one or more genes or proteins as determined at step (b).

30. The method of claims 28 or 29, wherein the method further comprises a step of providing a treatment of diabetes based on the diagnosis or prognosis of step (c).