Gene-expression profiling of laser-dissected islets and studies in deficient mice reveal chemokines as differential driving force of type 1 diabetes.

Although type 1 diabetes (T1D) results from the autoimmune destruction of the insulin-producing ß-cells, its treatment is largely restricted to exogenous insulin administration. Only few therapies targeting the autoaggressive immune system have been introduced into clinical practice or are considered in clinical trials. Here, we provide a gene expression profile of the islet microenvironment obtained by laser-dissection microscopy in an inducible mouse model. Thereby, we have identified novel targets for immune intervention. Increased gene expression of most inflammatory proteins was apparent at day 10 after T1D induction and largely paralleled the observed degree of insulitis. We further focused on genes involved in leukocyte migration, including chemokines and their receptors. Besides the critical chemokine CXCL10, we found several other chemokines upregulated locally in temporary or chronic manner. Localization of the chemokine ligand/receptor pairs to the islet microenvironment has been confirmed by RNAscope. Interference with the CXCL16-CXCR6 and CX3CL1-CX3CR1 axes, but not the CCL5-CCR1/3/5 axis, resulted in reduced insulitis and lower T1D incidence. Further, we found that the receptors for the differentially expressed chemokines CXCL10, CXCL16 and CX3CL1 are distributed unevenly among islet autoantigen-specific T cells, which explains why the interference with just one chemokine axis cannot completely abrogate insulitis and T1D.

Antibody Neutralization of CXCL10 in Vivo Is Dependent on Binding to Free and Not Endothelial-bound Chemokine: IMPLICATIONS FOR THE DESIGN OF A NEW GENERATION OF ANTI-CHEMOKINE THERAPEUTIC ANTIBODIES.

To improve our understanding of properties that confer successful inhibition of chemokines in vivo, we analyzed anti-murine CXCL10 monoclonal antibodies (mAb) having different characteristics. 1B6 displayed potent inhibition of cell recruitment in vitro with an IC50 of 0.5 nm but demonstrated little efficacy in various animal models of human disease. On the contrary, 1F11 showed efficacy in several models of inflammation yet was less potent at inhibiting chemotaxis in vitro with an IC50 of 21 nm Furthermore, we observed that 1B6 displayed a rapid dose-dependent clearance (t½ 10-60 h) in contrast to 1F11, which presented a dose-proportional pharmacokinetic profile and a half-life of 12 days. Moreover, 1B6 recognized glycosaminoglycan (GAG)-bound CXCL10, resulting in target-mediated clearance, which was corroborated using CXCL10-deficient mice. In contrast to 1B6, 1F11 inhibited the interaction of CXCL10 with GAGs, did not recognize GAG-bound CXCL10, and did not display target-mediated drug disposition. Confirming previous animal studies, 1B6 was poor at reversing glycemia in a model of type 1 diabetes, whereas 1F11 induced early and prolonged control of diabetes. Furthermore, when using 1A4, a subsequently generated anti-mCXCL10 mAb that shares the property with 1F11 of being unable to recognize CXCL10 immobilized on GAG, we observed a similar superior control of diabetes as compared with 1B6. We therefore concluded that targeting chemokines with antibodies such as 1B6 that recognize the more abundant GAG-bound form of the chemokine may not be the optimal strategy to achieve disease control.

Potent antitumor activity of anti-HER2 antibody-topoisomerase I inhibitor conjugate based on self-immolative dendritic dimeric-linker.

Antibody-drug conjugates (ADCs) are a rapidly expanding class of anticancer therapeutics, with 14 ADCs already approved worldwide. We developed unique linker technologies for the bioconjugation of drug molecules with controlled-release applications. We synthesized cathepsin-cleavable ADCs using a dimeric prodrug system based on a self-immolative dendritic scaffold, resulting in a high drug-antibody ratio (DAR) with the potential to reach 16 payloads due to its dendritic structure, increased stability in the circulation and efficient release profile of a highly cytotoxic payload at the targeted site. Using our novel cleavable linker technologies, we conjugated the anti-human epidermal growth factor receptor 2 (anti-HER2) antibody, trastuzumab, with topoisomerase I inhibitors, exatecan or belotecan. The newly synthesized ADCs were tested in vitro on mammary carcinoma cells overexpressing human HER2, demonstrating a substantial inhibitory effect on the proliferation of HER2-positive cells. Importantly, a single dose of our trastuzumab-based ADCs administered in vivo to mice bearing HER2-positive tumors, showed a dose-dependent inhibition of tumor growth and survival benefit, with the most potent antitumor effects observed at 10 mg/kg, which resulted in complete tumor regression and survival of 100% of the mice. Overall, our novel dendritic technologies using the protease-cleavable Val-Cit linker present an opportunity for the development of highly selective and potent controlled-released therapeutic payloads. This strategy could potentially lead to the development of novel and effective ADC technologies for patients diagnosed with HER2-positive cancers. Moreover, our proposed ADC linker technology can be implemented in additional medical conditions such as other malignancies as well as autoimmune diseases that overexpress targets, other than HER2.

Anti-CD3/Anti-CXCL10 Antibody Combination Therapy Induces a Persistent Remission of Type 1 Diabetes in Two Mouse Models.

Anti-CD3 therapy of type 1 diabetes results in a temporary halt of its pathogenesis but does not constitute a permanent cure. One problem is the reinfiltration of islets of Langerhans with regenerated, autoaggressive lymphocytes. We aimed at blocking such a reentry by neutralizing the key chemokine CXCL10. Combination therapy of diabetic RIP-LCMV and NOD mice with anti-CD3 and anti-CXCL10 antibodies caused a substantial remission of diabetes and was superior to monotherapy with anti-CD3 or anti-CXCL10 alone. The combination therapy prevented islet-specific T cells from reentering the islets of Langerhans and thereby blocked the autodestructive process. In addition, the local immune balance in the pancreas was shifted toward a regulatory phenotype. A sequential temporal inactivation of T cells and blockade of T-cell migration might constitute a novel therapy for patients with type 1 diabetes.

Functional redundancy of CXCR3/CXCL10 signaling in the recruitment of diabetogenic cytotoxic T lymphocytes to pancreatic islets in a virally induced autoimmune diabetes model.

Cytotoxic T lymphocytes (CTLs) constitute a major effector population in pancreatic islets from patients suffering from type 1 diabetes (T1D) and thus represent attractive targets for intervention. Some studies have suggested that blocking the interaction between the chemokine CXCL10 and its receptor CXCR3 on activated CTLs potently inhibits their recruitment and prevents ß-cell death. Since recent studies on human pancreata from T1D patients have indicated that both ligand and receptor are abundantly present, we reevaluated whether their interaction constitutes a pivotal node within the chemokine network associated with T1D. Our present data in a viral mouse model challenge the notion that specific blockade of the CXCL10/CXCR3 chemokine axis halts T1D onset and progression.