Objective
Type 1 diabetes (T1D) is characterized by the hyperglycemia resulting from autoimmune destruction of insulin-expressing beta-cells of the pancreas. No cure exists for this disease and despite progress in understanding islet pathology during T1D, key conceptual gaps still remain in identifying mechanisms that instigate immune cell infiltration and their recognition of beta cells. Here we hypothesize that treatment with an anti-Cadm1 mAb disrupts Cadm1-mediated immune cell interactions including binding of CD8+ T cells to islet endocrine and/or myeloid cells. We further hypothesize that reduced intercellular contact attenuates antigen presentation and subsequent T cell activation ultimately preventing the pancreatic beta-cell destruction that leads to autoimmune diabetes. To test these hypotheses, we will perform the following Aims:
Aim 1. To test whether early administration of an anti-Cadm1 monoclonal antibody provides sustained protection against autoimmune diabetes. Our preliminary results showed weekly administration of a Cadm1 mAb to NOD mice attenuated cytotoxic destruction of beta-cells suggesting Cadm1-mediated binding facilitate immune cell activation (via CRTAM on T cells). Here we will test whether weekly treatment with the Cadm1 mAb starting at age 4 weeks can provide indefinite protection from autoimmune destruction of pancreatic beta-cells in female NOD mice. In addition, we will test whether weekly treatment of the Cadm1 mAb any protection to diabetic NOD mice from complete beta-cell destruction. These experiments will test whether efficacy of the Cadm1 mAb is dependent upon the stage of disease at initial treatment.
Aim 2. To delineate the impact of the anti-Cadm1 monoclonal antibody on the immune cell repertoire during autoimmune diabetes. Preliminary results show administration of a Cadm1 mAb to NOD mice reduced the number of activated CD8+ T cells. To further understand the extent to which the Cadm1 mAb impacts activation of the immune cell repertoire, we will perform single cell RNA sequencing (scRNA seq) on CD45+ cells isolated from pancreatic islets of female NOD mice after treatment with the anti-Cadm1 antibody. In addition, we will evaluate whether Cadm1 mAb treatment alters immune cell numbers or their interaction with islet cells. These studies will address how the Cadm1 mAb affects specific immune cell populations (ie. T and B cell activation, regulatory T cells, myeloid cell number and antigen presentation).
Aim 3. To delineate the impact of soluble CADM1 on the human immune cell repertoire during autoimmune diabetes. Our preliminary results show the cleaved ectodomain of CADM1 (sCADM1) is detected in serum of NOD mice at age 7 weeks (during the pre-symptomatic stage) but is significantly diminished at age 21 weeks (after diabetes onset). Similarly, sCADM1 levels are decreased in newly-diagnosed T1D human subjects compared to non-diabetic control subjects. To further understand the functional relevance of sCADM1, we will perform scRNA seq on human PBMCs isolated from non-diabetic, auto-antibody-positive and T1D human subjects after treatment with sCADM1. These studies aim to establish how elevated levels of sCADM1 incur changes in the immune cell repertoire leading to immune cell activation prior to diabetes onset.
Impact: Completion of this work will establish the CADM1 mAb as a potential therapeutic strategy for attenuating the immune cell activation that causes autoimmune diabetes. These studies will also provide insight into whether treatment with this mAb at early stages of disease progression can block immune cell activation and beta cell destruction. Moreover, this work will elucidate the impact of both membrane-bound and sCADM1 on the immune cell repertoire using both mouse and human model systems. Successful completion of these Aims will further establish CADM1 as a model target for developing antibodies to suppress cytotoxic T cell activation in addition to serving as a bridge for identifying targetable co-factors that contribute to T1D pathogenesis.
Background Rationale
Cell adhesion molecule 1 (referred to as CADM1) is a cell surface protein of the Ig superfamily that mediates intercellular contact between dissimilar cell types including immune, neuronal and endocrine cells. In light of our observations identifying increased numbers of CADM1+ myeloid cells in islets of auto-antibody-positive (aAb+) and T1D human subjects, we hypothesized that CADM1 mediates binding between islet cells and cytotoxic T-cells leading to beta-cell destruction and autoimmune diabetes. To test this, we administered the anti-CADM1 monoclonal neutralizing antibody (CADM1 mAb) to female NOD/ShiLtJ mice, a model of autoimmune diabetes, starting at age 14 weeks. Preliminary results showed weekly treatment of NOD mice with the CADM1 mAb antibody prevented cytotoxic destruction of beta-cells and attenuated autoimmune diabetes onset in comparison to NOD mice treated with a control IgY antibody. In addition, treatment with the CADM1 mAb also reduced the number of activated CD8+ T cells in NOD mice. These results suggest the CADM1 mAb may disrupt CADM1-mediated intercellular binding and block autoimmune destruction of pancreatic beta-cells in NOD mice. Together, the rationale for the study of Cadm1 in the context of T1D is based on: 1) CADM1 enrichment in islet endocrine and myeloid cells during human T1D, 2) demonstration of CADM1 binding of the receptor CRTAM on CD8+ T cells, and 3) attenuation of beta-cell destruction, CD8+ T cell activation and diabetes onset in NOD mice after treatment with anti-CADM1 monoclonal neutralizing antibody.
Description of Project
Type 1 diabetes (T1D) is characterized by the hyperglycemia resulting from autoimmune destruction of insulin-expressing beta-cells of the pancreas. No cure exists for this disease and despite progress in understanding islet pathology during T1D, key conceptual gaps still remain in identifying mechanisms that instigate immune cell infiltration and their recognition of beta cells. Given the established role of autoreactive T-cells in mediating beta-cell destruction during T1D, continued emphasis remains on identifying the islet proteins that mediate immune cell infiltration that could potentially be targeted to both preserve pancreatic beta-cell mass, and prevent the immune response that initiates T1D pathogenesis.
Our previous work showed increased expression of the gene CADM1 in myeloid cells within the pancreatic islets of auto-antibody positive (aAb+) and in T1D human subjects. As a cell membrane surface protein, CADM1 mediates interaction between different cell types including immune cells and endocrine cells. Notably, CADM1 has been shown to act as a ligand for the receptor CRTAM present in CD8+ T-cells, a cell population widely known to infiltrate the pancreas and facilitate pancreatic beta-cell destruction during T1D. Here we hypothesize increased levels of CADM1 in endocrine cells and macrophages within the pancreatic islet facilitate recruitment of cytotoxic CD8+ T-cell populations. Our preliminary results show that treatment with a monoclonal ‘neutralizing’ antibody raised against CADM1 attenuates onset of autoimmune diabetes in mice. This project will elucidate the changes in the immune cell repertoire of the pancreas resulting from this antibody treatment during the pre-symptomatic phase of the disease. In this study we will ascertain how the CADM1 antibody affects T and B cell activation and whether there is a critical window for antibody treatment before disease onset to suppress the autoimmune attack on the pancreas. Successful outcomes of these preclinical studies will lay the foundation for the development of humanized CADM1 ‘neutralizing’ antibodies each targeting unique parts of the extracellular domain that could constitute an optimal strategy for preventing or potentially treating and curing individuals with T1D.
Anticipated Outcome
In these studies, we expect to observe that treatment with the CADM1 monoclonal antibody during the pre-symptomatic stage of autoimmune diabetes to be an effective strategy for preventing immune cell activation and subsequent pancreatic beta-cell destruction. We anticipate that early treatment in mice will attenuate T cell infiltration and binding of pancreatic endocrine cells as well as suppress the activation of CD8+ T cells. We expect the suppression of this immune cell population will provide sustained protection from autoimmune destruction of the insulin-producing beta-cells. Together these results will establish the CADM1 antibody as a model reagent for developing novel therapeutic and diagnostic strategies for ultimately preventing human T1D. Future studies will entail the development of humanized CADM1 antibodies that bind different regions of the extracellular domain of CADM1 to test whether blocking multiple domains of CADM1 confers enhanced efficacy in preventing immune cell destruction of the pancreas (via individual or combinatorial antibody approaches).
Relevance to T1D
Cell adhesion molecule 1 (referred to as human CADM1 and mouse Cadm1) is a cell surface protein of the Ig superfamily that mediates intercellular contact between dissimilar cell types including immune, neuronal and endocrine cells. In light of our observations identifying increased numbers of CADM1+ myeloid cells in islets of auto-antibody-positive (aAb+) and T1D human subjects, we hypothesized that CADM1 mediates binding between islet cells and cytotoxic T-cells leading to beta-cell destruction and autoimmune diabetes. To test this, we administered the anti-CADM1 monoclonal neutralizing antibody (CADM1 mAb) to female NOD/ShiLtJ mice, a model of autoimmune diabetes, starting at age 14 weeks. Preliminary results showed weekly treatment of NOD mice with the CADM1 mAb antibody prevented cytotoxic destruction of beta-cells and attenuated autoimmune diabetes onset in comparison to NOD mice treated with a control IgY antibody. In addition, treatment with the CADM1 mAb also reduced the number of activated CD8+ T cells in NOD mice. These results suggest the CADM1 mAb may disrupt Cadm1-mediated intercellular binding and block autoimmune destruction of pancreatic beta-cells in NOD mice. Together, the rationale for the study of Cadm1 in the context of T1D is based on: 1) CADM1 enrichment in islet endocrine and myeloid cells during human T1D, 2) demonstration of CADM1 binding of the receptor CRTAM on CD8+ T cells, and 3) attenuation of beta-cell destruction, CD8+ T cell activation and diabetes onset in NOD mice after treatment with anti-CADM1 monoclonal neutralizing antibody. Successful outcomes of these preclinical studies will lay the foundation for the development of humanized CADM1 ‘neutralizing’ antibodies each targeting unique parts of the extracellular domain that could constitute an optimal strategy for preventing or potentially treating and curing individuals with T1D.