BDC2.5 mimotope 1040-31

A primary initiating epitope in the NOD mouse model of Type 1 Diabetes (T1D) lies between residues 9 and 23 of the insulin B chain. The B:9-23 peptide can bind to the NOD MHC class II molecule (I-Ag7) in multiple registers, but only one, (register 3, R3), creates complexes able to stimulate the majority of pathogenic B:9-23-specific CD4+ T cells. Previously we generated a monoclonal antibody (mAb287) that targets this critical I-Ag7-B:9-23(R3) complex. When given weekly to pre-diabetic mice at either early or late stages of disease, mAb287 was able to delay or prevent T1D in the treated animals. Although the precise mechanism of action of mAb287 remains unclear, we hypothesized that it may involve deletion of antigen presenting cells (APCs) bearing the pathogenic IAg7-B:9-23(R3) complexes, and that this process might be rendered more efficient by re-directing cytotoxic T cells using a mAb287 chimeric antigen receptor (287-CAR). As anticipated, 287-CAR T cells secreted IFN-γ in response to stimulation by I-Ag7-B:9-23(R3) complexes expressed on artificial APCs, but not I-Ag7 loaded with other peptides, and killed the presenting cells in vitro. A single infusion of 287-CAR CD8+ T cells to young (5 week old) NOD mice significantly delayed the onset of overt hyperglycemia compared to untreated animals (p = 0.022). None of the 287-CAR CD8+ T cell treated mice developed diabetes before 18 weeks of age, while 29% of control-CAR T cell treated mice (p = 0.044) and 52% of the un-treated mice (p = 0.0001) had developed T1D by this time. However, the protection provided by 287-CAR CD8+ T cells declined with time, and no significant difference in overall incidence by 30 weeks between the 3 groups was observed. Mechanistic studies indicated that the adoptively transferred 287-CAR T cells selectively homed to pancreatic lymph nodes, and in some animals could persist for at least 1-2 weeks post-transfer, but were essentially undetectable 10-15 weeks later. Our study demonstrates that CAR T cells specific for a pathogenic MHC class II:peptide complex can be effective in vivo, but that a single infusion of the current iteration can only delay, but not prevent, the development of T1D. Future studies should therefore be directed towards optimizing strategies designed to improve the longevity of the transferred cells.

Type 1 diabetes is an autoimmune disorder caused by autoreactive T cells that mediate destruction of insulin-producing beta cells of the pancreas. Studies have shown that T cell tolerance can be restored by inducing a partial or altered signal through the TCR. To investigate the potential of bivalent peptide-MHC class II/Ig fusion proteins as therapeutics to restore Ag-specific tolerance, we have developed soluble peptide I-A(g7) dimers for use in the nonobese diabetic mouse model of diabetes. I-A(g7) dimers with a linked peptide specific for islet-reactive BDC2.5 TCR transgenic CD4(+) T cells were shown to specifically bind BDC2.5 T cells as well as a small population of Ag-specific T cells in nonobese diabetic mice. In vivo treatment with BDC2.5 peptide I-A(g7) dimers protected mice from diabetes mediated by the adoptive transfer of diabetogenic BDC2.5 CD4(+) T cells. The dimer therapy resulted in the activation and increased cell death of transferred BDC2.5 CD4(+) T cells. Surviving cells were hypoproliferative to challenge by Ag and produced increased levels of IL-10 and decreased levels of IFN-gamma compared with cells from control I-A(g7) dimer-treated mice. Anti-IL-10R therapy reversed the tolerogenic effects of the dimer. Thus, peptide I-A(g7) dimers induce tolerance of BDC2.5 TCR T cells through a combination of the induction of clonal anergy and anti-inflammatory cytokines.

Chimeric major histocompatibility complex (MHC) molecules supplemented with T cell receptor (TCR) signaling motifs function as activation receptors and can redirect gene-modified T cells against pathogenic CD8 T cells. We have shown that β2 microglobulin (β2m) operates as a universal signaling component of MHC-I molecules when fused with the CD3-ζ chain. Linking the H-2Kd-binding insulin B chain peptide insulin B chain, amino acids 15-23 (InsB15-23) to the N terminus of β2m/CD3-ζ, redirected polyclonal CD8 T cells against pathogenic CD8 T cells in a peptide-specific manner in the non-obese diabetic (NOD) mouse. Here, we describe mRNA electroporation for delivering peptide/β2m/CD3-ζ genes to a reporter T cell line and purified primary mouse CD8 T cells. The peptide/β2m/CD3-ζ products paired with endogenous MHC-I chains and transmitted strong activation signals upon MHC-I cross-linking. The reporter T cell line transfected with InsB15-23/β2m/CD3-ζ mRNA was activated by an InsB15-23-H-2Kd-specific CD8 T cell hybrid only when the transfected T cells expressed H-2Kd. Primary NOD CD8 T cells expressing either InsB15-23/β2m/CD3-ζ or islet-specific glucose-6-phosphatase catalytic subunit-related protein, amino acids 206-214 (IGRP206-214)/β2m/CD3-ζ killed their respective autoreactive CD8 T cell targets in vitro. Furthermore, transfer of primary CD8 T cells transfected with InsB15-23/β2m/CD3-ζ mRNA significantly reduced insulitis and protected NOD mice from diabetes. Our results demonstrate that mRNA encoding chimeric MHC-I receptors can redirect effector CD8 against diabetogenic CD8 T cells, offering a new approach for the treatment of type 1 diabetes.