A biomimetic five-module chimeric antigen receptor (^5MCAR) designed to target and eliminate antigen-specific T cells

Abstract

24T cells express clonotypic T cell receptors (TCRs) that recognize peptide antigens in the 25 context of class I or II MHC molecules (pMHCI/II). These receptor modules associate 26 with three signaling modules (CD3ge, de, and zz), and work in concert with a coreceptor 27 module (either CD8 or CD4), to drive T cell activation in response to pMHCI/II. Here we 28 describe a first generation biomimetic 5-module chimeric antigen receptor ( 5M CAR). We 29show that: (i) chimeric receptor modules built with the ectodomains of pMHCII assemble 30 with CD3 signaling modules into complexes that redirect cytotoxic T lymphocyte (CTL) 31 specificity and function in response to the clonotypic TCRs of pMHCII-specific CD4 + T 32 cells; and, (ii) surrogate coreceptor modules enhance the function of these complexes. 33Furthermore, we demonstrate that adoptively transferred 5M CAR-CTLs can mitigate type 34 I diabetes by targeting autoimmune CD4 + T cells in NOD mice. This work provides a 35 framework for the construction of biomimetic 5M CARs that can be used as tools to study 36 the impact of particular antigen-specific T cells in immune responses, and may hold 37 potential for ameliorating diseases mediated by pathogenic T cells. 38Main 39 T cells scan major histocompatibility complex (MHC) molecules on the surfaces of cells 40for the presence of peptide antigens (pMHC) derived from microbes, vaccines, or tumor 41 cells with their clonotypic T cell receptors (TCRs). If the dwell time of the TCR on the 42 pMHC is of sufficient duration, a T cell will become activated and differentiate to helper 43 (Th), cytotoxic (CTL), regulatory (Treg), or memory (Tm) T cells that are essential for 44 long-lived immunity 1,2 . CD4 + Th subsets provide help for effective CTL, B cell, and 45 innate immune cell function upon immunologic challenge, Tregs are crucial for 46 peripheral tolerance to self and commensal antigens, Tm allow for rapid antigen-specific 47 responses, and CTLs can eliminate infected or cancerous cells 3-6 . However, the activity 48 of each T cell subset can be counter-productive if conditions are such that they result in 49 the induction of allergies, asthma, autoimmunity, transplant rejection, or, in the case of 50Tregs, the protection of tumors. Considerable effort has thus been focused on 51 developing strategies to: determine how T cells of a particular pMHC-specificity impact 52 an immune response; enhance T cell responses to fight infections or tumors; or, 53 mitigate T cell-mediated pathologies. 54Chimeric antigen receptors (CARs) have gained attention as a technology that 55 can redirect T cell specificity and function for novel purposes (Fig. 1a). The archetypal 56 CAR design consists of a single-chain module (referred to here as 1M CARs) wherein 57 ligand specificity (e.g. tumor antigens) is usually conferred via an antibody-derived Fv, 58 while intracellular signaling is directed through a tandem array of known signaling 59 motifs 7,8 . Work to improve 1M CAR efficacy has resulted in the development and testing 60 of...

Antigen-specific depletion of CD4 + T cells by CAR T cells reveals distinct roles of higher- and lower-affinity TCRs during autoimmunity

“…3B), suggesting that the use of an intact MHCII molecule can facilitate CAR signaling by co-receptor engagement. This does not appear to be possible with the previously reported pMHCII-TCR fusion due to the lack of a CD4 binding site ( 23 ). In summary, these data suggest that pMHCII-TCR affinity dictates the efficiency of target cell recognition and killing by pMHCII-CAR T cells.…”

“…For example, in a partially humanized murine model of rheumatoid arthritis, pMHCII-CAR T cells composed of HLA-DR1 bound to a type II collagen peptide were effective at preventing collagen-induced arthritis ( 35 ). The pMHCII-CAR approach represents an alternative to a recent report using a MHCII-TCR fusion (5M)CAR to prevent autoimmune diabetes ( 23 ). A potential advantage of using the intact MHCII molecule over a MHCII-TCR fusion is the ease of constructing pMHCII-CARs using different MHCII alleles, which we did with murine I-A d and human HLA-DR4 sequences without modification as proof of principle.…”