A Novel Peptide-MHC Targeted Chimeric Antigen Receptor T Cell Forms a T Cell-like Immune Synapse

Abstract

Chimeric Antigen Receptor (CAR) T cell therapy is a promising form of adoptive cell therapy that re-engineers patient-derived T cells to express a hybrid receptor specific to a tumour-specific antigen of choice. Many well-characterised tumour antigens are intracellular and therefore not accessible to antibodies at the cell surface. Therefore, the ability to target peptide-MHC tumour targets with antibodies is key for wider applicability of CAR T cell therapy in cancer. One way to evaluate the effectiveness and efficiency of ligating tumour target cells is studying the immune synapse. Here we generated a second-generation CAR to targeting the HLA-A*02:01 restricted H3.3K27M epitope, identified as a possible therapeutic target in ~75% of diffuse midline gliomas, used as a model antigen to study the immune synapse. The pMHCI-specific CAR demonstrated specificity, potent activation, cytokine secretion and cytotoxic function. Furthermore, we characterised killing kinetics using live cell imaging as well as CAR synapse confocal imaging. Here we provide evidence of robust CAR targeting of a model peptide-MHC antigen and that, in contrast to protein-specific CARs, these CARs form a TCR-like immune synapse which facilitates TCR-like killing kinetics.

Bringing cell therapy to tumors: considerations for optimal CAR binder design

“…The scFv format permits facile reformatting of antibodies derived from conventional immunization campaigns. These are almost entirely, to date, raised against the extracellular domains of cell-surface proteins that are identified as viable tumor targets, though there are recent developments in generating scFvs that recognize intracellular tumor antigens with limited MHC restriction (which is a limiting factor in TCR-T cell therapy) through display technologies [ 37 , 38 ] or using structural analysis to drive rationale engineering to optimize the binder [ 39 , 40 ]. It remains to be seen how broadly applicable and indeed successful this approach will be.…”

Endogenous H3.3K27M derived peptide restricted to HLA-A∗02:01 is insufficient for immune-targeting in diffuse midline glioma

“…In our previous work, we developed a scFv antibody that targets the novel H3.3K27M-HLA-A∗02:01 peptide-MHC complex. 9 Among the reactive scFv reactive clones, we identified the most effective one, named A615, for further investigation. 9 The A615 scFv was then engineered with an avitag, allowing the production of A615-tetramer-mWasabi conjugates for easy detection using flow cytometry.…”

“…We have previously described the characterization of a novel H3.3K27M-HLA-A∗02:01 specific CAR and shown a functional immune synapse, characteristic of a classical TCR-like immune synapse structure. 9 In this study, we present the characterization of a novel single chain variable fragment (scFv) domain, named A615, targeting H3.3K27M-HLA-A∗02:01, which we engineered into second generation CD28ζ CARs.…”

Bispecific GPC3/PD‑1 CAR‑T cells for the treatment of HCC

“…CD3ζ) with or without 1 or 2 costimulatory molecules, and a transmembrane domain (15). CAR-T cells can distinguish the exact tumour-associated antigens (TAAs) in a manner independent of the major histocompatibility complex (16)(17)(18). Nowadays, although the curative effect of CAR-T cell therapy has been proven to treat hematological malignancies (19), it remains unsatisfactory in treating solid tumours (20,21).…”