An MHC-restricted antibody-based chimeric antigen receptor requires TCR-like affinity to maintain antigen specificity

Abstract

Chimeric antigen receptors (CARs) are synthetic receptors that usually redirect T cells to surface antigens independent of human leukocyte antigen (HLA). Here, we investigated a T cell receptor-like CAR based on an antibody that recognizes HLA-A*0201 presenting a peptide epitope derived from the cancer-testis antigen NY-ESO-1. We hypothesized that this CAR would efficiently redirect transduced T cells in an HLA-restricted, antigen-specific manner. However, we found that despite the specificity of the soluble Fab, the same antibody in the form of a CAR caused moderate lysis of HLA-A2 expressing targets independent of antigen owing to T cell avidity. We hypothesized that lowering the affinity of the CAR for HLA-A2 would improve its specificity. We undertook a rational approach of mutating residues that, in the crystal structure, were predicted to stabilize binding to HLA-A2. We found that one mutation (DN) lowered the affinity of the Fab to T cell receptor-range and restored the epitope specificity of the CAR. DN CAR T cells lysed native tumor targets in vitro, and, in a xenogeneic mouse model implanted with two human melanoma lines (A2+/NYESO+ and A2+/NYESO-), DN CAR T cells specifically migrated to, and delayed progression of, only the HLA-A2+/NY-ESO-1+ melanoma. Thus, although maintaining MHC-restricted antigen specificity required T cell receptor-like affinity that decreased potency, there is exciting potential for CARs to expand their repertoire to include a broad range of intracellular antigens.

Figure 1

T1/28z CAR construct. (a) SFG vector indicating LTRs, packaging signal (ψ), splice donor and splice acceptor sites, leader sequence, single-chain variable fragment of the T1 (HLA-A2/NYESO1-specific) antibody fused to human CD28 and human CD3zeta signaling domains. The ires-GFP domain is 3′. (b) Primary human T cells 5 days after transduction with T1/28z retroviral vector, stained with HLA-A2/NYESO pentamer. Cells are gated on FSC/SSC only. Chromium release cytotoxicity assay using T1/28z-transduced T cells as effectors against (c) T2 cells pulsed with 10 μg/ml of either NYESO1 or flu peptide. E:T ratios normalized to pentamer+ cells or (d) human melanoma lines SK Mel 37, SK Mel 23, or SK Mel 52. GFP, green fluorescent protein; E:T, effector:target.

Figure 2

Rationally targeted mutations designed to decrease binding of T1 to HLA-A2 alpha helix. (a) Crystal structure of T1 Fab binding HLA-A2/NYESO1, with highlighting of targeted amino acids. (b) A2/NYESO1 pentamer stains of primary human T cells 5 days after transduction with parental (T1), D53N mutant, Y34F, and DNYF mutations in the CAR. Fluorescence-activated cell sorting (FACS) plots are gated on FSC/SSC only. (c) Chromium release assays of corresponding CAR-transduced effectors against T2 cells pulsed with either flu or NYESO peptide as targets. Effector to target ratios are normalized to pentamer+ cells. CAR, Chimeric antigen receptor.

Figure 3

Direct measurement of affinity of the DN mutation in the Fab antibody by BIAcore. Sensograms of DN Fab run over a range of concentrations (18.5 nmol/l – 1187 nmol/l) of HLA-A2 presenting (a) native NYESO1 (SLLMWITQC) or (b) mutant C9V NYESO1 (SLLMWITQV) peptide, which avoids disulfide bonding. Data were fitted (red) in a BIAevaluation 3.0 following a simple 1:1 Langmuir binding model.

Figure 4

DN mutation restores epitope specificity of T1-antibody-based CAR-transduced T cells. Chromium release assays using (a) T1/28z CAR-transduced or (b) DN/28z CAR- transduced T cells as effectors against target T2 cells pulsed with titrating concentrations of NYESO1 peptide. (c) Expansion of T1/28z and DN/28z CAR-transduced T cells derived from an HLA-A2+ donor. T cells were rested for one week after transduction and then stimulated with artificial APCs. Growth of CD3+GFP+ cells was calculated as total number of lymphocytes × frequency of CD3+GFP+ cells as determined by fluorescence-activated cell sorting (FACS) staining. (d) Expansion of HLA-A2+ T cells in response to antigen. Data are shown as fold-increase over unstimulated cells. Mel 37 is the SK-Mel 37 tumor cell line (HLA-A2+, NY-ESO-1+); U266 is the multiple myeloma cell line (also HLA-A2+, NY-ESO-1+). Proliferation was measured 4 days after restimulation. (e)Absolute number of CD3+GFP+ T cells per μl of blood at two timepoints after injection of NSG-HLA-A2 transgenic mice with T1/28z-, DN/28z-, or untransduced HLA-A2+ T. CARs, Chimeric antigen receptors; GFP, green fluorescent protein.

Figure 5

Similar efficacy of T1/28z and DN/28z CAR-transduced T cells. A2/NYESO1 pentamer stains of T cells transduced with (a) T1/28z or (b) DN/28z CAR. MFI shown is mean fluorescence intensity of GFP+ cells. Plots shown are gated only on fsc/ssc. Chromium release assays of (c) T1/28z or (d) DN/28z CAR-transduced T cells against peptide-pulsed T2 cells and against native tumor targets (melanoma cell lines SK- Mel-37 and SK-Mel-23). DN/28z T cell culture was diluted with untransduced T cells to equalize the frequency of pentamer+ cells with the T1/28z T cell culture. Effector: target ratios are normalized to pentamer+ cells. CARs, Chimeric antigen receptors.

Figure 6

DN/28z CAR-T cells specifically traffic to and delay progression of an NY- ESO-1-positive melanoma tumor in a xenogeneic mouse model. Subcutaneous tumor implants of mice treated with escalating doses of DN/28z CAR-T cells (or CD19/28z CAR-T cells as a control) were harvested and stained for CD3 by immunohistochemistry. The number of CD3+ T cells infiltrating the (a) SK Mel 37 (NY-ESO-1 positive) and (b) SK Mel 23 (NY-ESO-1 negative) was quantified as cells per mm². Representative slides of the tumors from mice treated with 8 × 10⁶ CAR-T cells are shown (c,d). The volume of each tumor (e,f) over time in mice treated with the 8 × 10⁶ DN/28z CAR-T cells or control T cells is shown. Lines are plotted as mean ± SEM of five mice for each group. CAR, Chimeric antigen receptor; SEM, standard error of mean.