Engineering of an Avidity-Optimized CD19-Specific Parallel Chimeric Antigen Receptor That Delivers Dual CD28 and 4-1BB Co-Stimulation.

Leena Halim,Kushal K Das,John Maher,Reuben Benjamin,Andrea Candelli,Adam Ajina,David M Davies,Richard Dillon,Daniel Larcombe-Young

doi:10.3389/fimmu.2022.836549

Abstract

Co-stimulation is critical to the function of chimeric antigen receptor (CAR) T-cells. Previously, we demonstrated that dual co-stimulation can be effectively harnessed by a parallel (p)CAR architecture in which a CD28-containing second generation CAR is co-expressed with a 4-1BB containing chimeric co-stimulatory receptor (CCR). When compared to linear CARs, pCAR-engineered T-cells elicit superior anti-tumor activity in a range of pre-clinical models. Since CD19 is the best validated clinical target for cellular immunotherapy, we evaluated a panel of CD19-specific CAR and pCAR T-cells in this study. First, we generated a panel of single chain antibody fragments (scFvs) by alanine scanning mutagenesis of the CD19-specific FMC63 scFv (VH domain) and these were incorporated into second generation CD28+CD3ζ CARs. The resulting panel of CAR T-cells demonstrated a broad range of CD19 binding ability and avidity for CD19-expressing tumor cells. Each scFv-modified CAR was then converted into a pCAR by co-expression of an FMC63 scFv-targeted CCR with a 4-1BB endodomain. When compared to second generation CARs that contained an unmodified or mutated FMC63 scFv, each pCAR demonstrated a significant enhancement of tumor re-stimulation potential and IL-2 release, reduced exhaustion marker expression and enhanced therapeutic efficacy in mice with established Nalm-6 leukemic xenografts. These data reinforce the evidence that the pCAR platform delivers enhanced anti-tumor activity through effective provision of dual co-stimulation. Greatest anti-tumor activity was noted for intermediate avidity CAR T-cells and derived pCARs, raising the possibility that effector to target cell avidity is an important determinant of efficacy.

Highlights

Chimeric antigen receptors (CARs) are fusion molecules that redirect lymphocyte specificity against cell surface targets
We focused on CARs that contain a CD28+CD3z endodomain since CD19 expression is most commonly retained at the time of disease relapse, following infusion of these cells [16,17,18]
We have recently demonstrated superior anti-tumor function of pCAR T-cells, in which a CD28-containing 2G CAR is coexpressed with a 4-1BB-containing co-stimulatory receptor (CCR) [10]

Summary

Introduction

Chimeric antigen receptors (CARs) are fusion molecules that redirect lymphocyte specificity against cell surface targets. The key event that propelled this technology to become the largest growth area in immunooncology [1] was the inclusion of either CD28 or 4-1BB costimulatory elements within the CAR endodomain [2, 3]. Evaluation of these so-called second generation (2G) CARs in human T-cells revealed that they confer a significantly enhanced capacity to mediate target-dependent proliferation and cytokine release, when compared to first generation (1G) receptors that only deliver an activating signal [4, 5]. Given the therapeutic failure of earlier designs, this highlights the vital importance of co-stimulation in this clinical breakthrough

Methods

Results

Conclusion