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

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.

Functional avidity of anti-B7H3 CAR-T constructs predicts antigen density thresholds for triggering effector function

“…Several previous studies have investigated avidity of CAR-T cells using the Lumicks platform. For example, the group of Maher et al studied a range of anti-CD19 binders in the context of leukemia therapy using conventional 2 nd generation CARs co-expressing a 4-1BB chimeric costimulatory receptor 27 . Interestingly, intermediate avidity CAR-T were identified to have the greatest in vivo capacity to control tumor growth.…”

Succinylation-associated lncRNA signature to predict the prognosis of colon cancer based on integrative bioinformatics analysis

“…Co-stimulatory receptor (CCR) was found to be may contribute to target cell docking under some circumstances 35 . There is considerable evidence that combined provision of both CD28 and 4-1BB co-stimulation can synergistically enhance T-cell immune responses 36 , 37 .…”

Adoptive neoantigen-reactive T cell therapy: improvement strategies and current clinical researches

“…Using transgenic TCR, co-expressing CD8 αβ with TCR and upregulating adhesion molecules can enhance MHC-TCR binding avidity and elevate signal-transducing efficiency [87][88][89]. Besides, engineering co-stimulatory signals is proposed to prolong T cell persistence and enhance anti-tumor activity, which can be achieved by coupling T cell activating signals (CD3ζ) with co-stimulating signals (CD28, OX40, 4-1BB), or using chimeric switch receptors which link exodomain of CTLA-4, PD-1 or TIGHT to intradomain of CD28 [90][91][92][93][94][95]. In addition, engineering cytokine receptors represented by orthogonal IL-2 has been found to enhance T cell antitumor function while attenuating the side effects caused by cytokine pleiotropy [96][97][98].…”