Engineering Cytoplasmic Signaling of CD28ζ CARs for Improved Therapeutic Functions

Abstract

Chimeric antigen receptor modified T cells (CAR-T) have yielded impressive clinical outcomes in treating hematopoietic malignancies. However, relapses have occurred in a substantial number of patients and limited the development of CART therapy. Most underlying reasons for these relapses can be attributed to poor persistence and rapid exhaustion of CART cells in vivo. Despite multiple strategies having been developed, how to improve CART persistence or resist exhaustion while maintaining sufficient cytotoxic functions is still a great challenge. Here we discuss engineering cytoplasmic signaling as an important strategy for CAR optimization. This review summarizes recent advances showing that the anti-tumor function of CART cells can be improved by optimizing the CD3ζ domain or downstream signaling of CD28ζ CAR.

CAR-T Cell Performance: How to Improve Their Persistence?

“…CD3ζ ITAMs have different roles in the regulation of T-cell activation; for example, mutations of CD3ζ ITAM1 and ITAM2 significantly impaired signal transduction and induced cell death. However, mutation of CD3ζ ITAM3 did not induce cell death, but rather increased IL-2 secretion and MAPK phosphorylation ( 99 , 100 ). CD28ζ-based CAR-T cells that only contain the ITAM1 domain resulted in higher percentages of T SCM and T CM and a lower fraction of T EFF cells and yielded long-lasting and complete tumor remission in an in vivo animal model ( Figure 4 ) ( 101 ).…”

“…An ITAM domain consists of two consecutive YxxL/I motifs separated by a defined number of amino acids (YxxL/I-X6−8-YxxL/I) ( 98 ). TCR binding to the peptide–MHC complex leads to the activation of the Src family kinase Lck, which phosphorylates two tyrosine residues in each of the CD3ζ ITAMs ( 97 , 99 ). CD3ζ ITAMs have different roles in the regulation of T-cell activation; for example, mutations of CD3ζ ITAM1 and ITAM2 significantly impaired signal transduction and induced cell death.…”

“…The CD28 cytoplasmic domain contains a YMNM motif that gets phosphorylated upon binding to CD80/CD86 ligands, which can bind to Grb2/Gads through the asparagine residue ( 102 ). Besides, the CD28 proline-rich regions can interact with Itk, Tec, Lck, Grb2/Vav, and filamin A (FLNA) ( 99 , 103 ). CAR-T cells bearing a mutated CD28 domain (CD28-YMFM) promote a long-lasting antitumor control ( Figure 4 ).…”

TRAIL-R1-targeted chimeric antigen receptor T cells exhibit dual antitumor effects

“…Although, the role of costimulatory signals in CAR-T development is well-established, the optimal costimulatory domains for CAR-T cells remains to be defined, and should be evaluated case-by-case in order to fine-tune immunotherapy approaches [37,38]. Literature reported that the choice of 4-1BB signaling domain in CARs conferred improved selectivity for higher tumor antigen density, reduced T cell exhaustion phenotype and reduced basal T-cell activation [39,40].…”

Understanding CAR T cell-tumor interactions: Paving the way for successful clinical outcomes

“…20,[69][70][71][72] However, despite intensive research, the exact molecular mechanism of CAR-induced T cell activation is still not well understood. 73 It is known that signal transmission plays a key role in activation, but the mechanism of CAR signal transduction differs from that of the native TCR regarding its activation patters. 40,74 A side-by-side investigation using bulk and single-cell RNA sequencing showed distinct but similar transcriptional signatures between CD19-targeting CAR and TCR signaling.…”