Compromised antigen binding and signaling interfere with bispecific CD19 and CD79a chimeric antigen receptor function

Abstract

Therapy with CD19 directed chimeric antigen receptor (CAR) T cells has transformed the treatment of advanced B-cell malignancies. However, loss of or low antigen expression can enable tumor escape and limit the duration of responses achieved with CAR-T cell therapy. Engineering bispecific CAR-T cells that target two tumor antigens could overcome antigen negative escape. We found that CD79a and b, which are heterodimeric components of the B cell receptor, were expressed on 84.3% of lymphoma cases by immunohistochemistry, and that 87.3% of CD79ab positive tumors coexpressed CD19. We generated three bispecific permutations: tandem, bicistronic and pooled products of CD79a-CD19 or CD79b-CD19 CAR-T cells and showed that bispecific CAR-T cells prevented the outgrowth of antigen negative cells in a CD19-loss lymphoma xenograft model. However, the tandem and bicistronic CAR-T cells were less effective than monospecific CD19 or CD79a CAR-T cells for the treatment of tumors that only expressed CD19 or CD79, respectively. When compared to monospecific CAR-T cells, T cells expressing a tandem CAR exhibited reduced binding of each target antigen and T cells expressing a bicistronic CAR vector exhibited reduced phosphorylation of downstream CAR signaling molecules. Our study showed that despite added specificity, tandem and bicistronic CAR-T cells exhibit different defects that impair recognition of tumor cells expressing a single antigen. Our data provide support for targeting multiple B cell antigens to improve efficacy and identify areas for improvement of bispecific receptor designs.

Sensitive bispecific chimeric T cell receptors for cancer therapy

“…Improving efficacy requires the development of synthetic receptors that are highly sensitive and capable of recognizing multiple tumor antigens. Bispecific CARs have been designed for this purpose but often exhibit reduced sensitivity for each individual antigen, enabling escape of tumor cells with low antigen levels [ 8 , 43 ]. Here, we describe the design and function of chimeric TCRs that simultaneously target two tumor antigens with high sensitivity.…”

“…Bispecific targeting with CARs can also be achieved with a bicistronic vector or by the infusion of two mono-specific CAR products either at the same time or sequentially. These strategies weren’t formally compared here but would not be predicted to provide better antigen sensitivity, and in the case of two products, add manufacturing complexity and cost [ 43 , 57 ]. Surprisingly the superior in vivo efficacy of the Bi-ChTCR T cells was observed in the absence of providing additional costimulation either in trans or intrinsic to the ChTCR [ 30 , 31 , 58 ].…”

Novel and multiple targets for chimeric antigen receptor-based therapies in lymphoma

“…In preclinical testing, bbT369 was more potent than CD19-CAR T and induced longer remission ( 124 ). Dual-targeting CAR T against CD19 and either CD79a or CD79b CARs are developed ( 125 , 126 ). Leung, et al., demonstrated that CD19/79a(b)-CAR T induced longer tumor control than single-antigen targeting CAR T from preventing antigen-loss relapse, and targeting CD79a was more potent than C79b.…”

“…Leung, et al., demonstrated that CD19/79a(b)-CAR T induced longer tumor control than single-antigen targeting CAR T from preventing antigen-loss relapse, and targeting CD79a was more potent than C79b. However, bispecific CAR T with either tandem or bicistronic CAR structures had reduced activity against single-antigen positive cells due to compromised antigen binding and signaling, indicating the need to optimize structural design ( 126 ). Low-level aberrant CD79b expression monocytes, hematopoietic progenitor cells and T-cells could theoretically cause untoward hematologic toxicity, which will be elucidated by clinical trials ( 127 ).…”

Programming CAR T Cell Tumor Recognition: Tuned Antigen Sensing and Logic Gating

“…In a recent study, Leung and colleagues found that CD19 and CD79a cotargeting with Tan- or Dual-BBζ CARs superseded pooled monospecific CARs in preventing antigen escape ( 65 ). Despite improved tumor control in vivo , the Tan-CAR configuration compromised antigen binding compared with monospecific CARs, consistent with previous observations ( 61, 66 ), whereas Dual-CAR exhibited diminished signaling, possibly owing to competition for downstream signaling molecules ( 65 ). Dual-CARs that employ a common hinge/transmembrane component may heterodimerize and function as parallel CARs (see below).…”