Trispecific CD19-CD20-CD22–targeting duoCAR-T cells eliminate antigen-heterogeneous B cell tumors in preclinical models

Abstract

A substantial number of patients with leukemia and lymphoma treated with anti-CD19 or anti-CD22 monoCAR-T cell therapy relapse because of antigen loss or down-regulation. We hypothesized that B cell tumor antigen escape may be overcome by a chimeric antigen receptor (CAR) design that simultaneously targets three B cell leukemia antigens. We engineered trispecific duoCAR-T cells with lentiviral vectors encoding two CAR open reading frames that target CD19, CD20, and CD22. The duoCARs were composed of a CAR with a tandem CD19- and CD20-targeting binder, linked by the P2A self-cleaving peptide to a second CAR targeting CD22. Multiple combinations of intracellular T cell signaling motifs were evaluated. The most potent duoCAR architectures included those with ICOS, OX40, or CD27 signaling domains rather than those from CD28 or 4-1BB. We identified four optimal binder and signaling combinations that potently rejected xenografted leukemia and lymphoma tumors in vivo. Moreover, in mice bearing a mixture of B cell lymphoma lines composed of parental triple-positive cells, CD19-negative, CD20-negative, and CD22-negative variants, only the trispecific duoCAR-T cells rapidly and efficiently rejected the tumors. Each of the monoCAR-T cells failed to prevent tumor progression. Analysis of intracellular signaling profiles demonstrates that the distinct signaling of the intracellular domains used may contribute to these differential effects. Multispecific duoCAR-T cells are a promising strategy to prevent antigen loss–mediated relapse or the down-regulation of target antigen in patients with B cell malignancies.

Locally secreted BiTEs complement CAR T cells by enhancing solid tumor killing

“…However, loss of EGFRvIII did not have a significant influence on wild-type amplified EGFR expression in these tumors ( 18 ). A multivalent targeting strategy was demonstrated as a valid way in mitigating antigen loss to treat hematological and solid tumors ( 15, 17, 21–23 ). In this study, we also detected heterogeneously expressed EGFRvIII and EGFR in freshly resected glioma samples.…”

“…Despite the prominent effect achieved by CAR T cells and BiTEs in the treatment of leukemia and lymphoma, a significant number of treated patients have had limited antitumor activity or tumor relapse ( 1, 7, 14 ). Antigen loss or downregulation is a common mechanism of treatment resistance, which could be mitigated by combinatorial targeting of multiple antigens ( 15–17 ). Antigen loss was also observed in solid tumors after CAR T cell therapy.…”

Overcoming Tumor Heterogeneity by Ex Vivo Arming of T Cells Using Multiple Bispecific Antibodies

“…Multi-antigen targeted strategies have the potential to overcome these antigen escape mechanisms. Simultaneous targeting CD19/CD20 or CD19/CD20/CD22 using "OR" logic-gated tandem CAR T cells [25] reduced or prevented target antigen escape, offering an advantage over single CAR T cells or pooled populations of monospecific CAR T cells [26][27][28][29]. Given the minimal requirement of BsAb (only 500 to 5,000 molecules) per T cell for anti-tumor activity [13], multiple BsAbs built on the same IgG-[L]-scFv platform can be installed on each T cell before the maximum capacity is reached (30,000 to 56,000 molecules per T cells [13,23]).…”

Case Report: Post-CAR-T Infusion HBV Reactivation in Two Lymphoma Patients Despite Entecavir Preventive Therapy

“…In the past two decades, CAR T-cell therapy has been rapidly emerging as a promising novel treatment for hematological malignancies (6)(7)(8). CAR T-cell cocktail infusion strategy can reduce tumor antigen escape and improve therapeutic effects (12,13). Therefore, CAR T-cell cocktail therapy with anti-CD19 and anti-CD22 CAR T-cell infusion was selected for these two patients.…”