T Cells Expressing CD19/CD20 Bispecific Chimeric Antigen Receptors Prevent Antigen Escape by Malignant B Cells

Abstract

The adoptive transfer of T cells expressing anti-CD19 chimeric antigen receptors (CARs) has shown remarkable curative potential against advanced B-cell malignancies, but multiple trials have also reported patient relapses due to the emergence of CD19-negative leukemic cells. Here, we report the design and optimization of single-chain, bispecific CARs that trigger robust cytotoxicity against target cells expressing either CD19 or CD20, two clinically validated targets for B-cell malignancies. We determined the structural parameters required for efficient dual-antigen recognition, and we demonstrate that optimized bispecific CARs can control both wild-type B-cell lymphoma and CD19(-) mutants with equal efficiency in vivo To our knowledge, this is the first bispecific CAR capable of preventing antigen escape by performing true OR-gate signal computation on a clinically relevant pair of tumor-associated antigens. The CD19-OR-CD20 CAR is fully compatible with existing T-cell manufacturing procedures and implementable by current clinical protocols. These results present an effective solution to the challenge of antigen escape in CD19 CAR T-cell therapy, and they highlight the utility of structure-based rational design in the development of receptors with higher-level complexity. Cancer Immunol Res; 4(6); 498-508. ©2016 AACR

Fig. 1

Schematics of single-input and bi-specific CARs. A, a second-generation, single-input CAR. B, a bi-specific, OR-gate CAR. C, schematic of four OR-gate CARs containing variations in extracellular spacer length and ordering of scFv domains. Hinge, CH2, and CH3 are domains within human IgG4. CD28tm is the CD28 transmembrane domain. EGFRt was fused to all CAR constructs via a T2A cleavage peptide to facilitate staining for CAR expression and sorting.

Fig. 2

Functional CD19-OR-CD20 CARs can be constructed by linking scFv domains in tandem. A, IFNγ, TNFα, and IL2 production by T cells expressing single-input or OR-gate CARs. Cytokine levels in media were measured after a 24-h co-incubation with K562 target cells. B, target-cell lysis activity of CD19-OR-CD20 CARs following a 4-hour co-incubation with K562 target cells. Reported values are the mean of triplicates, with error bars indicating one standard deviation (SD). Results are representative of two independent experiments performed with CAR-T cells derived from two different donors.

Fig. 3

Rational structural modifications improve OR-gate CAR activity against mutant tumor cells. A, design of 20–19 Short CARs incorporating linkers with increased length and/or rigidity. B, CD69, CD137, and CD107a surface expression (in median fluorescence intensity; MFI) by CAR–T cells after a 24-h co-incubation with CD19⁻ Raji cells. C, IFNγ, TNFα, and IL2 production by the CAR–T cells in (B). D, cell lysis by single-input and OR-gate CAR–T cells after 4-h co-incubation with WT and CD19⁻ Raji cells. Reported values are the mean of triplicates, with error bars indicating one SD. P-values were calculated by two-tailed Student t test; *: P < 0.05; **: P < 0.01. Data in B-E are representative of two independent experiments performed with CAR-T cells derived from two different donors.

Fig. 4

CD19 and OR-gate CAR T-cells exhibit similar differentiation and cell proliferation following antigen stimulation. A, distribution of T-cell subtypes prior to co-incubation with WT Raji cell targets and 6 d post target-cell addition. Cells were surface-stained for CD45RA and CCR7 expression. B, T-cell proliferation after co-incubation with WT Raji cells. CD19 and OR-gate CAR–T cells show similar proliferation while CD20 CAR–T cells show significantly less proliferation compared to CD19 CAR–T cells (P < 0.01 for all time points except for day 6). C, expansion of mixed CD19 and CD20 CAR–T cells stimulated with WT Raji cells over 8 days. Values shown are the mean of triplicates, with error bars indicating one SD. P-values were calculated by two-tailed Student t test; n.s.: not significant (P > 0.1); *: P < 0.05; **: P < 0.01. Results are representative of three independent experiments using CAR-T cells derived from three different donors.

Fig. 5

OR-gate CAR–T cells maintain robust lysis capability through repeated antigen stimulation. Mock-transduced and CAR–T cells were co-incubated with WT Raji targets for 6 days, and then co-incubated with CD19⁻ mutant Raji targets for another 6 d. A, survival of WT Raji cells co-incubated with effector T cells. B, survival of CD19– Raji cells co-incubated with T cells that were previously challenged with WT Raji cells. (Note: mock-transduced T cells had been overwhelmed by WT Raji by day 6 and were not rechallenged with mutant Raji.) C, Exhaustion marker staining of CAR–T cells before antigen stimulation, 48 h after co-incubation with WT Raji (Day 2), and 48 h or 6 d after subsequent co-incubation with CD19⁻ Raji cells (Days 8 and 12, respectively). At each time point, cells were surface-stained for Lag-3, Tim-3, and PD-1 and then analyzed for the simultaneous expression of one, two or three markers. Values shown are the mean of triplicates, with error bars indicating one SD. Results are representative of three independent experiments using CAR-T cells derived from three different donors.

Fig. 6

OR-gate CARs abrogate the effects of antigen escape in vivo. A, tumor progression in NSG mice bearing WT or mixed (75% WT, 25% CD19–) Raji xenografts. Bioluminescence imaging was performed on days 6, 18, and 21 post tumor injection (T cells were injected on day 7). B, survival of mice bearing mixed Raji tumor xenografts and treated with T cells expressing no CAR, the single-input CD19 CAR, or OR-gate CARs. n = 5 in all test groups. P-values were calculated by log-rank test analysis; n.s.: not significant (P > 0.1); *: P < 0.1; **: P < 0.05. Results represent one independent trial.

Fig. 7

CD19 and OR-gate CAR–T cells persist, upregulate PD-1, and exhibit similar differentiation subtypes in vivo. A, presence of CAR–T cells in peripheral blood 3 days after T-cell injection. Cells from retro-orbital blood were stained for the expression of human CD8 and EGFRt, which was co-translated with each CAR as a T2A fusion. B, percentage of CAR–T cells present in the bone marrow collected at the time of sacrifice. C, PD-1 expression of CAR–T cells in the bone marrow of animals bearing WT Raji tumors. D, pre-injection and post-mortem T-cell subtype distributions. “WT Raji” and “Mixed Raji” labels indicate the type of tumor engrafted in the animal from which the T cells were harvested. P-values were calculated by two-tailed Student t test; n.s.: not significant (P > 0.1); *: P < 0.05; **: P < 0.01.