mSA2 affinity-enhanced biotin-binding CAR T cells for universal tumor targeting

Abstract

Chimeric antigen receptor T cells (CAR-Ts) are promising cancer therapeutics. However, since cancer cells can lose the CAR-targeted antigen and avoid destruction, targeting multiple antigens with multiple CARs has been proposed. We illustrate here a less cumbersome alternative, anti-tag CARs (AT-CARs) that bind to tags on tumor-targeting antibodies. We have created novel AT-CARs, using the affinity-enhanced monomeric streptavidin 2 (mSA2) biotin-binding domain that when expressed on T cells can target cancer cells coated with biotinylated antibodies. Human T cells expressing mSA2 CARs with CD28-CD3ζ and 4-1BB-CD3ζ signaling domains were activated by plate-immobilized biotin and by tumor cells coated with biotinylated antibodies against the tumor-associated antigens CD19 and CD20. Furthermore, mSA2 CAR T cells were capable of mediating cancer cell lysis and IFNγ production in an antibody dose-dependent manner. The mSA2 CAR is a universal AT-CAR that can be combined with biotinylated tumor-specific antibodies to potentially target many different tumor types.

Keywords: CAR; Chimeric antigen receptor; adoptive cell therapy; cancer immunotherapy; synthetic biology; tag-CAR.

Figure 1.

Schematic of the function and vector design of mSA2 anti-biotin CARs. (A) A tumor-specific antibody that is biotinylated encounters and binds to its target antigen on a tumor cell while simultaneously providing biotin as a new target. Avidin-bearing mSA2 CAR T cells recognize the biotin as their specific target, which leads to their activation, cytokine production and tumor cell lysis. (B) Designs of the mSA2-41BBζ and mSA2-CD28ζ CAR lentiviral expression constructs and the positive control FMC63-CD28ζ anti-CD19 CAR construct.

Figure 2.

Cell surface expression of mSA2 CARs and activation of primary human T cells. (A) Primary human T cells were transduced with lentiviruses encoding the mSA2–41BBζ CAR, the mSA2-CD28ζ CAR or the FMC63-CD28ζ CAR. After one stimulation cycle, cells were sorted by flow cytometry for TagBFP expression and stained with biotin-FITC or for CD4 and CD8 expression. (B) CAR T cells were plated in the presence of the indicated plate-immobilized biotinylated antibody for 18 hours and assayed by flow cytometry for T cell activation markers CD69, CD107a, and CD62L (down-regulated upon activation).

Figure 3.

mSA2 CAR T cells display biotinylated antibody dose-responsive effector functions. mSA2-CD28ζ or mSA2–41BBζ cells were incubated at an E:T ratio of 10:1 with CD20+ Raji tumor targets in the presence of increasing concentrations of biotinylated anti-CD20 antibody Rituximab for 18 hours. (A) Cells were stained for activation markers CD69 (upper panels) and CD62 L (lower panels) and evaluated by flow cytometry. (B) Supernatants from co-incubations were evaluated by ELISA for IFNɣ production. (C) Co-incubations were performed similar to (A) and (B) however target cells were labeled with Cell Trace Yellow were further evaluated by flow cytometry for viability by staining with Ghost Dye Red. For (B) and (C), multiple ANOVA comparisons were performed. As the data did not have homogeneity of variance (Levene's test), Tukey's HSD was used for post hoc analysis between antibody conditions. “*” denotes a significance of p < .01 and “NS” stands for not significant. Error bars represent standard deviations for n = 3 replicates.

Figure 4.

mSA2 CAR T cells display biotinylated-antibody mediated effector functions against various target cell lines. Tumor target cells, CAR T cells, and 5 µg/ml (33.33 nM) of the indicated antibodies were incubated for 18 hours and (A) cells were stained for the activation marker CD69 and evaluated by flow cytometry. (B) Supernatants from co-incubations were evaluated by ELISA for IFNɣ production. (C) Co-incubations were performed similar to (A) and (B) however target cells were labeled with Cell Trace Yellow were further evaluated by flow cytometry for viability by staining with Ghost Dye Red. For (B) and (C), multiple ANOVA comparisons were performed. As the data did not have homogeneity of variance (Levene's test), Tukey's HSD was used for post hoc analysis between antibody conditions. “*” denotes a significance of p<.01 and “NS” stands for not significant. Error bars represent standard deviations for n = 3 replicates.