Universal Chimeric Antigen Receptors for Multiplexed and Logical Control of T Cell Responses

Abstract

T cells expressing chimeric antigen receptors (CARs) are promising cancer therapeutic agents, with the prospect of becoming the ultimate smart cancer therapeutics. To expand the capability of CAR T cells, here, we present a split, universal, and programmable (SUPRA) CAR system that simultaneously encompasses multiple critical "upgrades," such as the ability to switch targets without re-engineering the T cells, finely tune T cell activation strength, and sense and logically respond to multiple antigens. These features are useful to combat relapse, mitigate over-activation, and enhance specificity. We test our SUPRA system against two different tumor models to demonstrate its broad utility and humanize its components to minimize potential immunogenicity concerns. Furthermore, we extend the orthogonal SUPRA CAR system to regulate different T cell subsets independently, demonstrating a dually inducible CAR system. Together, these SUPRA CARs illustrate that multiple advanced logic and control features can be implemented into a single, integrated system.

Keywords: CAR T therapy; cellular immunotherapy; chimeric antigen receptors; immune cell engineering; synthetic biology; systems biology.

Figure 1. Design and characterization of the SUPRA CAR system

(A) Comparison between the conventional CAR and SUPRA CAR design. A SUPRA CAR system is composed of a zipCAR and zipFv. A zipCAR has a leucine zipper as the extracellular portion of the CAR and zipFv has a scFv fused to a cognate leucine zipper that can bind to the leucine zipper on the zipCAR. (B) A SUPRA CAR system targeting multiple tumor antigens using different zipFvs. K562 cells expressing Her2, Mesothelin, or Axl were co-cultured in vitro with RR zipCAR expressing CD8+ human primary T cells (n=3, mean ± SD). (C) Variables explored for characterization of the SUPRA CAR system: (1) the affinity between leucine zipper pairs, (2) the affinity between tumor antigen and scFv, (3) the concentration of zipFv, and (4) the expression level of zipCAR. (D) Effect of concentration of three zipFvs with leucine zippers (SYN 3, SYN5, and EE) that have different affinities to RR zipCAR on the IFN-γ production by primary CD4+ T cells (n=3, mean ± SD). (E) Effect of zipper affinity, scFv-tumor affinity, and zipCAR expression level on the IFN-γ production by primary CD4+ T cells expressing RR zipCAR (n=3, mean).

Figure 2. Utilizing SUPRA CAR for OFF switch function and combinatorial antigens targeting

(A) (Left) A Schematic diagram of the SUPRA CAR system with an OFF switch zipFv. Three competitive leucine zippers that can bind to the EE leucine zipper with different affinities are used to tune the T cell activation level. (Right) A cytotoxicity plot demonstrating the effect of competitive zipFvs (n=3, mean ± SD). (B) A cytotoxicity plot of “OR” gate implementation of the SUPRA CAR system. Her2+/Axl+ K562 tumor cells were co-cultured with RR zipCAR expressing CD8+ T cells with different zipFv combinations (n=3, mean ± SD). (C) Using the SUPRA CAR system as cell selector. Cells either expressed Her2 or Her2 and Axl. Axl acted as a “safe marker” that can inhibit SUPRA CAR T cell activity (n=3, mean ± SD, statistical significance was determined by Student’s t test, *** = p ≤0.001).

Figure 3. In vivo activity of SUPRA CAR in SK-BR-3 and Jurkat xenograft models

(A) (Left) The tumor burden was quantified as the total flux (photons/sec) from the luciferase activity of each mouse using IVIS imaging. Compared to RR zipCAR or tumor only control groups, the RR zipCAR + EE zipFv group showed significantly reduced tumor burden, comparable to conventional Her2 CAR (red arrow indicates injection of engineered CD8+ T cells and highlighted region indicates injection of zipFv (every 2 days at 5mg/kg for 2 weeks)). (Right) Representative IVIS images of groups treated with (1) no T cells, (2) conventional Her2 CAR, (3) RR zipCAR, and (4) RR zipCAR with EE zipFv at day 41 (n=4, mean ± SEM). (B) Representative IVIS images of groups treated with (1) no T cells, (2) RR zipCAR, (3) α-Her2 RR zipFv with RR zipCAR (non-binding), and (4) α-Her2 EE zipFv with RR zipCAR (complete SUPRA) (day 57, Figure S4B). (C) Tumor burden as total flux (photons per sec) of each mouse shown in Figure 3B (n=4, mean ± SEM). (D) In vivo IFN-γ cytokine level after 24 hours of initial CD8+ T cells and zipFv injection (n=4, mean ± SD). (E) (Left) Jurkat tumor cells were injected intravenously on day 0 to immune-compromised NSG mice. At day 3, primary human CD8+ T cells expressing RR zipCAR were injected once (red arrow) with α-Her2-EE zipFv which was dosed every day for 6 days at 3mg/kg (highlighted). The tumor burden was quantified as the total flux (photons/sec) from the luciferase activity of each mouse using IVIS imaging. (Right) Representative IVIS images of groups treated with (1) no T cells, (2) conventional Her2 CAR, (3) RR zipCAR, and (4) RR zipCAR with EE zipFv at day 21 (n=4, mean ± SEM, statistical significance was determined by Student’s t test, *= p ≤0.05, ***= p ≤0.001).

Figure 4. In vivo control of cytokine production by the SUPRA CAR

(A) In vivo IFN-γ cytokine level at 24 hr. In vivo cytokine level increased in a dose-dependent manner (n=4, mean ± SD). (B) In vivo IFN-γ cytokine level at 24hr, demonstrating a leucine zipper affinity-dependent increase of in vivo IFN-γ cytokine (n=4, mean ± SD). (C) In vivo IFN-γ cytokine level demonstrating the effect of competitive zipFv (n=4, mean ± SD, the statistical significance was determined by Student’s t test, * = p ≤0.05, *** = p ≤0.001).

Figure 5. Controlling different signaling domains with orthogonal SUPRA CARs

(A) Design of orthogonal SUPRA CARs that control either CD3ζ or CD28/4-1BB signaling domains. The RR zipCAR and FOS zipCAR contain CD28/4-1BB co-stimulatory and CD3ζ signaling domain, respectively. α-Her2 zipFv binds to the FOS zipCAR and activates the CD3ζ domain, whereas the α-Axl zipFv binds to the RR zipCAR and activates CD28/4-1BB co-stimulatory domains. CD69 expression, IFN-γ, IL-2, and IL-4 secretion were measured as outputs. (B) Amount of each zipFv was varied to define signaling strength from each receptor. CD69 expression (left) and IFN-γ secretion (right) were measured (n=3, data are represented as mean).

Figure 6. Controlling different cell types with orthogonal SUPRA CARs

(A) Design of orthogonal SUPRA CARs that control either CD4+ or CD8+ human primary T cells. The RR zipCAR and FOS zipCAR control CD4+ and CD8+ T cells activity, respectively. α-Axl zipFv binds to RR zipCAR and activates CD4+ T cells. α-Her2 zipFv binds to the FOS zipCAR and activates CD8+ T cells. (B) The CD69 expression and (C) IFN-γ measurements showing independent control of CD4+ and CD8+ T cells with orthogonal SUPRA CARs (n=3, mean ± SD). (D) Forward- and side-scatter FACS plots of the cell mixture after 24 hours co-culture of T cells (both CD4+ and CD8+, blue) with Her2+/Axl+ K562 tumor cells (orange). Tumor cells are killed efficiently when CD8+ cytotoxic T cells were activated by the α-HER2 zipFv (representative of three biological replicates).

Figure 7. Engineering a prosthetic immune system with SUPRA CARs

(A) The SUPRA CAR system enables the flexible and advanced control of signaling in T cells, reminiscent of an audio mixing console for controlling audio signals. The affinity and dosage of each zipFv are like knobs and dials on the mixing console, which can be varied to achieve user-defined T cell activation levels. Different orthogonal pairs of SUPRA CARs are like different channels, which can be utilized to control different signaling pathways in same cells. (B) The SUPRA CAR system can also be implemented in other cell types, thus setting the foundation for creating a prosthetic immune system.