(D-156) Engineering controllable universal chimeric antigen receptor T cells for solid tumor treatment

Introduction:: Cell-based immunotherapies, specifically chimeric antigen receptor (CAR) T cell-based therapies, have exhibited remarkable success in treating hematological malignancies. However, they still face formidable challenges in treating solid tumors, including antigen escape and tumor heterogeneity, which are difficult to be overcome by single antigen-targeting CAR T cells. The universal (or switchable) CAR design, which targets a specific molecule that can be conjugated to the different antibodies thus allowing simultaneous targeting of different antigens with the same CAR, provides a potentially powerful strategy to address these issues. Nonetheless, the clinical use of universal CARs is hampered by two major issues: (1) the potential risk on immunogenicity associated with currently available universal CARs and (2) the increased risk of on-target off-tumor toxicity due to the broadened targeting ability, thus making normal tissues more susceptible to be targeted.

Here, we sought to (1) develop a novel universal CAR with low immunogenecity risk, and (2) develop a method to control the in vivo activivty of universal CAR in both space and time, thus providing safe and effective universal CAR based immunotherapy that can address the antigen escape and tumor heterogeneity issues (Fig.1a).


Materials and Methods:: For (1): we have developed a monobody with high affinity for phycoerythrin (PE) via directed evolution (named as PEbody). By combining PEbody with CD28 transmembrane and cytosolic domains, 4-1BB cytotoslic domain, and CD3z cytosolic domain, we have generated a third generation universal CAR (named as PEbody CAR, Fig. 1b-c). Unlike other universal CARS, as PEbody is derived from human type III fibronectin, the PEbody CAR is fully humanized; PE is also considered to have minimal immunogenecity with long history of applications, thus the PEbody CAR system should have minimal immunogenecity risks for patients.

For (2): remote-controllable CAR T cells, such as light- or focused ultrasound-inducible CAR T cells, have demonstrated great potentials in reducing the adverse effects of CAR T therapies including on-target off-tumor cytotoxicity and cytokine storm. To regulate the in vivo activity of PEbody CAR, here we adopted the tamoxifen-gated photo-activatible gene expression system (TamPA-Cre), and developed light-controllable PEbody CAR T cells (Fig. 2a). To further enhance the safety and persistance of the inducible PEbody CAR T cells, a new strategy featuring light-inducible synNotch-activated PEbody CAR-enhanced tumor remission (named as LI-SABER) was developed (Fig. 3a-b). The light-inducible antigen expression on tumor cells can define the CAR T activation region, and the synNotch mechanism can confine CAR T activity in the tumor region while providing long-term PEbody CAR expression on the engineered T cells.

Results, Conclusions, and Discussions:: Results and Discussion
(1) PEbody CAR. PEbdy CAR can be used to target different antigens and tumor cell types (Fig,1d).

(2) Light-inducible PEbody CAR T cells. TamPA-Cre can successfully control the PEbody CAR expression and cytotoxicity in T cells by light (Fig. 2b-c).

(3) Addressing antigen escape and tumor heterogeneity by light-inducible PEbody CAR T cells. An antigen escape model was generated based on CD19+ and CD19- Nalm6 cells (Fig. 3a). When antigen escape happened, the CD19-targeting PEbody CAR T cells showed reduced cytotoxicity, which can be fully restored by switching to the PE-conjugated CD20 antibody (Fig. 2d). In each case, the cytotoxicity of PEbody CAR T cells can be well regulated by light (Fig. 2e). Additionally, a heterogeneous tumor model was generated based on prostate cancer cell line PC-3 cells (PSMA+/CD19- and PSMA-/CD19+, 1:1 mixture). With antibody targeting either CD19 or PSMA, the PEbody CAR T cells were not able to kill both populations, while with both antibodies present, the PEbody CAR T cells showed increased cytotoxicity against the heterogeneous PC-3 cells (Fig. 2f).

(3) Light-inducible synNotch-activated PEbody CAR-enhanced tumor remission (LI-SABER). The LI-SABER system was demonstrated using engineered MDA-MB-231 cells (Fig. 3c). Furthermore, an in vivo tumor mouse model was generated with subcutaneous inoculation of non-engineered breast cancer cell line MDA cells on both flanks, with one side mimicing target tumor and the other side mimicing normal tissues with similar antigen profile (Fig. 3d, upper). To apply the LI-SABER system for tumor killing, tumor cells were transduced by adeno-associated virus, encoding the light-inducible antigen expression system, and synNotch-PEbody CAR T cells were injected via tailvein, followed by antibody injection which facilitates the PEbody CAR T cell killing (Fig. 3d, lower). Tumor remission was observed on the blue light-illuminated side (target tumor), while minimal cytotoxicity was observed on the other side (mimicing normal tissue), thus LI-SABER can provide safe and effective tumor control (Fig. 3e).

Conclusions:
(1) A fully humanized universal CAR was developed.
(2) Controllable universal CAR T cells can provide safe and effective treatment for solid tumors.

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