Logic-gated CAR-T cells safely target distant tumor cells

From the Riddell Laboratory, Clinical Research Division

In light of the successful FDA approval of CD19 chimeric antigen receptor or CAR-T cells, researchers are looking to expand the list of what antigens CAR-T cells can target. CAR-T cells utilize an antigen receptor consisting of a binding domain that allows the CAR-T cell to selectively bind target antigens, and a signaling domain that activates effector functions when the CAR-T encounters the antigen. Ideal target antigens for CAR-T cells are homogenously expressed within tumors, but not on normal tissues, which could cause toxicity if damaged by CAR-T cells.  Since loss of healthy B cells does not cause serious toxicity, an example of an acceptable target antigen is CD19, which is normally only expressed on malignant and healthy B cells.  Many target antigens are commonly expressed on epithelial tumors, but they are also expressed on some normal tissues. One attractive target is the receptor tyrosine kinase-like orphan receptor 1 (ROR1) that is expressed on many different solid tumors, including breast, lung, and ovarian cancer. However, ROR1 is also expressed on some normal cells, making toxicity a potential risk of targeting this molecule with CAR-T cells. In a study led by Dr. Shivani Srivastava from the Riddell laboratory (Clinical Research Division), a new approach to engineered ROR1 CAR-T cells that could target ROR1 expressing tumor cells while ignoring normal cells expressing ROR1 is described. In this study, published in the journal Cancer Cell, the authors developed what are known as logic-gated CAR T cells, meaning that they require the presence of two antigens to target cells for destruction. In these logic-gated CAR-T cells, the binding of one antigen by a synthetic Notch (synNotch) induces the expression of the CAR. Only upon expression of CAR can the CAR-T cells attack specific cells.

Logic-gated CAR-T cells exhibit anti-tumor activity without toxicity if tumor and normal tissue are separated.
Logic-gated CAR-T cells exhibit anti-tumor activity without toxicity if tumor and normal tissue are separated. Image provided by Dr. Shivani Srivastava

Because ROR1 is expressed on several normal tissues, the authors first tested the safety of ROR1 CAR-T cell therapy in a mouse model. They transduced murine T cells with a CAR specific to an epitope of ROR1 conserved in humans and mice. When the researchers transferred ROR1 CAR-T cells into mice pre-conditioned with radiation or cytotoxic chemotherapy to increase engraftment of the T cells, they found that the mice showed signs of toxicity, including weight loss and a decrease in red blood cells and platelets. They found that ROR1 CAR-T cells accumulated in the spleen and bone marrow where they attacked ROR1+ stromal cells critical for recovery from hematopoietic stress. The authors further characterized the ROR1+ stromal cells and found that they do not express EpCAM, an antigen expressed on murine mammary carcinoma cells known as 4T1 cells. They used this knowledge to design logic-gated ROR1 CAR-T cells with an EpCAM-specific synNotch receptor to control expression of the CAR. The authors tested these cells by transferring them into preconditioned mice with 4T1 tumors and found that they exhibited anti-tumor activity compared to untransduced control T-cells. In addition, the synNotch ROR1 CAR-T cells did not induce toxicity in bone marrow or spleen, showing that they were only activated in the tumor where there is high expression of EpCAM.

As the CAR in synNotch CAR-T cells has a half-life of around eight hours, the researchers wondered if the cells would cause toxicity if the tumor cells and other ROR1 expressing cells were in close proximity. They tested this by inoculating immunodeficient mice with a human lymphoma that expresses ROR1 and CD19 and metastasizes to the bone marrow. The authors transduced T cells with a synNotch receptor specific for human CD19 and a CAR specific for ROR1. While the CD19 synNotch ROR1 CAR-T cells demonstrated anti-tumor activity, they also caused toxicity. These synNotch CAR-T cells were unable to discriminate cancerous ROR1 expressing cells and normal stromal cells when the two were in close proximity. These results indicate that unless the tumor cells have metastasized to the bone marrow, logic-gated ROR1 CAR T cells may be safe to treat cancer in patients. These experiments demonstrate the power of logic-gating CARs, and future studies may continue to expand the repertoire of synNotch receptors. In addition, researchers may explore more complicated logic-gates that differentially affect the behavior of CAR T cells depending on which cells they encounter. Dr. Srivastava said, “We found that our logic-gated CAR-T cells could discriminate tumors from normal tissue well if the cells were spatially separated in different anatomical sites but failed to do so if the cells were highly co-localized in the same organ like the bone marrow. Our lab is working on developing other engineering strategies that would enable a T cell to tell apart tumor from normal cells even if the cells are right next to each other, which will be important for patients with advanced disease whose tumor has metastasized widely to different anatomical sites.”

This work was supported by the National Institutes of Health, Cancer Center Support Grant, and Juno Therapeutics.

Fred Hutch/UW Cancer Consortium members Drs. Jarrod Dudakov, Robert Pierce, and Stanley Riddell contributed to this research.

Srivastava S, Salter AI, Liggitt D, Yechan-Gunja S, Sarvothama M, Cooper K, Smythe KS, Dudakov JA, Pierce RH, Rader C, Riddell SR. 2019. Logic-Gated ROR1 Chimeric Antigen Receptor Expression Rescues T Cell-Mediated Toxicity to Normal Tissues and Enables Selective Tumor Targeting. Cancer Cell. 2019 Mar 18;35(3):489-503.e8. doi: 10.1016/j.ccell.2019.02.003.