Development of CAR-T Cell Constructs with Broad Anti-Tumor Tropism
Authors: Mishra Ameet K. , Kemler Iris , Dingli David 
Published: February 2021

Chimeric antigen receptor T (CAR-T) cell therapy is a transformative approach to cancer eradication. CAR-T is expensive in part due to the restricted use of each CAR construct for a specific set of tumors such as B cell lymphoma targeted with CD19 and multiple myeloma targeted with BCMA. A CAR construct with broad anti-tumor activity can be advantageous due to wide applicability and scalability of production. We show that CD126, the IL-6 receptor alpha, is an antigen that is expressed by many hematologic and solid malignancies including multiple myeloma, non-Hodgkin lymphoma, acute myeloid leukemia, pancreatic and prostate adenocarcinoma, non-small cell lung cancer and malignant melanoma amongst others. High CD126 expression is a negative prognostic marker in many malignancies. The two CD126 targeting CAR-T cell constructs contain the CD28 anchoring domain followed by 4-1BB and CD3 zeta signaling domain. Lentiviral vectors were generated with triple plasmid (CAR, psPAX2 and pMD2.G) transfection of 293T cells and the vector concentrated by ultracentrifugation and used to transduce human T cells. T cells were isolated from leuko-reduction cones using negative selection with magnetic beads. The transduction efficiency was around 60%. The T cells were activated with anti-CD3/CD28 beads and expanded for two weeks before using for downstream experiments. CD126 CAR-T cells are able to kill many tumor cells in an antigen specific manner and with an efficiency that is directly proportional to the cell surface expression of CD126 expression (rho = 0.6, p = 0.0019). The presence of soluble CD126 in the culture media did not interfere with CAR-T cell killing. The CAR-T constructs bind murine CD126. However, injection of CD126 targeting CAR-T cells in NSG mice did not lead to any evidence of hepatotoxicity and weight loss despite possible expression of this antigen on hepatocytes. In vivo studies in NSG mice with multiple myeloma (RPMI-8226) and prostate adenocarcinoma (DU-145) xenograft models (n=10 tumors per group) showed that the intravenously injected CD126 targeted CAR-T cells (107) infiltrated the tumors, expanded, produced human interferon gamma and killed the tumor cells (p&lt;0.001). Bioluminescence imaging showed control of tumor growth in the actively treated tumors compared to the controls (p&lt;0.05). At post mortem, mice injected with CD126 targeted CAR-T cells had smaller residual tumors compared to controls injected with non-engineered human T cells from the same donor. Binding of sIL-6R by CAR-T cells could mitigate cytokine release syndrome. In support of this, murine SAA-3 levels (the equivalent of human CRP) were lower in mice injected with CD126 CAR-T compared to controls (p&lt;0.05), suggesting that binding of sIL-6R by CAR-T cells could mitigate cytokine release syndrome. CD126 provides a novel therapeutic for CAR-T cells in a broad variety of tumors with low risk of toxicity.

Disclosures
Dingli: Apellis: Consultancy; Millenium: Consultancy; Janssen: Consultancy; Bristol Myers Squibb: Research Funding; Sanofi-Genzyme: Consultancy; Alexion: Consultancy; Rigel: Consultancy; Karyopharm Therapeutics: Research Funding.