Development of CT109-CAR-T, a novel CAR-T therapy for solid tumors manufactured at Point of Care (POC)

<h3>Background & Aim</h3> Carcinoembryonic antigen-related cell adhesion (CEACAM) molecules are complex macromolecules found to be over-expressed in various cancers linked to cancer development and progression. Orgenesis (ORGS) has developed two candidate CAR-T products (CT109-CAR-T), derived from the proprietary antibody (CT109). CT109 binds to a distinct epitope on CEACAM5 and CEACAM6 with no cross reactivity to other CEACAM family proteins. Our results suggest that CT109-CAR-T has high potency and specificity against CEACAM5/6 expressing target cells. Therefore, CT109-CAR-T has potential for treatment of solid tumors including pancreatic or colorectal cancers. <h3>Methodology</h3> PBMCs from 3 healthy donors were transduced using 3^rd generation lentiviruses encoding two CT109-CAR candidate constructs (LV-A, LV-B) and subsequently cultured in a G-Rex® to produce CT109-CAR-T cells. Critical quality attributes (CQAs) tested include cell growth, T-cell identity, and copy number. Activity was demonstrated through several potency-related CQA's including transduction rate, IFNg release, target dependent CAR-T cell proliferation, and cytotoxicity. <h3>Results</h3> Cell expansion (18-48-fold), viability (97-98%), and T-cell identity (>90% CD3+/CD45+) were within an acceptable range and similar between the two constructs. Transduction rate was higher with LV-A (22-47%) compared to LV-B (6.8-21%). CT109-CAR-T from both constructs proliferated in response to co-culture with antigen positive target cells (BxPC-3) at an E:T ratio of 1:1. IFNg release trended lower in LV-B CAR-T (Donor 1,2: 2471, 2127pg/mL) compared to LV-A CAR-T (Donor 1,2,3: ≥5000, 5852, 1033pg/mL) at E:T ratio 1:1. Importantly, significant cytotoxicity was observed for both CAR-T products using a qualified impedance-based killing assay. Cytolysis at 24-hours co-culture with BxPC-3 at 1:1 ratio was similar between the two constructs LV-A (Donor 1-3: 67-86%) and LV-B (Donor 1: 67%). <h3>Conclusion</h3> Significant process and analytical development were completed for the two candidate CT109-CAR-T therapies. It establishes ORGS point of care (POC) manufacturing platform is well positioned for production and product release can be completed with a small team in less than 36 hours by leveraging the qualified impedance-based killing assay which enables real-time remote monitoring by QC/QA and retrospective data analysis in an automated streamlined assay. The data supports the potential of CT109-CAR-T and future studies will evaluate the efficacy in animal models of solid tumors.