Engineering Next-Generation Mesothelin CAR-T cells by Harnessing Naïve and Early Memory T cells

Abstract

Background & Aim

CAR-T cells for the treatment of patients with solid tumors, including mesothelin CAR-Ts targeting mesothelioma, have shown limited clinical efficacy to date. It is hypothesized that one barrier to response is lack of T cell persistence, a quality associated with naïve and early memory T-cells. We therefore designed a GMP process to manufacture mesothelin CAR-Ts directly from these minimally differentiated T cells. CD62L is expressed on naïve, stem/central memory, and central memory T-cells but is also expressed on monocytes and B cells. Because monocytes can inhibit transduction and expansion, we depleted CD14⁺ monocytes prior to CD62L enrichment using a two-step magnetic selection.

Methodology

A total of 4 mononuclear cell (MNC) apheresis products from healthy donors were incubated with CD14 magnetic beads on the Sepax C-Pro, depleted for CD14⁺ monocytes on the CliniMACS Plus, and enriched for CD62L⁺ cells on the CliniMACS Prodigy. T-cells were activated using TransAct stimulation reagent plus IL-2, IL-7, and IL-15, followed by lentiviral transduction with a CAR expressing a novel scFv (hYP218) targeting the membrane-proximal domain of mesothelin. Cells were expanded on the CliniMACS Prodigy for 7-9 days, with a target harvest of day 7 for clinical products.

Results

Using the two-step magnetic selection process, we purified CD62L⁺ cells from an initial frequency of 54 - 76% CD62L⁺ of CD3⁺ to ≥99% CD62L⁺ of CD3⁺ (98% of total), while also depleting ≥94% of CD14⁺ monocytes, demonstrating successful purification. From 8.8 ± 1.5 × 10⁹ total MNCs, we isolated 1.8 ± 0.5 × 10⁹ CD62L⁺CD3⁺ T-cells, recovering 58% of CD62L⁺CD3⁺ cells on average during enrichment. We next initiated 55 × 10⁶ T-cells into culture, which expanded to 2.2 ± 0.4 × 10⁹ cells by day 7. T-cell purity increased from 83.1 ± 4.1% CD3⁺ on day 0 to above 96% on day 7 with 40-60% transduction at MOI=3.4. Approximately 98% of transduced T-cells expressed CD62L at harvest, suggesting that the naïve/early memory phenotype is maintained.

Conclusion

This innovative manufacturing process for generating mesothelin CAR-T cells demonstrates consistency and robustness, yielding a final product enriched with CD62L⁺ T cells, high transduction efficiency, and full compliance with all safety release criteria. This process will be utilized in a Phase 1 dose escalation study of patients with mesothelin-expressing tumors including mesothelioma as well as lung, thymic, colorectal, pancreatic, and gastric cancers.