Abstract P053

Development of CD30 CAR T-cells co-expressing an anti-PDL1.CD28 chimeric switch receptor for the treatment of r/r cHL

Introduction: CD30 is a validated therapeutic target in Hodgkin lymphoma (HL), particularly in relapsed and refractory cases. Chimeric antigen receptor (CAR) T-cell immunotherapy targeting CD30 demonstrates promising clinical activity. However, inhibitory signals mediated by the PD-1/PD-L1 pathway may limit the durability of response. In this study, we investigated the impact of co-expressing a chimeric switch receptor (CSR) targeting PD-L1 on the proliferative and cytotoxic activity of second-generation CD30-CAR T-cells.

Materials and Methods: To compare the in vitro proliferative and functional activity of four second-generation CD30-CAR T-cell constructs (4-1BBz), including a variant with an anti-PD-L1 CSR (KN035.CD28), during repeated restimulation of CD30+ HL tumor cell lines. The following CAR constructs were evaluated: hHRS3.S-BBz.EGFRt — short hinge region (IgG4), hHRS3 scFv; SGN30.S-BBz.EGFRt — an analogous construct with SGN30 scFv (the antigen-binding fragment is identical to brentuximab vedotin); hHRS3.L-BBz.EGFRt — long hinge region (CH2-CH3) with two point mutations L235E, N297Q, and an alpha-tag for CAR detection and selection using an anti-alpha-tag nanobody; hHRS3.S-BBz.KN035-CD28 — a CAR with a CSR that binds PD-L1 and utilizes CD28 signaling. T-cells from healthy donors were activated via CD3/CD28 and transduced with the corresponding lentiviruses. Expansion was performed over 15 days in the presence of IL-7, IL-15, and IL-21. To evaluate the durability of response, a rechallenge model was applied: CD30+ tumor cells HDLM-2 (CD30+/PD-L1+) and KM-H2 (CD30+/PD-L1-) were added every 3–4 days for a total of 5 rounds. Analysis included the quantification of CAR T-cells, tumor burden evaluation, and the calculation of AUC (area under the curve) for both proliferation and cytotoxicity.

Results: Among the evaluated constructs, CAR.CSR T-cells (hHRS3.S-BBz.KN035-CD28) induced the most pronounced expansion on HDLM-2, demonstrating a 17-fold increase by day 21. The remaining constructs reached peak expansion (5–10 fold) by day 10, followed by a subsequent decline. Under conditions of stimulation with the PD-L1-negative line (KM-H2), differences in activity between CAR.CSR and baseline CARs were less pronounced.

Conclusions: CD30-CAR T-cells co-expressing the CSR KN035.CD28 demonstrated a sustained proliferative advantage and enhanced control of PD-L1+ tumor cells in vitro.