Changes in Glycosylation on the Cell Surface Result in Highly Potent and Long-Lasting Allogeneic CAR-T Therapy in Patients

Background: Allogeneic CAR T-cell therapy faces challenges such as limited persistence and host rejection. Recent research has bolstered its viability by modifying genes like B2M, although new challenges, such as NK cell targeting, have surfaced. Through CRISPR screening, we have identified that knocking out an intramembrane-cleaving aspartyl protease prevents it from cutting glycosyltransferases, thereby influencing glycan composition. This alteration enhances glycosylation levels, equipping immune cells with a glycan shield that impacts immune responses and the initiation of activation-induced cell death (AICD). Thereby, it not only combats host rejection but also improves the potency of allo-CAR T cells. Engineered protease^KO/TCR^KO/anti-CD19 CAR T cells (ET-901) resisted allogeneic killing and maintained anti-tumor effects without causing GvHD, marking a significant advancement in allogeneic CAR T-cell therapy.

Methods: ET-901 is being evaluated in a phase 1 trial at a single center for patients with r/r B-NHL. The trial follows a 3+3 dose escalation design, with subsequent expansion at the recommended Phase 2 dose (RP2D). Eligible patients must have an ECOG performance status of 0-2 with no prior history of allo-HSCT or CAR-T cell therapy. The primary objectives of the trial are to determine the safety of ET-901 and establish the RP2D. Following lymphodepletion of fludarabine (30-50 mg/m²/d) and cyclophosphamide (500-1000 mg/m²/d) for a duration of 3 days, patients receive a single-dose infusion of ET-901 at escalating doses of 1*10e6/kg, 3*10e6/kg, and 10*10e6/kg. Pharmacokinetic samples were collected during the follow-up period.

Results：Out of the initial 20 screened patients, 9 were enrolled and underwent CAR-T infusion from September 6, 2023, to May 31, 2024. All enrolled 9 patients had advanced disease (Ann Arbor stage III-IV). The median number of prior treatment lines was 4 and two-thirds of patients had refractory disease. The median sum of product diameters (SPD) was 7,660 mm² (range: 1,710 to 43,400 mm²), and 4 patients had bulky disease (≥7∙5 cm). Notably, extranodal site involvement was observed in 5 out of 9 cases, complicating treatment with CAR-T therapy. No dose-limiting toxicities (DLTs) or GvHD were observed within 28 days post-infusion. Common adverse events among patients included lymphopenia, anemia, thrombocytopenia, and cytokine release syndrome (CRS), with grade 3 occurring in 3 patients (33%). CRS typically started 1 day after infusion and lasted a median of 7 days. Immune effector cell-associated neurotoxicity syndrome (ICANS) was observed in 3 patients (33%), with onset at a median of 5 days post-infusion, lasting 3 days. All events were resolved, and cytokine levels normalized within 10 days. The objective response rate was 100%, with 66.7% complete responses and 33.3% partial responses. Of the participants, 5 had ongoing responses, 3 experienced progressive disease.

The median peak CAR vector copy number in peripheral blood was 123,529.4 copies/μg. The peak level of CD3-CAR+T cells was 49.2 cells/μl, with the area under the curve (AUC) from days 0 to 28 being 104.5 cells/μl. CAR-T cell expansion correlated with dose and disease burden, peaking at 7 days post-infusion.

Conclusions: This study advances allogeneic CAR T-cell therapy for B-cell malignancies, addressing challenges such as limited persistence and rejection. Employing CRISPR to modify the certain protease enhances the resistance of T cells to immune attacks and AICD, maintaining their anti-tumor efficacy without causing GvHD. The phase 1 trial of ET-901 in relapsed/refractory B-NHL patients showed no dose-limiting toxicities, with observed treatment-related adverse events being manageable. All patients demonstrated objective responses, with significant CAR-T cell expansion, confirming the engineered cells' long-term immune privilege and activity. This represents a significant step in making allogeneic CAR T-cell therapy more viable and effective.

No relevant conflicts of interest to declare.