Ted J. Hofmann

Patients with relapsed/refractory T-cell acute lymphoblastic leukemia (T-ALL) have a dismal prognosis largely mediated by nonsustained responses to chemotherapy and few targeted therapy options. Surface antigen targets in T-ALL include CD2, CD5, CD7, and CD38; however, ongoing clinical development of these targets is challenged by (1) T-cell fratricide during manufacturing, (2) T-cell depletion during treatment, and (3) high frequency of target-negative relapse. Here, we use a combination of flow cytometry, single-cell genomics, and bulk RNA sequencing to identify CC chemokine receptor type 4 (CCR4) as a novel surface target in T-ALL. We analyzed the T-ALL microenvironment from 40 patients treated on the AALL0434 clinical trial and identified a subpopulation of bone marrow–enriched CCR4+ FOXP3+ T-regulatory cells that express immune checkpoints (PD-1 and TIGIT) and could be targeted with anti-CCR4 therapy. Finally, we describe the preclinical efficacy of an anti-CCR4 chimeric antigen receptor T-cell therapy in in vitro and in vivo models, paving the way for future translational efforts in chemotherapy-refractory T-ALL.

Patient demographics and clinical covariates from neuroblastoma tumor microarray.

Neuroblastoma cell surface GPC2 activates and induces proliferation of D3-GPC2 CAR-28z T cells.

D3-GPC2 CAR-BBz T cells from a second T cell donor are robustly and selectively activated by GPC2-high neuroblastoma cell lines.

D3-GPC2 CAR-28z T cells are robustly and selectively cytotoxic to GPC2-expressing neuroblastoma cells.

D3-GPC2 CAR T cells are not associated with any toxicity in murine models of neuroblastoma.

GPC2-expressing SCLCs activate and are susceptible to D3-GPC2 CAR T cell killing from a second T cell donor.

Purpose:We previously identified glypican 2 (GPC2) as a cell-surface MYCN-regulated neuroblastoma oncoprotein and developed a D3-GPC2 antibody that specifically binds a conformational, tumor-specific epitope conserved between mouse and human.Experimental Design:In this study, we sought to further validate GPC2 as an immunotherapeutic target and develop Investigational New Drug application–enabling data to support the clinical translation of D3-GPC2 chimeric antigen receptor (CAR) T cells.Results:Immunohistochemistry validated that GPC2 is widely expressed on human neuroblastomas, and flow cytometry showed high levels of cell-surface GPC2 on neuroblastoma cellular models. Second-generation D3-GPC2 CAR T cells with either a 4-1BB or CD28 co-stimulatory domain were selectively activated and induced potent neuroblastoma cell cytotoxicity in several complementary in vitro co-incubation assays. Conversely, no measurable cytotoxicity or D3-GPC2 CAR T-cell activation was observed in co-incubation studies with nine primary human normal tissue cell lines. Moreover, GPC2 CAR T cells induced significant regression of GPC2-expressing neuroblastoma xenografts. No GPC2 CAR-related toxicities were noted, including in comprehensive mouse necropsies performed after GPC2 CAR T-cell administration. Finally, to explore the potential broader clinical impact of GPC2 CAR T cells, we showed that they are also potently cytotoxic to preclinical models of GPC2-expressing small cell lung cancers.Conclusions:These data validate GPC2 as a bona fide CAR T-cell target in neuroblastoma and other cancers. The safety and preliminary efficacy of GPC2 CAR T cells are being tested in a first-in-human phase I clinical trial for children with relapsed/refractory neuroblastoma (NCT05650749).

Description of target cells for in vitro D3-GPC2 CAR T cell pharmacology assays.

Normal human cell lines used for D3-GPC2 CAR T cell in vitro toxicology assays.