-Author name in bold denotes the presenting author
-Asterisk * with author name denotes a Non-ASH member
Clinically Relevant Abstract denotes an abstract that is clinically relevant.

PhD Trainee denotes that this is a recommended PHD Trainee Session.

Ticketed Session denotes that this is a ticketed session.

885 Fratricide-Resistant Anti-CD2 Chimeric Antigen Receptor T-Cells with Endogenous CD2 Knockout Are Highly Effective Against T-Cell Neoplasms

Program: Oral and Poster Abstracts
Type: Oral
Session: 703. Cellular Immunotherapies: Basic and Translational: Innovative T Cell Therapies for Unexplored Frontiers
Hematology Disease Topics & Pathways:
Research, Lymphoid Leukemias, ALL, Biological therapies, Translational Research, Lymphomas, Chimeric Antigen Receptor (CAR)-T Cell Therapies, T Cell lymphoma, Diseases, aggressive lymphoma, Therapies, Lymphoid Malignancies, emerging technologies, Technology and Procedures
Monday, December 11, 2023: 3:15 PM

Mathew G. Angelos, MD, PhD1,2, Ruchi P. Patel, MS1, Yi-Hao Chiang, MD3*, Wei Xie, MD3*, Raymone Pajarillo, MS1*, Carolyn E. Shaw, MS3*, Ekta Singh, BS3*, Jason Xu, PhD4, Avery D. Posey, PhD3*, Alain Rook, MD5*, Antonia Rotolo, MD, PhD3*, Vinodh Pillai, MD, PhD6*, Stefano A. Pileri, MD, PhD7, David T. Teachey, MD4 and Marco Ruella, MD1,8

1Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA
2Division of Hematology and Oncology, University of Pennsylvania, Philadelphia, PA
3Center for Cellular Immunotherapies, University of Pennsylvania, Philadelphia, PA
4Division of Oncology, Children's Hospital of Philadelphia, Philadelphia, PA
5Division of Dermatology, University of Pennsylvania, Philadelphia, PA
6Division of Hematopathology, Children's Hospital of Philadelphia, Philadelphia, PA
7Division of Hematopathology, Istituto Europeo di Oncologia, Milano, Italy
8Lymphoma Program, University of Pennsylvania, Philadelphia, PA

T cell neoplasms, such as peripheral T cell lymphomas (PTCL) and acute T cell lymphoblastic leukemia (T-ALL), have some of the worst prognoses of all hematological malignancies. Poor outcomes are driven by a limited number of effective treatments, particularly in relapsed and refractory settings. While chimeric antigen receptor T cell (CAR-T) immunotherapy has been successful in other hematological diseases, its clinical use against T cell neoplasms is limited due to a lack of unique tumor-specific antigens. Additionally, CAR specificity against pan-T cell antigens causes CAR-T fratricide and insufficient manufacturability for therapeutic dosing. Early studies of CAR-T against CD5- and CD7-expressing T cell neoplasms have shown responses, however, 1) most patients ultimately relapse, and 2) in some patients, antigen negative relapse has occurred. To circumvent these obstacles, we hypothesized that targeting CD2, a pan-T cell antigen, using anti-CD2 CAR-T cells that are engineered to lack CD2, would permit effective CAR-T manufacturing against T cell neoplasms.

In a retrospective analysis of clinical flow cytometry diagnostic samples, we found CD2 expression in 25 of 33 (75.8%) and 37 of 51 (72.5%) pediatric PTCL and T-ALL cases, respectively. Similarly, we found 82 of 89 (92.1%) adult PTCL cases with CD2 immunohistochemical expression, which exceeded CD7 expression (38 of 90, 42.2%) that is currently being evaluated in early phase clinical trials. We optimized early CD2 knockout (KO) during CAR-T cell manufacturing from human donor T cells (KO efficiency: 81.2% ± 2.4, n=9 donors). We next engineered a best-in-class, 4-1BB-costimulated, second-generation CD2KO anti-CD2 CAR-T product (CART2) via lentiviral transduction (PANEL A) using seven distinct single-chain variable fragments (transduction efficiency: 60.2% ± 5.5, n=7 donors) based on cellular expansion and stressed in vivo efficacy against CD2+ T-ALL cells (Jurkat). There were no significant differences in population doublings during CART2 expansion as compared to both CD2 wild-type (CD2 WT) untransduced T cells (Mock UTD) and CD2KO untransduced T cells (CD2KO UTD) (6.20 ± 0.42 vs. 6.12 ± 0.62 vs. 6.67 ± 0.68, respectively, Day 15, n=3 donors), while CD2 WT CART2 cells failed to expand due to fratricide. CART2 cells had no significant differences in T cell memory phenotypes or T cell exhaustion markers, including PD1 and LAG3 expression, at the end of the expansion.

Expanded CART2 cells eradicated primary patient-derived CD2+ Sezary cells and CD2+ T-ALL leukemic blasts as measured by in vitro cytotoxicity assays. Cytotoxicity correlated with CD8+ CART2 cells expressing IL-2 (20.4% vs. 0.2% vs. 0.35%), TNFα (28.5% vs. 0.27% vs. 0.2%), and IFNγ (11.27% vs. 0.18% vs. 0.14%) relative to Mock UTD and CD2KO UTD controls stimulated with CD2+ Jurkat cells (n=2 donors). Importantly, we also found both CD2KO UTD and CART2 cells were capable of TNFα and IFNγ release upon interaction with cytomegalovirus and influenza peptides, suggesting that T cells with CD2 perturbations are still able to mediate effector T cell responses.

We further demonstrated that CART2 cells were highly effective in controlling systemic tumor burden of T-ALL patient-derived xenograft mouse (PDX) models and significantly improved median overall survival as compared to Mock UTD and CD2KO UTD controls (138 days vs. 30 days vs. 30 days, p=0.0007, Mantel-Cox) (PANEL B). Long-term tumor control was proportional to CART2 peripheral blood persistence at Day 28 (CD2KO UTD: 186.6 ± 83.7, n=5 vs. CART2: 936.3 ± 241.7, n=5) and Day 100 (CD2KO UTD: N/A vs. CART2: 514.0 ± 441, n=3). We also found that rescue infusion of CART2 could eliminate relapsed T-ALL in PDX mice previously in a remission after initial treatment with anti-CD5 CAR-T cells, with long-term persistence of CD2-negative T cells thereafter.

In conclusion, our data demonstrate that CART2 are manufacturable and highly efficacious against T-cell neoplasms. To our knowledge, this is the first report of prolonged efficacy using a CD2-targeted cellular monotherapy in human leukemia preclinical models. This product could be delivered alone or in combination with additional CARs for dual targeting, such as anti-CD5 or anti-CD7 CARs, to reduce the potential of antigen-negative escape. Further research is ongoing to study the effect of CD2 deletion in anti-CD19 CAR T cells and the possible implication for clinical translation.

Disclosures: Patel: viTToria biotherapeutics: Consultancy. Pileri: CELGENE: Other: Advisory board; ROCHE: Speakers Bureau; NANOSTRING: Other: Advisory Board; Stemline: Speakers Bureau; Diatech Pharmacogenetics: Consultancy; Beigene: Research Funding, Speakers Bureau; Eli Lilly: Speakers Bureau. Teachey: Jazz: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sobi: Membership on an entity's Board of Directors or advisory committees, Research Funding; Neoimmune Tech: Research Funding; BEAM: Research Funding. Ruella: viTToria biotherapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Scientific Founder, Research Funding; Bristol Myers Squibb: Consultancy; GlaxoSmithKline: Consultancy; AbClon: Consultancy, Research Funding; Bayer: Consultancy; NanoString: Consultancy, Research Funding; Beckman Coulter: Research Funding.

*signifies non-member of ASH