THE ROLE OF NEURAL CELL ADHESION MOLECULE (NCAM) IN THE RECOGNITION AND KILLING OF TUMOR BY CAR T CELLS
Poster #: 129
Session/Time: B
Author:
James Baodan Collins, BS, MS
Mentor:
Sarah Richman, MD, PhD
Research Type: Basic Science
Abstract
INTRODUCTION:
Chimeric antigen receptor (CAR) T cell therapy achieves remission in 60-90% of pediatric patients with refractory hematologic cancers but has shown limited efficacy in solid tumors. Neuroblastoma (NB), responsible for 15% of pediatric cancer deaths, has a five-year survival of <50% for high-risk patients. A key difference between hematologic and solid tumors is adhesion molecule biology. Blood cancers often express intercellular adhesion molecule-1 (ICAM-1), which stabilizes T cell activation via leukocyte function-associated antigen-1 (LFA-1). In contrast, NB expresses neural cell adhesion molecule (NCAM), which has been observed being incorporated into the immunological synapse of anti-NB CAR T cells. We hypothesize that the binding of NCAM on activated CAR T cells enhances NB killing in vitro
METHODS:
Four SH-SY5Y neuroblastoma cell lines were generated expressing GFP, full-length EGFR, and varying NCAM expression: (1) parental SH-SY5Y-EGFR, (2) ICAM-1 knock-in (ICAM+), (3) NCAM knockout (NCAM KO), and (4) NCAM KO with ICAM+ (NCAM KO ICAM+). Constructs were introduced via lentiviral transduction. EGFR-specific CARs included CD8 hinge and transmembrane domains with 4-1BB/CD3ζ signaling and mCherry reporter. Adhesion molecule expression was validated by flow cytometry. GFP-labeled NB cells were co-cultured with CAR or non-transduced T cells at effector-to-target ratios of 1:1 and 5:1 for 72 hours. Cytotoxicity was quantified by live-cell imaging (IncuCyte S3, Sartorius).
RESULTS:
NCAM expression on NB cells did not affect their growth rate when co-incubated with non-transduced CAR T cells. CAR T cells demonstrate less cytotoxicity on NB cells with no NCAM surface expression. Importantly, NCAM on tumor cells lead to increased cytotoxicity when co-incubated with 2224T CAR T cells expressing NCAM.
CONCLUSION:
NCAM enhances CAR T cell cytotoxicity in neuroblastoma, likely by stabilizing immune synapse formation. These findings identify NCAM as a potential modulator of CAR T activity in solid tumors lacking ICAM-1. Targeting NCAM may improve CAR T therapy for pediatric solid tumors, and further studies could elucidate NCAM's mechanistic role in CAR T cell activation.
Chimeric antigen receptor (CAR) T cell therapy achieves remission in 60-90% of pediatric patients with refractory hematologic cancers but has shown limited efficacy in solid tumors. Neuroblastoma (NB), responsible for 15% of pediatric cancer deaths, has a five-year survival of <50% for high-risk patients. A key difference between hematologic and solid tumors is adhesion molecule biology. Blood cancers often express intercellular adhesion molecule-1 (ICAM-1), which stabilizes T cell activation via leukocyte function-associated antigen-1 (LFA-1). In contrast, NB expresses neural cell adhesion molecule (NCAM), which has been observed being incorporated into the immunological synapse of anti-NB CAR T cells. We hypothesize that the binding of NCAM on activated CAR T cells enhances NB killing in vitro
METHODS:
Four SH-SY5Y neuroblastoma cell lines were generated expressing GFP, full-length EGFR, and varying NCAM expression: (1) parental SH-SY5Y-EGFR, (2) ICAM-1 knock-in (ICAM+), (3) NCAM knockout (NCAM KO), and (4) NCAM KO with ICAM+ (NCAM KO ICAM+). Constructs were introduced via lentiviral transduction. EGFR-specific CARs included CD8 hinge and transmembrane domains with 4-1BB/CD3ζ signaling and mCherry reporter. Adhesion molecule expression was validated by flow cytometry. GFP-labeled NB cells were co-cultured with CAR or non-transduced T cells at effector-to-target ratios of 1:1 and 5:1 for 72 hours. Cytotoxicity was quantified by live-cell imaging (IncuCyte S3, Sartorius).
RESULTS:
NCAM expression on NB cells did not affect their growth rate when co-incubated with non-transduced CAR T cells. CAR T cells demonstrate less cytotoxicity on NB cells with no NCAM surface expression. Importantly, NCAM on tumor cells lead to increased cytotoxicity when co-incubated with 2224T CAR T cells expressing NCAM.
CONCLUSION:
NCAM enhances CAR T cell cytotoxicity in neuroblastoma, likely by stabilizing immune synapse formation. These findings identify NCAM as a potential modulator of CAR T activity in solid tumors lacking ICAM-1. Targeting NCAM may improve CAR T therapy for pediatric solid tumors, and further studies could elucidate NCAM's mechanistic role in CAR T cell activation.