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Multiplex gene-editing strategy to engineer allogeneic EGFR-targeting CAR T-cells with improved efficacy against
Ryan Murray1,2, Md Raihan Chowdhury1, Nuria Roxana Botticello-Romero1
1New England Inflammation and Tissue Protection Institute, Department of Pharmaceutical Sciences, Bouvè College of Health Sciences, Northeastern University, Boston, MA, USA.
Abstract:
Chimeric Antigen Receptor (CAR) T cells have induced remarkable clinical responses in patients with hematological cancers. However, CAR T-cell therapies against solid tumors have not elicited similar outcomes since immunosuppressive barriers in the tumor microenvironment attenuate anti-tumor activity. Here, we describe a multifaceted approach to engineer allogeneic CAR T-cells resistant to both biochemical (hypoxia-adenosinergic) and immunological (PD-L1 and TGF-β) inhibitory signaling using an adenine base editor and a CRISPR-Cas12b nuclease. The resulting EGFR-targeting CAR T-cell product comprised a combination of six gene edits designed to evade allorejection (B2M, CIITA), prevent graft-versus-host disease (CD3E) and overcome biochemical (ADORA2A) and immunological (PDCD1, TGFBR2) barriers in solid tumor microenvironment of subcutaneously grown EGFR+ human lung tumor xenografts. This combinatorial genetic disruption enhances CAR T cell effector function and anti-tumor efficacy leading to improved tumor elimination and survival in xenograft and humanized mouse solid tumor models. Our strategy confers CAR T cells resistance to multiple clinically relevant inhibitory signaling pathways that are amplified in hypoxic tumor areas and may improve the therapeutic potential of CAR T-cells against solid tumors.
Insights
Engineered Chimeric Antigen Receptor (CAR) T-cells overcome solid tumor defenses by resisting immunosuppressive signals. This multi-gene editing approach enhances CAR T-cell efficacy against tumors, improving survival in preclinical models.
Area of Science:
- Immunology
- Cancer Biology
- Gene Editing Technologies
Background:
- Chimeric Antigen Receptor (CAR) T-cell therapy shows promise in hematological cancers but faces challenges in solid tumors due to immunosuppressive tumor microenvironments.
- Solid tumors create barriers, including hypoxia-adenosinergic and immunological signaling (PD-L1, TGF-β), which limit CAR T-cell anti-tumor activity.
- Overcoming these immunosuppressive mechanisms is crucial for advancing CAR T-cell therapy against solid tumors.
Purpose of the Study:
- To engineer allogeneic CAR T-cells resistant to both biochemical and immunological inhibitory signaling pathways within the solid tumor microenvironment.
- To develop a multifaceted gene-editing strategy to enhance CAR T-cell effector function and anti-tumor efficacy against EGFR-positive solid tumors.
- To evaluate the therapeutic potential of these enhanced CAR T-cells in preclinical solid tumor models.
Main Methods:
- Utilized an adenine base editor and CRISPR-Cas12b nuclease for precise gene editing in CAR T-cells.
- Introduced six genetic modifications to confer resistance to allorejection (B2M, CIITA), prevent graft-versus-host disease (CD3E), and overcome tumor microenvironment barriers (ADORA2A, PDCD1, TGFBR2).
- Tested the engineered EGFR-targeting CAR T-cells in EGFR+ human lung tumor xenografts and humanized mouse models.
Main Results:
- The combinatorial genetic disruption strategy successfully engineered CAR T-cells resistant to multiple inhibitory signaling pathways.
- Enhanced CAR T-cell effector function and anti-tumor activity were observed in preclinical models.
- Significant improvements in tumor elimination and prolonged survival were achieved in both xenograft and humanized mouse solid tumor models.
- The engineered CAR T-cells demonstrated resistance to hypoxia-adenosinergic, PD-L1, and TGF-β signaling.
Conclusions:
- This multifaceted gene-editing approach effectively confers CAR T-cell resistance to key inhibitory signaling pathways prevalent in solid tumor microenvironments.
- The engineered CAR T-cells exhibit enhanced anti-tumor efficacy, offering a promising strategy to improve CAR T-cell therapy for solid tumors.
- The developed strategy holds potential for advancing the therapeutic application of CAR T-cells against challenging solid tumor indications.
