Optimization of hypo-alloimmunogenic multispecific CAR-T and SARS-CoV-2-specific T cells for off-the-shelf adoptive cell therapy

King Pan Ng; Michaela Su-Fern Seng; Wing Leung

doi:10.1016/j.omtm.2025.101462

Optimization of hypo-alloimmunogenic multispecific CAR-T and SARS-CoV-2-specific T cells for off-the-shelf adoptive cell therapy

Mol Ther Methods Clin Dev. 2025 Apr 10;33(2):101462. doi: 10.1016/j.omtm.2025.101462. eCollection 2025 Jun 12.

Authors

King Pan Ng^{1

2}, Michaela Su-Fern Seng^{1

3

2}, Wing Leung^{1

3

2}

Affiliations

¹ Paediatric Bone Marrow Transplantation and Cell Therapy Centre, Children's Blood and Cancer Centre, KK Women's and Children's Hospital, SingHealth, Singapore, Singapore.
² Duke-NUS Medical School, Singapore, Singapore.
³ Department of Paediatric Haematology and Oncology, KK Women's and Children's Hospital, SingHealth, Singapore, Singapore.

Abstract

Despite promising results with chimeric antigen receptor modified T(CAR-T) cells and virus-specific T(VST) cells, both forms of therapy are limited by timely availability, affordability, in vivo persistency, and antigen escape. To overcome these barriers, we developed multitargeting hypo-alloimmunogenic CAR-T and VST for off-the-shelf administration. We generated bi-specific CAR-T against CD19 and CD22 and tri-specific VST against S, M, and N proteins of SARS-CoV-2 for real patient use. Portions of these clinical-grade products were collected for proof-of-concept laboratory studies mimicking off-the-shelf settings. By electroporation delivery of Cas9 nuclease/guide RNA (gRNA) ribonucleoprotein, we developed a single-step approach to knockout B2M, achieving efficient (>80% of cells) B2M null on both bi-specific CAR-T and tri-specific VST, leading to marked reduction of allo-immunogenicity with intact potency, antigen specificity, phenotypes, and proliferative potential. Using newly designed paired Cas9 nickases-AAV6 B2M site-specific knockin system, we further refined the approach to re-express HLA-E in B2M null bi-specific CAR-T cells (>50% of cells), offering protection from natural killer cytotoxicity. The editing was highly specific with minimal off-target effects. Our approach enables expedient production of clinical-grade, off-the-shelf, hypo-alloimmunogenic, multi-specific CAR-T and VST, with improved potential for long-term in vivo persistence, primary disease control, genome-safety, immediate availability, product homogeneity, and reduced cost.

Keywords: AAV; B2M; CD19/CD22; CRISPR-Cas9; HLA-E; SARS-CoV-2-specific T cells; adoptive T cell therapy; chimeric antigen receptor; knockin; paired Cas9 nickase.