Leucine Zipper-based Cell Sorting for Purification of Dual-vector-transduced Cells

Katerina Su; Bintao Wang; Adhithi Rajagopalan; Sophia Chen; Alexander P. Boardman; Marcel R. M. van den Brink; Scott E. James

doi:10.3791/68628

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Method Article

Leucine Zipper-based Cell Sorting for Purification of Dual-vector-transduced Cells

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DOI:

10.3791/68628

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October 24th, 2025

Katerina Su^*¹ , Bintao Wang^*¹ , Adhithi Rajagopalan¹ , Sophia Chen¹ , Alexander P. Boardman² , Marcel R. M. van den Brink^*¹ , Scott E. James^*¹^,³

¹City of Hope Los Angeles and National Medical Center, ²Lymphoma and Cellular Therapy Services, Memorial Sloan Kettering Cancer Center, ³Department of Hematology and Hematopoietic Cell Transplantation, City of Hope National Medical Center

^* These authors contributed equally

Summary

Here, we describe a protocol for selectively purifying dual-transduced T cells from a mixture of co-transduced T cells. This approach combines two retroviral vectors that each encode one component of a paired leucine zipper-based cell sorting methodology that enables selective immunomagnetic purification of dual-vector-transduced cells.

Abstract

Adoptive T cell therapies, including chimeric antigen receptor (CAR) T cells, have demonstrated impressive clinical activity against hematologic malignancies and are showing promise in treating solid tumor malignancies. Despite these successes, multiple mechanisms of resistance to T cell immunotherapy have been identified that limit therapeutic success, including loss of or weak expression of target antigens, development of T cell exhaustion, and the presence of an immunosuppressive tumor microenvironment. Multiple cell engineering strategies have been developed to overcome these mechanisms of resistance. However, multiple mechanisms of resistance can occur simultaneously, necessitating a combination of multiple engineering strategies to optimize anti-tumor activity. Vector packaging constraints limit the delivery of large amounts of genetic information to T cells and present a challenge in co-expressing multiple engineered constructs. Here, we describe a protocol for co-transducing T cells with two vectors to encode multiple transgenes, thereby increasing the number of engineered functions. By co-expressing a leucine zipper-based cell sorting methodology, termed Zip-sort, we direct selective immunomagnetic purification of dual-transduced cells that have incorporated two distinct vectors.

Introduction

CAR T cell therapy has revolutionized the treatment of B cell leukemia, lymphoma, and multiple myeloma¹^,². CAR T cells are also being investigated for solid tumor malignancies³. However, recurrent mechanisms for failure of CAR T cell therapy have been identified, including poor expression or loss of the CAR target antigen molecule, development of T cell dysfunction (exhaustion), active immune suppression by the tumor microenvironment, and limited T cell trafficking to and persistence in the tumor microenvironment³^,⁴^,<....