Next-generation all-in-one CRISPR/Cas9 multiply-edited CD30CAR-T cells: Potency despite risk of translocations

Background Chimeric antigen receptor (CAR)-T cells are therapeutic breakthroughs against advanced non-Hodgkin lymphomas and myelomas. On the other hand, no CAR-T cell product has been so far clinically approved for therapy of Hodgkin Lymphoma (HL), T cell lymphoma (TCL), or Epstein-Barr-Virus (EBV)-associated lymphoproliferative diseases (EBV-LPDs). CD30 (TNFRSF8) is commonly expressed on HL and on subsets of TCL and EBV-LPDs. CD30CAR-T cells generated via transduction with viral vectors have been tested in clinical trials, showing overall good responses against HL. CAR-T cells produced entirely with locus-specific gene editing methods are emerging as attractive next-generation engineered cell products for ease of multiple seamless cell modifications.

Using CRISPR/Cas9-mediated techniques, we optimized homology-directed repair templates (HDRTs) and performed all-in-one multiplex editing to knock-in (KI) CD30CAR within the TCRα constant (TRAC) locus and to simultaneously knock-out (KO) PD-1 or/and β2M. CD30CAR-T cells were tested in CD30+ cell models of HL, TCL, and EBV-LPDs.

We compared mouse versus human anti-CD30 scFv designs in HDRTs incorporating TRAC homology arms, FcIg spacer/detection domain, and CD28 / CD3ζ signaling domains. We obtained an average of 30% TRACKICD30CAR-T cells and efficient in vitro cytotoxicity with CD30+ cell targets. CARs incorporating the high-affinity humanized 5F11 scFv showed the highest CAR expression, and the editing templates were further modified to incorporate a truncated CD34 (tCD34) spacer/detection domain. 5F11-CD30CAR-tCD34-T cells showed high CAR-KI rates (approx. 50-80% 12-14 days after editing) and potency in vitro and in vivo. Subsequently, we tested all-in-one CAR KI with additional KOs by co-electroporation of guide RNAs (gRNAs) targeting the genes encoding PD-1 or /and β2M to improve function and allow for improved cell persistence in allogeneic recipients, respectively. Compared with CD30CAR-T cells, CD30CAR-β2MKO-T cells were similarly viable and functional and showed low risk of translocations. PD1KO enabled CD30CAR-T cells to produce higher levels of cytotoxic features upon exposure to targets. However, simultaneous β2MKO and PD-1KO compromised the expansion capacity of CD30CAR-T cells and resulted in detectable translocations.

Non-virally engineered 5F11-CD30CAR-T cells represent a novel cell therapy modality against CD30+ lymphomas. Multiplex editing remains to be optimized to avoid unwanted genomic alterations and chromosomal translocations. Competing Interest Statement R Stripecke, J-M Kleid and M Damrat have filed a patent application for the generation of CD30CAR-T cells targeting infections. The information provided in this publication is shared only for non-commercial purposes and should not be used, directly or indirectly, for any commercial purposes. R. Stripecke obtained research funding from The Jackson Laboratory to develop mouse models using the NSG-DKO strain. List of Abbreviations - β2M - Beta 2 microglobulin - CAR - Chimeric antigen receptor - CRISPR/Cas - Clustered regularly interspaced short palindromic repeats/Cas - EBV - Epstein-Barr virus - EBV-LPD - Epstein–Barr virus–associated lymphoproliferative diseases - FACS - Fluorescence-activated cell sorting - HIV - Human Immunodeficiency virus - HLA - Human leukocyte antigen - GMP - Good Manufacturing Practice - KI - Knock-in - KO - Knock-out - LCL - Lymphoblastoid cell line - PCR - Polymerase chain reaction - PD-1 - Programmed cell death protein 1 - PD-L1 - Programmed cell death 1 ligand 1 - TCL - T-cell lymphoma - TCR - T-cell receptor - TRAC - T-cell receptor alpha chain - WT - Wild-type