Efficient and Specific Multi-Locus Editing of Allogeneic CAR T Cells for Hypoimmunity during Large Scale Manufacture Using Cas12b

Introduction: Allogeneic CAR T cell therapies may offer key advantages over autologous therapies including choice of starting cells, better manufacturing control, and lower cost. However, the manufacture of these cells imposes a high demand on genome engineering specificity and efficiency, given the dual needs for product safety and yield. This challenge is amplified for newer generations of allogeneic CAR T cell designs that may offer improved performance but require more edits to produce, which may increase the potential of unintended genome modification and insufficient yield of fully engineered product. Here we have tested a novel editing platform, Cas12b, for the ability to mediate efficient and specific disruption of three genes (B2M, CIITA, and TRAC) during large scale manufacturing of CAR T cells with immune evasive properties and abrogated graft-vs-host response. We have characterized the resulting cell product for on-target modification efficiency, type and levels of unintended genome modification, and potency for tumor clearance in a Nalm6 humanized mouse model, with the aim of demonstrating suitability for translation to human studies.

Methods: Our manufacturing strategy to generate hypoimmune CAR T cells introduces five genetic modifications, using Cas12b for disruption of B2M, CIITA, and TCR expression, and lentiviral transduction to overexpress CD47 and introduce a CD19 CAR. T cells obtained from healthy donors were first transduced with lentiviral vector and then edited simultaneously to disrupt the B2M, CIITA and TRAC genes via electroporation of Cas12b mRNA and appropriate guides. Edited and purified cells were characterized via flow cytometry for efficiency of each genetic modification. Cells were further characterized for knockout levels via next generation sequencing (NGS) as well as vector copy number per diploid genome (VCN). Cells were characterized for viability, proliferation, and efficacy in a Nalm6 mouse model of tumor clearance. An initial assessment of chromosomal translocations was performed by G banding karyotype analysis. To assess editing specificity, each Cas12b guide was evaluated by Cas-OFFinder and an unbiased oligonucleotide duplex capture assay in T cells from multiple donors under conditions of high on-target modification. Candidate off-target sites were then examined for evidence of cleavage via PCR amplification from the manufactured cell product followed by NGS.

Results: Our manufacturing process yielded clinically relevant scales of cell product having > 85% B2M knockout, > 90% CIITA knockout, and < 1% of residual TCR expressing cells. VCN levels were < 5. Cell viability and proliferation were minimally impacted by the gene editing process. Cells exhibited robust anti-tumor properties with doses of 7 x 10⁶ hypoimmune CD19 CAR T cells (per animal) providing efficient killing in a Nalm6 mouse model. Initial karyotype analysis identified low levels of translocation between on-target sites with overall translocation levels otherwise not significantly affected compared to unedited cells. Our characterization of Cas12b in edited T cells across multiple donors revealed high specificity, consistent with previous reports (Nat Commun 10 212), with studies to date having yielded no evidence of cleavage at any candidate off-target site.

Summary: Cas12b-mediated multi-locus editing of allogeneic CAR T cells during large scale manufacture resulted in a high yield product with potent tumor clearance capabilities. Initial analysis of editing specificity across multiple donors and engineering runs identified no off-target sites, and early karyotype assessments indicated an overall level of chromosomal translocations consistent with prior experiences in the field. In companion in vivo studies, these hypoimmune CD19 CAR T cells have been shown to efficiently evade host immune response (see posters by Hu and Duback). Together, these data demonstrate a profile for potency, genome integrity, and manufacturing yield that supports progressing these cells into human studies.