Detection of Vector Copy Number in Bicistronic CD19xCD22 CAR T Cell Products with Digital PCR
Authors: Murphy Lindsey A. , Marians Russell , Kohler Mark Eric , Fry Terry J. , Winters Amanda C. 
Published: November 2021

Abstract
Chimeric antigen receptor (CAR) T cell therapy is a rapidly evolving immunotherapeutic treatment modality for adult and pediatric patients with a variety of cancers, which has been most extensively investigated in B-cell malignancies. Given that CAR T cell immunotherapy involves changing the genetic composition of a patient's T cells, this living drug presents unique safety and quality control challenges. Vector copy number (VCN), a measurement of transgene copies within a CAR T cell product, is a product-specific characteristic that must be quantified prior to patient administration as high VCN increases the risk of insertional mutagenesis.
Historically, VCN assessment in CAR T cell products has been performed via qPCR. qPCR is reliable along a broad range of concentrations but has inherent limitations in its lower limit of detection and limit of quantification. Digital PCR (dPCR) methods were developed for absolute quantification of target sequences by counting nucleic acid molecules encapsulated in discrete, volumetrically defined partitions. Advantages of dPCR compared to qPCR include simplicity, reproducibility, lower limit of detection, and definitive quantification.
In this present study, we developed an assay for analysis of the novel bicistronic UCD19x22 CAR T cell construct, which was developed in the laboratory of Dr. Terry Fry at the University of Colorado and will be moving in to clinical trials later this year. Custom primer-probe assays were designed using Primer Express v3.0.1 and the ThermoFisher Custom TaqMan Assay Design Tool. As an internal control, forward and reverse primers as well as a VIC-labeled probe specific to human albumin (NCBI gene 213, HGNC:399) were designed. Primers and a FAM-labeled probe assay, specific for the bicistronic CD19x22 CAR T cell product, were designed at the junction site between the two distinct CARs. This study compares two different digital PCR modalities: (1) droplet digital PCR (ddPCR) via the BioRad QX200 system which utilizes water-in-oil droplet partitions and (2) the QIAcuity digital PCR system utilizing a nanoplate-based partitioning platform. While dPCR is a newer methodology compared to ddPCR, the two apply parallel procedures, data generation, and analyses. The primer/probe assay was validated with qPCR, dPCR and ddPCR using patient samples from preclinical CAR T cell manufacturing production runs, as well as Jurkat cell subclones which stably express this bicistronic CAR T product.
We successfully developed an assay to specifically detect and quantify our bicistronic CD19xCD22 CAR transgene. ddPCR confirmed the specificity of this assay to detect only the bicistronic CAR product without any signal detected in samples containing untransduced T cells or T cells transduced with CD19 only CARs. Additionally, our assay gives accurate, precise, and reproducible CAR T cell VCN measurements across qPCR, dPCR, and ddPCR modalities. We demonstrate that digital PCR strategies can be utilized for absolute quantification of CAR transgenes and VCN measurements, and that specific assays can be developed for detection of unique constructs. Future studies will evaluate the utility of this assay with digital PCR modalities in measuring CAR T cell persistence in clinical trial patient samples after receiving this novel CAR T cell product.
Figure 1 Figure 1.

Disclosures
Fry: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company.