Reference gene selection for clinical chimeric antigen receptor T-cell product vector copy number assays

Abstract

Background aims: Reference genes are an essential part of clinical assays such as droplet digital polymerase chain reaction (ddPCR), which measure the number of copies of vector integrated into genetically engineered cells and the loss of plasmids in reprogrammed cells used in clinical cell therapies. Care should be taken to select reference genes, because it has been discovered that there may be thousands of variations in copy number from genomic segments among different individuals. In addition, within the same person in the context of cancer and other proliferative disorders, substantial parts of the genome also can differ in copy number between cells from diseased and healthy people. The purpose of this study was to identify reference genes that could be used for copy number variation analysis of transduced chimeric antigen receptor T cells and for plasmid loss analysis in induced pluripotent stem cells using ddPCR.

Methods: We used The Cancer Genome Atlas (TCGA) to evaluate candidate reference genes. If TCGA found a candidate gene to have low copy number variance in cancer, ddPCR was used to measure the copy numbers of the potential reference gene in cells from healthy subjects, cancer cell lines and patients with acute lymphocytic leukemia, lymphoma, multiple myeloma and human papillomavirus-associated cancers.

Results: In addition to the rPP30 gene, which we have has been using in our copy number assays, three other candidate reference genes were evaluated using TCGA, and this analysis found that none of the four gene regions (AGO1, AP3B1, MKL2 and rPP30) were amplified or deleted in all of the cancer cell types that are currently being treated with cellular therapies by our facility. The number of copies of the genes AP3B1, AGO1, rPP30 and MKL2 measured by ddPCR was similar among cells from healthy subjects. We found that AGO1 had copy number alteration in some of the clinical samples, and the number of copies of the genes AP3B1, MKL2 and rPP30 measured by ddPCR was similar among cells from patients with the cancer cell types that are currently being treated with genetically engineered T-cell therapies by our facility.

Conclusions: Based on our current results, the three genes, AP3B1, MKL2 and rPP30, are suitable for use as reference genes for assays measuring vector copy number in chimeric antigen receptor T cells produced from patients with acute leukemia, lymphoma, multiple myeloma and human papillomavirus-associated cancers. We will continue to evaluate AGO1 on our future samples.

Keywords: chimeric antigen receptor T-cells; droplet digital PCR; vector copy number.

Fig. 1.

Gene copy number results from the analysis of DNA from various cancer types. ddPCR analysis of copy numbers of AGO1, AP3B1, MKL2 and rPP30 was performed on liver carcinoma DNA from OriGene, K562 genomic DNA from Promega, lung carcinoma DNA from ATCC and normal human genomic DNA (from human blood buffy coat) from Sigma. The x-axis shows the different DNA samples and y-axis shows the copy numbers per microliter. Normal: normal human genomic DNA; Leukemia: the leukemia cell line K562.

Fig. 2.

Gene copy numbers analysis of DNA from normal donors. The number of copies of the four reference genes (AGO1, AP3B1, MKL2 and rPP30) were tested on three normal donor DNA samples: donor 071, donor 080 and donor 110. The x-axis shows the donor name, y-axis shows the copy numbers as copies per microliter.

Fig. 3.

Gene copy number analysis in genetically engineered T-cell products. Copy number of AGO1, AP3B1, MKL2 and rPP30 was tested in five different types of genetically engineered T cells. For each type of CAR T-cell three sets of CAR T-cells and associated untransduced T-cell samples were tested. HPV E7-TCR engineered T-cells and CD22-, CD30-, BCMA- and SLAMF7-CAR T-cells were evaluated. The x-axis indicates T cells transduced or not. The y-axis represents copy number per microliter. TR, transduced sample; UT, untransduced sample.

Fig. 4.

Gene copy numbers in genetically engineered T cells normalized to rPP30. Genetically engineered T-cell samples and the associated untransduced control T cells were analyzed for copy number of three reference genes (AP3B1, rPP30 and MKL2) and the values were normalized to rPP30. HPV E7 TCR engineered T cells and CD22-, CD30-, BCMA- and SLAMF7-CAR T-cells were evaluated. For each type of genetically engineered T-cell, samples from 3 patients were tested. TR, transduced sample; UT, untransduced sample.