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

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.

Genome-wide profiling of retroviral DNA integration and its effect on clinical pre-infusion CAR T-cell products.

BACKGROUND: Clinical CAR T-cell therapy using integrating vector systems represents a promising approach for the treatment of hematological malignancies. Lentiviral and γ-retroviral vectors are the most commonly used vectors in the manufacturing process. However, the integration pattern of these viral vectors and subsequent effect on CAR T-cell products is still unclear. METHODS: We used a modified viral integration sites analysis (VISA) pipeline to evaluate viral integration events around the whole genome in pre-infusion CAR T-cell products. We compared the differences of integration pattern between lentiviral and γ-retroviral products. We also explored whether the integration sites correlated with clinical outcomes. RESULTS: We found that γ-retroviral vectors were more likely to insert than lentiviral vectors into promoter, untranslated, and exon regions, while lentiviral vector integration sites were more likely to occur in intron and intergenic regions. Some integration events affected gene expression at the transcriptional and post-transcriptional level. Moreover, γ-retroviral vectors showed a stronger impact on the host transcriptome. Analysis of individuals with different clinical outcomes revealed genes with differential enrichment of integration events. These genes may affect biological functions by interrupting amino acid sequences and generating abnormal proteins, instead of by affecting mRNA expression. These results suggest that vector integration is associated with CAR T-cell efficacy and clinical responses. CONCLUSION: We found differences in integration patterns, insertion hotspots and effects on gene expression vary between lentiviral and γ-retroviral vectors used in CAR T-cell products and established a foundation upon which we can conduct further analyses.

High efficiency closed-system gene transfer using automated spinoculation.

BACKGROUND: Gene transfer is an important tool for cellular therapies. Lentiviral vectors are most effectively transferred into lymphocytes or hematopoietic progenitor cells using spinoculation. To enable cGMP (current Good Manufacturing Practice)-compliant cell therapy production, we developed and compared a closed-system spinoculation method that uses cell culture bags, and an automated closed system spinoculation method to decrease technician hands on time and reduce the likelihood for microbial contamination. METHODS: Sepax spinoculation, bag spinoculation, and static bag transduction without spinoculation were compared for lentiviral gene transfer in lymphocytes collected by apheresis. The lymphocytes were transduced once and cultured for 9 days. The lentiviral vectors tested encoded a CD19/CD22 Bispecific Chimeric Antigen Receptor (CAR), a FGFR4-CAR, or a CD22-CAR. Sepax spinoculation times were evaluated by testing against bag spinoculation and static transduction to optimize the Sepax spin time. The Sepax spinoculation was then used to test the transduction of different CAR vectors. The performance of the process using healthy donor and a patient sample was evaluated. Functional assessment was performed of the CD19/22 and CD22 CAR T-cells using killing assays against the NALM6 tumor cell line and cytokine secretion analysis. Finally, gene expression of the transduced T-cells was examined to determine if there were any major changes that may have occurred as a result of the spinoculation process. RESULTS: The process of spinoculation lead to significant enhancement in gene transfer. Sepax spinoculation using a 1-h spin time showed comparable transduction efficiency to the bag spinoculation, and much greater than the static bag transduction method (83.4%, 72.8%, 35.7% n = 3). The performance of three different methods were consistent for all lentiviral vectors tested and no significant difference was observed when using starting cells from healthy donor versus a patient sample. Sepax spinoculation does not affect the function of the CAR T-cells against tumor cells, as these cells appeared to kill target cells equally well. Spinoculation also does not appear to affect gene expression patterns that are necessary for imparting function on the cell. CONCLUSIONS: Closed system-bag spinoculation resulted in more efficient lymphocyte gene transfer than standard bag transductions without spinoculation. This method is effective for both retroviral and lentiviral vector gene transfer in lymphocytes and may be a feasible approach for gene transfer into other cell types including hematopoietic and myeloid progenitors. Sepax spinoculation further improved upon the process by offering an automated, closed system approach that significantly decreased hands-on time while also decreasing the risk of culture bag tears and microbial contamination.

Variation of the killer cell immunoglobulin-like receptors and HLA-C genes in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is an Epstein-Barrvirus (EBV)-associated malignancy. Previous studies have shown that NPC is associated with specific human leukocyte antigen (HLA) alleles which function in adaptive immunity to present viral and other antigens to the immune system. The role of innate immunity in NPC development is unknown. To determine whether innate immunity is associated with NPC, a case-control study was conducted among 295 Taiwanese NPC cases (99% EBV seropositive) and 252 community controls (29% EBV seropositive). Using high-resolution genotyping, we evaluated the variation of HLA class I alleles and killer cell immunoglobulin-like receptor (KIR) alleles. Located on the surface of natural killer (NK) cells and a subset of T cells, inhibitory KIRs diminish NK cytolysis of target cells upon binding to their HLA class I ligands and activating KIRs are thought to stimulate NK destruction of target cells. Our results suggest that an increasing number of activating KIRs may be associated with increasing NPC risk, particularly in individuals seropositive for anti-EBV antibodies known to be linked to NPC susceptibility (P(trend) = 0.07). Among EBV-seropositive individuals, carriers of > or =5 activating KIRs had a 3.4-fold increased risk of disease (95% confidence interval, 0.74-15.7) compared with individuals with no functional activating KIRs. In contrast, there was no clear evidence of risk associated with increasing numbers of inhibitory KIRs. When evaluating HLA-Cw alleles, we observed that carriers of HLA-Cw*0401 alleles were at a significantly reduced NPC risk (odds ratio, 0.46; 95% confidence intervals, 0.23-0.92), an effect that could not be explained by linkage disequilibrium with other NPC-associated HLA alleles. Our results suggest that KIR-mediated activation may be associated with NPC risk. As this finding is consistent with a recent report examining cervical cancer, a malignancy caused by human papillomavirus, the data raises the possibility that KIRs, and more generally innate immunity, may be involved in the pathogenesis of viral-associated cancers.