A Large Majority of Donor NeoR-Expressing Gene Modified T-Cells Circulating More Than 9 Years after Infusion with allo-BMT, and Capable of Being In-Vitro Expanded and Cloned, Contain a Large Deletion of the HS-tk Gene and Arise from One T-Cell Clone in Each Patient.

Abstract

We performed a clinical trial of HS-tk-expressing gene-modified T cell (GMTC) infusion at time of geno-identical allo-BMT. Among 12 patients (pts) initially included, 4 are still alive, off immunosuppression, free of chronic graft-versus-host disease (GvHD). More than 9 years after BMT, circulating GMTC can be found in all 4 patient's mononuclear cells (PBMC), as detected by HS-tk or NeoR quantitative PCR assays. Although a similar sensitivity for both assays, GMTC were detected at a lower level with the HS-tk vs NeoR PCR assay, suggesting HS-tk gene deletions. The aims of our study were todevelop a molecular tool in order to precisely identify the areas of deletion,characterize the mechanism of deletion anddetermine if the deletions were present in the packaging cell line genome or occurred ex-vivo during GMTC preparation or subsequently in-vivo.PBMC, harvested from the 4 pts (70, 106, 67 & 76 months post BMT) were polyclonally expanded, G418-selected and cloned. Of 32 NeoR+ clones, 31 (12, 3, 2 and 14 clones for pts #6, 7, 8 & 9, respectively) were HS-tk PCR negative, confirming the presence of HS-tk transgene deletions in a large majority (31/32) of long-term circulating Neo-R expressing GMTC. By contrast, all clones generated from freshly produced GMTC were found to be both NeoR+/HS-tk+, excluding an impact of the cloning process. Using a newly designed PCR-based transgene “walking” method (from the NeoR gene to the 5′LTR), we identified, among the 31 NeoR+HS-tk- clones, 4 different deletions, each one patient specific, involving the total HS-tk transgene and including partial flanking SV40 or packaging signal y sequences. These newly identified deletions differed from the spliced HS-tk form previously described (Garin et al., Blood 2001). Junction deletion areas were sequenced and involved, for 3 out of 4 patients, homologue sequence motifs (CCGCCC, AATTC and GATC respectively for pt# 6, 7 & 8), suggesting recombination mechanisms within the transgene sequences. With the help of deletion-specific PCR assays, we then confirmed that each deletion was patient-specific and not detected in the 3 other pts. Using more sensitive deletion-specific (nested) PCR assays, no deletions within packaging cell line DNA were detected. Nevertheless, these patient-specific deletions were found in GMTC products, albeit at a very low frequency, prior to infusion at time of BMT as well as 1, 3 and 6 years post infusion, independently of GCV treatment or anti-HS-tk immune response. Lastly, by means of LAM-PCR, we determined that all the clones of a same patient carried the same patient-specific insertional site, demonstrating that all the G418-reselected/cloned T cells of a patient derived from the same original GMTC. Overall, we establish thatpatient-specific HS-tk gene deletions occur within the integrated retroviral sequence in GMTC prior to infusion;HS-tk gene deletions are found in almost all Neo-R expressing long term circulating GMTC, thus strongly in favour of a in vivo selection advantage, by escaping an anti- HS-tk immune response and/or GCV treatment andunique patient-specific HS-tk gene deletions and retroviral insertion site in GMTC capable of in-vitro expansion/cloning.This last finding suggests an extremely rare initial gene deletion event and/or a possible in vivo growth/survival advantage of rare GMTC clones.