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Summary Based on promising pre-clinical data demonstrating the eradication of systemic B cell malignancies by CD19-targeted T lymphocytes in vivo in SCID beige mouse models, we are launching Phase 1 clinical trials in patients with chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL). We present here the validation of the bioprocess we developed for the production and expansion of clinical grade autologous T cells derived from patients with CLL. We demonstrate that T cells genetically modified with a replication-defective gammaretroviral vector derived from the Moloney murine leukemia virus encoding a chimeric antigen receptor (CAR) targeted to CD19 (1928z) can be expanded with Dynabeads® CD3/CD28. This bioprocess allows us to generate clinical doses of 1928z+ T cells in approximately 2 to 3 weeks in a large-scale semi-closed culture system using the Wave bioreactor. These 1928z+ T cells remain biologically functional not only in vitro but also in SCID beige mice bearing disseminated tumors. The validation requirements in terms of T cell expansion, T cell transduction with the 1928z CAR, biological activity, quality control testing and release criteria were met for all four validation runs using apheresis products from patients with CLL. Additionally, following expansion of the T cells, the diversity of the skewed Vβ T cell receptor repertoire was significantly restored. This validated process will be used in phase I clinical trials in patients with chemo-refractory CLL and in patients with relapsed ALL. It can also be adapted for other clinical trials involving the expansion and transduction of patient or donor T cells using any chimeric antigen receptor or T cell receptor.

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is the infectious cause of KS and is also linked to the pathogenesis of certain lymphoproliferations (4,14). It is proposed that KSHV latent proteins are directly involved in modulating signal transduction pathways and cellular circuits leading to uncontrolled cell proliferation (2).At the far right-hand end of the KSHV genome, open reading frame (ORF) K15 encodes a putative transmembrane protein in the same genomic location as the Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) (5,7,12,39). K15 resembles LMP2A not only in genomic location but also in its splicing pattern and predicted protein structure. Two highly divergent forms of K15 have been identified: the predominant (P) and minor (M) forms (5, 12, 39). These two alleles possess only 33% amino acid identity yet retain 12 membrane-spanning domains and a putative cytoplasmic signal-transducing carboxyl terminus (C terminus) (5). The C terminus of K15 has potential signaling motifs, including Src homology 2 and 3 binding domains (SH2-B and SH3-B, respectively) (12). A CD8-K15 C-terminal chimeric protein was shown to be constitutively tyrosine phosphorylated within the SH2-B motif (5). Like LMP2A, this CD8-K15 chimeric protein modulates B-cell receptor (BCR) signal transduction. The mechanism(s) of signal transduction is unknown but appears to be distinct from that of LMP2A and does not involve intracellular free calcium mobilization (5).In addition, the C terminus of K15 has sequences similar to those found in EBV LMP1, including a putative tumor necrosis factor receptor-associated factor (TRAF) binding site. K15 therefore appears to be a hybrid of a distant evolutionary relative of both EBV LMP1 and LMP2A (13). The putative C terminus of K15 has been shown to interact with the TRAFs (12), and we have also shown that K15 can indeed activate NF-B via this putative TRAF binding site (unpublished data). By way of activating NF-B, LMP1 of EBV plays an essential role in EBV-induced transformation of B lymphocytes (3,16,21). NF-B activation also appears to be essential for the proliferation potential of KSHV positive primary effusion lymphoma (PEL) cells (22), but whether all of this NF-B activity in PEL cells is due to K15 expression is not yet known.Although K15 mRNA has been demonstrated in PEL cells (5, 12, 39), it is not known whether the K15 protein is actually expressed in latently infected tumor cells. The size of endogenous protein, its exact subcellular localization, and its cellular binding partners have not previously been determined.We generated a monoclonal antibody (MAb) against K15 and show here that when K15 cDNA is ectopically expressed we detect the predicted 50-kDa form as well as a series of smaller proteolytically cleaved forms, of which the 35-and 23-kDa species are predominant. Deletion of the initiator AUG of the K15 ORF abolished protein expression, suggesting that the 50-kDa form of K15 is a precursor which is subsequently proteolytically processed into smaller species. We ...

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IntroductionLiver disease mortality and morbidity are rapidly rising and liver transplantation is limited by organ availability. Small scale human studies have shown that stem cell therapy is safe and feasible and has suggested clinical benefit. No published studies have yet examined the effect of stem cell therapy in a randomised controlled trial and evaluated the effect of repeated therapy.Methods and analysisPatients with liver cirrhosis will be randomised to one of three trial groups: group 1: Control group, Standard conservative management; group 2 treatment: granulocyte colony-stimulating factor (G-CSF; lenograstim) 15 µg/kg body weight daily on days 1–5; group 3 treatment: G-CSF 15 µg/kg body weight daily on days 1–5 followed by leukapheresis, isolation and aliquoting of CD133+ cells. Patients will receive an infusion of freshly isolated CD133+ cells immediately and frozen doses at days 30 and 60 via peripheral vein (0.2×106 cells/kg for each of the three doses). Primary objective is to demonstrate an improvement in the severity of liver disease over 3 months using either G-CSF alone or G-CSF followed by repeated infusions of haematopoietic stem cells compared with standard conservative management. The trial is powered to answer two hypotheses of each treatment compared to control but not powered to detect smaller expected differences between the two treatment groups. As such, the overall α=0.05 for the trial is split equally between the two hypotheses. Conventionally, to detect a relevant standardised effect size of 0.8 point reduction in Model for End-stage Liver Disease score using two-sided α=0.05(overall α=0.1 split equally between the two hypotheses) and 80% power requires 27 participants to be randomised per group (81 participants in total).Ethics and disseminationThe trial is registered at Current Controlled Trials on 18 November 2009 (ISRCTN number 91288089, EuDRACT number 2009-010335-41). The findings of this trial will be disseminated to patients and through peer-reviewed publications and international presentations.

The ability to genetically modify human T cells to target tumor antigens through retroviral gene transfer constitutes a potentially powerful approach to cancer immunotherapy. However, low transduction efficiencies may hamper the efficacy of such therapeutic strategies in the clinical setting. Most commonly, gammaretroviral gene transfer into T cells is conducted through spinoculation, that is, centrifugation of retroviral particles and T cells on RetroNectin-coated non-tissue culture vessels. Here we present data investigating the impact of temperature, speed, and frequency of spinoculation on T cell transduction efficiencies. We found that all three variables independently impacted gene transfer, with increasing temperature, speed, and frequency of spinoculation all enhancing the transduction of T cells. These improved conditions were additive, with the greatest proportion of transduced T cells being generated at the highest tested temperature and speed, after daily spinoculation for 2 to 3 days. Under these conditions, enhanced gene transfer was observed in T cells derived from healthy donors, using research-grade vector stocks. Whereas both RetroNectin and spinoculation were critical to optimal gene transduction, preloading of gammaretroviral particles before spinoculation did not enhance gene transfer. Significantly, application of these enhanced transduction conditions to T cells derived from previously treated patients with chronic lymphocytic leukemia allowed for adequate gene transfer under both small-scale and large-scale clinically applicable conditions using either preclinical or current Good Manufacturing Practice-grade gammaretroviral vector stocks.

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