Tumor response and endogenous immune reactivity after administration of HER2 CAR T cells in a child with metastatic rhabdomyosarcoma

Refractory metastatic rhabdomyosarcoma is largely incurable. Here we analyze the response of a child with refractory bone marrow metastatic rhabdomyosarcoma to autologous HER2 CAR T cells. Three cycles of HER2 CAR T cells given after lymphodepleting chemotherapy induces remission which is consolidated with four more CAR T-cell infusions without lymphodepletion. Longitudinal immune-monitoring reveals remodeling of the T-cell receptor repertoire with immunodominant clones and serum autoantibodies reactive to oncogenic signaling pathway proteins. The disease relapses in the bone marrow at six months off-therapy. A second remission is achieved after one cycle of lymphodepletion and HER2 CAR T cells. Response consolidation with additional CAR T-cell infusions includes pembrolizumab to improve their efficacy. The patient described here is a participant in an ongoing phase I trial (NCT00902044; active, not recruiting), and is 20 months off T-cell infusions with no detectable disease at the time of this report

Lentivector Producer Cell Lines with Stably Expressed Vesiculovirus Envelopes

Retroviral and lentiviral vectors often use the envelope G protein from the vesicular stomatitis virus Indiana strain (VSVind.G). However, lentivector producer cell lines that stably express VSVind.G have not been reported, presumably because of its cytotoxicity, preventing simple scale-up of vector production. Interestingly, we showed that VSVind.G and other vesiculovirus G from the VSV New Jersey strain (VSVnj), Cocal virus (COCV), and Piry virus (PIRYV) could be constitutively expressed and supported lentivector production for up to 10 weeks. All G-enveloped particles were robust, allowing concentration and freeze-thawing. COCV.G and PIRYV.G were resistant to complement inactivation, and, using chimeras between VSVind.G and COCV.G, the determinant for complement inactivation of VSVind.G was mapped to amino acid residues 136–370. Clonal packaging cell lines using COCV.G could be generated; however, during attempts to establish LV producer cells, vector superinfection was observed following the introduction of a lentivector genome. This could be prevented by culturing the cells with the antiviral drug nevirapine. As an alternative countermeasure, we demonstrated that functional lentivectors could be reconstituted by admixing supernatant from stable cells producing unenveloped virus with supernatant containing envelopes harvested from cells stably expressing VSVind.G, COCV.G, or PIRYV.G.NIBSC; EPSRC; UCL Cancer Institute Research Trust

Production of self-inactivating lentiviral vectors by constitutive packaging cell lines for gene therapy clinical applications

Lentiviral vectors (LVs) are useful experimental tools for stable gene delivery and have been used to treat human inherited genetic disorders and hematologic malignancies with promising results. Because some of the LV components are cytotoxic, transient plasmid transfection has been used to produce the large batches needed for clinical trials. However, this method is costly, poorly reproducible and hard to scale up. Generation of stable packaging cell lines (PCLs) that continuously produce LVs can potentially overcome these limitations. The WinPac-RDpro cell line was developed between Collins and Takeuchi laboratories in Division of Infection and Immunity, UCL by inserting a codon-optimized HIV-1 Gag-Pol expression cassette in a continuously expressed locus in 293FT cells using Cre recombinase-mediated cassette exchange (RMCE). Subsequently HIV-1 Rev and RDpro envelope expression cassettes were serially transfected. In this thesis, WinPac-RDpro cells were used to generate model producer cells by stably transfecting a plasmid expressing a SIN GFP-encoding LV. Vector titers in excess of 106 293T transducing units (TU)/ml could be repeatedly harvested from the final producer clones in a volume of >0.5 L even under reduced serum conditions. Titers could be increased to around 1 x 10^8 293T TU/ml by concentration using scalable tangential flow filtration (TFF). Additionally, these LVs efficiently transduced human T cells and CD34+ cells at low multiplicities of infection (MOI). Titers in excess of 10^6 TU/ml were achieved using an RMCE-based strategy that was aimed at introducing a SIN LV expression cassette at a pre-selected locus. Similar titers were also achieved by using a promoterless selectable marker cloned in cis to the vector genome expression cassette. Furthermore, the Cocal Virus G protein (COCV-G) was stably expressed in WinPac cells to generate WinPac-CVG cells. These packaging cells were able to support the production of COCV-G pseudotyped SIN LVs at high titers (up to 106 TU/ml) following transient supplementation of a SIN LV expression plasmid. The efficient and stable expression of SIN LV genomes in these cells is expected to facilitate high-titer production of vectors with favorable characteristics. In conclusion, the work presented here provides significant improvements to available LV production methods. This will be of use to all basic and clinical investigators who wish to produce large batches of LVs, and addresses an important issue that has hindered large-scale LV clinical testing and application

Intrapersonal intelligence: supporting students in the English classroom

This study investigated eight and nine year old children's capabilities to develop skills in the intrapersonal intelligence domain as defined by Howard Gardner. A group of twenty-seven, seven to nine year olds were introduced to a program specifically designed to foster their self-knowledge as learners and their self-management skills in the English learning environment. The students were introduced to activities that would help them to identify their own relative strengths and limitations and use this knowledge to negotiate a learning environment that would best suit their own learning needs. This program included developing skills in goal setting and identification of personal learning strategies. It also sought to improve work habits and student on- task behaviors and encourage self-monitoring, self-evaluation and self-reflection. The results obtained evidenced a considerable improvement in the students' self- knowledge and how this impacted on their perceptions of themselves as learners. The students grew increasingly aware of their own relative strengths and used this information to negotiate their learning environment, to identify strategies that worked for them and to take increasingly more responsibility for their own learning