To develop novel cell therapies to improve outcomes after stem cell and organ transplant.

My research to date has focussed primarily on improving outcomes after haemopoietic stem cell transplant and during the period of the award I will continue to work on the approaches outlined in section 2. However, during this award I aim to extend immunotherapy approaches to the solid organ transplant (SOT) setting. I am a national expert on EBVassociated post-transplant lymphoproliferative disease (PTLD) and lead clinician responsible for the management of this complication in SOT patients at Great Ormond St Hospital, working closely with the renal/heart/lung transplant teams. I regularly provide advice on management of patients with PTLD to colleagues around the UK and abroad. My group has developed a novel immunotherapy approach for the treatment of this disorder in SOT patients. The NIHR Research Professorship will enable me translate this pre-clinical work through the necessary scale-up to a clinical study to test this approach. My established track record in translational clinical studies on adoptive cellular immunotherapy, together with my close links with some of the leading paediatric SOT programmes in the UK and the world-class gene therapy programme at UCL/ICH make us uniquely positioned to perform clinical studies in this field. Together with the ongoing studies described in section 2, this work will (a) demonstrate the feasibility of translating cellular therapies from bench to bedside, extending this approach to organ transplant recipients (b) build further our existing capability at UCL ICH to deliver clinical studies of Cell and Gene therapy and (c) establish me as an international leader in the field of immunotherapy after SOT. Immunotherapy with EBV-specific cytotoxic T-lymphocytes resistant to calcineurin inhibitors for Lymphoproliferative Disease after Solid Organ Transplant Rationale: 4000 patients undergo solid organ transplant (SOT) per year in the UK and these patients are at high risk of developing malignancy. EBV driven PTLD is the commonest cancer observed in patients after SOT and we estimate there are 200 new cases/year in the UK. PTLD is thus a major disease burden in this patient group. PTLD arises from the uncontrolled proliferation of EBV-infected B-cells when the normal cellular immune response to EBV (EBV cytotoxic T-cells, CTL) is suppressed by drugs used to prevent rejection. The incidence of EBV-PTLD post SOT ranges from 1-20% and is highest when intensive immunosuppression is used (eg small bowel and lung transplant). Current therapies for PTLD are frequently ineffective and have significant toxicity. Reducing immunosuppression frequently results in graft rejection (indeed in one large series of patients with PTLD death from graft rejection was as frequent as death from disease). Rituximab is only effective in 50-60% of patients and is associated with a high relapse rate and chemotherapy is often poorly tolerated. Hence the outcome for patients with PTLD is poor with event free survival rates of 60-70%. Adoptive transfer of ex vivo–expanded donor-derived EBV-specific CTLs have been highly successful in preventing and treating EBV-PTLD in stem cell transplant (SCT) recipients. However, the results of similar approaches in the SOT setting have been much less impressive with limited expansion, persistence and efficacy. This difference is likely to represent the ongoing need for immunosuppression post-SOT, which often cannot be withdrawn because of the risk of rejection. The major immunosuppressive drug used after SOT is the calcineurin inhibitor, tacrolimus (FK506) and in vitro studies have shown that CTL do not proliferate or secrete cytokines in the presence of this drug. In work funded by the NIHR, using retroviral transfer of a dominant negative calcineurin mutant (CNA12) that does not bind FK506 but retains the ability to dephosphorylate NFAT, we have genetically engineered EBV CTL to be resistant to FK5069. As shown below, transduction of primary EBV CTL with retroviral vectors carrying CNA12 enabled CTL to proliferate and to secrete IFN-? in response to stimulation with EBV in the presence of supra-therapeutic doses of FK506, although they remain sensitive to cyclosporin. Transduced CTL had an unchanged immunophenotype, remained antigen-dependent and lysed autologous EBV-infected targets normally.We have recently demonstrated that human EBV-CTL transduced with such mutants lead to regression of EBV induced tumours in a ?-chain/RAG2-/C5- SCID-hu model of PTLD even in the presence of FK506.The standard methodology for generating EBV CTL takes many weeks and involves the use of live B95-8 EBV, with consequent manufacturing and regulatory complexity which has been a major barrier to the broader use of this strategy. To make this approach simpler and more broadly applicable, we have developed a rapid methodology for generation of FK506 resistant CTL using IFN-? capture following stimulation with a mix of peptides of the immunodominant EBV antigens. As shown below, when CTL generated using this method are transduced with a retroviral vector encoding our calcineurin mutants, they can proliferate and secrete IFN-? in the presence of FK506.Objectives: 1. To generate retroviral vectors encoding a calcineurin mutant conferring resistance to tacrolimus (CNA12) linked a minimal CD34 epitope (Q8) marker gene and a control vector encoding Q8 alone under Good Manufacturing Practice conditions. 2. To scale up and validate production EBV CTL transduced with these retroviral vectors from normal donors. 3. To conduct a clinical study based on the “double-marking” approach, comparing the survival and expansion of autologous EBV CTL transduced with CNB30 and control retroviral vectors. Page 16 of 36 Plan of Investigation: 1. Generation of retroviral vectors encoding a minimal CD34 epitope (Q8) alone or with CNA12: The CNA12 mutant differs from wild-type by only three residues. Consequently, we plan to co-express a marker gene Q8 consisting of a minimal 12 residue epitope from CD34 attached to the stalk and transmembrane regions of CD8 to facilitate producer line selection, measurement of transduction efficiency and tracking transduced CTLs in patient peripheral blood using flow cytometry. In the first year of the award, we will generate 2 retroviral vectors encoding (a) Q8 alone and (b) a bicistronic vector co-expressing a codon-optimised CNA12 with Q8 using the self-cleaving 2A sequence.The use of retroviral vectors, which integrate only in dividing T-cells will ensure selective transduction of EBV-specific CTL following antigenic stimulation. Using the PG13 packaging cell line, high titre stable producer clones of each vector will be generated under Good laboratory Practise (GLP) conditions at the Gene Vector Lab at UCL. We have extensive experience of generation of retroviral producer clones for clinical studies from our ongoing study of CD19 chimeric antigen receptor transfer. The supernatants obtained from these clones will be tested for their ability to transduce primary EBV CTL and enable them to proliferate and secrete IFN-? in the presence of FK506. We will develop and validate qPCR assays for the junctions between the retroviral packaging sequence and Q8/CNA12 transgenes enabling us to distinguish between cells transduced with each retrovirus. The optimal clones will then be transferred to our commercial partner EUFETS for generation and characterisation of clinical grade supernatants at the end of the first year of the award.2. Scale-up and dry runs: EBV CTL will be generated from buffy coats of normal EBV-seropositive donors by stimulation with a peptide mix of immunodominant EBV antigens followed by selection using the IFN- capture approach, as described by Moosman et al. We will collaborate with Prof Cliona Rooney's group (Houston, USA) to optimise large-scale generation of CTL in bioreactors. Following secondary stimulation of selected CTL with the peptide mix, they will be transduced with aliquots of our clinical grade retroviral vectors using retronectin and serum-free media under GMP conditions in the Gene Therapy Laboratory at Great Ormond St Hospital (GOSH). These dry runs will demonstrate the clinical feasibility of our approach and enable us to establish SOPs for generation of FK506 resistant CTL. Therapeutic doses (10e7/m2) of transduced EBV-CTLs will be generated and sampled for sterility/Mycoplasma, endotoxin, transduction efficiency, average copy number and replication competent retrovirus. We will flow cytometrically phenotype transduced CTL and assess the integration site profiles of the 2 vectors by LAM-PCR. The functionality of transduced CTL will be tested in Cr release cytotoxicity assays and by evaluating the ability of CTL transduced with the CNA12- Q8 vector but not the Q8 vector to proliferate and secrete IFN-? the presence of therapeutic levels of FK506. We will demonstrate that resistant CTL do not grow in the absence of antigenic stimulation and determine that cryopreserved transduced CTL show persistence of transgene expression over time. These studies will form the basis of our applications for ethical (GTAC) and regulatory (MHRA) approval for the proposed clinical study below. Based on our previous experience, we anticipate application for these approvals at the end of the second year of the award. 3. Clinical study comparing the survival and expansion of autologous EBV CTL transduced with CNA12 and control retroviral vectors: If our applications for ethical and regulatory approval are successful, in years 3-5 of the award, with our clinical collaborators at GOS and King’s College Hospital, we plan to test adoptive immunotherapy with autologous FK506 resistant EBV CTL in a clinical study in patients with PTLD. Study design 2 centre, open-label Phase I/II CTIMP. - Eligibility Paediatric solid organ transplant patients who develop EBV PTLD. - Trial Intervention Eligible patients will be leucapheresed prior to treatment with Rituximab. Other immunosuppressants will be reduced, but tacrolimus will be maintained at therapeutic levels. For each patient, both EBV CTL transduced with CNA12-Q8 and Q8 alone vectors will be generated under GMP conditions in the Gene Therapy laboratory at GOSH. Following completion of Rituximab therapy, patients will restaged. They will then be treated intravenously with 10e7/m2 CNA12- Q8 transduced EBV CTL and an equal dose of Q8 transduced EBV CTL accounting for transduction efficiency.- Assessment 1. Infusional toxicity will be assessed using the NCI Common criteria. 2. Blood samples will be taken monthly for 6 months and then 2 monthly until 1 year post-CTL infusion to assess EBV viral load by qPCR and in vitro EBV-specific T-cell responses by IFN-? ELISPOT assays 3. The persistence of transduced CTL will beassessed at the same time-points by flow cytometry for the Q8 epitope. The proportion of circulating Q8+ CTL transduced with the CNA12 and control vectors will be compared by qPCR of the junctional sequences between the retroviral packaging signal and either Q8 or CNA12. 4. Patients will be assessed monthly clinically and with appropriate organ-specific investigations for evidence of rejection, which if suspected will be confirmed histologically. We anticipate the risk of graft rejection is low because FK506 will be maintained at therapeutic levels but if this occurred, the CNA12 transduced CTL could be suppressed effectively with cyclosporin, providing an important safety strategy. - End-points 1. Primary: we will compare the in vivo survival and expansion of EBV CTL transduced with the 2 vectors. We hypothesize that CNA12-Q8 transduced CTL will show preferential expansion and prolonged persistence compared with Q8 transduced CTL because of ongoing FK506 therapy. 2. Secondary : the incidence of graft rejection and relapse rate at 1 and 2 years post CTL therapy will be determined and compared with historical data on patients treated with Rituximab alone. - Projected Outcomes This project will establish the feasibility/safety of immunotherapy with EBV CTL genetically modified to express CNA12 and will determine if this approach improves the in vivo persistence and expansion of adoptively transferred T-cells in the presence of ongoing immunosuppression. Such an approach could potentially be of major clinical benefit to both adult and paediatric patients undergoing organ transplantation by reducing the morbidity and mortality associated with PTLD without the need for withdrawal of the drugs used to prevent rejection. Further, this data may have important broader applicability as it represents a generic approach to enabling adoptively transferred T-cells to function in the face of ongoing immunosuppression. LaySummary>