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Advanced Teaching and TRaining for Adoptive Cell Therapy

Final Report Summary - ATTRACT (Advanced Teaching and TRaining for Adoptive Cell Therapy)

Considerable resources are devoted to fighting cancer throughout Europe, yet these efforts are not producing the results that might be expected. This is partly due to the fragmentation and duplication of research efforts within Member states and the lack of co-ordination at European level. However it can also be attributed to the need to retain and most importantly develop researchers within the EU, who have the research skills required to make a real contribution to cancer treatment. The need for quality researchers is particularly acute within the emerging interdisciplinary field of Adoptive Cell Therapy. T The ATTRACT comprises of thirteen partners, grouping together researchers working in high profile Universities, Research Institutes and industrial organizations. The consortium is based on on-going research collaborations: a relationship established within the 2005 FP6 Integrated Project ATTACK (Adoptive engineered T-cell Targeting to Activate Cancer Killing: LSh-CT-2005-018914). Through the execution of a collaborative research training programme, delivered by Europe’s leading academic and industrial experts in the field of Immunotherapy, the ATTRACT project equips selected researchers from a range of related backgrounds, with the interdisciplinary skills needed to not only initiate further development in Adoptive Cell Therapy, but also to become highly desirable candidates for future employment.

WP3 Objective: Homing and Persistence - to define the in vitro and in vivo parameters that will enhance the persistence and anti-tumor efficacy of re-directed T cells. Description of the Work Performed and Main Results Achieved: Different lymphopenia-inducing regimens were found to distinctly affect the balance between regulatory and effector CD4 T cells and with CD8 cells with high levels of busulphan/cyclosphosphamide resulting in an increased gender-dependent cytotoxicity. Nevertheless, we were able to establish a busulphan/cyclophosphamide pre-conditioning regimen promoting the persistence and efficacy of adoptively transferred cells. Less differentiated T cells (CD62L+/CCR7+) were found to enhance persistence and a host dendritic cell subset promoted their proliferation. T cells transduced with CX3CR1 showed significantly higher migration to a CX3CL1-exresssing colon tumor and this corresponded with increased anti-tumor efficacy. The combination of either IL-15/IL-21 or IL-7/IL-15 resulted in the expansion of T cells with a less differentiated phenotype and higher engraftment but under some conditions, could lead to the onset of xeno GvHD. Significant advances were made in promoting correct pairing of transgenic TCRs by cysteinization and codon optimization of the transgenic TCRs. Cells with dual CARs were developed and notably, each individual CAR was functional and the cells could eradicate tumors. Further work showed that a CD28-ζ costimulation domain was superior to CD28-ζ-OX40. Potential Impact and Use of the Findings: The findings from this WP will have, and indeed, have already impacted, on the development of anti-tumor immunotherapy trials with redirected T cells. This can be directly observed in the clinical melanoma and gastroesophageal trials proposed by the ATTACK2 consortium using redirected T cells. Furthermore, the data amassed here are critical for minimizing off-target and toxic effects.

WP4 Objective: Safety - Adoptive T cell therapy has multiple risks including unintended activation against healthy tissues and acute cytokine release syndrome. The objectives of this WP were to identify the potential for autoimmunity after short- and long-term persistence of gene-modified T cells, to determine the potential for sudden cytokine release of gene modified T cells and to define the risk of genotoxicity by retroviral gene transfer to T cells. Description of Work Performed and Main Results Achieved: (1) The newly developed RNAi-assisted TCR replacement strategy reduces the formation of mixed TCR dimers, and thereby severely reduces the risk of TI-GVHD in TCR gene therapy. (2) There is an affinity threshold in CAR mediated T cell activation above which increase in affinity does not produce increased T cell function. CD28 costimulation does not impact the threshold. Different T cell subsets like effector memory cells and cytokine induced killer cells have different requirements with respect to costimulation in order to persist in the long-term. The koff-rate of the TCR provides important therapeutic and diagnostic information on the in vivo quality of T cells. (3) T cells can be re-stimulated by application of nano-beads coated with an antibody recognizing the CAR. Repetitive restimulation of anti-CEA CAR T cells in vivo did not produce auto-immunity against healthy tissues. (4) Preferential outgrowth of TCR modified T cells with retroviral integration in specific loci appeared to occur in vivo. (5) No differences with respect to auto-immunity, in particular weight loss, between mice treated with T cells pre-exposed to IL2 vs IL15+IL21 were observed. Potential Impact and Use of the Findings: Analyses will have major impact on the design of future clinical trials using CAR and TCR modified T cells, respectively. Significant implications are: RNAi-assisted TCR replacement reduces the formation of mixed TCR dimers, and thereby severely reduces the risk of TI-GVHD in TCR gene therapy; combined CD28-OX40 costimulation is superior for central memory cells, but not for cyokine induced killer cells which require CD28 costimulation only; repeated anti-CEA CAR stimulation of engineered T cells did not induce auto-immune pathologies or a cytokine storm in the CEA transgenic mouse; no differences in safety issues with respect to weight loss between mice treated with T cells pre-exposed to IL2 vs IL15+IL21; optimized TCR will be designed with respect to the koff-rate in binding which provides important information on the in vivo quality of T cells.

WP5 Objective: Interaction with Immune System - Research activity within WP5 was focused on the understanding of the impact of ACT on immune competence and on the crosstalk between adoptively transferred gene-engineered T cells and the host immune system. In addition, the impact of ACT on antigenicity and immunogenicity of tumours in preclinical models of tumour disease, and the contribution of the microenvironment to the functionality of engineered T cells were investigated together with the possibility to identify combination strategies able to synergize with ACT. Final aim of WP5 was to ameliorate persistence, homing, survival and intra-tumoral effectors functions, and ultimately therapeutic efficacy of ACT. Description of the Work Performed and Main Results Achieved: The fate of TCR/CAR redirected T cells within lymphoid organs and solid tumours was traced by multicolour flow cytometry and immunofluorescence analyses in preclinical tumour models. Data on TCR and CAR redirected T cells support the ability of adoptively transferred T cells to recirculate within the lymphoid tissues and to relocalize within the tumor area. Infused TCR and CAR redirected T cells overcome immunological tolerance to studied tumors, infiltrate transformed tissues, and in some case lead to tumor eradication, ameliorating long-term survival. In some instances, tumor escape was found, in spite of preserved antigenicity of the tumor, calling for more efficient tumor debulking, possibly via the implementation of combined strategies. Genetic engineering of T cells, by vectors enabling better intracellular signalling, and making T cells insensitive to metabolites deprivation, as well as strategies able to redirect T cells to the tumor were implemented with improved anti-tumor efficacy of ACT. In addition, novel activation and culture conditions have been devised to ameliorate long-term survival of infused T cells, and by that enhance immune surveillance against recurrent disease. Finally, combination strategies including post ACT tumor-directed vaccination, and stromal-targeting reagents have been tested, and validated for preclinical efficacy. Potential Impact and Use of the Findings: Data generated in this work package provide the basis to design innovative clinical trials adopting TCR engineered T cells. It is expected that some of the strategies envisaged in the studied preclinical models will be on a fast track for clinical translation.

WP6 Objective: Identification of Novel Targets and Development of New Receptors - this Work Package aimed at the isolation and characterization of new receptors, whether it be chimeric antigen receptors (CARs) or T cell receptors (TCRs), against defined target molecules. Target molecules were, where possible, carefully selected based on tumor-restricted expression and immunogenicity. When receptors would meet criteria set for antigen-specific T cell responses, such receptors would be developed further for clinical application. Description of the Work Performed and Main Results Achieved: Selection and characterization of antibodies with specificities for various HLA-A2-restricted MAGE epitopes. It was observed that selections with anchor-modified peptides result in antibodies that do not recognize wild type peptide, a finding that favors inclusion of wild type peptide or antigen-positive tumor cells in library selections. Isolation and characterization of TCRs specific for multiple MAGE-C2 epitopes, NY-ESO1 and Smcy (all epitopes are HLA-A2-restricted). This work has in part been published in Straetemans T, Clin Dev Immunol, 2012, and Sommermeyer D, Int J Can, 2013. TCRs may possess different reactivities when isolated from T cell clones that are generated using different T cell priming protocols. Using different NY-ESO1 TCRs it was demonstrated that one should assess TCRs for multiple parameters prior to a decision regarding their clinical development. TCRs may recognize a newly formed epitope when isolated from T cells that are primed in vitro with autologous dendritic cells that express codon-optimized HPV E7 antigen. Although these TCRs are not applicable for clinical investigation, these findings point to the risk of obtaining unwanted T cell specificity when using codon-optimized antigens to prime T cells. Validation of LMP2/HLA-A2 TCR and its movement towards clinical development to treat EBV-associated lymphoma and/or nasopharyngeal carcinoma using funding from the Medical Research Council. Development of a TCR gene capture method that is based on indexing of deep-sequencing results and which allows semi-high throughput isolation of relevant TCR genes from small numbers of tumor-reactive T cells. With help of this method, a large number of patient-derived TCR chains specific for various Cancer Testis Antigens and neo-antigens has been obtained, and this work has been published in Linnemann C, Nat Med, 2013. Notably, the use of such TCR collections in autologous TCR gene therapy in humanized mouse models demonstrated to be feasible. Optimization of a transposon-based gene delivery system for which a patent application is filed. This gene delivery system, together with the isolated TCR chains, may form the required ‘ingredients’ to develop TCR gene therapy into a much more broadly applicable approach. Potential Impact and Use of the Findings: Findings in this Work Package impact the following aspects of the field of receptor gene therapy: A collection of antigens, including Cancer Testis Antigens and viral antigens, have been exploited to obtain antigen-specific CARs or TCRs, and some of these receptors represent promising candidates for further validations. Validations with a TCR against LMP2 have been completed and this TCR is now included in a planned clinical trial. With respect to the selection of receptors a number of rules have been established, such as a recommendation against selection of antibodies with modified peptides, or selection of TCRs with codon-optimized antigens. Importantly, it became evident when selecting candidate receptors that it is important to evaluate receptors at multiple levels prior to clinical development. New methods have been developed, such as TCR gene capture and optimized transposon gene delivery, which are expected to advance and accelerate the clinical translation of new TCRs. In fact, a series of patient-derived TCRs specific for Cancer Testis Antigens and neo-antigens have already been obtained using TCR gene capture and validated in humanized mice. The testing of a TCR directed against a human minor histocompatibility antigen, which has been obtained in this project, together with TCRs directed against tumor antigens, would potentially enhance the anti-tumor efficacy of T cell therapy.

WP7 Objective: Clinical Development of Adoptive Cellular Therapy - WP7 was largely concerned with the clinical end of adoptive T cell therapy and, specifically, the testing and development of systems that can produce more efficacious T-cells for clinical testing. Description of the Work Performed: focused upon the clinical delivery of T-cells for adoptive therapy applications. This has involved research level testing of T-cell production processes including the investigation of cytokine combinations for the improved ex vivo culture of T cells. Moreover, work within WP7 has also focused upon the more clinical end of cell production through the development and testing of closed system technology and testing of media / cytokine combinations. Description of the Main Results Achieved: Cell selection was one main area of focus with selection of naïve and central memory T cells investigated due to published work suggesting these cells display improved in vivo anti-tumour activity. These studies were combined with efforts to identify improved media and cytokine combinations to maintain the improved in vivo function of the T cell product. Erasmus and UniMAN have both published reports documenting the effects of ex vivo cytokines upon gene-modified T cell in vitro function and in vivo engraftment (Pouw et al. Mol Immunol 2010, Oroczo et al. Human Gene Therapy Methods 2013). Miltenyi have developed novel T cell stimulation reagents, cell selection technology and serum free media that have been tested by WP7 partners. It appears that the serum-free media is not optimal for gene-modified T cells growth and requires the addition of plasma. Miltenyi have developed the cell selection and culture aspects into one automated closed system (Prodigy). Molmed has systematically tested the various components used to generate T cells for transplant procedures resulting in an optimised protocol for the expansion and election of HSV-tk+ suicide gene expressing T-cells based upon selection of the CD271 marker. Potential Impact and Use of the Findings: CD62L selection and T-cell expansion in IL-7 / IL-15 has been proposed as a method to generate optimal T cells for therapy and this is now being tested in clinical trials under the EU FP7 programme ATTACK. Experiments performed during WP7 have aided the development of the Miltenyi prodigy system. Optimised methods for the production of HSV-tk+ T cells for transplant purposes have been developed and now will be tested for clinical application.

WP8 Objective: Clinical Trial Design - Design, Approval and Implementation of Clinical Trials of Cell therapy, Development and standardisation of assays for monitoring trials, Management of Patients receiving cell therapy / other complex immunotherapy. Description of Work Performed: Training provided in: design and submission for regulatory approval of an early phase clinical trial, protocol development, the clinical management of patients undergoing a variety of cell therapy trials, including management of toxicity, management of patients receiving high-dose interleukin-2, interpretation of laboratory data
Description of the Main Results Achieved: Successful validation runs achieved for the enrichment of naïve and central memory CD8+ T cell subsets. Standard operating procedures have been developed for the management of patients on cell therapy trials. Standard templates for trial related prescriptions developed. Potential Impact and Use of the Findings: Development of a clinical protocol for: (1) T cell subset isolation, transduction and expansion which will soon be used for a Phase I clinical trial to test the efficacy of the various T cell subsets transduced with cancer specific TCR in cancer patients (2) A randomised phase II study in metastastic melanoma to evaluate the efficacy of adoptive cellular therapy with tumour infiltrating lymphocytes (3) Phase II trial to assess the activity of NY-ESO-1 targeted T cells in advanced oesphago-gastric cancer.

Conclusions: ATTRACT extends the traditional academic research training setting, equipping researchers with the right combination of research-related and transferable competences in the field of T cell therapy of tumours. The project builds its training programme on a long term research approach and European disciplinary skill shortage in which newly emerging insights within adoptive cell therapy are further investigated, optimised and translated into clinical trials. The ATTRACT project is therefore recognised as an important milestone in immunotherapy and one that has stimulated significant activity and collaborative activity. The training programme has been greatly appreciated by the majority of those who have participated, including both fellows and supervisors and has allowed the ATTRACT Fellows to develop into independent, competitive researchers. All research objectives have been achieved within the allocated timing and with the resources available.