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2018 CRUK KHP Centre PhD Project Catalogue

Contents

1.1 Dissecting the heterogeneity of tumour-infiltrating phagocytes: implications for tissue remodelling and immunotherapy………………………………………………………………………………………………………………………………………2

1.2. Targeting the tumour microenvironment to suppress pancreatic cancer cell invasion……………………………………3 2.1. Identification of tumour-specific epitopes produced by proteasome-catalysed peptide

splicing and suitable for adoptive T cell immunotherapy………………………………………………………………………………..4

2.2. Dissecting alterations in immune and tumour signalling pathways in head and neck cancer induced by combined T4 CAR T-cell and pembrolizumab immunotherapy……………………………………………………………………5

2.3. Understanding and improving the immune-modulatory actions of chemotherapy in cancer…………………………..6 3.1 Defining cellular and molecular mechanisms underlying cancer immunotherapy-induced

auto-inflammatory syndromes…………………………………………………………………………………………………………………………7 3.2. Migrastatics – How do they affect the immune response and existing immunotherapies?.................................8 3.3. An Indirect Labelling Approach for In vivo Tracking of Therapeutic CAR T-cells with PET/SPECT Imaging

Techniques……………………………………………………………………………………………………………………………………………………….9

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1.1 Dissecting the heterogeneity of tumour-infiltrating phagocytes: implications for tissue remodelling and immunotherapy. Co-Supervisor 1: Pierre GUERMONPREZ, Research Division/Department: School of Immunology and Microbial Sciences E-mail: pierre.guermonprez@kcl.ac.uk Co-Supervisor 2: Tanya SHAW, Research Division/Department: School of Immunology and Microbial Sciences E-mail: tanya.shaw@kcl.ac.uk Project description: Tumour-infiltrating phagocytes (TIPs) are important components of the tumour microenvironment. TIPs release a variety of immuno-suppressive factors, thus dampening the adaptive immune response of Th1 and CD8+ cytotoxic lymphocytes to tumours. Most TIPs derive from infiltrating monocytes and accumulate either inside or at the invasive margin of tumours, where they both permit cancer cell growth directly, and indirectly influence tumour growth and metastasis by activating and causing important changes to the neighbouring connective tissue (tumour stroma). The mechanisms by which these cells affect tissue remodelling of tumour stroma are not well understood. Tumours secrete factors that actively instruct monocyte recruitment and differentiation into TIPs. For example, oncogenic variants of Kras driving lung cancer induce GM-CSF secretion that promotes recruitment and differentiation. However, how tumour-derived factors shape TIP phenotype and pro-tumorigenic function remains ill-defined. Scientific hypothesis: Cancer cells secrete growth factors that influence a TIP phenotype that is pro-tumourigenic. The tumour-promoting effects of TIPs can be direct, through communication with the cancer cells themselves, and indirect, through their immunosuppressive and tissue remodelling capabilities. Experimental plan: Overall, this project intends to characterize TIP diversity and function in relation with tumour-derived instructive factors. This investigation will be performed in the context of lung adenocarcinoma. Using a combination of mouse models of inducible oncogenes as well as transplanted human tumour lines in immune-deficient mice, we aim to:

1. define and characterize the heterogeneity of TIPs in relationship with tumour-derived hematopoietic growth factors using unbiased high dimensional flow cytometry and single cell transcriptome analysis.

2. decipher the molecular mechanisms by which diverse TIPs influence tissue remodelling and neoplastic growth.

3. design and evaluate TIP reprogramming immunotherapeutic interventions for efficient re-purposing of TIPs into anti-tumoural effectors (e.g. by blocking tumour-derived factors influencing TIP recruitment or activation).

References :

1. Menezes S, Melandri D, Anselmi G, Perchet T, Loschko J, Dubrot J, Patel R, Gautier EL, Hugues S, Longhi MP, Henry JY, Quezada SA, Lauvau G, Lennon-Duménil AM, Gutiérrez-Martínez E, Bessis A, Gomez-Perdiguero E, Jacome-Galarza CE, Garner H, Geissmann F, Golub R, Nussenzweig MC, Guermonprez P. The Heterogeneity of Ly6Chi Monocytes Controls Their Differentiation into iNOS+ Macrophages or Monocyte-Derived Dendritic Cells. Immunity. 2016 Dec 20;45(6):1205-1218.

2. R Mori, TJ Shaw, P Martin. Acute knockdown of inflammation-induced osteopontin leads to rapid repair and reduced scarring at the wound site. Journal of Experimental Medicine 2008, 205(1):43-51.

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1.2. Targeting the tumour microenvironment to suppress pancreatic cancer cell invasion Co-Supervisor 1: Claire Wells Research Division/Department: School of Cancer and Pharmaceutical Sciences Email: Claire.wells@kcl.ac.uk Co-Supervisor 2: Debashis Sarker Research Division/Department: School of Cancer and Pharmaceutical Sciences Email: debashis.sarker@kcl.ac.uk Project description: Pancreatic cancer (PC) survival is devastatingly low with few patients surviving more than 5 years post diagnosis due to the prevalence of metastatic disease. Currently there are no treatment options that specifically target metastasis and thus novel approaches are urgently required. Our lab has recently developed a 3D imaging platform incorporating both PC and stromal cells (pancreatic stellate cells and Thp-1 monocytes) based on a spheroid assay(1). Using this platform we have preliminary evidence that stromal pancreatic stellate cells can actively promote PC cell invasion whilst the presence of monocytes is prohibitive. These studies are complimented by our organotypic invasion assay(2). In parallel we have new data that suggests Thp-1 monocytes can supress the formation of invadopodia (1, 3) in PC cells. We now aim to better understand how stromal PC stellate cells and monocytes influence invasive behaviour, by testing PC cell responses in the presence/absence of stromal cells and with the use of conditioned medium. We hypothesise that factors released by the immune/ stromal cells significantly impact on PC cell behaviour and that these factors could represent novel therapeutic opportunities. Furthermore, we know that the stromal cells and monocytes express p21-activated kinase (PAK) a protein known to drive cellular migration(4). We now wish to understand if targeting PAK activity in the stromal/immune compartment might significantly influence PC cell invasion. The Wells lab has access to PAK inhibitor compounds developed by Cancer Research Technology and PAK laboratory reagents (5). In parallel we will also test an FDA approved library for compounds that can suppress stromal cell promoted invasion. The PhD student will be provided with full training in all of the cell culture and microscopy techniques required and will be fully integrated into the Wells Laboratory; attending regular lab meetings, journal clubs and research seminars. In parallel Dr Sarker can offer the student the possibility of attending a pancreatic cancer clinic and will oversee the translational direction of the project. Experimental plan: Year 1: Optimise imaging of tumour and stromal/immune cells. – determine how presence /absence stromal cells (or conditioned medium) influences tumour cell invasion. Include live imaging of invasion and detection of hypoxia within the tumour cell core. Year 2: Optimise PAK inhibitors/ PAK editing to monitor immune cell/stromal cell influence on invasion. Identify molecular mechanisms that are changing the behaviour of the PC cells Year 3: Continue molecular mechanism studies. Use an FDA approved library to monitor response of PDAC and stromal cells; 3D platform. Validate key findings with reference to patient material and in vivo testing. References 1. Nicholas NS, Pipili A, Lesjak MS, Ameer-Beg SM, Geh JL, Healy C, MacKenzie Ross AD, Parsons M, Nestle FO, Lacy KE and Wells CM. PAK4 suppresses PDZ-RhoGEF activity to drive invadopodia maturation in melanoma cells. Oncotarget. 2016;7(43):70881-97. 2. King H, Thillai K, Whale A, Arumugam P, Eldaly H, Kocher HM, et al. PAK4 interacts with p85 alpha: implications for pancreatic cancer cell migration. Scientific reports. 2017;7:42575. 3. Md Hashim NF, Nicholas NS, Dart AE, Kiriakidis S, Paleolog E, Wells CM. Hypoxia-induced invadopodia formation: a role for beta-PIX. Open biology. 2013;3(6):120159. 4. Thillai K, Lam H, Sarker D, Wells CM. Deciphering the link between PI3K and PAK: An opportunity to target key pathways in pancreatic cancer? Oncotarget. 2017;8(8):14173-91. 5. Whale AD, Dart A, Holt M, Jones GE, Wells CM. PAK4 kinase activity and somatic mutation promote carcinoma cell motility and influence inhibitor sensitivity. Oncogene. 2013;32(16):2114-20.

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2.1. Identification of tumour-specific epitopes produced by proteasome-catalysed peptide splicing and suitable for adoptive T cell immunotherapy. Co-supervisor 1: Michele Mishto Research Division/Department: School of Immunology and Microbial Sciences Email: michele.mishto@kcl.ac.uk Co-supervisor 2: Mark Peakman Research Division/Department: School of Immunology and Microbial Sciences Email: mark.peakman@kcl.ac.uk Project description: At the cutting edge of the research for developing more effective and less debilitating therapies against cancer there is the adoptive T cell therapy (ATT). Ideal targets of ATT are epitopes carrying cancer recurrent driver mutations. They are tumor-specific neoantigens, they are less easily dismissed by the tumor and they could be targeted by ATT in a large cohort of patients. These epitopes are produced by proteasome, bound to MHC-I complexes and recognized by cytotoxic CD8+ T lymphocytes (CTLs) (Mishto & Liepe, 2017). We recently showed that a particular type of antigenic peptides – i.e. proteasome-generated spliced peptides - are about one third of the antigenic peptide pool of human cells (Liepe et al., 2016) and can be presented onto MHC-I complex in amounts comparable to canonical non-spliced peptides (Ebstein et al., 2016; Liepe et al., 2016). We also know that proteasome-generated spliced epitopes can trigger in vivo specific CTL responses toward either tumor-associated or pathogen-derived antigens (Ebstein et al., 2016; Platteel et al., 2017), and can be targets for effective anti-cancer ATT (Dalet et al., 2011; Warren et al., 2006). The development of different methods for the identification of proteasome-generated spliced peptides that has been carried out by Dr. Mishto and his team in the last years (Ebstein et al., 2016; Liepe et al., 2015; Mishto & Liepe, 2017; Platteel et al., 2017) puts the PhD student in a privileged position for the identification of spliced epitopes carrying the most “hot” tumor mutations and suitable for ATT. Experimental plan: The PhD student will identify spliced peptides carrying recurrent cancer driver mutations (e.g. BRAF V600E, KRAS G12D) efficiently presented onto the predominant MHC-I variants by combining in silico analyses (in collaboration with the Max Planck Institute Göttingen, Germany), in vitro assays and mass spectrometry (Platteel et al., 2017). The PhD student will isolate, expand and characterise CTL clones specific for these spliced peptides by cell culture, ELISA and FACS analysis (Ebstein et al., 2016). The resulting CTL clones will be used to verify - by using ELISPOT, FACS and cytotoxic assays – the endogenous production of the spliced epitope candidates in cancer cell lines carrying the somatic mutations and expressing the corresponding MHC-I variant. From the CTL clones activated by the tumour cells the PhD student will isolate the specific TCRab genes, and clone them into delivery vectors, which could then be used in the future for ATT in patients carrying the given tumour-specific mutation and MHC-I variant. References

1. Dalet, A., Robbins, P. F., Stroobant, V., Vigneron, N., Li, Y. F., El-Gamil, M., Hanada, K. I., Yang, J. C., Rosenberg, S. A., and Van den Eynde, B. J. (2011). An antigenic peptide produced by reverse splicing and double asparagine deamidation. Proc Natl Acad Sci U S A 108, E323- E331.

2. Ebstein, F., Textoris-Taube, K., Keller, C., Golnik, R., Vigneron, N., Van den Eynde, B. J., Schuler-Thurner, B., Schadendorf, D., Lorenz, F. K., Uckert, W., et al. (2016). Proteasomes generate spliced epitopes by two different mechanisms and as efficiently as non-spliced epitopes. Sci Rep 6, 24032.

3. Liepe, J., Holzhutter, H. G., Bellavista, E., Kloetzel, P. M., Stumpf, M. P., and Mishto, M. (2015). Quantitative time-resolved analysis reveals intricate, differential regulation of standard- and immuno-proteasomes. Elife 4.

4. Liepe, J., Marino, F., Sidney, J., Jeko, A., Bunting, D. E., Sette, A., Kloetzel, P. M., Stumpf, M. P., Heck, A. J., and Mishto, M. (2016). A large fraction of HLA class I ligands are proteasome generated spliced peptides. Science 354, 354-358.

5. Mishto, M., and Liepe, J. (2017). Post-Translational Peptide Splicing and T Cell Responses. Trends Immunol. Platteel, A. C. M., Liepe, J., Textoris-Taube, K., Keller, C., Henklein, P., Schalkwijk, H. H., Cardoso, R., Kloetzel, P. M., Mishto, M., and Sijts, A. (2017). Multi-level Strategy for Identifying Proteasome-Catalyzed Spliced Epitopes Targeted by CD8+ T Cells during Bacterial Infection. Cell Rep 20, 1242-1253.

6. Warren, E. H., Vigneron, N. J., Gavin, M. A., Coulie, P. G., Stroobant, V., Dalet, A., Tykodi, S. S., Xuereb, S. M., Mito, J. K., Riddell, S. R., and Van den Eynde, B. J. (2006). An antigen produced by splicing of noncontiguous peptides in the reverse order. Science 313, 1444-1447.

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2.2. Dissecting alterations in immune and tumour signalling pathways in head and neck cancer induced by combined T4 CAR T-cell and pembrolizumab immunotherapy. Supervisor 1: Anita Grigoriadis Research Division/Department: School of Cancer and Pharmaceutical Sciences Email: anita.grigoriadis@kcl.ac.uk Supervisor 2: John Maher Research Division/Department: School of Cancer and Pharmaceutical Sciences Email: john.maher@kcl.ac.uk Supervisor 3: Selvam Thavaraj Research Division/Department: Mucosal and Salivary Biology, Dental Institute Email: selvam.thavaraj@kcl.ac.uk Project description (background and scientific hypothesis): Traditionally cancer has been managed with surgery, radiation therapy and molecular pharmaceuticals. This landscape has dramatically altered over the past 15 years through the clinical implementation of two breakthrough immunotherapeutic approaches, namely chimeric antigen receptor (CAR)-engineered T-cells and immune checkpoint inhibitors. In haematological cancers such as refractory acute lymphoblastic leukaemia, complete remission rates exceeding 80% are consistently achieved using CAR T-cell immunotherapy. However, solid tumours are much less responsive to this approach. We are pursuing a Phase I trial of intra-tumoural panErbB-targeted T4 CAR T-cell immunotherapy in patients with refractory squamous cell carcinoma of head and neck (SCCHN).1 Following administration of escalating doses from 10-300 million CAR T-cells, disease control has been achieved without dose-limiting toxicity in 8/12 patients (stable disease). In a similar patient population, immunotherapy with the PD1 immune checkpoint inhibitor, pembrolizumab has achieved a 16% response rate, accompanied by prolonged survival.2 We are about to combine these agents in the next phase of our ongoing clinical trial. We hypothesise that this combination will synergistically increase efficacy, acting through enhanced anti-tumour activity of both CAR T-cells and the endogenous T-cell compartment in SCCHN.3 Here, we propose to dissect such interactions and will also determine whether counterattack mechanisms are deployed that may hinder therapeutic activity. The aims of the study are:

• To characterise effects of the therapeutic intervention on tumour signalling pathways.

• To identify effects on stimulatory and suppressive innate and adaptive immune pathways operative within the tumour/ stromal landscape.

• To establish correlates of clinical response in the tumour transcriptome.

• To interrogate the relationship between alterations in endogenous T-cell repertoire and disease outcome. Samples to be collected:

• RNASeq will be used to determine gene expression profiles from serial tumour biopsies undertaken before, one week after, and two weeks after administration of T4 immunotherapy.

• ImmunoSeq analysis will be performed on tumour and serial peripheral blood samples to determine kinetics of clonality, evenness and diversity of endogenous and T-cell receptor (TCR) repertoires.

Experimental plan: Year 1: collate information available in public or in-house generated transcriptomic databases from SCCHN tumours. Year 2: analysis of transcriptomes from clinical trial patients; determine deregulated intra-tumoural and peri-tumoural signalling pathways. Year 3: Perform comprehensive TCR repertoire analyses of clinical trial patients’ data. Year 4: Cross-reference findings with individual and synergistic immunotherapy strategies to position combined T4 CAR T-cell/pembrolizumab immunotherapy in locally advanced or recurrent SCHHN. References: 1.van Schalkwyk MC, et al. Design of a Phase I Clinical Trial to Evaluate Intratumoral Delivery of ErbB-Targeted Chimeric Antigen Receptor T-Cells in Locally Advanced or Recurrent Head and Neck Cancer. Hum Gene Ther Clin Dev. 2013;24:134-142. 2.Seiwert TY, et al. Safety and clinical activity of pembrolizumab for treatment of recurrent or metastatic squamous cell carcinoma of the head and neck (KEYNOTE-012): an open-label, multicentre, phase 1b trial. Lancet Oncol. 2016;17:956-965. 3.Saloura V et al., Characterization of the T-Cell Receptor Repertoire and Immune Microenvironment in Patients with Locoregionally Advanced Squamous Cell Carcinoma of the Head and Neck. Clin Cancer Res. 2017;23:4897-4907.

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2.3. Understanding and improving the immune-modulatory actions of chemotherapy in cancer Co-Supervisor 1: James Arnold Research Division/Department: School of Cancer and Pharmaceutical Sciences Email: james.n.arnold@kcl.ac.uk Co-Supervisor 2: James Spicer Research Division/Department: School of Cancer and Pharmaceutical Sciences Email: james.spicer@kcl.ac.uk Project description Chemotherapy has been utilised for the treatment of cancer for nearly 70 years. These molecules were first administered to patients with cancer for their ability to target the cell cycle. However, chemotherapy can also act as an immunotherapy, stimulating immune surveillance through both priming anti-tumour CD8+ T cells and eliciting their infiltration into the tumour microenvironment. Despite this, in the majority of cases, chemotherapy alone does not represent a cure because remission is often followed by relapse. Immune checkpoint blockade therapies have been demonstrated in vivo to improve chemotherapy-elicited immune surveillance, suggesting that tumoral immune suppression can be one hurdle to efficiently harnessing the anti-cancer properties of chemotherapeutic agents. The proposed project will utilise spontaneous in vivo models of breast cancer to investigate the immune-modulatory effects of chemotherapeutic drugs used in the clinic. The project will focus on CD8+ T cells which infiltrate the tumour in response to chemotherapy. These cells will be characterized in detail and their ability to target the tumour investigated in vivo and ex vivo. This project will then consider translational opportunities for improving the efficacy of the chemotherapy-elicited anti-tumour immune response by directly targeting the CD8+ T cells, as well as the wider tumour microenvironment, with agents such as immune checkpoint inhibitors. It is hoped that through furthering our understanding of the nature of the CD8+ T cell response elicited by chemotherapy, and their interaction with the tumour microenvironment more widely, the therapeutic efficacy of chemotherapy in cancer can be improved. The techniques that this project will utilise include: Transcriptomic analyses, flow cytometry, confocal microscopy, quantitative reverse transcriptase PCR, Western blot, in vivo and ex vivo models, cell culture (primary and cell line), co-culture, and therapeutic interventions (small molecule inhibitors, neutralising antibodies). This project would be suitable for both clinical and non-clinical applicants.

Reference Bracci, L., G. Schiavoni, A. Sistigu, and F. Belardelli. 2014. Immune-based mechanisms of cytotoxic chemotherapy: implications for the design of novel and rationale-based combined treatments against cancer. Cell death and differentiation 21: 15-25.

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3.1 Defining cellular and molecular mechanisms underlying cancer immunotherapy-induced auto-inflammatory syndromes

Co-Supervisor 1: Leonie Taams Research Division/Department: School of Immunology and Microbial Sciences Email: leonie.taams@kcl.ac.uk Co-Supervisor 2: Sophie Papa Research School/Division: School of Cancer and Pharmaceutical Sciences Email: sophie.papa@kcl.ac.uk Co-Supervisor 3: Andrew P. Cope Research School/Division: Centre for Inflammation Biology and Cancer Immunology, School of Immunology and Microbial Sciences E-mail: andrew.cope@kcl.ac.uk Project description: Cancer therapy with immune checkpoint inhibitors is transforming the treatment of solid tumours such as melanoma. The rationale is based on breaking immune tolerance to tumour antigens, unleashing effector T cells to kill tumour antigen expressing cells. This is accomplished by inhibiting the function of the immune system’s own immunosuppressive “checkpoint” molecules, the best characterised being CTLA4 and PD-1. In spite of these breakthroughs, response rates to cancer immunotherapy are well below 60% across indication, highlighting a need to better understand responders and non-responder states. These therapies, at the same time, break tolerance to self antigens leading to an emerging group of auto-inflammatory syndromes comprising rapid onset (6-8 weeks), and often severe inflammatory disease targeting skin, gut, liver, joint or endocrine organs. Being one of the largest cancer immunotherapy centres in the UK offers an unparalleled opportunity to understand the delicate balance between tumour immunity and autoimmunity in cancer patients, with the explicit intention of preventing adverse immune reactions on the one hand, and uncovering new insights into the pathogenesis of autoimmunity on the other. Experimental plan: In this project the student will undertake deep immune phenotyping of peripheral blood and tissues from cancer patients before and after receiving checkpoint inhibitor therapy (Year 1), exploiting high-end flow and mass cytometry to map the very earliest events associated with the onset of immune mediated inflammatory syndromes. This will be complemented by analysis of blood transcriptomes (Year 2), seeking to understand the relationship between cellular and molecular signatures and specific disease phenotype (Years 1-3). References: 1. Papa S, van Schalkwyk M, Maher J. Clinical Evaluation of ErbB-Targeted CAR T-Cells, Following Intracavity Delivery in Patients with ErbB-Expressing Solid Tumors. Methods Mol Biol. 2015;1317:365-82. 2. Burn GL, Cornish GH, Potrzebowska K, Samuelsson M, Griffié J, Minoughan S, Yates M, Ashdown G, Pernodet N, Morrison VL, Sanchez-Blanco C, Purvis H, Clarke F, Brownlie RJ, Vyse TJ, Zamoyska R, Owen DM, Svensson LM, Cope AP. Superresolution imaging of the cytoplasmic phosphatase PTPN22 links integrin-mediated T cell adhesion with autoimmunity. Sci Signal. 2016 Oct 4;9(448):ra99. 3. Evans HG, Roostalu U, Walter GJ, Gullick NJ, Frederiksen KS, Roberts CA, Sumner J, Baeten DL, Gerwien JG, Cope AP, Geissmann F, Kirkham BW, Taams LS. TNF-α blockade induces IL-10 expression in human CD4+ T cells. Nat Commun. 2014;5:3199.

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3.2. Migrastatics – How do they affect the immune response and existing immunotherapies?

Co-Supervisor 1: Victoria Sanz-Moreno Research Division/Department: Randall Division of Cell and Molecular Biophysics, School of Basic and Medical Biosciences Email: victoria.sanz_moreno@kcl.ac.uk Co-Supervisor 2: Gilbert Fruhwirth Research Division/Department: Imaging Chemistry and Biology, School of Biomedical Engineering &Imaging Sciences Email: gilbert.fruhwirth@kcl.ac.uk Project description: Immune cells are naturally required to move around the body to detect and act on pathogens in an orchestrated manner (immune response). A hallmark of cancer is immune evasion by tumour cells. Current molecular immunotherapies intended to redirect the existing immune system to attack the tumour have been shown to be affected by development of drug resistance. Cellular immunotherapies, while promising in liquid cancers, face significant complications in solid tumours. Metastasis is the abnormal spread of cancer cells around the body, and is responsible for >90% of cancer mortality. To metastasize, cancer cells need to acquire abnormal migratory abilities via cytoskeletal remodelling1,2. We have recently defined migrastatic3 agents -chosen for their ability to interfere with all modes of cancer cell invasion - they are different from cytostatic drugs, mainly directed against cell proliferation. The effects of migrastatic drugs on molecular and cellular immunotherapies remain elusive despite the clinical need to carefully balance interference with cancer cell motility against protection of immune cell motility. In this project, we will focus on melanoma as it represents a particular metastatic cancer type that is already being treated clinically with certain types of immunotherapy. Experimental Plan: Objective 1: First we will systematically evaluate in vitro the effects of a panel of migrastatic drugs (e.g. inhibitors of the Rho Kinase-Myosin network1-3) on the motility of macrophages (M1, M2) and T-cells (CD4, CD8) as well as T-cell-derived cellular immunotherapeutics (CAR T-cells, enriched T-cells). Furthermore, we will assess if and how the migrastatic drugs alter immune cell:cancer cell interactions in co-culture experiments including different melanoma cell lines and the above immune cells and cellular immunotherapeutics (3D matrices combined with multi-colour cell tracking by fluorescence microscopy). Objective 2: We will use our new in vivo traceable melanoma models in different settings of immuno-competence and co-track adoptively transferred reporter gene labelled immune cell subpopulations in the presence or absence of migrastatic challenge. Labelled immune cell populations will originate either from commercially available mice (B6.CD4-GFP, B6.CX3CR1-GFP) or be isolated and transduced with fluorescence/radionuclide reporter genes. Metastasis and immune cell co-tracking will be achieved by serial multimodal whole-body in vivo radionuclide imaging4,5 and based on novel multiplex reporter gene imaging combinations (non-immunogenic reporter, tracer). All tumours will also be immunoprofiled using cytometric analysis and histologic assessment of immune cell infiltration/localization. Objective 3: We will evaluate the scope and clinical feasibility for migrastatic and immunotherapy co-administration through optimization of their courses of administration in the preclinical melanoma setting. If migrastatics’ effects should turn out detrimental for cellular immunotherapy motility, we will encapsulate the migrastatic drugs into liposomes5. References: 1. Cantelli G, Orgaz JL, Rodriguez-Hernandez I, Karagiannis P, Maiques O, Matias-Guiu X, Nestle FO, Marti RM , Karagiannis SN and Sanz-Moreno V (2015) TGF -induced transcription sustains amoeboid melanoma migration and dissemination. Curr Biol, 16;25 (22):2899-914. 2. Herraiz C, Calvo F, Pandya P, Cantelli G, Rodriguez-Hernandez I, Orgaz JL, Kang N, Chu T, Sahai E and Sanz-Moreno V (2016) Reactivation of p53 by a cytoskeletal sensor to control the balance between DNA damage and tumor dissemination. J Natl Cancer Inst. 2015, 13;108 (1). 3. Gandalovičová A, Rosel D, Fernandes M, Veselý P, Heneberg P, Čermák V, Petruželka L, Kumar S, Sanz-Moreno V *and Brábek J* (2017) *co-corresponding. MIGRASTATICS, the anti-metastatic and anti-invasion drugs: the promise and challenges. Trends in Cancer 3:391-406 4. Fruhwirth GO, Diocou S, Blower PJ, Ng T, Mullen GE. (2014) A whole-body dual-modality radionuclide optical strategy for preclinical imaging of metastasis and heterogeneous treatment response in different microenvironments. J Nucl Med. 55(4):686-94. 5. Diocou S, Volpe A, Jauregui-Osoro M, Boudjemeline M, Chuamsaamarkkee K, Man F, Blower PJ, Ng T, Mullen GED, Fruhwirth GO. (2017) [18F]tetrafluoroborate-PET/CT enables sensitive tumor and metastasis in vivo imaging in a sodium iodide symporter-expressing tumor model. Sci Rep. 19;7(1):946. 5. Edmonds S, Volpe A, Shmeeda H, Parente-Pereira AC, Radia R, Baguña-Torres J, Szanda I, Severin GW, Livieratos L, Blower PJ, Maher J, Fruhwirth GO, Gabizon A, T M de Rosales R. (2016) Exploiting the Metal-Chelating Properties of the Drug Cargo for In Vivo Positron Emission Tomography Imaging of Liposomal Nanomedicines. ACS Nano. 22;10(11):10294-10307.

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3.3. An Indirect Labelling Approach for In vivo Tracking of Therapeutic CAR T-cells with PET/SPECT Imaging Techniques

Co-Supervisor 1: Ran Yan Research Division/Department: Imaging Chemistry and Biology, School of Biomedical Engineering& Imaging Sciences Email: ran.yan@kcl.ac.uk Supervisor 2: John Maher Research Division/Department: School of Cancer and Pharmaceutical Sciences Email: john.maher@kcl.ac.uk

Background and hypothesis: Emerging as the fourth pillar of healthcare, cell-based therapies have shown great promise in cancer treatment. Exemplifying this, chimeric antigen receptor (CAR) technology is increasingly being harnessed to generate a novel T-cell based adoptive immunotherapeutic approaches for cancer and other disease types.1 Indeed, the first CAR T-cell therapeutic product received FDA approval in 2017, for treatment of paediatric acute lymphoblastic leukaemia. One fundamental challenge in the successful development and clinical application of cellular therapeutics is the need to better understand the in vivo behaviour of adoptively infused cell products. Imaging affords a non-invasive solution to dynamically track the persistence, migration, proliferation, and final fate of the infused cells on a patient-by-patient basis (Fig. 1A).2 Consequently, it is essential to incorporate cell tracking studies at the earliest stage of clinical development in order to quantify the migration of infused cells to target sites, in addition to tissues where toxicity may ensure. Recently, Maher (biomedical co-supervisor) has engineered a second-generation CAR named A20-28z that re-targets T-cells against tumour cells of diverse origin that aberrantly express the v 6 integrin.3 Myc epitope tags have been engineered into the hinge/spacer of this CAR which can be recognised by the 9E10 antibody (Fig. 1B&C). Given its small size and linear nature, the myc epitope tag can easily be engineered into any CAR design. Moreover, the anti-c-myc antibody 9E10 Fab fragment (45 kDa) was produced from E. coli with the retention of nanomolar binding affinity for the myc epitope. Consequently, the in vivo persistence and proliferation of CAR T-cells could in principle be detected with a radio-labelled anti-c-myc antibody 9E10 Fab fragment using nuclear imaging techniques. This interdisciplinary collaborative project aims to develop a generic indirect CAR T-cell labelling method to track the persistence and proliferation of myc tag-containing CAR T-cells in vivo with PET/SPECT imaging.

Experimental plan Year 1: A20-28z CAR T-cell generation. Production of anti-c-myc Fab fragment. Fab radiolabelling with 64Cu for PET, 99mTc- and 123I for SPECT. Year 2: Measurement of the binding affinity (EC50) of the radio-labelled anti-c-myc Fab fragments, quantification their CAR(A20-28z) T-cell uptake, and the antitumor cell effect of the radio-labelled CAR(A20-28z) T-cell in vitro. Year 3: In vivo tracking of CAR(A20-28z) T-cells in tumour-bearing mice following administration of 64Cu- 99mTc- or 123I-labelled anti-c-myc Fab fragments using PET/SPECT imaging. Year4: Completion of data collection & analysis. Thesis write up. Conference presentation. Manuscript(s) preparation.

References: Maher J. Immunotherapy of Malignant Disease Using Chimeric Antigen Receptor Engrafted T Cells; 2012, ISRN oncology, Volume 2012, P 23; Moritz F. Kircher, Sanjiv S. Gambhir & Jan Grimm; Noninvasive cell-tracking methods; Nature Reviews Clinical Oncology 2011, 8, 677-688. Whilding LM, Parente-Pereira AC, Zabinski T, Davies DM, Petrovic RMG, Kao YV, Saxena SA, Romain A, Costa-Guerra JA, Violette S, Itamochi H, Ghaem-Maghami S, Vallath S, Marshall JF, Maher J Targeting of aberrant αvβ6 integrin expression in solid tumors using chimeric antigen receptor-engineered T-cells. Molecular Therapy 2017, 25(1), 259-273.

Figure 1. A) above,MRI images of glioblastoma (1, 2) in a male patient; below, PET images of the accumula tionof CD8+ T cells in glioblastoma (1, 2) that were indirectly tracked by 18F-FHBG. B) the A20-28z CAR indicating thelocation of myc epitope tags on the T-cell surface. C) Detection of A20-28z-expressing CAR T-cells by flowcytometry using the anti-c-myc 9E10 antibody. In this example, cells were co-transducedwith a second retroviralvector that encodes green fluorescent protein (GFP) and renilla luciferase.3

A A BA BB C