Borsa di Studio “Damiano per l’Ematologia” 2022

Proclamazione del Vincitore

E’ risultato vincitore il progetto “ Study of the immunomodulatory effects of targeted therapies and their impact on the generation of effective CAR-T cells in patients with chronic lymphocytic leukemia”. L’autrice è la Dr.ssa Rebecca Jones, che svolgerà il suo lavoro presso la Sezione di Ematologia del Dipartimento di Biotecnologie Molecolari e Scienze per la Salute dell’Università degli Studi di Torino, diretta dal Prof. Dario Ferrero, sotto la supervisione della Dr.ssa Marta Coscia.

Testo del progetto

Study of the immunomodulatory effects of targeted therapies and their impact on the generation of effective CAR T cells in patients with chronic lymphocytic leukemia.

b) Introduction

Chronic Lymphocytic Leukemia (CLL) is the most diffused leukemia in the adults of the Western world. Patients affected by CLL present complex immune dysregulations and many studies have reported abnormalities in the absolute number and phenotype of immune cells. Among these alterations it was reported an inverted CD4:CD8 T cell ratio, reduction of naïve T cells and accumulation of terminally differentiated effector memory T cells [1]. Furthermore, other studies reported skewing from Th1 dominant response towards a Th2 response, impaired immune synapse formation and T cell functional exhaustion induced by chronic activation [2]. In CLL patients, T cells have been reported to express elevated inhibitory receptors on the cell surface (i.e. PD-1, CD160. CD244, TIGIT) [1] and regulatory T cells (Tregs) result expanded in blood and lymph nodes [3, 4]. Dysfunctions in the innate immune response have also been reported as dysfunctional Vγ9Vδ2 T cells and natural killer (NK) cells [5, 6]. Treatment approaches exploiting CAR T cells (T cells engineered to express a chimeric antigen receptor, CAR) are rapidly developing in the hematological field. However, in the setting of CLL, the efficacy of CAR T cell treatment appears suboptimal as compared to other diseases [7]. This may be, at least in part, attributable to immune system dysfunctions - particularly those affecting the T-cell compartment - which characterize patients with CLL and limit the generation and expansion of effective CAR T cells. It has been recently shown that in the small subset of complete-responding patients with CLL, the therapeutic efficacy of CAR T cells depends on the composition of the cellular product and on the intrinsic T cell fitness [8, 9]. Targeted drugs, such as ibrutinib and venetoclax, recently entered the therapeutic armamentarium of CLL, showing excellent results in terms of efficacy. Nevertheless, disease relapse still occurs, particularly in high-risk patients, who are therefore eligible for allogeneic transplant or treatment with CAR T cells. Interestingly, besides the direct anti-neoplastic effect, new targeted molecules may exert complex activities on the host immune system [10, 11]. However, little is known on the mechanisms underlying the potential impact of treatment with targeted drugs on the efficiency of manufacturing and the final anti-tumor efficacy of CAR T cells.

c) Aims of the project:

This project is articulated into two Tasks.

Task 1 aims at a better characterization of the immunomodulatory effects of ibrutinib and venetoclax in CLL, with a special focus on immunophenotypic and functional properties of T-cell compartment. To this aim, peripheral blood samples collected from CLL patients’ before and during treatment with ibrutin ib or venetoclax will be profiled in terms of CD4+ and CD8+ composition, distribution of differentiation subsets, T helper cell polarization and expression of activation markers, homing markers and immune checkpoint molecules. In addition, the proliferation ability of T cells in response to polyclonal stimuli will be evaluated.

Task 2 aims at assessing the impact of treatment with targeted agents (ibrutinib and venetoclax) on the generation of CAR T cells in patients with CLL in terms of manufacturing efficiency, immunophenotypic characteristics and functional properties. Peripheral blood samples from patients with CLL will be collected before starting therapy with ibrutinib or venetoclax, and during treatment. Anti-CD19 CAR T cells will be generated and tested for: (i) viability, expansion and generation efficiency; (ii) phenotypic characteristics, in terms of CD4/CD8 composition, differentiation subset distribution, exhaustion markers and expression of immune checkpoint molecules; (iii) in vitro functional properties, in terms of proliferation ability, cytokines production, cytotoxic activity and killing of target cells. CAR T cells produced from the same patient at different timepoints will be compared. Correlation with clinic-biological features of CLL patients will be correlated with i) phenotypic features of T cells and ii) phenotypic and functional properties of CAR T cells.

d) Methods:

All patients will provide written informed consent through an Institutional Review Board-approved protocol. Blood samples will be collected before and during (i.e. 6 months) the treatment. PBMC will be isolated by

density gradient centrifugation. Immunophenotypic characterization of T cells will be performed by immunostaining and flow cytometry (FC). The expression of population markers (i.e. CD4 and CD8), activation markers (i.e. CD25 and CD69) and immune checkpoint molecules (i.e. will be PD-1, Tim-3, CTLA-4, TIGIT) will be evaluated. The percentage of T-cell differentiation markers and T helper cell polarization will be evaluated by FC as well. T-cell proliferation will be measured by CFSE assay and FC.

For CAR T cells generation, T cells will be enriched by immunomagnetic bead method and they will be activated with anti-CD3 and anti-CD28 agonists. For lentiviral particles production, 293T cell line (packaging cells) will be co-transduced with (i) a transfer plasmid containing the anti-CD19 CAR construct, (ii) an envelope plasmid, and (iii) a packaging plasmid. The assembled lentivirus will be then harvested from the culture supernatants and will be used to transduce activated T cells. Transduced cells will be cultured and expanded in specific culture medium containing cytokines (i.e. IL-7 and IL-15) for approximately 14 days. Manufacturing efficiency rate will be evaluated measuring cell viability (the percentage of AnnexinV and Propidium Iodide negative cells measured by FC) and the percentage of transduction (evaluated by FC) of CAR T cells at the end of the expansion period (approximately day 14 of culture). In addition, the expansion rate will be evaluated by the ratio between T-cell count at the end of the culture and T-cell count at the beginning of manufacturing process. Cell viability, transduction efficiency and rate of expansion will be compared between CAR T cells generated from CLL patients before and during treatment with the targeted therapy. Quality controls will be performed to assess the validity of the process (e.g. T-cell enrichment evaluation and viability assessment). Cell viability will be evaluated by AnnexinV and Propidium Iodide staining. Phenotypic characteristics of CAR T cells will be assessed by FC as described above for T-cell phenotype. To test in vitro functional properties, CAR T cells will be exposed to target cells (CD19+ tumor cell lines) and evaluated for: (i) proliferation ability by CFSE assay, (ii) IFN-γ and TNF-α production by FC, (iii) cytotoxic activity by CD107a degranulation assay. CAR-T cell effector functions will be evaluated by a killing assay. Specific target cells will be labelled with a fluorescent dye and cultured with CAR T cells. At the end of the culture the viability of target cells will be assessed by FC (AnnexinV/Propidium Iodide assay) and the percentage of lysis will be calculated. The percentage of lysis obtained with CAR T cells generated from CLL patients before and during treatment with ibrutinib or venetoclax will be compared. Appropriate statistical tests will be applied to evaluate and compare biological parameters between different groups of patients. Statistical significance will be defined as a p value <0.05.

e) References:

[1] S. Man, P. Henley, Chronic lymphocytic leukaemia: the role of T cells in a B cell disease, British journal of haematology 186(2) (2019) 220-233.

[2] J.C. Riches, J.K. Davies, F. McClanahan, R. Fatah, S. Iqbal, S. Agrawal, A.G. Ramsay, J.G. Gribben, T cells from CLL patients exhibit features of T-cell exhaustion but retain capacity for cytokine production, Blood 121(9) (2013) 1612-21.

[3] G. D'Arena, L. Laurenti, M.M. Minervini, S. Deaglio, L. Bonello, L. De Martino, L. De Padua, L. Savino, M. Tarnani, V. De Feo, N. Cascavilla, Regulatory T-cell number is increased in chronic lymphocytic leukemia patients and correlates with progressive disease, Leukemia research 35(3) (2011) 363-8.

[4] S. De Matteis, C. Molinari, G. Abbati, T. Rossi, R. Napolitano, M. Ghetti, A.G.L. Di Rorà, G. Musuraca, A. Lucchesi, G.M. Rigolin, A. Cuneo, D. Calistri, P.P. Fattori, M. Bonafè, G. Martinelli, Immunosuppressive Treg cells acquire the phenotype of effector-T cells in chronic lymphocytic leukemia patients, Journal of translational medicine 16(1) (2018) 172.

[5] M. Coscia, C. Vitale, S. Peola, M. Foglietta, M. Rigoni, V. Griggio, B. Castella, D. Angelini, S. Chiaretti, C. Riganti, A. Guarini, D. Drandi, M. Ladetto, A. Bosia, R. Foà, L. Battistini, M. Boccadoro, J.J. Fournié, M. Massaia, Dysfunctional Vγ9Vδ2 T cells are negative prognosticators and markers of dysregulated mevalonate pathway activity in chronic lymphocytic leukemia cells, Blood 120(16) (2012) 3271-9.

[6] T. Hofland, E. Eldering, A.P. Kater, S.H. Tonino, Engaging Cytotoxic T and NK Cells for Immunotherapy in Chronic Lymphocytic Leukemia, International journal of molecular sciences 20(17) (2019).

[7] D.L. Porter, W.T. Hwang, N.V. Frey, S.F. Lacey, P.A. Shaw, A.W. Loren, A. Bagg, K.T. Marcucci, A. Shen, V. Gonzalez, D. Ambrose, S.A. Grupp, A. Chew, Z. Zheng, M.C. Milone, B.L. Levine, J.J. Melenhorst, C.H. June, Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia, Science translational medicine 7(303) (2015) 303ra139.

[8] C.J. Turtle, L.A. Hanafi, C. Berger, T.A. Gooley, S. Cherian, M. Hudecek, D. Sommermeyer, K. Melville, B. Pender, T.M. Budiarto, E. Robinson, N.N. Steevens, C. Chaney, L. Soma, X. Chen, C. Yeung, B. Wood, D. Li, J. Cao, S. Heimfeld, M.C. Jensen, S.R. Riddell, D.G. Maloney, CD19 CAR-T cells of defined CD4+:CD8+ composition in adult B cell ALL patients, The Journal of clinical investigation 126(6) (2016) 2123-38.

[9] J.A. Fraietta, S.F. Lacey, E.J. Orlando, I. Pruteanu-Malinici, M. Gohil, S. Lundh, A.C. Boesteanu, Y. Wang, R.S. O'Connor, W.T. Hwang, E. Pequignot, D.E. Ambrose, C. Zhang, N. Wilcox, F. Bedoya, C. Dorfmeier, F. Chen, L. Tian, H. Parakandi, M. Gupta, R.M. Young, F.B. Johnson, I. Kulikovskaya, L. Liu, J. Xu, S.H. Kassim, M.M. Davis, B.L. Levine, N.V. Frey, D.L. Siegel, A.C. Huang, E.J. Wherry, H. Bitter, J.L. Brogdon, D.L. Porter, C.H. June, J.J. Melenhorst, Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia, Nature medicine 24(5) (2018) 563-571.

[10] S.S. Gabriel, N. Bon, J. Chen, T. Wekerle, A. Bushell, T. Fehr, P.E. Cippà, Distinctive Expression of Bcl-2 Factors in Regulatory T Cells Determines a Pharmacological Target to Induce Immunological Tolerance, Frontiers in immunology 7 (2016) 73.

[11] V. Griggio, C. Vitale, M. Todaro, F.R. Mauro, C. Salvetti, D. Pietrasanta, I.D. Vincelli, L. Scarfo, G. Del Poeta, G. Gaidano, V. Gattei, R. Foà, M. Boccadoro, M. Coscia, Ibrutinib Treatment Mitigates Phenotypic Alterations of Non-Neoplastic Immune Cell Compartments in Chronic Lymphocytic Leukemia, Blood 132(Supplement 1) (2018) 4412-4412.

[12] J. Gauthier, A.V. Hirayama, J. Purushe, K.A. Hay, J. Lymp, D.H. Li, C.C.S. Yeung, A. Sheih, B.S. Pender, R.M. Hawkins, A. Vakil, T.D. Phi, R.N. Steinmetz, M. Shadman, S.R. Riddell, D.G. Maloney, C.J. Turtle, Feasibility and efficacy of CD19-targeted CAR T cells with concurrent ibrutinib for CLL after ibrutinib failure, Blood 135(19) (2020) 1650-1660.

[13] S.I. Gill, V. Vides, N.V. Frey, S. Metzger, M. O'Brien, E. Hexner, A.R. Mato, S.F. Lacey, J.J. Melenhorst, E. Pequignot, W.L. Gladney, W.-T. Hwang, A. Lamontagne, M. Davis, J.C. Byrd, S.J. Schuster, D.L. Siegel, R.E. Isaacs, C.H. June, D.L. Porter, Prospective Clinical Trial of Anti-CD19 CAR T Cells in Combination with Ibrutinib for the Treatment of Chronic Lymphocytic Leukemia Shows a High Response Rate, Blood 132(Supplement 1) (2018) 298-298.

Time to complete the project: 12 months. Co-author.