Daniel W. Hommes PROGRESS REPORT

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1 PROGRESS REPORT Progress report for the first year of the project entitled therapeutic use of regulatory T cells in inflammatory bowel diseases, Grant No. IBD-0295R. Therapeutic use of regulatory T cells in inflammatory bowel diseases Progress i) Describe the major achievements for this project to date. Major achievements for this project to date: The major achievements for this project to date are the identification of two novel antiinflammatory approaches with the capacity to ameliorate the intestinal inflammatory response and restore mucosal immune tolerance. Additionally in order to establish a foundation for the setup of a gene therapy approach as a novel treatment strategy in IBD we performed a study to characterize the pre-existing humoral responses to the adenoassociated-virus (AAV) capsid in IBD patients. Generation of inducible Treg (itreg) cells in vitro for a cell therapy based approach We have established an innovative and simple method to generate stable and functional itreg from both murine and human naïve (CD4+CD25-) T cells in vitro. This method consists of a two steps activation procedure with an initial PMA/ionomycin activation step followed by anti-cd3 stimulation in presence of a low dose of IL-2. This approach has the advantage of promoting the conversion of naive T cells to a high number of functional itreg and could be suitable for application under GMP conditions. Furthermore, by making use of substances that are well known, simple to obtain and relatively low priced this protocol could be easy to employ in a routine laboratory setting. This protocol has been described in two manuscripts namely Generation of stable, functional human regulatory T lymphocytes in vitro within 4 days by a two step activation protocol (Manuscript in preparation, van der Marel et al.) and Regulatory T lymphocytes generated in vitro by a two step activation protocol, ameliorate experimental colitis in vivo (Manuscript in preparation, Majowicz et al.), which will be submitted shortly. 1

2 Suppressive functions of the generated murine itreg cells were demonstrated in vivo in a mouse model of inflammatory disease (Inflammatory Bowel Disease model- CD45RB high ) (Majowicz et al. manuscript in preparation). Long term phenotype stability and functionality of the generated human itreg cells were demonstrated in vitro by use of flow cytometry techniques and mixed lymphocyte reactions respectively (van der Marel et al. manuscript submitted). Determination of the AAV serotype of choice for application as gene therapy vector to treat IBD patients A potential barrier to the use of AAV vectors in gene therapy is the pre-existing humoral immunity as a consequence of exposure to wild type AAV (1-3). Neutralizing antibodies (nab) made in response to exposure to either wild type AAV or AAV-based vectors have been associated with a partial to complete block of transduction (4;5). Therefore the characterization of the pre-existing nab titer against different AAV serotypes in future patient populations is important as it will permit to define the serotype of choice for further gene therapy applications. We have determined the prevalence and titers of pre-existing anti-aav nab for serotypes -1, -2, -3, -4, -5, -6 and -8 in a panel of IBD patients and healthy controls. This study has been reported in a publication (6). Our data can be used for future AAV based gene therapies as novel treatment approaches for IBD. The differences demonstrated between the IBD patient population and the healthy controls and our data concerning the presence of nabs and certain disease characteristics offer insight into the IBD pathogenesis (6). We show that against AAV4 and AAV5, there is a favorable pre-existing humoral immune status, based on the low titers of nabs found in the plasma of the positive samples (6). By consequent, AAV4 and AAV5 could be considered in a gene therapy approach in the IBD patient population (6). Stimulation of natural and inducible Treg cells in vivo using a gene therapy based approach 2

3 Tregitopes are regulatory T-cell epitopes found in the Fc region of IgG molecule that have been shown to have the property to induce the activity of the body s own Treg cells (7;8). We explored the potential of the Tregitope 167 (7;8) to induce Treg cells in vivo upon delivery by an AAV vector. AAV was used as a delivery vector as it has been shown to present a good safety profile and proved to be both safe and effective as a gene therapy vector in the clinic for the treatment of a broad range of diseases (9-16). To evaluate the therapeutic potential of AAV-mediated systemic delivery of Tregitopes, we used the TNBS (Trinitrobenzene sulfonate) induced model of colitis. AAV serotype 5 (AAV5) was used for the systemic delivery of the Tregitope in our pre-clinical work. We choose AAV5 for this purpose, since preexisting immunity to the viral capsid was low in the IBD population using the nab assay, as described (6). Systemic administration of AAV5 (CMV-Tregitope 167) was shown to ameliorate the clinical and histopathologic severity of the induced inflammatory colitis. The therapeutic effect was associated with an increase in expression of the regulatory T cell markers Foxp3 and GARP in the thymic CD4 + CD8 + lymphocyte population. ii) Describe any negative results or failed experiments during the period. We set out to develop an AAV transduction protocol efficient enough to transduce CD4+CD25- T cells in order to deliver Foxp3 and generate Treg cells in vitro. Multiple experiments under different experimental conditions were performed in order to transduce either murine or human CD4+CD25- T cells in vitro with AAV. The experimental conditions tested were based on literature searches and experimental observations. Those conditions are listed below: Different AAV serotypes (-1, -2, -5 and -6) were tested for their potential to transduce CD4+CD25- T cells in vitro. Different ratios of AAV genome copies per T cell were used, ranging from 1 x10 3 to 1 x10 6 genome copies/cell. 3

4 Viral infections: single infection and repeated infections were applied. CD4+CD25- T cells activated and not activated were used in the transduction experiments. Activation strategies included cytokines: IL-2 and IL-7; TCR stimulation: anti-cd3 and Ionomycin and PMA. Different combinations and different exposure times were applied. Different chemicals were used which demonstrated to improve viral transduction in vitro for other viral vectors or cell types i.e. polybrene (17), retronectin (18) and hydroxyurea (19). Overall, the transduction efficacy of CD4+CD25- T cells by the AAV vector was low (1%). AAV did not appear, in our experimental conditions, as the optimal viral vector for the transduction of naive CD4+CD25- T cells. The transduction efficacy was not high enough to generate sufficient numbers of Treg cells for future translational studies. These negative results led us to develop other approaches to generate Treg cells. b) What is the significance of the achievements to inflammatory bowel disease (IBD) diagnosis, therapy or prevention? The significance of the achievements to IBD relate to possible novel therapeutic approaches, which could aim at long term tolerance induction. Our study identified two novel anti-inflammatory strategies with the capacity to ameliorate the intestinal inflammatory response and restore mucosal immune tolerance. c) Research Plan Changes i) Did you modify the research plan? If so, why and in what way? ii) Were there any unexpected directions or changes in the project based on the data generated so far? The aim of the study describe in the original research project was to generate both murine and human Treg cells using AAV technology and to characterize these cells both in vitro and in vivo for immunosuppressive properties. However, as mentioned in the section: negative results, the transduction of both murine and human CD4+CD25- T cells with AAV vectors was not efficient enough. The quantity of Treg cells generated was too low to be suitable for therapeutic use. 4

5 Therefore, as mentioned in the section the major achievements for this project to date, we set out to explore alternative approaches which could lead to the generation of a quantity of Treg cells compatible with in vivo applications: As an in vitro alternative approach, we developed an activation protocol to promote the conversion of naive T cells to a high number of functional itreg. This method is composed of two steps of activation with an initial PMA/ionomycin activation step followed by anti-cd3 stimulation in presence of a low dose of Interleukin-2 (IL-2). This approach has the advantage to induce the conversion of naive T cells to a high number of functional itreg and could be suitable for application under GMP conditions. As an in vivo alternative approach, we explored the potential of regulatory T-cell epitopes (Tregitopes) found in the Fc region of IgG molecule to induce Treg cells in vivo upon delivery by an AAV vector. In this approach, systemic administration of AAV5 (CMV-Tregitope 167) was shown to ameliorate the clinical and histopathologic severity of the induced inflammatory colitis. The therapeutic effect was associated with an increase in expression of the Treg cell markers Foxp3 and GARP in the thymic CD4 + CD8 + lymphocyte population. iii) Would you have done your experiments any differently knowing what you know now? Based on the information acquired during the past year, we would propose to focus on methodology allowing the induction of itreg and the activation of naturally occurring Treg (ntreg) cells in vivo. A direct in vivo approach has the advantage to be more physiological and could be a more efficient strategy for the therapeutic use of Treg cells. The success of Tregitopes in suppressing experimental autoimmunity (7;8) may lead to their use as a therapy for IBD. Successful development of Tregitope based therapy would have a radical impact on the fields of autoimmunity, transplantation, and protein therapeutics (7;8). 5

6 d) Have there been any changes to the investigators originally slated to work on this project? If so, please explain. Gijs R van den Brink moved to a new position from the LUMC to the Academic Medical Center in Amsterdam (AMC) and is not implicated in this project anymore. Harald Petry and Valerie Ferreira (AMT) joined the project as investigators in addition to Daniel W. Hommes and Sander J. van Deventer is. The experimental work was performed by Sander van der Marel and Anna Majowicz. e) Indicate whether your study involves human subjects or animals and complete the following information: Murine studies (as was described in the original grant application for the first year of funding). i) the number of subjects originally described to be studied in the currently funded year; No specific number of mice experiments described in the original application ii) the number studied so far in the currently funded year; Two mouse studies were performed iii) the number to be completed during the balance of the currently funded year; Two mouse studies were completed iv) If the number of human or animal subjects to be completed during this currently funded year is either less or more than originally projected, explain why. Not applicable 6

7 f) List all publications, abstracts and presentations resulting from this grant. If you have not yet provided a copy of this material to the BMRP, attach one copy to this application. Publications related to the project 1. van der Marel S, Comijn EM, Verspaget HW, van Deventer S, van den Brink GR, Petry H, Hommes DW, Ferreira V. Neutralizing antibodies against adeno-associated viruses in inflammatory bowel disease patients: Implications for gene therapy. Inflamm Bowel Dis van der Marel S, Majowicz A, van Deventer S, Petry H, Hommes DW, Ferreira V. Gene and cell therapy based treatment strategies for inflammatory bowel diseases. World J Gastrointest Pathophysiol 2011;2: Generation of stable, functional human regulatory T lymphocytes in vitro within 4 days by a two step activation protocol (Manuscript in preparation, van der Marel et al.) 4. Regulatory T lymphocytes generated in vitro by a two step activation protocol, ameliorate experimental colitis in vivo (Manuscript in preparation, Majowicz et al.) 5. Adeno associated virus (AAV) mediated delivery of regulatory T-cell epitopes as a therapeutic approach for treatment of inflammatory bowel diseases (Manuscript in preparation, van der Marel et al.) Abstracts 1. DDW, May 2011, Chicago, US, Neutralizing antibodies against adeno-associated viruses (AAV) in inflammatory bowel disease patients: Implications for gene therapy. (Poster presentation, van der Marel et al.) 7

8 2. Immune Tolerance Networks, October 2011, Amsterdam, Netherlands, Generation of stable, functional regulatory T lymphocytes in vitro within 4 days by a two step activation protocol. (Poster presentation, van der Marel et al.) 3. DDW, May 2012, San Diego, US, AAV5 mediated delivery of Regulatory T-cell epitope 167 ameliorates acute experimental colitis and prevents a type 1 hypersensitivity reaction in a TNBS model of Crohn s disease. (Poster presentation, van der Marel et al.) Reference List (1) Boutin S, Monteilhet V, Veron P et al. Prevalence of serum IgG and neutralizing factors against adeno-associated virus (AAV) types 1, 2, 5, 6, 8, and 9 in the healthy population: implications for gene therapy using AAV vectors. Hum Gene Ther 2010;21(6): (2) Calcedo R, Vandenberghe LH, Gao G et al. Worldwide epidemiology of neutralizing antibodies to adeno-associated viruses. J Infect Dis 2009;199(3): (3) Halbert CL, Miller AD, McNamara S et al. Prevalence of neutralizing antibodies against adeno-associated virus (AAV) types 2, 5, and 6 in cystic fibrosis and normal populations: Implications for gene therapy using AAV vectors. Hum Gene Ther 2006;17(4): (4) Lin J, Calcedo R, Vandenberghe LH et al. Impact of preexisting vector immunity on the efficacy of adeno-associated virus-based HIV-1 Gag vaccines. Hum Gene Ther 2008;19(7): (5) Petry H, Brooks A, Orme A et al. Effect of viral dose on neutralizing antibody response and transgene expression after AAV1 vector re-administration in mice. Gene Ther 2008;15(1):

9 (6) van der Marel S, Comijn EM, Verspaget HW et al. Neutralizing antibodies against adeno-associated viruses in inflammatory bowel disease patients: Implications for gene therapy. Inflamm Bowel Dis (7) De Groot AS, Moise L, McMurry JA et al. Activation of natural regulatory T cells by IgG Fc-derived peptide "Tregitopes". Blood 2008;112(8): (8) Elyaman W, Khoury SJ, Scott DW et al. Potential application of tregitopes as immunomodulating agents in multiple sclerosis. Neurol Res Int 2011;2011: (9) Kaplitt MG, Feigin A, Tang C et al. Safety and tolerability of gene therapy with an adeno-associated virus (AAV) borne GAD gene for Parkinson's disease: an open label, phase I trial. Lancet 2007;369(9579): (10) Bainbridge JW, Smith AJ, Barker SS et al. Effect of gene therapy on visual function in Leber's congenital amaurosis. N Engl J Med 2008;358(21): (11) Cideciyan AV, Aleman TS, Boye SL et al. Human gene therapy for RPE65 isomerase deficiency activates the retinoid cycle of vision but with slow rod kinetics. Proc Natl Acad Sci U S A 2008;105(39): (12) Maguire AM, Simonelli F, Pierce EA et al. Safety and efficacy of gene transfer for Leber's congenital amaurosis. N Engl J Med 2008;358(21): (13) Stroes ES, Nierman MC, Meulenberg JJ et al. Intramuscular administration of AAV1-lipoprotein lipase S447X lowers triglycerides in lipoprotein lipasedeficient patients. Arterioscler Thromb Vasc Biol 2008;28(12): (14) Brantly ML, Chulay JD, Wang L et al. Sustained transgene expression despite T lymphocyte responses in a clinical trial of raav1-aat gene therapy. Proc Natl Acad Sci U S A 2009;106(38):

10 (15) Maguire AM, High KA, Auricchio A et al. Age-dependent effects of RPE65 gene therapy for Leber's congenital amaurosis: a phase 1 dose-escalation trial. Lancet 2009;374(9701): (16) Nathwani AC, Tuddenham EG, Rangarajan S et al. Adenovirus-Associated Virus Vector-Mediated Gene Transfer in Hemophilia B. N Engl J Med (17) Jacobsen F, Hirsch T, Mittler D et al. Polybrene improves transfection efficacy of recombinant replication-deficient adenovirus in cutaneous cells and burned skin. J Gene Med 2006;8(2): (18) Lamers CH, van EP, van Steenbergen SC et al. Retronectin-assisted retroviral transduction of primary human T lymphocytes under good manufacturing practice conditions: tissue culture bag critically determines cell yield. Cytotherapy 2008;10(4): (19) Russell DW, Alexander IE, Miller AD. DNA synthesis and topoisomerase inhibitors increase transduction by adeno-associated virus vectors. Proc Natl Acad Sci U S A 1995;92(12):