Early Human Development 90S1 (2014) S16–S18

Possible alternatives to antimicrobial therapies Marco Zecca *, Rita Maccario, Sabrina Basso, Patrizia Comoli Pediatric Hematology/Oncology and Cell Factory, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy

A R T I C L E Keywords: Transplantation T cell therapy Viral infections Fungal infections

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A B S T R A C T The care of immunosuppressed patients has constantly improved over the years, and pharmacologic developments contributed significantly to this success. However, despite these advances, current anti-infectious agents are limited in their efficacy by either weak specificity or side effects, including suppression of bone marrow function. Control of infection will ultimately depend on reconstitution of specific immunity. Thus, adoptive cellular immunotherapy represents an attractive, low-toxicity strategy to restore specific immune surveillance, and prevent/treat potentially life-threatening disease due to pathogens relevant to the immunosuppressed host. Evidence derived from trials conducted in recipients of hematopoietic stem cell transplantation indicate that adoptive transfer of antigen-specific T cells is a feasible and safe strategy to restore protective immunity and prevent or reverse virus-associated disease. Despite the great potential, immunotherapy for viral and fungal disease still has a marginal role in the management of immunosuppressed patients. This is due to limitations inherent to the technologies and products employed, and, more importantly, to the financial and structural requirements that are associated with GMP production. However, cell therapy offers a unique opportunity to restore antipathogen immune surveillance, and it is therefore conceivable that application of this strategy will increase in the next few years. © 2014 Elsevier Ireland Ltd. All rights reserved.

Among the novel biologic therapeutics that will increase our ability to cure infectious complications in immunocompromised individuals in the years to come, adoptive T cell therapy (ATCT) is one of the most promising approaches [1,2]. Although this is a complex and challenging field, there have been major advances in basic and translational research resulting in clinical trial activity that is now beginning to confirm this promise. Early trials conducted at the beginning of the 90s have focused on the treatment of viral infections and virus-related tumors developing in the immunocompromised host. Initial proof of concept for the utility of antiviral ATCT in hastening reconstitution of virusspecific immunity towards cytomegalovirus (CMV) was shown two decades ago by Riddell and Greenberg in Seattle [3]. However, translation into standard clinical application was hampered by the arduous, time-consuming and costly expansion protocols used at that time, which required up to 9 weeks of culture. From the year 2000, a number of groups developed culture techniques allowing more rapid generation of therapeutic products and a series of small phase I–II studies, which confirmed lower cell doses generated in 2–3 weeks of co-culture might elicit similar protective immunity. Whole viral particles have been gradually substituted by

* Corresponding author: Dr. Marco Zecca, Pediatric Hematology/Oncology, Fondazione IRCCS Policlinico San Matteo, V.le Golgi, 19, 27100 Pavia, Italy. Tel.: +39 0382-502.607; fax: +39 0382-501.251. E-mail address: [email protected] (M. Zecca). 0378-3782/$ – see front matter © 2014 Elsevier Ireland Ltd. All rights reserved.

immunodominant viral proteins, such as pp65 or IE1, as antigens stimulating CMV cytotoxic T-lymphocytes expansion [4–6]. In the same years, Epstein-Barr virus (EBV)-associated malignancies offered a unique model to develop T cell-based immune therapies, targeting viral antigens expressed on tumor cells [7]. In the last 20 years, EBV-specific cytotoxic T-lymphocytes (CTLs) have been successfully employed for the prophylaxis and treatment of EBV-related posttransplant lymphoproliferative disorders in more than 150 patients worldwide [8]. In our institution, since August 2001, we have monitored EBV DNA in peripheral blood of haplo-HSCT recipients by quantitative PCR weekly for the first 3 months, and monthly thereafter until 1 year posttransplant. Upon detection of increased positivity in 2 subsequent samples, patients were treated with rituximab (375 mg/m2 /dose × 2 doses). In the presence of relapsed viremia and clinical EBV disease, patients were rescued with EBV-specific CTLs. A total of 14 patients did not respond to rituximab treatment, and persisted with high viral loads (7 patients) or progressed to PTLD (7 patients). These patients received rescue therapy with donor EBV-specific CTLs, 1–3 doses of 0.3–0.5×106 cells/kg body weight. All patients with DNAemia showed viral clearance. Likewise, 6/7 patients with PTLD achieved durable complete remission, including a pt with NK/T PTLD of recipient origin, while 1 patient, who developed a B-cell PTLD of recipient origin, had disease progression, as CTLs of donor origin could not recognize patient’s tumor cells. No immediate adverse events were observed. No de novo GVHD developed, and only 1

M. Zecca et al. / Early Human Development 90S1 (2014) S16–S18

patient developed a GI tract hemorrhage due to tumor lysis, that required transfusion support and resolved spontaneously. Our data suggests that an EBV surveillance program and preemptive/early treatment of PTLD, including the use of virus-specific cytotoxic T-cells, in high-risk patients, may help reduce the incidence and mortality of PTLD in pediatric recipients of T-cell depleted, HLAhaploidentical HSCT from family donors [9]. Our group was among the first to apply ATCT in the setting of solid organ transplantation [10,11]. Such a strategy is being implemented for other viruses relevant to transplantation recipients. Since 2009, we have monitored adenovirus (AdV) DNA in peripheral blood of HLA-haploidentical HSCT recipients by qPCR, and AdV specific immunity by INF-γ ELISPOT analysis. Since anti-viral drugs have limited efficacy in the treatment of AdV-related pathology, in conjunction with the optimization of the PCR technology to detect AdV DNA and of the immunological assays, we validated a method for GMP expansion of AdV-specific T-cells. In the presence of persistent high/increasing viraemia, and/or clinical AdV disease, without specific immunity, patients were treated with cidofovir, and in case of non-response, rescued with AdV-specific T cell lines (TCL) (1-4 doses of 0.25×106 cells/kg). All patients with DNAemia showed viral clearance, and the patients with disease all achieved remission. No immediate adverse events or de novo GVHD were observed. One patient with a GI tract involvement experienced a GI hemorrhage, that required transfusion support. Our data suggests that an AdV surveillance program and preemptive/early treatment, including the use of virus-specific T-cells, may help reduce the incidence and mortality of AdV infection after HSCT [12,13]. More recently, methodologies have been developed by some groups for using genetically modified antigen presenting cells to generate CTLs from donor peripheral blood that target multiple viruses and showed that adoptively transferred donor-derived CTLs can reconstitute antiviral immunity to all three viruses and effectively treat established infections [14]. However, the time taken to prepare patient-specific products and the lack of virusspecific memory T cells in cord blood and seronegative donors restricts application. Thus, research has been focused on T-cell lines manufactured using methods that exclude viral components and utilize simplified manufacturing technology can be clinically effective. Another means of avoiding growing CTLs for individual patients is to bank lines that are then available as an “off the shelf” product of most closely HLA-matched allogeneic cytotoxic T lymphocyte lines. Pilot studies have been conducted both in Europe and the USA. The results are encouraging, although further improvements to the clinical protocols could increase efficacy [15]. In addition to prevention and treatment of viral complications, T cell lines have been successfully employed to treat fungal infections in HSCT recipients. In this setting, clinical trials of ATCT have so far provided only clear proofs-of-principle that clinical responses may be attainable. Further efforts are underway to design ATCT production protocols that are fully compliant to GMP requirements. In particular, studies are focusing on the switch from the use of fungal products to recombinant proteins/peptides as stimulating antigen [16,17]. ATCT offers a unique opportunity to restore anti-pathogen immune surveillance, and it is therefore conceivable that application of this strategy will increase in the next few years. Despite the great potential, immunotherapy still has a marginal role in the management of patients with infections. This is due to limitations inherent to the technologies and products employed, to study design and to the financial and structural burden that are associated with cell therapy. The critical need for randomised confirmatory studies, rather than small serial phase I–II evaluation of iterant selection strategies,

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has become increasingly clear. The design of such studies is reliant on a consideration of the patient groups most at risk of infection, the frequency of various infections in these groups, the relative limitations of ACT in certain clinical situations, and an understanding of the business models that might ultimately drive commercial development of these products. Finally, the extensive infrastructure needed for exploiting such approaches still restricts their use to academic centres with specific programs in the field. For progression to a widely used, effective and safe ATCT, cooperation in ATCT networks is crucial. Conflict of interest The authors have no conflicts of interest to disclose. Acknowledgments This work was supported in part by Progetti Ricerca Finalizzata, Ministero della Salute, to PC, MZ; AIRC (Associazione Italiana Ricerca sul Cancro) to PC; IRCCS Policlinico San Matteo, progetti Ricerca Corrente to PC and MZ; grant from Fondazione La Nuova Speranza Onlus to PC. References [1] Locatelli F, Comoli P, Montagna D, Rossi F, Daudt L, Maccario R. Innovative approaches of adoptive immune cell therapy in paediatric recipients of haematopoietic stem cell transplantation. Best Pract Res Clin Haematol 2004;17(3):479–92. [2] Leen AM, Tripic T, Rooney CM. Challenges of T cell therapies for virusassociated diseases after hematopoietic stem cell transplantation. Expert Opin Biol Ther 2010;10(3):337–51. [3] Walter EA, Greenberg PJ, Gilbert MJ, Finch RJ, Watanabe KS, Thomas ED, et al. Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor. N Engl J Med 1995;333(16):1038–44. [4] Einsele H, Roosnek E, Rufer N, Sinzger C, Riegler S, Löffler J, et al. Infusion of cytomegalovirus (CMV)-specific T cells for the treatment of CMV infection not responding to antiviral chemotherapy. Blood 2002;99(11):3916–22. [5] Peggs KS, Verfuerth S, Pizzey A, Khan N, Guiver M, Moss PA, et al. Adoptive cellular therapy for early cytomegalovirus infection after allogeneic stem-cell transplantation with virus-specific T-cell lines. Lancet 2003;362(9393):1375– 7. [6] Cobbold M, Khan N, Pourgheysari B, Tauro S, McDonald D, Osman H, et al. Adoptive transfer of cytomegalovirus-specific CTL to stem cell transplant patients after selection by HLA-peptide tetramers. J Exp Med 2005;202(3):379– 86. [7] Rooney CM, Smith CA, Ng CY, Loftin S, Li C, Krance RA, et al. Use of genemodified virus-specific T lymphocytes to control Epstein-Barr-virus-related lymphoproliferation. Lancet 1995;345(8941):9–13. [8] Bollard CM, Rooney CM, Heslop HE. T-cell therapy in the treatment of posttransplant lymphoproliferative disease. Nat Rev Clin Oncol 2012;9(9):510–9. [9] Comoli P, Basso S, Zecca M, Pagliara D, Baldanti F, Bernardo ME, et al. Preemptive therapy of EBV-related lymphoproliferative disease after pediatric haploidentical stem cell transplantation. Am J Transplant 2007;7(6):1648–55. [10] Comoli P, Labirio M, Basso S, Baldanti F, Grossi P, Furione M, et al. Infusion of autologous Epstein-Barr virus (EBV)-specific cytotoxic T cells for prevention of EBV-related lymphoproliferative disorder in solid organ transplant recipients with evidence of active virus replication. Blood 2002;99(7):2592–98. [11] Comoli P, Maccario R, Locatelli F, Valente U, Basso S, Garaventa A, et al. Treatment of EBV-related post-renal transplant lymphoproliferative disease with a tailored regimen including EBV-specific T cells. Am J Transplant 2005;5(6):1415–22. [12] Comoli P, Schilham MW, Basso S, van Vreeswijk T, Bernardo ME, Maccario R, et al. T-cell lines specific for peptides of adenovirus hexon protein and devoid of alloreactivity against recipient cells can be obtained from HLAhaploidentical donors. J Immunother 2008;31(6):529–36. [13] Comoli P, Basso S, Labirio M, Baldanti F, Maccario R, Locatelli F. T cell therapy of Epstein-Barr virus and adenovirus infections after hemopoietic stem cell transplant. Blood Cells Mol Dis 2008; 40(1):68–70. [14] Leen AM, Myers GD, Sili U, Huls MH, Weiss H, Leung KS, et al. Monoculturederived T lymphocytes specific for multiple viruses expand and produce clinically relevant effects in immunocompromised individuals. Nat Med 2006;12(10):1160–6. [15] Haque T, Wilkie GM, Jones MM, Higgins CD, Urquhart G, Wingate P, et

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al. Allogeneic cytotoxic T-cell therapy for EBV-positive posttransplantation lymphoproliferative disease: results of a phase 2 multicenter clinical trial. Blood 2007;110(4):1123–31. [16] Leen AM, Bollard CM, Mendizabal AM, Shpall EJ, Szabolcs P, Antin JH, et al. Multicenter study of banked third-party virus-specific T cells to treat

severe viral infections after hematopoietic stem cell transplantation. Blood 2013;121(26):5113–23. [17] Perruccio K, Tosti A, Burchielli E, Topini F, Ruggeri L, Carotti A, et al. Transferring functional immune responses to pathogens after haploidentical hematopoietic transplantation. Blood 2005;106(13):4397–406.

Possible alternatives to antimicrobial therapies.

The care of immunosuppressed patients has constantly improved over the years, and pharmacologic developments contributed significantly to this success...
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