Project: Validation of a novel powerful technology for clinical-scale isolation of T-cells for adenovirus therapy

ADENO-TAG aims at the development of a detection and therapy system wrapped-up in one single strategy for the non-toxic treatment of immune-compromised patients._x000D__x000D_In spite of substantial progress in the field of allogeneic hematopoietic stem cell transplantation (HSCT) and solid organ transplantation (SOT), patients are highly susceptible to a wide range of chemotherapy resistant viral infections that usually lead to severe complications. Despite the inconspicuous course of Adenovirus (ADV) infections in adults, the disease represents one of the most frequent complications after allogeneic HSCT in immune-compromised or immune-suppressed patients, with a high impact on morbidity (approx. 42%) and mortality (approx. 7%) in children (1). Thereby the spectrum of ADV-associated disease in transplanted patients ranges from mild enteritic or repiratory symptoms to severe haemorrhagic enteritis, hemorrhagic cystitis, nephritis, hepatitis, pneumonia, encephalitis, myocarditis nad lethal multiple organ involvement, frequently associated with hepatic failure (1). As a consequence, most immune-compromised patients are treated prophylactically, risking side effects of toxic antiviral drugs (2) e.g. nephrotoxicity and cytopenia._x000D__x000D_Cytotoxic T-cells, commonly known as “killer cells”, are capable of deactivating virally infected cells, thereby inducing an overall immune response against the virus. Evidence indicating that recovery of ADV specific T-cells correlates with the clearance of ADV infections has prompted several attempts to use ADV-specific T-cells for antiviral therapy (3-7). However, approaches for adoptive T-cell therapy and viral clearance so far show disadvantages in terms of costs/labour intensiveness and specificity, the latter increasing the risk of GvHD (graft versus host disease). Therefore, simplification of the production process of highly specific and functional ADV-specific T-cells (ADENO-TAG cells) will certainly fulfil the requirements of an affordable, specific and effective therapy as is needed for immune-compromised patients. _x000D__x000D_The day to day direct involvement with pediatric patients and the analysis of clinical samples we receive from Labdia is our motivation to develop a strategy to induce cellular immunity against viral infections without inducing GvHD in children. _x000D__x000D_IBA GmbH’s unique BioTAGnology’s MHC/peptide Streptamers are able to specifically bind ADV-specific T-cells and allow easy dissociation via biotin cleavage. Within the present project, two different isolation procedures for ADV-specific T-cells from healthy donors will be validated and the immunoactive properties of purified ADENO-TAG cells against ADV-peptide-presenting cells - are representative of ADV-infected cells in vivo (Figure 1) - will be assessed. Since all experiments will be performed under good manufacturing practice (GMP) conditions, upon availability of results fast translation into the clinic will be possible. _x000D__x000D_Our approach is based on existing promising results in a similar setting. In a clinical trial by Schmitt and Busch(8) Streptamer-isolated CMV-specific T-cells could clear CMV viremia in patients after HSCT. Strikingly, it was demonstrated that this approach is also feasible with immune incompatible donors (8 and 9)_x000D__x000D_IBA’s Streptamer technology in addition to Labdia’s knowhow and diagnostics services and our close interaction with the St. Anna Children´s Hospital constitute a small but highly specialized setup for the development of a detection and therapeutic approach wrapped-up in one strategy, considerably raising the prospects of success for this project._x000D__x000D_ADENO-TAG could bring substantial advantages for immune-compromised pediatric patients after HSCT or SOT and immune-deficient infections by improving survival rates and providing a non-toxic prophylactic treatment against the fast progression and devastating effects of ADV infections in this group of patients. This therapy approach undoubtedly represents a promising opportunity to further expand Labdia’s range of services among its primarily pediatric medical area, and to promote the establishment of IBA’s Streptamer technology within the diagnostics & therapeutic field._x000D_

Acronym	ADENOTAG (Reference Number: 5744)
Duration	01/06/2011 - 31/03/2013
Project Topic	ADENO-TAG is a highly specific, non-toxic therapeutic approach against Adenovirus disease that will benefit immune-compromised patients. Here, feasibility and GMP production of the unique streptamer technology and rapid T-cell amplification will be validated in order to prepare the clinical phase I.
Project Results (after finalisation)	The project ADENOTAG developed a detection and therapy system for the non-toxic treatment of immunocompromised patients, who are highly susceptible to chemotherapy resistant viral infections - in particular Adenovirus (ADV) infections - leading to life threatening complications and morbidities after allogeneic hematopoietic stem cell transplantation (HSCT) or solid organ transplantation. _x000D_To overcome ADV infections, this project evaluated a strategy to magnetically select highly ADV- specific cytotoxic T-cells (ADENO-TAG cells) which could be used for antiviral therapy without increasing the risk of Graft versus Host Disease (GvHD)._x000D_This was achieved by the cooperation between IBA GmbH from Germany, providing highly specific and reversible good manufacturing process (GMP)-conform major histocompatibility complex (MHC)/peptide multimers (“Streptamers”). Streptamers are able to specifically bind and consequently allow magnetic selection of ADV-specific T-cells from leukapheresis products which is termed “large scale selection”. _x000D_The general low frequency (<0.01 among CD3+ T cells) of virus-specific T-cells within blood products could negatively influence the purity of large scale selected T-cells. In order to increase the purity of ADV-specific T cells in the starting material, an in vitro expansion-protocol, - developed by Labdia Labordiagnostik GmbH, a non-profit spin off of the St Anna Children´s Cancer Research Association from Austria - was performed prior to the magnetic selection which is termed “small scale selection”. _x000D__x000D_The CO results of the project:_x000D_• The successful manufacture of 5 ADV- and 4 EBV-specific research- and clinical-grade Streptamers by IBA GmbH. Of note, only the B35 EBV-Streptamer could not be produced under clinical-grade conditions. _x000D_• All 5 ADV- and 5 EBV-specific research-grade Streptamers provided by IBA were able to detect virus-specific T cells derived from appropriate donors. _x000D_• Development and successful validation of a novel HLA-A*02-based ADV-specific Streptamer together with IBA and Stefan Stevanovic (Department of Immunoloy, Institute for Cell Biology, Eberhard-Karls-Universität Tübingen, Tübingen, Germany; not mentioned in the original application form)_x000D_• In 6/8 and 2/4 large scale selections, a clear population of ADV- and EBV-specific T cells were seen, respectively. However the percentage of ADV- and EBV-specific T cells among CD3+ T cells were very low with a median of 1.1% (range: 0-7.6%) for ADV and 14.4% (range: 0-24.2%) for EBV. In fact, purities below 10% will not be considered as safe for the infusion into patients._x000D_• Nevertheless, the use of multiple Streptamers (e.g. including ADV, EBV and CMV-Streptamers) per single selection would highly increase the purity and therefore enable a clinical use. _x000D_• To overcome the rather low purities after large scale selection, an in vitro expansion was performed prior to the small scale selection, which resulted in much higher purities (mean 59.5%, range 22.8-99%) and absolute cell numbers of ADV-specific T-cells compared to large-scale selections._x000D_• In addition, even the in vitro expansion alone without any further streptamer selection resulted in high 2-3 log increased frequencies of ADV-specific T cells, which were measured by streptamers. _x000D_• Strikingly, ELIspot analyses revealed that all large-scale selected ADV- and EBV-specific T cells, in vitro expanded and 2/3 of small scale selected ADV-specific T cells were functionally active, which was represented by the secretion of IFN-g via ELIspot. _x000D_• None of the clinical-grade magnetically selected virus-specific T cells showed bacterial contaminations, which is an important issue for further clinical trials. _x000D__x000D_As a specific “highlight” of the project, two HSCT-patients in the life threatening situation of increasing viral load despite virostatic treatment have been treated with GMP-compliant in vitro expanded ADV-specific T cells (without streptamer selection) in "named patient use" at t
Network	Eurostars
Call	Eurostars Cut-Off 4

Project partner

Number	Name	Role	Country
2	IBA GmbH	Partner	Germany
2	Labdia Labordiagnostik GmbH	Coordinator	Austria