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Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture – total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by >90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

3. For the preparation of responder cells use either previously frozen PBMCs or non-adherent mononuclear cells that remain after the DC selection (adherence or CD14 selection). Thaw the cells, transfer to prewarmed culture medium, wash once with CTL media. Resuspend the cells in CTL Media, count the cells and bring them to a concentration of 2x10 6 cells per ml. Take 15x10 6 cells or 7.5ml and supplement with 30000U IL4 (1000U/ml -final conc.) and 300ng IL7 (10ng/ml -final conc.). 4. Transfer 7.5ml of PBMC (15x10 6 cells) to the G-Rex and top up the bioreactor with CTL media to a total volume of 30ml. 5. Culture the G-Rex for 6-7 days in a 37°C/5% CO 2 humidified incubator.

T cell expansion
1. On day 6-7, aspirate 10ml of media, then mix the cells in the remaining 20ml of media with a 10ml pipette and count viable cells using trypan blue. If there are <50x10 6 replenish with fresh media + cytokines. If there are >50x10 6 cells remove 10ml of cell suspension, transfer to a new G-Rex, and then feed both G-Rexs with fresh CTL media + cytokines. 2. Culture for an additional 4-6 days. Once sufficient cells have been expanded, perform phenotypic and functional characterization of the CTL and cryopreserve excess for future use.

Representative Results:
A schematic of our FDA-approved multivirus-specific CTL generation process is shown in Figure 1. In contrast to convention multivirus CTL protocols which use adenovectors and EBV-LCL to stimulate virus-reactive T cells 2 we have replaced infectious virus material with DNA plasmids that encode multiple antigens derived from each of the viruses 3 .
To stimulate trivirus CTL we designed three multicistronic plasmids encoding Hexon and Penton of adenovirus, IE1 and pp65 of CMV, and EBNA1, LMP2, and BZLF1 of EBV. These antigens were chosen based on encouraging clinical results of our own and other groups showing that T cells directed against Adv-hexon and penton 2,[4][5][6] , and to CMV-1E1 and to CMV-pp65 are protective in vivo 7 . For EBV, EBNA1 is an immunodominant CD4+ T cell target antigen expressed in all EBVassociated malignancies and in normal EBV-infected B cells 8,9 , LMP2 is immunogenic across multiple HLA types and expressed in most EBV malignancies, 10,11 while BZLF1 encodes an immunodominant, immediate early lytic cycle antigen that stimulates both CD4+ and CD8+ T cells from most individuals and is likely important for the control of cells replicating virus 12 .
To further optimize our manufacturing methods we collaborated with Nature Technology who generated minimalized, antibiotic-free (FDA-compliant) plasmids for CTL stimulation 13,14 . Using this strategy we consistently achieve nucleofection efficiencies of >35% while maintaining high cell viability (data not shown) 3 . Figure 2 shows that the frequency of virus-specific T cells in response to optimized DNA plasmids as measured by IFNγ ELIspot, was greater than in response to conventional pShuttle-based expression plasmids expressing the same antigens (n=8 Adenovirus, n=4 CMV, and n=2 EBV). The optimal ratio of DC:PBMC was important for potent T cell stimulation as shown in Figure 3 where a ratio of 1:50 produced sub-optimal activation compared to a 1:20 S:R ratio (n=2 donors). Production of sufficient CTL numbers with broad antigen specificity is a pre-requisite for clinical efficacy against all three viruses. This is achieved by CTL culture in the G-Rex, which supports superior T cell expansion compared with conventional 24-well plates ( Figure 4A) 15 , while the addition of IL4 and IL7 to cultures increases the repertoire and specificity as shown in Figure 4B where the frequency of T cells reactive against the CMV-pp65-derived HLA-A2 resticted NLV peptide was assessed in cultures generated in the presence or absence of IL4 and/or IL7 16,17 . To assess the phenotype and functional capacity of the expanded cells we perform flow cytometric analysis, intracellular cytokine staining/IFNγ ELIspot, and Cr 51 release assays on the final product for cryopreservation/infusion. Typically the generated cells are polyclonal with a mixed population of CD4+ and CD8+ T cells with antigen-specificity detectable in both T cell compartments. The CTL are able to kill viral antigen-expressing target cells but not virus negative partially-HLA matched targets, indicating that they should not induce graft-versus-host disease (GvHD) in vivo ( Figure 5).  CMV-pp65 were used as targets. Alloreactivity was assessed using allogeneic PHA blasts as a target.

Discussion
Viral infections account for substantial morbidity and mortality in patients who are immunocompromised by their disease or its treatment. After HSCT, for example, infections caused by persistent herpesviruses such as EBV and CMV, as well as by respiratory viruses such as Respiratory Syncytial Virus (RSV), are well known, while the importance of infections caused by Adv, BK virus, and human herpesvirus (HHV)-6 have more recently been appreciated. While pharmacological agents are standard therapy for some infections, they have substantial toxicities, generate resistant variants, and are frequently ineffective. In contrast, virus-specific T cells derived from stem cell donors have proven safe and effective for the prevention and treatment of viral infection or disease in the hemopoietic stem cell transplant (HSCT) setting 2,5,6,[18][19][20][21] . However, the broader implementation of T cell immunotherapy is ultimately limited by the cost, complexity and time required for CTL production.
Our novel and rapid approach to generate multivirus CTL, described in the current manuscript, should substantially increase the feasibility of cytotoxic T cell therapy for viral diseases, enabling the strategy to become a standard of care for the immunocompromised host. The use of plasmid nucleofected DCs as APCs enables antigen presentation on both MHC class I and II without competition from viral vectors or indeed from multiple viral antigens being expressed within a single cell since different DC populations are utilized for each plasmid 3 . The use of IL-4/7 increases T cell survival and proliferation, which correspondingly helps increase the frequency and repertoire of responding antigen-specific T cells 16,17 . Finally, culture in the G-Rex dramatically reduces T cell apoptosis during culture. Gas exchange (O 2 in and CO 2 out) occurs across a gas permeable silicon membrane at the base of the flask, preventing hypoxia while allowing a greater depth of medium above the cells, providing more nutrients and diluting waste products. This platform can also be extended to additional viruses as when protective antigens are identified.