Serena K. Perna

Rapidly Generated Multivirus-specific Cytotoxic T Lymphocytes for the Prophylaxis and Treatment of Viral Infections

Severe and fatal viral infections remain common after hematopoietic stem cell transplantation. Adoptive transfer of cytotoxic T lymphocytes (CTLs) specific for Epstein–Barr virus (EBV), cytomegalovirus (CMV), and adenoviral antigens can treat infections that are impervious to conventional therapies, but broader implementation and extension to additional viruses is limited by competition between virus-derived antigens and time-consuming and laborious manufacturing procedures. We now describe a system that rapidly generates a single preparation of polyclonal (CD4+ and CD8+) CTLs that is consistently specific for 15 immunodominant and subdominant antigens derived from 7 viruses (EBV, CMV, Adenovirus (Adv), BK, human herpes virus (HHV)-6, respiratory syncytial virus (RSV), and Influenza) that commonly cause post-transplant morbidity and mortality. CTLs can be rapidly produced (10 days) by a single stimulation of donor peripheral blood mononuclear cells (PBMCs) with a peptide mixture spanning the target antigens in the presence of the potent prosurvival cytokines interleukin-4 (IL4) and IL7. This approach reduces the impact of antigenic competition with a consequent increase in the antigenic repertoire and frequency of virus-specific T cells. Our approach can be readily introduced into clinical practice and should be a cost-effective alternative to common antiviral prophylactic agents for allogeneic hematopoietic stem cell transplant (HSCT) recipients.

Interleukin 15 Provides Relief to CTLs from Regulatory T Cell–Mediated Inhibition: Implications for Adoptive T Cell–Based Therapies for Lymphoma

Purpose: Systemic administration of recombinant interleukin (IL)-2 is used to support the expansion and persistence of adoptively transferred antigen-specific CTLs in patients with cancer. However, IL-2 also expands regulatory T cells (Treg) that in turn impair the antitumor activity of CTLs. As recombinant IL-15 is approaching clinical applications, we assessed the effects of this cytokine on the proliferation and antitumor activity of CTLs in the presence of Tregs. We used the model of adoptive transfer of Epstein–Barr virus (EBV)-CTLs, as these cells induce responses in patients with EBV-associated Hodgkin lymphoma, and Tregs are frequently abundant in these patients. Experimental Design: Tregs were isolated from the peripheral blood of healthy donors and patients with Hodgkin lymphoma or from Hodgkin lymphoma tumors and assessed for their ability to inhibit the proliferation and antitumor activity of EBV-CTLs in the presence of IL-15 or IL-2. Specific molecular pathways activated by IL-15 were also explored. Results: We found that in the presence of Tregs, IL-15, but not IL-2, promoted the proliferation, effector function, and resistance to apoptosis of effectors T cells and EBV-CTLs. IL-15 did not reverse or block Tregs but instead preferentially supported the proliferation of CTLs and effector T cells as compared with Tregs. Conclusions: IL-15 selectively favors the survival, proliferation, and effector function of antigen-specific CTLs in the presence of Tregs, and thus IL-15, unlike IL-2, would have a significant impact in sustaining expansion and persistence of adoptively transferred CTLs in patients with cancer, including those infused with EBV-CTLs for treatment of EBV-associated malignancies. Clin Cancer Res; 19(1); 106–17. ©2012 AACR.

Elimination of Metastatic Melanoma Using Gold Nanoshell-Enabled Photothermal Therapy and Adoptive T Cell Transfer

Ablative treatments such as photothermal therapy (PTT) are attractive anticancer strategies because they debulk accessible tumor sites while simultaneously priming antitumor immune responses. However, the immune response following thermal ablation is often insufficient to treat metastatic disease. Here we demonstrate that PTT induces the expression of proinflammatory cytokines and chemokines and promotes the maturation of dendritic cells within tumor-draining lymph nodes, thereby priming antitumor T cell responses. Unexpectedly, however, these immunomodulatory effects were not beneficial to overall antitumor immunity. We found that PTT promoted the infiltration of secondary tumor sites by CD11b+Ly-6G/C+ myeloid-derived suppressor cells, consequently failing to slow the growth of poorly immunogenic B16-F10 tumors and enhancing the growth of distant lung metastases. To exploit the beneficial effects of PTT activity against local tumors and on antitumor immunity whilst avoiding the adverse consequences, we adoptively transferred gp100-specific pmel T cells following PTT. The combination of local control by PTT and systemic antitumor immune reactivity provided by adoptively transferred T cells prevented primary tumor recurrence post-ablation, inhibited tumor growth at distant sites, and abrogated the outgrowth of lung metastases. Hence, the combination of PTT and systemic immunotherapy prevented the adverse effects of PTT on metastatic tumor growth and optimized overall tumor control.

Early transduction produces highly functional chimeric antigen receptor-modified virus-specific T-cells with central memory markers: a Production Assistant for Cell Therapy (PACT) translational application

BackgroundVirus-specific T-cells (VSTs) proliferate exponentially after adoptive transfer into hematopoietic stem cell transplant (HSCT) recipients, eliminate virus infections, then persist and provide long-term protection from viral disease. If VSTs behaved similarly when modified with tumor-specific chimeric antigen receptors (CARs), they should have potent anti-tumor activity. This theory was evaluated by Cruz et al. in a previous clinical trial with CD19.CAR-modified VSTs, but there was little apparent expansion of these cells in patients. In that study, VSTs were gene-modified on day 19 of culture and we hypothesized that by this time, sufficient T-cell differentiation may have occurred to limit the subsequent proliferative capacity of the transduced T-cells. To facilitate the clinical testing of this hypothesis in a project supported by the NHLBI-PACT mechanism, we developed and optimized a good manufacturing practices (GMP) compliant method for the early transduction of VSTs directed to Epstein-Barr virus (EBV), Adenovirus (AdV) and cytomegalovirus (CMV) using a CAR directed to the tumor-associated antigen disialoganglioside (GD2).ResultsAd-CMVpp65-transduced EBV-LCLs effectively stimulated VSTs directed to all three viruses (triVSTs). Transduction efficiency on day three was increased in the presence of cytokines and high-speed centrifugation of retroviral supernatant onto retronectin-coated plates, so that under optimal conditions up to 88% of tetramer-positive VSTs expressed the GD2.CAR. The average transduction efficiency of early-and late transduced VSTs was 55 ± 4% and 22 ± 5% respectively, and early-transduced VSTs maintained higher frequencies of T cells with central memory or intermediate memory phenotypes. Early-transduced VSTs also had higher proliferative capacity and produced higher levels of TH1 cytokines IL-2, TNF-α, IFN-γ, MIP-1α, MIP-1β and other cytokines in vitro.ConclusionsWe developed a rapid and GMP compliant method for the early transduction of multivirus-specific T-cells that allowed stable expression of high levels of a tumor directed CAR. Since a proportion of early-transduced CAR-VSTs had a central memory phenotype, they should expand and persist in vivo, simultaneously protecting against infection and targeting residual malignancy. This manufacturing strategy is currently under clinical investigation in patients receiving allogeneic HSCT for relapsed neuroblastoma and B-cell malignancies (NCT01460901 using a GD2.CAR and NCT00840853 using a CD19.CAR).

Robust and cost effective expansion of human regulatory T cells highly functional in a xenograft model of graft versus host disease

The low frequency of naturally occurring regulatory T cells (nTregs) in peripheral blood and the suboptimal protocols available for their ex vivo expansion limit the development of clinical trials based on the adoptive transfer of these cells. We have, therefore, generated a simplified, robust and cost-effective platform for the large-scale expansion of nTregs using a gas permeable static culture flask (G-Rex) in compliance with Good Manufacturing Practice. More than 109 putative Tregs co-expressing CD25 and CD4 molecules (92±5%) and FoxP3 (69±19%) were obtained within 21 days of culture. Expanded Tregs showed potent regulatory activity in vitro (80±13% inhibition of CD8+ cell division) and in vivo (suppression or delay of graft-versus-host disease in a xenograft mouse model) indicating that the cost-effective and simplified production of nTregs we propose will facilitate the implementation of clinical trials based on their adoptive transfer.

Graft-versus-leukemia effect of HLA-haploidentical central-memory t-cells expanded with leukemic APCs and modified with a suicide gene

Allogeneic hematopoietic stem cell transplantation (HSCT) from a human leukocyte antigen (HLA)-haploidentical family donor (haplo-HSCT) is a readily available and potentially curative option for high-risk leukemia. In haplo-HSCT, alloreactivity plays a major role in the graft-versus-leukemia (GVL) effect, which, however, is frequently followed by relapse due to emerging leukemic cell variants that have lost the unshared HLA haplotype as a mechanism of immune escape. We report that stimulation of HLA-haploidentical donor T lymphocytes with leukemic antigen-presenting cells (L-APCs) expands a population of leukemia-reactive T cells, which, besides alloreactivity to unshared HLAs, contain leukemia-associated specificities restricted by shared HLAs. According to a preferential central-memory (T(CM)) phenotype and to high interleukin (IL)-7Rα expression, these T cells persist in vivo and sustain a major GVL effect in a clinically relevant xenograft model. Moreover, we demonstrate that modifying L-APC-expanded T cells to express the herpes simplex virus thymidine kinase (HSV-tk) suicide gene enables their elimination with the prodrug ganciclovir (GCV), therefore providing a safety switch in case of graft-versus-host disease (GVHD). These results warrant the clinical investigation of L-APC-expanded T cells modified with a suicide gene in the setting of haplo-HSCT.

722. Overcoming EBV Tumor Specific T-Cell Anergy in Rapidly-Generated EBVST-Cells for Adoptive Transfer Therapy

Up to 30% of Hodgkin and non-Hodgkin lymphomas carry the Epstein-Barr virus (EBV) genome and express the viral latency proteins EBNA1, LMP1, LMP2 and BARF1; a type 2 latency pattern of EBV gene expression. We previously reported that EBV-specific T-cells (EBVSTs) directed to LMP1 and LMP2 expanded from the blood of lymphoma patients produced complete tumor responses in over 50% of patients. To shorten and simplify the EBVST production time, we removed viral-vector components from our manufacturing process, replacing these components with dendritic cells pulsed with peptide libraries (pepmixes) spanning type 2 latency antigens. Responder T-cells are then expanded by restimulation with pepmix-pulsed, autologous, activated T-cells and HLA-negative K562 costimulatory cells in the presence of IL-4 and IL-7. Despite enhanced antigen specificity from EBVSTs generated from healthy donors, we were unable to consistently generate patient-derived EBVSTs with significant activity against the Type 2 antigens. We hypothesized that patient T-cells were anergized by their immunosuppressive tumors. IL-15 has been shown to rescue tolerant or anergized CD8+ T-cells and we found that substitution of IL-15 (5ng/mL) for IL-4 (in combination with IL-7 (10ng/mL)) improved CD4+ and CD8+ T-cells’ specificity for the EBV Type 2 latency antigens by up to 10-fold. By increasing the concentration of IL-15, we achieved significantly higher fold expansion (3 fold mean increase in absolute EBVST number) and further enhanced specificity for type 2 latency EBV antigens (high vs. low IL-15 concentration: EBNA1: 216±273 vs. 29±48, LMP1: 145±253 vs. 44±63, LMP2: 636±548 vs. 106±74 and BARF1: 80±100 vs. 29±22; SFC/105 cells; n=5). There was no increase in the absolute numbers of NK-cells despite high doses of IL-15. Enhanced antigen-specificity correlated with increased cytotoxic effect, with an increase in mean % specific lysis of EBV pepmix-pulsed autologous activated T-cell targets from 5 to 66% at a 20:1 E:T ratio in the low vs. high dose IL-15 conditions (n=5). We have infused EBVSTs manufactured using all three conditions into 17 patients with multiply-relapsed, EBV-positive lymphoma as adjuvant therapy after stem cell transplantation or chemotherapy in 8 patients and as treatment for disease in 9 patients. Of patients in remission at the time of infusion, two with IL-4/7-grown EBVSTs and 6 with IL-15/7 grown EBVSTs remain in remission. Of patients with disease at the time of infusion, one receiving IL-4/7-grown EBVSTs had stable disease and 3 had progressive disease, while of 5 patients with IL-15/7-grown EBVSTs, one had stable disease, one had a partial response, one had a complete response and two are too early to assess. We will continue to modify our manufacturing process in an attempt to further increase the specificity and function of EBVSTs from patients with relapsed or refractory lymphoma.

513. Establishment of a Highly Characterized Third-Party Virus-Specific T Lymphocyte Bank for Treatment of EBV+ Lymphoma

Although autologous or donor-derived EBV-specific T cells (EBVSTs) have clinical efficacy for Hodgkin and non-Hodgkin lymphoma, the lengthy manufacturing process and anergy of patient tumor-specific T cells reduce the feasibility of widespread use. An alternative approach is establishment of a pre-made third party bank that can provide access to EBVSTs for almost any patient. We previously evaluated multivirus-specific third-party T cells for refractory viral infections after HSCT. Of 50 recipients, 74% responded, even when T cells were HLA-matched at only a single allele. Although established banks consist primarily of donors from national marrow registries or family members according to HLA genotype, there are many limitations to this approach, including the generation of lines with limited specificity for the targeted antigens. To circumvent these drawbacks, we have generated our bank from blood bank-eligible donors, initially chosen based on racial diversity thus predicted to have HLA haplotypes representative of our diverse patient population and then selected for high specificity for the viral antigens targeted. Donors were subsequently screened for specificity to the EBV Type 2 latency antigens (LMP1/2, EBNA1, and BARF1) with IFN-γ Elispot assays using overlapping peptide libraries (pepmixes) spanning each antigen. The challenge for our study will be the accurate identification of the HLA alleles that restrict the EBV antigen-specific activity of each line to allow optimal assignation to patients with EBV+ lymphoma. To ensure that the best EBVST line is chosen for each recipient, it is important to ensure antigen-specific activity restricted by the alleles shared between donor and recipient. To characterize HLA restriction we use peptides or pepmix-pulsed PHA blasts or LCLs that are HLA matched at a single class I or II allele as target cells in cytotoxicity assays. We then constructed a database in which the HLA restriction of the antigen specificity of each T cell line is listed. The EBVSTs exhibited significant specificity (with >50% of donors recognizing 3 of the 4 antigens) and cytotoxicity to both pepmix-pulsed autologous activated T cells and HLA-matched EBV-lymphoblastoid cell lines that naturally express viral antigens. Based on data from our third party trivirus-specific T cell study, we expect that our projected bank of 30 highly characterized EBVSTs will cover >95% of referred patients (with matching at ≥ 2 class I and/or class II alleles) since our screening strategy ensures that a wide range of HLA haplotypes is covered. Additionally, given the significantly increased virus specificity of our highly characterized donors, we expect to see significant anti-tumor activity. This study will therefore allow us to determine whether third-party T cells can be effective outside the HSCT setting in patients who are not immunocompromised.

Abstract B17: Banked Epstein-Barr virus specific T-cells for treatment of EBV+ lymphoma

Standard intensive chemotherapy and hematopoietic stem cell transplant usually benefit patients with Hodgkin (HL) and Non-Hodgkin Lymphoma (NHL), but for those with T or NK-T NHL or who relapse, the prognosis is less favorable. Furthermore, substantially increased morbidity and mortality from the complications of aggressive chemotherapies is evident for many years, even in patients who are cured of their original disease. About 30% of all lymphomas carry the Epstein-Barr virus (EBV) genome and express 4 viral proteins (EBNA1, LMP1, LMP2 ± BARF1), a pattern termed Type 2 latency. T-cells specific for these antigens circulate with low frequency in healthy donors, but are rendered anergic in patients by their immunosuppressive tumors. We have previously shown that potent in vitro T-cell stimulation can reverse anergy. Virus-specific T-cells specific for the EBV antigens (EBVSTs) have produced complete remissions in 60% of patients with multiply relapsed EBV+ HL and NHL. Though successful, our earlier method was labor intensive and prolonged, and the requirement for live EBV and adenoviral (Ad) vectors increased costs and regulatory complexity. We have overcome these problems by replacing Ad vectors with peptide mixtures (pepmixes) and the EBV lymphoblastoid cell lines (LCL) with professional antigen presenting cells to provide costimulation. We therefore seek to produce a safe and effective immunotherapy for relapsed, refractory EBV+ lymphoma that can be implemented rapidly by creating and characterizing a bank of T-cells targeted to the four EBV antigens expressed in EBV+ HL and NHL. Thus far, we have successfully screened 30 eligible blood donors and manufactured several EBVST lines with specificity for the four Type 2 EBV latency proteins using the rapid pep-mix method. This strategy robustly and rapidly produces higher frequencies of EBV-specific T cells that kill LCLs that naturally express viral antigens, showing that the pepmixes are not recruiting low-avidity T-cells. T-cell lines will be selected and infused based on their ability to recognize EBV antigens through HLA alleles shared with the recipient. The in vivo expansion and persistence of the infused EBVSTs will be evaluated together with their ability to reactivate endogenous T-cells specific for non-viral tumor antigens. We hypothesize that allogeneic, partially HLA-matched T-cells can produce benefit in patients with lymphoma both by direct tumor cell killing and by creating a proinflammatory tumor microenvironment that reactivates and restores effective endogenous tumor-specific T-cells. Citation Format: Rayne H. Rouce, Serena K. Perna, Gayatri Vyas, Sandhya Sharma, Natalia Lapteva, Ann Leen, Cliona M. Rooney, Helen E. Heslop. Banked Epstein-Barr virus specific T-cells for treatment of EBV+ lymphoma. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B17.