Overlapping Peptide Libraries Research Articles

A Pilot Study of Adoptive Cellular Immunotherapy for Progressive Multifocal Leukoencephalopathy with Ex Vivo Generated Polyomavirus-Specific T-Cells

▪Background: Progressive Multifocal Encephalopathy (PML) is a subacute infection of the central nervous system (CNS) mediated by John Cunningham (JC) polyomavirus (PyV). The disease is frequently fatal, unless adaptive immunity to JC virus is restored. The disease typically occurs in immunosuppressed patients (e.g. HIV/AIDS). In the modern era, patients with hematological malignancies treated with rituximab and/or fludarabine or following immunosuppressive therapy for transplant or autoimmunity (e.g. multiple sclerosis (MS) or Crohn's disease) are also at risk. JC-PML can also occur in patients with genetic defects of immunity. Specific treatments do not exist and immunocompromised patients with history of cancer or genetic defects of immunity have no realistic chance for rapid recovery of their ability to fight infections. In subjects with Rituximab-related PML the case-fatality ratio is >90%. Survivors can be left with severe neurological disabilities.Methods: Based on our previously established procedure for generation of multi-virus virus-specific T cells using overlapping peptide libraries (pepmixes) as immunogens, we have developed dedicated polyomavirus-specific T cells (PyVST) targeting polyomavirus BK large T (LT) and Viral Protein 1 (VP1) antigens and highly cross-reactive with the structurally-homologous JC LT and VP1 proteins (see Figure). We hypothesized that adoptive transfer of donor-derived PyVSTs could be safely used for therapy of patients with refractory JC-PML. We have developed a pilot study to test the feasibility and safety of adoptive immunotherapy strategy using PyVSTs from the partially matched healthy 1st degree relative donors (sibling, parent or offspring; NIH study 16-N-0072). Adults diagnosed with PML who have no other treatment options were eligible. Patients with MS or HIV were excluded. PyVSTs were generated from donor blood leukocytes cultured for 14 days in G-rex flasks upon stimulation with BK LT and VP1 pepmixes. Subjects underwent baseline physical examination, MRI and lumbar puncture. Upon enrollment subjects received an intravenous infusion of a fixed dose of 1x10e6 (+/-10%) PyVST cells/kg. Safety monitoring period was 28 days after each infusion. Subjects were eligible for up to two additional infusions (dose 2x10e6 PyVSTs/kg) a minimum of 28 days apart if no toxicities were observed. Serial MRI and lumbar punctures were performed to monitor response. Subjects were followed for 12 months after the last infusion.Results: Nine subjects have been enrolled and treated with at least one infusion of PyVSTs under this protocol and the trial is still accruing subjects. No immediate infusion reactions or adverse events have been observed within the safety monitoring period. No CNS immune system reconstitution syndrome (IRIS) has occurred. One subject received a single infusion and withdrew from the study following Day 14 visit due to unwillingness to travel. One subject received two doses of PyVSTs with stabilization of the disease, not requiring the 3rd infusion. This subject was withdrawn from the study just prior to the 1 year follow up visit, as he was diagnosed with the stage IV lung cancer and entered hospice. Seven subjects received 3 infusions of PyVSTs. 3 of these 7 patients died of refractory PML, all >30 days after the 3rd dose of T cells. At the time of this report one subject completed the study at one year with stable PML. The remaining subjects are in the follow-up phase of the protocol.Conclusions: We report that partially matched T cells targeting PyVs generated from healthy related donors can be safely used for adoptive immunotherapy of severely immunocompromised patients with PML. Multiple infusions of T cells are very well tolerated at the doses of up to 2x10e6 PyVST cells/kg, displaying excellent safety profile without adverse events. Furthermore, our data suggest possible efficacy of this strategy as a life-saving therapy for patients who otherwise face a dismal prognosis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Very Early Adoptive Transfer of Ex Vivo Generated Multi-Virus Specific T Cells Is a Safe Strategy for Prevention of Viral Infection after Allogeneic T Cell Depleted Stem Cell Transplantation

▪Background: Reactivation of latent viruses post-allogeneic stem cell transplant (SCT) negatively affects outcomes and increases non-relapse mortality. We have reported over 80% early CMV reactivation rate and significant additional costs in recipients of T cell depleted (TCD) SCT. Ex vivo generated multi-virus specific T cells (MVSTs) are effective as a therapy of active infection, but have not been evaluated as prophylaxis early after transplant. In a Phase I study (NIH 14-H-0182) we transferred MVSTs targeting immunodominant viral proteins of CMV, Epstein-Barr virus (EBV), BK and adenovirus (Ad) immediately post SCT as a novel approach to prevent viral reactivation post-SCT.Methods: All subjects were enrolled in HLA-matched T cell depleted (TCD) transplant protocol (NIH 13-H-0144). MVSTs cells were manufactured from SCT sibling donors by stimulation of elutriated lymphocytes for 14 days with seven overlapping peptide libraries (pepmixes) pulsed onto autologous dendritic cells (DCs) in presence of IL-7, IL-15 and IL-2. MVSTs were infused as early as possible (day 0 to +60) post SCT. A Phase I 3+3 dose escalation design was used as follows: Cohort 1 - 1x10e5 total nucleated cells (TNC)/kg, Cohort 2 - 5x10e5 TNC/kg, Cohort 3 - 1x10e6 TNC/kg. Three additional subjects received MVST cells manufactured using pepmix-pulsed mononuclear cells as stimulators (Cohort 3A; 1x10e6TNC/kg) under an amended protocol. The primary safety endpoint at day 42 post infusion was the occurrence of dose limiting toxicity (DLT) defined as Grade IV GVHD or any other severe adverse even (SAE) deemed to be at least “probably” or “definitely” related to the MVST infusion. Patients were followed to day +100 post SCT for secondary outcomes of efficacy, immune reconstitution and GVHD biomarkers (ST2, REG3). CDR3 sequencing (ImmunoSEQ) was performed on selected MVST products and peripheral blood samples post MVST and compared to control SCT recipients. GVHD biomarkers were analyzed pre- and post-treatment.Results: Twelve subjects were treated: nine received MVSTs generated using DCs and three subjects using mononuclear cells (cohort 3A). Median time from SCT to MVST administration was 13 days (range D +2 to +52 post-SCT). Median time to MVST for subjects in Cohort 3A was 3 days (range 2-7). There were no immediate infusion-related adverse events or DLT at the highest dose level. De novo grade II-III aGVHD post-MVST infusion was seen in three subjects (one in Cohort 1 and two in Cohort 3A), but GVHD biomarker elevation predated MVST infusion. CMV reactivation post-MVST occurred in 6 out of 12 subjects (50%) vs. 45 out of 52 patients (50% vs 87%) in a historical group of recipients of T cell depleted SCT. In all cases CMV reactivation occurred in the context of high dose steroids and in two subjects MVSTs were derived from CMV seronegative donors with minimal anti-CMV activity. One subject experienced rapidly rising AdV viremia (asymptomatic) and received an additional infusion of MVSTs. We saw self-limiting low level EBV replication in 8 cases and one BK viremia, but no disease. CDR3 sequencing of MVST products and serial peripheral blood from subjects revealed a robust contribution of ex vivo expanded cells to the overall repertoire, in contrast to untreated controls where the repertoire of (sham) MVST cell products generated from the transplant donors did not significantly overlap with the immune repertoire in peripheral blood of TCD-SCT recipients in the early post SCT period. Only at day +180 some convergence of repertoires became visible indicating spontaneous immune reconstitution (Figure). Detailed CDR3 analysis of cytokine-captured CMV pp65 and IE-1 specific CD4+ and CD8+ T cells was performed in a representative subject clearly demonstrating the detrimental effect of high-dose steroids on frequency of anti-viral T cells, precipitating CMV reactivation.Conclusions: This is the first report demonstrating safety and feasibility of using MVST immediately post-SCT to rapidly reconstitute anti-viral immunity and ameliorate the detrimental impact of the early viral reactivation in SCT recipient. No DLTs were seen and a minimal risk of aGVHD was observed, as there was no correlation with GVHD biomarkers. As revealed by serial CDR3 sequencing, MVSTs robustly contributed to the T cell repertoire. Our results suggest efficacy of this strategy in reducing viral reactivation. A Phase II study is warranted. DisclosuresNo relevant conflicts of interest to declare.

Evaluation of emerging biomarkers for major organ toxicities and disease states

Zika virus (ZIKV) infection can cause severe birth defects in newborns with no effective currently available treatment. Adoptive transfer of virus-specific T cells has proven to be safe and effective for the prevention or treatment of many viral infections, and could represent a novel treatment approach for patients with ZIKV infection. However, extending this strategy to the ZIKV setting has been hampered by limited data on immunogenic T-cell antigens within ZIKV. Hence, we have generated ZIKV-specific T cells and characterized the cellular immune responses against ZIKV antigens.T-cell products were generated from peripheral blood of ZIKV-exposed donors, ZIKV-naive adult donors and umbilical cord blood by stimulation with pentadecamer (15mer) overlapping peptide libraries spanning four ZIKV polyproteins (C, M, E and NS1) using a Good Manufacturing Practice–compliant protocol.We successfully generated T cells targeting ZIKV antigens with clinically relevant numbers. The ex vivo–expanded T cells comprised both CD4+ and CD8+ T cells that were able to produce Th1-polarized effector cytokines and kill ZIKV-infected HLA-matched monocytes, confirming functionality of this unique T-cell product as a potential “off-the-shelf” therapeutic. Epitope mapping using peptide arrays identified several novel HLA class I and class II–restricted epitopes within NS1 antigen, which is essential for viral replication and immune evasion.Our findings demonstrate that it is feasible to generate potent ZIKV-specific T cells from a variety of cell sources including virus naïve donors for future clinical use in an “off-the-shelf” setting.

Generation of multivirus-specific T cells by a single stimulation of peripheral blood mononuclear cells with a peptide mixture using serum-free medium

Background: Restoration of virus-specific immunity by virus specific T cells (VSTs) offers an attractive alternative to conventional drugs, and can be highly effective in immunocompromised patients, including hematopoietic stem cell transplant (HSCT) recipients. However, conventional VSTs manufacture requires preparation of specialized antigen-presenting cells (APCs), prolonged ex vivo culture in serum-containing medium and antigen re-stimulation with viruses or viral vectors to provide viral antigens for presentation on APCs. Methods: To simplify this complex process, we developed a method to generate multiple VSTs by direct stimulation of peripheral blood mononuclear cells (PBMCs) with overlapping peptide libraries in serum-free medium. Results: We generated VSTs that targeted seven viruses (cytomegalovirus [CMV], Epstein-Barr virus [EBV], adenovirus [AdV], human herpesvirus 6 [HHV-6], BK virus [BKV], JC virus [JCV] and Varicella Zoster virus [VZV]) in a single line. The phenotype, growth and specificity of multiple VSTs produced in serum-free medium were equivalent to those generated in conventional serum-containing medium. Discussion: The use of serum-free medium allows this approach to be readily introduced to clinical practice with lower cost, greater reproducibility due to the absence of batch-to-batch variability in serum and without concerns for infectious agents in the serum used. This simplified approach will now be tested in recipients of Human Leukocyte Antigen (HLA)–matched sibling HSCT.

Clonal dynamics of adoptively transferred multi-virus specific T cells (MVST) in hematopoietic stem cell transplant patients

Abstract Hematopoietic stem cell transplant (HCT) patients often risk complications from viral infection due to loss of protective antiviral immunity. One strategy to overcome this hurdle is to supplement the HCT with multi-virus specific T cells (MVST). In phase I trial (NCT02231853), MVSTs were generated using HLA-matched donor-derived dendritic cells pulsed with viral overlapping peptide libraries (cytomegalovirus (CMV), Epstein-Barr virus, Adenovirus, and BK polyomavirus) and then co-cultured with donor lymphocytes. These cells were infused into the patient in early post-SCT. Using T cell receptor CDR3 immnuosequencing, we investigated the fate of adoptively transferred donor derived MVST in four donor-patient pairs. Relative clonal frequencies were analyzed in the peripheral blood and bone marrow day 60, 100, 180, and 1 year post transplant. The top 10 most frequent clones in the MVST product did not necessarily survive in the patients, and only minor subsets of MVST clonal sequences survived at day 100 (2.92–7.75%). However, these minor MVST clones quickly expanded in all four patients to constitute over >20% of peripheral blood T cells even 6 months to 1 year later. Two patients analyzed had MVST antiviral clones peak during and after detectable viremia or viriuria, but were asymptomatic. One patient received steroids for treatment of GVHD, which was accompanied by the decline of MVST clones and subsequent CMV reactivation. These results suggest that adoptive transfer of viral-specific T cells can promote reconstitution of the anti-viral T cell repertoire and these cells persist in patients long-term post transplant. Furthermore, the fittest clones are dynamically selected to serve as long term sentinels against viral reactivation.

Efficient generation of MCC oncoprotein-specific CD4+ T cells for potential adoptive immunotherapy

Abstract Merkel Cell Carcinoma (MCC) is an aggressive skin tumor caused by Merkel Cell Polyomavirus (MCV) in ~80% of cases that responds poorly to standard chemotherapies. Virus positive tumors have a low mutational burden, and are driven by alternatively spliced T-antigens (TAG), small T (sT) and truncated large T antigen (LTT). These intracellular proteins are constitutively expressed in MCC tumors and are required for tumor survival. We hypothesized that MCV TAG can be targeted by cytotoxic T lymphocytes for potential use as an adoptive T cell immunotherapy to treat MCC. Peripheral blood T cells were stimulated with autologous dendritic cells pulsed with overlapping peptide libraries derived from MCV sT and LTT in the presence of a pro-inflammatory cytokine cocktail (IL-1β, IL-6, IL-7, IL-15, IL-21, IL-23, and TGFβ). This culturing method increases yields by 6 fold of antigen-specific T cells that produce TNFα and IFNγ, from both MCC subjects and healthy donors. These expanded TAG-specific cells are predominately CD4+ cells of a memory phenotype that expresses low levels of PD-1, but no expression of IL-17A. Growing evidence suggests that CD4+ Th1 and Th17 cells can have potent anti-tumor activities, especially against MHC class II positive tumors. Three virus-positive and two virus-negative MCC cell lines were tested for HLA-DR expression by flow cytometry. We found that MCC cell lines, both virus positive and virus negative, can be induced to express HLA-DR in a dose-dependent fashion in response to IFNγ in vitro. These results suggest that MCC virus positive tumors can likely be directly recognized and destroyed by adoptively transferred Th1 or Th17 CD4+ T cells and offers a promising avenue for adoptive T cell immunotherapy as a MCC treatment.

Mapping Protective Regions on a Three-Dimensional Model of the Moraxella catarrhalis Vaccine Antigen Oligopeptide Permease A.

A vaccine against Moraxella catarrhalis would reduce tremendous morbidity, mortality, and financial burden by preventing otitis media in children and exacerbations of chronic obstructive pulmonary disease (COPD) in adults. Oligopeptide permease A (OppA) is a candidate vaccine antigen that is (i) a nutritional virulence factor expressed on the bacterial cell surface during infection, (ii) widely conserved among strains, (iii) highly immunogenic, and (iv) a protective antigen based on its capacity to induce protective responses in immunized animals. In the present study, we show that the antibodies to OppA following vaccination mediate accelerated clearance in animals after pulmonary challenge. To identify regions of OppA that bind protective antibodies, truncated constructs of OppA were engineered and studied to map regions of OppA with surface-accessible epitopes that bind high-avidity antibodies following vaccination. Protective epitopes were located in the N and C termini of the protein. Immunization of mice with constructs corresponding to these regions (T5 and T8) induced protective responses. Studies of overlapping peptide libraries of constructs T5 and T8 with OppA immune serum identified two discrete regions on each construct. These potentially protective regions were mapped on a three-dimensional computational model of OppA, where regions with solvent-accessible amino acids were identified as three potentially protective epitopes. In all, these studies revealed two regions with three specific epitopes in OppA that induce potentially protective antibody responses following vaccination. Detection of antibodies to these regions could serve to guide vaccine formulation and as a diagnostic tool for monitoring development of protective responses during clinical trials.

64 - Adoptive Immunotherapy with Rapidly-Generated Multivirus-Specific T Cells Against Adv, EBV, CMV, HHV6 and BK after Allogeneic Hematopoietic Stem Cell Transplant

Abstract Viral infections remain a significant cause of morbidity and mortality after allogeneic stem cell transplantation (HSCT). We now report on the outcomes of 67 patients infused with either donor-derived (n=21) or third party “off the shelf” (n=46) virus-specific T cells (VSTs) with activity against 5 common post-transplant viruses (EBV, CMV, Adv, BK, HHV6). We generated 84 clinical-grade VST lines by stimulating 30x106 PBMCs with overlapping peptide libraries spanning Adv (Hexon, Penton), CMV (pp65, IE1), EBV (LMP2, EBNA1, BZLF1), BKV (Large T, VP1) and HHV6 (U11, U14, U90) antigens. After a 9-11 day expansion phase in a G-Rex device in the presence of activating cytokines, a mean of 399x106 T cells (range 89-1006x106) were harvested. The lines were polyclonal, comprising both CD4+ (60±2%) and CD8+ (31±2%) cells and expressed central CD45RO+/CD62L+/CCR7+ (38±3%) and effector memory markers CD45RO+/CD62L-/CCR7- (10±1%). VST specificity was dependent on the donor's prior exposure to viruses; thus, 81/84 lines had activity against Adv (Hexon: 384±44; Penton: 293±50 SFC/2x105), 50/84 against CMV (IE1: 294±52; pp65 1009±194), 66/84 against EBV (LMP2: 153±26; EBNA1: 132±17; BZLF1: 91±25), 53/84 against BK (Large T: 134±22; VP1: 158±29) and 57/84 against HHV-6 (U90: 111±25; U14: 94±13; U11: 46±8). Twenty one allogeneic HSCT recipients were infused with donor-derived VSTs in a dose escalation study with informed consent; 4 on DL1 (5x106/m2), 4 on DL2 (1x107/m2) and 13 on DL3 (2x107/m2). Four patients received the cells prophylactically and remained free of viral infections up to 3 months post-infusion. The remaining patients were infused for one or more active infections. There were 4 cases of Grade I-II GvHD, of which only one required treatment with steroids. Based on viral load measurements and symptom assessment a single VST infusion successfully controlled active infections associated with all our targeted viruses (ORR 95%): CMV (7 CR, 1PR); EBV (7 CR, 1PR); Adv (6 CR); HHV6 (5 CR, 1PR, 1NR) and BKV (8 CR, 2 PR, 1 NR). Overall, we used VSTs to treat 7 cases of viral disease- EBV-PTLD (n=1), HHV-6 encephalitis (n=1), BKV hemorrhagic cystitis (HC) (n=5). The infused cells successfully cleared the EBV-PTLD (without rituximab) and HHV6 encephalitis, while all patients with BKV HC had marked improvement or resolution of symptoms post-VSTs. Based on the safety and efficacy profile of VSTs we extended our approach to third party recipients. To date we have treated 46 patients, all of whom had drug-refractory infections. Patients received 1-3 doses (fixed dose - 2x107 cells/m2) with 3rd party VSTs matching at one to six of eight HLA alleles. Despite the HLA disparity the cells have proven safe with three cases of de novo grade I-II GvHD documented. Third party VSTs successfully controlled active infections in 42 of 45 evaluable patients (ORR 94%): CMV (13 CR, 5 PR, 1 NR); EBV (2 CR); AdV (7 CR, 2 NR); BKV (5 CR, 14 PR); and HHV-6 (4 PR). Of note, 30 of 46 patients were treated for viral disease - CMV colitis (n=3), CMV encephalitis (n=1), EBV-PTLD (n=1), AdV respiratory tract infection (RTI) (n=2), AdV RTI and enteritis (n=1), AdV HC (n=1), HHV-6 encephalitis (n=2), BKV HC (n=17), BKV nephritis (n=2). All patients with BK-HC, 2 of 3 patients with CMV colitis, and the patients with CMV encephalitis, AdV RTI, enteritis and HC, and HHV-6 encephalitis had marked improvement or resolution of symptoms within 6 weeks following VST treatment. In more than 50% of responders (n=24) we detected an increase in the circulating frequency of VSTs post-infusion, which were confirmed to be 3rd party VST origin in 14 and remained detectable for up to 12 weeks post-infusion. Overall our experience with adoptively-transferred VSTs demonstrates not only the safety profile of the cells, even when administered as a 3rd party product, but also the efficacy of the infused cells in patients with up to 4 simultaneous/sequential infections, and HSCT recipients with virus associated disease. Disclosures Tzannou: ViraCyte LLC: Consultancy. Lulla: ASBMT Young Investigator Award: Research Funding; ASH Scholar Award: Research Funding; Leukemia Texas Research grant: Research Funding; Junior Faculty sees funding-Baylor College of Medicine: Research Funding; Lymphoma SPORE: Research Funding; Leukemia Texas: Membership on an entity's Board of Directors or advisory committees. Vera: Marker Therapeutics: Equity Ownership; ViraCyte LLC: Equity Ownership; Wilson Wolf Manufacturing: Consultancy. Rooney: ViraCyte LLC: Membership on an entity's Board of Directors or advisory committees. Heslop: Cell Medica: Patents & Royalties: EBV specific T cells, Research Funding; Viracyte: Equity Ownership; Marker Therapeutics: Equity Ownership; Celgene: Research Funding. Leen: ViraCyte LLC: Equity Ownership.

Overlapping Peptide Library to Map Qa-1 Epitopes in a Protein.

Qa-1 (HLA-E in human) belongs to a group of non-classical major histocompatibility complex 1b (MHC-Ib) molecules. Recent data suggest that Qa-1 molecules play important roles in surveying cells for structural and functional integrity, inducing immune regulation, and limiting immune responses to viral infections. Additionally, functional augmentation of Qa-1-restricted CD8+ T cells through epitope immunization has shown therapeutic effects in several autoimmune disease animal models, e.g. experimental allergic encephalomyelitis, collagen-induced arthritis, and non-obese diabetes. Therefore, there is an urgent need for a method that can efficiently and quickly identify functional Qa-1 epitopes in a protein. Here, we describe a protocol that utilizes Qa-1-restricted CD8+ T cell lines specific for an overlapping peptide (OLP) library for determining Qa-1 epitopes in a protein. This OLP library contains 15-mer overlapping peptides that cover the whole length of a protein, and adjacent peptides overlap by 11 amino acids. Using this protocol, we recently identified a 9-mer Qa-1 epitope in myelin oligodendrocyte glycoprotein (MOG). This newly mapped MOG Qa-1 epitope was shown to induce epitope-specific, Qa-1-restricted CD8+ T cells that enhanced myelin-specific immune regulation. Therefore, this protocol is useful for future investigation of novel targets and functions of Qa-1-restricted CD8+ T cells.

Overlapping Peptide Library to Map Qa-1 Epitopes in a Protein

Qa-1 (HLA-E in human) belongs to a group of non-classical major histocompatibility complex 1b (MHC-Ib) molecules. Recent data suggest that Qa-1 molecules play important roles in surveying cells for structural and functional integrity, inducing immune regulation, and limiting immune responses to viral infections. Additionally, functional augmentation of Qa-1-restricted CD8+ T cells through epitope immunization has shown therapeutic effects in several autoimmune disease animal models, e.g. experimental allergic encephalomyelitis, collagen-induced arthritis, and non-obese diabetes. Therefore, there is an urgent need for a method that can efficiently and quickly identify functional Qa-1 epitopes in a protein. Here, we describe a protocol that utilizes Qa-1-restricted CD8+ T cell lines specific for an overlapping peptide (OLP) library for determining Qa-1 epitopes in a protein. This OLP library contains 15-mer overlapping peptides that cover the whole length of a protein, and adjacent peptides overlap by 11 amino acids. Using this protocol, we recently identified a 9-mer Qa-1 epitope in myelin oligodendrocyte glycoprotein (MOG). This newly mapped MOG Qa-1 epitope was shown to induce epitope-specific, Qa-1-restricted CD8+ T cells that enhanced myelin-specific immune regulation. Therefore, this protocol is useful for future investigation of novel targets and functions of Qa-1-restricted CD8+ T cells.

Merkel Cell Carcinoma viral oncoprotein-specific polyclonal T cells can be generated from patients and from healthy donors

Abstract Truncated large T (LT-T) and small T (ST) antigens of Merkel Cell Polyomavirus (MCV) are viral oncoproteins critical for pathogenesis of Merkel Cell Carcinoma (MCC), making this malignancy an attractive target for immunotherapy. We explored feasibility of generating LT-T and ST specific T cells from 12 healthy donors and 4 patients with MCC. Manufactured overlapping peptide libraries (pepmixes) spanning entire LT-T and ST proteins were pulsed onto autologous dendritic cells or peripheral blood mononuclear cells and co-cultured with lymphocytes in presence of IL-2, IL-7 and IL-15. Analogous cultures were generated using BK and JC polyomavirus LT pepmixes and PRAME (MCC patients). On day 14, cells were tested for recognition of cognate pepmixes. T cells from 4/12 (33.3%) healthy donors released IFN-γ and/or TNFα (average 0.48 ± 0.144 %) in response to MCV T antigens, predominantly in CD4± cells. 2/4 patients with MCC had a robust T cell response against MCV LT (2.23 ± 0.65%), 3/4 patients responded to JCV LT (3.56 ± 1.92%), and 2/4 responded to PRAME (1.18 ± 0.707%). T cells specific for JC or BK LT antigens demonstrated mutual cross-reactivity, but negligible cross reactivity with MCV LT and vice versa, suggesting that the more abundant JC and BK-specific cells do not confer protection against MCC. Our data indicate that while MCC subjects have strong responses healthy donors harbor only rare memory T cells specific for MCC oncoproteins. Based on these findings we are developing highly-active clinical-grade MCC-specific T cells for adoptive immunotherapy.

Early Adoptive Transfer of Ex Vivo Generated Multi-Virus Specific T Cells Is a Safe Strategy to Prevent Viral Reactivation in Recipients of Allogeneic T Cell Depleted Stem Cell Transplant

Background: Allogeneic stem cell transplant (SCT) recipients suffer from a defective T cell mediated immunity causing potentially fatal reactivation of latent viruses. After T cell depleted SCT we have observed over 80% CMV reactivation and significant additional costs ($58-74k/patient). Despite aggressive monitoring and pre-emptive therapy, reactivation and/or positive CMV serology brings a significantly higher risk for non-relapse mortality (NRM). Adoptive transfer of ex vivo generated virus specific donor T cells is effective as a treatment of infection post-SCT but it has not been tested as a prophylaxis of early reactivation. Here in a Phase I study we transferred multi-virus specific T cells (MVSTs) immediately post SCT, targeting CMV, Ebstein-Barr virus (EBV), BK and adenovirus (Ad) as a novel strategy to prevent viral reactivation in the recipients of T cell depleted sibling HLA-matched SCT.Methods: Subjects were eligible if enrolled in HLA-matched T cell depleted transplant protocol (13-H-0144) and deemed at risk for CMV reactivation. MVST cells were manufactured from SCT sibling donors. Elutriated lymphocytes were stimulated with autologous dendritic cells (DCs) pulsed with seven overlapping peptide libraries (pepmixes) spanning the length of immunodominant proteins from CMV (pp65 and IE1), EBV (BZLF1 and EBNA1), BK (LT and VP1) and Ad5. Cultures were maintained in G-Rex flasks for 14 days in presence of IL-7, IL-15 and IL-2 (after 72hrs), tested for sterility, phenotype, potency and cryopreserved. MVST cells were thawed and administered intravenously as early as possible (day 0 to +60) post SCT. A Phase I 3+3 dose escalation design was used at the following dose levels: Cohort 1 - 1x10e5 total nucleated cells (TNC)/kg, Cohort 2 - 5x10e5 TNC/kg, Cohort 3 - 1x10e6 TNC/kg. The primary safety endpoint at day 42 post infusion was the occurrence of dose limiting toxicity (DLT), (Grade IV GVHD or any other severe adverse even (SAE) deemed to be at least "probably" or "definitely" related to the MVST infusion. Patients were followed to day +100 post SCT for secondary outcomes, including efficacy (Figure) and immune reactivity (for donor/recipient pairs).Results: MVST cells recognized the majority of pepmixes, were polyfunctional and robustly proliferated in response the cognate antigens, but minimally against allogeneic targets- suggesting a limited ability to induce GVHD. CDR3 sequencing of T cell repertoire showed a significant reduction in diversity and a striking dominance of a limited number of clonotypes in the final MVSTs. Nine subjects were enrolled and treated with MVST cells. MVSTs were successfully generated for all subjects, meeting the release criteria. Median time from SCT to MVST administration was 16 days (range D +6 to +52 post-SCT). Two subjects received MVST after day +30 due to cardiac instability and scheduling. There were no immediate infusion-related adverse events or DLT by day 42. One subject in cohort II developed a self-limiting grade I cytokine release syndrome in the setting of low-level EBV reactivation. One patient (cohort 1) developed de novo grade III aGVHD post-MVST infusion. CMV reactivation post-MVST occurred in 4 out of 8 evaluable subjects (50%) who completed D+100 post-SCT vs. 45 out of 52 patients (50% vs 87%; p value=0.031) in a historical cohort of recipients of T cell depleted SCT. In all cases CMV reactivation occurred during treatment with high dose steroids. In two cases MVST were generated from CMV seronegative donors and showed minimal activity against pp65 and IE1. In eight evaluable subjects who reached D+100 post-SCT there was no EBV-related disease, but we saw self-limiting low level EBV replication in 6 out of 8 cases. There were no cases of BK or Ad-related disease or viremia. ELISPOT analysis at D+100 revealed robust reconstitution of anti-viral immunity in analyzed recipients (vs. donors, Figure, B)Conclusions: This is the first report demonstrating that it is safe and feasible to use adoptively transferred allo-MVST immediately post-SCT to rapidly reconstitute anti-viral immunity and ameliorate the detrimental impact of the early viral reactivation in SCT recipient. No DLTs were seen. MVSTs had a markedly reduced allo-reactivity and carried a minimal risk of GVHD. Our results also suggest efficacy of this strategy in reducing viral reactivation. A Phase II portion of this study is currently enrolling patients. [Display omitted] DisclosuresSabatino:Kite: Employment, Equity Ownership.

Rapidly-Generated EBV-Specific T Cells (EBVST-cells) to Treat Type 2 Latency Lymphoma

Up to 30 % of Hodgkin and non-Hodgkin lymphomas carry the Epstein Barr virus (EBV) genome and express the viral latency type 2 proteins EBNA-1, LMP-1, LMP-2 and BARF-1 in a pattern termed Type 2 latency. We have previously shown that Epstein-Barr virus-specific T-cells (EBVSTs) can be expanded from the peripheral blood of lymphoma patients by stimulation with dendritic cells and autologous EBV-transformed lymphoblastoid cell lines (LCLs) modified with an adenoviral vector encoding LMP1 and LMP2. These EBVSTs induce complete clinical responses in over 50% of patients with active disease (Bollard et al J Clin Oncol 2014). Since the requirement for an LCL as an antigen-presenting cell added 3-4 months to the manufacturing time, we shortened and simplified the process. We replaced the LCL with autologous, activated T-cells together with HLA-negative K562 costimulatory cells as a source of antigen-presenting cell and replaced live virus (EBV) and adenoviral-vector components as a source of antigen with overlapping peptide libraries (pepmixes) spanning the all 4 Type 2 latency antigens (EBNA1, LMP1, LMP2 and BARF1). We also added IL4 and IL7, cytokines that increased the repertoire and expansion of EBVSTs as previously demonstrated by Gerdemann et al. (Mol Ther 2012) These changes reduced the manufacturing time to 3-4 weeks and increased the number of eligible patients, since patients with a short life expectancy were previously excluded and LCLs could not be generated from patients who had received the B-cell depleting antibody rituximab.However the majority of the first 14 EBVSTs generated from patients demonstrated low antigen-specificity. We hypothesized this poor response resulted from T-cell anergy and therefore replaced IL-4 with IL-15, which was reported to reverse tumor-specific anergy (Teague et al Nat Med 2006). This change increased the proliferative capacity and antigen specificity of patient EBVSTs by up to 10-fold, indicating a "rescue effect" of IL-15 on tolerant or anergized T-cells, and correlated with increased in vitro cytotoxicity against EBV antigen-positive target cells from 5% to 66%(n=5).In a Phase 1 clinical trial, we have infused 2 doses of EBVSTs generated with IL7 and IL15 into 24 patients with multiply-relapsed, EBV-positive Hodgkin or non-Hodgkin lymphoma, either as adjuvant therapy (after stem cell transplantation or chemotherapy (n=17) or as treatment for active disease (n=7). There were no immediate or delayed adverse events attributed to the cell infusion. Of 17 patients who received EBVSTs as adjuvant therapy, 15 remain in remission with follow up of 2 to 28 months while 2 subsequently progressed. Of 7 patients with active disease who received EBVSTs, two patients had complete responses (CRs) (sustained for 24+ and 18+ months so far), two had partial responses (PRs), one had stable disease and two patients progressed. The antitumor activity we observed was associated with increases not only in circulating EBVSTs in 14 of 22 patients followed for 3 months or more, but also in T-cells specific for one or more of the non-viral tumor associated antigens, MAGE-A4, SSX2, survivin, NY-ESO1 or PRAME. This antigen spreading was seen in 13 of the 14 patients with increased EBVSTs. In an effort to enhance T-cell mediated antitumor effects, we plan to incorporate PD-1 inhibition into the therapy for patients who have had a PR to this therapy, and evaluate lymphodepletion.This cell therapy product is also being tested in a multicenter Phase II trial (CITADEL, Cell Medica, NCT01948180), being run in the European Union, South Korea and the United States, in which patients with EBV positive NK-T lymphoma receive autologous EBVSTs grown with IL7 and IL15. Albeit early in the study, preliminary indications of activity have been observed. Of 2 evaluable patients with active disease at baseline, one had a PR at the 8 week follow up scan, based on independent radiological assessment, and of 2 patients with non-measurable disease at baseline, one remains in remission at 9+ month follow up. In addition, increases in circulating EBVSTs have been observed in 6 of 7 patients (minimum 2 week follow up). DisclosuresBollard:Cell Medica: Patents & Royalties. Inman:Cell Medica: Employment. Hodgkin:Cell Medica: Employment. Gunter:Cell Medica: Employment. Brenner:Viracyte: Equity Ownership; Cell Medica: Patents & Royalties. Heslop:Cell Medica: Patents & Royalties; Viracyte: Equity Ownership. Rooney:Viracyte: Equity Ownership; Cell Medica: Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties.

Safety and Preliminary Efficacy of "Ready to Administer" Cytomegalovirus (CMV)-Specific T Cells for the Treatment of Patients with Refractory CMV Infection

Despite advances in antiviral drugs, Cytomegalovirus (CMV) infections remain a significant cause of morbidity and mortality in immunocompromised individuals. We have recently demonstrated in hematopoietic stem cell transplant (HSCT) recipients that adoptively-transferred virus-specific T cells, generated from healthy 3rd party donors and administered as an "ready to administer" product, can be curative, even in patients with drug-refractory CMV infections. However, broader implementation has been hindered by the postulated need for extensive panels of T cell lines representing a diverse HLA profile, as well as the complexities of large scale manufacturing for widespread clinical application. To address these potential issues, we have developed a decision tool that identified a short list of donors who provide HLA coverage for >90% of the stem cell transplant population. Furthermore, to generate banks of CMV-specific T cells from these donors, we have created a simple, robust, and linearly scalable manufacturing process. To determine whether these advances would enable the widespread application of "ready to administer" T cells, we generated CMV cell banks (Viralym-C™) from 9 healthy donors selected by our decision tool, and initiated a fixed-dose (2x107 cells/m2) Phase I clinical trial for the treatment of drug-refractory CMV infections in pediatric and adult HSCT recipients.To generate the Viralym-C™ banks, we stimulated donor peripheral blood mononuclear cells (PBMCs) with overlapping peptide libraries spanning the immunodominant CMV antigens pp65 and IE1. Cells were subsequently expanded in a G-Rex device, resulting in a mean fold expansion of 103±12. The lines were polyclonal, comprising both CD4+ (21.3±6.7%) and CD8+ (74.8±6.9%) T cells, and expressed central CD45RO+/CD62L+ (58.5±4.2%) and effector memory markers CD45RO+/CD62L- (35.3±12.2%). Furthermore, the lines generated were specific for the target antigens (IE1: 419±100; pp65 1070±31 SFC/2x105, n=9).To date, we have screened 12 patients for study participation, and from our bank of just 9 lines we have successfully identified a suitable line for all patients within 24 hours. Of these, 6 patients have been infused; 5 received a single infusion and 1 patient required 2 infusions for sustained benefit. There were no immediate infusion-related toxicities; and despite the HLA disparity between the Viralym-C lines and the patients infused, there were no cases of de novo or recurrent graft versus host disease (GvHD). One patient developed a transient fever a few hours post-infusion, which spontaneously resolved. Based on viral load, measured by quantitative PCR, or symptom resolution (in patients with disease), Viralym-C™ cells controlled active infections in all 5 evaluable patients; 4 patients had complete responses, and 1 patient had a partial response within 4 weeks of cell infusion. One patient with CMV retinitis had complete resolution of symptoms following Viralym-C™ infusion.In conclusion, our results demonstrate the feasibility, preliminary safety and efficacy of "ready to administer" Viralym-C™ cells that have been generated from a small panel of healthy, eligible CMV seropositive donors identified by our decision support tool. These data suggest that cost-effective, broadly applicable T cell anti-viral therapy may be feasible for patients following HSCT and potentially other conditions. DisclosuresTzannou:ViraCyte LLC: Consultancy. Leen:ViraCyte LLC: Equity Ownership, Patents & Royalties. Kakarla:ViraCyte LLC: Employment.

Expanding the scope of WT1- and cyclin A1-specific TCR gene therapy for AML and other cancers

Abstract TCR gene therapy hastens the reproducible generation of tumor-specific T cells from patients, creating a promising avenue for treating hematological and solid tumors. Several hurdles remain to making this a broadly effective modality, including variable target antigen expression, risk of tumor antigenic escape, and current therapies limited almost exclusively to HLA-A0201+ patients. We previously characterized WT1 and cyclin A1 as favorable T cell immunotherapy targets, based on high expression in malignant versus normal cells and roles in oncogenesis, reducing likelihood of loss variants. We currently have a high-affinity HLA-A0201/WT1-specific TCR in Phase I trials for the treatment of AML and NSCLC, and are now focused on developing WT1- and cyclin A1-specific TCRs for ~7 common HLA types, aiming to cover >90% of patients. T cell lines were generated by stimulating donor PBMC with overlapping peptide libraries to identify new reactive epitopes, and the highest affinity T cell clones for each HLA/antigen combination identified by tetramer binding strength. TCRs were then cloned into lentiviral vectors for expression in CD8 T cells. We are currently in the process of selecting the best HLA-A0201/cyclin A1-specific TCR with which to proceed to clinical development. We are also continuing to optimize our epitope discovery protocol for the improved selection of TCRs for other HLA alleles. Safety and efficacy of selected TCRs are rigorously tested using bioinformatic screens, in vitro assays, and humanized MHC-I mouse models. Our goal is to create a molecular toolbox of off-the-shelf TCR reagents to facilitate rapid treatment of patients, using T cell immunotherapies targeted to their particular tumor antigenic profile and HLA type.

Definition of an 18-mer Synthetic Peptide Derived from the GB virus C E1 Protein as a New HIV-1 Entry Inhibitor

Background: A slower progression of AIDS and increased survival in GBV-C positive individuals, compared with GBV-C negative individuals has been demonstrated; while the loss of GBV-C viremia was closely associated with a rise in mortality and increased progression of AIDS. Following on from the previous reported studies that support the thesis that GBV-C E2 interferes with HIV-1 entry, in this work we try to determine the role of the GBV-C E1 protein in HIV-1 inhibition.Methods: The present work involves the construction of several overlapping peptide libraries scanning the GBV-C E1 protein and the evaluation of their anti-HIV activity.Results: Specifically, an 18-mer synthetic peptide from the GBV-C E1 protein, E1(139-156), showed similar antiviral activity against HIVs from viruses from clades A, B, C, D and AE. Competitive ELISA using specific gp41-targeting mAbs, fluorescence resonance energy transfer as well as haemolysis assays demonstrated that this E1 peptide sequence interacts with the highly conserved N-terminal region of the HIV-1 gp41 (the fusion peptide) which is essential for viral entry.Conclusions: We have defined a novel peptide lead compound and described the inhibitory role of a highly conserved fragment of the E1 protein.General Significance: The results together allow us to consider the non-pathogenic E1 GBV-C protein as an attractive source of peptides for the development of novel anti-HIV therapies.

Abstract IA22: Immunotherapy with virus-specific T cells

Abstract Epstein-Barr virus (EBV) reactivation post allogeneic hematopoietic stem cell transplantation (HSCT) can lead to the outgrowth of EBV-infected B cells and the development of post-transplant lymphoproliferative disease (EBV-PTLD). Since 1993 our group has used adoptive transfer of in vitro expanded EBV-specific T cells as a means to prevent or treat these EBV-driven lymphomas. In a series of Phase I and II clinical trials we have assessed the safety and clinical benefit associated with these transferred cells in allogeneic HSCT recipients. The T cell products infused were generated using 3 different manufacturing methodologies - [(i) EBV-transformed lymphoblastoid cell lines (EBV-LCLs) (~12 weeks manufacturing time), (ii) plasmid-nucloefected dendritic cells (DCs) (17 day manufacturing) and (iii) direct stimulation of PBMCs using overlapping peptide libraries (10 day manufacturing)] and were administered to either prevent (n=162) or treat (n=47) EBV reactivation/disease in a total of 209 allogeneic HSCT recipients ranging in age from 6 months to 63 years. Of 162 patients infused prophylactically only 1 (0.6%) developed EBV-PTLD, which occurred following the administration of steroids 3 weeks-post VST infusion. However, this patient responded to a second VST infusion after the steroids. 31 of 36 (86%) patients with elevated viral load (n=21) or biopsy-proven/probable LPD (n=15) achieved durable complete remissions and the infused cells persisted long term as demonstrated in 26 patients who received gene-marked VSTs that were detectable for up to 9 years post-infusion. To extend the approach to additional viruses we initially developed methodology for using genetically modified antigen presenting cells approach to generate VSTs from donor peripheral blood that target CMV, EBV and adenovirus and showed that adoptively transferred donor-derived VSTs can reconstitute antiviral immunity to all three viruses and effectively treat established infections. However, the time taken to prepare patient-specific products and the lack of virus-specific memory T cells in cord blood and seronegative donors restricts application. More recently we have evaluated whether T-cell lines manufactured using overlapping peptide pools and extended the specificity to include HHV6 and BK. When administered to 11 recipients of allogeneic transplants, 8 of whom had up to four active infections with the targeted viruses, these VSTs proved safe in all subjects and produced an overall 94% virological and clinical response rate that was sustained long-term. 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. We evaluated this strategy in a multicenter study through the NHLBI Specialized Centers for Cell-Based Therapy (SCCT) program in HSCT recipients who had viral reactivation or infection refractory to standard therapy. The overall cumulative incidence of first CR/PR in 50 patients based on viral load by day 42 was 74.0% (73.9% for CMV, 66.7% for EBV and 77.8% for adenovirus). In a follow up study using the peptide induced VSTs that recognize 5 viruses, based on viral load measurements by quantitative PCR a single VST infusion successfully controlled active infections in 19/21 evaluable patients. These results demonstrate the feasibility and safety of 3rd party multivirus-directed VSTs, generated by direct stimulation of PBMCs with synthetic peptides and administered as an off the shelf product. Citation Format: Helen E. Heslop, Ifigenia Tzannou, Bilal Omer, Malcolm K. Brenner, Ann M. Leen, Cliona M. Rooney. Immunotherapy with virus-specific T cells. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr IA22.

Adoptively-Transferred EBV-Specific T Cells to Prevent or Treat EBV-Related Lymphoproliferative Disease in Allogeneic HSCT Recipients - a Single Center Experience Spanning 22 Years

▪Epstein-Barr virus (EBV) reactivation post allogeneic hematopoietic stem cell transplantation (HSCT) can lead to the outgrowth of EBV-infected B cells and the development of post-transplant lymphoproliferative disease (EBV-PTLD). Since 1993 our group has used adoptive transfer of in vitro expanded EBV-specific T cells as a means to prevent or treat these EBV-driven lymphomas. In a series of Phase I and II clinical trials we have assessed the safety and clinical benefit associated with these transferred cells in allogeneic HSCT recipients. The T cell products infused were generated using 3 different manufacturing methodologies - [(i) EBV-transformed lymphoblastoid cell lines (EBV-LCLs) (~12 weeks manufacturing time), (ii) plasmid-nucloefected dendritic cells (DCs) (17 day manufacturing) and (iii) direct stimulation of PBMCs using overlapping peptide libraries (10 day manufacturing)] and were administered to either prevent (n=162) or treat (n=47) EBV reactivation/disease in a total of 209 allogeneic HSCT recipients ranging in age from 6 months to 63 years.198/209 patients were infused with donor-derived virus-specific T cells (VSTs) and administered at doses ranging from 5x106 to 1.2x108 cells/m2. Infusions were well tolerated, with acute graft versus host disease (aGvHD) grade I-II documented in just 10 patients, of which 2 cases were de novo. Chronic GvHD occurred in 13 patients and was extensive in 2. Other toxicities were seen only in patients with active disease and included localized swelling at the tumor site due to EBV-specific T cell infiltration (n=4), which was severe in 2 causing transient airway obstruction. An additional patient with bulky, Rituximab-resistant EBV-PTLD developed fever and sepsis-like signs post-infusion, coincident with a decrease in viral load, an increase in the frequency of circulating EBV-specific T cells and an elevation in plasma cytokines consistent with a cytokine release syndrome. However, the symptoms resolved within hours of administering steroids and an anti-TNF antibody and did not recur. Of 162 patients infused prophylactically only 1 (0.6%) developed EBV-PTLD, which occurred following the administration of steroids 3 weeks-post VST infusion. However, this patient responded to a second VST infusion after the steroids. 31 of 36 (86%) patients with elevated viral load (n=21) or biopsy-proven/probable LPD (n=15) achieved durable complete remissions and the infused cells persisted long term as demonstrated in 26 patients who received gene-marked VSTs that were detectable for up to 9 years post-infusion.Based on the safety and efficacy profile of donor-derived EBV-specific VSTs we extended our approach to provide an "off the shelf" product to third party recipients, generating and banking T cell lines from 88 consenting normal donors. To date we have treated 11 patients, all of whom had drug-refractory EBV infections. Two patients were treated for elevated viral loads while 9 had established PTLD. Patients received 1-5 doses (fixed dose - 2x107 cells/m2) with 3rd party VSTs matching at one to three of six HLA alleles. Despite the HLA disparity the cells have proven safe with a single case of grade I GvHD reported. Of the 11 patients infused, 8 responded to therapy with 5 complete and 3 partial responses for an overall response rate of 73%.Overall our experience with adoptively-transferred EBV-specific T cells over a 22-year period demonstrates the safety and efficacy of this approach for the prevention and treatment of post-transplant EBV disease. DisclosuresOff Label Use: Adoptively transfered T cells administered under an IND. Rooney:Celgene: Other: Collaborative research agreement; Cell Medica: Other: Licensing Agreement. Heslop:Cell Medica: Other: Licensing Agreement; Celgene: Other: Collaborative research agreement.

Safety, Maximum Tolerated Dose and Immunogenicity of CMV-MVA-Triplex in Healthy Volunteers with or without Prior Immunity to CMV and Vaccinia

In the era of preemptive antiviral therapy, cytomegalovirus (CMV) still remains the cause of major health complications, profound defects in immune reconstitution, and significant morbidity in post-transplant recovery of immune-compromised HCT recipients. Substituting antivirals with a vaccine that harnesses the native immune response to CMV may improve outcomes for HCT recipients.Modified Vaccinia Ankara (MVA), a vaccinia virus being investigated as a smallpox vaccine by the Defense Department, is a safe and robust delivery system to treat or prevent a wide range of diseases including HIV and cancer. Replication-defective MVA is safe, well tolerated and strongly immunogenic when given to HCT recipients or AIDS patients. We developed a multiple-antigen recombinant MVA with genes encoding 3 immunodominant CMV proteins: UL83 (pp65), UL123 (IE1-exon4), and UL122 (IE2-exon5) (CMV-MVA-Triplex), and established its pre-clinical safety and immunogenicity using humanized HLA transgenic mouse models and human PBMC from CMV-seropositive healthy volunteers and HCT recipients.Defining safety, persistence of the virus, maximum tolerated dose and immunogenicity of CMV-MVA-Triplex in healthy volunteers is a critical first step in its clinical development for HCT recipients as required by the FDA. In a Phase I trial (NCT01941056), these endpoints were evaluated in 24 healthy volunteers (age: 18-60), with or without prior immunity to CMV and vaccinia. Three escalating dose levels (DL) were administered intramuscularly (DL1=10xE7; DL2=5x10E7; DL3=5x10E8 pfu/dose) in 8 subjects/DL, with a booster injection 28 days later, and follow up for 1 year.As of July 2015, all 24 planned volunteers were enrolled, vaccinated and followed for at least 4 months. Vaccinations at all DL were well-tolerated, with only a few expected injection reactions and no SAE or dose limiting toxicities. Immunogenicity of the vaccine was evaluated by measuring the levels of the CD137 T-cell surface marker representing functional activation of PBL harvested from vaccinees and stimulated 24 hours with full-length pp65, IE-1 and IE2 overlapping peptide libraries, or direct measurement of CMV-specific T-cells using HLA multimers. CMV-MVA-Triplex induced robust expansion of pp65-, IE1- and IE2-specific CD8 and CD4 T-cells in vaccinated CMV-seropositives, at each DL (Cf. plot showing geometric mean with upper lower/limits of the pp65 T-cell levels for DL2 cohort). HLA multimers identified CMV-specific T-cells whose expansion closely followed CD137-activated CMV-specific T-cells in vaccinees with common HLA alleles which have a corresponding known CMV-CTL epitope (data to be presented).A statistical analysis performed using generalized estimated equations indicated that the post-vaccination levels of pp65-, IE1- or IE2-specific CD8 and CD4 T-cells were significantly increased, with p-values ranging from 3x10-5 to 0.025. For example, the pre-/post-vaccination median pp65-reactive CD4+ CD137+ T-cells rose from 1.3 to 4.4 cells/µL (p=3x10-5); and pp65-reactive CD8+ CD137+ T-cells rose from a median of 0.22 to 3.1 cells/µL (p=0.003). Importantly, robust immunity was detected in CMV-seronegatives (as shown in the plot for UPN 14 and 18), as well as in subjects who had received smallpox vaccinations. Elevated frequencies of CMV-specific CD4 and CD8 T cells for all 3 antigens plateaued after day 56, but in most cases remained elevated up to one year post-vaccination (data to be presented). Circulating MVA vector in blood was assessed by real-time PCR post-injection and showed only minimal residual vector DNA [10-30 gc/mL] in just 2 vaccinees in the DL3 cohort that disappeared within 3 months. These results provide evidence that CMV-seropositive HCT recipients, whether they receive stem cell product from CMV-seropositive or -seronegative donors could respond to CMV-MVA-Triplex by generating protective CMV-specific immunity.CMV-MVA-Triplex is the first vaccine against CMV that uses a recombinant MVA incorporating multiple CMV antigens, developed for HCT recipients, who are at risk for CMV reactivation. The safety and marked immunogenicity of CMV-MVA-Triplex in this Phase I trial, warrant testing of the vaccine in the HCT setting. A Phase 2 multicenter, placebo-controlled trial to assess CMV-MVA-Triplex in CMV seropositive recipients, receiving HCT from matched related or unrelated donors will start in Fall 2015. [Display omitted] [Display omitted] DisclosuresDiamond:Helocyte, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Administration of Most Closely HLA-Matched Multivirus-Specific T Cells for the Treatment of EBV, CMV, AdV, HHV6, and BKV Post Allogeneic Hematopoietic Stem Cell Transplant

▪Viral infections remain a significant cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). Adoptive immunotherapy with donor-derived virus-specific T cells (VSTs) has proven safe and effective for the prophylaxis and treatment of EBV, CMV, AdV, BKV and HHV-6 infections post-HSCT. However, broader application is restricted by the time taken to prepare patient-specific products and the lack of virus-specific T cell precursors in cord blood and seronegative donors. Thus, to assess whether 3rd party multivirus-directed VSTs could produce clinical benefit when administered as an "off the shelf" product to allogeneic HSCT recipients with refractory BKV, EBV, CMV, AdV and/or HHV-6 infections we initiated a Phase II clinical trial and now report interim results for 22 patients infused to date. We prepared a bank of 58 VST lines from individuals with common HLA polymorphisms by exposing donor PBMCs (3x107) to overlapping peptide libraries spanning AdV (Hexon, Penton), EBV (LMP2, EBNA1, BZLF1), CMV (pp65, IE1), BKV (Large T, VP1) and HHV-6 (U11, U14, U90) antigens followed by a 9-11 day expansion phase in a G-Rex device in the presence of IL4 and IL7. This produced a mean of 4.2±1x108 VSTs that were polyclonal, comprising both CD4+ (61±2.4%) and CD8+ (34±2.1%) cells that expressed central (CD45RO+/CD62L+/CCR7+ - 43±3.9%) and effector memory markers (CD45RO+/CD62L- - 10±1%). 56/58 lines had activity against AdV, 50/58 against EBV, 35/58 against BKV, 34/58 against HHV-6, and 33/58 against CMV.To date, 62 HSCT recipients have been screened for study participation and a potential line, based on target virus specificity through a shared allele and overall HLA match, was identified for 58. Of these, 22 patients with infections that were unresponsive to at least 2 lines of antiviral treatment have been infused (fixed dose level - 2x107 VSTs/m2) with VST lines matched at 1 to 6 HLA antigens. Seventeen received just 1 infusion, while 5 patients required 2 or more infusions for sustained benefit. Eight patients received VSTs for CMV infections, including 3 cases of CMV colitis, 8 for BKV (6 for cystitis, 2 for nephritis), 1 for HHV-6, 1 for EBV-PTLD, 1 for AdV, 1 for BKV and EBV, 1 for CMV and AdV and 1 for CMV and BKV infections. There were no immediate adverse effects related to infusion. Based on viral load measurements by quantitative PCR a single VST infusion successfully controlled active infections in 19/21 evaluable patients: CMV (4 CR, 5 PR, 1NR); EBV (2 CR); AdV (1 CR, 1 NR); BKV (1 CR, 7 PR); and HHV-6 (1 PR). Of note, all 6 patients with BK hemorrhagic cystitis and 2/3 patients with CMV colitis had marked improvement/resolution of symptoms following VST treatment. In 8 subjects who responded to VST therapy we saw an increase in virus-specific T cells post-infusion. These expanded cells were confirmed to be of 3rd party VST origin in 3 patients and persisted for up to 6 weeks post-infusion. Finally, despite the HLA disparity of VSTs and recipients, de novo GvHD occurred in only one subject, who developed Grade I skin GVHD 1 week post-infusion, which resolved with the administration of topical steroids. One additional patient had a flare of chronic skin GVHD coincident with tapering of immunosuppression and 1 patient developed a transient fever 5 hours post-infusion, which spontaneously resolved. These results demonstrate the feasibility and safety of 3rd party multivirus-directed VSTs, generated by direct stimulation of PBMCs with synthetic peptides and administered as an "off the shelf" product. The infused cells were capable of in vivo expansion in allogeneic HSCT recipients and proved clinically effective against refractory EBV, CMV and AdV infections and also controlled HHV-6 reactivation and BKV-associated hemorrhagic cystitis. DisclosuresOff Label Use: Adoptively transferred T cells administered under an IND. Brenner:Bluebird Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Other: Collaborative Research Agreement; Cell Medica: Other: Licensing Agreement. Rooney:Celgene: Other: Collaborative research agreement; Cell Medica: Other: Licensing Agreement. Heslop:Celgene: Other: Collaborative research agreement; Cell Medica: Other: Licensing Agreement.