ALBERT

Description: Adoptive immunotherapy with transplant donor derived virus specific T cells is an effective strategy for the treatment of CMV viremia and disease arising after an allogeneic hematopoietic stem cell (HSCT). This approach is not readily applicable if the donor is seronegative or not available to provide lymphocytes for in vitro expansion for CMV specific cytotoxic T lymphocytes (CMV-CTL) lines or if the CMV CTL lines derived from non-identical donors are restricted by non-shared HLA alleles. We and others have previously presented results indicating that treatment with in vitro expanded CMV-CTLs derived from an HLA partially matched third party donors can effectively treat CMV infections in the post-transplant setting. However, the patient population most likely to benefit from this approach has not been well defined. Patients at especially high risk of succumbing to CMV infection include those who acquire resistance to antiviral therapy. We now present results of treatment with banked off-the-shelf third party CMVpp65 specific CTLs in patients with genetically defined mutations in the UL54 DNA polymerase and UL97 kinase CMV genes predicting resistance to antiviral agents. Fifteen recipients of HSCT with mutations defined prior to the start of cell therapy were treated. All 15 had mutations associated with resistance to ganciclovir. Overall 5 had resistance to ganciclovir, foscarnet and cidofovir, 5 had resistance to ganciclovir and foscarnet, one had resistance to ganciclovir and cidofovir and 4 had resistance to ganciclovir alone. Third party CMV-CTLs were selected on the basis of HLA matching at high resolution at a minimum of 2/8 recipient alleles and HLA restriction of the T cells by one or more HLA alleles present in the patient. CMV-CTLS were selected from a bank of 132 lines generated under GMP conditions from normal HSCT donors specifically consented for use of their T cells in patients other than the designated transplant recipient. These 15 patients had a median age of 60.5 (7.4-70.1) years and started therapy with CMV-CTLs a median of 157 (70-564) days after reactivation of CMV. Four of these patients had CMV disease while 11 were treated for viremia alone. Prior therapy in this cohort included foscarnet (N=15) ganciclovir and/or valganciclovir (N=14) and cidofovir and/or brincidofovir (N=9). Each cycle of CMV-CTLs consisted of 3 weekly infusions of 1x106 T-cells/kg/infusion. These 15 patients received a median of 2 (1-3) cycles of CMV-CTLs. CMV-CTLs were well tolerated and there were limited toxicities. Six patients experienced AEs of which one grade 3 and one grade 4 AE were deemed possibly related to infusions with CMV-CTLs. Overall 11/15 (73.3%) patients responded to CMV-CTL therapy including 2 patients with disease with 6 CRs and 5 PRs. Overall survival at 6 months in the 11 responders and 4 non-responders was 72.7% and 25.0% respectively. Within the 6 month follow-up one of the 11 responding patients died of CMV while 3 of the 4 non-responding patients died of CMV. These results indicate that third party donor derived "off-the-shelf" CMV-CTLs can effectively treat CMV viremia or disease in patients not responding to antiviral therapy with demonstrated genetic resistance to antiviral agents. Figure 1. Figure 1. Disclosures Doubrovina: Atara Biotherapeutics: Consultancy, Research Funding. Hasan:Atara Biotherapeutics: Consultancy, Research Funding. Koehne:Atara Biotherapeutics: Consultancy.

Description: Despite advances in T-cell immunotherapy against Epstein-Barr virus (EBV)-infected lymphomas that express the full EBV latency III program, a critical barrier has been that most EBV+ lymphomas express the latency I program, in which the single Epstein-Barr nuclear antigen (EBNA1) is produced. EBNA1 is poorly immunogenic, enabling tumors to evade immune responses. Using a high-throughput screen, we identified decitabine as a potent inducer of immunogenic EBV antigens, including LMP1, EBNA2, and EBNA3C. Induction occurs at low doses and persists after removal of decitabine. Decitabine treatment of latency I EBV+ Burkitt lymphoma (BL) sensitized cells to lysis by EBV-specific cytotoxic T cells (EBV-CTLs). In latency I BL xenografts, decitabine followed by EBV-CTLs results in T-cell homing to tumors and inhibition of tumor growth. Collectively, these results identify key epigenetic factors required for latency restriction and highlight a novel therapeutic approach to sensitize EBV+ lymphomas to immunotherapy.

Description: Abstract 487 Adoptive transfer of donor-derived EBV-specific T-cells can induce complete and durable regressions of EBV lymphomas complicating allogeneic hematopoietic progenitor cell transplants (HSCT). However, this treatment has not been broadly applicable due to 1) lack of immediate access to such T-cells and 2) lack of adequate donor cells to generate the virus-specific T-cells required, either because of donor unavailability (e.g. cord blood or some unrelated donors) or seronegativity. To address this we have developed a bank of EBV-specific cells of defined HLA restriction, and are now evaluating the potential of these HLA-partially matched, third party EBV-specific T-cells. We here report results for the first 13 patients treated by this approach, including pts who developed clonal EBV lymphomas following HLA matched (n=2) or disparate (n=1) T-cell depleted HSCT (N=3) or HLA-disparate cord blood transplants (N=3); pts with chemotherapy refractory EBV PTLD (n=2) or EBV+ leiomyosarcomas (LS) (n=3) complicating organ transplants (N=5) and pts with EBV+ CNS lymphomas complicating AIDS or prolonged immunosuppression (N=2). These patients received infusions of third party EBV-CTLs after the failure of a median of 2 prior therapies (0 – 4) including rituximab in all but one case. Three patients had also failed prior infusions with autologous (1) or HSCT donor (2) EBV-CTLs. T-cells from consenting EBV seropositive HLA typed normal donors were sensitized in vitro for at least 28 days with irradiated autologous EBV BLCL transformed with the EBV strain B95.8, and were then tested for sterility virus-specificity and lack of alloreactivity and stored cryopreserved prior to use. The donors were selected based on matching for at least 2 HLA alleles and in most instances, known restriction of the EBV-specific T-cells for epitopes presented by HLA alleles shared by the EBV+ malignancy. Up to 4 courses of T-cells were administered in 3 weekly I.V. infusions of 1 × 106 T cells/kg, each course separated by a 3 week observation period. T cell infusions were well tolerated. One patient developed minor skin GvHD. No other patient developed GVHD or organ allograft rejection. Results for the 13 pts are summarized in Table 1. Those pts achieving CR, PR or SD had rapid increments in the frequencies of EBV CTLP detected that persisted 14–21 days after each course. Of the 4 pts with PD, none exhibited increases in the frequency of EBV CTLPs in the blood post T-cell infusion. No increase in EBV CTLPs was observed in a patient who progressed after treatment with EBV-specific T-cells from his haplotype disparate HSCT donor. In this case, the donor's EBV-specific T-cells were restricted by an HLA allele not shared by the host-type lymphoma. This patient was then treated with EBV-specific T-cells from a third party donor restricted by an HLA allele shared by the host-type lymphoma. These T-cells expanded post infusion, resulting in a durable CR. Overall, 9 of 13 survive either in CR or sustained PR or SD 〉6 to 〉 60 months post treatment. These results provide evidence that third party EBV-specific T-cells, that are partially HLA-matched, and appropriately HLA restricted can induce regressions of EBV-associated lymphomas, PTLD and LS complicating transplantation or prolonged immunodeficiency. A bank of such T-cells can provide an immediately accessible source of T-cells for adoptive therapy for a large proportion of patients developing these life threatening disorders. N CR PR SD PD Survival EBV+ Lymphoma Post HSCT or CBT 6 4 0 0 2 4 in CR EBV+ PTLD Post Organ Transplant 2 0 1 0 1 1 in PR EBV+ CNS Lymphoma in Immunodeficiency 2 1 0 0 1 1 in CR EBV+ Leiomyosarcoma Post Transplant 3 0 1 2 0 3 in PR or SD 13 5 3 2 4 9 Disclosures: No relevant conflicts of interest to declare.

Description: NF-kB plays important roles in immunity and oncogenesis, indicating that therapeutic targeting of this pathway could be beneficial in various clinical settings; however,an NF-kB-specific inhibitor does not exist in clinical practice to date. One approach toward development of such a compound is small-molecule-mediated direct inhibition of one or several members of the NF-kB family of transcription factors, a network that comprises five structurally related proteins including p50, p52, RelA, RelB and c-Rel. After screening of a library of 15,000 small molecules with a biochemical assay, we identified two scaffolds with inhibitory activity specific for the NF-kB subunit c-Rel. These scaffolds act as direct c-Rel inhibitors by modifying the conformation of the c-Rel protein, thus preventing DNA binding. We previously reported that in vitro treatment of T cells with the thiohydantoin IT-603 induces c-Rel deficiency, resulting in suppression of T cell alloactivation without compromising T cell activation triggered by recognition of tumor-associated or viral antigens (Shono et al., Cancer Discovery, 2014). Here, we for the first time demonstrate in vivo efficacy of a c-Rel inhibitor treatment regimen in mouse models of graft-versus-host disease (GVHD) and graft-versus-lymphoma (GVL), as well as xenograft models of human B cell lymphomas, revealing that inhibition of c-Rel activity allows not only for suppression of GVHD while retaining GVL activity, but it also mediates promising anti-lymphoma effects. We first show that the novel small molecule IT-901 is a more potent c-Rel inhibitor than IT-603 and has a superior pharmacokinetic profile. IT-901 displayed significantly improved in vivo efficacy, ameliorating GVHD while preserving the anti-lymphoma activity of T cells (Figure 1a,b). Recent genetic evidence has established a pathogenetic role for NF-kB signaling in lymphoid malignancies. We therefore sought to explore the potential of IT-901 for targeted therapy of human lymphomas. We analyzed six representative diffuse large B cell lymphoma (DLBCL) cell lines including activated B-like (ABC; HBL1, TMD8, U2932) and germinal center B-like (GCB; Ly19, SU-DHL4, SU-DHL8) cell lines for nuclear translocation of c-Rel and found that c-Rel was constitutively active in all cell lines. To examine if c-Rel inhibition with IT-901 alters cytokine production by DLBCL cells, we analyzed cytokine levels in the supernatant after in vitro incubation with IT-901. IT-901 treatment resulted in decreased levels of a wide range of cytokines in TMD8 cells, with the notable exceptions of interleukin 8 (IL-8), tumor necrosis factor (TNF)-α, and TNF-β (P

Description: Adoptive immunotherapy with transplant donor-derived virus specific T cells is an effective strategy for the treatment of CMV viremia and disease arising after an allogeneic hematopoietic stem cell (HSCT). At our center this approach has become a standard part of the armamentarium of CMV directed therapy. However, donor-derived CMVpp65-specific cytotoxic T cells (CMV-CTLs) are not available for patients transplanted from a seronegative or cord blood donor. In addition, in the HLA disparate transplant setting the CMV-CTL line derived from non-identical donors may be restricted by non-shared HLA alleles. For these patients as we have previously reported, treatment with in vitro expanded CMV-CTLs derived from an HLA partially matched third party donors is an option. One particularly difficult clinical situation is the treatment of patients who develop CMV disease involving the sanctuary sites of the central nervous system and retina. We have treated 12 patients with primary donor derived (n=1) or third party (n=11) CMV CTLs for retinitis (7) or meningitis/encephalitis (4) or both (1). Recipients of hematopoietic stem cell transplant (HSCT) had undergone T cell depleted (5) conventional (1) or cord blood (4) transplantation. One patient was treated after solid organ transplantation and one for HIV related CMV retinitis. Third party CMV CTLS were selected from a bank of 132 lines generated under GMP conditions from normal HSCT donors specifically consented for use of their T cells in patients other than their designated transplant recipient. Third party CMV-CTLs were selected on the basis of HLA matching at a minimum of 2/8 recipient alleles and HLA restriction of the T cells by one or more HLA alleles present in the patient. A total of 10 distinct third party CMV-CTL lines and one (1) donor derived line were used. Patients received infusions of CMV-CTLs after failing a median of 146 (43-419) days of prior therapy with a median of 4 anti-viral agents. Patients received 3 weekly infusions of CMV-CTLs at doses of 1 x 10e6 T cells/kg (n=10) 2 x 10e6 T cells/kg (n=1) and 0.5 x 10e6 T-cells/kg (n=1) and were eligible to receive additional cycles of cells if they had no toxicity five weeks after the start of cellular therapy. One patient was evaluated only for toxicity as efficacy was confounded by anti-viral therapy required for treatment of varicella zoster. Of the 11 evaluable patients, 7 achieved CR, 1 PR (clinical improvement in disease and a 3 log decrease in viral load) and 1 SD. All but one of these responses were durable. One patient who had achieved a CR had recurrence of low grade viremia which was not retreated due to his overall medical deterioration. The two patients with progression of disease ultimately died of CMV. Patients were monitored for expansion of CMV-CTLs by CMV-specific interferon gamma and where appropriate tetramer analysis. Responding patients consistently had detectable expansion of CMV-specific CTL populations. In addition, in one patient, undergoing serial lumbar punctures, we were able to detect third party CMV-CTLs in the CSF. There were limited toxicities associated with CMV-CTL infusions. No patient developed de novo GvHD or a flare of prior GvHD. This study demonstrates a high response rate among patients with otherwise refractory CMV chorioretinitis or meningoencephalitis following adoptive therapy with primary donor or third party CMV-CTLs; thus demonstrating the capacity of adoptively transferred CMV-CTLs to treat disease in these sanctuary sites without toxicity. When selected based on restriction through shared alleles, third party CMV-CTLs are effective despite significant HLA disparity. The bank of CMV specific T cells can provide an immediate source of HLA partially matched appropriately restricted T cells for adoptive immunotherapy to treat CMV disease affecting the CNS. This enables treatment early in the course of disease with CMV-CTL lines previously prepared and characterized in terms of HLA restriction. This approach is anticipated to maximize the response rate as well as minimize toxicity from anti-viral therapy. Disclosures Prockop: Atara Biotherapeutics: Other: I have no financial disclosures, but Atara Biotherapeutics has exercised a licensing agreement with Memorial Sloan Kettering Cancer Center and MSKCC and some investigators at MSKCC have a financial interest in Atara.. Hasan:Atara Biotherapeutics: Research Funding. O'Reilly:Atara Biotherapeutics: Research Funding.

Description: We evaluated HLA-compatible donor leukocyte infusions (DLIs) and HLA-compatible or HLA-disparate EBV-specific T cells (EBV-CTLs) in 49 hematopoietic cell transplantation recipients with biopsy-proven EBV-lymphoproliferative disease (EBV-LPD). DLIs and EBV-CTLs each induced durable complete or partial remissions in 73% and 68% of treated patients including 74% and 72% of patients surviving ≥ 8 days after infusion, respectively. Reversible acute GVHD occurred in recipients of DLIs (17%) but not EBV-CTLs. The probability of complete response was significantly lower among patients with multiorgan involvement. In responders, DLIs and EBV-CTLs regularly induced exponential increases in EBV-specific CTL precursor (EBV-CTLp) frequencies within 7-14 days, with subsequent clearance of EBV viremia and resolution of disease. In nonresponders, EBV-CTLps did not increase and EBV viremia persisted. Treatment failures were correlated with impaired T-cell recognition of tumor targets. Either donor-derived EBV-CTLs that had been sensitized with autologous BLCLs transformed by EBV strain B95.8 could not lyse spontaneous donor-derived EBV-transformed BLCLs expanded from the patient's blood or biopsied tumor or they failed to lyse their targets because they were selectively restricted by HLA alleles not shared by the EBV-LPD. Therefore, either unselected DLIs or EBV-specific CTLs can eradicate both untreated and Rituxan-resistant lymphomatous EBV-LPD, with failures ascribable to impaired T-cell recognition of tumor-associated viral antigens or their presenting HLA alleles.

Description: Abstract 351 We have generated donor-derived T-cell lines specific for CMVpp65 peptides for use in a phase I, dose escalation trial of adoptive immunotherapy. T-cells were sensitized by autologous monocyte-derived DCs loaded with a pool of 138 pentadecapeptides (15-mers), with 11 amino acid overlaps spanning the entire 561 amino acid sequence of the CMV protein pp65. The 138 pentadecapeptides were synthesized and the T-cells were sensitized under GMP conditions. In preclinical studies we have been able to generate CMVpp65 specific T-cell lines from each seropositive donor tested, irrespective of HLA genotype. During the culture period of 21–35 days, populations of T-cells specific for CMV-pp65 selectively expanded 200–300 fold, while T cells reactive against major or minor alloantigens were depleted. Thirteen pts with persistent CMV viremia, refractory to at least 2 weeks of therapeutic doses of ganciclovir or foscarnet, have been enrolled: 3 pts at a T cell dose of 5×105/kg, 3 pts at 1×106 T cells/kg, and 7 pts at 2×106 T cells/kg. CMV specific cytototoxic T lymphocytes (CTLs) were generated from HLA-identical unrelated donors (3 pts) or from HLA-identical siblings (10 pts). Two pts received conventional transplants after non-myeloablative conditioning; 11 pts received myeloablative conditioning and T-cell depleted transplants. Pts were eligible if they had persistent CMV viremia despite 2 weeks' treatment with antiviral drugs or had toxicities precluding further treatment with antiviral agents. Prior to infusion, T cell specificity against CMV was confirmed by cytotoxicity, intracellular interferon gamma (IFN-g) production, and MHC-tetramer staining (if available). The HLA-restrictions, epitope specificities, and TCR Vβ repertoires of the T-cell lines were also characterized before infusion. Cells were also assayed to establish lack of alloreactivity, microbiological sterility, and low endotoxin levels. All CTLs demonstrated cytolytic activity against peptide-loaded autologous PHA blasts but no cytotoxicity against non-pulsed HLA-matched or peptide-pulsed HLA-mismatched target cells. The proportion of CMVpp65-specific CD8+ cells in the infused T-cells, measured by intracellular IFN-g or MHC tetramers, ranged from 2 – 20 % or 4 – 70%, respectively. Post infusion, an increase in the absolute lymphocyte count correlated with an increase in CMV-specific T-cell frequencies to levels as high as 14% of CD8+ cells. In one pt, the CTLs were monitored and persisted for more than 2 years (10% of CD8+ cells) after the infusion. Notably, the same pp65-derived epitopes and HLA-restrictions which characterized the infused CTLs were detected in the pt specimens post infusion. The same TCR Vβ repertoires of the CMVpp65-specific CTLs infused were also detected post infusion. Donors for three of the treated pts expressed HLA-A*0201 and HLA-B*0701 alleles. Epitope-specific T cells for the HLA-A*0201-restricted NLVPMVATV peptide and the B*0701-restricted RPHERNGFTV peptide were detected and monitored in pre and post infusion T-cell populations in these three pts. In all three pts, the B*0701 restricted RPHERNGFTV emerged as the dominant T-cell population. All 13 pts tolerated the infusions well without acute toxicities. None developed symptoms of GvHD at the dose levels tested. Twelve of the 13 pts cleared CMV viremia by 2–8 weeks following the CTL infusions. One of the pts died six weeks after the CTL infusion of respiratory failure despite clearing CMV from blood and bronchial aspirates. Another pt who initially remained viremic following the CTL infusion was restarted on oral valganciclovir and subsequently cleared CMV viremia. Only one pt had persistent viremia and died of pneumonia 31 days after CTL infusion. The results from this trial demonstrate that donor T cells, sensitized with this pool of overlapping CMV pp65 pentadecapeptides, are safe and clear CMV viremia resistant to standard therapy. A larger phase II trial for the treatment of persistent CMV viremia and CMV infections is currently ongoing at MSKCC. Disclosures: No relevant conflicts of interest to declare.