Search

NF-κB plays a variety of roles in oncogenesis and immunity that may be beneficial for therapeutic targeting, but strategies to selectively inhibit NF-κB to exert antitumor activity have been elusive. Here, we describe IT-901, a bioactive naphthalenethiobarbiturate derivative that potently inhibits the NF-κB subunit c-Rel. IT-901 suppressed graft-versus-host disease while preserving graft-versus-lymphoma activity during allogeneic transplantation. Further preclinical assessment of IT-901 for the treatment of human B-cell lymphoma revealed antitumor properties in vitro and in vivo without restriction to NF-κB–dependent lymphoma. This nondiscriminatory, antilymphoma effect was attributed to modulation of the redox homeostasis in lymphoma cells resulting in oxidative stress. Moreover, NF-κB inhibition by IT-901 resulted in reduced stimulation of the oxidative stress response gene heme oxygenase-1, and we demonstrated that NF-κB inhibition exacerbated oxidative stress induction to inhibit growth of lymphoma cells. Notably, IT-901 did not elicit increased levels of reactive oxygen species in normal leukocytes, illustrating its cancer selective properties. Taken together, our results provide mechanistic insight and preclinical proof of concept for IT-901 as a novel therapeutic agent to treat human lymphoid tumors and ameliorate graft-versus-host disease. Cancer Res; 76(2); 377–89. ©2016 AACR.

Sequential imaging of genetically marked effector cells after adoptive transfer in vivo has greatly enhanced analyses of their biodistribution, growth, and activity both in animal models and in clinical trials of cellular immunotherapy. However, the immunogenicity of cells expressing xenogeneic reporter constructs limits their survival and clinical utility. To address this limitation, we have evaluated a human norepinephrine transporter (hNET) permitting imaging of transduced cells in vivo with a previously approved clinical grade radiolabeled probe, metaiodobenzylguanidine (MIBG). The hNET gene cDNA was cloned from the SK-N-SH cell line and inserted into a bicistronic retroviral vector also encoding green fluorescent protein. Following transfection, human EBV-specific T lymphocytes seemed fully functional in vitro and also selectively accumulated [123I]MIBG. In nonobese diabetic/severe combined immunodeficient mice bearing human EBV lymphoma xenografts, as few as 104 transduced T cells injected into the tumors could be imaged by single-photon emission computed tomography (SPECT) or positron emission tomography (PET) after i.v. infusion of [123I]MIBG or [124I]MIBG, respectively. When hNET+ EBV-specific T cells were infused i.v., their migration and specific accumulation in EBV+ tumors expressing their restricting HLA allele could be imaged by SPECT or PET over 28 days. Image intensity was closely correlated with the number of T cells accumulated in targeted tumors. The use of two reporter probes (MIBG and 2′-deoxy-2′-fluoro-β-d-arabinofuranosyl-5-iodouracil) permitted independent contemporaneous tracking of two distinct EBV-specific T-cell subpopulations expressing different reporter genes (hNET-CD4+ T cells and HSV-TK-CD8+ T cells) in the same animal using three-dimensional nuclear modalities (SPECT and PET). The hNET-based system described may thus have significant potential as a nonimmunogenic reporter for extended repeated quantitative in vivo imaging of transduced cells in man. [Cancer Res 2007;67(24):11959–69]

Adoptive transfer of transplant donor or third party donor derived CMV-specific T cells (CMV-CTL) can effectively treat CMV infections in HSCT recipients. In clinical trials, infusion of partially matched third party CMV-CTLs, has demonstrated high response rates against persistent CMV infection. T-cells (TC) generated in vitro or directly selected in vivo demonstrate a striking preponderance of specificity for 1-2 immunodominant (ID) epitopes presented by specific HLA alleles. ID epitopes elicit higher TC functional activity in vivo, compared to sub-dominant (SD) epitopes. The relative clinical efficacy of TC directed against ID versus SD epitopes in vivo remains undefined. Agents augmenting activity of TC responsive to SD epitopes are unexplored. When these alleles are co-inherited in humans, epitopes of CMVpp65 presented by HLA A*02:01 are ID over HLA A*24:02 presented epitopes. We describe an in vivo model to assess efficacy of CMV-CTLs using colon carcinoma cells (coca)transduced to express CMVpp65, as a surrogate system. HLA A*02:01 + and A*24:02 + human coca cells were transduced to express CMVpp65 and GFP-firefly luciferase (cocapp65). CMV-CTLs responding to either the A*0201 presented ID NLV epitope (A2-NLV) or the A*24:02 presented SD QYD epitope (A24-QYD) were generated from donors co-inheriting HLA A*02:01 and A*24:02 by in vitro stimulation using NIH 3T3 artificial antigen presenting cells, expressing HLA A*02:01 or A*24:02, B7.1, LFA-3, and ICAM1. Tumor cells (105 cells) were injected subcutaneously into groups of 5-6 NSG mice on the R flank, and 105 cells from a pp65 expressing melanoma cell line (melpp65), lacking expression of HLA A*02:01 or A*24:02 were injected on the L shoulder as control. 2 Groups each received 106 of tetramer+ A2-NLV or A24-QYD CMV-CTLs i.v per mouse; one of each CMV-CTL treated group also received 2 i.v doses ( 200µg /dose) of anti-PD1 antibody (Nivolumab-BMS) at day 2 and 7 post CTL infusion. Control groups received IL-2, with or without anti-PD1, or HLA mismatched CMV-CTLs. Tumor growth was monitored by bioluminescent imaging. CMV-CTLs responsive to SD A24-QYD epitope induced significant cocapp65 growth suppression compared to controls, but did not eradicate tumors in any animal. Combined treatment of A24-QYD CMV-CTLs with anti-PD-1 Ab induced complete cocapp65 eradication in 2 of 5 mice, with minute residual tumors in 3 mice. Treatment with ID A2-NLV CMV-CTLs induced complete cocapp65 eradication in 2 of 5 mice, and smaller residual tumors compared to SD A24-QYD CTL treatment. Combined treatment with anti-PD-1 and A2-NLV CMV-CTLs led to complete cocapp65 eradication in 3 of 5 mice, with minute tumors in 2 mice. Taken together, these data provide evidence that blocking the PD-1/PD-L1 interaction may significantly augment the antiviral activity of both ID and SD CMV-CTLs. Citation Format: Aisha N. Hasan, Annamalai Selvakumar, Tzu-Yun Kuo, Richard J. O'Reilly. Adoptively transferred CMV-specific T-cells recognizing dominant and sub-dominant pp65 epitopes demonstrate improved in vivo inhibition of tumor xenografts in combination with PD-1 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4978. doi:10.1158/1538-7445.AM2017-4978

Background: T cells can be genetically modified to target tumor antigens through the expression of a chimeric antigen receptor (CAR). CAR structure consists of two fundamental domains – the antigen binding portion (most commonly single chain variable fragment (scFv) derived from a monoclonal antibody) joined to one or more intracellular T cell signaling domains. To be effective CAR T cells must expand, persist, exhibit enduring anti-tumor cytotoxicity, withstand and/or counteract an immunosuppressive tumor microenvironment, and overcome targeted tumor antigen escape following infusion. T cells targeting the CD19 antigen is a novel therapeutic approach for patients with hematologic malignancies. To this end, we have previously demonstrated that CAR T cells have a significant clinical benefit in adult patients with relapsed or refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL). We now extend this novel treatment approach by testing of our CD19-specific CAR in a multicenter trial for children and young adults with relapsed CD19+ B-ALL. Patients and Methods: Pediatric and young-adult patients with very high risk (VHR) or R/R CD19+ B-ALL were eligible for enrollment and leukapheresis. Patients with R/R B-ALL were eligible for infusion if they demonstrated residual disease following salvage chemotherapy. Once eligibility was determined isolation and transduction of T cells was performed using a retroviral vector encoding a CD19-specific CAR comprised of a CD19-specific scFv and the CD28 and CD3ζ signaling domains (termed 19-28z). All patients received conditioning chemotherapy followed 2 days later by 1x106 3x106 19-28z CAR T cells/kg. The primary objective of the study was to evaluate the safety and anti-tumor activity of 19-28z CAR T cells. Post-treatment response was assessed at day 14-28 bone marrow aspirate including multiparameter flow cytometry for minimal residual disease (MRD). Results: To date, 28 patients with have been enrolled on protocol with a median age of 11.5 years (range 9 months to 20 years) at time of T cell collection. We have treated 11 patients with 19-28z CAR T cells with a median age 14.9 years (range 3-22 years). Patients received a dose of 1-3 x 10^6 CAR T cells/kg and complete response (complete remission or complete remission with incomplete count recovery) occurred in 7/11 (64%) patients. Significantly, correlations with response included lower disease burden (as assessed by bone marrow cellularity; p135 vs Conclusions: These early results demonstrate the feasibility and significant clinical impact of CAR T cells in patients with R/R B-ALL. In an effort to more rapidly generate statistically relevant data, demonstrate the exportability of this technology between academic institutions, and offer this therapeutic option to a broader number of pediatric patients with chemo-refractory B-ALL we have expanded this trial to include a collaborating institution. The objective of our trial is not to provide an intent-to-treat cohort, but rather demonstrate the tolerability of this technology in patients with relapsed B-ALL. Furthermore, patients meeting disease eligibility were not pre-screened for lymphocyte function prior to collection and/or treatment. Subsequent cohorts of patients will receive 19-28z CAR T cells and will be evaluated for toxicity, persistence of CAR T cells, and for anti-leukemic efficacy. Citation Format: Kevin J. Curran, Lewis B. Silverman, Rachel Kobos, Nancy A. Kernan, Steven P. Margossian, Jae H. Park, Craig S. Sauter, Victoria Szenes, Xiuyan Wang, Richard J. O'Reilly, Michel Sadelain, Isabelle Riviere, Reiner J. Brentjens. Chimeric antigen receptor T cells for cancer immunotherapy. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr IA07.

Background: Epstein-Barr Virus (EBV)-associated lymphoproliferative disorder (EBV-LPD) complicating allogeneic hematopoietic cell transplant (alloHCT) most frequently present as malignant, high grade, diffuse large B cell lymphomas (DLBCL) that do not respond to reduction in immune suppression. Rituximab induces remissions in approximately 55% of those with radiographically demonstrable disease. Median survival reported for rituximab-refractory EBV-LPD patients (pts) ranges from 16 - 56 days. There are few other treatment options. EBV-cytotoxic T lymphocytes (CTLs) offer a possible treatment approach. We evaluated the efficacy and safety of EBV-CTLs have been evaluated in 2 clinical trials in alloHCT recipients with EBV+ disease. Methods: As part of 2 ongoing clinical trials (95-024 and 11-130) 57 pts received EBV-CTLs derived from unrelated third-party donors (13 on 95-024 and 18 on 11-130) or primary stem cell donors (26 on 95-024). Fifty-one pts were treated for monomorphic DLBCL, 3 polymorphic, 1 NK/T cell lymphoma and 2 for viremia alone. Subjects on 11-130 (n = 18) had a median age of 52y, 8 were male, all had failed prior rituximab. Pts on 95-024 (n = 39) had a median age of 21y, 23 were male, and 28 had failed prior rituximab. Subjects in both studies received up to 5 cycles of EBV-CTL infusions; each cycle consisting of 1 or 2×106 cells/kg weekly for 3 weeks. Results: Of the 18 recipients of 3rd party EBV-CTLs on 11-130, 9 pts had complete response (CR), 3 had partial response (PR) and 1 had stable disease (SD) for a response rate of 67% and non-progression rate of 72%. The median duration of CR+PR was 318d. Kaplan-Meier (KM) overall survival (OS) was 71.8% at 1 and 2y. KM progression-free survival was 66.7% at 1y. Of 39 pts on 95-024, 23 had CR, 1 PR, and 3 had SD, for a response rate of 62% and non-progression rate of 69%. Strikingly, the 1y OS for rituximab-refractory pts in both studies was 50% and 49% for pts treated with 3rd party and transplant donor-derived EBV-CTLs, respectively. Four patients on 11-130 and 6 patients on 95-024 died within 2 months of the first EBV-CTL infusion due to progression of EBVLPD (N = 6), leukemic relapse (N = 2), or other causes (N = 2); no death was considered related to treatment. EBV-CTLs had low toxicity: no pts developed cytokine release syndrome or GvHD requiring systemic therapy. Conclusions: EBV-CTLs produce high response rates that are durable; pts who achieved CR had no relapses of EBV LPD. The OS in both studies far exceeded the survival reported for this patient population. EBV-CTLs had a favorable safety profile and were well tolerated. Citation Format: Susan E. Prockop, Ekaterina Doubrovina, Karim Baroudy, Farid Boulad, Ramzi Khalaf, Esperanza B. Papadopoulos, Craig Sauter, Victoria Szenes, Stephanie Suser, Gloria Wasilewski, Julianna Ruggierio, Richard J. O'Reilly. Epstein-Barr virus-specific cytotoxic T lymphocytes for treatment of rituximab-refractory Epstein-Barr virus-associated lymphoproliferative disorder. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr CT107. doi:10.1158/1538-7445.AM2015-CT107

The Wilms tumor protein, WT-1 is expressed in over 60% of serous adenocarcinomas of the ovary. Its expression has been hypothesized to be critical for the growth or survival of tumorigenic stem cells. In this study, we have assessed the capacity of in vitro generated T-cells specific for a series of immunogenic WT1 peptide epitopes presented by different HLA class I alleles to prevent the outgrowth of two human ovarian adenocarcinoma cell lines expressing either low (SKOV3-A2) or high (OVCAR3) levels of WT1 by FACS in NOD/SCID mice. For this study, epitope-specific HLA restricted WT1-CTLs were generated from PBMC of 4 normal donors by in vitro sensitization with autologous EBV BLCL loaded with a pool of 141 15-mers overlapping by 11aa and spanning the entire sequence of the WT1 protein. WT1-CTL restricted by HLA alleles expressed on the OVCAR3 and SKOV-3 lines were pre-incubated in vitro for 8 hours at different E:T ratios (0:1, 5:1, 10:1, 50:1, 100:1) with 0.05×10⁁6 ovarian carcinoma cells transduced to express a luciferase reporter gene. The cell mixtures were injected i.p. into NOD/SCID mice. Tumor growth was monitored weekly by intensity of their bioluminescent signal. In all animals injected with the tumor cells alone the bioluminescent signal could be detected in the abdomen by day 10-15 and increased steadily through 60 days of observation (by which time, all mice died). The tumor engraftment was either markedly inhibited (SKOV3-A2WT1low) or completely abrogated (in OVCAR-3WT1high) by pre-incubation at 100:1 E:T ratio and was correlated with higher survival of the animals (80%) over a period of 120 days. In the animals injected with tumor cells pre-incubated with the WT1-CTLs at a 50:1 and 10:1 E:T ratios, tumor growth was suppressed as reflected by weaker bioluminescent signal in the abdomen which increased much later in the course of the study. Pre-incubation of the tumor cells with WT1-CTL at a 5:1 E:T ratio did not significantly affect tumor engraftment and growth or survival of the mice. WT1-CTL specific for the 398-406LKTHTTRTHT epitope presented by the A0201 allele induced significantly greater suppression of tumor growth than T-cells specific for the (−125)-(−117)RQRPHPGAL peptide that can be presented by either HLA-A0301 or B0702. T cells specific for different WT1 epitopes administered i.v. at a dose of 2.5×10⁁6 cells/animal inhibited the growth of the pre-established OVCAR3 tumor xenografts inoculated i.p. as compared to the growth of the same tumors in control animals. Mice treated with WT-1 specific T-cells also had a significantly extended survival. Our results provide evidence that tumorigenic ovarian carcinoma cells expressing WT1+ are susceptible to eradication in vivo by high doses of WT1-CTL. However, the effectiveness of these T cells may differ depending on the level of WT1 expressed by the tumor cells and peptide epitope of WT1 targeted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5515. doi:10.1158/1538-7445.AM2011-5515