Cooperative Armoring of CAR and TCR T Cells by T Cell-Restricted IL15 and IL21 Universally Enhances Solid Tumor Efficacy.

PURPOSE: Chimeric antigen receptor (CAR) and T-cell receptor (TCR) T-cell therapies are effective in a subset of patients with solid tumors, but new approaches are needed to universally improve patient outcomes. Here, we developed a technology to leverage the cooperative effects of IL15 and IL21, two common cytokine-receptor gamma chain family members with distinct, pleiotropic effects on T cells and other lymphocytes, to enhance the efficacy of adoptive T cells. EXPERIMENTAL DESIGN: We designed vectors that induce the constitutive expression of either membrane-tethered IL15, IL21, or IL15/IL21. We used clinically relevant preclinical models of transgenic CARs and TCRs against pediatric and adult solid tumors to determine the effect of the membrane-tethered cytokines on engineered T cells for human administration. RESULTS: We found that self-delivery of these cytokines by CAR or TCR T cells prevents functional exhaustion by repeated stimulation and limits the emergence of dysfunctional natural killer (NK)-like T cells. Across different preclinical murine solid tumor models, we observed enhanced regression with each individual cytokine but the greatest antitumor efficacy when T cells were armored with both. CONCLUSIONS: The coexpression of membrane-tethered IL15 and IL21 represents a technology to enhance the resilience and function of engineered T cells against solid tumors and could be applicable to multiple therapy platforms and diseases. See related commentary by Ruffin et al., p. 1431.

Third-party cytomegalovirus-specific T cells improved survival in refractory cytomegalovirus viremia after hematopoietic transplant.

BackgroundRefractory CMV viremia and disease are associated with significant morbidity and mortality in recipients of hematopoietic stem cell transplant (HCT).MethodsIn phase I/II trials, we treated 67 subjects for CMV viremia or disease arising after HCT with adoptive transfer of banked, third-party, CMVpp65-sensitized T cells (CMVpp65-VSTs). All were evaluable for toxicity and 59 for response. Evaluable subjects had CMV disease or persisting viremia that had failed at least 2 weeks of induction therapy with a median of 3 antiviral drugs; 84.7% had more than 3 of 11 high-risk features. CMVpp65-VSTs were specific for 1 to 3 CMVpp65 epitopes, presented by a limited set of HLA class I or II alleles, and were selected based on high-resolution HLA matching at 2 of 10 HLA alleles and matching for subject and subject's HCT donor for 1 or more alleles through which the CMVpp65-VSTs were restricted.ResultsT cell infusions were well tolerated. Of 59 subjects evaluable for response, 38 (64%) achieved complete or durable partial responses.ConclusionsRecipients responding to CMVpp65VSTs experienced an improved overall survival. Of the risk factors evaluated, transplant type, recipient CD4+ and CD8+ T cell levels prior to adoptive therapy, and the HLA restriction of CMVpp65-VSTs infused each significantly affected responses. In addition, CMVpp65-specific T cells of HCT donor or recipient origin contributed to the durability of both complete and partial responses.Trial RegistrationNCT00674648; NCT01646645; NCT02136797 (NIH).FundingNIH (P01 CA23766, R21 CA162002 and P30 CA008748); Aubrey Fund; Claire Tow Foundation; Major Family Foundation; "Rick" Eisemann Pediatric Research Fund; Banbury Foundation; Edith Robertson Foundation; Larry Smead Foundation.

Dominant epitopes presented by prevalent HLA alleles permit wide use of banked CMVpp65 T cells in adoptive therapy.

We established and characterized a bank of 138 CMVpp65 peptide-specific T-cell (CMVpp65CTLs) lines from healthy marrow transplant donors who consented to their use for treatment of individuals other than their transplant recipient. CMVpp65CTL lines included 131 containing predominantly CD8+ T cells and 7 CD4+ T cells. CD8+ CMVpp65CTLs were specific for 1 to 3 epitopes each presented by one of only 34 of the 148 class I alleles in the bank. Similarly, the 7 predominantly CD4+ CMVpp65CTL lines were each specific for epitopes presented by 14 of 40 HLA DR alleles in the bank. Although the number of HLA alleles presenting CMV epitopes is low, their prevalence is high, permitting selection of CMVpp65CTLs restricted by an HLA allele shared by transplant recipient and hematopoietic cell transplant donor for >90% of an ethnogeographically diverse population of hematopoietic cell transplant recipients. Within individuals, responses to CMVpp65 peptides presented by different HLA alleles are hierarchical. Furthermore, within groups, epitopes presented by HLA B*07:02 and HLA A*02:01 consistently elicit immunodominant CMVpp65CTLs, irrespective of other HLA alleles inherited. All dominant CMVpp65CTLs exhibited HLA-restricted cytotoxicity against epitope loaded targets and usually cleared CMV infections. However, immunodominant CMVpp65CTLs responding to epitopes presented by certain HLA B*35 alleles were ineffective in lysing CMV-infected cells in vitro or controlling CMV infections post adoptive therapy. Analysis of the hierarchy of T-cell responses to CMVpp65, the HLA alleles presenting immunodominant CMVpp65 epitopes, and the responses they induce may lead to detailed algorithms for optimal choice of third-party CMVpp65CTLs for effective adoptive therapy.

Phase I dose escalation safety and feasibility study of autologous WT1-sensitized T cells for the treatment of patients with recurrent ovarian cancer.

BACKGROUND: This phase I dose escalation trial evaluated the feasibility of production, safety, maximum tolerated dose, and preliminary efficacy of autologous T cells sensitized with peptides encoding Wilms' tumor protein 1 (WT1) administered alone or following lymphodepleting chemotherapy, in the treatment of patients with recurrent WT1+ ovarian, primary peritoneal, or fallopian tube carcinomas. METHODS: A 3+3 dose escalation design was used to determine dose-limiting toxicity (DLT). In cohort I, patients received WT1-sensitized T cells dosed at 5×106/m2 (level I) without cyclophosphamide lymphodepletion. In cohorts II-IV, patients received lymphodepleting chemotherapy (a single intravenous dose of cyclophosphamide 750 mg/m2), 2 days prior to the first intravenous infusion of WT1-sensitized T cells administered at escalating doses (2×107/m2 (level II), 5×107/m2 (level III), and 1×108/m2 (level IV)). RESULTS: Twelve patients aged 23-72 years, with a median of 7 prior therapies (range 4-14), were treated on the study. No DLT was observed, even at the highest dose level of 1×108/m2 WT1-sensitized T cells tested. Common adverse events reported were grade 1-2 fatigue, fever, nausea, and headache. Median progression-free survival (PFS) was 1.8 months (95% CI, 0.8 to 2.6); 1 year PFS rate 8.3% (95% CI, 0.5 to 31.1). Median overall survival (OS) was 11.0 months (95% CI, 1.1 to 22.6); OS at 1 year was 41.7% (95% CI, 15.2% to 66.5%). Best response was stable disease in one patient (n=1) and progressive disease in the others (n=11). We observed a transient increase in the frequencies of WT1-specific cytotoxic T lymphocyte precursors (CTLp) in the peripheral blood of 9 of the 12 patients following WT1-sensitized T-cell infusion. CONCLUSION: We demonstrated the safety of administration of WT1-sensitized T cells and the short-term increase in the WT1 CTLp. However, at the low doses evaluated we did not observe therapeutic activity in recurrent ovarian cancer. In this heavily pretreated population, we encountered challenges in generating sufficient numbers of WT1-reactive cytotoxic T cells. Future studies employing WT1-specific T cells generated from lymphocytes are warranted but should be done earlier in the disease course and prior to intensive myelosuppressive therapy. TRIAL REGISTRATION NUMBER: NCT00562640. ONE-SENTENCE SUMMARY: The authors describe the first human application of autologous WT1-sensitized T cells in the treatment of patients with recurrent ovarian, primary peritoneal, and fallopian tube carcinomas.

Ocular Outcomes after Treatment of Cytomegalovirus Retinitis Using Adoptive Immunotherapy with Cytomegalovirus-Specific Cytotoxic T Lymphocytes.

PURPOSE: To describe ocular outcomes in eyes with cytomegalovirus (CMV) retinitis treated with adoptive immunotherapy using systemic administration of CMV-specific cytotoxic Tlymphocytes (CMV-specific CTLs). DESIGN: Retrospective cohort study. PARTICIPANTS: Patients with active CMV retinitis evaluated at a tertiary care academic center. METHODS: Treatment of CMV retinitis with standard-of-care therapy (systemic or intravitreal antivirals) or CMV-specific CTLs (with or without concurrent standard-of-care therapies). MAIN OUTCOME MEASURES: The electronic medical record was reviewed to determine baseline characteristics, treatment course, and ocular outcomes, including best-corrected visual acuity (BCVA), treatments administered (CMV-specific CTLs, systemic antivirals, intravitreal antivirals), resolution of CMV retinitis, any occurrence of immune recovery uveitis, cystoid macular edema, retinal detachment, or a combination thereof. RESULTS: Seven patients (3 of whom had bilateral disease [n = 10 eyes]) were treated with CMV-specific CTLs, whereas 20 patients (6 of whom had bilateral disease [n = 26 eyes]) received standard-of-care treatment. Indications for CMV-specific CTL therapy included persistent or progressive CMV retinitis (71.4% of patients); CMV UL54 or UL97 antiviral resistance mutations (42.9%); side effects or toxicity from antiviral agents (57.1%); patient intolerance to longstanding, frequent antiviral therapy for persistent retinitis (28.6%); or a combination thereof. Two patients (28.6%; 4 eyes [40%]) received CMV-specific CTL therapy without concurrent systemic or intravitreal antiviral therapy for active CMV retinitis, whereas 5 patients (71.4%; 6 eyes [60%]) continued to receive concurrent antiviral therapies. Resolution of CMV retinitis was achieved in 9 eyes (90%) treated with CMV-specific CTLs, with BCVA stabilizing (4 eyes [40%]) or improving (4 eyes [40%]) in 80% of eyes over an average follow-up of 33.4 months. Rates of immune recovery uveitis, new-onset cystoid macular edema, and retinal detachment were 0%, 10% (1 eye), and 20% (2 eyes), respectively. These outcomes compared favorably with a nonrandomized cohort of eyes treated with standard-of-care therapy alone, despite potentially worse baseline characteristics. CONCLUSIONS: CMV-specific CTL therapy may represent a novel monotherapy or adjunctive therapy, or both, for CMV retinitis, especially in eyes that are resistant, refractory, or intolerant of standard-of-care antiviral therapies. More generally, adoptive cell transfer and adoptive immunotherapy may have a role in refractory CMV retinitis. Larger prospective, randomized trials are necessary.

Adoptive therapy with CMV-specific cytotoxic T lymphocytes depends on baseline CD4+ immunity to mediate durable responses.

Adoptive cell therapy using cytomegalovirus (CMV)-specific cytotoxic T lymphocytes (CMV-CTLs) has demonstrated efficacy posttransplant. Despite the predicted limited engraftment of CMV-CTLs derived from third-party donors, partially matched third-party donor-derived CMV-CTLs have demonstrated similar response rates to those derived from primary hematopoietic cell transplantation donors. Little is known about the mechanisms through which adoptive cellular therapies mediate durable responses. We performed a retrospective analysis of patients receiving CMV-CTLs for treatment of CMV viremia and/or disease after allogeneic transplant between September of 2009 and January of 2018. We evaluated whether response to adoptively transferred CMV-CTLs correlated with immune reconstitution (IR), using validated CD4+ IR milestones of 50 × 106/L and 200 × 106/L. In this analysis, a cohort of 104 patients received CMV-CTLs derived from a primary transplant donor (n = 25), a third-party donor (n = 76), or both (n = 3). Response to therapy did not increase the likelihood of achieving CD4+ IR milestones at 1 (P = .53 and P > .99) or 2 months (P = .12 and P = .33). The origin of CMV-CTLs did not impact subsequent CD4+ IR. CMV-CTLs appeared to interact with host immunity in mediating responses. Recipients with a baseline CD4 >50 × 106/L had higher response to therapy (P = .02), improved overall survival (P < .001), and protection from CMV-related death (P = .002). Baseline endogenous immunity appears to improve CMV-related and overall survival in this cohort and can be an important marker at the initiation of therapy.

Epigenetic reprogramming sensitizes immunologically silent EBV+ lymphomas to virus-directed immunotherapy.

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.

Off-the-shelf EBV-specific T cell immunotherapy for rituximab-refractory EBV-associated lymphoma following transplantation.

BACKGROUNDAdoptive transfer of donor-derived EBV-specific cytotoxic T-lymphocytes (EBV-CTLs) can eradicate EBV-associated lymphomas (EBV-PTLD) after transplantation of hematopoietic cell (HCT) or solid organ (SOT) but is unavailable for most patients.METHODSWe developed a third-party, allogeneic, off-the-shelf bank of 330 GMP-grade EBV-CTL lines from specifically consented healthy HCT donors. We treated 46 recipients of HCT (n = 33) or SOT (n = 13) with established EBV-PTLD, who had failed rituximab therapy, with third-party EBV-CTLs. Treatment cycles consisted of 3 weekly infusions of EBV-CTLs and 3 weeks of observation.RESULTSEBV-CTLs did not induce significant toxicities. One patient developed grade I skin graft-versus-host disease. Complete remission (CR) or sustained partial remission (PR) was achieved in 68% of HCT recipients and 54% of SOT recipients. For patients who achieved CR/PR or stable disease after cycle 1, one year overall survival was 88.9% and 81.8%, respectively. In addition, 3 of 5 recipients with POD after a first cycle who received EBV-CTLs from a different donor achieved CR or durable PR (60%) and survived longer than 1 year. Maximal responses were achieved after a median of 2 cycles.CONCLUSIONThird-party EBV-CTLs of defined HLA restriction provide safe, immediately accessible treatment for EBV-PTLD. Secondary treatment with EBV-CTLs restricted by a different HLA allele (switch therapy) can also induce remissions if initial EBV-CTLs are ineffective. These results suggest a promising potential therapy for patients with rituximab-refractory EBV-associated lymphoma after transplantation.TRIAL REGISTRATIONPhase II protocols (NCT01498484 and NCT00002663) were approved by the Institutional Review Board at Memorial Sloan Kettering Cancer Center, the FDA, and the National Marrow Donor Program.FUNDINGThis work was supported by NIH grants CA23766 and R21CA162002, the Aubrey Fund, the Claire Tow Foundation, the Major Family Foundation, the Max Cure Foundation, the Richard "Rick" J. Eisemann Pediatric Research Fund, the Banbury Foundation, the Edith Robertson Foundation, and the Larry Smead Foundation. Atara Biotherapeutics licensed the bank of third-party EBV-CTLs from Memorial Sloan Kettering Cancer Center in June 2015.

Therapeutic advantages provided by banked virus-specific T-cells of defined HLA-restriction.

We have developed banks of EBV and CMV-specific T-cell lines generated from healthy seropositive third party donors and characterized them as to their HLA type, virus specificity, lack of alloreactivity, and HLA restriction. We here summarize results of studies employing these immediately accessible, broadly-applicable third party virus-specific T-cells for adoptive therapy of EBV lymphomas and CMV infections in allo-HCT recipients. We describe the characteristics contributing to their safety. We also discuss several distinctive advantages of banked third party virus-specific T-cells selected on the basis of their HLA restriction, particularly in the treatment of Rituximab-non-responsive EBV+ lymphomas and drug refractory CMV infections complicating HLA non-identical transplants.

Adoptive immunotherapy with haploidentical natural killer cells and Anti-GD2 monoclonal antibody m3F8 for resistant neuroblastoma: Results of a phase I study.

Natural killer (NK) cell-mediated antibody-dependent toxicity is a potent mechanism of action of the anti-GD2 murine monoclonal antibody 3F8 (m3F8). Killer immunoglobulin-like receptor (KIR) and HLA genotypes modulate NK activity and are key prognostic markers in m3F8-treated patients with neuroblastoma. Endogenous NK-cells are suppressed in the setting of high tumor burden and chemotherapy. Allogeneic NK-cells however, demonstrate potent anti-neuroblastoma activity. We report on the results of a phase I clinical trial of haploidentical NK-cells plus m3F8 administered to patients with high-risk neuroblastoma after conditioning chemotherapy. The primary objective was to determine the maximum tolerated NK-cell dose (MTD). Secondary objectives included assessing anti-neuroblastoma activity and its relationship to donor-recipient KIR/HLA genotypes, NK function, and donor NK chimerism. Patients received a lymphodepleting regimen prior to infusion of haploidentical CD3-CD56+ NK-cells, followed by m3F8. Overall and progression free survival (PFS) were assessed from the time of first NK-cell dose. Univariate Cox regression assessed relationship between dose and outcomes. Thirty-five patients received NK-cells at one of five dose levels ranging from <1×106 to 50×106 CD3-CD56+cells/kg. One patient experienced grade 3 hypertension and grade 4 pneumonitis. MTD was not reached. Ten patients (29%) had complete or partial response; 17 (47%) had no response; and eight (23%) had progressive disease. No relationship was found between response and KIR/HLA genotype or between response and FcγRIII receptor polymorphisms. Patients receiving >10×106 CD56+cells/kg had improved PFS (HR: 0.36, 95%CI: 0.15-0.87, p = 0.022). Patient NK-cells displayed high NKG2A expression, leading to inhibition by HLA-E-expressing neuroblastoma cells. Adoptive NK-cell therapy in combination with m3F8 is safe and has anti-neuroblastoma activity at higher cell doses.

PARP Inhibitors in Clinical Use Induce Genomic Instability in Normal Human Cells.

Poly(ADP-ribose) polymerases (PARPs) are the first proteins involved in cellular DNA repair pathways to be targeted by specific inhibitors for clinical benefit. Tumors harboring genetic defects in homologous recombination (HR), a DNA double-strand break (DSB) repair pathway, are hypersensitive to PARP inhibitors (PARPi). Early phase clinical trials with PARPi have been promising in patients with advanced BRCA1 or BRCA2-associated breast, ovary and prostate cancer and have led to limited approval for treatment of BRCA-deficient ovary cancer. Unlike HR-defective cells, HR-proficient cells manifest very low cytotoxicity when exposed to PARPi, although they mount a DNA damage response. However, the genotoxic effects on normal human cells when agents including PARPi disturb proficient cellular repair processes have not been substantially investigated. We quantified cytogenetic alterations of human cells, including primary lymphoid cells and non-tumorigenic and tumorigenic epithelial cell lines, exposed to PARPi at clinically relevant doses by both sister chromatid exchange (SCE) assays and chromosome spreading. As expected, both olaparib and veliparib effectively inhibited poly-ADP-ribosylation (PAR), and caused marked hypersensitivity in HR-deficient cells. Significant dose-dependent increases in SCEs were observed in normal and non-tumorigenic cells with minimal residual PAR activity. Clinically relevant doses of the FDA-approved olaparib led to a marked increase of SCEs (5-10-fold) and chromatid aberrations (2-6-fold). Furthermore, olaparib potentiated SCE induction by cisplatin in normal human cells. Our data have important implications for therapies with regard to sustained genotoxicity to normal cells. Genomic instability arising from PARPi warrants consideration, especially if these agents will be used in people with early stage cancers, in prevention strategies or for non-oncologic indications.

Characterization of a c-Rel Inhibitor That Mediates Anticancer Properties in Hematologic Malignancies by Blocking NF-κB-Controlled Oxidative Stress Responses.

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.

Therapeutic bispecific T-cell engager antibody targeting the intracellular oncoprotein WT1.

Intracellular tumor antigens presented on the cell surface in the context of human leukocyte antigen (HLA) molecules have been targeted by T cell-based therapies, but there has been little progress in developing small-molecule drugs or antibodies directed to these antigens. Here we describe a bispecific T-cell engager (BiTE) antibody derived from a T-cell receptor (TCR)-mimic monoclonal antibody (mAb) ESK1, which binds a peptide derived from the intracellular oncoprotein WT1 presented on HLA-A*02:01. Despite the very low density of the complexes at the cell surface, ESK1-BiTE selectively activated and induced proliferation of cytolytic human T cells that killed cells from multiple leukemias and solid tumors in vitro and in mice. We also discovered that in an autologous in vitro setting, ESK1-BiTE induced a robust secondary CD8 T-cell response specific for tumor-associated antigens other than WT1. Our study provides an approach that targets tumor-specific intracellular antigens without using cell therapy and suggests that epitope spreading could contribute to the therapeutic efficacy of this BiTE.

Immunotherapy with Donor T Cells Sensitized with Overlapping Pentadecapeptides for Treatment of Persistent Cytomegalovirus Infection or Viremia.

We conducted a phase I trial of allogeneic T cells sensitized in vitro against a pool of pentadecapeptides (15-mer peptides) spanning the sequence of CMVpp65 for adoptive therapy of 17 allogeneic hematopoietic cell transplant recipients with cytomegalovirus (CMV) viremia or clinical infection persisting despite prolonged treatment with antiviral drugs. All but 3 of the patients had received T cell-depleted transplants without graft-versus-host disease (GVHD) prophylaxis with immunosuppressive drugs after transplantation. The CMVpp65-specific T cells (CMVpp65CTLs) generated were oligoclonal and specific for only 1 to 3 epitopes, presented by a limited set of HLA class I or II alleles. T cell infusions were well tolerated without toxicity or GVHD. Of 17 patients treated with transplant donor (n = 16) or third-party (n = 1) CMVpp65CTLs, 15 cleared viremia, including 3 of 5 with overt disease. In responding patients, the CMVpp65CTLs infused consistently proliferated and could be detected by T cell receptor Vß usage in CMVpp65/HLA tetramer + populations for period of 120 days to up to 2 years after infusion. Thus, CMVpp65CTLs generated in response to synthetic 15-mer peptides of CMVpp65 are safe and can clear persistent CMV infections in the post-transplantation period.

A small-molecule c-Rel inhibitor reduces alloactivation of T cells without compromising antitumor activity.

Preventing unfavorable GVHD without inducing broad suppression of the immune system presents a major challenge of allogeneic hematopoietic stem cell transplantation (allo-HSCT). We developed a novel strategy to ameliorate GVHD while preserving graft-versus-tumor (GVT) activity by small molecule-based inhibition of the NF-κB family member c-Rel. Underlying mechanisms included reduced alloactivation, defective gut homing, and impaired negative feedback on interleukin (IL)-2 production, resulting in optimal IL-2 levels, which, in the absence of competition by effector T cells, translated into expansion of regulatory T cells. c-Rel activity was dispensable for antigen-specific T-cell receptor (TCR) activation, allowing c-Rel-deficient T cells to display normal GVT activity. In addition, inhibition of c-Rel activity reduced alloactivation without compromising antigen-specific cytotoxicity of human T cells. Finally, we were able to demonstrate the feasibility and efficacy of systemic c-Rel inhibitor administration. Our findings validate c-Rel as a promising target for immunomodulatory therapy and demonstrate the feasibility and efficacy of pharmaceutical inhibition of c-Rel activity.

Targeting the intracellular WT1 oncogene product with a therapeutic human antibody.

The Wilms tumor 1 (WT1) oncoprotein is an intracellular, oncogenic transcription factor that is overexpressed in a wide range of leukemias and solid cancers. RMFPNAPYL (RMF), a WT1-derived CD8+ T cell human leukocyte antigen (HLA)-A0201 epitope, is a validated target for T cell-based immunotherapy. Using phage display technology, we discovered a fully human "T cell receptor-like" monoclonal antibody (mAb), ESK1, specific for the WT1 RMF peptide/HLA-A0201 complex. ESK1 bound to several leukemia and solid tumor cell lines and primary leukemia cells, in a WT1- and HLA-A0201-restricted manner, with high avidity [dissociation constant (Kd)=0.1 nM]. ESK1 mediated antibody-dependent human effector cell cytotoxicity in vitro. Low doses of naked ESK1 antibody cleared established, disseminated, human acute lymphocytic leukemia and Philadelphia chromosome-positive leukemia in nonobese diabetic/severe combined immunodeficient γc-/- (NSG) mouse models. At therapeutic doses, no toxicity was seen in HLA-A0201 transgenic mice. ESK1 is a potential therapeutic agent for a wide range of cancers overexpressing the WT1 oncoprotein. This finding also provides preclinical validation for the strategy of developing therapeutic mAbs targeting intracellular oncogenic proteins.

Mapping of novel peptides of WT-1 and presenting HLA alleles that induce epitope-specific HLA-restricted T cells with cytotoxic activity against WT-1(+) leukemias.

The Wilms tumor protein (WT-1) is widely recognized as a tumor antigen that is expressed differentially by several malignancies. However, WT-1 peptides known to induce tumoricidal T cells are few. In the present study, we evaluated T-cell responses of 56 healthy donors to in vitro sensitization with autologous APCs loaded with a pool of overlapping 15-mer peptides spanning the sequence of WT-1. Thereafter, we mapped the WT-1 peptides eliciting responses in each individual, defined the immunogenic peptides, and identified their presenting HLA alleles. We report 41 previously unreported epitopes of WT-1: 5 presented by class II and 36 by class I alleles, including 10 that could be presented by more than 1 class I allele. IFNγ(+) T cells responding to 98% of the class I and 60% of the class II epitopes exhibited HLA-restricted cytotoxicity against peptide-loaded targets. T cells specific for 36 WT-1 peptides were evaluable for leukemocidal activity, of which 27 (75%) lysed WT-1(+) leukemic targets sharing their restricting HLA allele. Each epitope identified induced T-cell responses in most donors sharing the epitopes' presenting allele; these responses often exceeded responses to flanking peptides predicted to be more immunogenic. This series of immunogenic epitopes of WT-1 should prove useful for immunotherapies targeting WT-1(+) malignancies.

Adoptive immunotherapy with unselected or EBV-specific T cells for biopsy-proven EBV+ lymphomas after allogeneic hematopoietic cell transplantation.

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.

Novel strategies for adoptive therapy following HLA disparate transplants.

Transplants of SBA-E- allogeneic marrow or G-CSF mobilized CD34+ (ISOLEX) E- peripheral blood progenitor cells which are adequately depleted of T-cells, when administered without post-transplant immunosuppression now induce consistent engraftment with low incidences of acute and chronic GVHD both in HLA matched and HLA disparate recipients. Furthermore, the incidence of relapse post transplant is not increased in patients transplanted for AML, MDS or ALL. In our series, the incidence of severe infections in HLA-matched recipients of such T-cell depleted grafts also does not differ from that detected following similarly matched unmodified grafts. However, in recipients of HLA-haplotype disparate T-cell depleted grafts, the risk of lethal viral infections is increased and prolonged. In many cases, this risk is closely correlated with failures of immunodominant virus-specific donor T-cells transferred in the graft to recognize infected host cells because they are restricted by HLA alleles not shared by the host. To address this limitation, we have developed a panel of artificial antigen presenting cells, each expressing a single prevalent HLA-allele. Using this panel, we are able to selectively generate virus-specific cytotoxic T-cells of desired HLA restriction, to insure their effectiveness in HLA haplotype-disparate transplant recipients. We have also shown that partially HLA-matched, third party-derived EBV-specific T-cells, selected from our bank of previously generated and characterized GMP-grade cell lines on the basis of their HLA restriction, can induce durable remissions of rituximab-refractory EBV lymphomas. These approaches may thus provide new, immediately accessible resources for the generation and broad application of immune cell therapies to treat and prevent severe viral diseases post transplant.

Successful treatment of EBV-associated posttransplantation lymphoma after cord blood transplantation using third-party EBV-specific cytotoxic T lymphocytes.

Cellular therapy of Epstein-Barr virus (EBV)(+) posttransplantation lymphoproliferative diseases (PTLD) in cord blood transplant (CBT) recipients is limited by lack of donor access and the donor's naive neonatal immune system. We therefore used partially human leukocyte antigen-matched third-party in vitro expanded EBV-specific cytotoxic T lymphocytes (CTLs) to treat 2 CBT recipients with life-threatening, donor-derived monoclonal EBV(+) diffuse large B-cell lymphomas with extranodal involvement developing in the context of graft-versus-host disease. Both patients had failed immunosuppression taper and Rituximab. After 5 and 9 infusions of 10(6) EBV-CTL/kg, respectively, each patient achieved a sustained complete remission without toxicity or graft-versus-host disease. Each is alive without recurrence at 20 and 15 months, respectively, post-EBV-PTLD diagnosis. This approach demonstrates the efficacy of using "off-the-shelf," virus-specific third-party CTLs restricted by human leukocyte antigens expressed by the tumor to treat otherwise lethal EBV-PTLD. Such therapy may also be applicable to the treatment of other infections and residual or recurrent malignancy after CBT.