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Exploring the repertoire of peptides presented on major histocompatibility complexes (MHC) has been utilized to identify targets for immunotherapy in many hematological malignancies. However, such data have not been described systematically for diffuse large B-cell lymphomas (DLBCL), which might be explained by the profound downregulation of MHC expression in many DLBCLs, and in particular in the EZH2-mutated subgroup. Epigenetic drug treatment, especially in the context of interferon gamma (IFNg), restored MHC expression in DLBCL. DLBCL MHC-presented peptides were identified via mass spectrometry following tazemetostat or decitabine treatments alone, or in combination with IFNg. Such treatment synergistically increased MHC class I surface protein expression up to 50-fold and class II expression up to 3-fold. Peptides presented on MHC complexes increased to a similar extent for MHC class I and remained constant for class II. Overall, these treatments restored the diversity of the immunopeptidome to levels described in healthy B cells and allowed the systematic search for new targets for immunotherapy. Consequently, we identified multiple MHC ligands from regulator of G protein signaling 13 (RGS13) and E2F transcription factor 8 (E2F8) on different MHC alleles, none of which have been described in healthy tissues and therefore represent tumor-specific MHC ligands, which are unmasked only after drug treatment. Overall, our results show that EZH2 inhibition in combination with decitabine and IFNg can expand the repertoire of MHC ligands presented on DLBCLs by revealing cryptic epitopes, thus allowing the systematic analysis and identification of new potential immunotherapy targets.Key pointsCombination therapy of interferon gamma with epigenetic regulators leads to large increases in the immunopeptidome of DLBCL.HLA ligands from proteins RGS13 and E2F8 may provide DLBCL-specific targets for immunotherapy.

PurposeWilms’ tumor 1 (WT1) is overexpressed in several cancers, and WT1 expression levels are associated with poor prognosis. As a host protein that functions as an oncogene, it represents an important immunotherapeutic target. This study evaluated WT1 expression in Kaposi sarcoma (KS) tumors to assess whether immunotherapy targeting WT1 is a potential therapeutic approach for KS. We also investigated the role of the causal agent of KS, Kaposi sarcoma herpesvirus (KSHV/HHV-8) in regulating WT1 expression.Experimental designImmunohistochemistry for WT1, KSHV, and B and T cells subsets, followed by image analysis, was performed in 363 KS tumor biopsies. Expression of KSHV vFLIP was evaluated by immunofluorescence. Primary endothelial cell cultures and cell lines were infected with KSHV in vitro, or transduced with an inducible vFLIP vector and induced with doxycycline, and then assessed for WT1 expression. Binding of ESK-1, a T cell receptor mimic therapeutic antibody that recognizes WT1 peptides presented on MHC HLA-A0201, was assessed using flow cytometry.ResultsWe report overexpression of WT1 in KS tumors, which was associated with increased with increasing histopathologic stage and the proportion of KSHV-infected cells. Areas with high WT1 expression showed sparse T cell infiltrates. KSHV infection in vitro resulted in WT1 upregulation, mediated by the viral protein vFLIP, which resulted in stronger binding of ESK1.ConclusionsKS lesions express high levels of WT1, a process regulated by the KSHV-encoded vFLIP. These findings suggest that immunotherapy directed against WT1 may represent a therapeutic approach for this cancer.Translational RelevanceKaposi sarcoma (KS) is a vascular neoplasm caused by the Kaposi sarcoma herpesvirus (KSHV/HHV-8). People living with HIV are not only at a significantly higher risk of developing KS, but also often have a more aggressive clinical course. Although antiretroviral therapy may cause regression of HIV-associated KS lesions, advanced cases of KS also require chemotherapy, which is rarely curative. Wilms’ tumor 1 (WT1) has been reported to be overexpressed in various cancers, functioning as an oncogene and associated with a poor prognosis. WT1 is also an important immunotherapeutic target, with several WT1-directed therapies showing promising results in early clinical trials for leukemias and solid tumors. Here we report high expression of WT1 in KS, especially in higher histological stages. Our findings provide pre-clinical evidence that supports conducting anti-WT1 immunotherapy trials in KS, and evaluating WT1 expression as a potential biomarker to identify individuals most likely to benefit.

The use of T cells reactive with intracellular tumor-associated or tumor-specific antigens has been a promising strategy for cancer immunotherapies in the past three decades, but the approach has been constrained by a limited understanding of the T cell receptor’s (TCR) complex functions and specificities. Newer TCR and T cell-based approaches are in development, including engineered adoptive T cells with enhanced TCR affinities, TCR mimic antibodies, and T cell-redirecting bispecific agents. These new therapeutic modalities are exciting opportunities by which TCR recognition can be further exploited for therapeutic benefit. In this review we summarize the development of TCR-based therapeutic strategies and focus on balancing efficacy and potency versus specificity, and hence, possible toxicity, of these powerful therapeutic modalities.

Cyclin dependent kinase 4/6 inhibitors (CDK4/6i) lead to cell-cycle arrest but also trigger T cell-mediated immunity, which might be mediated by changes in human leukocyte antigen (HLA) ligands. We investigated the effects of CDK4/6i, abemaciclib and palbociclib, on the immunopeptidome at nontoxic levels in breast cancer cell lines by biochemical identification of HLA ligands followed by network analyses. This treatment led to upregulation of HLA and revealed hundreds of induced HLA ligands in breast cancer cell lines. These new ligands were significantly enriched for peptides derived from proteins involved in the “G1/S phase transition of cell cycle” including HLA ligands from CDK4/6, Cyclin D1 and the 26S regulatory proteasomal subunit 4 (PSMC1). Interestingly, peptides from proteins targeted by abemaciclib and palbociclib, were predicted to be the most likely to induce a T cell response. In strong contrast, peptides induced by solely one of the drugs had a lower T cell recognition score compared to the DMSO control suggesting that the observed effect is class dependent. This general hypothesis was exemplified by a peptide from PSMC1 which was among the HLA ligands with highest prediction scores and which elicited a T cell response in healthy donors. Overall, these data demonstrate that CDK4/6i treatment gives rise to drug-induced HLA ligands from G1/S phase transition, that have the highest chance for being recognized by T cells, thus providing evidence that inhibition of a distinct cellular process leads to increased presentation of the involved proteins that may be targeted by immunotherapeutic agents.

Identification of neoepitopes as tumor-specific targets remains challenging, especially for cancers with low mutational burden, such as ovarian cancer. To identify mutated human leukocyte antigen (HLA) ligands as potential targets for immunotherapy in ovarian cancer, we combined mass spectrometry analysis of the major histocompatibility complex (MHC) class I peptidomes of ovarian cancer cells with parallel sequencing of whole exome and RNA in a patient with high-grade serous ovarian cancer. Four of six predicted mutated epitopes capable of binding to HLA-A*02:01 induced peptide-specific T cell responses in blood from healthy donors. In contrast, all six peptides failed to induce autologous peptide-specific response by T cells in peripheral blood or tumor-infiltrating lymphocytes from ascites of the patient. Surprisingly, T cell responses against a low-affinity p53-mutant Y220C epitope were consistently detected in the patient with either unprimed or in vitro peptide-stimulated T cells even though the patient's primary tumor did not bear this mutation. Our results demonstrated that tumor heterogeneity and distinct immune microenvironments within a patient should be taken into consideration for identification of immunogenic neoantigens. T cell responses to a driver gene-derived p53 Y220C mutation in ovarian cancer warrant further study.

Chimeric antigen receptor (CAR) T cells represent a novel class of FDA-approved drugs with high efficacy against refractory B cell derived malignancies and potentially other cancer types. However, target selection for CAR T cell therapy remains challenging as cell surface proteins are not cancer-specific and therefore often not adaptable for CAR T cell therapy. In contrast, many intracellular proteins can be highly tumor specific and are targetable after proteasomal degradation and presentation on human leukocyte antigen (HLA) complexes recognized by T cell receptor mimic antibodies. This class of antibodies recognizes peptide:HLA complexes with a similar mode of recognition as a TCR, but with the clinical versatility and applicability of an antibody. To identify a tumor specific target that is presented as a peptide in conjunction with the highly prevalent HLA allele A*02:01, we immunopurified peptide:HLA complexes from various cancer cell lines of different origins, separated HLA ligands from complexes and identified their peptide sequences via mass spectrometry. Network analysis of the resulting HLA ligand datasets identified shared biological processes among the tumor cell lines that were not present in network analyses of published datasets of healthy human tissue HLA ligandomes. Through this filtering process several potential targets were identified and an HLA ligand derived from kinetochore NDC80 protein homolog (NDC80) was selected as a target. The NDC80 derived peptide was detected in over 90% of the A*02 positive cell lines tested and never reported to be present in HLA ligand datasets of healthy human tissues. Furthermore, NDC80 has been shown to be differentially expressed in malignant compared to adjacent non-malignant tissues and is associated with poor prognosis in many cancer types. After utilizing E-ALPHA®phage library screening, one clone (NDC80-L1) was selected as the lead TCR mimic antibody. Overall, NDC80-L1 showed high specificity for the target HLA:peptide complex in both antibody and CAR T cell format in vitro and demonstrated binding primarily to the central region of the HLA ligand as determined by alanine screening assays. The exquisite specificity of NDC80-L1 was further illustrated by NDC80 knockdown experiments as well as successful immunopurification of the target peptide together with no relevant off-targets from BV173 ALL cells in mass spectrometry assays. Given the high specificity, sensitivity was assessed primarily in a potent CAR T cell format: Multiple tumor cell lines of different origin (e.g. ALL, AML, lymphoma, melanoma, mesothelioma, pancreatic and thyroid cancer) were successfully killed in vitro by NDC80-L1 CAR T cells, but no toxicity towards A*02:01 positive CAR T cells, healthy PBMCs or NDC80 target negative cell lines was observed. Interestingly, NDC80-L1 CAR T cells demonstrated highest efficacy in hematological malignancies most likely correlating with elevated expression of antigen presentation machinery and rapid cell division which leads to strong surface expression of NDC80 peptides. In summary, CAR T cells directed against peptide/HLA-A*02 derived from the NDC80 protein effectively kill multiple cancer cell lines in vitro without evidence of relevant off-target killing. However, the improved killing especially against ALL, AML and lymphomas highlights the potential of these CAR T cells to preferentially eliminate cancer cells with high proliferative capacity. Future in vivo studies with CAR T cell and antibody format will further investigate this TCR mimic antibody's potential as a tumor-agnostic therapeutic agent. Disclosures Klatt: MSKCC/EUREKA: Patents & Royalties: MSKCC AND EUREKA THERAPUETICS HAVE FILED A PATENT FOR THIS ANTIBODY/SCFV. Yang:Eureka Therapuetics: Current Employment, Current equity holder in private company, Patents & Royalties: MSKCC and Eureka have filed patent for this TCRm and ScvF. Liu:Eureka Therapue: Current Employment, Current equity holder in private company, Patents & Royalties: Eureka Therapuetics and MSKCC have filed patent on this ScFV and TCRm. Dao:Eureka Therapeutics: Consultancy. Liu:Eureka Therapeutics: Current Employment, Current equity holder in private company, Patents & Royalties: Eureka Therapuetics and MSKCC have filed patent on this ScFV and TCRm. Scheinberg:Eureka Therapeutics: Consultancy, Current equity holder in private company, Patents & Royalties: Eureka Therapuetics and MSKCC have filed patent on this ScFV and TCRm; Actinium: Consultancy, Current equity holder in private company; Sellas: Consultancy, Current equity holder in private company; Contrafect: Current equity holder in private company; Arvenas: Current equity holder in private company; Sapience: Consultancy, Current equity holder in private company; Iovance: Current equity holder in private company; Oncopep: Consultancy; Pfizer: Consultancy, Current equity holder in private company; Lantheus: Current equity holder in private company; Enscyse: Current equity holder in private company.

Objectives Adoptive transfer of autologous Tcells specifically targeting neoepitopes derived from tumor mutations has achieved responses in melanoma and other cancers. We sought to identify ovarian cancer neoepitopes. Methods We collected tumor, blood and ascites specimens from ovarian cancer patients. We combined mass spectrometry analysis of MHC class1 peptidomes with parallel sequencing of whole exome and RNA of cancer cells, to identify neoepitopes. Missense mutations were identified by whole exome sequencing and epitopes confirmed by mass spectrometry from ascites. We selected peptides predicted to have high binding affinity to HLA-A*02:01, for in vitro Tcell priming. We first validated the immunogenicity of these peptides for priming Tcells with synthesized peptides in healthy HLA-A*02:01+ donors. Results 32 missense mutations were identified in patient’s ascites. 7 epitopes were confirmed by mass spectroscopy; 5 were predicted to have high binding affinity to HLA-A*02:01. 3/5 epitopes induced peptide-specific Tcell responses not cross-reactive with native sequences. However, these 3 peptides failed to induce autologous peptide-specific Tcell response in TILs to patient‘s ascites, nor to autologous tumor cells, suggesting immunosuppression. Interestingly, when autologous tumor cells were pre-treated with IFN-gamma, Tcell responses against tumor cells were observed. Conclusions We demonstrated effective T cell responses against tumor neoantigens, is not only dependent on stimulation of Tcells, but also on efficient epitope processing and presentation on cell surface. While efforts have been focused on activation of Tcell responses, such as immune checkpoint blockade, our study suggests that strategies to modulate tumor cells to efficiently present neoepitopes should be considered for successful immunotherapy against neoantigens.

A National Cancer Institute consensus study on prioritization of cancer antigens ranked the Wilms tumor 1 (WT1) protein as the top immunotherapy target in cancer. We previously reported a pilot study of a multivalent WT1 peptide vaccine (galinpepimut-S) in acute myeloid leukemia (AML) patients. We have now conducted a phase 2 study investigating this vaccine in adults with AML in first complete remission (CR1). Patients received 6 vaccinations administered over 10 weeks with the potential to receive 6 additional monthly doses if they remained in CR1. Immune responses (IRs) were evaluated after the 6th and 12th vaccinations by CD4+ T-cell proliferation, CD8+ T-cell interferon-γ secretion (enzyme-linked immunospot), or the CD8-relevant WT1 peptide major histocompatibility complex tetramer assay (HLA-A*02 patients only). Twenty-two patients (7 males; median age, 64 years) were treated. Fourteen patients (64%) completed ≥6 vaccinations, and 9 (41%) received all 12 vaccine doses. Fifteen patients (68%) relapsed, and 10 (46%) died. The vaccine was well tolerated, with the most common toxicities being grade 1/2 injection site reactions (46%), fatigue (32%), and skin induration (32%). Median disease-free survival from CR1 was 16.9 months, whereas the overall survival from diagnosis has not yet been reached but is estimated to be ≥67.6 months. Nine of 14 tested patients (64%) had an IR in ≥1 assay (CD4 or CD8). These results indicated that the WT1 vaccine was well tolerated, stimulated a specific IR, and was associated with survival in excess of 5 years in this cohort of patients. This trial was registered at www.clinicaltrials.gov as #NCT01266083.

T-cell responses against neoepitopes presented by human leukocyte antigen (HLA) complexes represent a critical effector of anticancer immunity. However, detection of neoepitopes by mass spectrometry is still challenging as is identification of the fraction of neoepitopes that elicit immune responses in vitro and in vivo. To address these problems, we developed a strategy to identify HLA ligands combining the peptide identification algorithm Byonic with the epitope binding predictor netMHCpan. We obtained up to 4-fold increases in unique HLA ligand identifications compared to standard approaches, false discovery rates below 0.6% and over 17,000 unique HLA ligand identifications in a single experiment. Even with only 10 million cancer cells we were able to detect over 5,000 unique HLA ligands, which allows our approach to be applied to small tumor samples in a clinical setting. Of note, phosphorylated as well as glycosylated HLA ligands were identified with the same accuracy and allowed a better characterization of the rules for presentation of post-translationally modified HLA ligands. Furthermore, by reanalyzing mass spectrometry samples with our novel approach, we confirmed high-confidence, rejected low-confidence and identified additional neoepitopes including the first-time detection of a phosphorylated neoepitope. Finally, we used our broadened knowledge of the immunopeptidome to create rules for prediction of non-immunogenicity of neoepitopes based on high biochemical similarity with unmutated HLA ligands, which showed high specificity and positive predictive value when validated with two datasets derived from neoepitope-based clinical studies. Altogether, our methods substantially improved sensitivity and specificity for detection of native, modified and mutated HLA ligands, propose an explanation for tolerance to neoepitopes, and should in combination facilitate the design of neoepitope-based therapies. Citation Format: Martin G. Klatt, Ron S. Gejman, Sung S. Moon, Tatyana S. Korontsvit, Tao Dao, David A. Scheinberg. A new high-performance HLA ligand identification strategy enables prediction of T-cell tolerance to neoepitopes [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr PR13.

The Wilms' tumor oncogene protein (WT1) is a highly validated tumor antigen for immunotherapy. WT1-targeted immunotherapy has been extensively explored in multiple human trials in various cancers. However, clinical investigations using WT1 epitopes have generally focused on two peptides, HLA-restricted to HLA-A*02:01 or HLA-A*24:02. The goal of this study was to identify new epitopes derived from WT1, to expand the potential use of WT1 as a target of immunotherapy. Using computer-based MHC-binding algorithms and in vitro validation of the T cell responses specific for the identified peptides, we found that a recently identified HLA-A*24:02-binding epitope (239–247), NQMNLGATL (NQM), was also a strong CD8+ T cell epitope for HLA-A*02:01 molecule. A peptide second position Q240L substitution (NLM) or Q240Y substitution (NYM), further enhanced the T cell responses in both HLA-A*02:01 positive and HLA-A*24:02 positive healthy donors. Importantly, T cells stimulated with the new analog peptides displayed heteroclitic cross-reactivity with the native NQM sequence and were able to kill HLA-matched WT1-positive tumor cell lines and primary leukemia blasts. In addition, longer native and heteroclitic HLA-DR.B1-binding peptides, comprising the nine amino acid NQM or NLM sequences, could induce T cell response that recognized the CD8+ epitope NQM, suggesting the processing and the presentation by HLA-A*02:01 molecules of the CD8+ T cell epitope embedded within it. Our studies suggest that the analog peptides NLM and NYM could be potential candidates for future immunotherapy targeting WT1 positive cancers in the context of HLA-A*02:01 and A*24:02 positive populations.

Adoptive transfer of in vivo generated antigen-specific donor-derived T-cells is increasingly recognized as an effective approach for the treatment or prevention of EBV lymphomas and cytomegalovirus infections complicating allogeneic hematopoietic cell transplants. This review examines evidence from preclinical experiments and initial clinical trials to critically assess both the potential and current limitations of adoptive transfer of donor T-cells sensitized to selected minor alloantigens of the host or to peptide epitopes of proteins, differentially expressed by clonogenic leukemia cells, such as the Wilms tumor protein, WT-1, as a strategy to treat or prevent recurrence of leukemia in the post-transplant period.

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.

The emerging heterogeneity of dendritic cells (DCs) mirrors their increasingly recognized division of labor at myriad control points in innate and acquired cellular immunity. We separately generated blood monocyte-derived DCs (moDCs), as well as Langerhans cells (LCs) and dermal-interstitial DCs (DDC-IDCs) from CD34+ hematopoietic progenitor cells. Differential expression of CD11b, CD52, CD91, and the CD1 isoforms proved useful in distinguishing these three DC types. All mature DCs uniformly expressed comparable levels of HLA-DR, CD83, CD80, and CD86, and were potent stimulators of allogeneic T cells after exposure either to recombinant human CD40L trimer or a combination of inflammatory cytokines with PGE2. moDCs, however, required 0.5–1 log greater numbers than LCs or DDC-IDCs to stimulate comparable T cell proliferation. Only moDCs secreted the bioactive heterodimer IL-12p70, and moDCs phagocytosed significantly more dying tumor cells than did either LCs or DDC-IDCs. LCs nevertheless proved superior to moDCs and DDC-IDCs in stimulating CTL against a recall viral Ag by presenting passively loaded peptide or against tumor Ag by cross-priming autologous CD8+ T cells. LCs also secreted significantly more IL-15 than did either moDCs or DDC-IDCs, which is especially important to the generation of CTL. These findings merit further comparisons in clinical trials designed to determine the physiologic relevance of these distinctions in activity between LCs and other DCs.

During peripheral T cell deletion, lymphocytes accumulate in nonlymphoid organs including the liver, a tissue that expresses the nonclassical, MHC-like molecule, CD1. Injection of anti-CD3 Ab results in T cell activation, which in normal mice is followed by peripheral T cell deletion. However, in CD1-deficient mice, the deletion of the activated T cells from the lymph nodes was impaired. This defect in peripheral T cell deletion was accompanied by attenuated accumulation of CD8+ T cells in the liver. In tetra-parental bone marrow chimeras, expression of CD1 on the T cells themselves was not required for T cell deletion, suggesting a role for CD1 on other cells with which the T cells interact. We tested whether this role was dependent on the Ag receptor-invariant, CD1-reactive subset of NK T cells using two other mutant mouse lines that lack most NK T cells, due to deletion of the genes encoding either β2-microglobulin or the TCR element Jα281. However, these mice had no abnormality of peripheral T cell deletion. These findings indicate a novel role for CD1 in T cell deletion, and show that CD1 functions in this process through mechanisms that does not involve the major, TCR-invariant set of NK T cells.

Introduction: The Wilms' tumor 1 (WT1) protein is a tumor associated antigen that is potentially targetable by immunotherapeutic approaches. We have demonstrated the overexpression of WT1 in myeloma cells by IHC and in HLA-A*0201+ pts by staining with a high-affinity fully human IgG1 mAb (ESK1) specific to the RMFPNAPYL/HLA-A*0201 complex on malignant plasma cells. We report initial results from pts with plasma cell leukemia (PCL) or relapsed/refractory multiple myeloma (rMM) who have been treated with CD34-selected allo transplants followed by the administration of donor-derived WT1-specific T-cell infusions to induce an immunotherapeutic effect. Methods: In situ expression of WT1 was assessed by IHC analyses using a sequential double staining technique of MoAbs specific for CD138 and WT1.For staining with the RMFPNAPYL/HLA-A*0201 complex, BM samples were blocked with human FcR Blocking Reagent and then directly stained with MoAbs specific for CD38, CD56, CD45 and ESK1 or its isotype control human IgG1 and were analyzed by flow cytometry. WT1-specific T cells were generated from the original stem cell donors by sensitization of CD3+ enriched T-cell fractions with autologous APCs loaded with the pool of overlapping pentadecapeptides of WT1 (Invitrogen, Boston, MA). Cells were propagated in vitro with weekly restimulation and supplementation with IL-2 beginning at day 10-16. After 35-49 days, T-cells were harvested, counted and tested for antigen specific cytotoxicity, HLA-restriction, lack of alloreactivity and sterility. Pts received CD34-selected PBSC allografts after myeloablative cytoreduction with busulfan, melphalan and fludarabine. Pts were treated with 3 infusions of donor-derived WT1-specific T-cell infusions (5x10e6 cells/kg) starting 6 weeks post allo HSCT and at 4 weekly thereafter. Results: Marrow from all pts with immunohistochemical documented plasma cell involvement stained positive for WT1 IHC while WT1 staining remained negative in pts in CR. Only pts expressing HLA-A*0201 that stained positively for WT1 by IHC also demonstrated expression of WT1 by the RMFPNAPYL/HLA-A*0201 complex, whereas pts lacking HLA-A*0201 but with active disease stained positive for WT1 IHC but not ESK1 staining. Of 7 pts, 3 PCL and 4 rMM, treated with WT1-specific T cells, 4 pts had persistent disease post CD34-selected allotransplant. Of these 4 pts 2 pts developed a striking rise of WT1-specific T-cell frequencies and developed a complete remission post WT1 CTL infusions lasting for >2years. Conclusion: WT1 is overexpressed on malignant plasma cells and serves as a target for potential immunotherapeutic approaches in pts with multiple myeloma. Pts with persistent PCL following CD34-selected allografts treated with adoptive transfer of donor-derived WT1-specific cytotoxic T cells can achieve long lasting remission underscoring the therapeutic potential of T-cells specific for immunogenic WT1 peptides expressed on malignant plasma cells. Disclosures O'Reilly: Atara Biotherapeutics: Research Funding.

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.

Immunosuppression by tumor-induced regulatory T (Treg) cells present major obstacles for successful immunotherapy. Treg expansion and its negative prognostic impact represent a general phenomenon seen in multiple types of cancer. Therefore, developing strategies for Treg depletion could enhance the reactivation of immune responses against the malignant tumor cells. However, the strategies of depleting Tregs have been hindered by the lack of specificity, which also leads to the depletion of anti-tumor effector cells. The transcription factor forkhead box p3 (FoxP3) is selectively expressed in and is essential to the suppressive function of Treg cells. FoxP3 thus would be an appealing target for eliminating Treg cells. However, Foxp3 is an undruggable intracellular protein. Here, we took a novel and unconventional approach to target Foxp3 by using a T cell receptor mimic (TCRm) mAb, which recognizes a human Foxp3-derived CD8 T cell epitope, presented by HLA-A*02:01 molecule. We show that the Foxp3 mAb specifically bound to CD4+CD25hiCD127lo Foxp3+ Treg cells from HLA-A0201 positive donors, and tumor cell lines co-expressing Foxp3 and HLA-A*02;01 molecule. Both afucosylated Fc enhanced human IgG1 and bispecific T cell engager formats of the Foxp3 mAb are able to kill in vitro-generated Treg clones from HLA-A0201+ donors and “Treg-like” cutaneous lymphoma cells (HLA-A*02:01+) that have a high level expression of CD4, CD25 and Fox3+. FoxP3-targeting antibodies could potentially be a novel approach in cancer immunotherapy by overcoming immunosuppression caused by Tregs and tumor cells expressing Foxp3. Citation Format: Tao Dao, Casey Jarvis, Andrew C. Scott, Tatyana Korontsvit, Victoria Zakhaleva, Dmitry Pankov, Manuel Direito de Morais Guerrerio, Melissa Mathias, Neal Cheng, Cheng Liu, David A. Scheinberg. Selective targeting of T regulatory cells by a TCR-mimic monoclonal antibody specific for foxp3-derived epitopes [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A058.

Adoptive therapy with chimeric antigen receptor (CAR) T cells specific for CD19 is clinically successful in a limited set of leukemias and most CARs studied are targeted against external antigens. Wilms Tumor Antigen 1 (WT1) protein (WT1) is an intracellular antigen overexpressed in many cancers, including leukemias and solid malignancies and is thus an appealing, broadly applicable target. We have engineered the first armored T cell receptor-mimic (TCRm) CAR against WT1. Derived from the ESK1 antibody, the second generation CAR, WT1-28z, is reactive with the RMFPNAPYL peptide of WT1 that is processed and presented on the surface of cells in the context of HLA-A*02:01. WT1-28z was further modified to secrete human IL-12 cytokine, thus creating the armored CAR WT1-28z/IL-12. T cells expressing WT1-28z or WT1-28z/IL-12 are cytotoxic against a range of both hematological and solid tumors. Importantly, both WT1-directed T cells are specific for the WT1-HLA-A*02:01 complex and are not reactive against cells that do not express both HLA-A*02:01 and WT1. In established SCID/Beige mouse models of either acute leukemia or ovarian cancer, one dose of WT1-28z T cells prolongs survival of mice over untreated or irrelevant antigen-specific CAR T cell treated mice. Furthermore, one dose of the armored WT1-28z/IL12 CAR T cells further significantly prolongs survival of mice in both models over WT1-28z CAR T cell treated mice, with a subset of mice whose disease was eradicated. The armored TCRm CAR T cells against WT1 are effective in eradicating disease in both hematologic and solid tumors and may hold great clinical potential to expand on the success of CAR T cell therapy.

Bi-specific T cell engager antibody (BiTE) therapy has recently emerged as an effective immunotherapy by redirecting polyclonal T cell cytotoxicity against cell surface protein on tumor cells. We generated the first BiTE construct derived from a TCR-mimic monoclonal antibody (mAb), ESK1, specific for a peptide from an intracellular oncoprotein, WT1, in the context of HLA-A*02:01 molecules. Despite the low density peptide/HLA-A2 complex on the cell surface, ESK-BiTE was able to selectively activate and induce proliferation of cytolytic human T cells to kill multiple leukemias and cancers in vitro and in mice. Surprisingly, we also discovered that in an autologous setting, ESK-BiTE induced a robust secondary CD8 T cell response specific for antigens other than WT1, including HLA-A2-restricted her2-neu-derived peptide 369-377, in patients with her2- positive ovarian cancer. Therefore, the study demonstrated a new vaccinal mechanism for BiTE mAb action that could contribute to more effective long-term therapeutic activity of BiTE's and further broaden their reach to other tumor antigens not previously known or originally targeted. Citation Format: Tao Dao, Dmitry Pankov, Andrew Scott, Tatyana Korontsvit, Victoriya Zakhaleva, Manuel Direito de Morais Guerrerio, Yiyang Xu, Jingyi Xiang, Su Yan, Nicholas Veomett, Nicholas Veomett, Leonid Dubrovsky, Michael Curcio, Ekaterina Doubrovina, Cheng Liu, Richard J. O'Reilly, David A. Scheinberg. Potent therapeutic and immunological effects of the first T-BiTE derived from a TCR-mimic antibody targeting intracellular oncoprotein WT1. [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 A055.

The production of interleukin-2 (IL-2) by phytohemaglutinin (PHA)-stimulated cells of human leukemia T cell line MOLT-16 can be significantly increased by interleukin-1 (IL-1) or interferon-alpha (IFN-alpha). The enhancing effect of IL-1 and IFN-alpha on IL-2 production was studied at the IL-2 mRNA level. We show that IL-1 enhances considerably and transiently, with the maximum level between 1 and 2 hr after stimulation, the expression of IL-2 mRNA in the PHA-stimulated cells. The level of IL-2 mRNA declined rapidly within 4 to 6 hr after stimulation in both PHA- and PHA plus IL-1-stimulated cell cultures. On the contrary, IFN-alpha does not elevate the level of IL-2 mRNA above the level in PHA-stimulated cultures, but maintains an enhanced level of IL-2 mRNA in the activated cells for more than 6 hr after stimulation. These observations correlate well with the kinetics of IL-2 protein production into the culture media. The results thus suggest that IL-1 and IFN-alpha may exert an enhancing effect on IL-2 production by distinct mechanisms. In addition, none of the five other lymphokines tested (i.e., IL-2, IL-3, IL-4, IL-5, and IL-6) had any significant effect on IL-2 mRNA expression in the activated MOLT-16 cells.

The production of interleukin 2 (IL-2) by phytohemagglutinin (PHA)-stimulated human leukemia T cell lines MOLT-13 and MOLT-14, the T cell receptor (TcR) gamma delta-bearing clones, was significantly increased by adding human natural interferon-gamma (IFN-gamma) to the cell cultures. In addition, the IL-2 production by PHA-stimulated MOLT-13 and MOLT-14 cells was also augmented by human natural tumor necrosis factor-alpha (TNF-alpha) in a dose-dependent manner but was inhibited by IFN-alpha even at low concentrations (10 IU/ml). This pattern of reactivity is different from the responsiveness of the MOLT-16 cells, a TcR alpha beta-bearing cell line, which respond to IFN-alpha and TNF-alpha, but not to IFN-gamma, by augmentation of IL-2 production. These results suggest that (1) the addition of IFN-gamma has a direct effect on PHA-stimulated MOLT-13 and MOLT-14, resulting in an augmentation of IL-2 production; (2) phenotypically different T cell lines vary in their susceptibility to IFN-alpha and IFN-gamma regulation; and (3) the augmentation of IL-2 production by TNF-alpha appears to have no correlation with phenotypically different T cell clones.

A pilot study was undertaken to assess the safety, activity, and immunogenicity of a polyvalent Wilms tumor gene 1 (WT1) peptide vaccine in patients with acute myeloid leukemia in complete remission but with molecular evidence of WT1 transcript. Patients received 6 vaccinations with 4 WT1 peptides (200 μg each) plus immune adjuvants over 12 weeks. Immune responses were evaluated by delayed-type hypersensitivity, CD4+ T-cell proliferation, CD3+ T-cell interferon-γ release, and WT1 peptide tetramer staining. Of the 9 evaluable patients, 7 completed 6 vaccinations and WT1-specific T-cell responses were noted in 7 of 8 patients. Three patients who were HLA-A0201-positive showed significant increase in interferon-γ–secreting cells and frequency of WT1 tetramer-positive CD8+ T cells. Three patients developed a delayed hypersensitivity reaction after vaccination. Definite related toxicities were minimal. With a mean follow-up of 30 plus or minus 8 months after diagnosis, median disease-free survival has not been reached. These preliminary data suggest that this polyvalent WT1 peptide vaccine can be administered safely to patients with a resulting immune response. Further studies are needed to establish the role of vaccination as viable postremission therapy for acute myeloid leukemia. This study was registered at www.clinicaltrials.gov as #NCT00398138.

Cyclin D1 is over-expressed in various human tumors and therefore can be a potential oncogenic target antigen. However, only a limited number of T cell epitopes has been characterized. We aimed at identifying human cyclin D1-derived peptides that include both CD4 and CD8 T cell epitopes and to test if such multi-epitope peptides could yield improved cytotoxic CD8 T cell responses as well as cytotoxic CD4 T cells. Five HLA-DR.B1-binding peptides containing multiple overlapping CD4 epitopes and HLA-A0201-restricted CD8 T cell epitopes were predicted by computer algorithms. Immunogenicity of the synthetic peptides was assessed by stimulating T cells from healthy donors in vitro and the epitope recognition was measured by IFN-gamma ELISPOT and (51)Chromium release assays. A HLA-DR.B1 peptide, designed "DR-1", in which a HLA-A0201-binding epitopes (D1-1) was imbedded, induced CD3 T cell responses against both DR-1 and D1-1 peptides in IFN-gamma ELISPOT assay. This suggested processing of the shorter D1-1 epitope from the DR-1 sequence. However, only DR-1-stimulated CD4 or CD3 T cells possessed cytotoxicity against peptide-pulsed autologous DCs and a cancer cell line, that expresses a high level of cyclin D1. Monoclonal antibody to HLA-DR abrogated the epitope-specific responses of both CD3 and CD4 T cells, demonstrating class II-mediated killing. Our studies suggest a possible role of CD4 T cells in anti-tumor immunity as cytotoxic effectors against HLA-DR expressing cancers and provide a rationale for designing peptide vaccines that include CD4 epitopes.

The development of cancer vaccines directed against myeloid leukaemias has been a research area of intense interest in the past decade. Both human studies in vitro and mouse models in vivo have demonstrated that leukaemia-associated antigens (LAAs), such as the fusion protein BCR-ABL, Wilms' tumour protein and proteinase 3, may serve as effective targets for cellular immunotherapy. Peptide-based vaccines are able to induce cytotoxic T-lymphocyte responses that kill leukaemia cells. Based on these results, pilot clinical trials have been initiated in chronic and acute myeloid leukaemia and other haematological malignancies, which include vaccination of patients with synthetic peptides derived from these LAAs. Results from these trials show that peptide vaccines are able to induce immune responses that are sometimes associated with clinical benefit. These early clinical results are promising and provide valuable information for future improvement of the vaccines. This chapter will focus mainly on discussing the preclinical studies of peptide vaccines in human systems, the results from clinical trials and the future prospects for vaccine therapy for myeloid leukaemia.

The major hurdle to the creation of cancer-specific monoclonal antibodies (mAb) exhibiting limited cross-reactivity with healthy human cells is the paucity of known tumor-specific or mutated protein epitopes expressed on the cancer cell surface. Mutated and overexpressed oncoproteins are typically cytoplasmic or nuclear. Cells can present peptides from these distinguishing proteins on their cell surface in the context of human leukocyte antigen (HLA). T cell receptor mimic (TCRm) mAb can be discovered that react specifically to these complexes, allowing for selective targeting of cancer cells. The state-of-the-art for TCRm and the challenges and opportunities are discussed. Several such TCRm are moving toward clinical trials now.

Purpose: Presentation of exogenous antigen by MHC class I molecules, or cross-presentation, is a property of dendritic cells, which is considered crucial for the priming of cytotoxic T-cell response to tumor antigens. However, the precise mechanisms of this process are not fully understood.Experimental Design and Results: We show here in a human in vitro system, using B lymphoma cells as a tumor model, that the cross-presentation of cell-associated antigens to T cells by dendritic cells requires “help” from natural killer cells. When autologous dendritic cells that had taken up apoptotic B lymphoma cells and induced to a fully mature state were used to stimulate nonadherent cells of peripheral blood mononuclear cells from healthy donors, they induced strong cytotoxicity against B lymphoma cells in a HLA-A0201-restricted manner. The cells failed to induce cytotoxicity, however, when purified T cells were used as effector cells. Depletion of CD56+ cells, but not CD14+ or CD19+ cells, abrogated the cytotoxicity of nonadherent cells, showing that the help was provided by natural killer cells. Further, when natural killer cells were present in the cultures, a strong and persistent production of interleukin-18, but not interleukin-12 and interleukin-15, was observed. Blocking interleukin-18 significantly reduced the cytotoxicity of nonadherent cells against B lymphoma cells.Conclusions: These results suggest that capture of tumor cells and a full maturation status of dendritic cells are not sufficient to cross-prime CD8 T cells. Effective cross-priming requires further activation of dendritic cells by natural killer cells and an abundant production of interleukin-18, which, along with other yet undefined mechanisms, contribute to the generation of CTL response against B-cell lymphoma.

The vast majority of cellular proteins, many of which are the most interesting and important molecules for regulating normal and neoplastic growth, lie within the cell, hidden from monoclonal antibody (mAb) therapeutics by the barriers of the plasma membrane. We recently demonstrated in vivo the successful treatment of several human cancers by use of a human “TCR-like” mAb to the intracellular oncogenic protein, WT1, a difficult to drug cancer target (Dao et al, Science Transl. Med. 5:176r33, 2013.) Mab have emerged in the last 15 years as some of the most important drugs for cancer, inflammatory diseases and infections. New methods to rapidly select mAb from libraries, to re-engineer mAb with new functionalities, and to make mAb fully human to reduce immunogenicity, has greatly expanded their versatility. The biochemical features of mAb (large size, charge and protein structure) are still an important impediment that restricts diffusion and penetration into cells. Therefore, the exquisite specificity of mAb is prevented from addressing some of the only truly specific cancer targets, such as mutated signaling molecules and transcription factors, fusion-protein oncogenes and many other tumor associated antigens. How does one use an antibody to reach these interesting targets and kill the cell? MAb have reached intra-cellular targets, but usually after the cell has lysed, releasing histones as an example, or tumor-associated vesicular cargo into the extracellular milieu such as in melanosomal granules, or exposing intracellular proteins by permeabilized membranes. Indeed, there is a FDA-approved mAb imaging agent, Prostascint, that reacts with an intracellular epitope, and thus is only exposed upon death of the cell. In this context, one alternative approach is to select intracellular antigenic targets that are exposed on the cell surface as part of the normal process of protein catabolism and presentation on MHC molecules. Intracellular proteins are usually degraded by the proteasome or endo/lysosomes, and the resulting specific peptide fragments bind to MHC class molecules. These peptide-MHC complexes are displayed at the cell surface where they provide targets for T cell recognition via peptide-MHC T cell receptor (TCR) interaction. The idea of using TCR-like mAb for studying immunobiology and ultimately treating cancer dates back more than a decade and has been nicely reviewed by Dohan and Reiter (Expert Rev Mol Med. 14:e6, 2012.). Even with the enlarging preclinical demonstration of such TCR-like therapeutic mAb, there remains considerable skepticism as to their promise. The first concern is the low target density on diseased cell surface. With tens of thousands of peptides processed for binding to MHC class molecules within the cell, the likelihood that any one peptide will be expressed on the cell surface in context of HLA molecules in large quantities is small. Many predict that fewer than 10 copies of an individual peptide MHC complex will be presented. That almost all of FDA-approved antibody drugs require tens of thousands of target molecules per cell makes it improbable for such a low density antigen to work. However, for TCR-based T-cell responses, this number appears adequate for effective killing of target cells, based on work from several laboratories. Then why is this not sufficient for mAb mediating human effectors? We observed clearance of disseminated human leukemias in NSG mice with as few as several hundred epitopes present per cell(Dao et al, Science Transl. Med. 5:176r33, 2013.). Moreover, new technology that brings together mAb specifity with T-cell potency have emerged that may make these prejudices against low density targets obsolete. For example, chimeric antigen receptor engineered T cells (CAR T cells) recognize mAb-specific surface targets yet kill cancer cells like a T cell, resulting in patient responses. In addition, bi-specific mAbs, which closely cross-link the target cell to an effector T cell, have also displayed exceptional potency in humans. Second, we and others have seen that the expression of peptide-MHC epitope is not always a few per cell surface, but can be on the order of 5-10,000, a level that is easily approached by conventional mAb therapy or antibody drug conjugates. For example, mAb directed to CD33 have been approved in the treatment of leukemia. Finally, it is possible to upregulate MHC by pharmacologic means, and this may be an approach if antigen density is truly limiting. Therefore, the risk for low antigen density should not discourage the development therapeutic reagents to these targets. A second hurdle was the notion that peptide-MHC complex internalize poorly or slowly, rendering the use of antibody drug conjugates or radio-conjugates problematic. While this may be true, TCR-like mAb immunotoxins have been shown to be effective in mouse cancer models. In addition, radio-conjugates do not need to be internalized and the potency of alpha-particle emitting and alpha- particle isotope generators, which can kill a cell with a single hit, are certainly potent enough for these epitope densities. A third criticism is the risk of cross-reactivity with human MHC expressed on all the nucleated cells. This would be a pharmacologic hurdle and possibly a risk for toxicity. Overcoming this problem can be achieved with increasing ease due to the use of phage-display technology and high stringencies in screening to select specific mAb. A final criticism of the TCR-like mAb approach is that the mAb is HLA-restricted, thereby limiting its use. This is a soft argument in the increasing context of “personalized medicine”, in which each drug will be directed to a subset of patients with a type of cancer. Importantly, given the dominance of just a few HLA worldwide, a TCR-like antibody to an important, widely expressed intracellular oncogenic protein, that crosses many tumor types, could easily still be used in many thousands of patients around the world. In conclusion, while there are still unanswered questions about the best use, most appropriate targets, and different formats of TCR-like mAb, the lure of finally killing a cell based on the tumor-specific recognition of one of the myriad of important intracellular proteins is strong. In addition, even currently drug-able targets might be suitable mAb targets, as small molecule inhibitors allow escape of the cell via compensatory pathways, relief of feedback inhibition, or mutation of the binding pocket. In contrast, a cytotoxic mAb recognizes the expression of the epitope and not its function, making escape unlikely via these mechanisms. The first human trials of such mAb drugs are likely within the next one to two years. Based on the great success of mAb therapy to date, the selectivity and lack of toxicity, versatility, and ease of development, we are confident that that the reach of the arms of mAb will get longer.

Using genetic and phenotypic analyses, we have analyzed the developmental pathway of mouse CD1d-restricted invariant NKT cells. We provide strong evidence that similar to conventional T cells, positive selection of NKT cells occurs during a CD4(+)CD8(+) stage. Later stages of NKT cell development involved the down-regulation of both TCR and CD4 levels and therefore diverge from conventional T cell development pathways. A unique and complete dependency for development on Fyn, a Src family kinase member, also distinguishes the NKT cell and conventional T cell populations.

We examined the specificity of positive and negative selection by using transgenic mice carrying a variant of the D10 TCR. We demonstrate that a point mutation at position 51 within the CDR2α segment significantly reduces the avidity of this TCR for its cognate ligand, but does not impact recognition of nonself MHC class II molecules. Although structural studies have suggested that this TCR site interacts with the MHC class II β-chain, the avidity of this TCR for its ligand and the function of the T cell can be reconstituted by a point mutation in the bound antigenic peptide. These data demonstrate that the bound peptide can indirectly alter TCR interactions by influencing MHC structure. Remarkably, reducing the avidity of this TCR for a specific antigenic peptide-MHC ligand has a dramatic impact on thymic selection. Positive selection of thymocytes expressing this TCR is nearly completely blocked, whereas negative selection on allogenic MHC class II molecules remains intact. Therefore, the recognition of self that promotes positive selection of the D10 TCR is highly peptide-specific.

The Wilms’ tumor oncogene protein (WT1) is an intracellular, oncogenic transcription factor that is over-expressed in a wide range of leukemias and solid cancers. RMFPNAPYL (RMF), a WT1-derived CD8 T cell HLA-A0201epitope, is a validated target for T cell-based immunotherapy. We generated a high affinity, fully human IgG1 mAb specific for the RMF/HLA-A0201 complex. The mAb shows potent anti-leukemia activity both in vitro and in vivo in mouse models. Bi-specific T cell engaging antibodies (BiTE) have been used effectively to target cell surface proteins and kill cancers. We have developed a new, potent form of the ESK mAb that is a bi-specific T cell engaging antibody (BiTE) specific for tumor cells coexpressing the intracellular oncoprotein, WT-1 and HLA A0201. ESK-BiTE and an irrelevant control BiTE were constructed with ESK1 scFv or irrelevant ScFv on one arm, and anti-CD3 ScFv fragment as the other arm. The BiTE constructs were expressed in CHO cells. Both the ESK-BiTE and the control BiTE bind human CD3 T cells. The ESK-BiTE selectively bind to leukemia cells that express both WT1 and HLA-A0201. The ESK-BiTE activated human resting T cells and EBV-specific T cells retargeting potent cytotoxicity against WT-1+ HLA A0201+ human leukemia cells in vitro. In an NSG mouse xenograft model, injection of ESK-BiTE (20 ug/ml) twice a week, following I.V. administration of 2x107 human EBV-specific T cells, once a week, significantly inhibited the growth of a previously established disseminated HLA A0201+, WT-1+ human Ph+ ALL, BV173 expressing luciferase, as measured by bioluminescence imaging. In a second NSG mouse model mice injected I.V. with an aggressive human AML, SET-2, on day 0, were treated on day 4 with ESK-Bite for 6 days consecutively at 20 ug/day together with EBV-specific T cells given twice a week. In this setting, the ESK-BiTE and T cells resulted in undetectable leukemic growth for 14 days post-leukemia inoculation, with a minimal tumor burden detected by day 18, while all control groups showed massive increases in leukemia burden by day 14. Mice bearing SET-2 leukemia, that received ESK-BiTE and T cells also showed longer survival and delayed limb paralysis. As expected, the human T cells, which were EBV-specific, did not induce signs of GVHD in mice. Our data provide evidence that ESK-BiTE is a potent and specific therapeutic agent against aggressive human leukemias expressing WT1 and HLA-A0201. This is the first study showing efficacy of a TCR-like BiTE antibody targeting an intracellular tumor antigen expressed at low density. Supported by the Leukemia and Lymphoma Society, NIH R01CA55349 and P01 23766. Disclosures: No relevant conflicts of interest to declare.

Hepatocytes constitutively express CD95 (also called Fas/APO-1) and are therefore potential targets for CD95-ligand (CD95L)-mediated injury. To study this mechanism of cell death in hepatocytes we developed an in vitro model of liver cell apoptosis using membrane-bound CD95L as the inducing agent. Primary mouse hepatocytes were cocultured with NIH 3T3 fibroblasts, stably transfected with mouse CD95L (FCD95L+). Fibroblasts stably transfected with vector only (FCD95L−) served as controls. Hepatocytes from mice expressing low levels of CD95 (Faslpr mice) served as controls for effects unrelated to CD95. Morphologic and biochemical studies indicate that CD95 is expressed in cultured mouse hepatocytes. Membrane-bound CD95 from transfected fibroblasts destroyed all cocultured hepatocytes within 24 hours in the absence of protein synthesis inhibitors. Characteristic features of apoptosis were observed in dying hepatocytes and occurred in the following sequence: formation of cytoplasmic blebs and nuclear condensation after 3 hours; nuclear fragmentation and DNA strand breaks after 4 hours. These changes were observed only when normal hepatocytes were cocultured with FCD95L+ and were not observed with FCD95L− or in hepatocytes from Faslpr mice. Anti-CD95 antibody (Jo2) evoked similar changes in hepatocytes, although to a much lesser extent. We conclude that coculture of mouse hepatocytes with FCD95L+ is a useful in vitro model for CD95-mediated apoptosis induced by CD95L. The high incidence of apoptosis caused by membrane-bound CD95L differs from the much smaller effects induced by the Jo2 antibody. In view of the high sensitivity of hepatocytes towards CD95L we speculate that CD95L-induced liver damage in vivo may be minimized by restricting exposure of hepatocytes to CD95L. (Hepatology 2000;32:779-785.)

Abstract 2599 The Wilms' tumor oncogene protein (WT1) is an intracellular, oncogenic transcription factor that is over-expressed in leukemias and a wide range of cancers. WT1 may be expressed in leukemia stem cells. RMFPNAPYL, “RMF”, a WT1-derived CD8 T cell epitope presented by HLA-A0201, is a validated target for T cell-based immunotherapy, used in multiple clinical trials of vaccines and cellular therapies. Therefore, we hypothesized that a “TCR-like antibody” specific for the WT1 peptide/HLA-A0201 complex might be an effective therapeutic agent. Using phage display technology, we generated 2 lead, high avidity (Kd < 0.2nM), fully human monoclonal antibodies (mAb) specific for the WT1 RMF peptide/HLA-A0201 complex. One version, ESK1, is a native human IgG1. A second version, ESKM, with enhanced antibody dependent human effector cell cytotoxicity (ADCC) function due to altered Fc glycolsylation was also prepared. ESK mAb bind to leukemia lines and other cancer cell lines, as well as primary leukemia cells that are both WT1+ and HLA-A0201+. In vitro, both ESK mAb mediated ADCC against CML cells, at concentrations below 3 ug/ml, but ESKM was about 8–10 fold more potent and could kill targets with far fewer peptide/MHC complexes expressed on the cell surface. At therapeutic doses of ESKM, there was no difference in biodistribution between the 2 mAb in C57BL6 mice or in mice that were transgenic for HLA-A0201. Low doses of ESK (25–100ug twice) effectively treated an established disseminated, ALL or human bcr/abl + lymphoid leukemias in a NSG mouse model (T, B and NK deficient) and prolonged survival. In mice, ESKM was slightly more effective than ESK1. An F(Ab')2 version of the antibody had no anti-tumor effect, indicating that an Fc-mediated mechanism plays the major role in therapy. When combined with a single infusion of human CD34-, CD3-, human NK and monocyte effectors in the NSG mice, therapeutic effects of the mAb were more pronounced and more durable. There was no therapeutic effect of either mAb on WT1 low/A0201 negative disseminated Daudi ALL in mice. There was no observed toxicity in HLA-A0201 transgenic mice at the therapeutic mAb dose and schedule. ESK mAb are potential therapeutic agents for ALL, CML, other leukemias and cancers over-expressing the WT1 oncoprotein. Its expression in early leukemia cells may allow for elimination of the progenitors. The data also provide proof of concept for developing therapeutic mAb targeting important intracellular oncogenic proteins. Disclosures: Yan: Eureka: Employment, Equity Ownership. Liu:Eureka: Employment, Equity Ownership.

The production of interleukin-2 (IL-2) by mitogen-stimulated human peripheral blood leukocytes (PBL) was significantly enhanced by a short pretreatment of PBL with direct current (DC) at low intensities. Exposure of PBL to DC of 1 mA for more than 10 min resulted in inhibition of IL-2 production which correlated with decreased proliferation capacity of the treated cells. DC had similar enhancing and inhibitory effects on IL-2 production by the mitogen-stimulated human leukemia T cell line MOLT-13. However, DC itself did not stimulate IL-2 production in PBL or in MOLT-13 cells. In two additional T cell lines tested, MOLT-16 and H-9, DC did not enhance mitogen-stimulated IL-2 production, but suppressed it when applied at 1 mA for more than 10 min. Both the mitogen-stimulated and the interleukin-1 (IL-1)-enhanced IL-2 production were completely inhibited by 1 mA of DC applied for 20 min. The observed effects of DC were due to a direct action of DC on the cells and not to possible effects of the secondary by-products of electrolysis. These results show that pretreatment of cells with DC can enhance or suppress IL-2 production depending on the intensity of DC and the type of IL-2 producing cell clones.

Abstract 1677 The Wilms' tumor oncogene protein (WT1) is an attractive target for immunotherapy for a wide range of leukemias and cancers. WT1 is an intracellular transcription factor that is widely expressed in acute and chronic leukemias, but not appreciably in normal adult tissues. WT1 has been implicated in oncogenesis and appears to be expressed in the CML stem cell. Thus far, the intracellular location of WT1 has limited its use as a cancer target to T cell-based vaccine therapies. Several such vaccines directed to WT1 peptides are in clinical trials worldwide. However, such approaches is likely to be weaker and slower acting than typical antibody therapies that act directly to kill cells, especially for a leukemia that is not in remission. The WT1 peptide (RMFPNAPYL) is presented by HLA-A0201 and induces cytotoxic CD8 T cells capable of killing WT1+ leukemia cells. Therefore, we hypothesized that a monoclonal antibody (mAb) specific for WT1 peptide/HLA-A2 complexes on the cell surface (a “TCR-like antibody”) could be an effective direct therapeutic agent alone, or armed with potent anti-cancer agents. In addition, such an antibody would provide a potential tool for identifying appropriate patients for clinical trials of both WT1 vaccine and WT1 mAb therapies, or as a prognostic tool. Using phage-display technology, we selected a number of human scFv phage mAbs specific for WT1 peptide RMFPNAPYL, when bound in the context of HLA-A0201 molecules. Bivalent, full-length human IgG1 mAbs were next constructed and tested for biochemical and immunological functions. The scFv phage mAbs bound specifically to T2 cells (TAP deficient) pulsed with WT1 peptide RMFPNAPYL, but not other HLA-A0201-binding peptides, demonstrating specific recognition of the WT1peptide/HLA-A0201 complex. Flow cytometry and radio-immunoassay was used to quantify sites, determine avidity, and show specificity. The IgG1 mAb showed high binding affinity (Kd= 0.2 nM) for the WT1 peptide/A0201 complex on live cells. The IgG1 mAb also recognized leukemia and tumor cell lines (n= 8 of 12) that expressed WT1, in an HLA-A0201, WT1 peptide-restricted manner, but not WT1-negative or HLA-A02-negative cells (n=13). The mAb was capable of killing WT1 peptide-pulsed T2 cells, as well as un-pulsed WT1+, A0201 + cancer cell lines, demonstrating presentation of the epitope in sufficient numbers for therapeutic attack. Complement mediated killing of cells was not seen. Alanine scanning of the epitope pointed to peptide position #1 as critical. In conclusion, cytotoxic human monoclonal antibodies targeting WT1 peptide/HLA complexes represent a novel therapeutic approach to target leukemias and solid tumors that over-express the intracellular WT1 oncoprotein. Disclosures: Yan: eureka therapeutics: Employment. Zhou:eureka therapeutics: Employment. Liu:eureka therapeutics: Employment.

WT1 is a transcription factor over-expressed in several types of leukemia and solid tumors which has the potential to serve as a target for immune based therapies. Previous studies have demonstrated the ability of WT1 peptides to induce specific HLA restricted T cell cytotoxic responses. In an effort to overcome immune tolerance associated with this self-antigen, we used computer prediction analysis to design heteroclitic peptides derived from WT1 protein sequences by substituting various amino acids at key HLA A0201 binding positions. A selected peptide (WT1-A1) was found to stabilize MHC class I molecules better than native sequences and was also able to cross react with native WT1 sequences to kill HLA matched tumor cells. A modified version of the heteroclitic peptide was also created by adding flanking amino acids (WT1-122A1) to the core sequence in an effort to extend reactivity of this peptide to also include a broad range of HLA class II molecules. These 2 peptides were then combined with 2 native WT1 sequences (WT1-427 & WT1-331) capable of recruiting CD4 T cell response, to form a polyvalent vaccine with the potential to react across a number of HLA-DR subtypes. A clinical pilot study was designed to test the safety, activity and immunogenicity of this vaccine in patients with Acute Myelogenous Leukemia (AML) in complete remission, but with molecular evidence of WT1 transcript as measured by quantitative polymerase chain reaction (RQ-PCR) or with advanced mesothelioma or non-small cell lung cancer (expressing WT-1 protein by immunohistochemistry). Each patient receives 6 vaccinations with the 4 WT1 peptides (200mcg each) plus the immune adjuvant Montanide over 12 weeks. In addition, GM-CSF injections are administered to local sites of inoculation 2 days before and on the day of vaccination. Study design called for 10 patients in each group (solid tumor; hematologic malignancy). Immune responses are evaluated by delayed-type hypersensitivity, CD4 T-cell proliferation, CD4 and CD8 T-cell interferon-gamma release, and WT1 peptide tetramer staining. In the AML patients, minimal residual disease was assessed by sequential RQ-PCR at baseline and after the 3rd and 6th vaccines. To date, 8 patients (4 AML, 3 mesothelioma, 1 non-small lung cancer) have been enrolled on study. Two of the mesothelioma patients discontinued the vaccines after 3 and 6 doses because of progression of disease. One other mesothelioma patient has stable disease following 10 vaccinations. Two of the 4 AML patients have relapsed while receiving the vaccine (one after 3rd dose, the other after the 6th). Each had evidence of increasing WT-1 transcript at the time of relapse. One other remains in CR and has a transcript level 50% of baseline. No significant adverse events have been observed to date in the patients undergoing vaccination. Three of 4 evaluable patients showed evidence of either a CD8 or CD4 immune response to the vaccinating peptides and native peptides in vitro. In summary, preliminary data suggest that this heteroclitic WT1 peptide vaccine can be administered safely to patients and that immune responses can be elicited.

Wilms tumor protein 1 (WT1) is a transcription factor over-expressed in several types of leukemias and solid tumors, making it an ideal target for immunotherapy. A number of class I binding WT1 peptides have been identified and shown to stimulate CD8+ T cells. These peptides are being tested as potential cancer vaccine candidates in a variety of clinical trials. However, the induction and maintenance of a robust memory CD8+ cytotoxic T cell response requires CD4+ T cell help. Herein we report the identification of three HLA Class II peptide epitopes of WT1 using the SYFPEITHI and RANKPEP predictive algorithms. Peptides 328–349 and 423–441 are able to stimulate a peptide specific CD4+ response that can recognize WT1 positive tumor cells in multiple HLA-DRB*1 settings, as determined by IFN-gamma ELISPOT assays. Due to the highly polymorphic nature of the HLA class II alleles, such broad reactivity is critical in the development of a universal therapeutic. In addition, we identified a WT1 CD4+ peptide epitope (122–140) that lies within close proximity to a previously identified CD8+ peptide epitope (126–134). Residue 126 was mutated from an Arginine (R) to a Tyrosine (Y) thereby embedding a synthetic immunogenic analog CD8+ peptide that was previously designed to improve immunogenicity and induce a potent CD8+ response. Mutated peptide 122–140 is able to induce a CD4+ and cytotoxic CD8+ WT1 specific T cell response that can recognize the native WT1 epitopes on the surface of human CML and solid tumor cells. Cross-priming experiments demonstrated that APCs pulsed with either CML or mesothelioma tumor lysates can process and present each of the CD4+ peptides identified. These studies provide the rationale for using the three WT1 CD4+ peptides in conjunction with CD8+ peptide epitopes to vaccinate patients with WT1 expressing cancers.

6514 Background: CML is characterized by a tumor-specific fusion oncoprotein, BCR-ABL. Peptides spanning the B3A2 breakpoint of BCR-ABL can elicit MHC restricted T cell responses and clinical responses. No such data have previously been reported for vaccines targeting B2A2. Methods: We initiated a clinical trial administering synthetic analog breakpoint specific peptide vaccines for either B3A2 or B2A2 breakpoints to CML patients with major or complete cytogenetic remission. Measurable minimal disease was determined by either quantitative polymerase chain reaction (RQ-PCR) or nested PCR for BCR-ABL. Vaccine was administered with GM-CSF and Montanide ISA 51 subcutaneously. Eleven vaccinations were planned over the course of 1 year with the first 5 doses administered bi-weekly. Results: Eleven of 20 planned patients have been accrued to the study. Eight had documented cytogenetic remission and 3 were BCR-ABL positive via FISH analysis. Seven of the 11 patients had measurable BCR-ABL transcript levels as determined by RQ-PCR and all 11 were positive using the nested technique. Of the 5 patients positive for BCR-ABL (by RQ-PCR) before vaccination, 3 converted from RQ-PCR positive to negative while the transcript levels in the other patients decreased by approximately 1 log after 5 doses. All 5 of these patients remained positive using the nested PCR technique. Vaccination was well tolerated with local skin reactions at the injection sites. Immunologic reactivity was assessed ex vivo by CD4 autologous proliferation assay and T cell interferon (IFN) secretion (ELISPOT) assay. Ten of 11 patients were immunologically unreactive to the peptides prior to the vaccinations. Following the 5th dose of vaccine, 8/8 patients (including 2 with B2A2) tested showed significant immunologic responses by one of the above assays. Conclusions: These preliminary results suggest vaccination with synthetic analog peptides derived from CML proteins results in immunologic responses and may be associated with molecular improvement. Clinical efficacy of these vaccines in reducing/ eliminating minimal disease has yet to be established. [Table: see text]

Imatinib mesylate is now standard treatment for chronic myelogenous leukemia. In spite of its high efficacy in controlling the disease, residual bcr-abl cells measured by RT-PCR are still detectable in more than 95% of the patients treated with this drug. In addition, recent reports describe the emergence of resistance to Imatinib mesylate. A major mechanism for the appearance of resistance, are point mutations changing amino acids in the bcr-abl tyrosine kinase that make imatinib ineffective. We hypothesized that some bcr-abl kinase mutations selected by imatinib would generate new immunogenic peptide-epitopes that could be recognized by the human immune system. For this purpose, we selected with predictive computer algorithms potentially immunogenic mutated amino acid sequences and synthetic analogues of mutated bcr-abl kinase sequences to study for immunogenicity and anti-leukemia activity in an in vitro human system. Initially we designed a number of synthetic peptides derived from the most frequent bcr-abl kinase mutations induced by imatinib in which single amino acid substitution was introduced at different MHC-binding positions. We investigated the most common mutations: E255V, E255K, Y253H, Y253F, F311L, T315I, M351T and H396P. Specific single and combined mutations improved the predicted binding of peptides to HLA A0201. Further additional single amino-acid substitutions on mutated peptides generated high predicted binding. Selected sequences were evaluated for eliciting MHC-restricted, peptide-specific CTL responses in an in vitro model using T-cells from different HLA A0201 healthy donors after stimulation with monocyte-derived DC. CTL lines were assessed by either IFN gamma ELISPOT or by a chromium51 release assay. Mutated derived peptides from E255K, E255V, Y253F and T315I were shown to be immunogenic in vitro by the specific release of IFN gamma. Furthermore, single amino-acid substitutions in key residues improved the immunogenicity of these peptides. Finally, T cell lines generated with optimized peptides derived from E255K and T315I (the most common imatinib-induced mutations) were able to kill peptide pulsed targets; more important, these T cell lines cross-reacted with the mutated peptides (a heteroclitic response). In conclusion, Imatinib-selected mutations of peptides from bcr-abl kinase have a better predicted binding than native kinase sequences. Selected peptides from the most common mutations are immunogenic in vitro in a HLA A0201 restricted manner. T cells generated with optimized peptides from these mutations are able to elicit heteroclitic peptide-specific cytotoxicity.

In order to analyze the cellular mechanism of immunosuppression during acute infection by Ascaris suum, cytokine and natural killer activities in A. suum infected mice were investigated.Ten days after infection with 103 or 104 embryonated A. suum eggs, Concanavalin A induced interleukin 2 (IL-2) production by splenocytes were reduced. At the same time, NK activity of splenocytes and the levels of serum interferon induced by Poly I. Poly C were also reduced. On the other hand, culture supernatants of peritoneal adherent cells from A. suum infected mice showed significantly high interleukin 1 activity which reached peak level on day 15 following lipopolysaccharide stimulation. Increase in the number of macrophages in the peritoneal cavity were also observed in this period. Furthermore, the culture supernatants of peritoneal adherent cells from 10-days A. suum infected mice exhibited a suppressive effect on both IL-2 production by normal splenocytes and IL-2 activity assayed using CTLL-2 cells. Dialysis and molecular filtration studies indicated that the factor with molecular weight below 10, 000 related to the suppressive effect. But there was no significant increase of prostaglandin E2 in the culture supernatants.These results suggest that activated macrophages may play an important part in the immunosuppression by producing a soluble suppressive factor during acute infection by A. suum.