ALBERT

Description: 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.

Description: Wilms tumor protein (WT1) is a transcription factor selectively over expressed in several types of leukemia and solid tumors, making it a promising potential target antigen for immunotherapy. Several open clinical trials use native or altered peptide sequences derived from the WT1 protein in order to overcome the weak immunogenicity of the self-antigen. Here we report a new strategy to circumvent tolerance by designing peptides that incorporate non-natural amino acids into the native sequence of WT1 peptides. Starting from the nonamer sequences WT1 187–195 and WT1 235–243, eight peptides containing natural amino acids and nine peptides in which different chemical modifications (fluorination, photo-reactive azido groups or benzophenone groups) were introduced at major histocompatibility complex (MHC) and T cell receptor binding positions, were synthesized. The new non-natural peptides could stabilize MHC class I molecules better than the native sequences and were also able to elicit strong specific T-cell responses. Photo-reactive peptides were additionally modified with biotin handles to allow streptavidin-biotin pull down and western blot analysis of kinetics of binding and catabolism. Upon UV irradiation, these peptides covalently bound to MHC molecules on the live cells; clearance of the peptide-MHC covalent complex occurred over 24 hours, consistent with the T2 thermo-stabilization data for the same peptide. Further catabolic studies may elucidate the important or novel cellular proteins involved in antigenic peptide processing and cross presentation and should aid in vaccine development. We are investigating whether covalent interaction with the MHC may lead to alterations in immune responses as well. T cells stimulated with one of the synthetic peptides (WT1J-W4WF) cross-reacted with the native WT1J sequence and were able to kill WT1 positive HLA-A0201 matched acute lymphoblastic leukemia cell lines. In conclusion, this study shows that peptides with non-natural amino acids can be successfully incorporated into T cell epitopes to provide increased immunogenicity and novel biological information.

Description: A tumor-specific, bcr-abl-derived fusion peptide vaccine can be safely administered to patients with chronic myelogenous leukemia (CML) and can elicit a bcr-abl peptide-specific T-cell immune response. In the present phase 2 trial, 14 patients with CML in chronic phase were vaccinated with 6 fusion peptides mixed with Quillaja saponaria (QS-21). No significant toxic effects were observed. In 14 of 14 patients, delayed-type hypersensitivity (DTH) and/or CD4 proliferative responses developed after beginning vaccinations, and 11 of 14 patients showed interferon-gamma (IFN-gamma) release by CD4 enzyme-linked immunospot (ELISPOT) at one or more time points. These responses were CD4+CD45RO+. A peptide-specific CD8+ interferon-gamma ELISPOT was found in 4 patients. Four patients in hematologic remission had a decrease in Philadelphia chromosome (Ph) percentage (3 concurrently receiving interferon-alpha and 1 on imatinib mesylate), and 3 patients in molecular relapse after allogenic transplantation became transiently polymerase chain reaction (PCR) negative after vaccination; 2 of these patients received concurrent donor lymphocyte infusion (DLI). All 5 patients on IFN-alpha ultimately reached a complete cytogenetic remission. In conclusion, a tumor-specific bcr-abl breakpoint peptide-derived vaccine can be safely administered and can reliably elicit measurable peptide-specific CD4 immune responses, including in patients after bone marrow transplantation, on interferon, or on imatinib mesylate. A relationship between the clinical responses and vaccination cannot be determined from this trial. (Blood. 2004;103:1037-1042)

Description: Acute myeloid leukemia (AML) is an aggressive hematological malignancy with a 5-year overall survival rate of less than 30% which causes over 10,000 deaths per year in the United States. Treatment options for this disease increasingly include epigenetic drugs, such as hypomethylating agents (e.g. decitabine) or histone deacetylase (HDAC) inhibitors (e.g. pracinostat) which can function via direct cytotoxic mechanisms and also through altered differentiation of AML blasts; immunomodulatory effects like reactivation and presentation of cancer testis antigens in context of human leukocyte antigen (HLA) complexes have been reported as well, which may result in clearance of cells via the adaptive immune system. However, the landscape of immunogenic T cell epitopes induced by these drugs might be even broader than reported since standard analyses only consider exonic protein sequences and do not take into account typically untranslated genomic regions. Recently, it has been shown that single and combination treatment of decitabine and pracinostat can induce cryptic transcription start sites in generally epigenetically repressed solitary long-terminal repeats (LTRs) of the LTR12C family which give rise to novel mRNAs and resulting protein variants. We hypothesized that the intronic parts of these gene products might provide a source of cryptic T cell epitopes with high immunogenic potential, which are induced through epigenetic drug treatment. To test this hypothesis, we treated 5 different AML cell lines (HL-60, U937, OCI-AML02, MOLM13, AML14) with (1) DMSO, (2) 500 nM decitabine or (3) a combination of 500 nM decitabine and 100 nM pracinostat for 72 hours to induce transcription of non-annotated transcription start sites. Subsequently, HLA class I complexes were immunopurified and peptides presented by these complexes isolated and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The activation of silenced genes by epigenetic drug treatment with either decitabine alone or the combination treatment yielded increases of about two-fold in the identified unique HLA ligands. This increase in peptide identifications also led to improved detection of cancer testis antigen-derived epitopes, as has been reported before. Intriguingly, by adding LTR12C derived sequences stretching from the published GATA2 specific binding site until the next genomic exon to the peptide search analyses we were able to identify several cryptic peptides from 4 out of 5 AML cell lines derived from these usually untranscribed genomic regions. The identifications were exclusively dependent on previous treatment with either decitabine alone or in combination with pracinostat. Though the immunogenicity of these HLA ligands has not been determined yet, we assume that due to their genetically repressed state in untreated cells, these new peptide sequences represent a new class of neoepitopes, with potential to be novel targets of existing T cells within patients or after augmentation by other immunotherapies. In summary, we demonstrated for the first time the induced presentation of epitopes from normally untranscribed LTR12C regions through epigenetic drug treatment and therefore provide a previously undescribed source of potential targets for immunotherapy in AML. Disclosures Scheinberg: Eureka: Consultancy; Ensyce: Consultancy.

Description: 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.

Description: Major Histocompatibility Complex class I (MHC-I) molecules present antigenic peptides to T cells on the cell surface as a prerequisite for stimulating cytotoxic T cell response. Thus, the ability to reliably identify the peptides that can bind to MHC molecules is of practical importance for rapid vaccine development. Several computer-based prediction methods have been applied to study the interaction of MHC class I/peptide binding. Here we have compared three of the most commonly used predictive algorithms BIMAS, SYFPEITHI and Rankpep with actual binding of HLA-A*0201 peptides in vitro. Forty six HLA-A*0201 peptides were selected from several target oncoproteins: Wilms’ tumor (WT1), native and imatinib- mutated bcr-abl p210 and JAK2 protein. Experimental peptide binding to HLA-A*0201 was assessed using a MHC stabilization assay on T2, TAP deficient cells. Peptides were considered to show positive in vitro binding if the mean fluorescence was at least 50 % of the binding of a high affinity reference peptide. Peptides qualified as positive in vitro if the BIMAS score was ≥ 100, the SYFPEITHI score ranked ≥ 24 or the Rankpep was ≥ 50. Results are summarized below: BIMAS SYFPEITHI RANKPEP Sensitivity 84 % 72 % 60 % Specificity 76 % 71 % 81 % Positive Predictive Value 84 % 72 % 60 % Negative Predictive Value 80 % 68 % 63 % Combining two or more computer methods did not appear to improve the predictive value. In conclusion, of the three predictive algorithms, the best correspondence with the actual MHC binding was demonstrated with the BIMAS algorithm. Predictive computer algorithms are important for preselection of potential T-cell epitope candidates for the application in vaccine design.

Description: 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.

Description: Intracellular processing of the breakpoint products of the bcr-abl fusion gene may generate novel peptides that, if capable of binding to HLA class I molecules, would be potential targets for a cytotoxic T lymphocyte (CTL) response. In humans, peptides derived from the e1a2 p190 breakpoint have generated peptide-specific MHC class II proliferative responses. Short peptides have shown binding to MHC class I molecules, although processing and presentation of endogenous p190 protein has not been shown. In a Kd Balb/c mouse model, we tested the hypothesis that single, key amino acids near the breakpoint could be the reason of lack of immunogenicity of the p190 breakpoint peptides. We synthesized a native peptide from the 190 breakpoint (AFHGDAEAL) as well as a synthetic mutated peptide (AYHGDAEAL), which showed excellent predicted binding on the BIMAS algorithm (1152 and 2880 respectively), although in vivo experiments did not show any specific CTL response. In order to assess if the lack of immunogenicity in vivo was due to the absence of binding to the MHC class I molecule rather than to poor TCR interactions, we designed a series of peptides where neutral amino acid, alanine, substitutions were introduced at different potential binding sites in the synthetic peptide: at position three (AYAGDAEAL), position four (AYHADAEAL), position five (AYHGAAEAL) and position seven (AYHGDAAAL). The binding of these altered peptides to H2 class I was assessed using a MHC stabilization assay on T2-Kd cells (TAP deficient cells). In spite of the good computer prediction for binding the MHC stabilization assay did not show evidence of binding of the native and synthetic peptides (