ALBERT

Description: Key Points This study shows the effective anticancer activity of a T-cell receptor mimic antibody targeting WT1 in resistant human Ph+ ALL. In combination with tyrosine kinase inhibitors, ESKM can result in cure of Ph+ ALL in murine models.

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: 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.

Description: 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.

Description: Adoptive transfer therapy of T cells expressing chimeric antigen receptors (CAR) against tumor-associated antigens has been shown to be clinically successful in a limited set of leukemia. However, novel antigen targets for both hematological and solid malignancies are required. Most CARs described thus far are targeted against external antigens on particular cell types. We have designed and engineered the first CAR T cell against a human intracellular protein, WT1. WT1 is overexpressed in many cancers, including acute and chronic leukemias and numerous solid tumors. Our TCRm CAR, derived from the ESK1 TCRm mAb, termed WT1 28z, is reactive with the RMFPNAPYL peptide of the WT1 protein that is processed and presented on the surface of cells in the context of HLA-A*02:01. WT1 28z expressing T cells have high expression of the CAR on their surface. They are cytotoxic in standard 51Cr assays against a range of cancer cell lines, including the megakaryoblastic cell line SET2, the acute myeloid leukemia (AML) cell line AML14, the multiple myeloma cell line KARPAS, and the ovarian cancer line, OVCAR3, as compared to CAR T cells against an irrelevant antigen. The WT1 28z CAR T cells are also cytotoxic against primary AML bone marrow blasts in this assay. When co-cultured with these primary cells or cancer cell lines, the WT1 28z CAR T cells have enhanced production of proinflammatory cytokines such as IFN-g, IL-2, and GM-CSF, as compared to irrelevant CAR T cells. Importantly, WT1 28z T cells are specific for the WT1-HLA-A*02:01 complex. The cells do not show cytotoxicity against cell lines or primary cells that are not both HLA-A*02:01- positive and WT1 positive. WT1 28z T cells are currently being tested alongside irrelevant antigen CAR T cells in AML and ovarian cancer murine models in vivo to assess efficacy, with the ultimate goal of translating this novel approach into the clinical setting for both hematological and solid cancers. The data provide the proof-of-concept that CAR T cells also may be directed at intracellular antigens. Disclosures Dao: Novartis: Patents & Royalties. Liu:Eureka: Employment, Inventor Other. Scheinberg:Novartis: Patents & Royalties. Brentjens:Juno Therapeutics: Consultancy, Scientific co-founder and Stock holder Other.

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: 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 3624 WT1 is a transcription factor which has been implicated in leukemogenesis and has been used as a marker of minimal residual disease (MRD). We previously demonstrated the feasibility of vaccinating AML patients in CR with a multivalent WT1 peptide vaccine and inducing immune responses. In an effort to further explore the safety and efficacy of this approach, we are conducting a Phase II study in which the vaccine is administered to AML patients in first CR and who completed all planned postremission chemotherapy. Eligible patients had WT1 transcript detectable by RT-PCR. The vaccine consisted of 4 native and derived WT1 peptides administered with the immune adjuvants Montanide and GM-CSF. Patients received 6 vaccinations over 10 weeks. Early toxicity was assessed at weeks 2 and 4. Immune responses were evaluated at week 12 by CD4+ T cell proliferation, CD3+ T cell interferon-g interferon release (ELISPOT) and WT1 peptide tetramer staining. Patients who were clinically stable and without disease recurrence could continue with up to 6 more vaccinations administered approximately every month. To date, 12 patients have been accrued to the study (6-M, 6-F; median age – 66 years, range 26–73 years). Cytogenetic subtypes varied among the study patients (Favorable-3, Intermediate-5, Unfavorable-4). The median time to vaccination after achieving CR was 7.5 months (range: 3–22 months). One patient was removed early because of relapse prior to receiving the first vaccination. Four patients have received at least 6 vaccines and 2 others have completed 12 vaccinations. Eight patients are alive without evidence of disease. One of these patients has an HLA-A02 subtype and was found to have developed T cells reactive with WT1-A (native peptide) HLA tetramers following 6 vaccinations which persisted (at a lower level) until after the 12th vaccination. Three patients relapsed during vaccination (after 1, 5 and 11 vaccines) and 2 of the 3 have died. Two of the relapsed patients who had sample available for immunologic evaluation, did not develop a CD4+ response to any of the peptides tested. Two other patients discontinued vaccination because of toxicity (hypersensitivity/pain with GM-CSF administration). Both remain in CR. No episodes of anaphylaxis or generalized urticaria were observed. Neither median disease free survival nor overall survival has been reached in this small cohort of patients. These preliminary findings demonstrate that the WT1 peptide vaccine is relatively well tolerated and has immunologic activity. Trial accrual is ongoing and further follow-up is required before any beneficial effect on outcome can be determined. Disclosures: Scheinberg: Formula Pharma: WT1 Vaccine inventor, Patent held by MSKCC and Licensed to Formula Pharma, WT1 Vaccine inventor, Patent held by MSKCC and Licensed to Formula Pharma Patents & Royalties.