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: 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: In vivo biopanning with phage displayed peptide libraries has generated a group of peptide probes which bind selectively to the surface of atherosclerotic plaque endothelium. The highest affinity peptide, EKO130, binds to the 78 kDa glucose regulated protein (Grp78). Grp78 has been demonstrated to play a role in numerous pathological processes as well as a possible role in the local cell surface regulation of the coagulation cascade. The goal of this study is to determine the role of Grp78 in coagulation including plasma clotting, factor Xa (Xa) generation, and tissue factor (TF) gene expression. siRNA mediated inhibition of Grp78 results in a marked increase in TF gene expression in bEND.3 endothelial cells and RAW macrophage-like cells. Antibody mediated inhibition of cell surface Grp78 results in increased TF procoagulant activity and TF-dependent Xa generation in both the endothelial and macrophage cell types. These studies are consistent with results from another laboratory demonstrating that Grp78 over-expression inhibits TF mediated initiation and support of the coagulation protease cascade. Thus, our work indicates that Grp78 suppresses TF at both the functional and molecular level by inhibiting both its thrombogenic potential and gene expression.

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: Acute and chronic leukemias, including CD34+ CML stem cells, overexpress the Wilms tumor gene 1 (WT1) protein, making WT1 an attractive therapeutic target. ESKM is a fully human IgG1 antibody that targets a 9 amino acid sequence (RMF) of the protein WT1 in the context of HLA-A0201, allowing it to target an undruggable, widely expressed, intracellular oncogene product. BV173 is an HLA-A0201+, human Ph+ ALL cell line that expresses WT1, and tagged by our lab with luciferase. We engineered a tyrosine kinase inhibitor (TKI) resistant BV173-R cell line by transducing BV173 with the resistant T315I Bcr-Abl plasmid. Antibody-dependent cellular cytotoxicity (ADCC) was evaluated in vitro by chromium release assay, utilizing human PBMC effectors. Tumor growth in vivo was assessed in NOD/SCID gamma (NSG) mice with bioluminescence imaging (BLI). RT-PCR was used to evaluate minimal residual disease in mice with negative BLI signal at the end of therapy. Imatinib, dasatinib, and ponatinib were used at up to maximally tolerated doses, given IP once daily. ESKM was administered at 100 µg twice weekly IP. ESKM mediated ADCC against both BV173 and BV173-R cell lines in vitro. In a BV173 engrafted human leukemia xenograft model, ESKM was more potent than imatinib, with median tumor growth reduction of 78% vs 52%. Combination of imatinib and ESKM therapy resulted in a 94% reduction in leukemic growth. High dose dasatinib (40 mg/kg daily) was more potent than ESKM, but discontinuation of therapy due to dasatinib toxicity resulted in relapse. Combination with ESKM therapy with dasatinib resulted in cure in 75% of mice, confirmed by bone marrow RT-PCR three weeks after termination of therapy. For mice cytoreduced with dasatinib followed by consolidation therapy with ESKM, delayed relapse was observed, but no cures. ESKM was highly superior to imatinib and dasatinib against the T315I BV173-R leukemia in vivo. Cures were not achieved with combination therapy of ESKM and either first or second generation TKIs against resistant T315I leukemia. Ponatinib at 10 mg/kg had higher efficacy than ESKM alone against BV173-R, but mice treated with combination of ESKM and ponatinib had superior tumor reduction. CONCLUSION: ESKM is an effective therapeutic antibody for sensitive and T315I Ph+ ALL. Resistant T315I Ph+ leukemic growth is inhibited more effectively by ESKM therapy compared to imatinib and dasatinib, and combination therapy with ESKM is superior to ponatinib. Supported by the Leukemia and Lymphoma Society, NIH R01CA55349, P01 23766 and T32CA62948-18. Disclosures: Yan: Eureka Therapeutics: Employment. Liu:Eureka Therapeutics: Employment, Equity Ownership.

Description: Recently, a new class of RNAs has been identified that, like mRNAs, are usually transcribed by RNA polymerase II, but lack significant translated open reading frames. They are non-coding RNAs (ncRNAs) that exert roles directly as RNAs, and function as genetic regulators, or riboregulators. In the present study, we discovered a novel very large ncRNA. From differential gene profiling of CD31 positive tumor microvascular endothelial cells from a murine colon carcinoma, we amplified an ~380 bp nucleotide sequence, DP100. This RNA was present in tumor cells as well as tumor vascular endothelium. The full-length DP100 transcript is an ~7 Kb RNA lacking an open reading frame set for more than 66 amino acids. Also the hypothetically predicted proteins lacked significant similarity to known proteins, consistent with identity of DP100 as an unusually large non-coding RNA transcript. Further bioinformatic search demonstrated that the full-length DP100 sequence is highly homologous to the human alpha gene, which encodes an 8.5 Kb, non-coding RNA transcript, and is located in a region implicated in chromosomal abnormalities of human tumors. The full-length DP100, here designated the mouse alpha gene, is evolutionally highly conserved among vertebrates, suggesting its functional significance. The role for this ncRNA in cellular behavior is under investigation.

Description: 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.