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PDF file - 78KB, ESKM has a modified Fc glycosylation pattern, altering binding to FcgammaRs but not to the RMF/A2 target. Where applicable, data points are averages of each group, and error bars represent SEM. (A) Comparison of the oligosaccharide profile of ESK1 and ESKM. Peak assignment is based on the retention time and the monosaccharide composition analysis. G# indicates the number of terminal galactoses, F indicates presence of core fucose, Hex5GlcNAc2 denotes (GlcNAc)2 core with terminal Hexose 5 glycan structure (terminating in mannose and/or glucose). (B-C) Representative binding curves of ESK1 and ESKM against human FcgammaRI, FcgammaRIIa, FcgammaRIIb, FcgammaRIIIa - 158V, FcgammaRIIIa -158F, and FcRn (B), and mouse FcgammaRIIb and FcgammaRIV (C). (D) 125 I-labeled ESK1 and ESKM mAbs were titrated against JMN cells. All curves were fit with a non-linear single-site total binding saturation curve, and Kd was calculated using Prism software.

PDF file - 145KB, All human antibodies tested accumulated more in spleens of HLA-A2+ transgenic mice, but ESK1 did not bind specifically to isolated HLA-A2+ spleen, bone marrow or thymus cells. (A) Accumulation of 125 I-labeled antibodies in spleens of C57BL6/J or HLA-A2+ transgenic mice relative to antibody level in the blood. Mice were injected retroorbitally with 2microg indicated antibody, then sacrificed after 24 hours for blood and spleen collection. (B) Specific binding of 125I-labeled ESK1 to bone marrow, spleen, or thymus cells isolated from C57BL6/J or HLA-A2+ transgenic mice. Tissues were collected from 2 (C57) or 3 (HLA-A2+ transgenic) mice, then bound by 1microg/mL 125 I-labeled ESK1 either alone or after blocking with 50-fold excess unlabeled ESK1. Specific binding was determined, and #ESK1 bound per cell was calculated.

PDF file - 38KB, Histopathologic report from ESKM- or isotype control human IgG1-treated HLA-A*02:01+ transgenic mice.

Purpose: RMFPNAPYL (RMF), a Wilms' tumor gene 1 (WT1)–derived CD8 T-cell epitope presented by HLA-A*02:01, is a validated target for T-cell–based immunotherapy. We previously reported ESK1, a high avidity (Kd < 0.2 nmol/L), fully-human monoclonal antibody (mAb) specific for the WT1 RMF peptide/HLA-A*02:01 complex, which selectively bound and killed WT1+ and HLA-A*02:01+ leukemia and solid tumor cell lines.Experimental Design: We engineered a second-generation mAb, ESKM, to have enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) function due to altered Fc glycosylation. ESKM was compared with native ESK1 in binding assays, in vitro ADCC assays, and mesothelioma and leukemia therapeutic models and pharmacokinetic studies in mice. ESKM toxicity was assessed in HLA-A*02:01+ transgenic mice.Results: ESK antibodies mediated ADCC against hematopoietic and solid tumor cells at concentrations below 1 μg/mL, but ESKM was about 5- to 10-fold more potent in vitro against multiple cancer cell lines. ESKM was more potent in vivo against JMN mesothelioma, and effective against SET2 AML and fresh ALL xenografts. ESKM had a shortened half-life (4.9 days vs. 6.5 days), but an identical biodistribution pattern in C57BL/6J mice. At therapeutic doses of ESKM, there was no difference in half-life or biodistribution in HLA-A*02:01+ transgenic mice compared with the parent strain. Importantly, therapeutic doses of ESKM in these mice caused no depletion of total WBCs or hematopoetic stem cells, or pathologic tissue damage.Conclusions: The data provide proof of concept that an Fc-enhanced mAb can improve efficacy against a low-density, tumor-specific, peptide/MHC target, and support further development of this mAb against an important intracellular oncogenic protein. Clin Cancer Res; 20(15); 4036–46. ©2014 AACR.

PDF file - 183KB, (A) ESKM reduced individual tumor burden during the treatment course in 3 of 5 mice. Alternate representation of data in Figure 2A, where signal for each mouse is normalized to starting luminescence. (B) ESKM had no effect against JMN in a NOG mouse model. (C) Comparison of BV173 (Ph+ ALL) and SET2 (AML) cell lines in NSG mice.

PDF file - 142KB, Human PBMCs were separated into effector cell populations and tested for in vitro ADCC activity against BV173 cells, mediated by ESKM or isotype control mAb. Only fractionated NK cells showed activity relative to isotype control antibody. % Lysis is calculated based on spontaneous release (target cells, no effectors or mAb) as 0% and maximal release (target cells + SDS) as 100%.

Purpose Transarterial chemoembolization (TACE) is the standard of care for patients with intermediate-stage hepatocellular carcinoma (HCC). Lenvatinib, a multikinase inhibitor, and pembrolizumab, a PD-1 inhibitor, have shown efficacy and tolerability in patients with HCC, and adding this combination to TACE may enhance clinical benefit. Protocol LEAP-012 is a prospective, double-blind randomized phase 3 study. Adults with confirmed HCC localized to the liver without portal vein thrombosis and not amenable to curative treatment, ≥ 1 measurable tumor per Response Evaluation Criteria in Solid Tumors 1.1 (RECIST 1.1), Eastern Cooperative Oncology Group performance status 0 or 1, Child–Pugh class A and no previous systemic treatment for HCC are eligible. Patients will be randomly assigned to lenvatinib once daily plus pembrolizumab every 6 weeks plus TACE or placebos plus TACE. Dual primary endpoints are overall survival and progression-free survival per RECIST 1.1 by blinded independent central review (BICR). Secondary endpoints are progression-free survival, objective response rate, disease control rate, duration of response and time to progression per modified RECIST by BICR; objective response rate, disease control rate, duration of response and time to progression per RECIST 1.1 by BICR; and safety. Statistics The planned sample size, 950 patients, was calculated to permit accumulation of sufficient overall survival events in 5 years to achieve 90% power for the overall survival primary endpoint. Discussion LEAP-012 will evaluate the clinical benefit of adding lenvatinib plus pembrolizumab to TACE in patients with intermediate-stage HCC not amenable to curative treatment. ClinicalTrials.gov NCT04246177.

506 Background: In LEAP-002 (NCT03713593), len + pembro achieved a median OS of 21.2 mo vs 19.0 mo with len + pbo with manageable safety in both arms in 1L aHCC, although the significance thresholds for OS and PFS were not met. We present here the results of prespecified exploratory patient (pt)-reported outcomes (PROs). Methods: 794 pts were randomized. PROs were assessed by EORTC QLQ-C30, EuroQol-5D5L (EQ5D-5L), and EORTC QLQ-HCC18 questionnaires. Analysis population for HRQoL endpoints included pts who received ≥1 dose of study Tx and completed ≥1 HRQoL assessment. Least squares mean (LSM) score changes from baseline (BL) to wk 27 were compared using a constrained longitudinal data analysis model (covariates: Tx, time, Tx by time interaction, and stratification factors). Kaplan-Meier method was used to estimate time to deterioration (TTD, time to 1st onset of ≥10 [out of 100] deterioration from BL in a given scale or subscale/confirmed by a 2nd adjacent ≥10 deterioration from BL) for EORTC QLQ-C30 global health status (GHS)/QoL, physical functioning (PF), and EORTC QLQ-HCC18 abdominal swelling, pain and fatigue, as reported by pts. Stratified Cox proportional hazards model was used to assess the magnitude of the Tx difference (HR) between arms in TTD. Results: Compliance with PRO assessments was >91% from BL until wk 27 in both arms. From BL to wk 27, LSM changes in GHS/QoL, PF, role functioning (RF), QLQ-HCC18 functional/domain scores and EQ5D-5L VAS were generally similar between the two arms (Table). Treatment with len + pembro delayed deterioration of patient-reported quality of life vs len + pbo (median TTD, 11.5 vs 4.3 mo; HR, 0.80; 95% CI, 0.65-0.98). LSM score difference (95% CI) was 0.5 (-2.5, 3.4) for GHS/QoL, -1.7 (-4.5, 1.1) for PF, -1.0 (-4.9, 2.9) for RF, 1.0 (-2.2, 4.3) for abdominal swelling, 2.0 (-1.2, 5.2) for fatigue, 2.0 (-1.0, 5.0) for pain, and 0.6 (-2.0, 3.2) for EQ5D-5L VAS. Median TTD in QLQ-C30 PF (5.7 vs 9.1 mo; HR, 1.14; 95% CI, 0.93-1.41) and QLQ-HCC18 abdominal swelling (NR vs NR; HR, 1.19; 95% CI, 0.89-1.60) /fatigue (2.9 vs 2.8 mo; HR, 0.98; 95% CI, 0.81-1.19)/pain (9.6 vs 7.9 mo; HR, 0.93; 95% CI, 0.75-1.16) was similar between the arms. Conclusions: Len + pembro generally preserved HRQoL vs len + pbo in 1L for aHCC. Combined with efficacy and safety results from LEAP-002, these findings support continued development of len + pembro in HCC. Clinical trial information: NCT03713593 . [Table: see text]

TPS494 Background: Limited treatment options are available for patients with intermediate HCC. Lenvatinib, a potent multikinase inhibitor, and pembrolizumab, a PD-1 inhibitor, are approved first- and second-line therapies for advanced HCC, respectively. The LEAP-012 study (NCT04246177) is investigating lenvatinib plus pembrolizumab in combination with TACE versus placebo plus TACE in patients with intermediate-stage HCC. Methods: LEAP-012 is a randomized, double-blind, phase 3 study. Adults with confirmed HCC localized to the liver without portal vein thrombosis and not amenable to curative treatment, ≥1 measurable lesion per RECIST v1.1, Eastern Cooperative Oncology Group performance status of 0 or 1, and no previous systemic treatment for HCC are eligible. Patients will be randomly assigned to receive lenvatinib 8 mg (body weight < 60 kg) or 12 mg (body weight ≥60 kg) orally once daily plus pembrolizumab 400 mg intravenously (IV) every 6 weeks (Q6W) plus TACE or placebo orally once daily plus placebo IV Q6W plus TACE. Response will be assessed by imaging every 9 weeks; safety will be assessed throughout the study and up to 90 days after the end of treatment. Dual primary end points are overall survival and progression-free survival (PFS) per RECIST v1.1 by blinded independent central review (BICR). Secondary end points are PFS, objective response rate (ORR), disease control rate (DCR), duration of response (DOR), and time to progression (TTP) per modified RECIST by BICR; ORR, DCR, DOR, and TTP per RECIST v1.1 by BICR; and safety. Exploratory end points are PFS, ORR, DCR, DOR, TTP, and time from randomization to second/subsequent disease progression after initiation of new anticancer therapy or death from any cause, whichever occurs first, per RECIST v1.1 by investigator review, identification of molecular biomarkers, and health-related quality of life. Recruitment began in April 2020, and the planned sample size is 950 patients. The results of the LEAP-012 study will show the clinical benefit of adding lenvatinib plus pembrolizumab to the current standard of care TACE for patients with intermediate-stage HCC not amenable to curative treatment. Clinical trial information: NCT04246177.

PURPOSE The immunomodulatory effect of lenvatinib (a multikinase inhibitor) on tumor microenvironments may contribute to antitumor activity when combined with programmed death receptor-1 (PD-1) signaling inhibitors in hepatocellular carcinoma (HCC). We report results from a phase Ib study of lenvatinib plus pembrolizumab (an anti–PD-1 antibody) in unresectable HCC (uHCC). PATIENTS AND METHODS In this open-label multicenter study, patients with uHCC received lenvatinib (bodyweight ≥ 60 kg, 12 mg; < 60 kg, 8 mg) orally daily and pembrolizumab 200 mg intravenously on day 1 of a 21-day cycle. The study included a dose-limiting toxicity (DLT) phase and an expansion phase (first-line patients). Primary objectives were safety/tolerability (DLT phase), and objective response rate (ORR) and duration of response (DOR) by modified RECIST (mRECIST) and RECIST version 1.1 (v1.1) per independent imaging review (IIR; expansion phase). RESULTS A total of 104 patients were enrolled. No DLTs were reported (n = 6) in the DLT phase; 100 patients (expansion phase; included n = 2 from DLT phase) had received no prior systemic therapy and had Barcelona Clinic Liver Cancer stage B (n = 29) or C disease (n = 71). At data cutoff, 37% of patients remained on treatment. Median duration of follow-up was 10.6 months (95% CI, 9.2 to 11.5 months). Confirmed ORRs by IIR were 46.0% (95% CI, 36.0% to 56.3%) per mRECIST and 36.0% (95% CI, 26.6% to 46.2%) per RECIST v1.1. Median DORs by IIR were 8.6 months (95% CI, 6.9 months to not estimable [NE]) per mRECIST and 12.6 months (95% CI, 6.9 months to NE) per RECIST v1.1. Median progression-free survival by IIR was 9.3 months per mRECIST and 8.6 months per RECIST v1.1. Median overall survival was 22 months. Grade ≥ 3 treatment-related adverse events occurred in 67% (grade 5, 3%) of patients. No new safety signals were identified. CONCLUSION Lenvatinib plus pembrolizumab has promising antitumor activity in uHCC. Toxicities were manageable, with no unexpected safety signals.

4084 Background: In a phase 1b trial (NCT03006926), L + P had promising antitumor activity as first-line (1L) therapy in uHCC. We present exploratory biomarker analyses of circulating angiogenic factors and cytokines/chemokines related to the mechanism of action of L + P (ie, pharmacodynamic [PD] biomarkers), as well as biomarker correlations with clinical outcomes in patients (pts) with uHCC, from this trial. Methods: Pts received lenvatinib 12 mg/d (bodyweight [BW] >60 kg) or 8 mg/d (BW < 60 kg) PO + pembrolizumab 200 mg IV Q3W. Tumors were assessed using mRECIST or RECIST v1.1 per independent imaging review. Peripheral blood samples were collected before administration of study drug at baseline, cycle (C) 2, day (D) 1, C3D1, C4D1, and off-treatment. 43 Biomarkers were assayed in serum from 100 1L uHCC pts (excluding 4 pts from the dose-limiting toxicity part of the trial with prior sorafenib). Of these 43, 31 biomarkers (for which ≤20% of samples had measurements above/below the quantification limit of the assay) were included in the analyses. Changes in biomarker levels from baseline were evaluated via 1-sample Wilcoxon signed-rank test. Associations were explored between changes in biomarker levels and maximum tumor shrinkage (MTS) via the Spearman’s rank correlation test, objective response (OR; complete response + partial response) via the Wilcoxon rank sum test, and PFS via Cox regression analysis and log rank test. Data cutoff date for clinical endpoints was 7 August 2020. Results: Levels of PD biomarkers related to angiogenic signaling (VEGF increase/ANG2 decrease), FGF signaling (increase in FGF23/FGF19), and IFNγ signaling (increase in IFNγ, CXCL9/10/11) were changed significantly (adjusted P< 0.05) with L + P (C2D1–C4D1; except for FGF19 at C3D1). Significant decreases of TIMP1 and increases of MCP1 were observed at C4D1 during treatment; these were associated with greater MTS. Greater decreases in TIMP1 and greater increases in MCP1 were observed in pts with OR vs others. Changes in levels of the PD biomarkers ANG2, IL10, and VEGFR2 were found to be associated with PFS by dichotomized analysis. With tertile 2 cutoff, median PFS for pts in the group with greater decreases of ANG2 was 13.9 months vs 9.6 months for pts in the group with lesser decreases of ANG2 (unadjusted P= 0.002; HR 2.65, 95% CI 1.39–5.08). Conclusions: These are the first exploratory biomarker analyses for the single-arm study of L + P in pts with uHCC. Changes in serum biomarkers associated with angiogenic-, FGF-, and IFNγ-signaling pathways indicated target engagement of L + P. Decreases in TIMP1 and increases in MCP1 were associated with MTS and OR. Associations were found between longer PFS and a greater decrease in levels of ANG2. Angiogenesis inhibition and modulation of cancer immune response were observed with L + P. Further validation from independent studies is warranted. Clinical trial information: NCT03006926.

4519 Background: LEN is a multikinase inhibitor of VEGFR 1–3, FGFR 1–4, PDGFRα, RET, and KIT, approved for first line (1L) treatment of uHCC. PEMBRO, an anti-PD-1 monoclonal antibody, was granted accelerated approval for the treatment of patients (pts) with HCC after sorafenib therapy. We assessed the safety and efficacy of LEN + PEMBRO in uHCC. Methods: In this phase 1b trial (NCT03006926), pts received LEN 12 mg/day (bodyweight [BW] ≥60 kg) or 8 mg/day (BW

Background LEN is a multikinase inhibitor of VEGFR 1–3, FGFR 1–4, PDGFRα, RET, and KIT. PEMBRO is an anti-PD-1 monoclonal antibody. LEN monotherapy is approved for first-line treatment of uHCC. PEMBRO monotherapy has shown significant efficacy in the second-line treatment of HCC. We report updated results from a phase 1b trial of LEN + PEMBRO in uHCC. Methods In this open-label, phase 1b study of LEN + PEMBRO in uHCC, 104 patients (pts) with BCLC stage B (not amenable for transarterial chemoembolization) or C, Child-Pugh class A, and ECOG PS≤1 received LEN (body wt≥60kg: 12mg/day; Results Pts received LEN + PEMBRO (DLT-evaluation part, n=6; expansion part, n=61). At data cutoff (June 30, 2019), 34 (50.7%) pts remained on study treatment; median duration of follow-up was 11.7 months (95% CI, 7.8-17.6). Serious adverse events occurred in 42 (62.7%) pts; no new safety signals emerged. Updated safety data will be provided. ORR (including unconfirmed responses) was 44.8% (Table) and compared favorably with LEN arm of REFLECT trial (24.1%; Kudo M. Lancet. 2018). Median DOR was 18.7 months (95% CI, 6.9-NE). Conclusions LEN + PEMBRO showed promising antitumor activity and an acceptable safety profile in pts with uHCC. Blinded independent central review is in progress. A phase 3 trial (LEAP-002; NCT03713593) is ongoing to assess LEN + PEMBRO vs LEN alone as first-line therapy for pts with uHCC.Table747PTableResponse Parameter, n (%)LEN + PEMBRO (n=67)mRECIST Per Investigator ORR (CR + PR + unconfirmed PR or CR)a Best Overall Response CR. PRa Stable disease Progressive disease Unknown/not evaluable 30 (44.8) 4 (6.0) 26 (38.8) 25 (37.3) 6 (9.0) 6 (9.0) aIncludes unconfirmed partial responses (2 patients). CR, complete response; (m)RECIST, (modified) Response Evaluation Criteria In Solid Tumors [Lencioni et al. Semin Liver Dis. 2010;30:52-60]; ORR, objective response rate; PR, partial response. Clinical trial identification NCT03006926; Release date: December 30, 2016. Editorial acknowledgement Medical writing support was provided by April Suriano, PhD, of Oxford PharmaGenesis, Newtown, PA, USA and was funded by Eisai Inc. Legal entity responsible for the study Eisai Inc. Funding Eisai Inc. and Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA. Disclosure J. Llovet: Advisory / Consultancy, Research grant / Funding (self): Bayer; Advisory / Consultancy, Research grant / Funding (self): Eisai; Advisory / Consultancy, Research grant / Funding (self): Bristol-Myers Squibb; Advisory / Consultancy, Research grant / Funding (self): Ipsen; Advisory / Consultancy: Eli Lilly; Advisory / Consultancy: Celsion Corporation; Advisory / Consultancy: Exelixis; Advisory / Consultancy: Merck; Advisory / Consultancy: Glycotest; Advisory / Consultancy: Navigant; Advisory / Consultancy: Leerink Swann LLC; Advisory / Consultancy: Midatech Ltd; Advisory / Consultancy: Fortess Biotech; Advisory / Consultancy: Sprink Pharmaceuticals; Advisory / Consultancy: Nucleix; Advisory / Consultancy: Can-Fite Biopharma. K.V. Shepard: Full / Part-time employment: Eisai Inc. R.S. Finn: Advisory / Consultancy, Research grant / Funding (institution): Bayer; Advisory / Consultancy, Research grant / Funding (institution): Bristol-Myers Squibb; Advisory / Consultancy, Research grant / Funding (institution): Eisai; Advisory / Consultancy: Genentech/Roche; Advisory / Consultancy, Research grant / Funding (institution): Lilly; Advisory / Consultancy, Research grant / Funding (institution): Merck; Advisory / Consultancy, Research grant / Funding (institution): Novartis; Advisory / Consultancy, Research grant / Funding (institution): Pfizer. M. Ikeda: Honoraria (self): Abbott, Bayer, Bristol-Myers Squibb, Dainippon Sumitomo Pharma, Eisai, Lilly, Nobelpharma, Novartis, Otsuka, Taiho Pharmaceutical, Teijin Pharma, Yakult Honsha; Advisory / Consultancy: Bayer, Daiichi Sankyo, Eisai, Kyowa Hakko Kirin, MSD, NanoCarrier, Novartis, Shire, Teijin Pharma, Lilly; Research grant / Funding (self): ASLAN Pharmaceuticals, AstraZeneca, Baxalta/Shire, Bayer, Bristol-Myers Squibb, Chugai Pharma, Eisai, Kowa, Kyowa Hakko Kirin, Lilly, Merck Serono, MSD, NanoCarrier, Novartis, Ono Pharmaceutical, Taiho Pharmaceutical, Takara Bio, Yakult Honsha. M. Sung: Advisory / Consultancy: Bayer; Advisory / Consultancy: Eisai; Advisory / Consultancy: Exelixis. A.D. Baron: Speaker Bureau / Expert testimony: Amgen; Speaker Bureau / Expert testimony: Bristol-Myers Squibb; Speaker Bureau / Expert testimony: Genentech/Roche; Speaker Bureau / Expert testimony: Lilly; Speaker Bureau / Expert testimony: Merck. M. Kudo: Honoraria (self), Research grant / Funding (self): AbbVie; Honoraria (self), Advisory / Consultancy: Bayer; Honoraria (self), Advisory / Consultancy, Research grant / Funding (self): Bristol-Myers Squibb; Honoraria (self): EA Pharma; Honoraria (self), Advisory / Consultancy: Eisai; Honoraria (self): Gilead Sciences; Honoraria (self): Merck Serono; Honoraria (self), Advisory / Consultancy: MSD; Honoraria (self): Novartis; Honoraria (self): Pfizer; Honoraria (self), Research grant / Funding (self): Taiho Pharmaceutical; Research grant / Funding (self): Astellas Pharma; Research grant / Funding (self): Chugai Pharma; Research grant / Funding (self): Daiichi Sankyo; Research grant / Funding (self): Otsuka. T. Okusaka: Honoraria (self): AstraZeneca, Bayer, Bristol-Myers Squibb, Celgene, Chugai Pharma, Daiichi Sankyo, EA Pharma, Eisai, Fujifilm, Lilly Japan, Nippon Chemiphar, Nobelpharma, Novartis, Ono Pharmaceutical, Pfizer, Sumitomo Dainippon, Taiho Pharmaceutical, Teijin Pharma, Yakult; Advisory / Consultancy: Daiichi Sankyo, Sumitomo Dainippon, Taiho Pharmaceutical, Zeria Pharmaceutical; research funding from AstraZeneca, Baxter, Bayer, Chugai Pharma, Dainippon Sumitomo Pharma, Eisai, Kowa, Kyowa Hakko Kirin, Lilly Japan, Merck Serono, NanoCarrier, Nippon Boeh. M. Kobayashi: Speaker Bureau / Expert testimony: Eisai Inc. H. Kumada: Honoraria (self): AbbVie; Honoraria (self): Bristol-Myers Squibb; Honoraria (self): Gilead Sciences; Honoraria (self): MSD; Honoraria (self): Sumitomo; Honoraria (self): Dainippon. S. Kaneko: Honoraria (self): EA Pharma, MSD, Aska Pharma, Astellas Pharma, AbbVie, Eisai, Eidia, Otsuka, Gilead Sciences, Sysmex Corporation, Daiichi Sankyo, Taisho Toyama, Sumitomo Dainippon, Taiho, Takeda, Chugai, Bayer, Bristol-Myers, Mylan EPD, Miyarisan, Mochida, Kowa, Mitsubis; Research grant / Funding (self): Gilead Sciences, AbbVie, Daiichi Sankyo, Astellas Pharma, Takeda, Nippon Boehringer Ingelheim, MSD, Ono Pharma, Pfizer, Shionogi, Teijin Pharma, Mitsubishi Tanabe, Zeria, Abbott Vascular Japan, Taisho Toyama, Novartis, Terumo Corporation, Taiho, Novo Nord. K. Mamontov: Honoraria (self): Bayer; Honoraria (self): Eisai; Honoraria (self): Merck; Honoraria (self): MSD; Honoraria (self): Medtronic; Honoraria (self): Roche. T. Meyer: Advisory / Consultancy: Bristol-Myers Squibb; Advisory / Consultancy: Bayer; Advisory / Consultancy: Eisai; Advisory / Consultancy: BTG; Advisory / Consultancy: AstraZeneca; Advisory / Consultancy: BeiGene; Advisory / Consultancy: Tarveda; Advisory / Consultancy: MSD. K. Mody: Full / Part-time employment: Eisai Inc. T. Kubota: Full / Part-time employment: Eisai Co., Ltd. K. Saito: Full / Part-time employment: Eisai Inc. A.B. Siegel: Full / Part-time employment: Merck. L. Dubrovsky: Full / Part-time employment: Merck. A.X. Zhu: Advisory / Consultancy: AstraZeneca; Advisory / Consultancy, Research grant / Funding (institution): Bayer; Advisory / Consultancy, Research grant / Funding (institution): Bristol-Myers Squibb; Advisory / Consultancy: Eisai; Advisory / Consultancy: Exelixis; Advisory / Consultancy, Research grant / Funding (institution): Merck; Advisory / Consultancy, Research grant / Funding (institution): Lilly; Advisory / Consultancy, Research grant / Funding (institution): Novartis; Advisory / Consultancy: Sanofi. All other authors have declared no conflicts of interest.

Acute and chronic leukemias, including CD34(+) CML cells, demonstrate increased expression of the Wilms tumor gene 1 product (WT1), making WT1 an attractive therapeutic target. However, WT1 is a currently undruggable, intracellular protein. ESKM is a human IgG1 T-cell receptor mimic monoclonal antibody directed to a 9-amino acid sequence of WT1 in the context of cell surface HLA-A*02. ESKM was therapeutically effective, alone and in combination with tyrosine kinase inhibitors (TKIs), against Philadelphia chromosome-positive acute leukemia in murine models, including a leukemia with the most common, pan-TKI, gatekeeper resistance mutation, T315I. ESKM was superior to the first-generation TKI, imatinib. Combination therapy with ESKM and TKIs was superior to either drug alone, capable of curing mice. ESKM showed no toxicity to human HLA-A*02:01(+) stem cells under the conditions of this murine model. These features of ESKM make it a promising nontoxic therapeutic agent for sensitive and resistant Ph(+) leukemias.

TPS4152 Background: Len, an inhibitor of VEGF receptors 1-3, FGF receptors 1-4, PDGF receptor α, RET, and KIT, is approved for first-line treatment of unresectable HCC (uHCC) based on the open-label phase 3 REFLECT study in which len showed noninferior overall survival (OS) and significantly improved objective response rate (ORR), progression-free survival (PFS), and time-to-progression (TTP) vs sorafenib. In the phase 2 KEYNOTE-224 study of pembro (a PD-1 inhibitor) as second-line treatment of advanced HCC, pembro showed meaningful clinical efficacy in pts previously treated with sorafenib, with median PFS 4.9 mo, median OS 12.9 mo, and a manageable safety profile. In results from the phase 1b KEYNOTE-524 trial, len+pembro was well-tolerated, with promising antitumor activity in pts with uHCC. LEAP-002 is a phase 3 study to evaluate the safety and efficacy of len+pembro vs len+placebo as first-line therapy for advanced HCC. Methods: Eligible pts are ≥18 y and have HCC confirmed by radiology, histology, or cytology; ECOG PS 0/1; BCLC stage C or stage B disease not amenable to locoregional therapy or curative treatment approach; CP class A liver score within 7 days before study; and ≥1 measurable lesion by RECIST v1.1. Pts with past or ongoing HCV infection and those with controlled HBV are eligible. 750 pts will be randomized 1:1 to receive len 12 mg (body weight [BW] ≥60 kg) or 8 mg (BW 400 ng/mL; and ECOG PS 0/1. Primary end points are PFS per RECIST v1.1 by blinded independent central review (BICR) and OS. Secondary end points are ORR, duration of response, disease control rate, and TTP per RECIST v1.1 by BICR, efficacy per modified RECIST, pharmacokinetics, and safety. Imaging assessments will be performed Q9W on study. AEs will be graded per CTCAE v4.0 and monitored up to 90 days after last dose. Clinical trial information: NCT03713593.

Background High-dose chemotherapy followed by autologous hematopoietic cell transplantation (HCT) is used in many therapeutic protocols for pediatric intra- and extra-cranial solid tumors. HCT can be curative, but is associated with significant toxicity. Procedure Between January 2001 and June 2009, 92 solid tumor patients (age 6 months to 27 years) underwent 94 autologous apheresis procedures at Children's National Medical Center. Out of that group, 71 patients, who underwent 162 autologous HCT, were analyzed for transplant outcomes. Multiple variable modeling was used to identify independent variables related to transplant toxicity outcome measures, such as bacteremia, intensive care admission, and death. Other outcome measures (time to pre-apheresis peripheral blood CD34+ count, product yield, and time to engraftment) were also analyzed. Independent variables included patient-specific variables (age, weight, tumor type, chemotherapy administered, and primary vs. relapsed disease) and harvest or transplant-related variables (total white blood cell and CD34+ cell counts prior to transplant, and quantity of total nucleated cells and CD34+ cells infused during transplant). Results Transplant toxicity was significantly greater in younger patients (P = 0.001) and in neuroblastoma patients (P = 0.003). The time to neutrophil engraftment, controlling for weight, age, and chemotherapy, was positively related to absolute CD34+ cells/kg infused (P = 0.01). The time to CD34+ recovery pre-apheresis was affected by patient diagnosis (P = 0.05). Conclusions Younger patients had increased transplant toxicity, with infants

Purpose: To characterize the relationship between CD34+ collection efficiency and blood volumes processed in pediatric patients undergoing autologous peripheral blood stem cell (PBSC) collection. Methods: Retrospective 8-year (2001–2009) study of pediatric patients (n = 79) with neuroblastoma and central nervous system (CNS) tumors undergoing first day of autologous PBSC harvest using MNC program on the COBE Spectra (Caridian BCT, Lakewood, CO) was performed. Patients undergoing 0 to 2.9 BV (standard volume), 3 to 6 BV (large volume), and greater than 6 BV (ultra large volume) harvests were evaluated for CD34+ collection efficiency, diagnosis (neuroblastoma vs. nonneuroblastoma), disease type (primary vs. relapse), mobilization regimen, granulocyte colony stimulating factor (GCSF) dose, and apheresis complications. Results: CD34+ collection efficiencies (CE) for neuroblastoma patients were 67%, 50%, and 53% for standard (n = 14), large (n = 9), and ultra large (n = 5) volume harvests, respectively. Similarly, patients with nonneuroblastoma diagnoses had CD34+ CE of 63%, 55%, and 65% for low (n = 19), large (n = 27), and ultra large (n = 5) volume harvests, respectively. Weight, granulocyte colony stimulating factor (G-CSF) stimulation, type of mobilization, and apheresis complications (normalized by run time) were similar between the standard, large, and ultra large volume groups in patients with either neuroblastoma or nonneuroblastoma diagnoses. Conclusions: CD34+ collection efficiency in pediatric autologous PBSC collection on the first day of harvest does not decrease with higher numbers of blood volumes processed in patients with either neuroblastoma or nonneuroblastoma primary disease. These results indirectly indicate bone marrow CD34+ cell mobilization occurs with longer apheresis procedures in pediatric patients. J. Clin. Apheresis, 2011. © 2011 Wiley-Liss, Inc.

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.

To quantify the relationship between sequence and transmembrane dimer stability, a systematic mutagenesis and thermodynamic study of the protein-protein interaction residues in the glycophorin A transmembrane helix-helix dimer was carried out. The results demonstrate that the glycophorin A transmembrane sequence dimerizes when its GxxxG motif is abolished by mutation to large aliphatic residues, suggesting that the sequence encodes an intrinsic propensity to self-associate independent of a GxxxG motif. In the presence of an intact GxxxG motif, the glycophorin A dimer stability can be modulated over a span of -0.5 kcal mol(-1) to +3.2 kcal mol(-1) by mutating the surrounding sequence context. Thus, these flanking residues play an active role in determining the transmembrane dimer stability. To assess the structural consequences of the thermodynamic effects of mutations, molecular models of mutant transmembrane domains were constructed, and a structure-based parameterization of the free energy change due to mutation was carried out. The changes in association free energy for glycophorin A mutants can be explained primarily by changes in packing interactions at the protein-protein interface. The energy cost of removing favorable van der Waals interactions was found to be 0.039 kcal mol(-1) per A2 of favorable occluded surface area. The value corresponds well with estimates for mutations in bacteriorhodopsin as well as for those mutations in the interiors of soluble proteins that create packing defects.

Preferentially expressed antigen in melanoma (PRAME) is a cancer-testis antigen that is expressed in many cancers and leukemias. In healthy tissue, PRAME expression is limited to the testes and ovaries, making it a highly attractive cancer target. PRAME is an intracellular protein that cannot currently be drugged. After proteasomal processing, the PRAME300-309 peptide ALYVDSLFFL (ALY) is presented in the context of human leukocyte antigen HLA-A*02:01 molecules for recognition by the T cell receptor (TCR) of cytotoxic T cells. Here, we have described Pr20, a TCR mimic (TCRm) human IgG1 antibody that recognizes the cell-surface ALY peptide/HLA-A2 complex. Pr20 is an immunological tool and potential therapeutic agent. Pr20 bound to PRAME+HLA-A2+ cancers. An afucosylated Fc form (Pr20M) directed antibody-dependent cellular cytotoxicity against PRAME+HLA-A2+ leukemia cells and was therapeutically effective against mouse xenograft models of human leukemia. In some tumors, Pr20 binding markedly increased upon IFN-γ treatment, mediated by induction of the immunoproteasome catalytic subunit β5i. The immunoproteasome reduced internal destructive cleavages within the ALY epitope compared with the constitutive proteasome. The data provide rationale for developing TCRm antibodies as therapeutic agents for cancer, offer mechanistic insight on proteasomal regulation of tumor-associated peptide/HLA antigen complexes, and yield possible therapeutic solutions to target antigens with ultra-low surface presentation.

Purpose: RMFPNAPYL (RMF), a Wilms' tumor gene 1 (WT1)–derived CD8 T-cell epitope presented by HLA-A*02:01, is a validated target for T-cell–based immunotherapy. We previously reported ESK1, a high avidity (Kd < 0.2 nmol/L), fully-human monoclonal antibody (mAb) specific for the WT1 RMF peptide/HLA-A*02:01 complex, which selectively bound and killed WT1+ and HLA-A*02:01+ leukemia and solid tumor cell lines. Experimental Design: We engineered a second-generation mAb, ESKM, to have enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) function due to altered Fc glycosylation. ESKM was compared with native ESK1 in binding assays, in vitro ADCC assays, and mesothelioma and leukemia therapeutic models and pharmacokinetic studies in mice. ESKM toxicity was assessed in HLA-A*02:01+ transgenic mice. Results: ESK antibodies mediated ADCC against hematopoietic and solid tumor cells at concentrations below 1 μg/mL, but ESKM was about 5- to 10-fold more potent in vitro against multiple cancer cell lines. ESKM was more potent in vivo against JMN mesothelioma, and effective against SET2 AML and fresh ALL xenografts. ESKM had a shortened half-life (4.9 days vs. 6.5 days), but an identical biodistribution pattern in C57BL/6J mice. At therapeutic doses of ESKM, there was no difference in half-life or biodistribution in HLA-A*02:01+ transgenic mice compared with the parent strain. Importantly, therapeutic doses of ESKM in these mice caused no depletion of total WBCs or hematopoetic stem cells, or pathologic tissue damage. Conclusions: The data provide proof of concept that an Fc-enhanced mAb can improve efficacy against a low-density, tumor-specific, peptide/MHC target, and support further development of this mAb against an important intracellular oncogenic protein. Clin Cancer Res; 20(15); 4036–46. ©2014 AACR.

Specific immunotherapy for acute leukemia remains a great unmet need. Native unmodified monoclonal antibody therapies, while promising, are inadequately effective for these malignancies, and multiple mechanisms for failure have been described. Antibody-dependent cellular cytotoxicity or phagocytosis is the primary modality of mAb-mediated cell killing in vivo, but ultimately leads to relapse of the leukemias, in model systems and in humans. By use of a T-cell receptor mimic mAb ESKM, derived against a WT1 peptide expressed in complex with HLA-A*02:01, whose only mechanism of therapeutic action is ADCC, we evaluated the mechanisms of leukemic relapse from its potent therapeutic action in mouse xenograft models of human leukemia. Leukemia escape was not associated with loss of the antigenic target, downregulation of cell surface HLA, antibody pharmacokinetic or biodistribution issues, or development of leukemia cell-intrinsic resistance to ADCC. Interestingly, the rapidity of leukemic growth determined whether leukemia was able to evade cytotoxicity independent of the presence of sufficient effector cells. By engineering leukemia cells with upregulated p27Kip1 and slower cell cycling times, we show that relapse was inversely correlated with growth rates resulting in the eventual inadequacy of effector to target ratio. Moreover, lack of migration of effector cells into lymphomatous pockets of ALL also allowed local escape. Successful leukemia therapy with mAb might therefore be improved in similar situations by combination with measures to reduce burden and slow leukemia cell growth.

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.

Preferentially expressed antigen in melanoma (PRAME) is a well-validated target for T cell-based immunotherapy in leukemias and solid tumors. PRAME is a retinoic acid receptor binding protein that prevents retinoic acid-mediated differentiation, proliferation arrest, and apoptosis. As a cancer-testis antigen, PRAME has limited expression in healthy adult tissue that is restricted to the testes, ovaries, and endometrium. However, PRAME is over-expressed in multiple cancers including ALL, AML, melanomas, and breast cancers, making it a specific and highly attractive therapeutic target. PRAME is an intracellular protein making it impossible to target using traditional antibodies and it is not currently druggable. After proteasomal processing, the PRAME300-309 peptide is presented on the cell surface in the context of HLA*A02:01 molecules, for recognition by CD8 T cells. We therefore hypothesized that a TCR-mimic (TCRm) monoclonal antibody that recognizes surface PRAME300-309 presented by HLA*A02:01 could have therapeutic activity. Here, we describe Pr20, a therapeutic TCRm antibody, specific for the PRAME300-309 peptide in complex with HLA*A02:01, identified through a human scFv phage display library screen. Pr20 was engineered into full length human IgG1. Pr20 exhibited specific binding to PRAME300-309 -pulsed TAP-deficient T2 cells and bound PRAME+/ HLA*A02:01+ Ph+ ALL and AML, demonstrating that endogenously presented PRAME300-309 could be recognized by Pr20. Pr20 was determined to have 4 nM binding affinity by scatchard plot analysis. The specific epitope was mapped using alanine substitutions of non-anchor residues in the PRAME300-309 peptide and determined to primarily require the C-terminal residues. Pr20M, an afucosylated form of the antibody with enhanced Fc binding, mediated antibody-dependent cellular cytotoxicity (ADCC) in-vitro in a PRAME+/ HLA*A02:01+ restricted manner. Pharmacokinetic studies in C57BL/6 mice indicated that Pr20M was stable in-vivo and biodistribution studies in HLA*A02:01 transgenic mice suggested that there was no significant antibody sink. Pr20M was therapeutically active in established xenograft leukemia models in NSG mice (T, B, and NK-deficient). Interestingly, Pr20 binding to PRAME+/HLA*A02:01+ melanomas was minimally detectable, but was dramatically increased upon treatment with IFNγ, which also led to an increased sensitivity to ADCC. The data provide rationale for developing TCRm antibodies against intracellular oncoproteins as therapeutics. Disclosures No relevant conflicts of interest to declare.

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.

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.

3047 Background: Acute and chronic leukemias demonstrate significantly increased expression of the Wilms tumor gene 1 (WT1) product, including CD34+ CML stem cells, making WT1 an attractive therapeutic target. However, WT1 protein is intracellular and currently un-druggable. 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. Methods: BV173 is an HLA-A0201 positive Ph+ ALL cell line. It over-expresses WT1 and binds strongly to ESKM. We evaluated the in vitro and in vivo efficacy of ESKM in combination with TKIs. Antibody-dependent cell-mediated cytotoxicity (ADCC) was evaluated in vitro by chromium release assay, utilizing human PBMC effectors. In vivo tumor growth was assessed in NSG mice bearing disseminated luciferase tagged BV173 with bioluminescence imaging and flow cytometry of the bone marrow after sacrifice. Imatinib was used at maximum tolerated doses for these mice as determined in pilot studies. Results: The addition of imatinib in vitro did not affect the ability of ESKM to perform ADCC. The BV173 engrafted NSG mice treated with ESKM with and without TKIs showed tumor regression one week after beginning therapy, clearing leukemia from the liver and spleen. Mice relapsed primarily in the bone marrow, with increasing luciferase signal after two weeks of therapy. Compared to untreated control animals, after 5 weeks of therapy, imatinib alone only reduced tumor growth by 45%; ESKM alone reduced growth by 81%, and the combination of ESKM and imatinib reduced growth by more than 95%. Flow cytometry of cells remaining after treatment showed binding of ESKM, suggesting escape was not due to down regulation of the epitope. Conclusions: In this mouse model of Ph+ ALL, ESKM antibody therapy is superior to imatinib and the combination of both modalities is additive. This antibody is efficacious in vitro and in vivo against WT1 overexpressing leukemias, in context of HLA-A0201. This combination holds promise as a therapy for leukemias in patients who are HLA-A0201 positive, with the potential of improved cytoreduction in patients with Ph+ leukemias.