Abstract: TPS7071 Background: The CD33 antigen is expressed on myeloblasts in approximately 75% of patients with myelodysplastic syndromes (MDS) and is also expressed on immunosuppressive myeloid-derived suppressor cells (MDSCs) that are prevalent in MDS. AMV564 is a novel bivalent, bispecific (2:2) T-cell engager that binds both CD33 and the invariant CD3ε on T-cells with strong avidity. AMV564 has significant cytotoxic activity against CD33-postive cells, including MDSCs, in animal models, in vitro studies, and in patients (Reusch et al. Clin Can Res 2016 and List et al. ASH 2017). Preliminary results from the phase 1 acute myeloid leukemia (AML) trial have demonstrated safety and activity in patients with AML arising from prior MDS (Westervelt et al. ASH 2018). Methods: AMV564-201 is an open label, phase 1, multicenter, dose-escalation with expansion trial of AMV564 in patients with intermediate-2 or high-risk MDS who are refractory to or relapsed from hypomethylating agents (HMAs) or intensive chemotherapy. The key objectives of the dose-escalation stage of the study are to characterize the safety and tolerability of AMV564, identify a maximum tolerated dose and/or a dose-schedule to evaluate in the dose expansion portion of the study. In the dose expansion stage of the study, the safety and tolerability of AMV564 will be further characterized in addition to a preliminary assessment of efficacy. Other objectives include characterization of AMV564 pharmacokinetics, pharmacodynamics, and immunogenic potential. Approximately 80 patients with intermediate-2 or high-risk MDS will be enrolled. The Dose Escalation Stage will include up to approximately 30 patients, depending on the dose at which the MTD is determined for each schedule, and approximately 50 additional patients will be enrolled in the Expansion Stage. AMV564 will be administered daily as a continuous intravenous (CIV) infusion over a period of 24 hours for 14 days in each cycle. Each cycle will be approximately 4 weeks. A lead-in dose regimen will be utilized for all patients treated at dose levels at or above 50 mcg/day. Patients will receive up to 4 cycles of AMV564 treatment at the assigned dose level. Clinical trial information: NCT03516591.

Abstract: Background: AMV564 is a novel bivalent, bispecific (2x2) CD33/CD3 targeted immunotherapy that binds both CD33 and the invariant CD3ε on T-cell receptors with strong avidity, thus creating an immune synapse between CD33-expressing cells and T cells, initiating T-cell directed lysis of CD33 expressing cells, and inducing expansion, differentiation and proliferation of T cells. AMV564 is being evaluated in clinical trials for patients with acute myeloid leukemia (AML, NCT03144245). Previously, we demonstrated that the parent molecule, T564, had specific, T-cell mediated cytotoxic activity against a KG-1 CD33+ cell line in vitro and eliminated blasts in an autologous AML patient-derived xenograft mouse model (Eissenberg, et al. 2015 ASCO). Here we investigated factors that may contribute to antileukemic activity of AMV564 in another, more aggressive, preclinical model of AML. Methods: Disseminated activity studies in NOD scid gamma mice (NSG) were performed by injecting MOLM13 AML cells transduced with click beetle red luciferase and green fluorescent protein (MOLM13-CG) in the tail vein. In a series of experiments, AMV564-mediated clearance of MOLM13-CG cells and overall mouse survival was determined. Tumor cells (either 3.3 x 103 or 1 x 105) were engrafted in non-conditioned NSG mice for 3-7 days. Variables included dose of AMV564 (0.5 to 25 mcg, i.v.), number of AMV564 cycles (1 or 2) and cycle length (4-5 days), number of T cell injections (1 or 2), and total number of human T cells administered (0.2 to 1.2 x 107). Tumor burden was serially measured by bioluminescence (BLI) and survival measured for 51 days. Results: Mouse survival was greatest when AMV564 was injected along with a total number of T cells ≥ 8 x 106 regardless of the number of T cell injections or their timing relative to tumor injection. AMV564 mediated up to a 3-fold increase in survival time compared to untreated mice. In the experiment shown in Figure 1, untreated mice died at day 28, yet about half of the treated mice were still alive when the experiment was terminated on day 51. In the absence of AMV564 (i.e., T cell treatment only), animal survival and tumor burden were similar to that of untreated animals. In the same experiment we administered, AMV564 daily from days 7-11 after MOLM13-CG infusion along with one injection of 8 x 106 human T cells on day 7, then measured tumor burden by BLI signal (Figure 2). By day 14 we observed a median 4.0 log or 4.8 log reduction in tumor using 5 or 25 mcg AMV564. In fact by day 14, 4 of 10 mice treated with 25 mcg AMV564 and 2 of 10 treated with 5 mcg had no detectable signal above background. No rebound in tumor burden occurred in these mice throughout the 51-day study. Conclusions: AMV564 is a potent and effective immunotherapy against an aggressive human AML cell line in NSG mice. AMV564 very effectively prolonged survival and dramatically reduced tumor in the bone marrow and peripheral blood. These results are consistent with our previous preclinical data with primary AML and support the testing of this reagent in patients with relapsed and refractory AML in the clinic. Disclosures Eissenberg: Amphivena Therapeutics: Research Funding; Novimmune: Research Funding. Rettig:Novimmune: Research Funding; Amphivena Therapeutics: Research Funding. Fox:Sunesis Pharmaceuticals: Employment; Amphivena Therapeutics: Employment. Guenot:Amphivena Therapeutics, Inc: Employment.

Abstract: Purpose: Randomized studies with gemtuzumab ozogamicin have validated CD33 as a target for antigen-specific immunotherapy of acute myelogenous leukemia (AML). Here, we investigated the potential of CD33/CD3-directed tandem diabodies (TandAbs) as novel treatment approach for AML. These tetravalent bispecific antibodies provide two binding sites for each antigen to maintain the avidity of a bivalent antibody and have a molecular weight exceeding the renal clearance threshold, thus offering a longer half-life compared to smaller antibody constructs. Experimental Design: We constructed a series of TandAbs composed of anti-CD33 and anti-CD3 variable domains of diverse binding affinities and profiled their functional properties in CD33+ human leukemia cell lines, xenograft models, and AML patient samples. Results: Our studies demonstrated that several CD33/CD3 TandAbs could induce potent, dose-dependent cytolysis of CD33+ AML cell lines. This effect was modulated by the effector-to-target cell ratio and strictly required the presence of T cells. Activation and proliferation of T cells and maximal AML cell cytolysis correlated with high avidity to both CD33 and CD3. High-avidity TandAbs were broadly active in primary specimens from patients with newly diagnosed or relapsed/refractory AML in vitro, with cytotoxic properties independent of CD33 receptor density and cytogenetic risk. Tumor growth delay and inhibition were observed in both prophylactic and established HL-60 xenograft models in immunodeficient mice. Conclusions: Our data show high efficacy of CD33/CD3 TandAbs in various preclinical models of human AML. Together, these findings support further study of CD33/CD3 TandAbs as novel immunotherapeutics for patients with AML. Clin Cancer Res; 22(23); 5829–38. ©2016 AACR.

Abstract: Background: AMV564 is a novel bivalent, bispecific (2:2) CD33/CD3 T-cell engager that binds CD33 on target cells and CD3 on T-cells leading to T-cell-directed lysis of CD33+ leukemic blasts and myeloid derived suppressor cells (MDSCs), as well as T-cell expansion, differentiation and proliferation. By design, AMV564 has reduced clearance and therefore has a longer half-life (t1/2) than monovalent, bispecific T-cell engagers. In preclinical investigations using both leukemic cell lines and primary cells from AML patients, AMV564 eliminated myeloid blasts with picomolar potency and broad activity independent of cytogenetic or molecular abnormalities, CD33 expression level, and disease stage, with no nonspecific activation of T cells (Reusch U et al. Clin Cancer Res. 2016;22:5829-38). Methods: This is an ongoing Phase 1 study with a 3+3 dose-escalation design (NCT03144245). The primary objectives of this study are to characterize the safety, tolerability, and preliminary anti-leukemic activity of AMV564. Evaluation of pharmacokinetics (PK), cytokine changes, and immunophenotyping are secondary objectives. Key inclusion/exclusion criteria are: adults with relapsed/refractory AML after 1-2 prior induction regimens (with a standard anthracycline-based regimen or hypomethylating agent) and no more than 2 prior salvage regimens. AMV564 is administered by continuous intravenous infusion (CIV) for 14 consecutive days over a 28 day cycle, with prophylactic antiemetics, antipyretics, and antihistamines. AMV564 and cytokine (IL2, IL4, IL6, IL8, IL10, TNF-α, and IFN-γ) concentrations were measured by validated immunoassays. T-cell activation was measured using flow cytometry to quantify T cells expressing CD25, CD38, CD69, or HLA-DR. Results: To date, 36 patients (20 male/16 female) with a median age of 71 years (range 24-85 years) have been enrolled in 10 dose cohorts from 0.5 to 300 mcg/day. Twenty-four patients (67%) had secondary AML and/or adverse cytogenetics, including 9 patients (25%) with a TP53 mutation. Fourteen patients (39%) had received at least 1 prior salvage regimen and 23 (64%) had received prior intensive chemotherapy, including 13 patients (36%) who had received a high-dose (≥ 1 g/m2) cytarabine-based regimen and 1 patient (3%) with prior allogeneic stem cell transplant. At the time of this analysis, 36 patients were evaluable for safety and 35 patients were evaluable for activity. No dose-limiting toxicities were reported. Median duration of treatment was 20 days (range 3-204 days). Using a lead-in dose escalation schedule, no Grade 3 or higher cytokine release syndrome has been observed. The most common Grade ≥3 treatment-emergent AE has been anemia, reported in 4 (11%) patients. No patient has died within 30 days of treatment initiation. Bone marrow blast reductions have been observed in 17 (49%) of 35 efficacy evaluable patients. Objective responses have been observed including 1 complete response (CR) during cycle 1 at the 200 mcg/day assigned dose, 1 CRi (CR with incomplete hematologic recovery) during cycle 2 at the 150 mcg/day assigned dose, and 1 partial response (PR) during cycle 1 at the 100 mcg/day assigned dose. In addition, 3 patients had hematologic improvement in neutrophil counts. AMV564 PK was dose proportional through the 100 mcg/day dose level with a terminal half-life of 2-3 days. Serum concentrations increased gradually, with times to steady-state concentration of 3-7 days. T-cell redistribution from the periphery upon initiation of dosing (consistent with T-cell activation) was observed, as was evidence of increased bone marrow T-cells with repeated cycles of treatment. Conclusions: AMV564 is well-tolerated and demonstrates anti-leukemic activity through T-cell engagement. Disclosures Cortes: Sun Pharma: Research Funding; Forma Therapeutics: Consultancy, Honoraria, Research Funding; Merus: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; BiolineRx: Consultancy; Immunogen: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Astellas Pharma: Consultancy, Honoraria, Research Funding; Jazz Pharmaceuticals: Consultancy, Research Funding; Biopath Holdings: Consultancy, Honoraria; Daiichi Sankyo: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding. Altman:Novartis: Consultancy; Cancer Expert Now: Consultancy; Agios: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Glycomimetics: Consultancy, Honoraria, Other: Data Safety and Monitoring Committee; Theradex: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; PeerView: Speakers Bureau; prIME Oncology: Speakers Bureau; France Foundation: Speakers Bureau; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Biosight: Other: US Lead. Oehler:Pfizer Inc.: Research Funding; Blueprint Medicines: Consultancy; NCCN: Consultancy. Gojo:Abbvie: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Merck: Research Funding; Juno: Research Funding; Amgen Inc: Consultancy, Honoraria, Research Funding; Amphivena: Research Funding. Guenot:Amphivena Therapeutics, Inc.: Employment. Chun:Amphivena Therapeutics, Inc: Employment. Roboz:Celltrion: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bayer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Argenx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amphivena: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Actinium: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Eisai: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees; Otsuka: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sandoz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Trovagene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees.