T lymphocytes can be effectively recruited for ex vivo and in vivo lysis of AML blasts by a novel CD33/CD3-bispecific BiTE antibody construct

Abstract

Patients with acute myelogenous leukemia (AML) are in high need of novel targeted therapies. Here we explored the ex vivo activity of AMG330, a novel T-cell-engaging BiTE (bi-specific T-cell engagers) antibody (Ab) construct, that is bispecific for the myeloid differentiation antigen, CD33 and CD3, in primary samples from AML patients (N=23) and AML cell lines. KG-1 and U937 cells were lysed in co-culture with healthy donor T-cells at AMG330 concentrations as low as 0.1 ng/ml (1.8 pM). T-cells derived from AML patient samples were found to be as active in redirected lysis by AMG330 as T-cells from healthy donors. In an autologous setting, AMG330 could activate and expand T-cells in primary AML patient samples, and effectively mediated the redirected lysis of AML blasts and normal myeloid cells. A deficiency in target-cell lysis was only observed in samples with very low initial effector-to-target (E:T) ratio. However, this could be overcome if previously stimulated autologous T-cells were tested in patient samples at a higher E:T ratio. In vivo experiments in immunodeficient mice demonstrated significant inhibition of tumor growth by AMG330 and an inducible infiltration of human T-cells into subcutaneous HL60 tumors. The activities of the CD33/CD3-bispecific BiTE Ab construct AMG330 warrant further development for the treatment of AML.

Cellular determinants for preclinical activity of a novel CD33/CD3 bispecific T-cell engager (BiTE) antibody, AMG 330, against human AML

“…12,13 As prior in vitro investigations of the cytotoxic effect of AMG 330 demonstrated that the T cell killing of targets increased over time, 11 we first screened for activity at 24 hours. At this time point, very little specific cytotoxicity could be detected even at higher doses of AMG 330 (data not shown).…”

“…14 We used HL-60, ML-1, NB4, and TF-1 cells as additional models because of their CD33 expression and, in the case of HL-60, ML-1, and NB4 cells, our previous studies showing sensitivity to CD33-targeted therapy with GO. 12,13 As prior in vitro investigations of the cytotoxic effect of AMG 330 demonstrated that the T cell killing of targets increased over time, 11 we first screened for activity at 24 hours. At this time point, very little specific cytotoxicity could be detected even at higher doses of AMG 330 (data not shown).…”

“…AMG 330 has shown activity against AML blasts in initial preclinical studies but the critical cellular characteristics for the cytolytic activity have not been explored in detail. 11 Herein, we tested potential variables that may modulate the in vitro cytotoxicity of AMG 330 against human AML, using well-defined AML cell lines and genetically engineered sublines, and then conducted proof-of-principle studies in diagnostic specimens obtained from patients with AML.…”

Preclinical Characterization of AMG 330, a CD3/CD33-Bispecific T-Cell–Engaging Antibody with Potential for Treatment of Acute Myelogenous Leukemia

“…AMG 330 has previously shown robust ex vivo activity in peripheral blood mononuclear cell (PBMC) samples from patients with AML by redirecting autologous T cells for lysis of AML blasts at low endogenous effector-to-target (E:T) ratios (18). Here, we have characterized AMG 330 in detail for its dual binding activity, CD33 epitope recognition, stability in serum, cross-reactivity with a non-human primate species, potency of redirected lysis of multiple human AML cell lines, potency at reduced E:T ratios, sensitivity toward shed CD33, T-cell activation, and impact of intravenously (i.v.)…”

Dual-targeting triplebody 33-16-123 (SPM-2) mediates effective redirected lysis of primary blasts from patients with a broad range of AML subtypes in combination with natural killer cells

“…15 However, in spite of these limitations, the agent produced clear clinical benefits, including long-lasting treatment successes in particular after administration in fractionated doses to limit toxicity, probably due to its ability to eliminate some of the relapse-initiating LSCs. 16,17 Another CD33-directed antibody-drug conjugate, SGN-CD33A (Vadustuximab Talirine), is in clinical development, 18,19 as well as bispecific T cell engagers (BiTEs; AMG 330 and others), 20–24 and tetravalent tandabs targeting CD33 and engaging T cells as cytolytic effectors (AMV 564). 25 Finally, genetically engineered T cells equipped with transgenic chimeric antigen receptors (CARs) specific for CD33 showed anti-leukemic efficacy in xeno-transplanted mice and were tested in an AML-patient.…”