T-cell ligands modulate the cytolytic activity of the CD33/CD3 BiTE antibody construct, AMG 330

Abstract

Preclinical and emerging clinical studies demonstrate that bispecific T-cell engaging (BiTE) antibody constructs can potently lyse targeted tumor cells, but the determinants for their activity remain incompletely understood. Using human acute myeloid leukemia (AML) cell lines engineered to overexpress individual T-cell ligands, we found that expression of the inhibitory ligands, PD-L1 and PD-L2, reduced the cytolytic activity of the BiTE antibody construct targeting CD33, AMG 330; conversely, expression of the activating ligands, CD80 and CD86, augmented the cytotoxic activity of AMG 330. Consistent with these findings, treatment with an activating antibody directed at the co-stimulatory T-cell receptor, CD28, significantly increased AMG 330-induced cytotoxicity in human AML cell lines. Using specimens from 12 patients with newly diagnosed or relapsed/refractory AML, we found that activation of CD28 also increased the activity of AMG 330 in primary human AML cells (P=0.023). Together, our findings indicate that T-cell ligands and co-receptors modulate the anti-tumor activity of the CD33/CD3 BiTE antibody construct, AMG 330. These findings suggest that such ligands/co-receptors could serve as biomarkers of response and that co-treatment strategies with pharmacological modulators of T-cell receptor signaling could be utilized to further enhance the activity of this targeted therapeutic.

Characterization of CD33/CD3 Tetravalent Bispecific Tandem Diabodies (TandAbs) for the Treatment of Acute Myeloid Leukemia

“…These analyses demonstrated that the TandAbs not only activated T-lymphocytes but also re-directed polyclonal T cells from healthy donors as well as autologous T cells from patients with AML to effectively lyse CD33 þ AML cells even at low E:T cell ratios. The anti-AML effect of these TandAbs was dependent on antibody concentration and E:T cell ratio, and required the presence of both T cells and CD33 þ target cells-mechanistic determinants that are similar to those identified recently for small CD33/CD3-directed bispecific antibodies (8)(9)(10)(11)(12)(13)(14)(15)(16)(17). Importantly the TandAbs' avidity of CD3 and CD33 strongly influenced the degree of cytotoxic activity in that, for high antileukemia potency, high avidity to both CD33 and CD3 was required.…”

“…S1 for analytic strategy; refs. 13,16,17). After 48 hours, cell numbers and viability, using DAPI to detect non-viable cells, were determined using a LSRII cytometer (BD Biosciences) and analyzed with FlowJo.…”

“…After 48 hours, cell numbers and viability, using DAPI to detect non-viable cells, were determined using a LSRII cytometer (BD Biosciences) and analyzed with FlowJo. Results from cytotoxicity assays are presented as the mean percentage of specific cytotoxicity AE SEM (13,16,17).…”

“…Recent preclinical data with small bispecific antibodies that combine the variable fragments (Fvs) of the two antibodies on one polypeptide chain, including AMG 330, a CD33/CD3 bivalent bispecific T-cell-engaging (BiTE) antibody, demonstrate that such constructs can bring polyclonal CD3 þ T cells in close proximity to CD33 þ AML cells, trigger lymphocyte activation, and then lead to efficient cytolysis of tumor cells at low effector-to-target (E:T) cell ratios (8)(9)(10)(11)(12)(13)(14)(15)(16)(17). However, because of their low molecular weight of approximately 55 kDa, these and similar antibody constructs are readily excreted by the kidneys, resulting in short half-lives and need for prolonged continuous intravenous administration (18)(19)(20).…”

In Vitro and In Vivo Efficacy of a Novel CD33-Targeted Thorium-227 Conjugate for the Treatment of Acute Myeloid Leukemia

“…Furthermore, AMG330, a CD33/CD3-directed bispecific T-cell engager antibody (BiTE; refs. [10][11][12][13], has entered clinical testing and chimeric antigen receptors T-cell approaches (CAR-T) are additionally feeding the pipeline for the treatment of AML (14,15). All of these immunotherapeutic agents are targeting the sialoadhesin receptor CD33 (Siglec-3), a 67-kDa protein, expressed on leukemic blasts of AML patients (16).…”