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

Abstract

There is high demand for novel therapeutic options for patients with acute myelogenous leukemia (AML). One possible approach is the bispecific T-cell-engaging (BiTE, a registered trademark of Amgen) antibody AMG 330 with dual specificity for CD3 and the sialic acid-binding lectin CD33 (SIGLEC-3), which is frequently expressed on the surface of AML blasts and leukemic stem cells. AMG 330 binds with low nanomolar affinity to CD33 and CD3e of both human and cynomolgus monkey origin. Eleven human AML cell lines expressing between 14,400 and 56,700 CD33 molecules per cell were all potently lysed with EC 50 values ranging between 0.4 pmol/L and 3 pmol/L (18-149 pg/mL) by previously resting, AMG 330-redirected T cells. Complete lysis was achieved after 40 hours of incubation. In the presence of AML cells, AMG 330 specifically induced expression of CD69 and CD25 as well as release of IFN-g, TNF, interleukin (IL)-2, IL-10, and IL-6. Ex vivo, AMG 330 mediated autologous depletion of CD33-positive cells from cynomolgous monkey bone marrow aspirates. Soluble CD33 at concentrations found in bone marrow of patients with AML did not significantly affect activities of AMG 330. Neoexpression of CD33 on newly activated T cells was negligible as it was limited to 6% of T cells in only three out of ten human donors tested. Daily intravenous administration with as low as 0.002 mg/kg AMG 330 significantly prolonged survival of immunodeficient mice adoptively transferred with human MOLM-13 AML cells and human T cells. AMG 330 warrants further development as a potential therapy for AML. Mol Cancer Ther; 13(6); 1549-57. Ó2014 AACR.

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.…”

“…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).…”

A novel BCMA/CD3 bispecific T-cell engager for the treatment of multiple myeloma induces selective lysis in vitro and in vivo

“…Purification of the monomeric protein was performed in a two-step process using immobilized nickel chelate chromatography followed by size exclusion chromatography, as described for other BiTE antibody constructs. 26,27 Cell lines and cell culture Cell lines were obtained from ATCC and DSMZ. The cells were cultured as described in the supplier's description and tested to exclude mycoplasma contamination.…”

Development of a Target cell-Biologics-Effector cell (TBE) complex-based cell killing model to characterize target cell depletion by T cell redirecting bispecific agents

“…The binding affinity values of blinatumomab to CD3 and CD19 were 260 nM and 1.49 nM, respectively. 23 The binding affinity values of AMG 330 to CD3 and CD33 were 5.1 nM and 8.0 nM, respectively 57 . The binding affinity values of P-cadherin LP-DART to CD3 and P-cadherin were 11.4 nM and 0.47 nM, respectively.…”