Abstract
Abstract Despite significant advances in the treatment of B-cell lymphoma, in many cases the disease remains untreatable or displays recurrence after initial response to therapy. The recent clinical success of bi-specific molecules capable of redirecting T-lymphocyte-mediated cytotoxicity (Bi-specific T-cell Engagers, BiTEs) against B-cell lymphoma provides a feasible therapeutic option. We have developed an alternate bi-specific platform of Dual-Affinity Re-Targeting (DART) molecules that is well suited for clinical applications. Compared to BiTEs, which comprise a pair of single-chain Fv domains in a single polypeptide, DARTs contain two separate chains associated into a heterodimeric, covalently linked, ‘diabody’ structure. We show here that DARTs are very stable in vitro and can be built to recruit and retarget different effector cells, including T cells or CD16- (Fc RIII)-positive cells, against B-cell-lineage targets. A CD3xCD19 DART capable of recruiting cytotoxic T cells against CD19-positive targets mediated extremely potent killing of human peripheral blood B lymphocytes or B-cell lymphoma lines in vitro, with an EC50 that compares favorably to that of a BiTE with identical Fv sequences. Redirected killing of Daudi, Raji, or Nalm6 lymphoma lines was observed in the picomolar range using either freshly purified T cells or peripheral blood mononuclear cells (PBMC) as effector populations. In contrast, no killing was observed against a CD19-negative cell line (RPMI-8226) with either resting or stimulated effector populations, indicating a requirement for CD19 engagement for T-cell-mediated killing by the CD3xCD19 DART molecule. Furthermore, a DART constructed with a humanized version of a pan anti-TCR-beta antibody (TCRxCD19 DART) was as efficient against CD19-positive targets as the CD3-based DART. A CD16xCD32B DART was also constructed to redirect CD16-positive NK and/or mononuclear phagocyte effector cells against CD32B-positive B cells, displaying potent killing against Daudi, Raji or RPMI-8226 cells but not against a CD32B-negative cell line (Ramos). Both TCR- and CD16-based DARTs show potent activity in vivo in several mouse models, including autologous B-cell depletion in huCD32B-transgenic mice, tumor reduction in human Raji Burkitt's lymphoma cell xenografts and Raji cell implants in NOD/SCID mice reconstituted with human PBMC. In conclusion, DARTs represent a robust and flexible platform to recruit different cell populations via various cell surface targets for re-directed killing applications against B-cell malignancies. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C180.
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