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.