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A Divalent Hapten-Peptide Induces Apoptosis in Human Non–Hodgkin Lymphoma Cell Lines Targeted by Anti-CD20 x Anti-Hapten Bispecific Antibodies

Authors' Affiliations: ¹ Center for Molecular Medicine and Immunology, Belleville, New Jersey, ² IBC Pharmaceuticals, Inc., and ³ Immunomedics, Inc., Morris Plains, New Jersey

Requests for reprints: Robert M. Sharkey, Center for Molecular Medicine and Immunology, 520 Belleville Avenue, Belleville, NJ 07109. Phone: 973-844-7121; Fax: 973-844-7020; E-mail: rmsharkey{at}gscancer.org.

Purpose: Bispecific antibody (bsMAb) pretargeting procedures use divalent hapten-peptides to stabilize the binding of the hapten-peptide on tumor cells by a process known as the affinity enhancement system. The goal of this study was to determine if a divalent hapten-peptide could induce apoptosis by cross-linking bsMAb bound to CD20.

Methods: Three forms of bsMAbs were prepared by coupling the IgG, F(ab')₂, or Fab' of a humanized anti-CD20 antibody to a Fab' of a murine antibody directed against the hapten histamine-succinyl-glycine (HSG). A recombinant bsMAb with divalent binding to CD20 and monovalent binding to HSG was also examined. Induction of apoptosis on SU-DHL-6, RL, and Ramos cells was examined by propidium iodide staining, caspase-3 activation, and mitochondrial membrane potential collapse, and compared with induction by cross-linking an anti-CD20 IgG with an antispecies antibody.

Results: The various forms of bsMAb had differing baseline levels of apoptosis in the absence of the divalent HSG peptide. The addition of the divalent HSG peptide significantly increased the level of apoptosis seen with the Fab' x Fab' bsMAb by 2.2- to 3.9-fold, as well as the F(ab')₂ x Fab', IgG x Fab', and the recombinant bsMAbs by 1.5-fold.

Conclusions: The addition of a divalent HSG peptide to various forms of bispecific anti-CD20 MAbs could enhance apoptotic signaling in several lymphoma cells. This effect was more consistently measured when the orientation of the anti–hapten-binding arm of the bsMAb was well defined, such as in the Fab' x Fab' and recombinant forms of bsMAb.