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

Abstract

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 ¶ Â Fab ¶ bsMAb by 2.2-to 3.9-fold, as well as the F(ab ¶) 2 Â Fab ¶, IgG Â Fab ¶, and the recombinant bsMAbs by f1.5-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 ¶ Â Fab ¶ and recombinant forms of bsMAb.

Improved Therapeutic Results by Pretargeted Radioimmunotherapy of Non–Hodgkin's Lymphoma with a New Recombinant, Trivalent, Anti-CD20, Bispecific Antibody

“…Other controls included 90 Y-IMP-288 alone, 90 Y-IMP-288 pretargeted with the irrelevant TF2 anti-carcinoembryonic antigen (CEA) bsMAb, and non-radiolabeled IMP-288 pretargeted with TF4 (i.e., TF4 and IMP-288 doses equivalent to 0.5 mCi 90 Y-IMP-288 treatment). The last control group was included because in vitro studies had indicated that the divalent IMP-288 hapten-peptide could induce apoptosis in a lymphoma cell line as a consequence of crosslinking TF4 on the cell surface (39). All syringes were read for 90 Y-activity in a calibrated dose calibrator (Capintec CRC-15R Ramsey, NJ).…”

Pretargeted Versus Directly Targeted Radioimmunotherapy Combined with Anti-CD20 Antibody Consolidation Therapy of Non-Hodgkin Lymphoma

“…Although in vitro studies have shown that the divalent hapten-peptide can cross-link an anti-CD20 · antihapten bsMAb and enhance apoptosis, in vivo studies in nude mice bearing established tumors, versus untreated animals, indicated no significant therapeutic improvement of the bsMAb with the unlabeled hapten-peptide (9,14). Therefore, the antitumor activity elicited with PT-RAIT is associated solely with the radiation.…”