Abstract 4572: Heterodimerization of CD74 and CD20 by two hexavalent, bispecific, anti-CD20/CD74 antibodies leads to potent cytotoxicity in mantle cell lymphoma
Authors: Gupta Pankaj , Rossi Edmund A. , Goldenberg David M. , Chang Chien-Hsing 
Published: June 2012

Abstract
Hexavalent antibodies (HexAbs) comprising four Fabs of one specificity linked to a full IgG of the same or a different specificity can be successfully generated by the Dock-and-Lock (DNL) method to retain the binding activity of each constitutive Fab. Whereas HexAbs made from veltuzumab (anti-CD20) and epratuzumab (anti-CD22) show direct toxicity in several B-cell non-Hodgkin lymphomas (NHL) in vitro, they are ineffective against mantle cell lymphoma (MCL) lines expressing similar levels of CD20 and CD22. In this study, we describe the properties of two novel HexAbs, one comprising veltuzumab linked to four Fabs of milatuzumab (anti-CD74), designated 20-(74)-(74), and the other comprising milatuzumab linked to four Fabs of veltuzumab, designated 74-(20)-(20). Both 20-(74)-(74) and 74-(20)-(20) potently inhibited the growth of MCL lines (JeKo-1, Granta-519, and Mino), chronic lymphocytic leukemia lines (WAC and MEC), and NHL lines (Daudi and Raji) at 10 nM in the MTS cell proliferation assay, without the need for crosslinking by a secondary antibody. In contrast, anti-proliferative activity was noted in Jeko-1 and other cell lines for the parental antibodies or the two monospecific HexAbs, 74-(74)-(74) and 20-(20)-(20), only in the presence of a secondary antibody to effect hypercrosslinking, suggesting the requirement of heterodimerization of CD74 and CD20 for the observed cytotoxicity. Additional experiments showed the two anti-CD20/CD74 HexAbs induced substantial apoptosis (25 to 30%) in Jeko-1 and Granta-519, which appeared to accompany significant changes in mitochondrial membrane potential, detectable generation of reactive oxygen species, pronounced phosphorylation of ERK and JNK MAP kinases, and downregulation of Bcl-xL, but not with activation of caspases or the involvement of autophagy. A key differentiation of the anti-CD20/CD74 HexAbs from the anti-CD20/CD22 HexAbs is the ability of the former to induce strong homotypic adhesion of the MCL lines, but not the latter or the respective parental antibodies. Immunofluorescence revealed 74-(20)-(20) internalized slower than 20-(74)-(74), which may account for the consistently higher potency of 74-(20)-(20) than 20-(74)-(74). We also found that pretreatment with C16, an agonistic anti-CD74 antibody known to promote cell proliferation, or macrophage migration-inhibitory factor (MIF), the pro-survival ligand of CD74, did not abrogate the growth inhibition induced by the anti-CD20/CD74 HexAbs. The potent in vitro cytotoxicity of the anti-CD20/CD74 HexAbs against MCL lines warrants their further evaluation in B-cell lymphomas that are refractory or poorly responsive to anti-CD20 or anti-CD22 antibodies.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4572. doi:10.1158/1538-7445.AM2011-4572