Phage Display–Derived Human Monoclonal Antibodies Isolated by Binding to the Surface of Live Primary Breast Cancer Cells Recognize GRP78

Abstract

Clinical trials using monoclonal antibodies (mAb) against cell-surface markers have yielded encouraging therapeutic results in several cancer types. Generally, however, anticancer antibodies are only efficient against a subpopulation of cancers, and there is a strong need for identification of novel targets and human antibodies against them. We have isolated single-chain human mAbs from a large naïve antibody phage display library by panning on a single-cell suspension of freshly isolated live cancer cells from a human breast cancer specimen, and these antibodies were shown to specifically recognize cancer-associated cell-surface proteins. One of the isolated human antibody fragments, Ab39, recognizes a cellsurface antigen expressed on a subpopulation of cancer cell lines of different origins. Immunohistochemical analysis of a large panel of cancerous and normal tissues showed that Ab39 bound strongly to several cancers, including 45% breast carcinomas, 35% lung cancers, and 86% melanomas, but showed no or weak binding to normal tissues. A yeast twohybrid screen of a large human testis cDNA library identified the glucose-regulated protein of 78 kDa (GRP78) as the antigen recognized by Ab39. The interaction was confirmed by colocalization studies and antibody competition experiments that also mapped the epitope recognized by Ab39 to the COOH terminus of GRP78. The expression of GRP78 on the surface of cancer cells, but not normal cells, makes it an attractive target for cancer therapies including mAb-based immunotherapy. Our results suggest that the human antibody Ab39 may be a useful starting point for further genetic optimization that could render it a useful diagnostic and therapeutic reagent for a variety of cancers. [Cancer Res 2007;67(19):9507-17]

Cell Surface Relocalization of the Endoplasmic Reticulum Chaperone and Unfolded Protein Response Regulator GRP78/BiP

“…The discovery of patient antisera specifically recognizing O-linked GRP78 on the surface of malignant cells (34) and the discovery that C terminus modification of GRP78 accounts for cancer-specific antibody specificity (35) prompted us to test the hypothesis that the putative O-linked glycosylation site of GRP78 is within close proximity of the KDEL motif and that modification of this site could mask the KDEL motif and allow GRP78 to escape the KDEL retrieval system. Our results showed no significant change in the level or the electrophoretic mobility of surface GRP78 regardless of whether the putative glycosylation site was intact or mutated in three different cell types.…”

“…Cell Surface Localization-Recent reports suggest the existence of an O-linked glycosylated form of GRP78 at the cell surface, and the site was implicated at the C terminus of GRP78 (34,35). Analysis of potential O-linked glycosylation sites on human GRP78 by the Net OGly 3.1 program revealed the strongest site at threonine 648 with close proximity to the KDEL motif at the C terminus of GRP78 (Fig.…”

“…This finding raises the possibility of targeting tumor cells via a specific cell surface variant of GRP78 (34). Further, phage display-derived human monoclonal antibodies isolated by binding to the surface of live primary breast cancer cells recognize a modified form of surface GRP78 at the C terminus (35). Despite these advances, the extent and mechanism of relocalization of GRP78/BiP from the ER to the cell surface at the biochemical level is not well understood.…”

Targeted Cancer Therapeutics

“…The power of structural biology coupled with fragment-based chemistry is leading to the rapid production of lead compounds with improved drug-like properties, increasing the probability of better lead optimization and ultimately more effective compounds (25). Similarly, phage displays can be used to identify antibodies against targets of interest, thereby accelerating the process of hybridoma creation and screening of thousand of clones (26).…”

Endoplasmic Reticulum Chaperones at the Tumor Cell Surface and in the Extracellular Space

“…Glykosylace nebo jiná modifikace tak může maskovat signální tetrapeptid KDEL a zabránit tak jeho vazbě s receptorem. U proteinu GRP78 bylo v blízkosti KDEL sekvence identifikováno potenciální glykosylační místo (Thr648) [24,25]. Současně byly popsány další odlišně lokalizované posttranslačně modifikované varianty kalretikulinu v buňce zahrnující arginylovanou formu na buněčné membráně a v cytozolu [26] a citrullinovanou formu související s imunitní aktivitou u revmatoidní artritidy [27,28].…”