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The antitumor monoclonal antibody 806 recognizes a high-mannose form of the EGF receptor that reaches the cell surface when cells over-express the receptor

Terrance G. Johns^*,1, Ira Mellman, Glenn A. Cartwright^*, Gerd Ritter, Lloyd J. Old, Antony W. Burgess and Andrew M. Scott^*

^* Ludwig Institute for Cancer Research, Tumour Targeting Program, Austin Hospital, Heidelberg, Australia and
Epithelial Biochemistry Laboratory, Royal Melbourne Hospital, Parkville, Australia;
Department of Cell Biology, Ludwig Institute for Cancer Research, Yale University School of Medicine, New Haven, Connecticut, USA;
Ludwig Institute for Cancer Research, New York Branch at Memorial Sloan-Kettering Cancer Center, New York, New York, USA

¹ Correspondence: Oncogenic Signalling Laboratory, Ludwig Institute for Cancer Research, Austin Hospital Level 6, Studley Road, Heidelberg 3084, Australia. E-mail: terry.johns{at}ludwig.edu.au

SPECIFIC AIMS

mAb 806 is a novel EGFR antibody with anti-tumor activity that was selected because of its specificity for a cancer-associated truncation of the EGFR known as the de2-7 EGFR. mAb 806 binds a subset of the wild-type (wt) EGFR when overexpressed, but does not bind the wt EGFR expressed in normal tissues. In this study, we conducted a detailed biochemical analysis of the de2-7 and wt EGFR recognized by mAb 806 in order to further define the unique specificity of this antibody.

PRINCIPAL FINDINGS

1. Immunoprecipitation of the wt EGFR by mAb 806 from cells overexpressing the receptor confirmed that this antibody only binds to a portion of the EGFR
The EGFR immunoprecipitated by mAb 806 appears to run faster on SDS-PAGE than the bulk of the receptor, suggesting it may be under-glycosylated. Pharmacological inhibition of glycosylation dramatically increased mAb 806 reactivity in cells overexpressing the EGFR.

2. Pulse/chase experiments in cells expressing the de2-7 EGFR or overexpressing the wt EGFR clearly showed that mAb 806 preferentially recognizes a form of the EGFR found at an early stage of processing

3. The EGFR immunoprecipitated by mAb 806 is sensitive to endoglycosidase H, an enzyme that removes the N-linked high-mannose moieties but not complex carbohydrates from glycosylated proteins
Thus, mAb 806 preferentially binds the immature high-mannose wt and de2-7 EGFR precursors normally located in the endoplasmic reticulum.

4. Using the unique specificity of mAb 806, we clearly demonstrated the presence of these high-mannose EGFR precursors on the cell surface
Since mAb 806 can only bind to a partially activated form of the wt EGFR, these high-mannose forms of the EGFR may possess unique signaling properties.

5. The presence of the high-mannose wt EGFR on the surface of cells that overexpress the receptor contributes to the unique specificity of mAb 806
Given that this subset of the wt EGFR may play an unique role in EGFR signaling, the ability of mAb 806 to target these receptors could be a factor in the anti-tumor activity exhibited by this antibody.

CONCLUSIONS AND SIGNIFICANCE

The wt EGFR immunoprecipitated by mAb 806 from three different cell lines overexpressing the EGFR migrated faster than the bulk of the receptors and was sensitive to endoglycosidase H digestion, demonstrating that it corresponds to the immature high-mannose EGFR located within the ER (Fig. 1 ). mAb 806 preferentially recognized the lower band of the de2-7 EGFR doublet, which was also sensitive to endoglycosidase H digestion (Fig. 1) . Thus, mAb 806 preferentially binds under-glycosylated forms of the de2-7 and wt EGFR. Immunoprecipitation of cell lysates with mAb 806 after surface labeling definitively demonstrated that the high-mannose forms of the wt and de2-7 EGFR are located on the cell surface. While some cell surface receptors routinely contain high-mannose structures, to the best of our knowledge this is the first demonstration that immature high-mannose forms of a receptor can be misdirected to the cell surface in significant amounts (Fig. 2 ). We recently demonstrated that the mAb 806 epitope is restricted to a short cysteine loop of the EGFR (amino acids 287–302) that is only available for antibody binding in a transitional form of the receptor, which occurs as the receptor changes from its inactive tethered conformation to a dimeric untethered form. This would suggest that the high-mannose forms of the wt EGFR are also untethered and so may contribute to the spontaneous EGFR signaling reported in cells overexpressing the receptor. These precursor forms of the EGFR thus represent novel tumor targets and contribute to the exceptional selectivity of mAb 806 for EGFR when overexpressed in cancer cells (Fig. 3 ) Because this observation is likely to apply to other receptors overexpressed in cancer, they suggest a strategy for developing antitumor antibodies even when the target receptor is expressed in normal tissue.

View larger version (41K): [in this window] [in a new window]   Figure 1. U87MG.2-7 cells express de2-7 EGFR; U87.wtEGFR, A431, and HN5 all overexpress the wt EGFR. Endoglycosidase H (Endo H) is an enzyme that removes the N-linked high-mannose moieties but not complex carbohydrates from glycosylated proteins. EGFR molecules containing high-mannose structures are thought to reside in the endoplasmic reticulum. a) The EGFR was immunoprecipitated by different anti-EGFR antibodies as indicated from 35S-labeled U87MG.2–7 (upper panel), U87MG.wtEGFR (middle panel) or A431 (lower panel) cells. The resulting immunoprecipitates were subjected to sham (–) or Endo H (+) digestion, resolved by SDS-PAGE (3–8%) and detected by autoradiography. The EGFR and its Endo H product are indicated by arrows. b) Lysates from HN5 35S-labeled cells were prepared, then immunoprecipitated and analyzed by Endo H as described in panel a to confirm our observations.

View larger version (33K): [in this window] [in a new window]   Figure 2. Immunoprecipitation after cell surface labeling. a) Lysates from A431 cells surface-labeled with biotin were immunoprecipitated with mAb 528 or 806 and subjected to sham (–) or Endo H (+) digestion. After SDS-PAGE (3–8%) of the immunoprecipitates biotin-labeled bands were detected by immunoblotting with streptavidin-HRP. The Endo H product is indicated by arrows. b) As for (a) except U87MG.2-7 were used. c) Lysates from A431 (left panel) and U87MG.2-7 (right panel) cells surface labeled with 125I were immunoprecipitated with mAb 806 and subjected to sham (–) or Endo H (+) digestion. After SDS-PAGE (3–8%) of the immunoprecipitates bands were detected by autoradiography. Thus, the high-mannose de2-7 and wt EGFR can be found on the cell surface (i.e., biotin or iodine-labeled material sensitive to Endo H).

View larger version (25K): [in this window] [in a new window]   Figure 3. Normal cells: the nascent 130 kDa EGFR polypeptide is cotranslationally modified by the addition of 8, or sometimes 9, N-linked oligosaccharide chains to yield a 160 kDa protein. This immature form of the receptor is located within the endoplasmic reticulum (ER) and is sensitive to Endo H. The immature EGFR is exported from the ER to the Golgi complex, where it acquires resistance to Endo H digestion due to trimming and terminal glycosylation of its N-linked chains. The mature receptor is then delivered to the plasma membrane as a 170 kDa species. The predominant form of the wt EGFR on the cell surface is an autoinhibited or tethered conformation. Tumor cells: in cells overexpressing the wt EGFR or expressing the de2-7 EGFR, some of high-mannose forms the EGFR fail to undergo further glycosylation in the Golgi, yet remain undetected by the quality control mechanisms found in this organelle. Small amounts of this inappropriately glycosylated material reach the cell surface where they contribute to mAb 806 reactivity. Since these underglycosylated receptors must be untethered in order for mAb 806 to bind, it is possible they have a distinct and biologically important function.

FOOTNOTES

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