All human cancer and normal cell lines were purchased from ATCC. Human breast cancer cell lines MCF-7 and SK-BR-3 and primary human fibroblasts are among those used in this study. SK-BR-3 were cultured in McCoy's 5a medium supplemented with L-glutamine and 10% FBS. MCF-7 was grown in MEM medium supplemented with L-glutamine, non-essential aminoacids, 10% FBS and 0.01 mg/ml bovine insulin. Fibroblasts were cultured in DMEM, supplemented with L-glutamine and 10% FBS. Other normal and neoplastic cell lines were cultured according to the conditions recommended by the ATCC. Hybridoma clones were produced and cultured according to previously described techniques 25.

The isotype of human Abs was determined by ELISA using murine anti-human isotype-specific MAbs to μ-, γ-, κ- and λ-chains (Sigma, USA) and goat anti-mouse Ig (25 μg/mL) conjugated to peroxidase and absorbed with human Ig.

Cells were plated on ethanol pre-treated cover slips (Fisher, USA) and placed in 6-well plates (Falcon, USA) in culture medium. After 24 hours the cover slips with attached cells were repeatedly washed in PBS and fixed in ethanol. Following fixation and repeated washes, cover slips were incubated with the primary and secondary antibodies according to standard protocols, stained with propidium iodide 1 μg/ml and analyzed by confocal fluorescent microscopy using a Zeiss Axiovert 100 TV microscope and Zeiss software. For immunohistochemistry randomly selected 5 μm thick sections of paraffin embedded breast cancer tissue were used. Endogenous peroxidase activity was blocked by incubation of slides in 3% H₂O₂ in methanol. Following washing, tissue slides were blocked with 5% normal goat serum in PBS. Monovalent Fab fragments of goat anti-human IgM+IgG (Jackson Immunoresearch Laboratories, Inc.), in blocking solution, was then applied for secondary blocking. After 3 washes in PBS, the human monoclonal antibody was applied at an approximate concentration of 5 ug/ml. The slides were then washed and incubated with a second FITC conjugated antibody to human κ-light chains (Sigma, USA) and propidium iodide at 1 μg/ml. Following a few washes, mounting medium (Biomeda, USA) and cover slips were applied and sections were analyzed by standard fluorescent microscopy.

Cells were lysed with freshly prepared ice cold lysis buffer [20 mM Tris-HCl, pH 7.6, 420 mM NaCl, 0.25% NP40, 2 mM phenylmethylsulfonyl fluoride, 1 ug/ml leupeptin, 250 U/ml Trasylol (aprotinin)] and stored at -80°C or used immediately. Protein concentration was determined with the BioRad Protein Detection Reagent (BioRad). Tissue samples were mechanically homogenized on ice, spun down at 3000 g for 30 min at 4°C and the lower (non lipid) phase was used for further analysis.

Equal amounts of protein were separated on 10% SDS polyacrylamide gels and either Coomassie blue or silver stained according to established techniques 27. Following electrophoresis, the proteins in the gel were transferred to a nitrocellulose membrane using a variation of the methods of Towbin 28 and Burnette 29, and following blocking, probed with relevant primary and HRP-conjugated secondary antibody. Membranes were processed using an enhanced chemiluminescence kit (ECL, Amersham), and visualized on Kodak BioMax MR-1 film. The immunoblotting with recombinant GIPC1 protein was carried out as previously described 26.

Antibody 27.F7 was labeled with Na-¹²⁵I (specific activity 17.4 mCi/mg) (New England Nuclear, MA, USA) using Iodogen as previously described 30. The resulting specific activity of ¹²⁵I-27.F7 was (100 mCi/mmol). SK-BR-3 cells were grown in 24-well plates supplemented with DMEM media with 10% FCS and used in these experiments at subconfluent phase at a density of 2 × 10⁵ cells per well. Cell monolayers were suspended with trypsin, cooled to 4°C by placing them on ice and washed twice with PBS containing 1% BSA (Sigma, USA). The cells (50,000 cells per sample) were blocked with 1% BSA-PBS for 1 hour at 4°C followed by incubation with ¹²⁵I-27.F7 (approx. 10⁵ cpm per sample) in the presence of increasing concentrations of cold unlabeled 27.F7 (ranging 0.1 – 200 ng/ml) for 1 hour at 4°C. After incubation the samples were applied to Millipore filters using Millipore 96-well membrane plates (Millipore, USA). The wells were broken off and counted individually in a Cobra γ-counter (Hewlett Packard, USA). Maximum binding, B_max, was determined by incubating varying numbers of cells (ranging from 1.25 × 10⁴ to 32 × 10⁵ cells) with radiolabeled 27.F7 antibody. Nonspecific binding of the tracer was determined in the presence of an excessive amount of unlabeled antibody (500 μg/ml) and was generally less than 5% of maximum binding. All experimental measurements of K_a and the number of antigen targets per cell were done in triplicate. Analysis of the data was performed according to previously described methods 313233.

RNA was purified from SK-BR-3 cells according to standard protocols. Preparation of mRNA was performed by oligo dT chromatography with a kit from Stratagene (La Jolla, CA). A lambda phage expression library was prepared in lambda ZAP (Stratagene, La Jolla, CA) and plaque lifts were screened with human monoclonal 27.B1 and 27.F7 according to previously described methods 2134. Phage plaques that were positive on the first screened were picked and two rounds of plaque purification was performed to ensure that they were true positives. Positive lambda phage clones were autoexcised according to the protocol provided by Stratagene, grown as plasmids according to standard protocols and sequenced to identify the cDNA inserts.

Total cellular RNA was isolated by the Guanidinium/Phenol extraction method and Northern blotting was performed as previously described 3536. Briefly, 15 μg of RNA is denatured and electrophoresed in a 1.2% Agarose gel along with 3.5% formaldehyde, transferred to a nylon membrane and hybridized sequentially with ³²P-labeled cDNA probes. The GIPC1 cDNA fragment that we isolated using SEREX technology was used for the gene specific probe and a cDNA fragment of the GAPDH gene was used as an internal control to normalize expression. Following hybridization, the filters were washed and exposed for autoradiography.

We previously described the construction of a unique fusion Partner cell line, MFP-2, and its use for the immortalization of both human peripheral blood and lymph node B-lymphocytes 2425. MFP-2 was employed for the generation of hybridoma cells from lymphocytes of breast cancer patients that produce autologous anti-breast cancer specific antibodies. The results of that study are described elsewhere 25. One of these native human monoclonal antibodies, designated 27.B1 (IgM, k), was chosen for further study. It demonstrated an intensely positive reactivity with two human breast cancer cell lines, SK-BR-3 and MCF-7 and no reaction with normal diploid primary human fibroblasts as tested by cELISA (In cELISA whole cells are used in place of a purified antigen as in ELISA) 25. Confocal microscopy with 27.B1 demonstrated the presence of the target antigen throughout the cytosol and in addition staining of the membrane was especially strong (see Figure 1). Furthermore, 27.B1 stained both primary and metastatic breast cancer with a high specificity and sensitivity 25. These results along with a more detailed immunocytochemical and immunohistochemical analysis are described elsewhere 25.

To identify the size of the 27.B1 target protein, Western blot analysis was performed using whole cell lysates from different cell lines and tissues. Cell lysates prepared from human breast cancer, normal breast tissue, human prostate cancer and two human fibroblast cell lines were run on PAGE under reducing conditions and blotted with fhMAb 27.B1. The antibody reacted with a protein band of approximately 42 kDa molecular weight that is detectable primarily in breast cancer cells (see Figure 2-A). There was no detectable immunoreactivity with the human fibroblasts' lysates and prostate cancer cell line LnCaP whereas only traces of immunoreactivity were detected to prostate cancer cell lines PC3 and DU-145 (data not shown). The protein band revealed by 27.B1 appeared as doublet with a dominant band that migrates slower on a gel. The doublet pattern was not the same in all 27.B1 positive cells; MCF-7 cells displayed the higher molecular weight band in much greater abundance, whereas SK-BR-3 showed both bands in more equivalent intensity (data not shown). Western blot analysis of the same cell lysates under non-reducing conditions displayed no difference in staining pattern, indicating that accessibility of the epitope bound by 27.B1 is disulfide bond independent and likely conformation independent.

Interestingly, we determined that another antibody, 27.F7, which was previously identified as binding breast cancer cells and tissue with high specificity and sensitivity 25, identified the same 42 kDa molecular weight doublet as 27.B1. Furthermore, this antibody detected the bands in the doublet with the same variability in different cell lines as 27.B1. To test the epitope specificity of the two antibodies one of them, 27.F7 was radiolabeled with ¹²⁵I and competitive Western blotting was performed with both antibodies (data not shown). Pretreatment of a SK-BR-3 cell lysate blot with unlabeled 27.F7 inhibited binding of ¹²⁵I-labeled 27.F7, whereas unlabeled 27.B1 antibody did not inhibit binding. This suggests that if these two antibodies are binding the same protein they likely bind different epitopes.

To identify the molecular target(s) for 27.B1 and 27.F7, SEREX technology was applied as previously described 212534 to a cDNA expression library prepared from SK-BR-3 mRNA. Expression clones staining positively with 27.F7 and 27.B1 were selected and the cDNA sequences were found to encode the protein known as GIPC1, following a BLAST algorithm homology search 37. This protein was previously identified as being involved in the regulation of G protein signaling 38. The sequence of the cloned cDNA inserts are identical to the respective sequence reported in GenBank for GIPC1.

To confirm that the 42 kDa band demonstrated in Figure 2-A is indeed GIPC-1, we performed immunoblotting with recombinant GIPC-1 protein (Figure 2-B). For this purpose, 27.B1 antibody was preincubated with a bacterial expressed and refolded recombinant GIPC1 protein prior to blotting (Figure 2-B, lane 1) and compared to non-preincubated control (Figure 2-B, lane 2). These results demonstrate that fhMAb 27.B1 binding to the same 42 kDa band was specifically inhibited by the recombinant protein and this confirmed the specificity of this antibody to the GIPC1 antigen.

Scatchard analysis of ¹²⁵I-27.F7 binding to SK-BR-3 cells revealed a two-mode pattern of binding, which matches a model with binders of two different avidities (see Figure 3) 31. One type of bound ligand is represented by approximately 20% of all 27.F7 targets and binds the antibody with high avidity (K_a = 4.2 × 10¹¹ M^-1). The second type of ligand binds with a lower avidity (K_a = 3.3 × 10⁹ M^-1) and constitutes about 80% of the total antigen molecules. An estimate for the total number of antigen molecules per cell is approximately 3 × 10⁵ target GIPC1 molecules per cell. The identification of two binding avidities may be related to the fact that human monoclonal antibody 27.F7 (and 27.B1) identifies a two band doublet on a Western blot (see Figure 2). This suggests that one of the bands may be a ligand of higher avidity while the second one is of lower avidity. The doublet itself has been previously reported although the reason for two bands has not been determined 38. It might be explained by an alternative start codon or posttranslational modification.

The strong staining by the human anti-GIPC1 monoclonal antibodies, 27.B1 and 27.F7, on breast cancer cell lines and absent staining of normal cells 25 suggests that the protein might be up-regulated. To semi-quantitatively examine gene expression we performed Northern blot analysis to determine if a higher level of GIPC1 specific mRNA was indeed present in the breast cancer cell lines relative to normal cell lines. RNA from a variety of breast cancer cell lines along with non-neoplastic cell lines was blotted and GAPDH expression was monitored as an internal control to normalize expression. The results are depicted in Figure 4. They indicate that breast cancer cells indeed have increased expression of GIPC1 specific RNA relative to that of normal cell lines.