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Updated: Aug 14, 2026
Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood
Published on: February 6, 2018
Characterization of a monoclonal antibody against human prolactin receptors
R Banerjee1, E Ginsburg, B K Vonderhaar
1Laboratory of Tumor Immunology and Biology, National Cancer Institute, Bethesda, MD 20892.
Abstract:
Tamoxifen is the first line of therapy for most human breast cancers. It not only works through the estrogen receptor but also can directly affect the binding of prolactin to its receptor. To define this latter mechanism, the nature of the prolactin receptor needs to be clearly defined. Monoclonal antibody (MAb) B6.2, and IgG1 raised against a membrane-enriched fraction from metastatic human breast cancer cells, was as effective as polyclonal anti-prolactin receptor antibody in inhibiting the binding of prolactin to membranes from human tissue and to T47D human breast cancer cells. Control MAbs, MOPC-2I and the anti-NCA B1.1 MAb, had no effect on binding. Epidermal growth-factor receptors on these same cells were unaffected by B6.2. Prolactin-induced growth of the T47D cells was blocked by addition of B6.2 to the media while the control antibodies were without effect. Specific binding of B6.2 to the cells was completely inhibited by prolactin. Binding of both prolactin and B6.2 was inhibited by growing the T47D cells in the presence of tunicamycin A1 under conditions where protein synthesis was not affected but glycosylation of proteins was. An affinity column of B6.2 was used to purify its antigen from T47D cells. The primary purification product, a M(r) 90,000 protein, specifically bound the lactogenic hormones human prolactin, human growth hormone and ovine prolactin but not the somatogenic hormone, bovine growth hormone and was precipitated by the polyclonal anti-prolactin receptor antibody but not by control MAbs. When tryptic and V8 digests of the B6.2 antigen and purified prolactin receptors were compared, identical electrophoretic profiles were obtained. Mouse 3T3 cells, when stably transfected with the gene for the long form of the human prolactin receptor, reacted with B6.2 and polyclonal anti-prolactin receptor antibody. Parental 3T3 cells, devoid of prolactin receptors, were negative for all antibodies tested. Thus, MAb B6.2 provides a useful tool for further studies on purification and characterization of these receptors from human tissues and may provide new insights into treatment for breast cancer.
Insights
A new monoclonal antibody (MAb) B6.2 effectively targets the prolactin receptor, a key factor in breast cancer. This antibody aids in purifying and characterizing the receptor, potentially leading to new breast cancer treatments.
Area of Science:
- Endocrinology
- Oncology
- Immunology
Background:
- Tamoxifen is a primary breast cancer therapy, acting via estrogen receptors and potentially prolactin receptors.
- Understanding prolactin receptor interactions is crucial for defining tamoxifen's full mechanism of action.
- The exact nature of the prolactin receptor in human breast cancer requires further definition.
Purpose of the Study:
- To characterize the prolactin receptor using a newly developed monoclonal antibody (MAb) B6.2.
- To investigate the role of the prolactin receptor in human breast cancer cell growth.
- To utilize MAb B6.2 for the purification and identification of the prolactin receptor.
Main Methods:
- Monoclonal antibody (MAb) B6.2 was generated against metastatic human breast cancer cell membranes.
- Inhibition assays were performed to assess prolactin binding to cells and membranes in the presence of antibodies.
- Affinity chromatography using MAb B6.2 was employed to purify the prolactin receptor antigen.
- Tryptic and V8 digests were compared to confirm receptor identity.
- Transfection studies in mouse 3T3 cells were conducted to validate antibody specificity.
Main Results:
- MAb B6.2 effectively inhibited prolactin binding to human breast cancer cells and tissues, similar to polyclonal anti-prolactin receptor antibodies.
- MAb B6.2 blocked prolactin-induced growth of T47D breast cancer cells.
- The primary purified antigen using MAb B6.2 was a 90,000 M(r) protein that specifically bound lactogenic hormones and was recognized by anti-prolactin receptor antibodies.
- Digest comparisons and transfection studies confirmed MAb B6.2 specifically targets the human prolactin receptor.
Conclusions:
- MAb B6.2 is a specific and effective tool for studying human prolactin receptors.
- This antibody facilitates the purification and characterization of prolactin receptors from human breast cancer tissues.
- Further research using MAb B6.2 may offer new therapeutic strategies for breast cancer treatment.
