Bixia Ge

Cancer cell expressions of immunoglobulin heavy chains with unique carbohydrate-associated biomarker

OC-3-VGH ovarian cancer cell line and numerous others from different human tissue origins were studied for their respective expressions of immunoglobulins as well as carbohydrate-associated epitope(s) recognized by RP215 monoclonal antibody. With no exceptions, all the cancer cell lines studied so far express human immunoglobulin G (IgG) heavy chains when determined by Western blot or nested RT-PCR with appropriate primers in the constant region. By Western blot assay, it was also shown that greater than 90% of cancer cell lines expressed RP215-specific epitope(s) on the detected heavy chain molecules. Further studies with OC-3-VGH cancer cells revealed the expressions of all immunoglobulin classes, subclasses, heavy as well as light chains. The primary structure of the IgG heavy chains expressed by single cloned cells of this cancer cell line was elucidated. It was shown to be homologous to that of normal human IgG1 heavy chain derived from B cells, except with high content of serine/threonine residues in the variable region. Expressions of other immunoglobulin-related genes were also detected. Widespread expressions of immunoglobulin heavy chains among cancer cells as well as the frequent presence of unique carbohydrate-associated epitope(s) recognized by RP215 monoclonal antibody might have important biological implications during carcinogenesis and applications in immunodiagnostics and antibody-based anti-cancer drug developments.

Inhibition of in vitro tumor cell growth by RP215 monoclonal antibody and antibodies raised against its anti-idiotype antibodies

RP215 monoclonal antibody (Mab) was shown to recognize carbohydrate-associated epitope(s) in the heavy chains of cancer cell-expressed immunoglobulins, designated in general as CA215 pan cancer marker. Growth inhibitions of tumor cells in vitro by RP215 Mab and antibodies against its anti-idiotype (anti-id) antibodies were investigated. Polyclonal rabbit anti-id antibodies and the corresponding rat anti-id Mabs were generated and characterized. Following immunizations in mice, antisera raised against anti-id antibodies were analyzed by typical immunoassays. It was observed that mouse anti-anti-id sera (Ab3) revealed binding affinity and specificity to CA215 that are comparable to those of RP215. Both RP215 and Ab3 were shown to induce apoptosis of cultured cancer cells in vitro by TUNEL and MTT assays. These experimental observations were consistent with that of in vivo tumor growth inhibition by RP215 in previous nude mouse experiments. Therefore, heterologous or homologous anti-id antibodies of RP215 that contain the internal image of its specific epitope in CA215 may serve as effective anti-cancer vaccines for therapeutic treatments of various cancers in humans. The relative stability of RP215-specific carbohydrate-associated epitope was compared to that of human IgG at extreme pH’s (≤2 or ≥12) or following NaIO4 treatments. The major molecular forms of CA215 were further documented with various enzyme immunoassays and found to have similar secondary structures to those of normal human immunoglobulin G.

Widespread expressions of immunoglobulin superfamily proteins in cancer cells

RP215 monoclonal antibody (Mab) was shown to recognize a carbohydrate-associated epitope of cancer cell–expressed glycoproteins, known as CA215. Extensive MALDI-TOF MS analysis was performed to search for the molecular identity of CA215. Besides immunoglobulin (Ig) heavy chains, homology to human T-cell receptors (TCR) and Ig-like cell adhesion molecules was also detected. By using RT-PCR and cDNA sequencing, it was observed that as many as 80% of cancer cell lines showed significant levels of gene expressions of TCR-α and TCR-β. Selected Ig-like cell adhesion molecules such as CD47, CD54, CD58 and CD 147 were also highly expressed among all the cell lines tested. In contrast, co-receptors and co-stimulators of TCR such as CD3, CD4 and CD8 were rarely expressed demonstrating the non-functional nature of TCR in cancer cells. Results of immunohistochemical staining and Western blot assays of cancer cell lines as well as cancerous tissue sections were consistent with these observations. Anti-TCR and anti-human IgG antibodies were shown to induce complement-dependent cytotoxicity and apoptosis of cultured cancer cells indicating the surface nature of Ig-like proteins. Based on these experimental observations, it was hypothesized that the expressions of these immunoglobulin superfamily (IgSF) proteins may be relevant to the immune protection and proliferations of cancer cells during carcinogenesis or cancer progression. Surface-bound TCR-like proteins as well as immunoglobulins may be the potential targets for RP215-based anti-cancer drugs.

CA215 and GnRH receptor as targets for cancer therapy

Two monoclonal antibodies (Mabs), RP215 and GHR106, were selected for the preclinical evaluations of anti-cancer drugs targeting various human cancers including those of the ovary, cervix, lung, and liver. Both Mabs were shown to react with pan cancer markers, which are over-expressed on the surface of almost all human cancers. RP215 Mab was shown to react with the carbohydrate-associated epitope(s) of cancer cell–expressed glycoproteins, mainly consisting of immunoglobulin superfamily (IgSF) proteins and mucins, generally known as CA215. GHR106 Mab was generated against the extracellular domain of human GnRH receptor, which is also highly expressed on the cancer cell surface. Preclinical studies were performed to evaluate the efficacy of these two Mabs as anti-cancer drugs for treating human cancers. High tumor specificity of RP215 Mab was demonstrated with immunohistochemical staining studies of various cancer cell lines, as well as normal and cancerous tissue sections. These two Mabs were shown to induce apoptosis as well as complement-dependent cytotoxicity upon treatment to many cultured cancer cells. Significant dose-dependent growth inhibition of tumor cells from several different tissue origins were demonstrated by nude mouse experiments. It was further demonstrated that GHR106 Mab can function as long-acting GnRH analogs in its biological actions. Efforts were made to generate human/mouse chimeric forms of the GHR106 Mab. Based on the results of these preclinical studies, we believe that these two Mabs, in chimeric or humanized forms, can be developed into suitable therapeutic agents for treatment of human cancers as anti-cancer drugs.

Molecular and Immuno-Characteristics of Immunoglobulin-like Glycoproteins in Cancer Cell-expressed Biomarker, CA215

RP215 monoclonal antibody (Mab) was shown to recognize a specific carbohydrate-associated epitope found in cancer cell-expressed glycoproteins, known as CA215. The membrane-bound and soluble forms of CA215 were detected in almost all of the cancer cells in humans, but rarely found in normal tissues. Through MALDI-TOF MS analysis, it has been reported previously that as much as 40% of the detected tryptic peptides of CA215 showed high degrees of sequence homology to those found in immunoglobulin heavy chains. The cancer cell-derived immunoglobulins were further purified from CA215 by affinity column-linked with goat anti-human IgG for molecular characterizations. Semi-quantitative RT-PCR was used to determine the mRNA levels of various immunoglobulin genes expressed by cancer cells of single or multi-cell origins and compared with those found in normal human serum. The stability of CA215 was investigated under different experimental conditions. It was observed that the RP215-specific epitope in CA215 is stable at neutral pH, in human serum or in mice (half life of 5–18 days), but unstable at extreme pH’s (pH ≤ 2.0; pH ≥ 12.0) or high temperatures. Enzyme immunoassays were performed with several secondary antibody probes related to human IgG. It was demonstrated that cancer cell-expressed immunoglobulins with RP215-specific epitope have much lower immunoactivity than that of normal human IgG (≤ 5%), despite the fact that both showed almost identical amino acid sequence in the respective Fc region reported previously. This could be the result of aberrant glycosylation of CA215 in cancer cells. Aberrant glycosylation of glycoproteins may have important biological implications on the proliferation of cancer cells in vitro or in vivo.

Carbohydrate-associated Immunodominant Epitope(s) of CA215

RP215 monoclonal antibody (Mab) was initially generated against OC-3-VGH ovarian cancer cells and was shown to react with a cancer-associated carbohydrate epitope in glycoproteins designated as CA215. Additional five high affinity Mabs, designated as RCA-10, -100, -104, -110 and -111, respectively, were generated by using affinity-purified CA215 as the immunogen in this study. All RCA Mabs were found to recognize periodate-sensitive carbohydrate-associated epitope(s) and to pair with RP215 in typical sandwich enzyme immunoassays for the quantification of CA215. When compared with those of RP215, the amino acid sequence homology of the Fab regions ranged from 100% for RCA-100 to 65% for RCA-110, based on which 3 distinct Mab groups were categorized. In vitro TUNEL apoptosis and complement-dependent cytotoxicity assays were performed with these Mabs and found to have comparable inhibitory efficacy to cancer cells. Results of biochemical and immunological assays revealed that RP215, RCA-100 and RCA-10 react with the linear carbohydrate-associated epitope, whereas the others recognize the conformational form of the epitope in CA215. This study has suggested that the unique carbohydrate-associated epitope(s) is immunodominant in mice when immunized with CA215. It remains to be demonstrated if the differential anti-cancer efficacy exists among the distinct groups of these anti-CA215 Mabs.

Monoclonal Anti-idiotype Antibodies against Carbohydrate- associate Epitope for Anti-Cancer Vaccine Development

Background: RP215 monoclonal antibody (Mab) was shown to react specifically with the unique carbohydrate associated epitope located in the variable regions of cancer cell-expressed immunoglobulins known as CA215. This Mab was shown to inhibit the growth of a variety of cancer cells in vitro or in vivo. Anti-idiotype (anti-id) Mabs against RP215 were generated and characterized for future development of epitope-specific anti-cancer vaccines in humans. Results: Following successful immunization of rats with F(ab’)2 fragments of RP215, rat anti-id Mabs were established for biochemical and immunological characterization. Subsequent immunizations of mice with purified rat anti-id Mab revealed significant anti-anti-id antibody (Ab3) responses. In immunohistochemical studies, both Ab3 and RP215 reacted positively with various cancer cells lines and with comparable staining intensities. Both RP215 and Ab3 inhibited significantly the growth of cancer cells in vitro by means of TUNEL assay. To document that the RP215-specific epitope is carbohydrate-associated, the absence of RP215-epitope expressions upon culturing of cancer cells in serumfree medium was demonstrated. Experiments confirmed that the expression of RP215-specific epitope was dependent on the presence of carbohydrate precursors in culture media. Conclusion: Judging from these observations, anti-id Mabs which carry the internal image of RP215-specific carbohydrate-associated epitope can be suitable candidates for anti-cancer vaccine development in humans.

Two Distinct Humanized Monoclonal Antibodies for Immunotherapy of Ovarian Cancer

Two monoclonal antibodies (Mab) were selected for development of anti-cancer drugs targeting ovarian cancer. RP215 Mab was shown to react with a carbohydrate-associated epitope detected mainly in the heavy chains of cancer cell-expressed immunoglobulins which are essential for the growth/proliferation of almost all human cancer cells. GHR106 Mab, on the other hand, reacts specifically with GnRH receptor on the surface of almost all cancer cells. In this review, efforts were made to use an ovarian cancer cell line, OC-3-VGH as the experimental model to study these two Mabs in murine and humanized isoforms including humanization, comparative biochemical and immunological characterizations. Surface binding of either of these two Mabs can result in apoptosis and complement-dependent cellular cytotoxicity to this ovarian cancer cell line and others. Both murine Mabs were humanized and shown to be bioequivalent with respect to affinity and biospecificity to their murine counterparts through extensive biochemical/immunological studies. Therefore, preclinical and clinical studies were warranted to continue the investigations of these two potential anti-cancer drug candidates for therapeutic treatments of ovarian cancer.