Gregory Lee

Myelin degeneration in multiple system atrophy detected by unique antibodies.

A rabbit antiserum (anti-EP), induced against a synthetic peptide corresponding to residues 68 to 86 of guinea pig myelin basic protein, powerfully immunostained abnormal-appearing oligodendrocytic processes and cell bodies in demyelinating areas associated with multiple system atrophy (MSA). However, as we reported previously, the antiserum, which is highly specific for the sequence QDENPVV corresponding to human myelin basic protein residues 82 to 88, failed to recognize any structures in normal human brain. QD-9, a mouse monoclonal antibody raised against human myelin basic protein residues 69 to 88, which also recognizes specifically the epitope QDENPVV, gave the same results as did anti-EP. The unusual epitope recognized by anti-EP/QD-9 antibodies appears to be accessible in areas of myelin degeneration, and the antibodies have been shown to detect such areas in multiple sclerosis and infarcted brains. These antibodies detect myelin degeneration more widely than previous conventional methods. The present study emphasizes the importance of myelin degeneration in the pathogenesis of multiple system atrophy.

Molecular identity of a pan cancer marker, CA215

The molecular nature of cancer-associated antigen, CA215 which reacts with RP215 monoclonal antibody and its unique epitope(s) was characterized. RP215 was initially selected and produced from one of 3,000 hybridomas which were generated from mice immunized with the cell extract of OC-3-VGH ovarian cancer cells. This cancer-associated antigen from various sources including cancer cell extract, shed culture medium and affinity-purified forms was analyzed by MALDI-TOF MS (Matrix Adsorption Laser Desorption Ionization-Time of Flight Mass Spectrometry), Western blot, carbohydrate profiling as well as enzyme immunoassays. The results of this study showed that CA215 is homologous to the heavy chains of human immunoglobulins with molecular sizes ranging from 50 to 70 KDa, when probed with RP215 or anti-human immunoglobulin G, A or M. Treatments of cancer cells with NaIO4 drastically reduce RP215 binding to the carbohydrate-associated epitope(s) of CA215 located on the variable domain of the human immunoglobulin heavy chains. Further studies indicated that CA215 is predominantly expressed by cancer cells in both secreted and membrane-bound monomeric forms. The carbohydrate-associated epitope(s) with pH-sensitive immunoactivity appear to be present only in cancer cell-derived immunoglobulins, but not in normal human immunoglobulins. Compared to normal immunoglobulin G, CA215 contains a significantly higher percentage of N-acetyl and N-glycoyl neuraminic acid (28% vs. 8%) in the O-linked glycans, but a lower content of N-acetylglucosamine (28% vs. 41%) in the N-linked ones. It was concluded from this study that RP215 reacts specifically with carbohydrate-associated epitope(s) of immunoglobulin heavy chains expressed by various human cancer cells.

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.

Preclinical assessment of anti-cancer drugs by using RP215 monoclonal antibody

RP215 monoclonal antibody was originally generated against OC-3-VGH ovarian cancer cells. It was shown to recognize specifically a carbohydrate-associated epitope(s) of cancer cell-expressed immunoglobulin heavy chains designated as CA215. Previous studies suggest that CA215 is expressed by all human cancer cell lines and tissues in both membrane bound and secreted forms. It may be an ideal target for therapeutic treatments of human cancers with humanized RP215-related antibodies. Based on the results of large scale immunohistochemical studies (50-100 cases each), the following types of cancers revealed high percentage(s) of positive staining with RP215: esophagus (76%), stomach (50%), colon (44%), ovary (64%), breast (32%), lung (31%), cervix (84%) and endometrium (78%). Nude mouse experiments were performed to determine if RP215 has any inhibitory effect on the growth of cancer cells in vivo. Following injections of a single dose (10 mg/kg) of I131-labeled RP215 (specific activity, 12.5 µCi/mg), the tumor size (OC-3-VGH ovarian cancer cells) was reduced to 30% of the untreated control within two weeks. By injecting the same dose, the unlabeled RP215 also reduced the tumor size to 50% of the control during the same period. The antibody treatments were found to have little effect on the body weight as well as apparent toxicity of these animals. To proceed with clinical trial studies of RP215-based anti-cancer drugs, chimeric form of this monoclonal antibody was generated and characterized. Through our effort, the “proof of concept” of anti-cancer drugs development was clearly established for the next stages of clinical trial studies.

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.

Peptide Mapping and Glycoanalysis of Cancer Cell–Expressed Glycoproteins CA215 Recognized by RP215 Monoclonal Antibody

RP215 monoclonal antibody was shown to react with carbohydrate-associated epitope(s) in cancer cell–expressed glycoproteins known as CA215 based on indirect experimental evidences. Efforts have been made to identify glycans in CA215 that may be involved in the epitope recognition. More than 100 tryptic peptides, derived from affinity-purified CA215, consist mainly of immunoglobulin superfamily (IgSF) proteins (∼60%), mucins (∼7%), and others. Glycoanalysis was performed with affinity-purified CA215 from two cancer cell lines, including (1) N- and O-linked glycan profilings and linked glycoanalysis, (2) glycosylation site mappings, and (3) treatments with selected glycolytic enzymes. High mannose and complex bisecting structures with terminal sialic acid (NeuAc or NeuGc) were detected in N-glycans, whereas as many as 10 O-glycans structurally similar to those of mucins were identified. Through glycosylation site mappings, two N-linked and six out of eight O-linked glycans were detected and matched almost 100% with human immunoglobulin heavy chains. Treatments with several glycolytic enzymes were found to have little effect on the immunoactivity of the RP215-epitope. The same activity was also not affected by the cancer cell culture in human serum instead of bovine serum, indicating that NeuAc and NeuGc are not involved in epitope recognition. The immunoassay results also suggested that the affinity-purified cancer cell–expressed immunoglobulins revealed similar structures and immunoactivities to those of normal human immunoglobulins, except that two additional O-glycans were detected in the former. Supplemental materials are available for this article. Go to the publishers online edition of Journal of Carbohydrate Chemistry to view the free supplemental file.

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.

Aberrant high expression of immunoglobulin G in epithelial stem/progenitor-like cells contributes to tumor initiation and metastasis

High expression of immunoglobulin G (IgG) in many non-B cell malignancies and its non-conventional roles in promoting proliferation and survival of cancer cells have been demonstrated. However, the precise function of non-B IgG remains incompletely understood. Here we define the antigen specificity of RP215, a monoclonal antibody that specifically recognizes the IgG in cancer cells. Using RP215, our study shows that IgG is overexpressed in cancer cells of epithelial lineage, especially cells with cancer stem/progenitor cell-like features. The RP215-recognized IgG is primarily localized on the cell surface, particularly lamellipodia-like structures. Cells with high IgG display higher migration, increased invasiveness and metastasis, and enhanced self-renewal and tumorgenecity ability in vitro and in vivo. Importantly, depletion of IgG in breast cancer leads to reduced adhesion, invasion and self-renewal and increased apoptosis of cancer cells. We conclude that high expression of IgG is a novel biomarker of tumor progression, metastasis and cancer stem cell maintenance and demonstrate the potential therapeutic benefits of RP215-recognized IgG targeted strategy.

IgG and IgA with Potential Microbial-Binding Activity Are Expressed by Normal Human Skin Epidermal Cells

The innate immune system of the skin is thought to depend largely on a multi-layered mechanical barrier supplemented by epidermis-derived antimicrobial peptides. To date, there are no reports of antimicrobial antibody secretion by the epidermis. In this study, we report the expression of functional immunoglobulin G (IgG) and immunoglobulin A (IgA), previously thought to be only produced by B cells, in normal human epidermal cells and the human keratinocyte line HaCaT. While B cells express a fully diverse Ig, epidermal cell-expressed IgG or IgA showed one or two conservative VHDJH rearrangements in each individual. These unique VDJ rearrangements in epidermal cells were found neither in the B cell-derived Ig VDJ databases published by others nor in our positive controls. IgG and IgA from epidermal cells of the same individual had different VDJ rearrangement patterns. IgG was found primarily in prickle cells, and IgA was mainly detected in basal cells. Both epidermal cell-derived IgG and IgA showed potential antibody activity by binding pathogens like Staphylococcus aureus, the most common pathogenic skin bacteria, but the microbial-binding profile was different. Our data indicates that normal human epidermal cells spontaneously express IgG and IgA, and we speculate that these Igs participate in skin innate immunity.

Binding of the monoclonal antibody RP215 to immunoglobulin G in metastatic lung adenocarcinomas is correlated with poor prognosis

Cancer cell‐derived immunoglobulin (Ig)G (cancer‐IgG) has been found to be involved in the pathogenesis and progression of many cancers, including lung cancer. The aim of the present study was to investigate the relationship between cancer‐IgG expression in lung adenocarcinoma (ADC) and clinicopathological characteristics and clinical outcome.