Mark C. Glassy

Novel Ganglioside Antigen Identified by B Cells in Human Medullary Breast Carcinomas: The Proof of Principle Concerning the Tumor-Infiltrating B Lymphocytes

The potential tumor-recognizing capacity of B cells infiltrating human breast carcinoma is an important aspect of breast cancer biology. As an experimental system, we used human medullary breast carcinoma because of its heavy B lymphocytic infiltration paralleled to a relatively better prognosis. Ig-rearranged V region VH-JH, Vκ-Jκ, and Vλ-Jλ genes, amplified by RT-PCR of the infiltrating B cells, were cloned, sequenced, and subjected to a comparative DNA analysis. A combinatorial single-chain variable fragment Ab minilibrary was constructed out of randomly selected VH and Vκ clones and tested for binding activity. Our data analysis revealed that some of the VH-JH, Vκ-Jκ, and Vλ-Jλ region sequences were being assigned to clusters with oligoclonal predominance, while other characteristics of the Ab repertoire were defined also. A tumor-restricted binder clone could be selected out of the single-chain variable fragment κ minilibrary tested against membrane fractions of primary breast tumor cells and tumor cell lines, the VH of which proved to be the overexpressed VH3-1 cluster. The specific binding was confirmed by FACS analysis with primary breast carcinoma cells and MDA-MB 231 cell line. ELISA and thin layer chromatography dot-blot experiments showed this target Ag to be a ganglioside D3 (GD3). Our results are a proof of principle about the capacity of B cells infiltrating breast carcinomas to reveal key cancer-related Ags, such as the GD3. GD3-specific Abs may influence tumor cell progression and could be used for further development of diagnostic and/or therapeutic purposes.

Immunoincompetence in cancer patients. Assessment by in vitro stimulation tests and quantification of lymphocyte subpopulations

The authors performed a variety of lymphocyte‐stimulation tests and quantified several lymphocyte subpopulations in 73 healthy controls and 72 patients with advanced cancer who were no longer receiving anticancer therapy. As a group, cancer patients had fewer lymphocytes and helper cells, but a greater proportion of suppressor cells and Ia+ cells than controls. The ratio of helper to suppressor cells was lower in the cancer group. Uptake of 125I‐uridine was markedly depressed in cancer patients in the face of stimulation with various plant lectins, foreign lymphocytes, and varicella—zoster antigen. There was little correlation between any of the stimulation tests and any of the lymphocyte subpopulation proportions or numbers. The two tests that were most frequently abnormally low among the cancer patients were percent lymphocytes and number of helper cells (81% each). The most frequently abnormal functional assay in patients was pokeweed mitogen stimulation (59%). Three separate statistical methods selected the combination of percent lymphocytes, percent Ia+ cells, percent suppressor cells, number of helper cells, and pokeweed mitogen stimulation as being the best predictors of cancer/immunoincompetent status. This study confirms the breadth of immunoincompetence in advanced cancer patients as defined by in vitro techniques. A smaller battery of tests can be useful in monitoring the immune status of such patients, especially during therapy with proposed immune modulators.

A Human Monoclonal Antibody Reactive with Human Prostate

A human immunoglobulin M monoclonal antibody secreting hybridoma, termed MHG7, has been isolated and characterized for its reactivity against human prostate cells. Lymphocytes isolated from a regional draining lymph node of a patient with prostate carcinoma were fused with murine P3-NS1-Ag4-1 myeloma cells. Supernatants from the generated mouse-human somatic cell hybrids were first screened for human immunoglobulin production by an enzyme immunoassay. The identified human immunoglobulin-secreting hybridomas were expanded for further analysis and their supernatants screened by enzyme immunoassay against a panel of prostate cell lines. The human immunoglobulin M monoclonal antibody MHG7, in addition to reacting with prostate cell lines, also reacted with prostate carcinoma cells and benign prostatic hypertrophy cells on both frozen and paraffin embedded tissue sections. These data suggest that regional draining lymph nodes of prostate carcinoma patients can be used as a source of human lymphocytes for generating human immunoglobulin-secreting hybridomas reactive with human prostate cells.

Development of a rapid microELISA assay for screening hybridoma supernatants for murine monoclonal antibodies

A micro enzyme-linked immunosorbent assay (ELISA) utilizing a filtration method has been developed which allows the rapid, simple, and sensitive detection of monoclonal antibodies that recognize either soluble or cell surface antigens. This assay involves the immobilization of target cells (or soluble antigen) onto glass fiber filter discs followed by an incubation with the test hybridoma supernatant and subsequent analysis by ELISA. A specially designed 96-well filtration device is employed which serves both as an incubation chamber and as a filtration manifold. This microELISA requires small volumes of antiserum, few target cells, and can be completed in less than 2 h. This assay is well suited for the rapid screening of murine hybridoma supernatants and can be adapted to detect monoclonal antibodies from other species.

Immunodetection of cell-bound antigens using both mouse and human monoclonal antibodies.

A micro enzyme-linked immunoassay (EIA) has been developed for the rapid and sensitive detection of either human or mouse monoclonal antibodies reactive with cell bound antigens. Whole intact cells are immobilized onto 96-well flat bottom microtiter plates by drying in an oven at 37 degrees C overnight prior to the start of the assay. This method of attachment was suitable for all cell types tested, regardless of origin, size and chromosomal content. The dried cells were then rehydrated, incubated with the appropriate test hybridoma supernatant, followed with subsequent analysis by EIA. The plates can be stored at 4 degrees C up to 1 month for future EIA analysis. This assay offers high sensitivity, requires only small amounts of target cells and test hybridoma supernatant, and can be completed within 3 h. This EIA is well suited for the rapid screening of large numbers of hybridoma supernatants and can also be adapted to include cells of any species, providing the appropriate antibody reagents are available.

An enzyme immunofiltration assay useful for detecting human monoclonal antibody

A microenzyme-linked immunoassay (EIA) utilizing an immunofiltration manifold has been developed which provides a rapid, simple, and sensitive method of detecting human monoclonal antibody class, concentration, and specificity. In this assay either whole cells or soluble antigens were immobilized on glass fiber filters followed by incubating with the test human hybridoma supernatant with subsequent analysis by EIA. A specially designed 96-chamber immunofiltration plate is employed which serves as both an incubation chamber and as a filtration manifold. The assay described is unique in that small volumes of human hybridoma supernatant are required, crude preparation of only a few target cells are needed, labile cell surface antigens are preserved and it can be completed in 3 h. This assay is well suited for the rapid screening of large numbers of human hybridoma supernatants.

ADCC of Cultured Human Melanoma Cells: Analysis with Monoclonal Antibodies to Human Melanoma-associated Antigens

Four monoclonal antibodies to human melanoma‐associated antigens (MAA) were found to be able to mediate specific cell‐dependent lysis (ADCC) of cultured human melanoma cells. The extent of specific lysis of melanoma cells was influenced by the effector to target cell ratio, the amount of antibody added to the reaction mixture, and the incubation time. Cultured melanoma cells treated for 16 h with puromycin or cycloheximide (final concentration, 1.0 μg/ml) displayed increased Susceptibility to ADCC even though the cell surface expression of MAA was not changed. On the other hand, treatment of melanoma cells with tunicamycin (final concentration, 1.0 μg/ml) reduced the expression of MAA but did not affect susceptibility to ADCC. These findings suggest that other properties of the target cell membrane besides antigen density play a role in the outcome of ADCC reaction.

Production methods for generating human monoclonal antibodies

The technology explosion that mushroomed from the uses and applications of monoclonal antibodies (MAbs; Re~. 1) .shows. no signs of slowing down. Virtually the entIre bIOmedIcal arena has been transformed with the introduction of the MAb as a routine reagent in both the laboratory and clinic. The promise of MAbs has been their pote~tial selectivity and exquisite specificity. One of the ObVIOUS goals of MAb technology is to develop clinically useful human MAbs (HMAbs). Historically, the first immortalized human cell secreting specific antibody was generated by infection with a ~-tropic Epstein-Barr virus (EBV) in 19772 and the first true human-human hybridoma secreting specific antibody was described in 1980.3 Although the HMAb field was slow to take off (see below), the progress made wit~ murine MAbs has been astonishing. Clearly, the antIbody of the 1980s was of mouse origin. Whatever c.ould h~v~ been done to a MAb was attempted at this tIme: chmcal protocols, simple and elaborate chemical modifications, and some very interesting molecular biology approaches. Unfortunately, the clinical usefulness of murine MAbs has been very disappointing, due for the most part to th~ innate foreigness and immunogenicity of the mouse Immunoglobulin protein, resulting in allergic respon~es,. hepati~ dysfunctions, and immune complex formatIon m the kIdney. With the ultimate aim of trying to control and eventually eradicate human diseases thro~gh immunomodulation and to improve our ability to diagnose abnormalities, it is believed that HMAbs will prove far superior than those of murine origin. H~Abs should be more effective because of their ability to mteract more appropriately with human complement or human effector cells. In addition, HMAbs would not only be less immunogenic, but they may also recognize a se~ of allogenic antigens that are not recognized by munne MAbs. As such, the antibody of the 1990s will

Serological, functional, and immunochemical characterization of a monoclonal antibody (MoAb Q2/70) to human Ia-like antigens

Serological and immunochemical studies showed that monoclonal antibody Q2/70 (MoAb Q2/70), produced by the hybridoma technique, is specific for human Ia-like antigens. This antibody recognizes an antigenic determinant which is different from those defining the serologic polymorphism of Ia-like antigens, and is expressed on subsets of human Ia-like molecules and on lymphoid cells from other species. MoAb Q2/70 inhibits unidirectional MLRs* between allogenic human lymphocytes, but not between murine and human lymphocytes. In ADCC* assays. MoAb Q2/70 mediates lysis of cultured human B lymphoid cells RPMI 4098, effected by murine splenocytes. The antibody is suitable to isolate immunologically functional B lymphocytes from human peripheral blood.

Genetically Stable Human Hybridomas Secreting Tumor-Reactive Human Monoclonal IgM

Human lymphocytes obtained from regional draining lymph nodes of patients with cancers of the cervix, kidney, prostate, and vulva were immortalized by polyethylene glycol-mediated somatic cell hybridization with either human UC 729-6 or murine P3-NS-1-Ag4-1. Four reactive human IgM-secreting hybridomas, termed CLNH5, MHG7, VLN1H12, and WLNA67 were isolated and characterized. Hybrids obtained by fusions with UC 729-6 have remained tetraploid for over 18 months, have doubling times from 25-35 hours, and have continuously secreted approximately 0.5-5.0 micrograms IgM/10(6) cells/ml per day. MHG7, a mouse-human hybrid, required subcloning every 4-6 months to maintain human IgM secretion. Binding of these human monoclonal antibodies (MoAbs) against a panel of cell lines was assessed by an enzyme-linked immunoassay (EIA). CLNH5 reacted with carcinomas of the cervix, lung, and vulva. MHG7 reacted with carcinomas of the prostate, stomach, and vulva. VLN1H12 reacted with carcinomas of the cervix, lung, prostate, stomach, and vulva. WLNA6 reacted strongly with a carcinoma of the lung. All four human MoAbs failed to react by EIA with hematopoietic cells or normal fibroblast cell lines. The data suggest that regional draining lymph nodes of cancer patients have been primed to produce antibodies against antigens associated with tumor cells and that UC 729-6 served as a genetically suitable vector for the capture and immortalization of these Ig-secreting B lymphocytes.