Alison Estabrook

Diagnosis of cutaneous T cell lymphoma by use of monoclonal antibodies reactive with tumor-associated antigens.

Two murine monoclonal antibodies (BE1 and BE2), produced by using leukemic helper T cells from a patient with cutaneous T-cell lymphoma (CTCL) as immunogens, reacted selectively with CTCL lymphocytes and some transformed cultured lymphocytes, as determined by radioimmunoassay (RIA) and indirect immunofluorescence (IIF). BE1 reacted significantly (P less than or equal to 0.001) with leukemic CTCL lymphocytes and with CTCL cells from infiltrated lymph nodes (RIA, mean +/- SD = 776 +/- 275 cpm), as compared with background counts (263 +/- 68). BE1 binding to normal blood mononuclear cells (RIA, mean +/- SD = 283 +/- 58 cpm) was indistinguishable from background. BE1 also reacted with Epstein-Barr virus (EBV)-transformed B-cell lines (RIA, mean +/- SD = 794 +/- 230) and some long-term T-cell lines. BE1 did not react with the majority of lymphoid cell lines or tumor cell lines tested. BE1 also did not react with any normal tissues screened by IIF. BE1 precipitated a molecule from CTCL cells that, under reducing conditions, has two components with molecular mass of 27,200 and 25,800 D. BE2 also reacted significantly (P less than or equal to 0.001) with CTCL cells from two of four patients (RIA, mean +/- SD = 519 +/- 113 cpm). The binding of BE2 to normal mononuclear cells was indistinguishable from background (309 +/- 38 cpm). BE2 also reacted with an antigen present on EBV-B-cell lines (RIA, mean +/- SD = 654 +/- 194) and MOLT 3 and HUT 78 T-cell lines. BE2 reacted with an antigen expressed on a subpopulation of lymphocytes from five of eight patients with B-cell CLL studied by IIF (mean +/- SD = 18 +/- 6). Other long-term T-cell lines and tumor cell lines studied by IIF were unreactive with BE2. BE2 did not react with any of the normal tissues studied. BE2 precipitated a molecule (78,000 D) from CTCL cells and EBV-B cells with a single component under reducing conditions. Immunoperoxidase-labeled BE1 and BE2 reacted with CTCL cells in frozen sections of infiltrated lymph nodes and skin. In addition, BE1 and BE2 reacted with blood lymphocytes from 16 of 21 patients whose CTCL had otherwise been considered localized to skin. These two monoclonal antibodies react with tumor antigens associated with CTCL and appear to be useful in the diagnosis of this disorder.

The extent and distribution of cancer in breasts with palpable primary tumors

The term multicentricity has been employed to describe cancer cells beyond the borders of the primary tumor. However, it is not clear if there are multiple independent sites of origin or if the process simply represents spread of the cancer. The present study was designed to examine the distribution and extent of cancer in the breast and identify factors that bear on these events. All mastectomy specimens between 1980 and 1983 were systematically examined by means of multiple sections. One hundred seventy-nine of 657 patients (27%) were found to have separate foci. The most common histologic type (invasive ductal) was least likely to have multifocal disease (19%), while it was extremely common in the small group of patients with intraductal lesions (81%). Size was a factor in ductal but not in lobular lesions. Ninety per cent of the secondary foci were found in close proximity to the primary, suggesting spread rather than multicentricity. This implies a more limited and predictable distribution of cancer cells and opens the way to more rational selection and surgical preparation of patients for breast preservation.

Isolation of Native Human Monoclonal Autoantibodies to Breast Cancer

Using a unique fusion partner cell line, MFP-2, and B-lymphocytes from breast cancer patients, we developed a set of fully human monoclonal antibodies (MAbs) that bind with high specificity and sensitivity to breast cancer cells. Immunofluorescent staining of normal tissues, primary tumors, and metastatic lymph nodes demonstrates that these antibodies are specific for breast cancer of autologous and allogeneic origin. We have also determined that many of the antibodies selected based on specific binding to breast cancer cells and tissue also bind prostate cancer cells and tissue with high specificity and sensitivity. The targets of these antibodies have been localized to the cytoplasm and membrane. Biological assays for internalization and cytotoxicity demonstrated the ability of three antibodies to rapidly internalize. Our study demonstrates that isolation of native human MAbs from the natural antibody repertoire, targeted to cancer cells, is feasible and may provide a source of tools for immunotherapy.

A human monoclonal autoantibody to breast cancer identifies the PDZ domain containing protein GIPC1 as a novel breast cancer-associated antigen

BackgroundWe have been studying the native autoimmune response to cancer through the isolation of human monoclonal antibodies that are cancer specific from cancer patients. To facilitate this work we previously developed a fusion partner cell line for human lymphocytes, MFP-2, that fuses efficiently with both human lymph node lymphocytes and peripheral blood lymphocytes. Using this unique trioma fusion partner cell line we isolated a panel of autologous human monoclonal antibodies, from both peripheral blood and lymph node lymphocytes, which are representative of the native repertoire of anti-cancer specific antibodies from breast cancer patients.MethodsThe current study employs immunocytochemistry, immunohistochemistry, Western blot analysis as well as Northern blots, Scatchard binding studies and finally SEREX analysis for target antigen identification.ResultsBy application of an expression cloning technique known as SEREX, we determined that the target antigen for two monoclonal antibodies, 27.B1 and 27.F7, derived from lymph node B-cells of a breast cancer patient, is the PDZ domain-containing protein known as GIPC1. This protein is highly expressed not only in cultured human breast cancer cells, but also in primary and metastatic tumor tissues and its overexpression appears to be cancer cell specific. Confocal microscopy revealed cell membrane and cytoplasmic localization of the target protein, which is consistent with previous studies of this protein.ConclusionWe have determined that GIPC1 is a novel breast cancer-associated immunogenic antigen that is overexpressed in breast cancer. Its role, however, in the initiation and/or progression of breast cancer remains unclear and needs further clarification.

Phototoxic liposomes coupled to an antibody that alone cannot modulate its cell-surface antigen kill selected target cells

SummaryMolecules such as antibodies that bind to cell surfaces can be used to deliver cytotoxic drugs to selected cells. To be effective the drug must usually be taken into the cells by endocytosis. In this study a T-cell line (CCRFCEM) was effectively killed by liposomes carrying a photosensitizer and bearing the antibody OKT4 (anti-CD4). The unconjugated antibody does not induce antigenic modulation in the target cells, an indication of the absence of endocytosis, and would therefore not normally have been selected as an agent for drug delivery. It cannot, however, be concluded with certainty that the conjugates act at the cell surface and several alternative explanations of their efficacy are offered.

Modulation of the antigenic phenotype of human breast carcinoma cells by modifiers of protein kinase C activity and recombinant human interferons

SummaryIn the present study we have analyzed the effect of a synthetic protein kinase C (PKC) activator 3-(N-acetylamino)-5-(N-decyl-N-methylamino)-benzyl alcohol (ADMB) and the natural PKC-activating tumor-promoting agents 12-O-tetradecanoylphorbol 13-acetate (TPA) and mezerein on the antigenic phenotype of T47D human breast carcinoma cells. All three agents increased the surface expression of the tumor-associated antigen BCA 225 and various cellular antigens, including HLA class II antigens, intercellular adhesion molecule 1 (ICAM-1) and c-erbB-2. Expression of the same antigens was also upregulated to various extents in T47D cells by recombinant fibroblast (IFNβ) and immune (IFNγ) interferon. Shedding of BCA 225 from T47D cells was induced by TPA, mezerein, IFNβ and IFNγ, whereas ADMB did not display this activity. The ability of ADMB, TPA and mezerein to modulate the antigenic phenotype of T47D cells appears to involve a PKC-mediated pathway, since the PKC inhibitor, H-7, eliminates antigenic modulation. In contrast, the ability of IFNβ and IFNγ to enhance the synthesis, expression and shedding of BCA 225, as well as to enhance HLA class II antigens, c-erbB-2 and ICAM-1 expression, was either unchanged or modestly reduced by simultaneous exposure to H-7. Analysis of steady-state mRNA levels for HLA class I antigens, HLA class II-DRβ antigen, ICAM-1 and c-erbB-2 indicated that the ability of H-7 to inhibit expression of these antigens in TPA-, mezerein- and ADMB-treated cells was not a consequence of a reduction in the steady-state levels of mRNAs for these antigens. The results of the present investigation indicate that the biochemical pathways mediating enhanced antigenic expression in T47D cells induced by TPA, mezerein and the synthetic PKC activator ADMB are different from those induced by recombinant interferons. Furthermore, up-regulation of antigenic expression in T47D cells can occur by a PKC-dependent or a PKC-independent pathway.