R. W. Baldwin

Immunological aspects of chemical carcinogenesis.

Publisher Summary This chapter focuses on the degree of involvement of immune mechanisms in modifying tumor formation and growth during chemical carcinogenesis. During the past two decades, the concept that tumors express antigens against which the host is capable of evoking an immune response has been firmly established. The validity of these studies, as well as comparable findings with MC-induced rat sarcomas (Baldwin, 1955), was critically analyzed by Prehn and Main (1957) who conclusively showed that the induction of immunity to transplanted sarcoma grafts was a tumor-specific phenomenon because immunized mice could still accept skin grafts from the tumor donor, precluding the involvement of an alloantigenic immune response. Evidence shows that tumor rejection antigens are specific and permanent, or quasi-permanent, characteristics of cells transformed by chemical carcinogens. Chemically induced tumors also express antigens normally present in embryonic, but not adult, tissue that may be viewed as the products of dedifferentiation processes induced by the carcinogen, and concurrently there is frequently a deletion of normal tissue antigens. The immunological responses to neoantigens expressed on preneoplastic and neoplastic cells can influence, either positively or negatively, the course of chemical carcinogenesis.

RADIOIMMUNODETECTION OF HUMAN COLORECTAL CANCERS BY AN ANTI-TUMOUR MONOCLONAL ANTIBODY

In 10 out of 11 patients with colorectal cancer radiolabelled antitumour monoclonal antibody (791T/36) was localised to the tumour. The mean tumour to non-tumour uptake ratio of antibody demonstrated by imaging with a gamma camera was 4.4/1 after subtraction of background radioactivity. The antibody did not localise in one patient who had received radiotherapy to his tumour two weeks previously. In 5 patients with primary neoplasms localisation of the antibody was confirmed by further imaging of the resected specimens and in-vitro radioactivity counting of the tumour and comparison with the activity in adjacent normal colon.

Immunological and structural features of the protein core of human polymorphic epithelial mucin

The protein core of high mol. wt polymorphic epithelial mucin (PEM--approximately 400 kDa glycoprotein) which is associated with breast carcinomas, consists of a repeating 20 amino acid peptide motif [Gendler et al. (1988) J. biol. Chem. 263, 12,820-12,823]. Monoclonal antibodies C595 (anti-urinary mucin) and NCRC-11 (anti-breast carcinoma cells), and other antibodies against human milk fat globule membranes, were found to recognize determinants present within this 20 amino acid peptide. A model of the peptide was developed based on hydropathicity and structure prediction calculations and these indicated that the repeated structure is dominated by a hydrophilic domain of seven amino acids, extending into two flanking beta turns. NMR analysis of the 20 amino acid peptide was undertaken to probe the secondary structure. Epitope mapping experiments involving solid phase synthesis of overlapping heptapeptides in the repeat unit identified the minimum structures for antibody binding as Arg-Pro-Ala-Pro and Arg-Pro-Ala for the C595 and NCRC-11 antibodies, respectively. These determinants were found within the predicted hydrophilic turn region domain of the peptide. The epitopes for six other PEM-reactive monoclonal antibodies were also determined to reside within the predicted hydrophilic turn domain. This evidence is in accord with the disposition of this region of the PEM peptide core being at the exterior of the glycoprotein where it would be accessible to antibody recognition and binding events.

Association between clinical symptoms and lymphocyte abnormalities in a population with chronic domestic exposure to industrial solvent-contaminated domestic water supply and a high incidence of leukaemia

SummaryAn unusually high incidence of leukaemia and recurrent infections was noted in children exposed in utero to domestic water supply contaminated with industrial solvents including trichloroethylene, perchloroethylene and 1,2-transdichloroethylene. Medical and laboratory investigations were carried out on 28 family members of the patients with leukaemia with particular emphasis on the immunological system to determine if they displayed symptoms associated with acute or chronic exposure to these chlorinated hydrocarbons. The principal organ systems affected were neurological, immunological and cardiological. Damage to these systems was found in all subjects by history, physical and laboratory parameters. Damage to the immunological system was manifest by altered ratios of T lymphocyte subpopulations, increased incidence of auto-antibodies, increased infections and recurrent rashes.

Tumour aneuploidy, prognostic parameters and survival in primary breast cancer

Cellular DNA content of primary tumours from 280 patients with operable breast cancer was determined by flow cytometry using nuclei from paraffin sections stained with DAPI, and 199 of these patients were followed for 8-13 years after surgery. Tumours from 67 patients have also been analyzed for their DNA content using single cell suspensions from fresh tumour tissue stained with mithramycin and ethidium bromide, and the results compared with those obtained from paraffin blocks of the same tumours. Overall 60% of the tumours contained cells with abnormal DNA content (DNA-aneuploid populations). Survival and disease free interval were not significantly different in patients with DNA-diploid and DNA-aneuploid tumours when analysed by Mantels life table method. There was however, an early advantage for patients with DNA-diploid tumours: during the first 30 months after surgery DNA-aneuploidy was associated with higher rate of recurrence and shorter survival. DNA-aneuploidy was strongly related to histological grade. Thus 11/49 (22%) grade I, 60/102 (59%) grade II, and 96/129 (74%) grade III tumours were DNA-aneuploid. Although there was no significant difference in survival of patients with DNA-diploid and DNA-aneuploid tumours overall, there appears to be an unexpected association between DNA-aneuploidy and better survival in grade II patients (P less than 0.01); a similar trend was observed for grade I patients. Although the proportion of DNA-aneuploid tumours was similar in oestrogen receptor positive and negative tumours, DNA-aneuploidy was associated with lower levels of oestrogen receptors in comparison to DNA-diploid tumours. Comparison between the modal DNA values of fresh and paraffin embedded samples showed high rate of comparability (64/67, P less than 0.0001).

Antitumor properties of vindesine-monoclonal antibody conjugates

SummaryThe anticancer alkaloid vindesine (VDS) was conjugated to four mouse monoclonal antibodies recognizing human tumor-associated antigens. The antibodies were 96.5 (antimelanoma, IgG2a); 791T/36 (antiosteogenic sarcoma, IgG2b); 11.285.14, and 14.95.55 (anticarcinoembryonic antigen, IgG1 and IgG2a respectively). Conjugates VDS-96.5 and VDS-791T/36 were tested in vitro and shown to be specifically cytotoxic for target cells expressing the appropriate antigen. The in vivo effects of the antibodies and conjugates were tested against human tumor xenografts in athymic or immunodeprived mice using multiple treatments. Conjugate VDS-96.5 retarded the initial growth of a melanoma xenograft, whereas free antibody was without effect. Similarly, VDS-791T/36 but not free antibody retarded the growth of osteogenic sarcoma 791T. The most marked antitumor effects observed were those obtained with VDS conjugates of the anti-CEA antibodies against a colorectal tumor xenograft. Antibody 14.95.55 suppressed tumor growth both alone and as a VDS conjugate, whereas 11.285.14 produced only a slight effect alone but an almost complete and lasting suppression of tumor growth as a VDS conjugate. Free VDS had little effect at nontoxic levels. Acute studies showed that VDS-11.285.14 conjugate was considerably less toxic than free VDS in Balb/c mice.

A monoclonal antibody, NCRC-11, raised to human breast carcinoma. 1. Production and immunohistological characterization

The production of a mouse monoclonal IgM antibody, NCRC‐11, raised against human breast carcinoma is described. It has been characterized immunohistologically. The antigen recognised has a wide but highly specific distribution in normal tissues, being virtually confined to the surface of certain epithelial cell types. It is found in some forms of epithelial metaplasia and most epithelial malignancies, particularly adenocarcinomas. The heterogeneity of staining in mammary carcinomas is outlined and is of particular interest. The immunohistological staining distribution of NCRC‐11 is similar to other antibodies, including anti‐epithelial membrane antigen, which were raised against human milk fat globule membrane. A competition experiment with some of these antibodies, using a flow cytofluorimeter, showed competition with one antibody, LICR LON/M8.

Selective cytotoxicity against human tumour cells by a vindesine-monoclonal antibody conjugate.

The anti-mitotic drug vindesine was coupled chemically to a monoclonal antibody raised originally against the human osteogenic sarcoma cell line, 791T. The cytotoxicity of the conjugate in vitro was tested, in comparison with free vindesine, against sarcoma 791T and other antigenically cross-reactive osteogenic sarcoma-cell lines, and also against tumour cell lines which have no detectable reaction with the monoclonal antibody. Continuous exposure of cultured 791T cells indicated that the vindesine was partially inactivated following conjugation since the conjugate was less toxic than the free drug. However, antibody-binding activity was essentially preserved following conjugation. Despite diminished drug activity in the conjugate, assays designed to mimic antibody binding to tumour in which target cells were treated with conjugate and washed before culture, showed selective cytotoxicity for osteogenic sarcoma lines with little or no effect on non-cross reactive control cells. In comparison, free vindesine was toxic equally for all cell lines and free antibody was non-toxic. These studies indicate that conjugation of a cytotoxic agent to a monoclonal antibody can confer on that agent selectivity for a particular target cell type which is recognised by the antibody.

Tumour localization of monoclonal antibody against a rat mammary carcinoma and suppression of tumour growth with adriamycin-antibody conjugates

SummaryMonoclonal antibody to the rat mammary carcinoma Sp4 isolated from hybridoma supernatants and labelled with 125I showed preferential in vivo localization into subcutaneous growths of Sp4 compared with normal tissues and a range of other transplanted tumours. No specific localization was seen with 125I-labelled normal rat immunoglobulin. Adriamycin conjugated to monoclonal antibody significantly retarded Sp4 tumour growth at 1/25th of the effective dose of the free drug, and in some cases brought about total tumour regression. Normal rat immunoglobulin-adriamycin conjugates were relatively ineffective. These studies indicate that monoclonal antibody directed against tumour cell surface antigens may be highly effective for tumour targeting of therapeutic agents.

Differences in tumour and normal tissue concentrations of iodine- and indium-labelled monoclonal antibody

The blood, tumour and whole-body levels and survivals of 131I- and 111In-labelled monoclonal antibody (791T/36) have been compared in mice with human tumour xenografts. The blood levels and survivals of 131I- and 111In-labelled antibody were similar when expressed as proportions of the injected doses. However, the whole-body survival of 111In following administration of 111In-labelled antibody was over twice as long as that of 131I after administration of 131I-labelled antibody, principally because free 131I was rapidly excreted but free 111In was retained, primarily in liver, spleen and kidneys. Consequently, when expressed in relation to the whole body, blood levels of 131In became progressively lower than those of 131I following administration of labelled antibodies. In mice with human tumour xenografts the proportion of the injected dose of 111In from 111In-labelled antibody deposited in tumour tissue was 4–5 times higher than that of 131I from 131I-labelled antibody. When compared with the whole-body levels of radiolabel the difference was less marked, although 111In accumulation in tumour was more rapid. The higher levels and longer retention of 111In in tumour produced tumour-to-blood ratios that were 7–8 times those achieved with 131I-labelled antibody.