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Xenografts of 3 human malignant cell lines in congenitally athymic nude mice have been examined for susceptibility to BCG. Growth of all 3 tumours, a bladder carcinoma, a melanoma and a colon carcinoma, was suppressed when cells were injected in admixture with BCG. Distant injection of BCG was ineffective. Mice with progressive growths had no detectable anti-human antibody, and rejection of cells and BCG failed to confer protection against subsequent tumour challenge. These studies indicate that human malignant cells are susceptible to local BCG-activated host responses, and that athymic mouse xenografts may be a useful model for assessing the response of human tumours to such agents.

Subcutaneous growth of immunogenic chemically induced rat sarcomata and a hepatoma was restricted when cells were injected into syngeneic animals in admixture with MER. Rats rejecting mixed inocula were immune to further challenge with the same tumour. Growth of a chemically induced mammary carcinoma which lacks detectable immunogenicity was suppressed when low cell inocula were injected in admixture with MER or intact BCG organisms, although animals were not immune to re-challenge. These studies indicate that clinically MER may be a suitable alternative to BCG for contact suppression of tumour growth or incorporation into tumour cell:adjuvant vaccines for active immunotherapy.

A 51Cr-release test was developed for the detection of cell-mediated cytotoxicity against transplanted solid chemically induced rat tumours, and the findings were compared with those obtained in parallel tests using a microcytotoxicity assay. It was necessary to incorporate several modifications of the original Brunner assay (Brunner et al., 1968) in order to increase the sensitivity of the test as applied to long-term tumour lines maintained as glass-adherent cultures. These included: 1. preincubation of labelled tumour cells at 37 degrees C for 3-4 h before addition of effector cells, 2. preincubation of effector cells at 37 degrees C for 12 h before addition to target cells, 3. upon admixture of target and effector cells, intimate cell contact was established by gentle centrifugation, 4. after incubation of target and effector cells at 37 degrees C, a further incubation for 1 h at 45 degrees C was included to complete the release of 51Cr from non-viable cells. Maximal cytotoxicity of tumour-bearer effector cells was detected after 12 h incubation of lymphoid and target cells at a ratio of 200:1. Spleen cells from tumour-bearing rats during the first 2 weeks of tumour growth exhibited the same pattern of reactivity in the 51Cr-release test and the microcytotoxicity assay, although significant reduction in target-cell numbers was more frequently recorded using the microcytotoxicity assay. Tumour-bearer spleen cells showed reactivity against the homologous tumour as well as against unrelated tumours. Using either assay, pre-exposure of effector cells to 3M KC1 extracts of tumour was found to inhibit their effect on tumour cells most frequently in tests in which the effector-cell donor, soluble extract and target cell were of the same tumour line.

Wistar rats were sensitized to rat embryonic tissue by immunization with irradiated (5000 rad) rat embryo cells (2 X 10(6) s.c. + 1 X 10(6) i.p.) derived from embryos aged 14-15 days, or by implantation of irradiated (5000 rad) tissue grafts from these embryos. Three to five immunizations were given at weekly intervals, and the rats were then challenged subcutaneously 7-10 days after the final inoculum with minimal tumour-producing tumour cell doses. Immunization with irradiated rat embryo cells failed to influence the growth and development of tumour cells prepared from hepatoma D23 and D30, sarcoma Mc57, mammary carcinoma AAF57 or cells prepared from spontaneously arising mammary carcinomata Sp4 and Sp15. Using adoptive transfer techniques, lymphoid cells from embryo-sensitized rats, when used in a 3000 : 1 ratio (lymphoid cells : tumour cells), were shown effectively to retard the growth of hepatoma D23 in 3 out of 7 experiments performed. Similar adoptive transfer procedures proved ineffective in preventing the growth of mammary carcinoma AAF57. Using in vitro cytotoxicity tests, lymph node cells and spleen cells from embryo-immunized rats were shown to be cytotoxic for several rat tumour cell targets : hepatoma D23 (7/10 tests), sarcoma Mc7 (8/12 tests), mammary carcinoma AAF57 (2/2 tests) and Sp4 (3/4 tests), and for 14-15-day-old rat embryo cells (5/10 tests). In comparative tests lymphoid cells were relatively non-cytotoxic for 20-day-old rat embryo cells (1/6 tests) or cells prepared from adult rat lung or kidney (1/10 tests). The role of embryonic antigen(s) in tumour rejection is discussed.

2-Acetylaminofluorene induced mammary and ear duct carcinomata and spontaneously arising mammary carcinomata and sarcomata were shown to express embryonic antigens at the cell surface by their reaction with serum from multiparous female rats. These observations with essentially non-immunogenic tumours are comparable with early findings showing that embryonic antigens are also expressed on aminoazo dye induced rat hepatomata, and sarcomata induced with 3-methylcholanthrene. Re-expression of embryonal components, therefore, may be a concomitant of neoplastic transformation.

Murine monoclonal antibodies directed against tumour associated antigens are potentially useful in tumour diagnosis and therapy. However, all the antigens they recognise may be heterogeneously expressed on tumours and this may allow escape of cells from therapy if a single monoclonal antibody is used. One approach is to use combinations of monoclonal antibodies recognising complementary cell surface antigens. A flow cytometric method which allows accurate quantitation of the intensity of staining and the percentage of fresh primary tumour cells binding a series of monoclonal antibodies has therefore been developed. This allows calculations as the number of drug molecules which could be potentially delivered by each monoclonal antibody and the optimal combination of antibodies which should be used. Monoclonal antibodies recognising Y hapten (C14), CEA (228, 161) and 791T-p72 antigen (791T/36) have been screened as a possible combination for colorectal cancer. There was inter-tumour variation in the binding of all the monoclonal antibodies although combinations could reduce or abrogate this problem. A combination of the monoclonal antibodies C14, 228, 791T/36 and 161 would recognise 100% of tumours. Sixty per cent of tumours bound all four antibodies, 78% any three, 90% any two and 100% any one antibody. There was also intra-tumour variation in the number of tumour cells per lesion that were recognised, the best monoclonal antibody, 161, stained a mean of 59% of cells per tumour whereas the anti-cytokeratin monoclonal antibody stained a mean of 74% of cells per tumour. An increased intensity of staining of tumour membranes was observed when a combination of C14 and 228 was used compared to binding of individual antibodies. Furthermore there was still no significant binding to normal colon membranes. Combinations of monoclonal antibodies which recognise a high percentage of tumours are likely to be necessary for monoclonal antibody drug targeting to prevent tumour recurrence and/or metastases.

A bispecific monoclonal antibody, reactive with methotrexate (MTX) and a tumour associated antigen (gp72) has been produced by fusing spleen cells from MTX immunised mice with 791T/36/3 (anti-gp72) hybridoma. The hybrid antibody was purified from anti-MTX and anti-gp72 antibodies present in the hybridoma culture supernatant by combinations of affinity chromatography on a MTX-agarose immunoabsorbent and stepwise acid elution from Sepharose-Protein A. A particular feature of the present antibody is that it reacts with conjugated MTX; this would allow in vivo targeting of conjugates, increasing many fold the number of molecules of drug carried or localising to pre-targeted antibody. Dual binding between tumour cell surface antigen and MTX was demonstrated by the ability of the hybrid antibody to bridge between tumour cells and MTX as MTX-HSA conjugate, reaction here being detected by flow cytofluorimetry. Purified hybrid antibody specifically enhanced the in vitro cytotoxicity of MTX-HSA for gp72 positive tumour cells.

The Y haptenic blood group determinant is expressed on the cells of malignant gastrointestinal tumours and on the normal gastrointestinal epithelium of individuals who can secrete blood group antigens. an enzyme-linked immunosorbent assay incorporating the high affinity biotin-avidin interaction was developed to measure the serum levels of antigens bearing the Y hapten in patients with colorectal cancer. The specificity of the assays was between 88 and 93% and the sensitivity in detecting extensive disease was between 24 and 34%. However, up to 67% of patients with local or abdominal recurrent disease secreted antigens expressing the Y hapten, whereas only 30% of patients with overt hepatic metastases secreted a similar antigen. Recognition of antigens bearing the Y hapten may therefore be useful in detecting early local relapse of colorectal cancer when a second operation to excise the recurrence may be possible.

A flow cytometric technique has been established for accurately quantitating the cell surface density of MHC antigens and the percentage of cells expressing MHC antigens in 38 colorectal tumours. Thirty-four percent of tumours were partially or completely negative for HLA-ABC antigen expression. Although the quantity of HLA-ABC antigens varied widely, there was no correlation between the density of HLA-ABC antigens, or the percentage of cells expressing these antigens and clinicopathological stage. Fifty percent of the colorectal tumours expressed HLA-DR with varying antigen densities. All of the poorly differentiated tumours expressed HLA-DR but there was no correlation between expression of HLA-DR and clinicopathological stage. The aneuploid tumours expressed more HLA-ABC and HLA-DR antigens on a higher percentage of cells than the diploid tumours. Abnormal expression of the tumour associated antigens CEA, Y haptenic blood group and 791T p72 also correlated with expression of HLA-ABC and HLA-DR antigens on colorectal tumours. The majority of early derived in vitro dividing cells failed to express both HLA-ABC and HLA-DR antigens although they expressed high levels of tumour associated antigens. If there is a correlation between in vitro and in vivo growth perhaps tumours are maintained and seeded by MHC antigen negative cells.

A major problem in the chemotherapy of colorectal cancers is their resistance to most cytotoxic drugs which may be due to insufficient cellular transport. Drugs conjugated to monoclonal antibodies recognising tumour antigens may overcome these difficulties by providing access of active agents to the tumour cells. The anti-tumour monoclonal antibody shown to localise in patients with colorectal cancer, 791T/36, has been investigated as a potential targeting antibody. Eight cell lines were established from surgically resected material and were shown to bind 791T/36 antibody. They were screened for their sensitivity to methotrexate, 5-fluorouracil and daunomycin. Although 5-fluorouracil is the drug of choice for chemotherapy of colorectal cancer it was the most cytotoxic drug in only 2 of the 8 cell lines. Only the 4 cell lines which were resistant to methotrexate showed less cytotoxicity with methotrexate than 5-fluorouracil. The cell lines which were resistant to methotrexate were more sensitive to 791T/36-methotrexate conjugates. Daunomycin was the most cytotoxic drug in 4 of the 8 cell lines. However, a similar cytotoxicity was observed for free drug and 791T/36 daunomycin in the two lines tested. Selective monoclonal antibody drug conjugates may offer a solution to treatment of tumours which are resistant to classical chemotherapeutic agents. This is the first report to show that newly established cell lines that are resistant to classical chemotherapeutic agents are rendered sensitive when the drug enters the cell as a drug monoclonal antibody carrier.

Immunocytochemical staining with monoclonal antibody NCRC 11 of formalin fixed paraffin embedded tumour tissue has been studied in 444 cases of primary breast cancer with a minimum follow period of 6 years. The relationship between extent of staining, assessed on a four point scale, and patient survival has been confirmed. There are significant relationships between staining and both histological grade and oestrogen receptor status. No association has been shown between staining and lymph node stage or tumour size. Simplification of staining assessment by modification to two staining groups still allows significant separation of patients into prognostic groups and incorporation into an existing prognostic index.

Urinary mucins which express determinants for the anti-breast carcinoma monoclonal antibody, NCRC-11 (IgM), closely resemble the mammary mucins found in milk fat globules and carcinomas. An IgG3 monoclonal antibody, C595, was prepared against urinary mucins isolated on a NCRC-11 antibody affinity column, and this 'second generation' antibody was shown to have a very similar pattern of reactivity to the original NCRC-11 antibody. By immunohistology, the profile of reactivity of both antibodies with tumour and normal tissue specimens was virtually identical. Both antibodies reacted with epithelial mucins isolated from breast tumours or normal urine using an NCRC-11 antibody affinity column, although the antibodies were unreactive with other antigen preparations. Heterologous immunoradiometric assays ('sandwich' tests) confirmed that NCRC-11 and C595 epitopes were co-expressed on the same molecule. C595 antibodies inhibited the binding of radiolabelled NCRC-11 antibodies to antigen, suggesting that the two epitopes were in close topographical proximity. The protein core of the mammary mucins has recently been shown to consist predominantly of a repeated 20 amino acid sequence (Gendler et al., 1988). Peptides with this complete sequence and small fragments were synthesised, and the C595 antibody was found to recognise an epitope within this repeat. The ability to identify and synthesise monoclonal antibody-defined determinants, as well as those in the adjacent or overlapping sequences within the protein core of epithelial mucins, is viewed as a strategy for facilitating the production of antibodies of new and novel specificity to complement the panels of existing anti-breast cancer reagents.

The anti-breast carcinoma monoclonal antibody (MAb), NCRC-11 defines a polymorphic epithelial mucin (PEM) which is elevated in the circulation of advanced breast carcinoma patients. Here we describe the purification and partial characterisation of this component from patients' sera and its use in the production of a second generation MAb, C568 (IgM). Pooled sera was fractionated by immunoaffinity and size-exclusion chromatography and the purity of preparations assessed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Serum-derived PEM shows a similar pattern of electrophoretic mobility to PEM isolated from primary breast tumour tissue and migrates as several bands in 4% SDS polyacrylamide gels (Mr greater than 400,000). The epitope expression of PEMs isolated from either source is also similar, with both bearing topographically distinct determinants for several anti-mucin MAbs. The immunoreactivities of antibodies C568 and NCRC-11 were unaffected by boiling, reduction and alkylation, or by enzyme desialylation of PEM. Periodate oxidation and proteolytic digestion have suggested that the antigenic determinant for C568 is carbohydrate in nature whilst that of NCRC-11 is peptidic. In accord with the mucinous nature of the molecule, serum-derived PEM is susceptible to reductive beta-elimination, elutes in the void volume of a Sepharose CL-4B column and has a buoyant density of 1.45 g ml-1.

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 Mantel's 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).

Reactivity of the monoclonal antibody 791T/36 with secondary malignant deposits has been investigated in frozen sections of 74 human liver biopsy specimens. There was no reactivity with hepatocytes but in some instances binding to fibrous tissues and in one case to portal tract lymphocytes was observed. Sections from 9 biopsy specimens contained malignant deposits. In seven of these 791T/36 bound either to malignant cells or to pseudoacinar contents (3 colorectal adenocarcinomas; 1 probable pancreatic adenocarcinoma; 1 medullary cell carcinoma of thyroid; 1 oat cell carcinoma of bronchus and 1 deposit of nodular sclerosing Hodgkins Disease). Two undifferentiated tumours (1 gastric adenocarcinoma and 1 oat cell bronchial carcinoma) showed no antibody binding. The histological pattern of reactivity previously reported with primary tumours appears to be similar in secondary deposits. A wider range of tumours than recognised hitherto binds 791T/36. Whether the binding to fibrous tissue seen in some instances is sufficient to cause diagnostic uncertainty when 791T/36 is used for scanning requires further investigation.

NCRC-11 is an IgM monoclonal antibody which defines an antigen found in most epithelial malignancies. The antigen has previously been shown to be a high mol. wt. glycoprotein (greater than 400,000) and in this study, antigen preparations were isolated by immunoadsorbent chromatography from ovarian mucinous and ovarian serous cyst adenocarcinoma and from breast carcinoma. Other monoclonal antibodies, against products in normal human milk, and antibodies of the Ca series (Bramwell et al., 1985) reacted with all three antigen preparations. Tests involving epitope mapping were performed to probe the relationships of the various epitopes to that defined by the NCRC-11 antibody, and, of note, the three antigen preparations from different tumour sources were remarkably similar with respect to their relative levels of epitope expression and to their topographical distribution of epitopes. The major differences in epitope expression could be attributed to the degree of sialylation in the three antigens. The antigens from ovarian tumours expressed I(Ma) blood group determinants (defined by the antibody LICR-LON-M18) which were partially masked by sialic acid. With NCRC-11 defined antigen from breast carcinoma, this determinant was totally masked by sialic acid although neuraminidase treatment clearly exposed epitopes reactive with M18 antibodies.

Cells from two adenocarcinomas, an adenoma and a metastatic node were isolated in soft agar. Expression of antigens, CEA, Y haptenic blood group and 791T-p72, defined by a range of candidate antibodies for tumour targeting was assessed. All of the cells expressed low levels of CEA but high levels of the Y haptenic blood group antigen although there was enormous inter and intraclonal variation. Of particular interest was the membrane expression of 791T-p72 antigen on all of the dividing tumour cells as previous studies had shown that 791T/36 antibody reacted with tumour stromal elements rather than malignant cell surfaces. The DNA content was abnormal in all of the cells whether they were derived from diploid or aneuploid primary tumours. They all grew readily in athymic mice and at least one monoclonal antibody, 791T/36, localised efficiently within these xenografts. Clonogenic cells therefore expressed the three tumour-associated antigens, several at higher levels than observed in the primary tumour. Monoclonal antibody 'cocktails' should therefore allow antibody mediated drug cytotoxicity to be effective at eradicating rapidly dividing tumour cells.

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.

Two mouse monoclonal antibodies against the human osteogenic sarcoma 791T were examined for their capacity to exert complement-dependent cytotoxicity against a panel of human tumour cell lines. Cytotoxicity was most evident against the immunizing tumour 791T although significant reactivity was directed against other osteogenic sarcomas. In admixture, the 2 antibodies displayed synergism in their cytotoxicity although this was only demonstrable with defined ranges of antibody concentration. The cytotoxicity of these antibodies was dependent upon the use of rabbit serum as complement and no tumour-cell lysis was produced using human, guinea-pig or mouse serum complement. The more potent cytotoxic antibody failed to modify the outgrowth of 791T tumour xenografts in immunodeprived mice even though localization of antibody at the tumour site has been demonstrated (Pimm et al., 1982).

Papain-solubilized tumour-specific antigens from the aminoazo dye-induced rat hepatoma D23 were purified by a combination of lectin affinity and immunoadsorbent column chromatography. Isolated antigens were radio-iodinated using three procedures and analysed for their reaction with specific antibodies in syngeneic immune sera by double-antibody co-precipitation tests and by the rebinding of labelled antigens to specific and non-relevant antibodies immobilized on Sepharose-4B. Soluble hepatoma D23-specific antigens were labile to radiolabelling, and for optimal retention of serological reactivity it was necessary to protect the antigenic determinant by performing the chloramine T method of iodination with antigen bound to the immunoadsorbent followed by elution from the solid phase with 3M NaSCN. Immunoadsorption chromatography indicated that one consequence of radiolabelling hepatoma D23-specific antigen with 125I was a reduction in the affinity of the labelled antigen for its syngeneic specific antibody.

Cells of osteogenic sarcoma line 791T were treated in vitro with a selectively cytotoxic methotrexate-human serum albumin-monoclonal antibody conjugate at concentrations which were toxic but allowed the “escape” of a small number of tumour cell colonies (less than 0.3% compared with controls). These colonies were propagated as clones in order to test their expression of the monoclonal antibody ( 791T /36)-defined antigen and their resistance to methotrexate (MTX) by comparison with parental cells. Most of the conjugate-treated clones were incapable of prolonged growth and died out, in contrast to untreated 791T clones which virtually always grow progressively. Only four treated clones grew at rates comparable with the parental line. Flow cytofluorometric analysis indicated that the surviving clones expressed normal or enhanced amounts of 791T /36-defined antigen and clonogenic assays demonstrated that they were sensitive to cytotoxicity by MTX. As could be predicted from these results, further exposure to the conjugate inhibited growth of the clones at doses comparable with those active against parental 791T cells. It is concluded that tumour cell clones emerging after exposure to a toxic concentration of a drug-antibody conjugate are not necessarily modified resistant clones, but may have severely impaired long-term growth potential or be susceptible to further contact with the same conjugate.

Cytoplasts and karyoplasts were obtained by ultracentrifugation of Hepatoma D23 cells on a Ficoll gradient containing cytochalasin B. Their nuclear and protein content and their metabolic activity were determined. Three i.p. injections of 2.3 x 10(7) cytoplasts were unable to protect syngeneic WAB/Not rats against an s.c. challenge of 10(4) D23 cells, whereas a similar amount of karyoplasts, or 3 injections of 10(6) irradiated D23 cells, were fully protective. Ability of cytoplasts to act as primary or secondary immunogen were also studied, and compared to that of 0.01% glutaraldehyde-treated cells, 43 degrees C heat-treated cells and 3M KCl-soluble extracts, these preparations also being of weak immunogenicity. Only heat-treated cells behaved as a primary immunogen, whereas none of the preparations provided a secondary stimulation. Moreover, when these preparations were fed in vitro to peritoneal-exudate cells before their injection into rats, cytoplasts and glutaraldehyde-treated cells showed no immunogenicity, whereas heat-treated cells induced full protection against tumour challenge. Therefore, in this tumour model, the in vivo persistence of immunogen and the presence of a nucleus are likely to be crucial in inducing transplantation resistance to tumour.

It has previously been shown that rat embryo cells treated in vitro with 3-methylcholanthrene (MCA) elicit antibodies in syngeneic rats which react specifically against established MCA-induced sarcomas. To examine the possibility that clonal amplification of one or a few antigenic, preneoplastic clones is responsible for the previously observed specific antibody responses, MCA-treated rat embryo cells have been subjected to 150 Gy of gamma-irradiation before injection into host animals. The resulting antisera were screened for reactivity against a panel of established syngeneic tumours by membrane immunofluorescence and an isotopic antiglobulin test. A positive reaction was observed between an antiserum pool raised against gamma-irradiated MCA-treated cells and the cells of an immunogenic spontaneous mammary carcinoma. Antiserum to gamma-irradiated control (acetone-treated) cells was negative. Thus gamma-irradiation of carcinogen-treated cells before injection failed to abolish specific antibody responses in immunized rats. To investigate further the relationships between cell-carcinogen interaction, neoantigen induction and malignancy, the cells of a non-immunogenic, spontaneous mammary carcinoma were treated with MCA in vitro, and antisera against treated and untreated cells were tested against a panel of established tumours. A positive membrane-immunofluorescence reaction was obtained with an antiserum to MCA-treated cells, but not to untreated cells against an aminoazodye-induced hepatoma, indicating that the previously non-immunogenic mammary carcinoma cells had acquired new antigenic specificities as a consequence of carcinogen treatment.

Monoclonal antibody against an osteogenic-sarcoma cell line (791T) was prepared by production and cloning of a somatic-cell hybrid between the mouse myeloma P3-NS1 and spleen cells from 791T-immunized mice. Three clones of hybridoma producing antibody against 791T, as detected by 125I-labelled Protein A binding, were tested against a range of normal and tumour cell targets to determine the pattern of expression of the antigen detected. The 3 clones had identical activity. They reacted strongly against 791T cells and another osteogenic sarcoma, 788T, and more weakly against a further 2 from a total panel of 10 osteogenic-sarcoma lines. The antibody was negative for fibroblasts from the donor of 791T, and for other fibroblasts, human red blood cells, human peripheral mononuclear cells and sheep red blood cells. When tested against a panel of unrelated tumours, they reacted against individual cell lines derived from carcinomas of colon, lung, bladder and cervix. These cross-reactions were not observed with other colon or lung carcinomas, and it is suggested that the antibody was reacting with a tumour-associated antigen expressed randomly on different tumour types, rather than specifically on osteogenic sarcomas.

The serum of athymic nude mice bearing rat tumour xenografts has been examined for tumour-specific antigen. With a sarcoma and a hepatoma, tumour-specific antigen expression continued in xenograft growths, and sera of tumour-bearing mice contained free antigen, assayed by its ability to neutralise reactivity of tumour-immune rat sera against tumour target cells in an indirect membrane-immunofluorescence test. In contrast, no anti-rat antibody was detectable in sera of mice bearing the xenografts, or rejecting cells injected in admixture with BCG.

A bispecific monoclonal antibody recognising both carcinoembryonic antigen (CEA) and ricin toxin A chain (RTA) was tested for its ability to target recombinant RTA (r-RTA) to CEA-expressing tumour cells, alone and in combination with ricin B chain (RTB). The antibody, 636 (Robins et al., 1990), induced significant RTA cytotoxicity against MKN45 gastric carcinoma cells which express high levels of CEA, using the r-RTA at a concentration below that known to be intrinsically cytotoxic. The addition of ricin toxin B chain (RTB) also potentiated cytotoxicity of r-RTA, and there was an additive increase in potentiation against CEA-positive cells when both RTB and 636 were included. The bispecific antibody restored potentiation by RTB after blocking of its binding site with excess galactose, and also the cytotoxic activity of whole ricin which had been blocked with galactose. It was concluded that the 636 bispecific antibody was highly effective in targeting the toxic moiety of the molecule to CEA-expressing cells, and allowed exploitation of the additional ability of the B chain to facilitate cellular incorporation. The facilitating function of the B chain was equally effective whether or not its lectin site was active.

Incubation in vitro of lymphnode cells (LNC) from rats bearing a transplanted syngeneic methylcholanthrene-induced sarcoma (Mc7) resulted in the generation of a potent cytotoxic activity. Four to seven days' culture was required for development of cytotoxic activity, which was shown to be mediated by a heat-stable soluble factor. The cytotoxicity was not detectable in a 3 h or 15 h 51Cr-release assay, but was demonstrated in a 48 h microcytotoxicity assay, where post-labeling with isotopically labelled cell precursors was used to quantitate cell survival. The cytotoxicity of the cultured tumour-bearer LNC and their supernatant factor was shown to be cross-reactive for tumour cell lines other than sarcoma Mc7, and was also expressed against adult or embryonic fibroblasts.

gamma-Irradiated rat hepatoma cells are immunogenic in syngeneic WAB/Not rats, so that immunized animals are protected against tumour-cell challenge and circulating tumour-specific antibody is produced. Treatment of the immunizing cells with glutaraldehyde at concentrations of 0.001% or greater for 30 min rendered these cells non-protective in tumour-rejection tests and no longer able to induce significant formation of specific antibody. However, tumour-specific antigens were shown to be expressed upon treated cells; they specifically bound tumour-specific antibody from syngeneic immune sera assessed in indirect membrane-immunofluorescence tests. Also, these cells specifically absorbed antibody from immune or tumour-bearer sera, as demonstrated in the indirect membrane-immunofluorescence test or a complement-dependent 51Cr-release test. Alloantigen expression was not influenced by glutaraldehyde treatment, although glutaraldehyde-treated hepatoma cells failed to induce alloantibody formation in KX/Not rats. Polyacrylamide-gel electrophoresis of treated cells, surface-labelled with 125I, indicated that extensive cross-linking of the surface protein occurred as a result of glutaraldehyde treatment. The present findings establish that although the expression of a tumour-specific antigen is necessary for the induction of immuno-protection against tumour-cell challenge, this alone is not a sufficient condition for eliciting tumour immunity.

I.p. immunization with gamma-irradiated hepatoma cells induces resistance to s.c. tumour-cell challenge in syngeneic WAB/Not rats. Mild heat treatment of these cells (greater than 41 degrees C for 30 min) destroyed this immunoprotective effect, but did not abolish tumour-specific antibody production in treated rats. The binding of syngeneic and alloantibodies to surface antigens expressed on hepatoma cells was unaffected by heat treatment. Thus, heat-treated gamma-irradiated hepatoma cells retain a serologically defined tumour-specific antigen but are unable to elicit immunoprotection. By examining the incorporation of radioactive precursors into DNA, RNA and protein in heat-treated cells, it was determined that above 41 degrees C there was a significant decrease in metabolic activity. It is postulated that for the effective induction of transplantation immunity to tumours, tumour-specific antigens should be present on the surface membranes of a metabolically active cell. This hypothesis accounts for the absence or marked reduction of immunoprotection induced by inviable or glutaraldehyde-treated cells, isolated cell membranes and soluble tumour extracts which retain serologically defined tumour-specific antigens.

Intrapleurally injected cells of an ascitic rat tumour produced intrapleural effusions and solid pleural deposits. BCG, or its methanol extraction residue (MER) injected into the pleural space, suppressed tumour development and prolonged survival. Treatment was effective if given a few days before or after tumour injection. In contrast, active specific immunotherapy by repeated s.c. injection of viable or radiation-attenuated tumour cells in admixture with BCG was unsuccessful, and did not improve the response to intrapleural BCG treatment.

Intrapleural growth of transplanted rat tumours was prevented or retarded by intrapleural administration of double-stranded RNA. A similar suppression of growth was achieved with peitoneal tumours by the intraperitoneal injection of the compound. These studies indicate the possible potential of this form of treatment of thoracic and peritoneal tumours for clinical application in the treatment of mesothelioma.

Growth of transplanted rat tumours was retarded and in some cases completely suppressed when cells were injected subcutaneously in admixture with double stranded RNA (ds-RNA). This response required intimate contact between ds-RNA and tumour cells and systemic treatment with the agent failed to prevent progressive growth of a range of rat tumours. Direct cytotoxic effects of ds-RNA may contribute to tumour suppression since the compound was cytotoxic in vitro for cultured tumour cells. The involvement of host factors is suggested, however, by the in vivo tests showing variations in susceptibility to ds-RNA mediated tumour suppression similar to that previously observed with bacterial adjuvants.

The tumour specific antigens and tumour associated embryonic antigens expressed on chemically induced rat hepatomata and sarcomata have been shown to be different by means of blocking antibody studies. Serum from multiparous female rats contained blocking antibody which protected both tumour and embryo cells from the in vitro cytotoxic effect of lymph node cells from multiparous donors. These sera did not, however, block the cytotoxicity of lymph node cells from tumour immune rats for cells of the immunizing tumour. In addition, the tumour specific rejection antigens and embryonic antigens have been shown to have dissimilar locations in tumour cells.

ICRF 159 and Triton WR 1339 have been examined for their ability to suppress subcutaneous growth and pulmonary metastases from a transplanted rat epithelioma. Neither compound influenced subcutaneous tumour development or reduced the propensity to metastasize when administered in regimens reported to suppress pulmonary, lymph node or intracerebral metastases in other experimental system.

Subcellular fractions from an aminoazo dye induced rat hepatoma (D23) were examined for their ability to evoke rejection responses in syngeneic hosts to transplanted tumour cells and to induce the production of humoral antibody. Membrane fractions isolated by zonal centrifugation and displaying an increased activity of tumour specific antigen (Price and Baldwin, 1974), as well as crude membrane fractions and purified tumour cell ghosts, all elicited tumour specific antibody demonstrable by membrane immunofluorescence staining of viable hepatoma D23 cells. Tumour cell nuclei or soluble cytoplasmic protein were, however, lacking in this capacity. Resistance to tumour cell challenge was not observed in rats treated with any of the hepatoma D23 subcellular fractions administered by various routes either alone or in admixture with bacterial adjuvants. These findings are relevant to current views that tumour immunity may be more optimally achieved by inoculation of intact (viable or attenuated) tumour cells.

Membrane fractions were isolated from homogenates of an aminoazo dye induced rat hepatoma (hepatoma D23) by sucrose density gradient centrifugation in zonal rotors. The membrane fractions retained tumour specific antigenic determinants and exhibited an increased antigenic activity over other subcellular membrane fractions, as defined by their capacity to quantitatively neutralize the membrane immunofluorescence staining of viable hepatoma D23 cells by antibody in tumour immune serum. In contrast, no antigenic activity was found to be associated with purified hepatoma D23 nuclei or nuclear membranes as evaluated by the in vitro antigen assay.The two methods described for the isolation of hepatoma D23 membranes have been developed for the large scale fractionation of tumour homogenates in order that further studies upon the nature and immunogenicity of membrane associated tumour specific antigens may be resolved using defined membrane preparations of increased antigenic activity.