A rat yolk-sac antigen defined by monoclonal antibodies

The distribution of a rat yolk-sac antigen (Rat YSA-I) as defined by a monoclonal antibody raised against rat yolk-sac carcinoma cells is described. The antigen is present on rat yolk-sac carcinomas, on visceral yolk-sac endoderm and on embryonal endoderm of 9-day-old embryos. It is not present on a variety of rat tumors other than yolk-sac carcinomas and not detectable on pre-implantation embryos, inner cell mass and fetal endoderm. Rat YSA-I differs from alpha-fetoprotein and Forssman antigen and is species-specific. In adult rats the only cells displaying this antigen are the spermatozoa and certain cells of the spermatogenic lineage.     

Rat yolk-sac antigen-2 defined by monoclonal antibodies

The distribution of the rat yolk-sac antigen-2 (Rat YSA-2) as defined by a monoclonal antibody raised against rat yolk-sac carcinoma cells is described. The antigen is present on rat yolk-sac carcinoma, on parietal yolk-sac endoderm and on the epithelium of fetal and adult gut and of the adult proximal kidney tubules. It is not present on a variety of rat tumors other than yolk-sac carcinomas and not detectable on pre-implantation and post-implantation embryos. Rat YSA-2 differs from YSA-I, other stage-specific embryonic antigens, basement membrane antigens, intestinal and tubular antigens.     

Tumour antigens and alloantigens. II. Lack of association of rat hepatoma-D23-specific antigen with beta 2 microglobulin.

It has previously been shown that the tumour-specific antigen of the chemically-induced rat hepatoma D23 has determinants recognised by alloantisera raised against normal syngeneic liver and spleen. However, no reactions were observed with alloantisera raised against syngeneic erythrocytes suggesting that the alloantigeneic determinant responsible for this cross-reactivity is not a major serologically-defined histocompatibility antigen. This concept has been further examined using a defined turkey anti-rat beta 2 microglobulin antiserum. This antiserum failed to block the binding of syngeneic antihepatoma D23 sera to hepatoma D23 target cells as assessed using membrane immunofluorescence and complement-dependant 51Cr release tests. Furthermore, immune precipitates formed from soluble tumour extracts containing hepatoma D23 specific antigen with turkey anti-rat beta 2 microglobulin failed to generate a tumour-specific antibody response in syngeneic rats althouth a cross-reactive antiserum was produced following immunization of allogeneic rats with the immune precipitate.     

Characterization of a membrane-associated glycoprotein (gp 43) on human hepatocellular carcinoma by a monoclonal antibody.

A monoclonal antibody, Hepama-1, produced by immunizing mice with cells of a human hepatocellular carcinoma cell line, has been used to identify and characterize a previously unreported antigen present on the surface of human hepatocellular carcinoma cells. The antigen occurred on the membranes of human hepatoma cell lines and tumor biopsies but was not detectable in tumors of other origin or normal tissues. Binding was determined by enzyme-linked immunoabsorbent assay and immunofluorescence on cell lines and by immunoperoxidase staining of tissue sections. In immunofluorescence studies, Hepama-1 antibodies stained five out of six human hepatoma cell lines, showed only slight binding to breast tumor cell lines, but failed to stain colon tumor or normal cell lines. The antihepatoma antibody exhibited positive immunoperoxidase staining of human liver tumor sections but did not stain tumors of other origin. Hepama-1 bound specifically to a membrane glycoprotein with an approximate molecular weight of 43,000. Western blot and solid phase enzyme-linked immunoabsorbent assay analysis showed that the 43-kD antigen occurred on five of six human hepatoma cell lines and was expressed by every human hepatocellular carcinoma biopsy tested. This cell surface molecule represents a potentially useful target for immunotherapy and localization of human hepatocellular carcinomas.     

Isolation of membrane-associated tumour-specific antigen from an aminoazo-dye-induced rat hepatoma

Tumour-specific antigen on the surface of cells of a transplanted rat hepatoma (D23), originally induced by 4-dimethylaminoazobenzene, was assayed by its capacity to absorb antibody directed specifically against this component and demonstrable by membrane immunofluorescence tests with viable hepatoma cells in suspension. Quantitative assay of antigen in subcellular fractions of tumour, in comparison with that on intact cells, was obtained by this method, the specificity of antibody absorption being confirmed by the lack of effect of intact cells or subcellular fractions of other rat hepatomas and unrelated tumours. Homogenization of hepatoma cells by nitrogen cavitation resulted in the release of membrane fractions retaining hepatoma-D23-specific antigen, the recovery being 13 to 17% of that expressed on intact tumour cells. Antigen was equally distributed between large-particle (sedimented at 10,000 × g) and small-particle (sedimented at 105,000 × g) fractions, indicating that the method of cell rupture produced antigen-associated membrane fragments of varying size. No antigenic activity was detectable in soluble cytoplasmic fractions of the tumour. These studies demonstrate that tumour-specific antigen can be isolated in membrane fragments with the same individual specificity as that demonstrated by immunofluorescence staining of intact hepatoma cells with immune serum or by the induction of tumour immunity.     

Tumour antigens and alloantigens. I. Relation of a rat tumour-specific antigen with normal alloantigens of the host strain.

It has been shown that tumour-specific antigen from a chemically-induced rat hepatoma is capable of binding to immunoadsorbent columns made from the Ig fractions of antisera raised in allogeneic animals against a variety of normal tissues. This reactivity is observed with antisera directed against normal syngeneic liver, spleen and lymph-node cells but it is not detected in normal sera, F, hybrid sera or an antiserum directed against syngeneic erythrocytes. This phenomenon is specific since soluble extracts of normal syngeneic liver, but not of normal altogeneic liver, are capable of inhibiting the binding of hepatoma-specific antigen to an immunoadsorbent column of Ig from an allogeneic anti-normal-liver antiserum. Syngeneic and allogeneic rats were immunized with immune complexes made from xenogeneic sera, alloantisera or syngeneic immune serum and soluble extracts of normal tissue and affinity-chromatography-purified hepatoma-specific antigen. The resulting sera were examined by means of the membrane immunofluorescence test for reactivity against a panel of syngeneic and allogeneic cells. Xeno-antisera directed against normal syngeneic tissue precipitated with purified hepatoma antigen and this material generated tumour-specific antibody following immunization into syngeneic rats. The phenomenon of cross-reactivity of one chemically-induced rat hepatoma-specific antigen with normal syngeneic alloantigens is not a unique phenomenon as preliminary results indicate that two other immunologically distinct hepatomas show similar characteristics.     

Preparation of aminoazo dye induced rat hepatoma membrane fractions retaining tumour specific antigen

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.     

Antitumour antibodies induced by rat embryo cells and spontaneous mammary carcinoma cells treated with 3-methylcholanthrene.

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.     

Specific glycolipid antigen(s) in SV40-transformed cell membranes

A glycolipid extract was prepared from an SV40-transformed hamster cell line (EH-SV) according to the Folch partition procedure. The glycolipids from the aqueous layer were incorporated in liposomal membranes composed of lecithin/sphingomyelin/cholesterol (1:1:2 by weight). This liposomal preparation was inoculated in Syrian hamsters to raise immune sera. The sera were absorbed with trypsinized "normal" hamster cells (EH-N) and tested on various cell lines by the indirect immunofluorescence technique. When used for staining living cells, the immune serum produced a distinct cell-surface fluorescence with SV40-transformed cell lines regardless of the cell origin (e.g., rat or hamster). No reaction was observed with heterologous Py-transformed cell lines, spontaneously transformed cells, or sera from non-immunized hamsters. When used for staining acetone-fixed cells, the antiglycolipid serum reacted specifically with a thermostable antigen in the nuclear envelope and the cytoplasm of SV40-transformed cells. The sera lack interfering SV40 T reactivity. The results indicate the presence of related SV40-specific glycolipid antigen(s) in the plasma membrane, the nuclear membrane and probably other endomembranes of SV40-transformed cells.     

Immunohistochemical characterization of two monoclonal antibodies, P25.48 and P25.91, which define a new prostate-specific antigen

We have generated two new mouse monoclonal antibodies against prostate cancer. P25.48 (IgG3) and P25.91 (IgG2a) were derived from a fusion using fresh prostate cancer cells as the immunogen. Initial screening was performed by indirect immunofluorescence on frozen tissue sections of prostate cancer specimens. The specificity analysis was performed by indirect immunoperoxidase on frozen sections of normal tissues and benign and malignant prostate tissues. P25.48 and P25.91 did not react with any benign prostatic tissues (0 of 17), but reacted with a subset of the malignant prostatic tissues. Five specimens of well-differentiated carcinoma were tested and did not react with P25.48 or with P25.91. Of 16 higher grade specimens, nine reacted with both P25.48 and P25.91, one reacted with P25.48 only, and one reacted with P25.91 only. In most positive cases, the reactivity was heterogenous. P25.48 and P25.91 showed a very restricted pattern of reactivity in nonprostatic tissues. Of 50 normal specimens, only one breast specimen showed some reactivity. None of the nine fetal tissues or of the 15 malignant tissues tested reacted with these monoclonal antibodies. The pattern of reactivity of P25.48 and P25.91 suggests that they recognize the same antigen. This antigen is selectively expressed by malignant prostatic epithelium. In addition, it appears to be distinct from all other previously described prostate cancer-associated antigens.     

A plasma membrane antigen highly associated with oat-cell carcinoma of the lung and undetectable in normal adult tissue.

The plasma membrane antigens of an oat-cell carcinoma of the lung were studied to determine if any antigens absent from normal adult tissue could be identified. Rabbit and monkey antisera were prepared to a highly purified plasma membrane fraction of an oat-cell carcinoma of the lung. The specificities of the antisera were studied by the indirect immunofluorescence method on frozen section substrate. The rabbit antiserum, after absorption with normal lung, liver, colon and peripheral nerve homogenates and extracts, failed to react with any detectable normal adult tissue. The absorbed anti-serum did react with 7 of 7 oat-cell carcinomas of the lung, but failed to react with any of 7 adeno-carcinomas of the lung, 6 epidermoid carcinomas of the lung, 7 colon carcinomas, 8 breast carcinomas, 4 kidney carcinomas, and 1 pancreatic carcinoma. The unabsorbed monkey antiserum failed to react with any detectable normal adult tissue, and had a tumor reactivity pattern nearly identical to that of the absorbed rabbit antiserum. Thus similar results were obtained with antisera from two different species. It is concluded that oat-cell carcinomas of the lung express a plasma membrane antigen(s) undetectable in normal adult tissue and highly associated with this tumor type.     

Tumour rejection in rats sensitized to embryonic tissue. I. Rejection of tumour cells implanted s.c. and detection of cytotoxic lymphoid cells.

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.     

Characterization, isolation and amino terminal sequencing of a rat colon carcinoma-associated antigen

Monoclonal antibodies have been raised against a cell line derived from a dimethylhydrazine-induced rat colon carcinoma. One of these antibodies (MAb E4) has previously been shown to react slightly with normal small intestine and colon, and not with other normal tissues as determined by immunohistochemistry. Using Western immunoblotting we confirmed this tumor specificity. Therefore, the Mr of approx. 66,000 glycosylated antigen (pE4) recognized by MAb E4 appeared to be a potential marker of colon carcinoma. Fifteen human tumor cell lines were tested by flow cytometry for the expression of pE4. This antigen was not detected on these cells. In the rat colon carcinoma cell, pE4 was exclusively found on the cell membrane. pE4 was purified to near homogeneity by immunoaffinity chromatography. The first 20 N-terminal amino acids were identified. Comparison with the NBRF data bank did not reveal a complete homology with known sequenced proteins but similarities were found with the mouse L3T4 precursor, the env polyprotein of human immunodeficiency virus type I, flagellin from Halobacterium halobium and the gp30 from hepatitis B surface antigen. Homology was always found in transmembranous or hydrophobic domains of these proteins. By indirect immunofluorescence analysis of adherent cells and size exclusion chromatography under native conditions, pE4 was found to interact with other molecules and perhaps to be involved in intercellular contact.     

Detection of circulating hepatoma D23 antigen and immune complexes in tumour bearer serum

Serum from rats bearing progressively growing aminoazo dye-induced rat hepatomata has been fractionated by Sephadex G150 gel filtration chromatography and isolated fractions have been examined by indirect membrane immunofluorescence techniques to detect tumour specific antigen and antibody. Hepatoma D23-specific antigenic activity was associated with material (of approximate molecular weight <150,000) isolated in the included volume of the gel at pH 7·3. The fraction excluded from the gel (of approximate molecular weight >150,000) was adjusted to pH 3·0 and further separated by Sephadex G150 gel filtration chromatography at pH 3·0 into gel included and excluded fractions. Hepatoma D23 specific antibody, demonstrable by membrane immunofluorescence staining of hepatoma D23 cells, was found to be eluted in the excluded volume and specific antigenic activity was retarded into the included volume of the gel. These results indicate that hepatoma D23 bearer serum contains free circulating tumour specific antigen in excess, together with specific immune complexes. The presence of these factors in tumour bearer serum is discussed in terms of “blocking” phenomena whereby serum factors may protect tumour cells from sensitized lymphocyte cytotoxic attack.     

A novel monoclonal antibody (7B10) with differential reactivity between human mammary carcinoma and normal breast

A monoclonal antibody (7B10) which displays differential reactivity with breast carcinomas compared to benign lesions or normal breast tissue was selected by fusion of spleen cells from BALB/c mice immunized with the T47D human mammary carcinoma cell line. The antigen, recognized by 7B10 on T47D cells, appeared to be both surface and cytoplasm localized, as demonstrated by indirect immunofluorescence, immunoperoxidase, and electron microscopy studies. This antibody (IgG1) bound with four human breast cancer cell lines (T47D, MCF7, ZR-75-1, and HSL53) which express estrogen receptors. No binding was observed with cancer cell lines of other origin or with normal cells. In vivo, by immunoperoxidase staining of frozen sections of normal breast, the antigen recognized by 7B10 appeared to be located on epithelial cell membranes, whereas in benign and malignant mammary disorders, staining also involved the cytoplasm, as confirmed by electron microscopy on fresh cancer tissue. On formalin-fixed, paraffin-embedded sections, cytoplasmic staining was detected in breast cancer, but no immunostaining was observed with benign lesions or normal breast. In paraffin sections, most normal tissues investigated did not react with 7B10 antibody. However, ducts in the parotid gland, tubules in the kidney and some biliary ducts, and apocrine glands in the skin showed irregular, diffuse weak staining. 7B10 was unreactive with adenocarcinomas of origin other than breast, except for some cells in ovarian clear cell carcinoma. No reactivity was observed with squamous carcinomas, lymphomas, or melanomas. The antigen recognized by 7B10 appeared to be a Mr 32,000 protein, as identified by immunoprecipitation from extracts of T47D after labeling with [35S]methionine. Since the antigen was present only on the membrane of differentiated normal mammary epithelial cells, and was also expressed in the cytoplasm of tumor cells, it may be of interest in immunological studies of mammary epithelial cell differentiation. Moreover, since in formalin-fixed tissues immunostaining is virtually confined to mammary carcinomas, monoclonal antibody 7B10 may have diagnostic applications in breast cancer.     

A Novel Human Monoclonal Antibody Directed to a Tumor-associated Antigen

Twelve human monoclonal antibodies (HuMAb) were established by the fusion of (mouse × human) heteromyeloma cells with B-lymphoblastoid cells derived from the regional lymph nodes of three patients with squamous cell carcinoma of the lung. They were tested for reactivity to two kinds of proteins (purified protein derivatives and bovine serum albumin) by ELISA, Sq-19 (squamous cell carcinoma) culture cells by indirect membrane immunofluorescence tests, and Sq-19 tumor xenograft by immunohistological study. Among them, one HuMAb 904F (IgM, λ) was selected. In indirect membrane immunofluorescence tests, this 904F antibody reacted with various kinds of cell lines, e.g. lung cancer, esophageal cancer, endometrial cancer, and stomach cancer. It did not react with malignant hematopoietic and diploid fibroblast cell lines. Immunohistologically, it stained the tumor nests of squamous cell carcinoma, adenocarcinoma, and large cell carcinoma of the lung. It also stained those of esophagus and colon, but not those of small cell carcinoma of lung, or stomach. On frozen-section specimens of normal tissues from various organs, it showed only limited areas of positive staining. Limited positive findings were observed at a reticular zone of the adrenal gland, at the esophagus as weak staining, and at islets of the pancreas as very weak staining. Western blotting analysis demonstrated that it recognized a 54 kDa trypsin-sensitive molecule which is expressed on the surface of tumor cells. These results suggest the 904F monoclonal antibody detects a novel tumor-associated antigen which is recognized by the human immune system.     

The SV40 S antigen; A carcinoembryonic-type antigen of the hamster?

In addition to hamster tumor cells induced or transformed by SV40, hamster anti-SV40 S (Surface) antiserum also reacted with non-SV40-exposed cell lines spontaneously induced (BHK 21) or transformed by heterologous oncogenic DNA and RNA viruses in the indirect membrane fluorescent antibody test. The antiserum titered equally with both BHK and SV40-transformed cells and the reaction could be absorbed with either of these cell lines, or with hamster embryo. The antiserum also reacted with early hamster embryo cells, although various organ cells from late fetuses, newborns and adults were negative. Mouse cell lines spontaneously induced (BALB/c-3T3) or transformed by SV40 and adenovirus 12 were also non-reactive. The results suggest that the antigen in question is a hamster somatic antigen present during embryonic life and derepressed in lines of actively growing cells.     

Fractionation of plasma membrane-associated tumour-specific antigen from an aminoazo dye-induced rat hepatoma

Tumour-specific antigens from an aminoazo dye-induced rat hepatoma were liberated in a soluble form following limited papain digestion of tumour cell membrane preparations. Fractionation of the soluble extract by DEAE-cellulose chromatography, gradient centrifugation and preparative electrophoresis yielded a major antigenic fraction, with an approximate molecular weight of 55,000, together with a range of other minor antigenic components of larger molecular weight. All preparations retained the capacity to inhibit the reaction of antibody in tumour-immune sera with the plasma membrane of viable hepatoma target cells in the indirect immunofluorescence test. It is considered that the defined antigen fractions isolated provide material suitable for analysing the nature of tumour antigen expression in chemical carcinogenesis and for evaluating the involvement of antigen-antibody interaction in tumour immunity.     

Immunogenicity of rat hepatoma membrane fractions

The principal expression of immunity elicited in syngeneic rats immunized with rat hepatoma membrane fractions was the development of a tumour specific antibody response. This antibody was demonstrable by membrane immunofluorescence staining of viable hepatoma cells in suspension and the sera exhibited complement dependent cytotoxicity for cultured hepatoma cells. In the absence of complement, however, membrane immune sera were highly “blocking”, protecting plated hepatoma cells from attack by sensitized lymph node cells. The cell mediated immune response elicited by hepatoma membrane immunization was weak, as evaluated by the colony inhibitory activity of lymph node cells for hepatoma cells in vitro or the adoptive transfer of immunity with peritoneal exudate cells. Correlated with this overall pattern of immune response, membrane immunization did not elicit tumour rejection reactions. These findings are relevant to current views that humoral factors operate antagonistically to limit cell mediated immunity to tumours. A further relevant feature was the observation that membrane immunization, eliciting a prominent humoral immune reaction, conditioned the recipients so that they subsequently failed to elicit a tumour rejection immunity on treatment with irradiated tumour cells.     

Tumor-associated membrane sialoglycoprotein on human small cell lung carcinoma identified by the IgG2a monoclonal antibody SWA20

A mouse IgG2a monoclonal antibody, SWA20, defining a tumor-associated cell surface antigen on small cell carcinoma of the lung (SCC) was generated. The reactivity of the antibody with cell lines was examined by indirect immunofluorescence staining and solid phase radioimmunoassay and the reactivity with tissues by immunoperoxidase staining. The antibody reacts with a proportion of small cell carcinoma cell lines (4 of 8) and tissues (7 of 12), but not with other pulmonary or extrapulmonary cell lines (0 of 30) or tumor tissues (0 of 78). The antibody was unreactive with primary cultures of normal bronchial epithelial cells, RBC, and WBC. Immunoperoxidase staining of normal tissues showed rare antigen-positive cells in suprabasal layers of bronchial epithelium and less than 10% of positive cells in colon epithelium. Immunoblots of SCC extracts demonstrated antibody reactivity with a doublet band at Mr 40,000, a broader band at Mr 100,000, and a band at Mr 180,000. The antigen was not present in crude lipid extracts of SCC cells. Solid phase radioimmunoassays and immunoblots showed binding competition with the lectin Triticum vulgaris, sensitivity of the antigen to neuraminidase, and a partial sensitivity to treatment with periodate. The antigen was coexpressed on SCC cell lines with the antigen sGP90-135 defined first by antibody LAM8 (R. Waibel, C. J. O'Hara, and R. A. Stahel. Cancer Res., 47:3766-3770, 1987) but differed from it by lack of reactivity with Lea-positive saliva and partial resistance to periodate treatment. There was no binding competition between radiolabeled antibodies SWA20 and LAM8 to SCC target cells. The IgG2a antibody SWA20 identifies a previously undescribed tumor-associated surface membrane antigen, sGP100, expressed selectively on a proportion of SCC.