Systemic adoptive transfer of immunity to 13762A rat mammary adenocarcinoma.

Rats cured of metastatic 13762A rat mammary adenocarcinoma by Corynebacterium parvum immunotherapy possess strong tumor-specific rejection immunity. Systemic adoptive transfer of lymphoreticular cells from cured rat donors conferred protective immunity on naive recipients. Fewer oil-induced peritoneal exudate cells than lymph node cells were required to transfer tumor rejection immunity. The adoptive immunity was specific since it strongly inhibited 13762A tumor growth but did not inhibit the growth of the antigenically unrelated R3230AC rat mammary tumor. Rats sensitized to the bacterial immune stimulant used to effect cure were unsuitable donors of PEC capable of transferring tumor rejection immunity. Macrophages or bone marrow-derived lymphocytes from immune donors did not transfer immunity. Treatment of peritoneal exudate with a xenogeneic antiserum specific for rat thymus-derived lymphocytes significantly reduced the efficiency of transfer. We conclude that thymus-derived lymphocytes from rats cured of 13762A tumor were required for the adoptive transfer of tumor-specific rejection immunity.

Regulation of the expression of adoptive tumor rejection immunity by recipient cyclophosphamide-sensitive cells.

Peritoneal exudate T-cells from rats immune to 13762A rat mammary tumor conferred specific tumor rejection immunity on normal recipients. The efficiency of systemic adoptive transfer of tumor rejection immunity with immune peritoneal exudate T-cells was improved by cyclophosphamide (CY) pretreatment of recipients. Optimal potentiation was obtained with a dose of 50 or 100 mg CY per kg body weight given the day before transfer. CY pretreatment of recipients was effective 1 to 3 days prior to transfer. The CY-potentiating effect was lost with longer intervals between CY administration and transfer, indicating recipient recovery. CY pretreatment enabled recipients to reject greater numbers (100 times) of tumor cells and inhibited tumor challenge established before systemic adoptive transfer. The CY-induced potentiation of systemic transfer of tumor immunity was reversed by i.v.-administered normal spleen. We concluded that CY-sensitive host regulatory cells restricted the expression of adoptive tumor rejection immunity. Control of the activity of these regulatory cells allows increased efficacy of effector T-lymphocytes in this system.

Conditions for effective Bacillus Calmette-Guérin immunotherapy of postsurgical metastases of 13762A rat mammary adenocarcinoma.

We evaluated critical variables in Bacillus Calmette-Guérin (BCG) immunotherapy of residual 13762A rat mammary adenocarcinoma. BCG was given intratumorally on Day 7 of tumor growth and followed by primary tumor excisions on Day 20. Untreated animals died on about Day 40 with axillary nodal and pulmonary parenchymal metastases. BCG-treated animals experienced prolonged survival, and some were cured. The highest dose (5.0 X 10(7) colony-forming units) of BCG was more effective than the lowest (0.5 X 10(7) colony-forming units), but 1,500 micrograms Corynebacterium parvum were more effective than even the highest BCG dose. Previous sensitization to BCG did not improve the effects of BCG treatment. BCG treatment was effective when given on Day 7 and sometimes as late as Day 12 or 17, but C. parvum was ineffective if given after Day 7. Repeated injections of BCG or C. parvum were not more effective than single injections were. Rats cured of residual 13762A tumor by BCG treatment were strongly and specifically immune to rechallenge. We concluded that a high dose (5.0 X 10(7) colony-forming units) of BCG given early (7 days) was the most effective presurgical treatment of 13762A metastases. Repeated injections or host presensitization to BCG did not improve the benefits.

Differential induction by interferons of major histocompatibility complex-encoded and non-major histocompatibility complex-encoded antigens in human breast and ovarian carcinoma cell lines.

Treatment of cancer cells with interferons can modulate expression of cell surface antigens, particularly those of the major histocompatibility complex (MHC). To examine the effect of recombinant gamma- and alpha-interferons on expression of non-MHC antigens, murine monoclonal antibodies have been used to quantitate 14 distinct tumor-associated cell surface antigens from five breast cancer cell lines and five ovarian cancer cell lines using a live cell radioimmunoassay. Both Class I and Class II MHC antigens could be augmented or induced with gamma-interferon. Significantly increased expression of MHC antigens was observed in nine of 10 cell lines with induction indices as high as 11-fold. When 17 non-MHC epitopes were measured on 10 cell lines, minimal (1.3-2.7-fold) induction was observed in 10 of the 170 instances evaluated. Expression of only two epitopes, 2G3 and 735B11, was increased on more than one cell line. On six cell lines expression of non-MHC epitopes could not be increased. Consequently, among many different cell surface determinants, interferons produced a highly selective augmentation or induction of MHC antigens, whereas augmentation or induction of other tumor-associated antigens was apparently restricted to a few epitopes.

Use of immunotoxins in combination to inhibit clonogenic growth of human breast carcinoma cells.

Substantial heterogeneity has been observed in the expression of individual antigens within tumor cell populations. Immunotoxins which bind to different cell surface antigens might exert additive or synergistic cytotoxicity when used in combination to eliminate all clonogenic cells within a tumor. Immunotoxins have been prepared by conjugating recombinantly derived toxin A chain to different monoclonal reagents which recognize cell surface determinants of Mr 42,000 (317G5), 55,000 (260F9), and 200,000 (741F8). Each immunotoxin was evaluated for binding, internalization, and cytotoxicity with four breast cancer cell lines. Each of the three immunotoxins bound to the SKBr3 cell line and exerted antitumor activity in a limiting dilution clonogenic assay. Simultaneous treatment with two immunotoxins produced additive antitumor activity with each of the possible combinations. Additive binding could be demonstrated by immunofluorescent techniques, however, with only one of three combinations. With two of the three combinations, subpopulations of tumor cells could be identified which lacked one or the other antigenic determinant but not both. Consequently, log-additive antitumor activity was produced by immunotoxins in combination, and heterogeneity of antigenic targets may have contributed to the combined cytotoxicity in some but not all cases.

Requirements for the internalization of a murine monoclonal antibody directed against the HER-2/neu gene product c-erbB-2.

A murine monoclonal antibody, TA1, is directed against an epitope on the extracellular domain of the HER-2/neu (c-erbB-2) gene product. Requirements for TA1-induced internalization of c-erbB-2 have been studied using the SKBr3 human breast cancer cell line and several rat fibroblast cell lines that express either wild-type or mutant human c-erbB-2. Internalization of TA1 was monitored by assaying protease-resistant uptake of 125I-labeled TA1, by electron microscopy of gold-labeled TA1, and by inhibition of clonogenic growth of cells incubated with TA1 that had been conjugated with blocked ricin. Similar rates of internalization of TA1 were observed in SKBr3 and in rat fibroblasts that expressed human c-erbB-2. The route of endocytosis was the same as that observed with antibodies against other membrane receptors. Anti-c-erbB-2 and anti-transferrin receptor cointernalized through clathrin-coated pits, coated vesicles, endosomes, and multivesicular bodies. Products of mutant c-erbB-2 that lacked a portion of the tyrosine kinase domain or that lacked most of the cytoplasmic domain were endocytosed in the presence of TA1 as promptly as the wild-type c-erbB-2 product. Slightly more rapid internalization of TA1 was observed in rat cells that expressed c-erbB-2 with a single point mutation in the transmembrane domain. Taken together, our data suggest that neither the intracytoplasmic domain nor receptor phosphorylation is required for antibody-mediated endocytosis of c-erbB-2.

Cytotoxic T-lymphocytes derived from patients with breast adenocarcinoma recognize an epitope present on the protein core of a mucin molecule preferentially expressed by malignant cells.

A population of tumor-reactive cytotoxic T-cells can be propagated from tumor-draining lymph nodes of patients with breast adenocarcinoma. These T-cells specifically recognize breast and pancreatic tumor cells in a major histocompatibility complex (MHC)-unrestricted fashion but not other tumors of epithelial origin or the natural killer target K562. The tumor-specific but MHC-unrestricted lytic activity of these cytotoxic T-lymphocytes (CTLs) is mediated through the alpha/beta T-cell receptor. The molecule recognized by these CTLs is ductal epithelial mucin produced by breast and pancreatic adenocarcinomas. The protein core of the mucin consists of multiple tandem repeats of a 20-amino acid sequence. Antibody SM3, directed against a determinant on the mucin protein core preferentially expressed on malignant cells is able to significantly inhibit lysis of tumor cells by the CTL, while other antibodies binding to different core epitopes are not. Normal breast epithelial lines, which also express mucin but not the SM3 epitope, are not lysed by these tumor-reactive CTLs or act as cold target inhibitors of lysis of tumor lines. The data suggest that the highly repetitive nature of the mucin allows cross-linking of the T-cell receptor on mucin-specific T-cells and therefore accounts for the lack of MHC restriction seen in this system. They further suggest that the mucin core epitope recognized on tumor cells is not expressed on normal epithelial cells in a manner that can be recognized by tumor-reactive CTLs. These findings support the role of mucins as important tumor-associated antigens mediating the cellular response to certain human cancers and suggest that epithelial mucin core sequences might form the basis for an effective vaccine to augment the antitumor immune response.

Immunohistochemical expression of CA 125 in endometrial adenocarcinoma: correlation of antigen expression with metastatic potential.

Immunohistochemical localization of CA 125 using murine monoclonal antibody OC 125 was performed on fresh frozen tissue from 44 endometrial adenocarcinomas and 26 benign endometria. Immunohistochemical evaluation incorporated both intensity and distribution of staining (CA 125 HSCORE). Thirty-seven cancers (84%) and 23 benign endometria (88%) expressed immunohistochemically detectable CA 125. Staining was confined to epithelial cells and was present both on the cell membrane and in the cytoplasm. Among the 44 endometrial cancers, CA 125 HSCORE did not correlate with histological grade, depth of myometrial invasion or estrogen/progesterone receptor levels. Following surgical staging, 13 patients (30%) were found to have extrauterine metastatic disease. The median CA 125 HSCORE of patients with metastatic disease (2.25) was significantly higher than that of patients with disease confined to the uterus (0.6) (P less than 0.001). In addition, high CA 125 HSCORE also correlated with the presence of lymph node metastasis (P less than 0.001). The results of this study suggest that high CA 125 expression by endometrial adenocarcinomas is associated with increased metastatic potential.

Tumor necrosis factor alpha as an autocrine and paracrine growth factor for ovarian cancer: monokine induction of tumor cell proliferation and tumor necrosis factor alpha expression.

Ovarian tumor cells produce macrophage colony stimulating factor, a potent chemoattractant for monocytes. Monocytes and macrophages produce tumor necrosis factor alpha (TNF-alpha) and interleukin 1 alpha or interleukin 1 beta (IL-1 beta) that can stimulate ovarian tumor cell growth. The present study has explored whether paracrine stimulation by monocyte derived cytokines might induce autocrine growth stimulation of normal and malignant ovarian epithelial cells. Endogenous expression of TNF-alpha mRNA was detected in ascites ovarian cancer cells isolated directly from patients, but not in established cultures of normal or malignant ovarian epithelial cells. When ascites tumor cells were cultured for 7 days, TNF-alpha expression ceased but could be reinduced by treatment with TNF-alpha or IL-1 beta. Ascites fluid contained concentrations of the cytokines that could mediate these effects. Similarly, treatment of normal or malignant ovarian epithelial cells with purified recombinant IL-1 beta or TNF-alpha induced transcription of TNF-alpha mRNA within 1 h. TNF-alpha protein could be detected by enzyme-linked immunosorbent assay in conditioned medium from IL-1 beta treated ovarian cancer cells. [3H]thymidine incorporation by normal or malignant ovarian epithelial cells was stimulated by a 24-h incubation with IL-1 beta or TNF-alpha. Stimulation of proliferation by IL-1 beta could be partially blocked by an antibody against TNF-alpha or by soluble TNF-alpha-receptor. Thus, TNF-alpha may function as both an autocrine and a paracrine growth factor in ovarian cancer.

Cell surface density of p185(c-erbB-2) determines susceptibility to anti-p185(c-erbB-2)-ricin A chain (RTA) immunotoxin therapy alone and in combination with anti-p170(EGFR)-RTA in ovarian cancer cells.

Approximately 30% of ovarian and breast cancers overexpress p185(c-erbB-2) with as many as 10(6) receptors/cell. Normal cells have as few as 10(4) receptors/cell. We have examined the susceptibility of SKOv3 human ovarian cancer cells to anti-c-erbB2 antibodies and immunotoxins as a function of c-erbB-2 density on the cell surface. A panel of SKOv3 clones that expressed different densities of p185(c-erbB-2) receptor were generated through transfection with the c-erbB-2 gene. A significant correlation was found between p185(c-erbB-2) density and susceptibility to killing by anti-p185(c-erbB-2)-ricin A chain (anti-p185(c-erbB-2)-RTA) immunotoxins. With 10(5) copies/cell of p185(c-erbB-2), <10% of clonogenic ovarian cancer cells could be eliminated, whereas in clones that expressed 10(6) copies/cell of p185(c-erbB-2), 99.9% of clonogenic tumor cells were killed. In cell lines that overexpressed p185(c-erbB-2) and also expressed p170(EGFR), anti-p185(cerbB-2)-RTA and anti-p170(EGFR)-RTA immunotoxins exerted synergistic cytotoxicity. Treatment with the two immunotoxins could eliminate 99.99% of clonogenic cells. Importantly, tumor cells that had survived first treatment with anti-p185(c-erbB2)-RTA alone still retained sensitivity to repeat treatment with the same immunotoxin and also proved susceptible to the synergistic cytotoxicity of anti-p185(cerbB-2)-RTA in combination with anti-p170(EGFR)-RTA. Growth characteristics of the clones expressing various levels of p185(c-erbB-2) were also studied. No correlation was found between p185(c-erbB-2) expression levels and the rate of anchorage-dependent growth, anchorage-independent growth, or in vivo growth in nude mice.

Synergistic interaction between anti-p185HER-2 ricin A chain immunotoxins and radionuclide conjugates for inhibiting growth of ovarian and breast cancer cells that overexpress HER-2.

Radionuclide conjugates or ricin A chain (RTA) immunotoxins that target pl85HER-2 have partially inhibited the growth of human ovarian cancer xenografts in athymic mice but generally have not cured mice bearing human tumor transplants. The present study was undertaken to explore whether a combination of ionizing radiation and an immunotoxin could exert additive or synergistic cytotoxicity in culture and in vivo against cancer cells that overexpress p185HER-2. In cell culture, treatment with 200-2000 cGy external beam irradiation followed by incubation with TA1-anti-pl85mHER2-RTA immunotoxin (TA1-RTA) produced synergistic inhibition of clonogenic growth of ovarian and breast cancer cells that expressed > 10(6) pl85HER-2 receptors/cell. The effect on cell survival correlated with an inhibition of DNA repair. A prior study (F. J. Xu et al, Nucl. Med. Biol., 24: 451-460, 1997) compared the biodistribution of radionuclide conjugates prepared with monoclonal antibodies that bind to different epitopes on the extracellular domain of pl85HER-2 and found optimal tumor uptake with the 520C9 antibody, which did not compete with TA1 for binding to the receptor. In this report, the TA1-RTA immunotoxin and the 131I-labeled 520C9 radionuclide conjugate could each inhibit the growth of clone-9002-18 xenografts in athymic mice but did not yield long-term survivors using maximally tolerated doses of each agent. When TA1-RTA and 131I-labeled 520C9 were used in combination, a greater inhibition of tumor growth was obtained than with either single agent. Similarly, survival with the combined treatment was significantly prolonged (P = 0.004) relative to treatment with immunotoxin or radionuclide conjugate alone. After treatment with an optimal combination of immunotoxin and radionuclide conjugate, 50% of mice survived >300 days, whereas controls succumbed with a median survival of 36 days. These results suggest that combinations of immunotoxins and radionuclide conjugates deserve further evaluation for the treatment of cancers that overexpress pl85HER-2.

Induction of HLA-specific CTL to nonimmunogenic, heat-inactivated lymphocytes by interleukin 2.

Human lymphocytes that have been heat-inactivated (1 hr, 45 degrees C) were used as stimulator cells in a model system to study the requirements of allogeneic T cell activation in vitro. Cytotoxic T lymphocytes were not generated in either primary or secondary mixed lymphocyte cultures after exposure to heated stimulator cells. Successful reconstitution of cytolytic activity in primary cultures was achieved by the addition of rIL-2. Further, cytotoxic T cell lines could be maintained in culture for several weeks by stimulation with heated allogeneic cells and periodic addition of exogenous IL-2. The cytotoxic T cells generated in primary cultures or in the T cell lines were specific for the HLA class I antigens of the stimulating cells. Thus, the combination of heated cells and IL-2 stimulated antigen-specific cytotoxic cells, and not merely lymphokine-activated killers. Although IL-2 production appeared to be a crucial missing component of MLCs with heated lymphocytes, the addition of IL-1, a factor known to act as a second signal for stimulating IL-2 production, did not reconstitute cytolytic activity. These results indicate that (1) heat treatment does not appreciably affect class I structure; (2) HLA class I/T cell receptor interactions are intact, resulting in responsiveness to IL-2 but not IL-1; and (3) heating creates a defect that has a minimal effect on CTL precursor activation but does disrupt a T helper cell/stimulator cell interaction critical for IL-2 production.

The role of peptide growth factors in epithelial ovarian cancer.

We studied the effect of epidermal growth factor, platelet-derived growth factor, fibroblast growth factor, and transforming growth factor-beta on proliferation of four epithelial ovarian cancer cell lines (OVCA 420, OVCA 429, OVCA 432, and OVCA 433). Epidermal growth factor stimulated growth of OVCA 429 cells (P = .0001) and OVCA 433 cells (P = .0002). Platelet-derived growth factor did not stimulate growth of any of the cell lines. Fibroblast growth factor stimulated growth of OVCA 420 cells (P = .003). Transforming growth factor-beta inhibited growth of OVCA 420 cells (P = .0001), OVCA 432 cells (P = .003), and OVCA 433 cells (P = .004). To detect production of known growth factors by the cancer cell lines, we tested the effect of cancer cell-conditioned media on proliferation of cell lines known to respond to growth factors. Only media exposed to OVCA 433 cells were found to contain activity that mimicked one of the known growth factors (transforming growth factor-beta). These results suggest that individual ovarian cancers vary widely in their response to and production of known peptide growth factors. Finally, we found that OVCA 429-conditioned medium significantly inhibited proliferation of mitogen-stimulated lymphocytes (P less than .0001). The characteristics of this immunosuppressive factor were distinct from those of transforming growth factor-beta. Production of this factor by an immortalized cell line provides a unique opportunity to identify an immunosuppressive substance associated with ovarian cancer.

Radioiodinated antibody targeting of the HER-2/neu oncoprotein.

The HER-2/neu oncogene encodes a 185 kDa phosphoglycoprotein that is overexpressed in breast, ovarian and other cancers. Seven monoclonal antibodies reactive with oncoprotein were labeled with 131I. In vitro experiments with SKOv3 9002-18 cells determined binding affinity, internalization and degradation. The biodistribution of these antibodies in comparison to 125I-labeled nonspecific antibody was measured in athymic mice with SKOv3 9002-18 ovarian carcinoma xenografts. Antibody 520C9 exhibited the highest and most specific retention in tumor, peaking at 17.4 +/- 5.6% ID/g at 24 h.

Coexpression of the HER-2 gene product, p185HER-2, and epidermal growth factor receptor, p170EGF-R, on epithelial ovarian cancers and normal tissues.

Monoclonal antibodies (MAbs) and immunoconjugates reactive with different antigens expressed by neoplastic cells can inhibit tumor growth. Use of these agents in combination with one another or with chemotherapy can exert additive or synergistic cytotoxicity against tumor cells. An augmented therapeutic activity with favorable therapeutic index might be attained when coexpression is observed on tumor cells, but not in normal tissues. In this study frozen sections of 19 ovarian cancers (2 stage I, 10 stage III, 2 stage IV, and 5 recurrent), as well as 29 normal tissues, were evaluated by immunohistochemistry using 11 distinct MAbs against HER-2/p185 and 2 antibodies against EGF-R/p170 to assess coexpression of these receptors. HER-2/p185 expression was detected in 5 to 100% of ovarian cancers and 0 to 50% of normal ovarian epithelia, depending on the antibody used. EGF-R/p170 expression was detected in approximately 70% of cancers and 40% of normal ovaries by both antibodies. Coexpression of p185 and p170 was observed in 47-68% of ovarian cancers and 9-18% of normal ovarian epithelial specimens depending upon the combination of antibodies used. Staining of 273 specimens from 29 normal tissues indicated that coexpression of HER-2 and EGF-R is rare. Normal tissues that coexpressed both receptors in > or =50% of the cases included cervix, endometrium, esophagus, skin, and prostate. These data confirm that HER-2 and EGF-R are more frequently expressed in advanced ovarian cancers than in normal ovarian epithelium and a significant fraction of these tumors coexpress both HER-2 and EGF-R.