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John W Greiner, Y Maurice Morillon II, Jeffrey Schlom Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USACorrespondence: Jeffrey SchlomLaboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, 10 Center Drive, Room 8B09, Bethesda, MD, 20892, USATel +1 240-858-3463Fax +1 240-541-4558Email schlomj@mail.nih.govAbstract: NHS-IL12 is a novel immunocytokine designed for delivery of IL-12 to the tumor microenvironment (TME). NHS-IL12 consists of two molecules of IL-12 fused to a human IgG1 (NHS76) recognizing DNA/histone complexes, which are often exposed in the necrotic portions of tumors. Preclinical studies demonstrated the tumor-targeting ability and longer plasma half-life for NHS-IL12 when compared with recombinant IL-12 (rIL-12). NHS-IL12 outperformed rIL-12 in enhancing the proliferation and activation of immune as well as antigen-presenting cells, resulting in a more robust primary immune response. NHS-IL12 also reduced the number and function of suppressive myeloid cells (myeloid derived suppressor cells/macrophages) within the TME. In a murine bladder tumor model, NHS-IL12 administration led to a coordinated increase in host immunity with a reduction of immunosuppressive myeloid cells in the TME resulting in substantial reduction in tumor growth. Several preclinical studies have demonstrated increased overall anti-tumor efficacy when NHS-IL12 was combined with either immune-based therapeutics or chemotherapeutic approaches.Keywords: interleukin-12, NHS-IL12, immunotherapy, cancer, immuno-oncology, review

The prevalence of obesity, an established epidemiological risk factor for many cancers, has risen steadily for the past several decades in the US and many other countries. Particularly alarming are the increasing rates of obesity among children, portending continuing increases in the rates of obesity and obesity-related cancers for many years to come. Modulation of energy balance, via increased physical activity, has been shown in numerous comprehensive epidemiological reviews to reduce cancer risk. Unfortunately, the effects and mechanistic targets of physical activity interventions on the carcinogenesis process have not been thoroughly characterized. Studies to date suggest that exercise can exert its cancer-preventive effects at many stages during the process of carcinogenesis, including both tumour initiation and progression. As discussed in this review, exercise may be altering tumour initiation events by modifying carcinogen activation, specifically by enhancing the cytochrome P450 system and by enhancing selective enzymes in the carcinogen detoxification pathway, including, but not limited to, glutathione-S-transferases. Furthermore, exercise may reduce oxidative damage by increasing a variety of anti-oxidant enzymes, enhancing DNA repair systems and improving intracellular protein repair systems. In addition to altering processes related to tumour initiation, exercise may also exert a cancer-preventive effect by dampening the processes involved in the promotion and progression stages of carcinogenesis, including scavenging reactive oxygen species (ROS); altering cell proliferation, apoptosis and differentiation; decreasing inflammation; enhancing immune function; and suppressing angiogenesis. A paucity of data exists as to whether exercise may be working as an anti-promotion strategy via altering ROS in initiated or preneoplastic models; therefore, no conclusions can be made about this possible mechanism. The studies directly examining cell proliferation and apoptosis have shown that exercise can enhance both processes, which is difficult to interpret in the context of carcinogenesis. Studies examining the relationship between exercise and chronic inflammation suggest that exercise may reduce pro-inflammatory mediators and reduce the state of low-grade, chronic inflammation. Additionally, exercise has been shown to enhance components of the innate immune response (i.e. macrophage and natural killer cell function). Finally, only a limited number of studies have explored the relationship between exercise and angiogenesis; therefore, no conclusions can be made currently about the role of exercise in the angiogenesis process as it relates to tumour progression. In summary, exercise can alter biological processes that contribute to both anti-initiation and anti-progression events in the carcinogenesis process. However, more sophisticated, detailed studies are needed to examine each of the potential mechanisms contributing to an exercise-induced decrease in carcinogenesis in order to determine the minimum dose, duration and frequency of exercise needed to yield significant cancer-preventive effects, and whether exercise can be used prescriptively to reverse the obesity-induced physiological changes that increase cancer risk. [ABSTRACT FROM AUTHOR]

Investigation of the efficacy and mechanisms of human immuno-oncology agents has been hampered due to species-specific differences when utilizing preclinical mouse models. Peripheral blood mononuclear cell (PBMC) humanized mice provide a platform for investigating the modulation of the human immune-mediated antitumor response while circumventing the limitations of syngeneic model systems. Use of humanized mice has been stymied by model-specific limitations, some of which include the development of graft versus host disease, technical difficulty and cost associated with each humanized animal, and insufficient engraftment of some human immune subsets. Recent advances have addressed many of these limitations from which have emerged humanized models that are more clinically relevant. This review characterizes the expanded usage, advantages and limitations of humanized mice and provides insights into the development of the next generation of murine humanized models to further inform clinical applications of cancer immunotherapeutic agents., (Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

The lack of serial biopsies in patients with a range of carcinomas has been one obstacle in our understanding of the mechanism of action of immuno-oncology agents as well as the elucidation of mechanisms of resistance to these novel therapeutics. While much information can be obtained from studies conducted with syngeneic mouse models, these models have limitations, including that both tumor and immune cells being targeted are murine and that many of the immuno-oncology agents being evaluated are human proteins, and thus multiple administrations are hampered by host xenogeneic responses. Some of these limitations are being overcome by the use of humanized mouse models where human peripheral blood mononuclear cells (PBMC) are engrafted into immunosuppressed mouse strains. Bintrafusp alfa (M7824) is an innovative first-in-class bifunctional fusion protein composed of the extracellular domain of the TGF-βRII to function as a TGF-β "trap" fused to a human IgG1 antibody blocking PD-L1. A phase I clinical trial of bintrafusp alfa showed promising anti-tumor efficacy in heavily pretreated advanced solid tumors, and multiple clinical studies are currently ongoing. There is still much to learn regarding the mechanism of action of bintrafusp alfa, including its effects on both human immune cells in the periphery and in the tumor microenvironment (TME), and any temporal effects upon multiple administrations. By using the NSG-β2m -/- mouse strain humanized with PBMC, we demonstrate here for the first time: (a) the effects of bintrafusp alfa administration on human immune cells in the periphery vs. the TME using three different human xenograft models; (b) temporal effects upon multiple administrations of bintrafusp alfa; (c) phenotypic changes induced in the TME, and (d) variations observed in the use of multiple different PBMC donors. Also discussed are the similarities and differences in the data thus far obtained employing murine syngeneic models, from clinical trials, and in the use of this humanized mouse model. The results described here may guide the future use of this agent or similar immunotherapy agents as monotherapies or in combination therapy studies., (Copyright © 2020 Morillon, Smalley Rumfield, Pellom, Sabzevari, Roller, Horn, Jochems, Palena, Greiner and Schlom.)

Background: While significant strides in the treatment of metastatic bladder cancer have been made with immune checkpoint inhibitors, the treatment of carcinoma in situ and non-muscle invasive, non-metastatic (superficial) human urothelial carcinoma, also termed non-muscle invasive bladder cancer (NMIBC), remains intractable with bacillus Calmette-Guerin (BCG) employed as the standard of care. In this study, an immunocytokine, NHS-muIL12, which consists of two molecules of murine IL-12 fused to NHS76, a tumor necrosis-targeting human IgG1, was examined as an immunotherapeutic in an orthotopic MB49 luc bladder tumor model., Methods: The antitumor activity of systemic administration of NHS-muIL12 was investigated on MB49 luc tumors, an aggressive, bioluminescent orthotopic bladder cancer model. Temporal studies were carried out on MB49 luc bladder tumors harvested during various time points during NHS-muIL12 treatment and cellular changes associated with the reduction in tumor burden following NHS-muIL12 were determined by flow cytometry. Effects of those changes on the proliferation/activation of lymphoid cells were also determined., Results: Studies revealed a significant reduction in MB49 luc bladder tumor burden occurring between days 3 and 6 after the third and final systemic administration of NHS-muIL12. Temporal analyses of the MB49 luc bladder tumor microenvironment (TME) initially revealed a large accumulation of myeloid-derived suppressor cells (MDSCs) and macrophages that elicited potent immunosuppression. Immunosuppression was characterized by the inability of CD4 + and CD8 + T cells to respond to broad-based immune stimulants. NHS-muIL12 administration resulted in temporal-dependent reductions in the number of MDSCs, macrophages and tumor-associated TGF-β, which culminated in a re-ignition of CD4 + and CD8 + T cells to elicit potent antitumor responses against MB49 luc bladder tumors., Conclusions: These findings provide strong evidence that the systemic administration of an immunocytokine consisting of a tumor-targeting Ig through recognition of DNA and DNA-histone complexes coupled to muIL-12 can effectively target the bladder TME; this significantly reduces the myeloid cellular compartment and reverts an immunosuppressive to an immunopermissive TME, ultimately resulting in antitumor effects. These studies provide further rationale for the employment of NHS-IL12 as an immunomodulator and clinical immunotherapeutic for NMIBC.

Immunotherapy was significantly enhanced in a murine tumor model by combining a vaccine with a fusion protein designed to target the glucocorticoid-induced tumor necrosis factor (TNF) receptor related gene (GITR) on the surface of T cells. The recombinant poxvirus-based vaccine platform included Modified Vaccinia virus Ankara (rMVA) and fowlpox (rF) vectors as the driver immunogens both engineered to express the human carcinoembryonic antigen (CEA) and three murine costimulatory molecules B7.1, ICAM-1, LFA-3 (designated TRICOM). In previous studies, mice expressing human CEA as a transgene (CEA.Tg mice) vaccinated with rMVA/rF-CEA-TRICOM overcame CEA immune tolerance by inducing anti-CEA‒specific immunity and regression of CEA-expressing tumors. The murine GITR ligand fusion protein (mGITRL-FP) consisted of a mouse IgG2a Fc region, a yeast-derived coiled GCN4 pII and the extracellular GITR-binding domain of murine GITR ligand. The design maximized valency and the potential to agonize the GITR receptor. Combined treatment of the vaccine and mGITRL-FP mediated a more robust tumor regression, leading to sustained improvement in overall survival. The enhanced immunotherapeutic effect was linked to the generation of a strong CD8 + T cell antitumor immune response. A treatment schedule with mGITRL-FP administered prior to the priming rMVA-CEA-TRICOM vaccination was of paramount importance. The mechanism of action for the enhanced antitumor effects resided in the depletion of immune cells, particularly FoxP3 + regulatory T cells, that express high GITR levels following activation. The results provide evidence that targeting GITR with mGITRL-FP in concert with a cancer vaccine represents a potential novel approach to more effective immunotherapy., Competing Interests: CONFLICTS OF INTEREST NCI authors do not have any potential conflicts of interest to disclose. Scott A. Hammond and Nicholas M. Durham are employees of MedImmune.

The combined therapeutic potential of an immunocytokine designed to deliver IL-12 to the necrotic regions of solid tumors with an anti-PD-L1 antibody that disrupts the immunosuppressive PD-1/PD-L1 axis yielded a combinatorial benefit in multiple murine tumor models. The murine version of the immunocytokine, NHS-muIL12, consists of an antibody (NHS76) recognizing DNA/DNA-histone complexes, fused with two molecules of murine IL-12 (NHS-muIL12). By its recognition of exposed DNA, NHS-muIL12 targets IL-12 to the necrotic portions of tumors; it has a longer plasma half-life and better antitumor efficacy against murine tumors than recombinant murine IL-12. It is shown here that NHS-muIL12, in an IFN-γ‒dependent mechanism, upregulates mPD-L1 expression on mouse tumors, which could be construed as an immunosuppressive action. Yet concurrent therapy with NHS-muIL12 and an anti-PD-L1 antibody resulted in additive/synergistic antitumor effects in PD-L1‒expressing subcutaneously transplanted tumors (MC38, MB49) and in an intravesical bladder tumor model (MB49). Antitumor efficacy correlated with (a) with a higher frequency of tumor antigen-specific splenic CD8+ T cells and (b) enhanced T cell activation over a wide range of NHS-muIL12 concentrations. These findings suggest that combining NHS-muIL12 and an anti-PD-L1 antibody enhances T cell activation and T cell effector functions within the tumor microenvironment, significantly improving overall tumor regression. These results should provide the rationale to examine the combination of these agents in clinical studies.

Natural killer (NK) cells are known to play a role in mediating innate immunity, in enhancing adaptive immune responses, and have been implicated in mediating anti-tumor responses via antibody-dependent cell-mediated cytotoxicity (ADCC) by reactivity of CD16 with the Fc region of human IgG1 antibodies. The NK-92 cell line, derived from a lymphoma patient, has previously been well characterized and adoptive transfer of irradiated NK-92 cells has demonstrated safety and shown preliminary evidence of clinical benefit in cancer patients. The NK-92 cell line, devoid of CD16, has now been engineered to express the high affinity (ha) CD16 V158 FcγRIIIa receptor, as well as engineered to express IL-2; IL-2 has been shown to replenish the granular stock of NK cells, leading to enhanced perforin- and granzyme-mediated lysis of tumor cells. The studies reported here show high levels of granzyme in haNK cells, and demonstrate the effects of irradiation of haNK cells on multiple phenotypic markers, viability, IL-2 production, and lysis of a spectrum of human tumor cells. Studies also compare endogenous irradiated haNK lysis of tumor cells with that of irradiated haNK-mediated ADCC using cetuximab, trastuzumab and pertuzumab monoclonal antibodies. These studies thus provide the rationale for the potential use of irradiated haNK cells in adoptive transfer studies for a range of human tumor types. Moreover, since only approximately 10% of humans are homozygous for the high affinity V CD16 allele, these studies also provide the rationale for the use of irradiated haNK cells in combination with IgG1 anti-tumor monoclonal antibodies.

The transcription factor brachyury is a major driver of epithelial to mesenchymal transition in human carcinoma cells. It is overexpressed in several human tumor types versus normal adult tissues, except for testes and thyroid. Overexpression is associated with drug resistance and poor prognosis. Previous studies identified a brachyury HLA-A2 cytotoxic T-lymphocyte epitope. The studies reported here describe an enhancer epitope of brachyury. Compared to the native epitope, the agonist epitope: (a) has enhanced binding to MHC class I, (b) increased the IFN-γ production from brachyury-specific T cells, (c) generated brachyury-specific T cells with greater levels of perforin and increased proliferation, (d) generated T cells more proficient at lysing human carcinoma cells endogenously expressing the native epitope, and (e) achieved greater brachyury-specific T-cell responses in vivo in HLA-A2 transgenic mice. These studies also report the generation of a heat-killed recombinant Saccharomyces cerevisiae (yeast) vector expressing the full-length brachyury gene encoding the agonist epitope. Compared to yeast-brachyury (native) devoid of the agonist epitope, the yeast-brachyury (agonist) enhanced the activation of brachyury-specific T cells, which efficiently lysed human carcinoma cells. In addition to providing the rationale for the recombinant yeast-brachyury (agonist) as a potential vaccine in cancer therapy, these studies also provide the rationale for the use of the agonist in (a) dendritic cell (DC) vaccines, (b) adjuvant or liposomal vaccines, (c) recombinant viral and/or bacterial vaccines, (d) protein/polypeptide vaccines, (e) activation of T cells ex vivo in adoptive therapy protocols, and (f) generation of genetically engineered targeted T cells.

Targeted delivery of IL-12 might turn this cytokine into a safer, more effective cancer therapeutic. Here we describe a novel immunocytokine, NHS-IL12, consisting of two molecules of IL-12 fused to a tumor necrosis-targeting human IgG1 (NHS76). The addition of the human IgG1 moiety resulted in a longer plasma half-life of NHS-IL12 than recombinant IL-12, and a selective targeting to murine tumors in vivo. Data from both in vitro assays using human PBMCs and in vivo primate studies showed that IFN-gamma production by immune cells is attenuated following treatment with the immunocytokine, suggesting an improved toxicity profile than seen with recombinant IL-12 alone. NHS-IL12 was superior to recombinant IL-12 when evaluated as an anti-tumor agent in three murine tumor models. Mechanistic studies utilizing immune cell subset-depleting antibodies, flow cytometric methods, and in vitro cytotoxicity and ELISA assays all indicated that the anti-tumor effects of NHS-IL12 were primarily CD8+ T cell-dependent and likely IL-12-mediated. Combining NHS-IL12 treatment with a cancer vaccine, radiation, or chemotherapy resulted in greater anti-tumor effects than each individual therapy alone. These preclinical findings provide a rationale for the clinical testing of this immunocytokine, both as a single agent and in combination with vaccines, radiation and chemotherapy.

Vaccines based on recombinant proteins require adjuvant systems in order to generate Th1-type immune responses. We have developed a vaccine adjuvant system using a viscous chitosan solution and interleukin (IL)-12, a Th1-inducing cytokine. The chitosan solution is designed to create a depot of antigen and IL-12 at a subcutaneous injection site. We measured the in vivo immune response of a vaccine containing 0.25, 1, or 4 μg murine IL-12 and 75 μg ovalbumin (OVA), formulated in a 1.5% chitosan glutamate solution. The chitosan/IL-12/OVA vaccine, in comparison to chitosan/OVA, IL-12/OVA, or OVA alone, elicited greater antigen-specific CD4(+) and CD8(+) T-cell responses, as determined by CD4(+) splenocyte proliferation, Th1 cytokine release, CD8(+) T-cell interferon-γ release, and MHC class I peptide pentamer staining. The combination of chitosan and IL-12 also enhanced IgG2a and IgG2b antibody responses to OVA. Co-formulation of chitosan and IL-12 thus promoted the generation of a Th1 immune response to a model protein vaccine., (Published by Elsevier Ltd.)

(51)Cr-prelabelled colon cancer cells (simulating 'circulating tumor cells', CTCs) were added to human peripheral blood and exposed to staurosporine (ST) to increase carcinoembryonic antigen (CEA) expression. CTCs were captured with immunomagnetic beads coated with Ber-EP4 monoclonal antibody, recognizing the common epithelial antigen present in the majority of cancer cells of epithelial origin, with capture efficiency of more than 80%. Moreover, ST treatment increased CEA expression without compromising Ber-EP4 capture efficiency. In a pilot clinical study on 37 patients, CTCs were captured using Ber-EP4 beads, and recognized by RT-PCR set for CEA or cytokeratin-19 (CK) mRNA detection. The results showed that: (a) the percentage of CEA-positive CTCs (CTC(CEA), 54.1%) was lower than that of CK-positive CTCs (CTC(CK), 70.3%); (b) in vitro ST treatment converted a significant number of CTC(CEA)-negative into CTC(CEA)-positive cases. Therefore, immunomagnetic capture combined with exposure to ST provides a feasible and sensitive technique for the detection of functionally-active CTCs responsive to ST-mediated CEA up-regulation.

Intravesical BCG has been used successfully to treat superficial bladder cancer for three decades. However, 20% to 30% of patients will fail initial BCG therapy and 30% to 50% of patients will develop recurrent tumors within 5 years. Alternative or complementary strategies for the management of superficial bladder cancer are needed. Interleukin-12 (IL-12) is a potent T(H)1 cytokine with robust antitumor activity and the ability to potentiate immunologic memory. Unfortunately, intravesical IL-12 did not show antitumor efficacy in a recent clinical study of patients with recurrent superficial bladder cancer. We hypothesized that coformulation of IL-12 with chitosan, a biocompatible, mucoadhesive polysaccharide, could improve intravesical IL-12 delivery and provide an effective and durable alternative for the treatment of superficial bladder cancer. In antitumor studies, 88% to 100% of mice bearing orthotopic bladder tumors were cured after four intravesical treatments with chitosan/IL-12. In contrast, only 38% to 60% of mice treated with IL-12 alone and 0% treated with BCG were cured. Antitumor responses following chitosan/IL-12 treatments were durable and provided complete protection from intravesical tumor rechallenge. Urinary cytokine analysis showed that chitosan/IL-12 induced multiple T(H)1 cytokines at levels significantly higher than either IL-12 alone or BCG. Immunohistochemistry revealed moderate to intense tumor infiltration by T cells and macrophages following chitosan/IL-12 treatments. Bladder submucosa from cured mice contained residual populations of immune cells that returned to baseline levels after several months. Intravesical chitosan/IL-12 is a well-tolerated, effective immunotherapy that deserves further consideration for testing in humans for the management of superficial bladder cancer.

Purpose: IFN-alpha is a pleiotropic cytokine possessing immunomodulatory properties that may improve the efficacy of therapeutic cancer vaccines. The aim of this study was to evaluate the effectiveness and compatibility of combining recombinant IFN-alpha with poxvirus vaccines targeting the human carcinoembryonic antigen (CEA) in murine models of colorectal and pancreatic adenocarcinomas, where CEA is a self-antigen., Experimental Design: The phenotypic and functional effects of IFN-alpha were evaluated in the draining inguinal lymph nodes of tumor-free mice. We studied the effect of the site of IFN-alpha administration (local versus distal) on antigen-specific immune responses to poxvirus vaccination. Mechanistic studies were conducted to assess the efficacy of IFN-alpha and CEA-directed poxvirus vaccines in tumor-bearing CEA transgenic mice., Results: We identified a dose and schedule of IFN-alpha that induced a locoregional expansion of the draining inguinal lymph nodes and improved cellular cytotoxicity (natural killer and CD8(+)) and antigen presentation. Suppression of the vaccinia virus was avoided by administering IFN-alpha distal to the site of vaccination. The combination of IFN-alpha and vaccine inhibited tumor growth, improved survival, and elicited CEA-specific CTL responses in mice with CEA(+) adenocarcinomas. In mice with pancreatic tumors, IFN-alpha slowed tumor growth, induced CTL activity, and increased CD8(+) tumor-infiltrating lymphocytes., Conclusions: These data suggest that IFN-alpha can be used as a biological response modifier with antigen-directed poxvirus vaccines to yield significant therapeutic antitumor immune responses. This study provides the rationale and mechanistic insights to support a clinical trial of this immunotherapeutic strategy in patients with CEA-expressing carcinomas.

Purpose: Saccharomyces cerevisiae, a nonpathogenic yeast, has been used previously as a vehicle to elicit immune responses to foreign antigens, and tumor-associated antigens, and has been shown to reduce tumor burden in mice. Studies were designed to determine if vaccination of human carcinoembryonic antigen (CEA)-transgenic (CEA-Tg) mice (where CEA is a self-antigen) with a recombinant S. cerevisiae construct expressing human CEA (yeast-CEA) elicits CEA-specific T-cell responses and antitumor activity., Experimental Design: CEA-Tg mice were vaccinated with yeast-CEA, and CD4(+) and CD8(+) T-cell responses were assessed after one and multiple administrations or vaccinations at multiple sites per administration. Antitumor activity was determined by tumor growth and overall survival in both pulmonary metastasis and s.c. pancreatic tumor models., Results: These studies demonstrate that recombinant yeast can break tolerance and that (a) yeast-CEA constructs elicit both CEA-specific CD4(+) and CD8(+) T-cell responses; (b) repeated yeast-CEA administration causes increased antigen-specific T-cell responses after each vaccination; (c) vaccination with yeast-CEA at multiple sites induces a greater T-cell response than the same dose given at a single site; and (d) tumor-bearing mice vaccinated with yeast-CEA show a reduction in tumor burden and increased overall survival compared to mock-treated or control yeast-vaccinated mice in both pulmonary metastasis and s.c. pancreatic tumor models., Conclusions: Vaccination with a heat-killed recombinant yeast expressing the tumor-associated antigen CEA induces CEA-specific immune responses, reduces tumor burden, and extends overall survival in CEA-Tg mice. These studies thus form the rationale for the incorporation of recombinant yeast-CEA and other recombinant yeast constructs in cancer immunotherapy protocols.

The prevalence of obesity, an established risk factor for several chronic diseases, including cancer, has risen dramatically over the past 4 decades. Dietary change and/or increased physical activity are the most commonly recommended lifestyle-based strategies for preventing or reversing obesity. One of several physiological systems that may be enhanced by dietary change and exercise is the immune system. In this study, we examined the effects of energy restriction (ER; 30% reduction relative to control energy intake) and/or exercise (EX; voluntary wheel running) on systemic and mucosal immune function. Female C57BL/6 mice were randomized into 4 treatment conditions: 1) controls consumed ad libitum (AL); 2) AL with access to running wheels (AL + EX); 3) 30% ER; and 4) 30% ER with access to running wheels (ER + EX). Both ER and EX reduced spleen weight and the number of splenic T and B lymphocytes (P < 0.05). ER enhanced natural killer (NK) cell function, but reduced concanavalin A (Con A)-induced T-cell proliferation (P < 0.05). In contrast, EX enhanced Con A-induced proliferation and cytokine production from Peyer's patch cells (P < 0.05). These data suggest that ER and EX enhance some, but not all, components of the immune system and are likely working via different biological mechanisms to regulate NK and T-cell function.

The present study was designed to determine whether: (a) chronic administration of dietary celecoxib (Celebrex), a potent nonsteroidal anti-inflammatory drug, which targets the cyclooxygenase-2 (COX-2) enzyme, negatively impacts host immunity; and (b) celecoxib can be coupled with a poxvirus-based vaccine to impact tumor burden in a murine tumor model of spontaneous adenomatous polyposis coli. Naive mice fed the celecoxib-supplemented diets developed eosinophilia with lowered plasma prostaglandin E(2) levels and reduced COX-2 mRNA expression levels in their splenic T cells. Responses of splenic T, B, and natural killer cells to broad-based and antigen-specific stimuli were, for the most part, unchanged in those mice as well as COX-2 knockout mice; exceptions included: (a) reduced IFN-gamma production by concanavalin A- or antigen-stimulated T cells; and (b) heightened lipopolysaccharide response of naive B cells from mice fed a diet supplemented with 1000 ppm of celecoxib. When transgenic mice that express the human carcinoembryonic antigen (CEA) gene (CEA transgenic) were bred with mice bearing a mutation in the Apc(Delta850) gene (multiple intestinal neoplasia mice), the progeny (CEA transgenic/multiple intestinal neoplasia) spontaneously develop multiple intestinal neoplasms that overexpress CEA and COX-2. Beginning at 30 days of age, the administration of a diversified prime/boost recombinant CEA-poxvirus-based vaccine regimen or celecoxib (1000 ppm)-supplemented diet reduced the number of intestinal neoplasms by 54% and 65%, respectively. Combining the CEA-based vaccine with the celecoxib-supplemented diet reduced tumor burden by 95% and significantly improved overall long-term survival. Both tumor reduction and improved overall survival were achieved without any evidence of autoimmunity directed at CEA-expressing or other normal tissues. Celecoxib is prescribed for the treatment of familial adenomatous polyposis in humans, and the CEA-based vaccines have been well tolerated and capable of eliciting anti-CEA host immune responses in early clinical studies. The results suggest that the administration of a recombinant poxvirus-based vaccine is compatible with celecoxib, and this combined chemoimmuno-based approach might lead to an additive therapeutic antitumor benefit not only in patients diagnosed with familial adenomatous polyposis but, perhaps, in other preventive settings in which COX-2 overexpression is associated with progression from premalignancy to neoplasia.

Human tumor-associated antigens (TAAs) are weak immunogens. One strategy for increasing the immunogenicity of TAAs is to generate altered peptide ligands. In the studies reported here, microarray technology has been used to compare gene expression profiles of a human T cell line that was derived from the peripheral blood of a cancer patient vaccinated with a carcinoembryonic antigen (CEA)-based vaccine. We compared the gene expression profiles of this CEA-specific CD8 T cell line when (a) stimulated with the native peptide used to derive this line vs. no peptide, and (b) stimulated with its TCR enhancer agonist epitope vs. no peptide. The results demonstrate that the effect on the T cell line, when stimulated with the agonist peptide, is not an enhanced quantitative expression of the same genes or gene sets induced by the native peptide, but is rather a nearly reciprocal upregulation of different gene sets. The gene for the chemokine lymphotactin, which was overexpressed only in T cells stimulated with the agonist peptide, stood out above all others. This finding was extended using other T cell lines, and another set of agonist and native peptides from another TAA. ELISPOT and ELISA assays for lymphotactin confirmed and extended these findings. These studies suggest a potential role for lymphotactin in the T-cell activation processes and subsequent anti-tumor events.

By virtue of its tissue-specific expression, carcinoembryonic antigen (CEA) is an important self, tumor-associated antigen, which is expressed by different human adenocarcinomas and also serves as a target for active-specific immunotherapy. Similar to humans, CEA expression in mice transgenic for the human CEA gene (CEA.Tg) occurs predominantly along the gastrointestinal tract. CEA.Tg mice were crossed with mice bearing a mutation in the Apc gene (MIN mice), and the CEA.Tg/MIN progeny developed multiple intestinal neoplasms, which overexpress CEA to levels that are reminiscent of those reported for tubulovillous intestinal adenomas from patients. CEA.Tg/MIN mice were vaccinated with an aggressive diversified prime/boost vaccine regimen: (a) a primary vaccine consisting of recombinant vaccinia virus-expressing CEA and a triad of costimulatory molecules (TRICOM): B7.1, ICAM-1, and LFA-3 (rV-CEA-TRICOM); and (b) a booster vaccine using CEA-TRICOM in a recombinant avipox (fowlpox) virus (rF-CEA-TRICOM). Granulocyte/macrophage colony-stimulating factor was administered as a biological adjuvant with all vaccinations, either as a recombinant protein (with rV-CEA-TRICOM) or as a recombinant avipox virus (with rF-CEA-TRICOM). That vaccine regimen generated strong CEA-specific host immune responses in CEA.Tg/MIN mice, which resulted in (a) a delayed onset of adult anemia and weight loss, (b) a significant reduction in the number of intestinal tumors, and (c) improved overall survival. No evidence of autoimmunity directed against normal tissues expressing CEA was observed in mice in which the CEA-based vaccine significantly reduced intestinal tumor load. The CEA.Tg/MIN mice present a clinically relevant model in which different CEA-based vaccine strategies can be tested on the spontaneous onset of intestinal tumorigenesis.

Human carcinoembryonic antigen (CEA) is an oncofetal glycoprotein overexpression of which by gastrointestinal carcinomas is well known. Expression of CEA in head and neck cancer (HNC) is not widely recognized. It is important to note that most of these studies used polyclonal antibodies that may have cross-reactivity with CEA-related antigens. Currently, CEA is being evaluated in preclinical and clinical studies as a target for specific immunotherapy against gastrointestinal adenocarcinomas that express the antigen. This study was conducted to evaluate CEA as a potential target for specific immunotherapy against HNC. Immunohistochemical analysis of tumor tissue from 69 cases of squamous cell carcinoma (SCC) of the head and neck using a CEA-specific monoclonal antibody (COL-1) showed the majority to be positive for CEA. Tumor cell lines derived from human HNC were screened for CEA transcripts using nested reverse transcription-PCR. Constitutive expression of CEA mRNA was detected in 7 of 10 HNC lines. CEA protein was detectable in lysates from all 7 of the lines by quantitative fluoroimmunometry. SDS-PAGE/Western blot analysis of cell lysates from these lines showed a COL-1 immunoreactive product with a molecular weight equivalent to that of CEA. Cell surface expression of CEA was low for the SCC lines; however, there was moderate to strong cytoplasmic staining intensity for all of the CEA(+) HNC lines by immunocytochemistry. Additional supportive evidence for CEA as a target was demonstrated by the presence of cytolytic activity of an HLA-A2-restricted/CEA-epitope-specific human CTL against a CEA-overexpressing HNC-derived SCC line. These results suggest that CEA may be considered as a possible target for specific vaccine-mediated immunotherapy against HNCs.

This study describes a CD8(+) T-cell line specific for a MHC class I-restricted carcinoembryonic antigen (CEA) epitope, residues 526-533, isolated from CEA transgenic (CEA.Tg) mice immunized with a recombinant vaccinia-CEA vaccine. Incubation of splenocytes from the immune CEA.Tg mice with the CEA(526-533) peptide resulted in the outgrowth of low-avidity CD8(+) T cells, which produced IFN-gamma and mediated perforin-dependent tumor cell lysis. However, the CEA peptide-specific T cells killed CEA-expressing murine colorectal tumor cells only after pretreatment of the targets with murine IFN-gamma (muIFN-gamma), and lysis was H-2D(b)-restricted and involved the Fas-FasL-mediated cytotoxic pathway. When the CEA peptide-specific T cells were used as in vivo effectors in adoptive T-cell transfer studies, muIFN-gamma treatment of the CEA.Tg mice was again required for T-cell-dependent growth suppression of CEA-expressing metastatic tumors. The results indicate that (a) vaccination of mice carrying the human CEA gene with recombinant vaccinia-CEA generates a CEA epitope-specific, CD8-dependent CTL response, (b) CEA, a normal, tissue-specific antigen, can also serve as a target for antitumor immunity after the adoptive transfer of CEA peptide-specific T cells, and (c) muIFN-gamma might be an effective cancer vaccine adjuvant by virtue of its ability to augment the susceptibility of tumor targets to cell-mediated lysis.

The use of reverse transcription-PCR (RT-PCR) to analyze cells in the blood of cancer patients for the detection of mRNA expressed in tumor cells has implications for both the prognosis and the monitoring of cancer patients for the efficacy of established or experimental therapies. Carcinoembryonic antigen (CEA) is expressed on approximately 95% of colorectal, gastric, and pancreatic tumors, and on the majority of breast, non-small cell lung, and head and neck carcinomas. CEA shed in serum is useful as a marker in only approximately 50% of colorectal cancer patients and rarely is shed by some other carcinoma types. RT-PCR has been used previously to detect CEA mRNA in cells in the blood and lymph nodes of cancer patients. Under the assay conditions validated in the studies reported here, 34 of 51 (67%) patients with different stages of colorectal cancer had blood cells that were positive by RT-PCR for CEA mRNA, whereas none of 18 patients with colonic polyps were positive; 2 of 60 apparently healthy individuals (who were age and sex matched with the carcinoma patients and were part of a colon cancer screening program as controls) were marginally positive. The results of CEA PCR in the blood of the carcinoma patients and the other groups showed strong statistical correlation with the disease (P2 < 0.0001). Analyses were carried out to detect both serum CEA protein levels and CEA mRNA in blood cells of colorectal carcinoma patients by RT-PCR. For all stages of disease, 18 of 51 patients (35%) were positive for serum CEA, whereas 35 of 51 (69%) were positive by RT-PCR. More importantly, only 5 of 23 (20%) of stage B and C colorectal cancer patients were positive for serum CEA, whereas 16 of 23 (70%) were positive by RT-PCR. The use of two other serum markers (CA19.9 and CA72-4) for colorectal cancer in combination with serum CEA scored two additional patients as positive; both were positive by RT-PCR for CEA mRNA. Pilot long-term longitudinal studies conducted before and after surgery identified some patients with CEA mRNA in blood cells that were negative for all serum markers, who eventually developed clinical metastatic disease. The studies reported here are the first to correlate RT-PCR results for CEA mRNA in blood cells with one or more serum markers for patients with different stages of colorectal cancer, and are the first long-term longitudinal studies to use RT-PCR to detect CEA mRNA in blood cells of cancer patients. Larger cohorts will be required in future studies to define the impact, if any, of this technology on prognosis and/or disease monitoring.

Recombinant avian poxviruses [fowlpox and canarypox (ALVAC)], restricted for replication in nonavian cell substrates and expressing granulocyte/macrophage-colony stimulating factor (avipox-GM-CSF), were evaluated for their ability to enrich an immunization site with antigen-presenting cells (APCs) and, in turn, function as biological vaccine adjuvants. Avipox-GM-CSF administered as a single s.c. injection significantly enhanced the percentage and absolute number of APCs in the regional lymph nodes that drain the injection site. Both the magnitude and duration of the cellular and phenotypic increases within the lymph nodes induced by the avipox-GM-CSF viruses were significantly (P < 0.05) greater than those measured in mice treated with four daily injections of recombinant GM-CSF protein. Temporal studies revealed that the APC enrichment of regional lymph nodes was sustained for 21-28 days after injection of the recombinant avipox virus expressing GM-CSF and, moreover, three injections of the recombinant virus could be given without any appreciable loss of in vivo bioactivity. Mice expressing human carcinoembryonic antigen (CEA) as a transgene (CEA.Tg) developed CEA-specific humoral and cell-mediated immunity after being immunized with avipox-CEA. The coadministration of recombinant avipox viruses expressing CEA and GM-CSF significantly enhanced CEA-specific host immunity with an accompanying immunotherapeutic response in tumor-bearing CEA.Tg mice. The optimal use of avipox-GM-CSF, in terms of dose and dose schedule, especially when used with different immunogens, remains to be determined. Nonetheless, the present findings demonstrate: (a) the effective delivery of GM-CSF to an immunization site using a recombinant avian poxvirus; (b) the compatibility of delivering an antigen and GM-CSF in replication-defective viruses to enhance antigen-specific immunity; and (c) the combined use of recombinant avipox viruses expressing CEA and GM-CSF to generate antitumor immunity directed at a self tumor antigen.

Repeated subcutaneous (s.c.) injections of recombinant granulocyte-macrophage colony-stimulating factor (recGM-CSF) for 4-5 days can enrich an immunization site with antigen-presenting cells (APC), which has been correlated with improved immune responses in experimental and clinical studies. A recombinant vaccinia virus encoding the GM-CSF gene (rV-GM-CSF) has been developed and can generate specific antitumour immunity in a whole tumour cell vaccine. In the present study, we examined whether rV-GM-CSF could produce and release GM-CSF locally which, in turn, might enrich a site of immunization for APC as previously shown for recGM-CSF. S.c. injection of rV-GM-CSF significantly (P<0.05) enhanced the percentage and overall number of APC, measured by class II expression levels, in the regional lymph nodes that drain the injection site. Dose- and temporal-dependent studies showed class II expression levels in the draining lymph nodes were maximally enhanced 5-7 days after a single injection of 10(7)plaque-forming units (pfu) of rV-GM-CSF. Flow cytometry revealed that the increase in class II expression resulted from (i) a higher class II expression level on CD19(+)B cells and (ii) an increase in the number of CD11c(+)/class II(+)professional APC within the draining lymph nodes. Moreover, isolation of lymph nodes from rV-GM-CSF-treated mice revealed their capacity to support higher levels of antigen-specific T cell proliferation and allospecific cytotoxic responses. A comparison between a single injection of rV-GM-CSF and a 4-day course of recGM-CSF revealed comparable changes in class II expression and functional T cell assays. GM-CSF can be delivered in a recombinant poxvirus, and the local production of the cytokine results in cellular and phenotypic changes that are similar to those of recGM-CSF. The ability to utilize rV-GM-CSF as a single inoculum may be more compatible with traditional immunization strategies., (Copyright 2000 Academic Press.)

Cytogenetic alterations associated with different stages in carcinogenesis can be distinguished in cultured human or rodent cells transformed by carcinogenic agents. Three tumorigenic rat mammary epithelial cell lines transformed in vitro with 7,12, -dimethylbenz[a]anthracene alone or in combination with 12-O-tetradecanoylphorbol-13-acetate were examined cytogenetically. Non-random alterations consisting of translocations involving the short arm of chromosome 3 and trisomy of chromosomes 14 and X were identified in all three lines. Deletion and inversion of chromosome 1 with the breakpoint at band 1q22 and a duplication 1q 32-43 and trisomy of chromosome 2 were observed in two cell lines. The accumulation of structural alterations and chromosome imbalances during the process of cell immortalization and acquisition of tumorigenicity are required for normal rat mammary cells to become malignant. Unbalanced translocations of chromosome 3 resulting in loss of the short arm had the breakpoint at 3p11. This site is a hotspot of breakage and recombination in various rat tumors and may represent a region of tumor suppressor gene critical to the development of rat mammary tumors, as well as other types of tumors.

Human carcinoembryonic antigen (CEA) is a well-characterized oncofetal glycoprotein whose overexpression by human carcinomas has been a target for cancer immunotherapy. Transgenic mice that express CEA as a self-antigen with a tissue distribution similar to that of humans have been developed. This study investigates: (a) the responsiveness of the CEA transgenic (CEA.Tg) mice to endogenous CEA or CEA administered as a whole protein in adjuvant; and (b) whether the presentation of CEA as a recombinant vaccinia virus could generate CEA-specific host immunity. By and large, the CEA.Tg mice were unresponsive to CEA, as shown by the lack of detectable CEA-specific serum antibodies and the inability to prime an in vitro splenic T-cell response to CEA. Furthermore, the administration of whole CEA protein in adjuvant to CEA.Tg mice failed to elicit either anti-CEA IgG titers or CEA-specific T-cell responses. Only weak anti-CEA IgM antibody titers were found in those mice. In contrast, CEA.Tg mice immunized with recombinant vaccinia virus expressing CEA generated relatively strong anti-CEA IgG antibody titers and demonstrated evidence of immunoglobulin class switching. These mice also developed T(H)1-type CEA-specific CD4+ responses and CEA peptide-specific cytotoxicity. The ability to generate CEA-specific host immunity correlated with protection of the CEA.Tg mice against a challenge with CEA-expressing tumor cells. Protection against tumor growth was accomplished with no apparent immune response directed at CEA-positive normal tissue. The results demonstrate the ability to generate an effective antitumor immune response to a tumor self-antigen by immunization with a recombinant vaccinia virus. CEA.Tg mice should be an excellent experimental model to study the effects of more aggressive immunization schemes directed at established tumors with the possible development of accompanying autoimmune responses involving normal tissues.

Preclinical studies have demonstrated that recombinant IFN-alpha (rIFN-alpha) can enhance the tumor associated glycoprotein 72 (TAG-72) on tumors. To determine whether rIFN-alpha could enhance TAG-72 expression in vivo in patients, 15 women with breast cancer were randomized to receive daily injections of rIFN-alpha (3 x 10(6) units/m2 for 14 days) beginning on day 1 (group 1 = 7 patients) or on day 6 (group 2 = 8 patients). On day 3, all patients received a 10-20-mCi tracer dose of 131I-CC49, a high-affinity murine monoclonal antibody reactive against TAG-72, followed by a therapy dose of 60-75 mCi/m2 of 131I-CC49 on day 6. Whole body and single-photon emission computed tomography scans along with whole blood pharmacokinetics were performed following tracer and treatment phases. Hematological toxicity was considerable; reversible grade 3-4 neutropenia and thrombocytopenia was observed in 12 of 15 patients. Twelve of 14 patients tested developed human antimouse antibodies 3-6 weeks after treatment. For group 1 patients, whole blood residence time increased significantly between that predicted from the tracer doses and therapy doses (42.6 +/- 4.7 versus 51.5 +/- 4.8 h, respectively; P < 0.01). The calculated radiation absorbed dose to red marrow from therapy compared to tracer activity was also significantly higher for this group (1.25 +/- 0.35 versus 1. 07 +/- 0.26 cGy/mCi; P < 0.05). Treatment with rIFN-alpha was found to enhance TAG-72 expression in tumors from patients receiving rIFN-alpha (group 1) by 46 +/- 19% (P < 0.05) compared to only 1.3 +/- 0.95% in patients not initially receiving IFN (group 2). The uptake of CC49 in tumors was also significantly increased in rIFN-alpha-treated patients. One partial and two minor tumor responses were seen. In summary, rIFN-alpha treatment altered the pharmacokinetics and tumor uptake of 131I-CC49 in patients at the expense of increased toxicity.

Colorectal tissue biopsies were obtained from 110 patients diagnosed with primary colorectal carcinoma (tumor and normal colonic mucosa samples), 20 patients diagnosed with benign colorectal disease, and 31 healthy donors. The level of expression of tumor-associated glycoprotein 72 (TAG-72) was quantitatively measured in each sample using a double-determinant RIA with monoclonal antibodies B72.3 and CC49 and detecting the sialyl-Tn epitope; this assay was termed CA 72-4. Statistical analysis revealed a significant (approximately 10-fold) increase of TAG-72 expression in the colon tumor biopsies when compared with the expression in normal colonic mucosa from the same patients. A regression analysis revealed a significant correlation (r = 0.459; P < 0.001) between TAG-72 levels measured in biopsies from the tumor lesions and those found in the corresponding normal colonic mucosa. Furthermore, regression analysis showed a significant positive correlation between TAG-72 levels in the tumors and sera of the same patients (r = 0.491; P < 0.001). TAG-72 levels in normal colonic mucosa from healthy donors and patients diagnosed with colorectal cancer were compared. TAG-72 expression was 5-fold higher in the normal mucosa from the colorectal carcinoma patients. No relationship between TAG-72 tumor tissue content and stage of disease was found. Moreover, the correlation between TAG-72 distribution and degree of tumor differentiation observed (P < 0.05) was not any more evident when mucinous carcinomas were excluded. Finally, the results provide further evidence that TAG-72 may be considered an important early marker for colorectal cancer and/or other dysplastic colonic diseases. The statistical correlation between TAG-72 levels in tumors and circulating TAG-72 indicates that patients with elevated levels of serum TAG-72, as measured by the CA 72-4 assay, would be most suited for diagnostic and/or therapeutic intervention with the anti-TAG-72 monoclonal antibodies B72.3 or CC49 or vaccine trials using the sialyl-Tn epitope.

Interleukin-2 (IL-2) has been an effective immune modulator in several active-specific immunotherapy experimental protocols using either viral or oncolysate-based vaccines. In this report, data indicate that IL-2 administration can appreciably augment the therapeutic effect of a single immunization of a recombinant vaccinia virus-carcinoembryonic antigen (rV-CEA) vaccine using a CEA-expressing syngeneic experimental murine model system. A single rV-CEA immunization of C57BL/6 mice bearing palpable CEA-positive colon adenocarcinoma tumors results in complete tumor regression in approximately 20% of the mice. The addition of a course of low-dose IL-2 results in complete tumor regression in 60-70% of the mice. Moreover, the combination of rV-CEA and IL-2 induces systemic immunity, which protects those tumor-free mice from subsequent rechallenge with the CEA-expressing tumor cells. No such tumor regression or protection was observed in those mice immunized with the wild-type vaccinia vaccine (V-Wyeth) alone or with IL-2 administration alone. Cellular immune assays revealed that the addition of IL-2 to rV-CEA immunization significantly increased the CEA-specific T-cell proliferative responses as well as the cytolytic T-cell responses when compared with rV-CEA immunization alone. The enhanced CEA-specific immune response, coupled with the improved experimental therapeutic outcome following IL-2 administration, suggests that treatment with that cytokine may effectively substitute for multiple rV-CEA immunizations in active-specific immunotherapy clinical protocols directed at CEA-expressing tumors.

Previous studies have shown that (a) single-chain antibody binding proteins, or sFvs, localize experimental tumor xenografts (D.E. Milenic et al, Cancer Res., 51: 6363-6371, 1991) and (b) the administration of gamma-interferon (IFN-gamma) increases the expression of a high molecular weight glycoprotein, tumor-associated glycoprotein 72 (TAG-72), which improves mAb-based tumor targeting as well as radioimmunotherapy (J. W. Greiner et al., Cancer Res., 53: 600-608, 1993). The present experimental study was designed to determine whether exploiting those two observations in combination could augment tumor detection. Initial results revealed significant localization of a single-chain antibody binding protein of CC49 (i.e., CC49 sFv), a second generation anti-TAG-72 mAb, to human colon tumor xenografts (HT-29), which express low constitutive TAG-72 levels. IFN-gamma treatment of mice bearing HT-29 tumors significantly increased TAG-72 levels in the tumor xenografts. Increased TAG-72 expression was accompanied by a 2-4-fold augmentation of CC49 sFv localized to the HT-29 tumors, measured by direct quantitation of 125I-labeled CC49 sFv tumor deposition as well as tumor:normal tissue ratios. Enhanced CC49 sFv tumor localization improved HT-29 tumor visualization by external scintigraphy as well as when using a hand-held gamma-detecting probe to discriminate between normal (i.e., heart, hind leg) and tumor tissue. The gamma-detecting probe was the same as that used intraoperatively with 125I-labeled CC49 IgG to identify occult tumors in patients. The present experimental findings indicate that the efficiency by which 125I-labeled CC49 sFv localizes tumor in vivo can be enhanced with IFN-gamma. Results of the present study suggest that (a) the incorporation of an IFN-gamma treatment schema prior to radioimmunscintigraphy may increase the signal from the tumor site(s), thus providing a better discrimination between tumor and background, and (b) combining 125I-labeled CC49 sFv with IFN-gamma will not only reduce the time interval between antibody injection and surgery, but will also increase the efficiency of tumor localization using the intraoperative gamma-detecting probe.

When administered as single agents, both interferon gamma (IFN-gamma) and interleukin 6 (IL-6) significantly increase carcinoembryonic antigen (CEA) and HLA class I antigen expression on the surface of human colorectal tumour cells. Studies were carried out to determine whether by combining those cytokines a synergistic enhancement of CEA and HLA expression could result. The findings revealed that the administration of 20 units IFN-gamma along with 1.7 ng IL-6, concentrations of each cytokine that individually induced minimal antigenic changes, together synergistically increased CEA and HLA class I as well as induced qualitative changes in HLA expression on WiDr human colon carcinoma cells. The magnitude of the synergistic increases in CEA and HLA class I expression were reminiscent of the level of antigen augmentation observed when administering 20- to 100-fold higher amounts of each cytokine as a single agent. Also, the addition of IL-6 potentiated the IFN-gamma induction of HLA class II expression. The combined administration of IL-6 potentiated the IFN-gamma did not have any additive or synergistic effects on the growth suppression of those tumour cells. Interestingly, utilization of specific neutralizing antibodies for type I interferons abrogated the increases of CEA and HLA expression seen with IL-6 treatment alone or in combination with IFN-gamma. Moreover, reverse transcriptase/polymerase chain reaction analyses revealed a constitutive expression as well as a temporal increase of IFN-beta mRNA transcripts in colon tumour cells treated with IL-6. Therefore, the findings provide indirect evidence that IFN-beta production seems to play a critical role in the ability of IL-6 to upregulate antigen expression alone or in combination with IFN-gamma. These findings provide insight into cytokine combinations that synergistically upregulate tumour-associated and normal HLA antigen expression on the surface of human tumour cells. Those results provide the rationale for the combined use of such cytokines to heighten tumour cell recognition in monoclonal antibody- or cell-mediated-based immunotherapeutic approaches.

An M(r) 110,000 antigen was initially described in human gastric carcinoma cells by its cross-reactivity with anti-carcinoembryonic antigen (CEA) monoclonal antibodies, as well as the ability of gamma-interferon to increase its level of expression. We describe the molecular cloning and sequence analyses of overlapping clones that constitute a full-length complementary DNA that encodes for the entire M(r) 110,000 molecule. The 1.5-kilobase message encodes for a 407-amino acid polypeptide whose structural analysis was consistent with an integral membrane glycoprotein. In particular, the extracellular domain was rich in serine and threonine residues at which carbohydrate substitution is likely through O- and N-linked glycosylation. This would explain the higher molecular weight of the antigen whose polypeptide backbone is approximately M(r) 42,000. Further computer-aided sequence analyses revealed no significant homology with any member of the CEA gene family. The cross-reactivity with anti-CEA monoclonal antibodies may be explained by the presence of CEA and normal cross-reacting antigen homologous sites proximal to the transmembrane region. No sequence homology was found with any known protein. Thus, the M(r) 110,000 molecule represents a potentially novel cell membrane glycoprotein whose possible role in human cancer and/or as a gamma-interferon-inducible gene product warrants further investigation.

The moderately differentiated human colon tumor cell line, HT-29, constitutively expresses low levels of the high molecular weight mucin, tumor-associated glycoprotein 72 (TAG-72), and the M(r) 180,000 carcinoembryonic antigen (CEA) when grown as s.c. tumors in athymic mice. We report that the in vivo administration of gamma-interferon (IFN-gamma) resulted in a time- and dose-dependent increase in both TAG-72 and CEA expression in the HT-29 tumors. Immunohistochemical staining revealed a more homogeneous TAG-72-positive tumor cell population after IFN-gamma. Furthermore, both anti-TAG-72 and anti-CEA monoclonal antibodies (MAbs) showed enhanced localization to the HT-29 tumors in mice treated with IFN-gamma. Using that experimental model, subsequent studies presented evidence showing that the combination of IFN-gamma with 131I-CC49, an anti-TAG-72 MAb, resulted in a statistically significant improvement in therapeutic efficacy when compared with 131I-CC49 alone. For example, treatment with 300 microCi of 131I-CC49 initially suppressed HT-29 tumor growth; however, that reduction in tumor growth was transient as evidenced by the emergence of additional tumor growth at later time points. In contrast, an 8-day treatment with IFN-gamma in combination with 300 microCi 131I-CC49 resulted in sustained suppression of HT-29 tumor growth. Thus, IFN-gamma in vivo can substantially increase the TAG-72 expression in human colon tumor xenografts which leads to an increased tumor localization of anti-TAG-72 MAbs and seems to be responsible for the enhanced antitumor effects when IFN-gamma was combined with 131I-CC49. The results provide further evidence for including a biological response modifier, such as IFN-gamma, which can increase the expression of specific tumor antigens (i.e., TAG-72 and CEA) subsequently leading to a dramatic improvement in the antitumor efficacy of a radionuclide-conjugated MAb.

The presence of three distinct serum markers of carcinoma, tumor-associated glycoprotein 72 (TAG-72; as measured by the CA 72-4 assay), CA 19-9, and carcinoembryonic antigen (CEA), was evaluated in 194 patients diagnosed with either malignant (n = 94) or benign (n = 100) gastric disease. Of the 94 patients diagnosed with gastric carcinoma, the percentage of patients whose serum samples were positive for TAG-72, CA 19-9, or CEA was 42.6, 31.9, and 20.2%, respectively. Furthermore, fewer false positive samples were observed for TAG-72 than either CA 19-9 or CEA. The analysis of serum TAG-72, CA 19-9, and CEA levels in patients diagnosed with early (stage I and II) versus advanced (stage III and IV) disease revealed a significantly higher level of TAG-72 and CA 19-9 in the serum of patients with advanced stage gastric carcinoma. The serum samples were also analyzed to determine whether any advantage might be gained by simultaneously measuring two or more of the tumor markers. The data clearly indicate that the measurement of TAG-72 with CA 19-9 significantly increased the percentage of gastric carcinoma patients with positive serum levels of either antigen. This advantage was achieved with no significant increase in the number of false positives. Twenty-one patients were followed postsurgically for up to 3 years to determine whether the appearance or reappearance of TAG-72, CA 19-9, or CEA accurately predicted disease recurrence. Positive serum TAG-72 levels correlated with disease recurrence in 7 of 10 patients, compared with 5 and 2 patients for CA 19-9 and CEA, respectively. The findings suggest that serum TAG-72 as measured by the CA 72-4 assay may be a useful marker for late stage gastric carcinoma and its measurement alone or in combination with CA 19-9 may have utility in the clinical management of gastric carcinoma.

A novel gene product which is immunologically related to carcinoembryonic antigen (CEA) and constitutively expressed by six of eight human gastric carcinoma cell lines is described. The antigen was initially identified by the differential binding patterns of four monoclonal antibodies (MAbs) which recognize the putative Mr 180,000 CEA and/or the Mr 90,000 CEA-related gene product, NCA (normal cross-reacting antigen). Western blot analyses of partially purified membrane fractions prepared from Hs 746T gastric carcinoma cells identified an Mr 110,000 antigen. Northern blot analyses using CEA- and NCA-specific complementary DNA probes did not identify any specific CEA or NCA transcripts in polyadenylate-selected mRNA isolated from the Hs 746T cells. Likewise, a probe designed to hybridize with different CEA-related family members failed to identify a CEA-related message in the Hs 746T cells. Subsequent studies revealed that interferon-gamma (IFN-gamma) treatment substantially increased the level of expression of the Mr 110,000 antigen on the Hs 746T and five other gastric cell types that constitutively expressed the antigen. IFN-gamma treatment also de novo induced the expression of the Mr 110,000 antigen on the surface of GaCa gastric carcinoma cells. A high percentage of Hs 746T (i.e., greater than 85%) and GaCa (approximately 75%) gastric carcinoma cells expressed the Mr 110,000 antigen after IFN-gamma treatment; yet, neither cell type expressed CEA or NCA as measured by the binding of the anti-CEA MAb, COL-1, or B6.2, an anti-NCA MAb. In contrast to CEA and NCA, phosphatidylinositol phospholipase C treatment failed to release the Mr 110,000 antigen from the surface of the Hs 746T or IFN-gamma-treated GaCa cells, suggesting that membrane attachment of this novel antigen is not via a glycosyl-phosphatidylinositol anchor. Finally, primers that amplify the 420 base pairs of the immunoglobulin-like domain of CEA and NCA detected an appropriately sized product in untreated as well as IFN-gamma-treated GaCa cells using the polymerase chain reaction method. Thus, a potentially novel gene product coding for an Mr 110,000 antigen that is strongly upregulated by IFN-gamma has been identified in human gastric carcinoma cells. Immunologically, the antigen shares reactive epitopes with CEA and its related NCA gene product; however, Northern blot analyses, polymerase chain reaction, and phosphatidylinositol phospholipase C results suggest that the antigen may be, at best, a distant relative of the CEA gene family.

Sera were collected from 111 patients diagnosed with adenocarcinoma or nonadenocarcinoma malignancies who received different schedules of interferon (IFN)-gamma or IFN-beta ser alone or in combination. Serum carcinoembryonic antigen (CEA) and tumor-associated glycoprotein-72 (TAG-72) antigen levels were measured to determine whether interferon could enhance the tumor shedding and, thereby, the serum level of either tumor antigen. Less than 10% of the sera samples from patients diagnosed with nonadenocarcinoma malignancies (e.g., hairy cell leukemia, melanoma) had positive titers of TAG-72 or CEA, and interferon neither increased nor resulted in the appearance of either tumor antigen in those sera. In contrast, 59.2% and 75.4% of the patients with adenocarcinoma had positive serum levels of TAG-72 and CEA, respectively, prior to interferon. IFN-gamma and IFN-beta ser alone or in combination significantly increased serum TAG-72 or CEA in approximately 65% of those patients. The results suggest that interferon administration to patients with adenocarcinoma can result in increased serum levels of selected tumor-associated antigens used in the diagnosis of malignancy. These preliminary findings may be important in the development of new strategies to obtain more sensitive tumor antigen serum assays for the diagnosis and monitoring for disease progression of adenocarcinoma.

Monoclonal antibody (MAb) D612 recognizes an antigen expressed on the cell surface of normal and malignant gastrointestinal epithelium. It is a murine IgG2a/kappa which has been previously shown to mediate killing of human colon carcinoma cells using human effector cells (which could be enhanced in the presence of interleukin-2). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of MAb D612 immunoprecipitates of extracts of L-[35S]methionine-, L-[3H]leucine-, and D-[3H]glucosamine-labeled human colon carcinoma cells showed that the D612 antigen is a Mr 48,000 glycoprotein. Similar estimates of molecular mass were obtained from SDS-PAGE analyses of MAb D612 immuno-precipitates of radioiodinated extracts of surgically resected colon carcinoma and adjacent normal colonic mucosa. D612 antigen was not detectable in immunoprecipitates of supernatant media from radiolabeled cell cultures, suggesting that the antigen is not readily shed from the surface of cultured cells. The D612 antigen was shown to be clearly distinct from previously described gastrointestinal carcinoma-associated glycoproteins: the D612 antigen shows a migration pattern of SDS-PAGE distinct from those of the antigens recognized by MAbs KS1/4 and GA733, and reciprocal immunodepletion analyses of D-[3H]glucosamine-labeled colon carcinoma cells utilizing MAbs D612 and GA733 revealed no cross-reactivity between these antibodies. Similarly, competitive binding studies between MAbs 17-1A and KS1/4 and MAb D612 revealed no similarity between the epitopes recognized by MAb D612 and MAbs 17-1A and KS1/4. MAbs D612 and 17-1A were also titered in immunoperoxidase staining assays on serial frozen sections of normal and malignant colon. MAb D612 showed a higher titer of immunostaining reactivity with both normal and malignant colon than did MAb 17-1A. MAb D612 showed roughly equivalent immunostaining titers against normal and malignant colon; whereas MAb 17-1A showed higher titer of immunostaining reactivity against the normal colon tissue than against the malignant colon. Flow cytometric analysis of phosphatidylinositol-specific phospholipase C-treated colon carcinoma cells revealed no loss of D612 antigen from the cell surface, suggesting that the mechanisms of attachment of the D612 antigen to the cell surface does not involve linkage to a phosphatidylinositol glycan.(ABSTRACT TRUNCATED AT 400 WORDS)

The regulation of carcinoembryonic antigen (CEA) expression by recombinant human interferon-gamma (IFN-gamma) was studied in a series of 7 human colorectal tumor cell lines at various stages of differentiation. Two of the colorectal cell lines were poorly differentiated and did not constitutively express CEA. IFN-gamma treatment, however, induced CEA expression in one of those lines (i.e., DLD-1) as evidenced by the appearance of CEA-related mRNA transcripts, as well as the cell surface expression of the antigen as measured by flow cytometry and radioimmunoassay. In the highly differentiated colorectal tumor cell lines, IFN-gamma treatment resulted in no detectable change in CEA content in whole cell extracts or in the percentage of cells positive for cell surface CEA expression. In fact, IFN-gamma treatment of the highly differentiated LS174T cell line not only failed to alter CEA expression, but also failed to induce class II human leukocyte antigen expression. Therefore, the highly differentiated LS174T cell line and the poorly differentiated MIP cell line represent colorectal tumor cell types that are unresponsive to the ability of IFN-gamma to induce alterations in tumor (i.e., CEA) or normal (i.e., class I and class II human leucocyte antigen) surface antigen expression. The most responsive of human colorectal tumor cells to the ability of IFN-gamma to alter CEA expression were the moderately differentiated cell lines (i.e., HT-29, WiDr, etc.). IFN-gamma treatment of those cell types increased the CEA content in cell extracts by 300-400%, and increased the percentage of cells positive for surface CEA expression from 30-45% to greater than 80%. The effect of IFN-gamma treatment on 2',5'-oligoadenylate synthetase (2'-5' A) activity was also studied using 4 of the 7 colorectal cell lines. Constitutive 2'-5' A activity varied approximately 14-fold and was not correlated with degree of cellular differentiation. IFN-gamma treatment increased 2'-5' A activity in all 4 colorectal tumor cells tested. In particular, the ability to enhance 2'-5' A activity in the MIP and LS174T cells, 2 colorectal tumor cell types that previously were shown to be unresponsive to IFN-gamma-mediated changes in their antigenic phenotype, clearly separates cellular events regulating 2'-5' A activity from those involved in regulating cell surface antigen expression. The findings also suggested that the regulation of CEA expression by IFN-gamma is not related to the degree of cellular differentiation and, furthermore, provide some insight into which human tumor cell populations may be the most amenable to tumor antigen augmentation by IFN-gamma in an adjuvant setting with a monoclonal antibody.

Tumor targeting by monoclonal antibodies (MAbs) can be enhanced by (a) increasing the percentage of injected dose taken up by the tumor and/or (b) increasing the tumor:nontumor ratios. Several groups have demonstrated that one can increase tumor to nontumor ratios by the use of antibody fragments or the administration of second antibodies. Several other modalities are also possible: (a) the use of recombinant interferons to up-regulate the expression of specific tumor associated antigens such as carcinoembryonic antigen or TAG-72 on the surface of carcinoma cells and thus increase MAb tumor binding has proved successful in both in vitro and in vivo studies; (b) the intracavitary administration of MAbs. Recent studies have demonstrated that when radiolabeled B72.3 is administered i.p. to patients with carcinoma of the peritoneal cavity, it localizes tumor masses with greater efficiency than does concurrent i.v. administered antibody. Studies involving the comparative pharmacology of intracavitary administration of radiolabeled MAb in patients and several animal models will be discussed; (c) it has been reported that prior exposure of hepatoma to external beam radiation will increase radiolabeled MAb tumor targeting. We and others have not been able to duplicate this phenomenon with a human colon cancer xenograft model and radiolabeled MAbs to two different colon carcinoma associated antigens. The possible reasons for these differences will be discussed; (d) the cloning and expression of recombinant MAbs with human constant regions and subsequent size modification constructs will also undoubtedly alter the pharmacology of MAb tumor binding in both diagnostic and therapeutic applications.

The temporal aspects of the anticarcinogenic action of cortisol were investigated by measurement of the frequency of carcinogen-induced morphological transformation of Syrian hamster embryo cells relative to treatment regimen. Treatment of hamster cells with 10(-5) to 10(-11) M cortisol, dexamethasone, corticosterone, 17 beta-estradiol, progesterone, or testosterone did not result in morphological transformation. However, cortisol or dexamethasone treatment beginning 48 hr before the carcinogen caused a dose-dependent reduction in the transformation frequency associated with benzo(a)pyrene [B(a)P] or ultraviolet irradiation treatment. The frequency of B(a)P transformation was reduced 54, 66, and 84% by 10(-11), 10(-10), and 10(-9) M cortisol, respectively, without altering colony formation efficiency. Dexamethasone was equipotent as cortisol as an inhibitor of B(s)P, N-methyl-N'-nitro-N-nitrosoguanidine, or ultraviolet irradiation-induced transformation. Sex steroids, non-steroid antiinflammatory compounds, and steroids without antiinflammatory activity also reduced the transformation frequency but only at concentrations that reduced colony formation. Cortisol inhibition of transformation increased as steroid exposure occurred closer (48, 24, and 8 hr) yet prior to B(a)P treatment. In contrast, if cortisol was added simultaneously with or up to 72 hr after B(a)P, the frequency of transformation was unaffected. Dexamethasone addition after B(a)P, however, reduced the transformation frequency. Thus, physiological concentrations of cortisol when present prior to carcinogen exposure can prevent carcinogen-induced morphological transformation. The ability of cortisol to inhibit ultraviolet irradiation-induced transformation indicates that the anticarcinogenic activity may be mediated by steroid-induced cellular changes which are independent to metabolic activation of the carcinogen.

The effects of recombinant human leukocyte (clone A) interferon (rHu-IFN-alpha A) were investigated on the expression of monoclonal antibody (MAb)-defined tumor antigens expressed on human mammary and colon carcinomas. The rHu-IFN-alpha A treatment substantially increased the localization of radiolabeled MAb B6.2-F(ab')2 to the transplantable Clouser human mammary carcinoma, as well as to the moderately differentiated human colon xenograft WiDr, when grown as s.c. tumors in athymic mice. In contrast, human tumor cell lines (i.e., LS174T, A375, etc.) that were unresponsive to the antigen-augmenting ability of rHu-IFN-alpha A in vitro were also unresponsive in vivo, indicating a possible method of screening carcinoma cell populations for subsequent rHu-IFN-alpha A adjuvant therapy prior to MAb administration. The method of delivery of rHu-IFN-alpha A was also studied. The i.m. route resulted in a 3-4 h plasma half-life for rHu-IFN-alpha A. The administration of rHu-IFN-alpha A via an osmotic pump resulted in a stable circulating plasma titer of 400-800 antiviral units/ml for 7 days. Utilizing delivery of rHu-IFN-alpha A by the constant infusion route, it was found that the increase in localization of 125I-B6.2-F(ab')2 was dependent on (1) the length of time of treatment and (2) the circulating plasma rHu-IFN-alpha A levels. These results thus provide information useful for subsequent studies to determine the potential efficacy of adjuvant rHu-IFN-alpha A treatment for MAb-targeted tumor diagnosis and treatment.

Treatment of human breast or colon carcinoma cells with recombinantly derived human leukocyte (clone A) interferon (IFN-alpha A) increases the surface expression of specific tumor-associated antigens (TAAs) recognized by monoclonal antibodies (MAbs). The MAbs used, B1.1, B6.2, and B72.3, recognize three distinct TAAs, i.e., the Mr 180,000 carcinoembryonic antigen, a Mr 90,000, and a Mr 220,000 to 400,000 glycoprotein, respectively. The binding of the MAbs to the surface of tumor cells increased in a dose-dependent manner, with optimal levels of TAA enhancement at 100 to 1,000 units IFN-alpha A/ml. Higher concentrations of IFN-alpha A that were cytostatic or cytotoxic were also less effective in enhancing TAA expression. Human melanoma (A375) cells and normal fibroblasts (WI-38 and Flow 4000) do not express any of the three TAAs, either before or after interferon treatment. The ability of IFN-alpha A to increase the expression of TAAs on human carcinoma cells was also temporally dependent, with optimal enhancement occurring after 16 to 24 hr. The enhancement of specific TAAs at the surface of the carcinoma cells by IFN-alpha A was confirmed, using fluorescence-activated cell sorter analysis. These data demonstrate that the IFN-alpha A-mediated increase of surface antigen is a result of both an accumulation of more antigen per cell, and an increase in the percentage of cells expressing the antigen. The ability of recombinant interferon to enhance specific TAAs on human carcinoma cells may be exploited in designing protocols for the in situ detection and therapy of human carcinoma lesions by MAbs, as well as in further defining the role of specific TAAs in the expression of the transformed phenotype.

Epithelial cells isolated from the mammary glands of virgin Sprague-Dawley rats and treated with 7,12-dimethylbenz[a] anthracene (DMBA) acquire an indefinite life span and anchorage-independent (AI) growth and form carcinomas in athymic nu/nu mice. Epithelial cells separated from fibroblasts and lipocytes by density-gradient centrifugation after collagenase digestion of the fat pads are grown in a hormone-supplemented medium. Control mammary epithelial cells survived approximately 30 days. After 2 days in culture, the mammary epithelial cells were treated with DMBA (1 microM) for 24 hr allowing for maximum oxidative metabolism of the hydrocarbon. DMBA-treated cells acquired an extended life span and grew in AI medium; however, in most cases, they were nontumorigenic and eventually ceased dividing. A pool of mammary epithelial cells, ME 10CL1, treated with DMBA has grown indefinitely, exhibited AI growth, and after 195 days in culture formed adenocarcinomas when 5 X 10(6) cells were injected into athymic nu/nu mice. When the tumor promoter, 12-O-tetra-decanoylphorbol-13-acetate (100 ng/ml), was added to another pool (ME 11CL2) of DMBA-treated mammary epithelial cells which had been in culture for 110 days, an irreversible increase in cell growth rate and a significant morphological alteration resulted. The 12-O-tetradecanoylphorbol-13-acetate-treated cells also formed colonies in AI medium after 140 days and poorly differentiated carcinomas in athymic nu/nu mice. Inhibition of tumor cell proliferation by tamoxifen is consistent with the mammary origin of the epithelial cells and suggests the presence of a viable estrogen receptor. The results demonstrate in vitro neoplastic transformation of rat mammary epithelial cells by DMBA or promotion of DMBA-initiated cells by 12-O-tetradecanoylphorbol-13-acetate resulting in two different epithelial tumor cell lines.

Rat mammary epithelial cells grown in primary culture contain the microsomal enzyme, aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH), which catalyses the oxidative conversion of polycyclic aromatic hydrocarbons (PAH) to more polar derivatives. Constitutive AHH activity, measured with an established fluorometric method, was 46 pmol/mg protein/h in homogenates of rat mammary epithelial cells after 5 days in culture. The addition of dimethylbenz[a]anthracene (DMBA), benz[a]anthracene (BA), or 3-methylcholanthrene (3-MC) to the cell culture medium increased AHH activity 5.3-, 4.7- and 2.4-fold, respectively. Kinetic studies revealed that maximal hydroxylase induction occurred 16 h after 1 micro M DMBA was added to the culture medium. The decay of the DMBA-induced hydroxylase was biphasic: one component had a t1/2 of 15--30 min and another a t1/2 of 4 h. Norepinephrine, 17 beta-estradiol and 5,6-benzoflavone also increased AHH activity in mammary epithelial cells in vitro, however, sodium phenobarbital had no effect. Fetal bovine serum (FBS), previously shown to be a potent in vitro inducer of AHH activity, had no effect on either constitutive or DMBA-induced mammary epithelial hydroxylase activities following treatment with 1% activated charcoal. Metyrapone and 7,8-benzoflavone, inhibitors of microsomal mixed function oxidase activity, reduced both constitutive and DMBA-induced AHH activities when added to homogenates of untreated and DMBA-treated mammary epithelial cells. The addition of 7,8-benzoflavone reduced both constitutive and DMBA-induced hydroxylase activities by approx. 80%, whereas metyrapone addition inhibited these activities by 20%. The study demonstrates several in vitro factors which alter AHH activity in primary cultures of rat mammary epithelial cells.

Heterogeneity in the expression of tumor-associated antigens, as defined by the binding of monoclonal antibodies, is a characteristic common to most, if not all, human carcinoma cell populations. Antigen-negative cells within the population can escape detection and therapy by their failure to bind the appropriate antibody. Therefore, the extent of antigenic heterogeneity is an important consideration when designing protocols for the management of cancer by administration of monoclonal antibodies. One approach to counteracting the effect of antigenic heterogeneity is the use of clone A of recombinant human leukocyte interferon (Hu-IFN-alpha A). Administration of Hu-IFN-alpha A in vivo effectively increased the amount of tumor antigen expressed by a human colon xenograft in situ and augmented the localization of a radiolabeled monoclonal antibody to the tumor site. Concomitant administration of Hu-IFN-alpha A and monoclonal antibody may thus be effective in overcoming the antigenic heterogeneity of carcinoma cell populations and in enhancing the efficacy of monoclonal antibodies in the detection and treatment of carcinoma lesions.

Using an assay based on the binding of a carcinoembryonic antigen (CEA)-specific monoclonal antibody, we have examined the expression of carcinoembryonic antigen genes in human colon tumor and normal fibroblast cell lines. CEA expression was not detectable in the normal fibroblast cell lines, whereas varying levels of high CEA expression were found in the colon tumor cell lines LS-174T, GEO, and WIDR. We have used a 550-base pair CEA probe derived from cloned complementary DNA to carry out Southern analysis of the DNA isolated from the normal and colon tumor cell lines. At high stringency, the CEA probe detected seven BamHI fragments in all DNAs analyzed. At low stringency, however, 14 BamHI fragments ranging from 1.5 to 23 kilobases were detected. Results of the Southern analysis demonstrate no amplification or rearrangement of the CEA genes in tumor cells. We used methylation-sensitive restriction endonucleases, HpaII and HhaI, to compare the degree of methylation of CEA family of genes in normal and colon tumor cell lines. Our results demonstrate that the CEA family of genes exists in a state of hypermethylation in the normal cell lines. In contrast, the CEA gene(s) are relatively hypomethylated in the tumor cell lines, suggesting a correlation between the state of methylation and degree of expression of the CEA gene(s). A comparison of the state of methylation of the CEA gene(s) in cells before and after treatment with the gamma-interferon (which up-regulates CEA steady-state mRNA levels) showed no detectable difference in the degree of DNA methylation. The segments of CEA genes that are hypermethylated in normal cells, but are hypomethylated in tumor cells, were also identified. Thus, these studies may help identify the sites of methylation that are crucial for the control of CEA gene regulation.

Human leukocyte (alpha) interferon (IFN-alpha) is composed of a multigene family within which at least eight different species have been expressed in Escherichia coli, isolated, and shown to exert a wide range of biological activities on different human target cells. In this study we utilized eight species of IFN-alpha (A, B, C, D, F, I, J, and K) and investigated their respective capabilities to alter the proliferation of a human breast carcinoma cell line (MCF-7). The antigens studied were all constitutively expressed on the MCF-7 cell surface: the Mr 180,000 carcinoembryonic antigen; a high molecular weight (greater than 10(6] glycoprotein, termed tumor-associated glycoprotein 72; and a major HLA histocompatibility antigen. The level of expression of each antigen was measured by the binding of monoclonal antibodies B1.1, B72.3, and W6/32, respectively. A high degree of diversity was found among the various IFN-alpha species with respect to their ability to enhance antigen expression and inhibit MCF-7 cell growth. The two most potent species, IFN-alpha A and IFN-alpha B, were found to increase the expression of tumor antigens as well as the HLA determinant by 2-5-fold. In contrast, IFN-alpha D and IFN-alpha J were virtually inactive in altering antigen expression but did inhibit the growth of MCF-7 cells. The remaining IFN-alpha species, -alpha C, -alpha F, -alpha I, and -alpha K, exerted an intermediate range of activities for both antigen enhancement and inhibition of MCF-7 cell growth. The relative ability of each species of IFN-alpha to inhibit MCF-7 cell growth appeared to be independent of their effectiveness in augmenting antigen expression. IFN-alpha D and IFN-alpha J, the two species that failed to alter tumor antigen expression, did, however, seem to interact with the interferon receptor since they inhibited MCF-7 cell growth and competed with other IFN-alpha species for the increase in carcinoembryonic antigen, tumor-associated glycoprotein 72, or HLA expression. A comparison of the concentrations of each IFN-alpha necessary to enhance antigen expression revealed that the surface HLA determinant was approximately 10-fold more sensitive to enhancement than was the tumor antigen, carcinoembryonic antigen. The individual members of the IFN-alpha family thus differ extensively in their ability to alter the level of antigen expression on the surface of MCF-7 breast carcinoma cells.(ABSTRACT TRUNCATED AT 400 WORDS)