Showing papers on "Tumor antigen published in 1998"

Simultaneous Humoral and Cellular Immune Response against Cancer–Testis Antigen NY-ESO-1: Definition of Human Histocompatibility Leukocyte Antigen (HLA)-A2–binding Peptide Epitopes

Abstract: A growing number of human tumor antigens have been described that can be recognized by cytotoxic T lymphocytes (CTLs) in a major histocompatibility complex (MHC) class I–restricted fashion. Serological screening of cDNA expression libraries, SEREX, has recently been shown to provide another route for defining immunogenic human tumor antigens. The detection of antibody responses against known CTL-defined tumor antigens, e.g., MAGE-1 and tyrosinase, raised the question whether antibody and CTL responses against a defined tumor antigen can occur simultaneously in a single patient. In this paper, we report on a melanoma patient with a high-titer antibody response against the “cancer–testis” antigen NY-ESO-1. Concurrently, a strong MHC class I–restricted CTL reactivity against the autologous NY-ESO-1–positive tumor cell line was found. A stable CTL line (NW38-IVS-1) was established from this patient that reacted with autologous melanoma cells and with allogeneic human histocompatibility leukocyte antigen (HLA)-A2−, NY-ESO-1–positive, but not NY-ESO-1–negative, melanoma cells. Screening of NY-ESO-1 transfectants with NW38-IVS-1 revealed NY-ESO-1 as the relevant CTL target presented by HLA-A2. Computer calculation identified 26 peptides with HLA-A2–binding motifs encoded by NY-ESO-1. Of these, three peptides were efficiently recognized by NW38-IVS-1. Thus, we show that antigen-specific humoral and cellular immune responses against human tumor antigens may occur simultaneously. In addition, our analysis provides a general strategy for identifying the CTL-recognizing peptides of tumor antigens initially defined by autologous antibody.

DNA vaccines with single-chain Fv fused to fragment C of tetanus toxin induce protective immunity against lymphoma and myeloma

Abstract: Vaccination with idiotypic protein protects against B-cell lymphoma, mainly through anti-idiotypic antibody. For use in patients, DNA vaccines containing single-chain Fv derived from tumor provide a convenient alternative vaccine delivery system. However, single-chain Fv sequence alone induces low anti-idiotypic response and poor protection against lymphoma. Fusion of the gene encoding fragment C of tetanus toxin to single-chain Fv substantially promotes the anti-idiotypic response and induces strong protection against B-cell lymphoma. The same fusion design also induces protective immunity against a surface Ig-negative myeloma. These findings indicate that fusion to a pathogen sequence allows a tumor antigen to engage diverse immune mechanisms that suppress growth. This fusion design has the added advantage of overcoming potential tolerance to tumor that may exist in patients.

Immunizing Patients With Metastatic Melanoma Using Recombinant Adenoviruses Encoding MART-1 or gp100 Melanoma Antigens

Abstract: The cloning and characterization of the genes encoding melanoma-associated antigens recognized by human T cells have opened new possibilities for the development of active immunization strategies for the treatment of patients with metastatic melanoma (1,2). Two immuno-dominant antigens, MART-1/MelanA and gp100, were recognized by the majority of tumor-infiltrating lymphocytes (TILs) obtained from HLA-A2-positive patients with metastatic melanoma (3-6). In prior studies (7-9), we have reported the initial results of immunization of patients with melanoma with immunodominant peptides obtained from the MART-1 or gp100 proteins incorporated in incomplete Freund's adjuvant (IFA) and have demonstrated that antitumor precursor cells are generated in the peripheral blood of immunized patients when comparing preimmunization and postimmunization samples. These studies suggested that improved response rates were seen when peptide immunization was followed by the administration of interleukin 2 (IL-2) (9). In murine models, immunization with recombinant adenoviruses, vaccinia viruses, and fowlpox viruses encoding model tumor antigens generated antitumor responses that were capable of significantly reducing the number of established pulmonary micrometastases (10,11). These preclinical studies have stimulated efforts to develop immunization strategies against tumor-associated antigens in humans using recombinant viruses. Adenoviruses are attractive candidates for use in the development of human vaccines and for human gene therapy because the adenovirus genome can be readily manipulated by recombinant DNA techniques and inserts of foreign genes are stably integrated [reviewed in (12,13)]. The incorporation of large DNA fragments into adenovirus requires the deletion of wild-type viral DNA sequences. Most commonly, DNA sequences from the E1, E3, or E4 regions are deleted, which results in a virus deficient in viral replication. Administration of adenoviruses has been shown to be safe, and vaccines consisting of unattenuated adenovirus have been administered to millions of military recruits over the past several decades (14,15). Recombinant adenoviruses have been used as vectors for gene therapy in patients with a variety of diseases (16-24) or as vaccines to raise cellular or antibody reactivity against infectious agents (12,13,25-27). In our own preclinical study (10), we demonstrated that immunization with a recombinant adenovirus expressing the model tumor antigen, β-galactosidase, could produce specific cytolytic T cells and could reduce established metastases in tumor-bearing mice that could be enhanced by the concomitant administration of IL-2. Thus, recombinant adenoviruses were generated expressing the MART-1 and gp100 melanoma-associated antigens and the characteristics of these viruses were determined (28). We have now conducted phase I clinical trials in patients with metastatic melanoma who received active immunization with multiple doses of these recombinant adenoviruses. The immunologic, therapeutic, and safety aspects of these immunizations in humans constitute the subject of this report.

p53-dependent cell death/apoptosis is required for a productive adenovirus infection

Abstract: The p53 tumor suppressor protein binds to both cellular and viral proteins, which influence its biological activity One such protein is the large E1b tumor antigen (E1b58kDa) from adenoviruses (Ads), which abrogates the ability of p53 to transactivate various promoters This inactivation of p53 function is believed to be the mechanism by which E1b58kDa contributes to the cell transformation process Although the p53-E1b58kDa complex occurs during infection and is conserved among different serotypes, there are limited data demonstrating that it has a role in virus replication However, loss of p53 expression occurs after adenovirus infection of human cells and an E1b58kDa deletion mutant (Onyx-015, also called dl 1520) selectively replicates in p53-defective cells These (and other) data indicate a plausible hypothesis is that loss of p53 function may be conducive to efficient adenovirus replication However, wild-type (wt) Ad5 grows more efficiently in cells expressing a wt p53 protein These studies indicate that the hypothesis may be an oversimplification Here, we show that cells expressing wt p53, as well as p53-defective cells, allow adenovirus replication, but only cells expressing wt p53 show evidence of virus-induced cytopathic effect This correlates with the ability of adenovirus to induce cell death Our data indicate that p53 plays a necessary part in mediating cellular destruction to allow a productive adenovirus infection In contrast, p53-deficient cells are less sensitive to the cytolytic effects of adenovirus and as such raise questions about the use of E1b58kDa-deficient adenoviruses in tumor therapy

A Breast and Melanoma-Shared Tumor Antigen: T Cell Responses to Antigenic Peptides Translated from Different Open Reading Frames

Abstract: Infusion of TIL586 along with IL-2 into the autologous patient with metastatic melanoma resulted in the objective regression of tumor. Here, we report that screening a cDNA library from the 586mel cell line using CTL clones derived from TIL586 resulted in the isolation of a gene, CAG-3 (cancer Ag gene 3). Sequence analysis revealed that CAG-3 encodes an open reading frame identical to NY-ESO-1, which was recently reported to be recognized by autologous serum from a patient with esophageal cancer. Thus, NY-ESO-1 appears to be an immune target for both Ab- and T cell-mediated responses. Significantly, NY-ESO-1-specific CTL clones were capable of recognizing two HLA-A31-positive fresh and cultured breast tumors. To our knowledge, this represents the first direct demonstration that tumor-specific CTL clones can recognize both breast and melanoma tumor cells. A 10-mer antigenic peptide ESO10-53 (ASGPGGGAPR) was identified from the normal open reading frame of NY-ESO-1 based on its ability to sensitize HLA-A31-positive target cells for cytokine release and specific lysis. Interestingly, two additional CTL clones that were sensitized with NY-ESO-1 recognized two overlapping antigenic peptides derived from an alternative open reading frame of the same gene. These findings indicate that CTLs simultaneously responded to two different gene products translated from the normal and alternative reading frames of the same gene. Understanding of this mechanism by which the alternative reading frame is translated may have important implications in tumor immunology.

Heteroclitic Immunization Induces Tumor Immunity

Abstract: In tumor transplantation models in mice, cytotoxic T lymphocytes (CTLs) are typically the primary effector cells. CTLs recognize major histocompatibility complex (MHC) class I–associated peptides expressed by tumors, leading to tumor rejection. Peptides presented by cancer cells can originate from viral proteins, normal self-proteins regulated during differentiation, or altered proteins derived from genetic alterations. However, many tumor peptides recognized by CTLs are poor immunogens, unable to induce activation and differentiation of effector CTLs. We used MHC binding motifs and the knowledge of class I:peptide:TCR structure to design heteroclitic CTL vaccines that exploit the expression of poorly immunogenic tumor peptides. The in vivo potency of this approach was demonstrated using viral and self-(differentiation) antigens as models. First, a synthetic variant of a viral antigen was expressed as a tumor antigen, and heteroclitic immunization with peptides and DNA was used to protect against tumor challenge and elicit regression of 3-d tumors. Second, a peptide from a relevant self-antigen of the tyrosinase family expressed by melanoma cells was used to design a heteroclitic peptide vaccine that successfully induced tumor protection. These results establish the in vivo applicability of heteroclitic immunization against tumors, including immunity to poorly immunogenic self-proteins.

Expression of SSX genes in human tumors

Abstract: The HOM-MEL-40 antigen which is encoded by the SSX-2 gene was originally detected as a tumor antigen recognized by autologous IgG antibodies in a melanoma patient. Expression analysis demonstrated that SSX-2 is a member of the recently described cancer/testis antigen (CTA) class as it is expressed in a variety of different human neoplasms, but not in normal tissues with the exception of testis and a weak expression in the thyroid. Further studies demonstrated that SSX-2 belongs to a gene family consisting of at least 5 homologous genes. We now report the analysis of the expression of all 5 SSX genes in 325 specimens of human neoplasms from various histological origins, using reverse transcription polymerase chain reaction (RT-PCR). SSX-1, -2, and -4 were found to be expressed in 8%, 15% and 15%, of the tumors, respectively, while the expression of the SSX-5 gene was rare (7/325), and SSX-3 expression was not detected. For defined tumor types, expression of at least one of the SSX family members was most frequently observed in head and neck cancer (75%), followed by ovarian cancer (50%), malignant melanoma (43%), lymphoma (36%), colorectal cancer (27%) and breast cancer (23%), while leukemias and the few cases of leiomyosarcomas, seminomas and thyroid cancers were found not to express any SSX gene.

A Novel Hydrophobized Polysaccharide/Oncoprotein Complex Vaccine Induces in Vitro and in Vivo Cellular and Humoral Immune Responses against HER2-expressing Murine Sarcomas

Abstract: To elicit specific cellular immune responses against cancer, the development of efficient devices to deliver tumor antigen peptides to the MHC class I pathway constitutes a central issue. We report here a novel formula of hydrophobized polysaccharide nanoparticles, which can deliver a HER2 oncoprotein containing an epitope peptide to the MHC class I pathway. A protein consisting of the 147 amino-terminal amino acids of oncogene erbB-2/neu/HER2 (HER2) was complexed with two kinds of hydrophobized polysaccharides, cholesteryl group-bearing mannan (CHM) and cholesteryl group-bearing pullulan (CHP), to form nanoparticles (CHM-HER2 and CHP-HER2). CHM-HER2 and CHP-HER2 were able to induce CD3+/CD8+ CTLs against HER2-transfected syngeneic fibrosarcoma cell lines. In contrast, the oncoprotein alone failed to do so. These CTLs were Kd-restricted and specifically recognized a peptide (position 63-71) that was a part of a truncated HER2 protein used as an immunogen. In addition, vaccination by CHM-HER2 complexes led to a strongly enhanced production of IgG antibodies against HER2, whereas vaccination with HER2 proteins alone resulted in a production of antibodies at a marginal level. Mice immunized with CHM-HER2 or CHP-HER2 before tumor challenge successfully rejected HER2-transfected tumors. The complete rejection of tumors also occurred when CHM-HER2 was applied not later than 3 days after tumor implantation. In the effector phase of in vivo tumor rejection, CD8+ T cells played a major role. The results suggest that a sort of hydrophobized polysaccharide may help soluble proteins to induce cellular immunity as well enhance humoral immunity; hence, such a novel vaccine may be of potential benefit to cancer prevention and cancer therapy.

Cytotoxic T cell response against the chimeric ETV6-AML1 protein in childhood acute lymphoblastic leukemia.

Abstract: Cytotoxic T lymphocytes (CTL) are potent effector cells that could provide long term antitumor immunity if induced by appropriate vaccines. CTL recognize 8-14 amino acid-long peptides processed intracellularly and presented by MHC class I molecules. A well-characterized example of a potential tumor antigen in childhood pre-B Acute Lymphoblastic Leukemia (ALL) results from the chromosomal translocation 12;21 leading to the fusion of the ETV6 and AML1 genes. This translocation is observed in > 25% of ALL-patients. In this study, we have examined whether the chimeric ETV6-AML1 protein could serve as a tumor specific antigen for CTL in HLA-A2.1 individuals. We have identified a nonapeptide (RIAECILGM), encoded by the fusion region of the ETV6-AML1 protein, that binds to HLA-A2.1 molecules and induces specific primary CTL in peripheral blood lymphocytes from healthy donors. These CTL specifically lysed HLA-A2.1 tumor cells endogeneously expressing the ETV6-AML fusion protein. CTL with similar functional capacities were found with high frequencies and cloned from one patient's bone marrow indicating that ETV6-AML1-specific anti-ALL CTL are, at least in some patients, spontaneously stimulated and might participate to host antileukemia defense.

Optimization of the sequence of antigen loading and CD40-ligand-induced maturation of dendritic cells.

Abstract: Dendritic cells (DCs), matured by CD40-ligand (CD40L), undergo marked changes in their ability to process and present antigen, resulting in augmented lymphocyte stimulatory activity. We demonstrate that the form of the tumor antigen (peptide or genetic material) used to load the DCs dictates the required sequence of antigen loading and maturation for antitumor immunotherapy. Optimal stimulation of carcinoembryonic antigen (CEA)-specific CTLs by peptide-loaded DCs occurs when DCs from cancer patients are matured with CD40L before exposure to CEA peptide, whereas optimal stimulation by RNA-transfected DCs occurs when the DCs are loaded with CEA RNA before maturation with CD40L.

Naturally processed class II epitope from the tumor antigen MUC1 primes human CD4+ T cells.

Abstract: Epithelial cell mucin MUC1 is expressed on adenocarcinomas in an underglycosylated form that serves as a tumor antigen in breast, pancreatic, ovarian, and other tumors. Two predominant MUC1-specific immune responses are found in patients: CD8+ CTLs, which recognize tandemly repeated epitopes on the MUC1 protein core, and IgM antibodies. There have been no reports to date of MUC1-specific CD4+ T-helper cells in cancer patients. We show here that MUC1-specific CD4+ T cells are neither deleted nor tolerized and that CD4+ T cell responses can be generated when an appropriate soluble form of MUC1 is used. Naive CD4+ T cells from healthy donors were primed in vitro to a synthetic MUC1 peptide of 100 amino acids, representing five unglycosylated tandem repeats, presented by dendritic cells. They produced IFN-gamma and had moderate cytolytic activity. We identified one core peptide sequence, PGSTAPPAHGVT, that elicits this response when it is presented by HLA-DR3.

HER-2/neu oncogenic protein: issues in vaccine development.

Abstract: Vaccine studies using whole tumor cells or heterogeneous mixtures of tumor antigens provide intriguing evidence that cancer vaccines might be effective. Now it is possible to test vaccines composed of well-characterized proteins and peptides. Testing vaccine formulations composed of known and defined antigens will allow a more precise determination as to why vaccines work when they work, and why they do not work when they fail. The demonstration that human malignancy is immunogenic and the definition of human tumor antigens has set the stage for a new generation of cancer vaccines directly targeting immunogenic cancer-related peptides and proteins. Many newly defined tumor antigens are self proteins. As an example, screening existent immunity in human melanoma has identified responses to nonmutated self proteins: MAGE, MART, gp100, and tyrosinase. Tolerance to self antigens now emerges as a possible mechanism of tumor immune escape. A new puzzle has emerged for tumor immunologists to solve; how to harness immunity to "self" tumor antigens for cancer therapeutics.

Methods for identifying ligand specific binding molecules

Abstract: The present invention provides a method for identifying a binding molecule having selective affinity for a ligand. The method consists of selectively immobilizing a diverse population of binding molecules to a solid support, simultaneously contacting the diverse population immobilized on the solid support with two or more ligands and determining at least one binding molecule which selectively binds to one or more of the ligands. The invention additionally provides a method for identifying an antibody having selective affinity for a tumor antigen. The method consists of selectively immobilizing a diverse population of antibodies to a solid support, simultaneously contacting the diverse population immobilized on the solid support with two or more tumor antigens and determining at least one antibody which selectively binds to one or more of the tumor antigens. The invention also provides an isolated binding polypeptide selective for a tumor antigen. Further provided by the present invention is a Complementarity Determining Region (CDR) or functional fragment thereof of an antibody selective for a tumor antigen.

p53 in Tumor Progression: Life, Death, and Everything

Abstract: Publisher Summary The p53 tumor suppressor protein was first described in 1979 as a protein that binds SV40 virus large T antigen and independently as a tumor antigen. The chapter introduces p53 in tumor progression, and it focuses on the role of p53 in tumor progression. In particular, the mechanisms of p53's control of the cell cycle, genomic stability, and apoptosis are reviewed and the way loss of these functions plays a role in tumor cell progression. The chapter discusses the biochemical activities of p53. The chapter discusses p53 and cell cycle control and describes the control of G1/S by p53, control of G2/M by p53, and non transcriptional controls of cell cycle by p53. p53 and Apoptosis is discussed in the chapter, where p53 control of apoptosis in tumors, p53 transactivation and apoptosis, and non transactivator function in apoptosis by p53 are described in detail. The chapter describes p53 and tumor progression, apoptosis and tumor progression, growth control and tumor progression, and genomic instability and tumor progression.

Down regulation of major histocompatibility complex class I expression in mammary carcinoma of HER‐2/neu transgenic mice

Abstract: Transgenic mice carrying the HER-2/neu proto-oncogene under tissue-specific transcriptional control of a mammary tumor virus long terminal repeat (Tg-MMTVneu mice) spontaneously develop mammary carcinomas. HER-2/neu is a tumor antigen that can be recognized by cytotoxic T lymphocytes if tumor cells present the appropriate major histocompatibility complex (MHC) class I glycoproteins. The purpose of this work was to assess whether mammary carcinomas arising in Tg-MMTVneu mice correctly expressed MHC (H-2q) class I gene products. We analyzed by flow cytometry 51 primary tumors from 19 transgenic mice. About one-half of the tumors showed a reduced expression of class I antigens. All tumors were highly positive for membrane neu. Some mice had multiple mammary carcinomas with widely different MHC expression levels, and most mice had at least one tumor with a low expression. Treatment with γ-interferon of carcinoma cells cultured in vitro induced a strong re-expression of H-2q antigens. Our results suggest that the immune response activated in vivo by HER-2/neu-positive tumors can lead to the emergence of escape variants characterized by a down-regulation of MHC class I products. Int. J. Cancer 77:937–941, 1998.© 1998 Wiley-Liss, Inc.

The shared tumor-specific antigen encoded by mouse gene P1A is a target not only for cytolytic T lymphocytes but also for tumor rejection.

Abstract: A number of human tumor antigens have been characterized recently using cytolytic T lymphocytes (CTL) as screening tools. Some of them are encoded by MAGE-type genes, which are silent in normal tissues except in male germ cells, but are activated in a variety of tumors. These tumor-specific shared antigens appear to be promising targets for cancer immunotherapy. However, the choice of these antigens as targets has been questioned because of the lack of direct evidence that in vivo responses against such antigens can lead to tumor rejection. The antigen encoded by the mouse gene P1A represents the only available animal model system for MAGE-type tumor antigens. We show here that mice immunized by injection of L1210 leukemia cells expressing P1A and B7-1 (L1210.P1A.B7-1) are efficiently protected against a challenge with a lethal dose of mastocytoma P815 tumor cells, which express P1A. Mice immunized with L1210 cells expressing B7-1 but not P1A were not protected. Furthermore, we observed that P1A-transgenic mice, which are tolerant to P1A, were not protected after immunization with L1210.P1A.B7-1. These results demonstrate that the immune response to P1A is the major component of the tumor rejection response observed in normal mice, and support the use of tumor-specific shared antigens as targets for the immunotherapy of human cancer.

The clinical value of squamous cell carcinoma antigen in cancer of the uterine cervix

Abstract: A review is given of the clinical use and interpretation of serum tumor marker levels during the treatment of patients with cancer of the uterine cervix. Pretreatment serum squamous cell carcinoma (SC

Construction and characterization of a triple-recombinant vaccinia virus encoding B7-1, interleukin 12, and a model tumor antigen

Abstract: Tumor antigens that can induce specific cytotoxic T-lymphocyte responses to tumor cells have been identified and cloned (1-3). Tumors that express such antigens infrequently induce cytotoxic T-lymphocyte responses adequate for their destruction, possibly because they lack essential immunologic cofactors. Two such factors, B7-1 (4,5) and interleukin 12 (IL-12) (6), have been expressed separately in recombinant vaccinia virus and appear to improve the efficacy of tumor immunotherapy. Vaccinia virus is a well-characterized expression vector (7,8) that has been used to express a wide variety of recombinant proteins (9). Furthermore, this virus is an ideal vector for the induction of efficient cytotoxic T-lymphocyte responses to recombinant proteins (10), inducing specific protective and therapeutic immune responses to both viral and cellular tumor antigens (11-13). The costimulatory molecule B7-1 is found on the surface of professional antigen-presenting cells, such as dendritic cells, and interacts with its ligands CD28 and cytotoxic T-lymphocyte antigen-4 expressed on most T cells. The simultaneous interactions of 1) the complex containing the peptide and the major histocompatibility complex with a specific T-cell receptor and of 2) B7-1 with CD28 are essential for the effective stimulation of antigen-specific cytotoxic T lymphocytes, mediated, in part, by the up-regulation and stabilization of interleukin 2 messenger RNA (14). Stimulation via the T-cell receptor without costimulation can result in T-cell anergy or apoptosis. Although many tumors express major histocompatibility complex class I molecules and are able to present antigen, most do not provide costimulation. Transfection of tumor cells with B7-1 can effectively stimulate immunotherapeutic responses (15). The cytokine IL-12 is a heterodimer composed of two glycoproteins, p40 and p35, and is expressed primarily by activated B cells, monocytes, and macrophages (16). This immunomodulatory cytokine has a variety of functions, including the induction of nonspecific natural killer cells and the maturation of CD8+ T cells into antigen-specific cytotoxic T lymphocytes (16). In addition, IL-12 can stimulate type 1 CD4+ helper T cells that can lead to the production of interferon gamma and the induction of a cell-mediated response that is essential in the vaccine-based immunotherapy of established tumors. Inoculation of mice with a combination of irradiated tumor cells and fibroblast cells expressing IL-12 induced partial protection to subsequent challenge with nonirradiated tumor cells, thus implicating IL-12 as a potential immune adjuvant in the immunotherapy of cancer (17). IL-12 given exogenously at high levels has also been shown to have effective antitumor activity in animal models (18). Several reports (19-21) indicate that the effects of IL-12 are greatly enhanced when B7-1 and IL-12 are added together. To create a cytokine microenvironment favorable to the antigen-specific activation of cytotoxic T lymphocytes, we examined the efficacy of IL-12 when endogenously expressed by a recombinant vaccinia virus or administered exogenously alone or in combination with B7-1, which augments antitumor responses (5). We have built a series of recombinant viruses that contain as many as five transgenes inserted into three loci of the viral genome to test the effectiveness of recombinant immunogens expressing a model antigen, IL-12, and B7-1. We tested these immunogens by treating a murine colon carcinoma cell line that had been transduced with a model tumor antigen, Escherichia coli β-galactosidase, to create a tumor line called CT26.C25 (22-24). The construction of these complex viruses required the development of a cassette system that uses three loci within the vaccinia viral genome—loci for hemagglutinin, thymidine kinase, and viral protein 37. We inserted up to five foreign genes into the viral genome by homologous recombination. The genes inserted were as follows: E. coli lacZ (that encodes β-galactosidase, the model antigen), E. coli gpt (xanthine-guanine phosphoribosyltransferase, a selection gene), murine B7-1, and the two subunits of murine IL-12 (p35 and p40). In this study, we explored the toxicity and efficacy of a triple-recombinant vaccinia virus expressing a model tumor-associated antigen alone or in combination with IL-12 and B7-1. We explored the possibility that recombinant viruses encoding specific target antigens and multiple immunostimulatory molecules may enhance the design of recombinant immunogens.

High homogeneity of MAGE, BAGE, GAGE, tyrosinase and Melan-A/MART-1 gene expression in clusters of multiple simultaneous metastases of human melanoma: implications for protocol design of therapeutic antigen-specific vaccination strategies.

Abstract: Human melanoma cells express several antigens which are recognized by autologous and specific CTL clones in association with HLA-class-I molecules. Many of these antigens represent suitable targets for tumor immunotherapy, since their expression in human melanoma cells is common and highly specific. In order to achieve real clinical success with therapeutic vaccination strategies, one important requirement is the expression of the target antigen by all the tumor lesions of a patient. We have studied this issue by assessing, through an RT-PCR approach, the expression of MAGE-1, MAGE-2, MAGE-3, BAGE, GAGE-1/2, Tyrosinase and Melan-A/MART-1 genes in 17 clusters of simultaneous in-transit or regional lymph-node metastases collected from 15 stage-III and 1 stage-IV (AJCC/UICC pTNM system) melanoma patients. In 14 out of 17 clusters of simultaneous metastatic lesions (82%), the homogeneity in the pattern of gene expression within the cluster was complete. Heterogeneity within the same cluster was observed in only 3 out of 17 clusters (18%) and represented only minor features. Our data reveal that, in AJCC-stage-III melanoma patients, different but simultaneous metastatic lesions express the same pattern of antigen-coding genes. These observations have 2 main clinical implications: (i) the antigenic characterization of one single and easily accessible lesion allows identification of optimal targets for an active antigen-specific immunotherapy treatment; (ii) almost all the metastatic lesions are expected to be hit by the immune response eventually induced against the tumor antigen. Moreover, these data suggest that active specific immunotherapy directed against MAGE-1, MAGE-3, BAGE, GAGE-1/2, Melan-A/MART-1 and Tyrosinase antigens could be exploited as an adjuvant treatment to surgery in high-risk AJCC-stage-III-melanoma patients. Int. J. Cancer 77:200–204, 1998.© 1998 Wiley-Liss, Inc.

Irradiated tumor cells adenovirally engineered to secrete granulocyte/macrophage-colony-stimulating factor establish antitumor immunity and eliminate pre-existing tumors in syngeneic mice

Abstract: The specific aim of this study was to examine the prophylactic as well as the therapeutic efficacies of irradiated mouse CT26 colon cancer cells, infected with recombinant adenoviruses harboring cDNAs specific for granulocyte macrophage-colony-stimulating factor (GM-CSF), interferon (IFN-gamma) and monocyte chemotactic protein1 (MCP-1). Results showed that tumor cells secrete the respective cytokines for several days after infection and subsequent irradiation. Vaccination with irradiated GM-CSF-secreting CT26 cells protected 90% of syngeneic mice challenged with live parental cells. On the other hand, vaccination with irradiated IFNgamma or MCP-1-secreting CT26 cells totally failed to protect mice from tumor development after challenge with parental cells. None of the tumor-free mice initially vaccinated with irradiated GM-CSF-producing CT26 cells developed tumor upon repeated challenge with parental cells during the entire observation period. The establishment of specific and long-lasting antitumor immunity following vaccination with GM-CSF-producing tumor cells requires the simultaneous presence of GM-CSF and tumor antigen at the vaccine site. Depletion of CD8+ cells, but not CD4+ cells, blocked the vaccine efficacy of GM-CSF-producing tumor cells. Subcutaneous injection of irradiated GM-CSF-producing CT26 cells also effectively prevented the growth of a small load of parental tumor that was implanted 3 days earlier or the development of metastatic foci in the lung from intravenously injected parental cells either 7 days before or 3 days after vaccination. Our data thus show that, in these experimental tumor models, subcutaneous injection of irradiated tumor cells adenovirally, transduced with the GM-CSF gene leads not only to prevention of growth of subsequently implanted tumor but also to elimination of pre-existing and metastatic tumors.

Tumor antigen presentation: changing the rules.

Abstract: Cell-based tumor vaccines have been developed on the basis of the hypothesis that tumor cells can be genetically modified to present antigen to T lymphocytes directly. Contrary to expectations, cross-priming is the predominant pathway for activation of tumor-specific CD8+ T cells, while direct presentation of antigen dominates activation of tumor-specific CD4+ T cells. These results pose interesting paradoxes for the generation of immune responses, and have definite implications for the development of anti-cancer vaccines.

Development of a retrovirus-based complementary DNA expression system for the cloning of tumor antigens.

Abstract: A new retroviral system has been developed for the generation of a cDNA library and the functional cloning of tumor antigens. These retroviral vectors contain a cytomegalovirus promoter in the 5′ long terminal repeat, an extended packaging signal for rapid production of high-titer retroviral particles, and many convenient cloning sites for cDNA library construction. The vesicular stomatitis virus G protein has been used to generate pseudotype retroviral particles to enable efficient viral infection. Using this system, viral titers in the range of 106 colony-forming units/ml could be generated routinely, and a high transduction efficiency in human primary cells, including fibroblasts, was achieved. In addition, a new procedure has been devised for screening a retrovirus-based cDNA library without a functional selection. The utility of this system was demonstrated by constructing a retrovirus-based cDNA library and re-isolating the NY-ESO-1 tumor antigen from a cDNA library using an antigen-specific CTL. This approach can facilitate the identification of novel tumor antigens recognized by T cells without knowledge of MHC class I restriction elements and is generally applicable for the isolation of any gene as long as a biological assay is available.

Tumor pretargeting for radioimmunodetection and radioimmunotherapy

Abstract: UNLABELLED The limited success of the sole use of monoclonal antibodies for cancer detection and treatment has led to the development of multistep methods using antibodies in conjunction with low molecular weight agents. For tumor pretargeting, it is important to optimize dose and schedule of relevant agents and to understand barriers to targeted delivery. Here, we address these issues for the anti-carcinoembryonic antigen bifunctional antibody-hapten and the streptavidinylated antibody-biotin systems using a recently developed physiologically based pharmacokinetic model. METHODS For baseline conditions of a standard 70-kg man with a 20-g tumor embedded in the liver, the model was used in conjunction with the Medical Internal Radiation Dosimetry schema to: estimate absorbed doses in tumor and normal tissues; determine the dose dependence of effector agent accumulation in tumor; simulate tumor-to-background effector agent uptake ratio; and calculate the therapeutic ratio for different antibody forms and radionuclides. Alternative drug administration schemes and variable tumor physiological conditions were considered. RESULTS Model simulations showed that 131I-labeled biotin with the streptavidinylated F(ab')2 provided the highest therapeutic ratio under the optimized conditions. The simulations also showed that biotin with the bifunctional streptavidinylated immunoglobulin G provided the highest tumor-to-liver uptake ratio during the early period. Sensitivity analysis showed that antibody extravasation was the major factor limiting the accretion of the effector agent in tumor, whereas antigen expression in normal tissues and tumor antigen shedding had little effect on the absorbed doses. CONCLUSION Tumor pretargeting should provide a definite advantage over direct antibody targeting with up to a 200% increase in tumor-to-background ratio in radioimmunodetection and up to a 76% increase in tumor-to-bone marrow therapeutic ratio in radioimmunotherapy. Rapid antibody clearance from the bloodstream before effector agent injection is expected to improve the therapeutic ratio marginally (3%-10%). However, continuous plasmapheresis dramatically increased the tumor-to-background ratio by a factor of 10 in RAID and the tumor-to-bone marrow therapeutic ratio by more than 110% for short-lived radionuclides in RAIT. Apart from drastic measures such as extended plasmapheresis, pretargeting selectivity was neither sensitive enough for radioimmunodetection nor effective enough for radioimmunotherapy in patients with typical solid tumors even using the optimized protocols.

Peptide mimics of a tumor antigen induce functional cytotoxic T cells

Abstract: The ability to mimic peptide/peptide and/or peptide/carbohydrate structures may be important in generating cross-reactive antibodies for autoimmune and other diseases. We show that the peptide sequence DAHWESWL can mimic the conformation of the unrelated MUC1 peptide SAPDTRPAP(G). Mice immunized with mannan-MUC1-peptides make cytotoxic T lymphocytes (CTLs) and are protected from MUC1+ tumors. We show that the same specific anti-MUCI responses can be produced by immunizing with the DAHWESWL peptide; furthermore, specific tumor protection is obtained in a manner similar to that with MUC1 immunization. The DAHWESWL peptide immunization leads to CTLs that recognize H2Dd and H2Ld but not H2b or human leukocyte antigens–group A (HLA-A)*0201 presented MUC1 peptides. However, mutation of the DAHWESWL peptide to a more HLA-A*0201–compatible structure with appropriate anchors (DLHWASWV), leads to the production of CTLs in HLA-A*0201 mice.

Class II-transfected tumor cells directly present endogenous antigen to CD4+ T cells in vitro and are APCs for tumor-encoded antigens in vivo.

Abstract: We have previously demonstrated that class II-transfected tumor cells are very effective immunogens that protect against wild-type primary and metastatic tumor and, if supertransfected with genes encoding co-stimulatory molecules, are immunotherapeutic agents that successfully treat mice with established solid tumor. These results are consistent with our hypothesis that the class II-transfected tumor cells act as antigen-presenting cells (APCs) that directly activate tumor-specific CD4+ T cells; however, direct data supporting this hypothesis are lacking. In the present study, we test this hypothesis using class II-transfected tumor cells supertransfected with the hen egg lysozyme gene as a surrogate tumor antigen. In vitro antigen presentation assays demonstrate that class II-transfected tumor cells present to CD4+ T cells endogenously encoded tumor antigen, provided they do not co-express the class II-associated invariant chain. In vivo experiments using genetically marked tumor cells and host APCs demonstrate that both class II-transfected tumor cells and host cells are APCs for tumor-encoded antigens, although tumor cells appear to dominate the response. These results support the hypothesis that the immunogenicity and therapeutic value of class II-transfected tumor cells stem from their ability to function as APCs for tumor-encoded antigens and directly activate tumor-specific CD4+ T lymphocytes.

Stuck in the MUC on the long and winding road.

Abstract: Antibodies that cross react with the tumor antigen MUC1 switch a cellular immune response to a humoral one with implications for the immunotherapy of cancer (pages 315–320).