Showing papers on "Tumor antigen published in 1997"

Immunostimulatory oligodeoxynucleotides containing the CpG motif are effective as immune adjuvants in tumor antigen immunization

Abstract: Recent advances in our understanding of the immune response are allowing for the logical design of new approaches to cancer immunization. One area of interest is the development of new immune adjuvants. Immunostimulatory oligodeoxynucleotides containing the CpG motif (CpG ODN) can induce production of a wide variety of cytokines and activate B cells, monocytes, dendritic cells, and NK cells. Using the 38C13 B cell lymphoma model, we assessed whether CpG ODN can function as immune adjuvants in tumor antigen immunization. The idiotype served as the tumor antigen. Select CpG ODN were as effective as complete Freund’s adjuvant at inducing an antigen-specific antibody response but were associated with less toxicity. These CpG ODN induced a higher titer of antigen-specific IgG2a than did complete Freund’s adjuvant, suggesting an enhanced TH1 response. Mice immunized with CpG ODN as an adjuvant were protected from tumor challenge to a degree similar to that seen in mice immunized with complete Freund’s adjuvant. We conclude that CpG ODN are effective as immune adjuvants and are attractive as part of a tumor immunization strategy.

A CASP-8 Mutation Recognized by Cytolytic T Lymphocytes on a Human Head and Neck Carcinoma

Abstract: Of the antigens recognized on human tumors by autologous cytolytic T lymphocytes, all those defined thus far have been identified on melanoma or renal cell carcinoma. We report here the identification of an antigen recognized by autologous cytolytic T lymphocytes on a human squamous cell carcinoma of the oral cavity. The antigen is encoded by a mutated form of the CASP-8 gene. This gene, also named FLICE or MACH, codes for protease caspase-8, which is required for induction of apoptosis through the Fas receptor and tumor necrosis factor receptor-1. The mutation, which was found in the tumor cells but not in the normal cells of the patient, modifies the stop codon and adds an Alu repeat to the coding region, thereby lengthening the protein by 88 amino acids. The ability of the altered protein to trigger apoptosis appears to be reduced relative to the normal caspase-8. The antigenic peptide is a nonamer presented by HLA-B*3503. The five last amino acids are encoded by the extension of the reading frame caused by the mutation. This, together with previous observations of CDK4 and beta-catenin mutations, suggests that a significant fraction of the point mutations generating a tumor antigen also play a role in the tumoral transformation or progression.

Self Antigens Expressed by Solid Tumors Do Not Efficiently Stimulate Naive or Activated T Cells: Implications for Immunotherapy

Abstract: Induction and maintenance of cytotoxic T lymphocyte (CTL) activity specific for a primary endogenous tumor was investigated in vivo. The simian virus 40 T antigen (Tag) expressed under the control of the rat insulin promoter (RIP) induced pancreatic β-cell tumors producing insulin, causing progressive hypoglycemia. As an endogenous tumor antigen, the lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) was introduced also under the control of the RIP. No significant spontaneous CTL activation against GP was observed. However, LCMV infection induced an antitumor CTL response which efficiently reduced the tumor mass, resulting in temporarily normalized blood glucose levels and prolonged survival of double transgenic RIP(GP × Tag2) mice (137 ± 18 d) as opposed to control RIP-Tag2 mice (88 ± 8 d). Surprisingly, the tumor-specific CTL response was not sustained despite the facts that the tumor cells continued to express MHC class I and LCMV-GP–specific CTLs were present and not tolerized. Subsequent adoptive transfer of virus activated spleen cells into RIP(GP × Tag2) mice further prolonged survival (168 ± 11 d), demonstrating continued expression of the LCMV-GP tumor antigen and MHC class I. The data show that the tumor did not spontaneously induce or maintain an activated CTL response, revealing a profound lack of immunogenicity in vivo. Therefore, repetitive immunizations are necessary for prolonged antitumor immunotherapy. In addition, the data suggest that the risk for induction of chronic autoimmune diseases is limited, which may encourage immunotherapy against antigens selectively but not exclusively expressed by the tumor.

Regression of tumors in mice vaccinated with professional antigen-presenting cells pulsed with tumor extracts.

Abstract: Vaccination with tumor extracts circumvents the need to identify specific tumor rejection antigens and extends the use of active immunotherapy to the vast majority of cancers, in which specific tumor antigens have not yet been identified. In this study we examined the efficacy of tumor vaccines comprised of unfractionated tumor material presented by professional antigen-presenting cells (APC): dendritic cells (DC) or macrophages (Mo). To enhance the relevance of these studies for human patients we used 2 poorly immunogenic murine tumor models and evaluated and effectiveness of the vaccination protocols in tumor-bearing animals. APC (in particular DC) pulsed with unfractionated extracts from these “poorly immunogenic” tumors were highly effective in eliciting tumor-specific cytotoxic T lymphocytes. A measurable CTL response could be detected after even a single immunization with tumor extract-pulsed DC. DC or Mo pulsed with tumor extract were also effective vaccines in tumor-bearing animals. In the murine bladder tumor (MBT-2) model a modest extension of survival and 45% cure rate was seen in the animal groups immunized with DC or Mo pulsed with MBT-2 tumor extract. DC or Mo pulsed with B16/F10.9 tumor extract were also remarkably effective in the B16 melanoma lung metastasis model, as shown by the observation that treatment with APC caused a significant reduction in lung metastases. Cumulatively, the CTL and immunotherapy data from the two murine tumor systems suggest that APC (in particular DC) pulsed with unfractionated cell extracts as a source of tumor antigen may be equally or more effective than genetically modified tumor vaccines. Int. J. Cancer 70:706–715, 1997. © 1997 Wiley-Liss, Inc.

HER-2/neu protein: a target for antigen-specific immunotherapy of human cancer.

Abstract: The HER-2/neu oncogenic protein is a tumor antigen. Some patients with cancer have a preexistent immune response directed against the HER-2/neu protein. Effective cancer vaccines targeting HER-2/neu will be able to boost this immunity to potentially therapeutic levels. In addition, HER-2/neu-directed monoclonal antibody therapy has been effective in eradicating malignancy in animal models and has shown benefit in the treatment of human HER-2/neu-overexpressing cancers. This review outlines studies that define HER-2/neu-specific immunity in patients with cancer and overviews the current vaccine strategies for generating or augmenting neu-specific immunity. The potential problems associated with eliciting HER-2/neu-specific immunity are addressed, including the question of precipitating autoimmune toxicity against this "self" -protein and the mechanisms of immunological escape that may play a role in preventing effective function of the HER-2/neu-specific immune response. Finally, antibody-based HER-2/neu-directed therapies are overviewed. HER-2/neu is a prototype antigen for groups investigating innovative modifications of monoclonal antibody technology, and cutting edge therapies targeting this antigen are being contemplated for clinical use in the treatment of human malignancy. Immune-based treatments designed to target the HER-2/neu oncogenic protein will soon give the clinical oncologist new therapeutic weapons, directed against a biologically relevant tumor-related protein, with which to fight cancer.

Identification of subdominant CTL epitopes of the GP100 melanoma-associated tumor antigen by primary in vitro immunization with peptide-pulsed dendritic cells.

Abstract: The gp100 melanoma-associated tumor Ag was selected as a model system to study the diversity of human antitumor cytotoxic T cell responses. First, peptides corresponding to dominant gp100 HLA-A2.1-restricted CTL epitopes were tested using lymphocytes from normal volunteers and an in vitro priming protocol that uses peptide-pulsed dendritic cells as APCs and IL-7 and IL-10 as immune-enhancing cytokines. High CTL activity toward both peptide-pulsed target cells and gp100+ melanoma cells was obtained with four out of five peptides tested. Second, HLA-A2.1-binding peptides from gp100 that do not appear to represent CTL epitopes in melanoma patients were also tested for their capacity to induce CTL using the in vitro priming protocol. Three of six peptides tested induced CTL in lymphocytes from normal volunteers. One of these peptides was also immunogenic for lymphocytes derived from a melanoma patient in remission. Because these three CTL epitopes were not recognized in the natural immune response in melanoma patients but do appear as immunogens when peptides are used to induce the T cell response, they may be considered as typical "subdominant" epitopes. The results are discussed in the context of the usefulness of this approach to detail the immunologic potential of a given tumor-associated Ag and its relevance for the design of effective immune-based therapies.

Tumor‐infiltrating dendritic cells are defective in their antigen‐presenting function and inducible B7 expression in rats

Abstract: Tumors are tolerated by the immune system notwithstanding the expression of tumor-associated antigens. PROb tumor cells, derived from a rat colon carcinoma, are rejected by tumor-immune hosts but give rise to progressive tumors in naive hosts. Paradoxically, these tumors are heavily infiltrated by dendritic cells that express MHC class II and ICAM-1. These tumor-infiltrating dendritic cells (TiDCs) could be expected to process and present to T cells the antigens released by the adjacent tumor cells. Indeed, we report here that TiDCs, compared with splenic dendritic cells, are poor stimulators of primary allogeneic T-cell proliferation and cytokine [interleukin-2 (IL-2) and interferon-gamma] production. Most of them (89-97%) do not express B7, an essential co-stimulatory signal for T cells, even after a culture period allowing B7 up-regulation on epidermal Langerhans cells. GM-CSF in association with tumor necrosis factor-alpha or IL-4, or cell-associated CD40-ligand, all known to be potent stimulators of B7 expression on other dendritic cells, did not restore B7 expression by TiDCs. After a first exposure to TiDCs, allogeneic T-cell response to a second challenge to splenic dendritic cells was decreased. The failure of most dendritic cells infiltrating PROb tumors to express B7, even after stimulation, may contribute to their poor capacity to stimulate T cells and could play a role in the immune tolerance allowing tumor growth.

Molecular detection of tumor-associated antigens shared by human cutaneous melanomas and gliomas.

Abstract: Both melanocytes and glial cells are derived embryologically from the neural ectoderm. Their malignant transformed counterparts, melanoma and glioma cells, respectively, may share common antigens. Numerous tumor-associated antigens have been identified in melanomas but only a few a gliomas. Using an established reverse transcriptase polymerase chain reaction plus Southern blot assay, we compared the mRNA expression of melanoma-associated antigens (MAAs) of melanomas to brain tumors primarily derived from glial cells. The MAAs studied included tyrosinase (Tyr), tyrosinase-related protein-1 and -2 (TRP-1 and TRP-2), gp100, human melanoma antigen-encoding genes 1 and 3 (MAGE-1 and MAGE-3), and melanotransferrin (p97). Glioblastoma multiforme (n = 21), anaplastic astrocytoma (n = 3), ependymoma (n = 2), meningioma (n = 3), oligodendroglioma (n = 1), and melanoma (n = 12) tumor specimens were assayed for MAA mRNA expression. Glioblastoma multiforme, astrocytoma, and melanoma cell lines were also assayed. We observed that individual MAA mRNAs were expressed in these brain tumors and cell lines at varying frequencies. The melanogenesis-pathway-related MAAs Tyr, TRP-1, TRP-2, and gp100 mRNAs were also expressed at different levels in normal brain tissues but at a much lower frequency than in glioblastoma multiforme and melanoma. MAGE-1 and MAGE-3 mRNA were expressed in different types of tumor specimens and cell lines but never in normal brain tissue. Tumor antigen p97 was expressed in all types of tumors and also in normal brain tissues. These studies demonstrate that melanomas and primary brain tumors express common MAAs and could be exploited in patients with malignant glioma by active specific immunotherapy against these common MAAs.

Dendritic cells transduced with an adenoviral vector encoding a model tumor-associated antigen for tumor vaccination.

Abstract: Evaluation of the potential role of dendritic cells (DCs) as adjuvants for tumor vaccination has focused primarily on techniques that load DCs with peptide tumor antigens. Our aim has been to optimize the induction of antitumor immunity by enhancing the ability of DCs to present tumor-associated antigens endogenously to the afferent lymphatic system in the appropriate major histocompatibility complex (MHC)-restricted context. We have used replication-defective adenovirus vectors (Ads) to transduce DCs with various genes, including tumor antigen genes. We found that 90% of murine bone marrow derived-DCs could be infected with an Ad vector expressing the β-galactosidase gene and still retain their physiologic and phenotypic characteristics. Furthermore, we demonstrated that transgene expression was detectable in the spleen for at least 3 days following intravenous injection of Ad-transduced DCs. Using a polyoma middle T (PymT) transgenic murine mammary carcinoma model, we have shown that a single i...

Bone Marrow-Derived Dendritic Cells Serve as Potent Adjuvants for Peptide-Based Antitumor Vaccines

Abstract: Dendritic cells (DCs) are considered the most effective antigen-presenting cells (APCs) for primary immune responses. Since presentation of antigens to the immune system by appropriate professional APCs is critical to elicit a strong immune reaction and DCs seem to be quantitatively and functionally defective in the tumor host, DCs hold great promise to improve cancer vaccines. Even though they are found in lymphoid organs, skin and mucosa, the difficulty of generating large numbers of DCs has been a major limitation for their use in vaccine studies. A simple method for obtaining DCs from mouse bone marrow cells cultured in the presence of GM-CSF + interleukin 4 is now available. In four different tumor models, mice injected with DCs grown in GM-CSF plus interleukin 4 and prepulsed with a cytotoxic T lymphocyte-recognized tumor peptide epitope developed a specific cytotoxic T lymphocyte response and were protected against a subsequent tumor challenge with tumor cells expressing the relevant tumor antigen. Moreover, treatment of day 5-14 tumors with peptide-pulsed DCs resulted in sustained tumor regression in five different tumor models. These results suggest that presentation of tumor antigens to the immune system by professional APCs is a promising method to circumvent tumor-mediated immunosuppression and is the basis for ongoing clinical trials of cancer immunotherapy with tumor peptide-pulsed DCs.

Pre-existing immunity to adenovirus does not prevent tumor regression following intratumoral administration of a vector expressing IL-12 but inhibits virus dissemination

Abstract: Adenovirus (Ad) vectors are being intensively studied as vehicles for cancer gene therapy. We have been exploring the benefits of direct intratumoral injection of Ads expressing cytokines for immunotherapy. Our previous work demonstrated that therapy using a vector expressing interleukin-12 (AdmIL-12.1) produced regressions in approximately 80% of treated tumors supporting further preclinical investigations. Recent reports have shown that immunity to Ad can be a major limiting factor in Ad-mediated gene transfer. As most animal studies with Ad vectors have involved nonimmune hosts, it remains difficult to predict how effective these treatments will be in humans, where the majority of individuals have had previous exposure to Ad. To address this question, we compared the effectiveness of the AdmIL-12.1 cancer therapy in naive and Ad-immune mice. We found that both groups responded equally well to treatment and that the response to AdmIL-12.1 in both groups resulted in the generation of CTL reactive against tumor antigen indicating that antitumor immunity was achieved. Peak transgene expression in the tumor was only reduced by 2.4 fold in Ad-immune animals compared with nonimmune mice. It was also observed that in naive animals, the virus disseminated from the site of the tumor following injection and by 72 h substantial transgene expression was detected in peripheral organs, most notably the liver. Transgene expression in the liver of Ad-immune animals was reduced by greater than 1000-fold relative to that in naive mice. These results strongly support the clinical utility of Ad-based cancer gene therapy and suggest that Ad immunity may be advantageous in that it is not a complete block to gene transfer in the tumor and it greatly reduces virus dissemination.

Cloning and characterization of the genes encoding the murine homologues of the human melanoma antigens MART1 and gp100.

Abstract: The recent identification of genes encoding melanoma-associated antigens has opened new possibilities for the development of cancer vaccines designed to cause the rejection of established tumors. To develop a syngeneic animal model for evaluating antigen-specific vaccines in cancer therapy, the murine homologues of the human melanoma antigens MART1 and gp100, which were specifically recognized by tumor-infiltrating lymphocytes from patients with melanoma, were cloned and sequenced from a murine B16 melanoma cDNA library. The open reading frames of murine MART1 and gp100 encode proteins of 113- and 626-amino acids with 68.8 and 77% identity to the respective human proteins. Comparison of the DNA sequences of the murine MART1 genes, derived from normal melanocytes, the immortalized nontumorgenic melanocyte line Melan-a and the B16 melanoma, showed all to be identical. Northern and Western blot analyses confirmed that both genes encoded products that were melanocyte lineage proteins. Mice immunized with murine MART1 or gp100 using recombinant vaccinia virus failed to produce any detectable T-cell responses or protective immunity against B16 melanoma. In contrast, immunization of mice with human gp100 using recombinant adenoviruses elicited T cells specific for hgp100, but these T cells also cross reacted with B16 tumor in vitro and induced significant but weak protection against B16 challenge. Immunization with human and mouse gp100 together [adenovirus type 2 (Ad2)-hgp100 plus recombinant vaccinia virus (rVV)-mgp100], or immunization with human gp100 (Ad2-hgp100) and boosting with heterologous vector (rVV-hgp100 or rVV-mgp100) or homologous vector (Ad2-hgp100), did not significantly enhance the protective response against B16 melanoma. These results may suggest that immunization with heterologous tumor antigen, rather than self, may be more effective as an immunotherapeutic reagent in designing antigen-specific cancer vaccines.

Preferential expression of novel MUC1 tumor antigen isoforms in human epithelial tumors and their tumor-potentiating function

Abstract: The human MUC1 gene expresses at least 2 type 1 membrane proteins: MUC1/REP, a polymorphic high m.w. MUC1 glycoprotein often highly expressed in breast cancer tissues and containing a variable number of tandem 20 amino acid repeat units, and the MUC1/Y protein, which lacks this repeat array and, therefore, is not polymorphic. Despite their documented importance in signal transduction processes, the relative expression of the 2 isoforms in epithelial tumors is unknown. Using antibody reagents which recognize different MUC1 domains, the expression of these isoforms in malignant epithelial cells has been evaluated. A comparison of the amounts of the 2 isoforms revealed preferential expression of the novel MUC1/Y protein in breast cancer tissue samples. Furthermore, although the MUC1/REP protein is almost undetectable in HeLa cervical adenocarcinoma epithelial cells, the MUC1/Y isoform is extensively expressed in these cells. The presence of the MUC1/Y sequence as well as that of an additional tandem-repeat-array-lacking isoform, designated MUC1/X, were demonstrated by reverse transcriptase PCR amplification of RNA extracted from HeLa and ovarian carcinoma cells. It has been shown previously that the MUC1 cytoplasmic domain interacts with the SH2 domain containing GRB2 protein, which transduces signals to ras, a protein which in its activated form can lead to cell transformation. We present here data demonstrating that MUC1/Y isoform expression increases the tumorigenic potential of DA3 mouse mammary epithelial cells; in contrast, potentiation of tumorigenicity is not observed with MUC1/REP expression. Our studies thus demonstrate that expression of the MUC1 gene in epithelial tumors can give rise to substantial levels of MUC1 proteins devoid of the tandem repeat array, which are generated by alternative splicing mechanisms.

Genomic cloning and localization of CTAG, a gene encoding an autoimmunogenic cancer-testis antigen NY-ESO-1 to human chromosome Xq28

Abstract: CTAG was initially cloned from an esophageal squamous cell carcinoma cDNA expression library by immunoscreening with autologous patient's serum. CTAG mRNA is expressed in a proportion of human cancers in a lineage-nonspecific fashion, whereas its expression in normal tissues is restricted to testis and ovary only. This expression pattern suggests that the CTAG product (NY-ESO-1) is an aberrantly activated tumor antigen and can potentially be an antigenic target for tumor vaccination. In the present study, we isolated human genomic clones of CTAG and established its genomic organization. By somatic cell hybrid studies and fluorescence in-situ hybridization, we localized this gene to chromosome Xq28, a region that also contains members of MAGE, a gene family that encodes several immunogenic tumor antigens with the characteristic cancer/testis expression pattern.

Tissue-specific expression of the human prostate-specific antigen gene in transgenic mice: implications for tolerance and immunotherapy.

Abstract: Human prostate-specific antigen (PSA) has been widely used as a serum marker for cancer of the prostate. The cell type-specific expression of PSA also makes it a potential tumor antigen for prostate cancer immunotherapy. Study of the immunological aspects of PSA within either normal or malignant prostate tissue has been hampered by the lack of a mouse model, because no PSA counterpart has been identified in mice. Using a 14-kb genomic DNA region that encompasses the entire human PSA gene and adjacent flanking sequences, we generated a series of human PSA transgenic mice. In the six independent lines of transgenic mice generated, the expression of the human PSA transgene, driven by its own cis-acting regulatory elements, is specifically targeted to the prostate. Tissue distribution analysis demonstrated that PSA transgene expression closely follows the human expression pattern. Immunohistochemical analysis of the prostate tissue also showed that the expression of the PSA transgene is confined to the ductal epithelial cells. Despite expressing PSA as a self-antigen in the prostate, these transgenic mice were able to mount a cytotoxic immune response against PSA expressed by tumor cells, indicating that expression of the transgene has not resulted in complete nonresponsiveness. This transgenic mouse model will provide a well defined system to gain an insight into the mechanisms of nonresponsiveness to PSA, ultimately leading to strategies for immunotherapy of human prostate cancer using PSA as the target antigen.

Influence of gene-modified (IL-7, IL-4, and B7) tumor cell vaccines on tumor antigen presentation.

Abstract: Tumor cells genetically modified to coexpress certain cytokines (such as IL-7 or IL-4) and B7.1 have increased immunogenicity. Since tumor Ags can be presented either directly by tumor cells or indirectly by host APC (cross-priming), we asked whether B7.1 and IL-7 or IL-4 complemented each other by improving preferentially one or both pathways of Ag presentation. We used TS/A (H-2d) tumor cells and their IL-7, B7, and IL-7/B7 transfectants, and MCA205 (H-2b) tumor cells and their IL-4 and B7 transfectants. beta-galactosidase (beta-gal) was chosen as surrogate tumor Ag. beta-gal has different predominant MHC class I epitopes in H-2d and H-2b mice. Immunization of (H-2b x d)F1 mice with TS/A/beta-gal transfectants showed that both IL-7 and B7.1 and, as control, granulocyte-macrophage CSF augmented cross-priming and rejection of a challenge with MCA205/beta-gal (H-2b). Similarly, immunization with MCA205/beta-gal B7.1 or IL-4 transfectants enhanced cross-priming and rejection of a challenge with TS/A/beta-gal. beta-gal-specific rejection was confirmed by CTL assay. However, direct Ag presentation by tumor cells was enhanced only by B7.1, and not IL-7. For this study, H-2b nu/nu mice reconstituted with F1 lymphocytes were immunized with H-2d TS/A/beta-gal transfectants and challenged with TS/A/beta-gal. In conclusion, indirect Ag presentation was augmented by B7, IL-7, and IL-4, while direct Ag presentation was improved only by B7.

Genetic Immunotherapy of Established Tumors with Adenovirus-Murine Granulocyte-Macrophage Colony-Stimulating Factor

Abstract: Increased local production of granulocyte-macrophage colony-stimulating factor (GM-CSF) by genetically modified tumor cells can induce specific antitumor cellular immunity. We constructed a recombinant adenovirus expressing murine GM-CSF and tested it for therapeutic efficacy in a syngeneic murine lung cancer model system. In vitro transduction of Lewis lung carcinoma cells with adenovirus-mGM-CSF suppressed tumor formation in syngenic mice (C57BL/6), and transduced and irradiated Lewis lung carcinoma cells induced regression of pre-established wild-type tumors without in vitro selection for transductants. Low, but significant, levels of specific antitumor cytotoxic T lymphocytes (CTL) were observed in mice inoculated with GM-CSF but not with reporter virus-transduced tumor cells. GM-CSF-transduced cells induced the accumulation of dendritic cells at the site of tumor, consistent with a mechanism involving improved tumor antigen presentation. These data suggest that transduction of tumor cells with recombinant GM-CSF adenovirus may be an effective and practical cancer gene therapeutic strategy.

Escape from host-antitumor immunity.

Abstract: Cancer cells may express proteins recognizable by the individual's immune system as foreign because they are either tumor-specific or not expressed at high levels in normal tissues to which the host is tolerant. There is now much evidence that tumors can be immunogenic, that is, that they frequently express antigens in a form recognizable by the host immune system. This has been shown not only in experimental animals but also for spontaneously occurring human tumors. Tumors therefore may progress by evolving variants that can evade immune responses or by developing other strategies to "escape" the immune response. The purpose of this review is to consider the current status of knowledge concerning these different tumor escape strategies.

Isolation of Novel HLA-DR Restricted Potential Tumor-associated Antigens from the Melanoma Cell Line FM3

Abstract: Endogenous peptides bound to the constitutively expressed MHC class II molecules HLA-DR and HLA-DQ of the melanoma cell line FM3 were examined By a combination of analytical methods (narrow bore and capillary reversed-phase high-performance liquid chromatography with subsequent spotting on polyvinylidene difluoride membranes, matrix-assisted laser desorption ionization mass spectrometry, and Edman microsequencing), we were able to isolate and identify a panel of HLA-DR4/2 (HLA-DRB1*0401/0201/DRB5*0101)-associated self-peptides from the melanoma cell line FM3 Among ubiquitously HLA-DR-associated peptides such as peptides from the class II-associated invariant chain peptide region of the invariant chain, HLA-class I, the transferrin receptor, and the IFN-γ receptor, we identified several potential tumor-associated antigens stemming from the MHC class I-restricted tumor antigen gp100, the Ca2+-binding protein annexin II, and proteins from the hsp70 family Chinese hamster ovary cells cotransfected with HLA-DRA, DRB1*0401, and CD80 genes were shown specifically to prime T lymphocytes from HLA-DRB1*0401 donors to the annexin II and gp100 peptides These results demonstrate that MHC class II molecules expressed by melanoma cells potentially present a variety of novel antigens to the immune system, some of which could be exploited for immunotherapy

Cancer immunotherapy using tumor cells combined with mixed lymphocytes

Abstract: This invention comprises cellular vaccines and methods of using them in cancer immunotherapy, particularly in humans. The vaccines comprise stimulated lymphocytes allogeneic to the subject being treated, along with a source of tumor-associated antigen. The allogeneic lymphocytes can be stimulated by combining or coculturing them with leukocytes obtained from the subject to be treated or form another third-party donor. Tumor antigen may be provided in the form of primary tumor cells, tumor cell lines or tumor extracts prepared from the subject. Stimulated allogeneic lymphocytes and tumor antigen are combined and administered at a site distant from the primary tumor, in order to prime or boost a systemic cellular anti-tumor immune response. This approach overcomes the natural refractory nature of the immune system to stimulation by tumor antigens, generating a host response and potentially improving the clinical outcome.

The expression of mouse gene P1A in testis does not prevent safe induction of cytolytic T cells against a P1A‐encoded tumor antigen

Abstract: Tumor antigen P815AB is recognized by cytolytic T lymphocytes (CTL) on mouse mastocytoma P815. This antigen is encoded by P1A, a gene activated in several tumors but silent in normal tissues except for testis and placenta. Notwithstanding the expression of P1A in testis, we found that male mice mounted P815AB-specific CTL responses as efficiently as females. The responding males remained fertile and no autoimmune lesions were observed in their testes. By immunohistochemistry with a rabbit antiserum directed against the P1A protein, we identified spermatogonia as the testicular cells expressing P1A. The absence of MHC class-I molecules on spermatogonia could be one of the mechanisms of protection against testicular autoimmunity, as the antigenic peptide should not be displayed at the cell surface. Human genes MAGE, BAGE and GAGE, which also code for tumor antigens recognized by autologous CTL, are not expressed in normal tissues other than testis. The results obtained in mice with antigen P815AB suggest that immunization of human males with such antigens will not generate autoimmune side-effects. Although P1A is strongly expressed in placenta, we also found that gestation did not prevent generation of CTL responses against antigen P815AB, and that such CTL responses did not affect gestation outcome. We identified labyrinthine trophoblasts as the placental cells expressing P1A. Again, the absence of MHC class-I molecules on these cells provides a plausible explanation for placental protection, although other mechanisms may also play a role.

Transloading of tumor antigen-derived peptides into antigen-presenting cells

Abstract: The discovery of a steadily growing number of tumor antigens (TAs) has made generic, cell-free, peptide-based cancer vaccines a possible alternative to cytokine-transfected autologous cellular cancer vaccines. The major drawback of peptide vaccines, however, is the poor immunogenicity of peptides. It is commonly thought that for the induction of an effective anticancer immune response, antigen-presenting cells (APCs) have to display TA-derived peptides to T lymphocytes. Polycationic amino acids have been employed in the past to enhance transport of proteins into cells. In a systematic study, the ability of different cationic polymers to transfer fluorescence-tagged peptides to APCs was investigated. We were able to show that several compounds enhance uptake of fluorescence-labeled peptides by APCs to different degrees. The most efficient compound identified, polyarginine (pArg), enhanced peptide delivery by more than 2 logs as compared with cells treated with peptide alone, whereas polylysine (pLys) treatment resulted in approximately 10-fold increased levels of fluorescence. Augmentation of peptide uptake was concentration-dependent, and the molecular weight of pArg or pLys also influenced peptide delivery. Furthermore, highly negatively charged peptides appear to be delivered with higher efficiency, although neutral peptides were also taken up at enhanced rates. Whereas peptide uptake mediated by pLys appears to be due to an at least transient permeabilization of cell membranes, peptide delivery in the presence of pArg may rely on endocytic processes. TA-derived peptides applied as cancer vaccines in conjunction with polycations afforded antitumor protection in animal models.

Cell-free tumor antigen peptide-based cancer vaccines.

Abstract: The central role that tumor antigen-derived peptides play in induction of antitumor immunity makes them ideal candidates for peptide-based cancer vaccines. We have demonstrated that “transloading” is an efficient strategy for importing short peptide ligands into antigen-presenting cells in vitro. Postulating that the transloading procedure might effect peptide uptake by antigen-presenting cells in vivo as well, we tested this approach for the generation of peptide-based cancer vaccines. In the P815 mastocytoma system, we vaccinated mice by s.c. injection of a single, known natural peptide derived from JAK-1 kinase. Whereas vaccination with peptide alone or mixed with incomplete Freund’s adjuvant was ineffective, application of the peptide in conjunction with the polycation poly-l-lysine protected a significant number of animals against tumor challenge. Dependent upon the type of poly-l-lysine applied, protection against tumor take was comparable to that achieved with irradiated whole-cell vaccines, genetically modified to secrete granulocyte–macrophage colony-stimulating factor. In the murine melanoma M-3, a combination of four putative tumor antigen-derived peptides was tested as a cancer vaccine. Administered in combination with polycations, these peptides evoked potent antitumor immunity that could not be obtained with the peptides alone or peptides emulsified in incomplete Freund’s adjuvant. However, peptide–polycation vaccines applied to the M-3 model were not as efficient as cellular control vaccines, consisting of irradiated interleukin 2 or granulocyte–macrophage colony-stimulating factor-secreting tumor cells.

Different cytokine profiles released by CD4+ and CD8+ tumor-draining lymph node cells involved in mediating tumor regression

Abstract: We have previously demonstrated that the growth of weakly immunogenic murine sarcomas leads to the induction of immunologically specific pre-effector cells in tumor-draining lymph nodes (TDLN) The in vitro activation of TDLN cells with anti-CD3 monoclonal antibodies (mAbs) and interleukin-2 (IL-2) resulted in the acquisition of effector function as measured by tumor regression in the adoptive immunotherapy of pulmonary metastases Further studies were performed to characterize the mechanisms associated with in vivo tumor reactivity mediated by activated TDLN cells By positive selection, CD4+ and CD8+ T cells were purified and activated by the anti-CD3/IL-2 method CD8+, but not CD4+, cells manifested tumor-specific granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon-gamma (IFN-gamma) release in vitro, and elicited tumor regression in vivo By contrast, only activated CD4+ were found to release significant amounts of IL-2 in response to tumor antigen but did not mediate tumor regression in vivo Mixing the two purified populations enhanced the antitumor activity of the CD8+ T cells In culture, IL-2 was found to augment the relative amount of tumor-specific release of GM-CSF and IFN-gamma by activated TDLN cells We found that the tumor-specific release of GM-CSF and IFN-gamma by activated lymphocytes was strongly associated with the in vivo therapeutic efficacy of these cells Evidence in support of this included the following: (1) cytokine release of TDLN derived after different durations of tumor growth correlated with tumor reactivity in adoptive transfer studies, (2) cytokine release of T cells derived from different lymphoid organs corresponded with tumor reactivity in adoptive transfer, and (3) in vivo administration of neutralizing mAb to IFN-gamma and GM-CSF significantly inhibited the antitumor reactivity of TDLN cells These studies document the contributory roles of IFN-gamma, GM-CSF, and IL-2 released by activated CD4+ and CD8+ T cells involved in tumor regression

Regulation of tumor antigen presentation by urocanic acid.

Abstract: Urocanic acid (UCA) accumulates in the epidermis after deamination of histidine. UCA isomerizes from the trans to the cis form upon exposure to environmental UV radiation. Cis-UCA is immunosuppressive in several models. Topically applied cis-UCA was reported to enhance the cutaneous tumor yield in chronically UV-irradiated mice, suggesting involvement of cis-UCA in photocarcinogenesis. Since Langerhans cells (LC) are capable of presenting tumor-associated Ags (TAA) for primary and secondary tumor-immune responses, we examined the effects of trans- and cis-UCA on LC tumor Ag presentation in a model of immunity to the S1509a spindle cell tumor (H-2a). In this system, induction of immunity requires exposure of LC to granulocyte-macrophage CSF. Naive CAF1 (H-2(a/d)) mice were immunized against S1509a by injection with granulocyte-macrophage CSF-exposed and TAA-pulsed epidermal cells (EC), as assessed by growth inhibition of inoculated tumor cells. Incubation of EC in cis-, but not trans-UCA completely inhibited Ag presentation in this system. Neither histamine antagonists nor indomethacin reversed these effects of cis-UCA. The ability of trans- and cis-UCA to modulate EC presentation of TAA for secondary immune responses was also examined. EC were pulsed with TAA in vitro and then injected into hind footpads of tumor-immune mice. After 24 h, footpad swelling was assessed as a measure of delayed-type hypersensitivity. Incubation with cis-, but again not trans-UCA before TAA exposure significantly inhibited elicitation of delayed-type hypersensitivity. These data indicate that cis-UCA may be an important regulator of LC Ag-presenting function in tumor-immune responses, and thus may play a role in photocarcinogenesis.

T cells recognize the VH complementarity-determining region 3 of the idiotypic protein of B cell non-Hodgkin's lymphoma.

Abstract: The idiotypic protein expressed by B lymphoma cells is a clone-specific tumor antigen which may be suitable for immune targeting by T cells. In this study, we cloned the immunoglobulin heavy chain variable gene (VH) of the idiotypic protein from a patient with B cell lymphoma and used a synthetic peptide of 22 amino acids corresponding to the VH complementarity-determining region (CDR)-3 of the idiotypic protein to investigate whether autologous T cells could recognize this unique peptide. We demonstrated that autologous T cells possessing both CD4+ and CD8+ phenotypes could be propagated. The T cells were able to proliferate, secrete cytokines, and lyse autologous cells presensitized with the specific peptide in a major histocompatibility complex-dependent manner. Moreover, these CDR3 peptide-primed T cells were also able to kill autologous lymphoma cells. Our results therefore offer new perspectives for specific therapeutic vaccination for the treatment of B cell lymphoma.