Expression of aberrantly glycosylated tumor mucin-1 on human DC after transduction with a fiber-modified adenoviral vector.

BACKGROUND: DC-presenting tumor Ag are currently being developed to be used as a vaccine in human cancer immunotherapy. To increase chances for successful therapy it is important to deliver full-length tumor Ag instead of loading single peptides. METHODS: In this study we used a fiber-modified adenoviral vector (rAd5F35) containing full-length tumor Ag cDNA to transduce human monocyte (Mo)-derived DC in vitro. Cells were efficiently transduced and survived for at least 3 days after adenoviral transduction. Phenotype and function after maturation of Mo-DC were not impaired by infection with adenovirus particles. Expression of the tumor-associated Ag mucin-1 (MUC1) was detected using MAb defining different MUC1 glycoforms. RESULTS: Non-transduced mature Mo-DC express endogenous MUC1 with normal glycosylation. After transduction with the rAd5F35-MUC1 adenoviral vector, Mo-DC also expressed MUC1 with tumor-associated glycosylation (Tn and T glycoforms), although no changes in mRNA levels of relevant glycosyltransferases could be demonstrated. DISCUSSION: The presence of aberrantly glycosylated MUC1 may influence Ag presentation of the tumor glycoforms of MUC1 to immune cells, affecting tumor cell killing. These findings could be highly relevant to developing strategies for cancer immunotherapy based on DC vaccines using MUC1 as tumor Ag.

A MAGE-3 peptide presented by HLA-B44 is also recognized by cytolytic T lymphocytes on HLA-B18.

Antigens encoded by MAGE genes are of particular interest for cancer immunotherapy because of their tumoral specificity and because they are shared by many tumors. Antigenic peptide MEVDPIGHLY, which is encoded by MAGE-3 and is known to be presented by human leukocyte antigen (HLA)-B44, is currently being used in therapeutic vaccination trials. We report here that a cytolytic T lymphocyte (CTL) clone, which is restricted by HLA-B*1801, recognizes the same peptide and, importantly, lyzes HLA-B18 tumor cells expressing MAGE-3. These results imply that the use of peptide MEVDPIGHLY can now be extended to HLA-B18 patients. We also provide evidence that, under limiting amounts of protein MAGE-3, HLA B*1801 and B*4403 compete for binding to the peptide.

Induction of cytolytic T lymphocytes by immunization of mice with an adenovirus containing a mouse homolog of the human MAGE-A genes.

The genes of the MAGE-A family code for antigens that are strictly tumor-specific and are shared by many human tumors. Melanoma patients have been immunized against these antigens and some tumor regressions have been observed. However, no unequivocal evidence of cytolytic T cell responses has been obtained by analyzing the blood lymphocytes of these patients. Hence it was considered worthwhile to examine in mouse systems whether or not immunization against antigens derived from the mouse Mage homologs can produce cytolytic T cell responses. We have identified an antigenic peptide encoded by mouse gene Mage-a2, and here we show that immunization of DBA/2 mice with a recombinant adenovirus containing either just the sequence encoding this peptide or a large part of the Mage-a2 coding sequence produces strong cytolytic T cell responses. The Mage-a2 system should prove useful for the comparison of vaccination modalities that could be applied to human patients in therapeutic vaccination trials with MAGE antigens.

A MAGE-A4 peptide presented by HLA-A2 is recognized by cytolytic T lymphocytes.

The MAGE-encoded antigens that are recognized by cytolytic T lymphocytes (CTL) are shared by many tumors and are strictly tumor specific. Clinical trials involving therapeutic vaccination of cancer patients with MAGE antigenic peptides or proteins are in progress. To increase the range of patients eligible for therapy with peptides, it is important to identify additional MAGE epitopes. We have used a method to identify CTL epitopes, which selects naturally processed peptides. CD8(+) T cells, obtained from individuals without cancer, were stimulated with autologous dendritic cells infected with a recombinant adenovirus containing the MAGE-A4 coding sequence. Responder cell microcultures that specifically lysed autologous EBV-transformed B cells infected with vaccinia-MAGE-A4 were cloned using autologous stimulator cells infected with a Yersinia enterocolitica carrying the MAGE-A4 sequence. An anti-MAGE-A4 CTL clone was obtained and the epitope was found to be decapeptide GVYDGREHTV (amino acids 230-239) presented by HLA-A2 molecules. The CTL clone lysed HLA-A2 tumor cells expressing MAGE-A4. This is the first reported antigenic peptide encoded by MAGE-A4. It may be valuable for cancer immunotherapy because MAGE-A4 is expressed in 51% of lung carcinomas and 63% of esophageal carcinomas, whereas about 50% of Caucasians and Asians express HLA-A2.

Thrombopoietin (TPO) knockout phenotype induced by cross-reactive antibodies against TPO following injection of mice with recombinant adenovirus encoding human TPO.

Adenovirus vectors have emerged as potent agents for gene transfer. Immune response against the vector and the encoded protein is one of the major factors in the transient expression following in vivo gene transfer. A single injection of an adenovirus encoding human thrombopoietin (TPO) into mice induced transient thrombocytosis, followed by a chronic immune thrombocytopenia. Thrombocytopenic mice had anti-human TPO Abs of the IgG2a and IgG1 isotypes. Thrombocytopenic mice sera neutralized more efficiently human than murine TPO, and exhibited no detectable anti-murine TPO Abs. Despite their low affinity for murine TPO, anti-TPO Abs induced a TPO knockout-like phenotype, i.e., low number of marrow megakaryocytes and of all kinds of hemopoietic progenitors. Hybridomas derived from a thrombocytopenic mouse revealed cross-reactivity of all of the secreted anti-TPO Ab isotypes. Mice subjected to myelosuppression after virus injection showed that anti-human TPO of IgG1 and IgG2a isotypes disappeared. Thus, sustained human TPO production was responsible for platelet elevation for at least 5 mo. Compelling results showed that elevated IgG2a/IgG2b ratios are always associated with thrombocytopenia, whereas low ratios are associated with tolerance or normal platelet counts. Finally, we hypothesize that in humans some chronic thrombocytopenia associated with a low TPO plasma level are due to anti-TPO Abs.

Active specific T-cell-based immunotherapy for cancer: nucleic acids, peptides, whole native proteins, recombinant viruses, with dendritic cell adjuvants or whole tumor cell-based vaccines. Principles and future prospects.

Whereas tumor cells are poor immunogens, recombinant tumor cells or dendritic cells as well as engineered viruses have been demonstrated to elicit specific antitumor immune responses leading to tumor growth suppression and long-lasting immunity in mouse tumor models. Single cytotoxic T lymphocyte-defined epitope-based strategies have proved useful for immunization in tumor-bearing mice. This strategy is under investigation in human melanoma, along with adjuvants such as cytokines or dendritic cells. Flt3L is an in vivo dendritic-cell growth factor that offers new prospects in the field of active specific immunotherapy. These immunotherapeutic approaches are being tested in clinical trials, and may open up novel avenues for disease-free patients with poor prognostic factors.

IL-2 gene delivery within an established murine tumor causes its regression without proliferation of preexisting antitumor-specific CTL.

Regression of P815 tumors established on naive syngeneic mice can be obtained by the intratumoral injection of a single dose of an adenoviral vector expressing the IL-2 gene (Ad.IL2). Injection triggers local IL-2 production for at least 10 days. We measured a number of immunologic parameters in situ following intratumoral Ad.IL2 treatment. We also analyzed the situation of regression obtained upon challenge with P815 cells of mice previously immunized against the tumor and compared both systems. While IFN-gamma messenger RNA expression was found to be elevated in both situations of tumor regression, the level of infiltration by tumor-specific CTL was different. A small amount of tumor-specific CD8+ T cells were present in growing, untreated tumors. Such cells are found in much larger numbers in tumors rejected upon challenge, consistent with a CTL-mediated rejection. In contrast, they were found not to proliferate following Ad.IL2 injection. The latter caused an increased infiltration of a polyclonal, presumably nonspecific, T cell population. These results suggest that the initial regression of established P815 tumors following Ad.IL2 treatment in vivo is mostly due to nonspecific effectors.

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

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.

Induction of a cytolytic T-cell response in mice with a recombinant adenovirus coding for tumor antigen P815A.

We investigated the efficacy of a recombinant adenovirus in inducing a cytolytic T-lymphocyte (CTL) response in mice against tumor antigen P815A, which is present on mouse mastocytoma P815. The recombinant adenoviral vector (Adeno.PIA) contained the sequence coding for the antigenic nonapeptide which binds to the H-2.Ld molecule to form antigen P815A. We verified that murine cells infected in vitro with Adeno. PIA were lysed by an anti-P815A CTL clone. Mice then received a single intradermal injection of Adeno. PIA, and after a few weeks their spleen cells were stimulated in vitro with tumor cells expressing antigen P815A. An anti-P815A CTL response was observed with the spleen lymphocytes of nearly all the mice, providing the lymphocytes were re-stimulated in vitro with cells expressing both P815A and co-stimulatory molecule B7.1. When the stimulatory cells did not express B7.1, a specific CTL response was observed in only 45% of the mice, and it was less intense. The Adeno. P1A viral vector was unable to raise an anti-P815A response in mice that had been previously infected with a recombinant adenovirus carrying the beta-galactosidase gene or with a defective adenovirus. We conclude that adenoviral vectors may be very useful for the priming of cytolytic T-cell responses directed against human tumor antigens. Other modes of immunization may be necessary to boost the responses induced with adenoviral vectors.

Strategies for cancer gene therapy using adenoviral vectors.

Modification of tumor cells using gene transfer either to enhance host immunity or to act directly on tumor cells is being intensively studied in animal models. Remarkable results have yielded to approved clinical protocols in the treatment of cancer patients using this approach. Several methods of gene delivery have been developed. This article is particularly devoted to the interest of the use of adenoviral vectors in the different strategies of cancer gene therapy.

Complete recovery of mice from a pre-established tumor by direct intratumoral delivery of an adenovirus vector harboring the murine IL-2 gene.

Direct introduction of exogenous genes into pre-existent tumors could provide an effective therapeutic approach for treatment of localized tumors. In this report we show that direct intratumoral delivery in animals of a replication-deficient adenovirus vector harboring the murine interleukin (IL)-2 gene (AD-mIL2) causes complete disappearance of P815 murine mastocytoma tumors in up to 75% of cases. Histological analysis of treated tumors revealed the presence of several zones of necrosis and the infiltration of macrophages and T cells. Moreover, the successfully treated animals develop a long lasting state of immunity during which further challenges with the tumor cells are rejected. To our knowledge this is the first successful in vivo treatment of an established tumor using adenoviral gene therapy methods.

Adenoviral interleukin-2 gene transfer into P815 tumor cells abrogates tumorigenicity and induces antitumoral immunity in mice.

The murine mastocytoma cell line P815 was used as a model to evaluate the effect on its tumorigenic capacity following interleukin-2 (IL-2) gene transfer into the tumor cells using a replication-defective adenovirus vector. The data show that P815 cells infected in vitro with this recombinant adenovirus secreted significant amounts of functional IL-2 as tested on CTL-L2 cells. Furthermore, when injected into syngeneic DBA/2 mice, the tumorigenic phenotype is lost in up to 80% of the animals. The rejection of the infected cells was host dependent, because co-injection at the same site or concomitant injection at the opposite side of the animal with a tumorigenic dose of noninfected P815 cells did not lead to tumor development in 50-70% of the mice. Moreover, protected animals developed a long-lasting state of immunization against the P815 tumor cells and their splenocytes were able to transfer the immunity to syngeneic naive recipients.