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Cancer Vaccines
(Released January 2006)

 
  by Preeti Gokal Kochar  

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  1. The rationale for combined chemo/immunotherapy using a Toll-like receptor 3 (TLR3) agonist and tumour-derived exosomes in advanced ovarian cancer

    M. Adams.

    Vaccine, Vol. 23, No. 17-18, Mar 2005, pp. 2374-2378.

    A clinical trial employing an immunotherapeutic approach based on the use of a Toll-like receptor 3 (TLR3) agonist and tumour-derived exosomes carrying tumour-associated antigens is planned in advanced ovarian cancer in conjunction with conventional first line chemotherapy. Most patients with ovarian cancer present with advanced disease and despite high initial response rate to chemotherapy the majority will relapse within 2 years with poor overall survival. Tumour antigen-specific T cells are naturally occurring in ovarian cancer patients and T cell infiltration of the tumour is highly prognostic. Novel immunotherapy to expand and activate tumour antigen-specific T cells combined with adjuvant treatment to overcome tumour-induced immunosuppression is considered to be therapeutically beneficial. The rationale for adopting such a combined approach is discussed here.

  2. Migration of dendritic cell based cancer vaccines: in vivo veritas?

    G. J. Adema, I. J. M. De Vries, C. J. Punt and C. G. Figdor.

    Current opinion in immunology, Vol. 17, No. 2, Apr 2005, pp. 170-174.

    Ex vivo generated cancer vaccines based on dendritic cells (DCs) are currently applied in the clinic. The migration of DCs from the tissues to the lymph nodes is tightly controlled and involves many different mediators and their receptors. A recent study demonstrated that the rate of migration of antigen-bearing DCs in situ from the skin to the lymph node is 100-fold higher than previously estimated. The migration of ex vivo generated DCs is rather inefficient but can be improved by pre-conditioning of the vaccine injection site with inflammatory cytokines. An alternative approach that is currently being explored is to target tumor antigens directly to DCs in situ, thereby exploiting the intricate migratory capacity of DCs in vivo. Recent advances have been made in understanding DC migration in the context of DC-based vaccines.

  3. Regulators of Apoptosis: Suitable Targets for Immune Therapy of Cancer

    Mads Hald Andersen, Juergen C. Becker and Per thor Straten.

    Nature Reviews: Drug Discovery, Vol. 4, No. 5, May 2005, pp. 399-409.

    Harnessing the immune system in the battle against cancer has been the focus of tremendous research efforts during the past two decades. Several means for achieving this goal, including adoptive transfer of tumour-reactive T cells, systemic or localized administration of immune modulating cytokines and the use of 'therapeutic' vaccines, have been explored. Anti-apoptotic molecules that enhance the survival of cancer cells and facilitate their escape from cytotoxic therapies represent prime candidates as vaccination antigens. Notably, spontaneous cellular immune responses against these proteins have frequently been identified in cancer patients. Here, we summarize current knowledge of IAP and BCL2 family proteins as T-cell antigens, report the results of the first explorative trial using these antigens in therapeutic vaccinations against cancer and discuss future opportunities. The clinical application of immunotherapy against cancer is rapidly moving forward in multiple areas, including the adoptive transfer of antitumour- reactive T cells and the use of 'therapeutic' vaccines. The latter aims at inducing cytotoxic T lymphocytes (CTLs) specific for tumour-associated antigens presented by cancer cells in the context of human leukocyte antigen (HLA) molecules. Surprisingly, until recently only limited attention has been focused on identifying the most suitable targets for the induction of clinically relevant anticancer immune responses, the delineation of the most effective epitopes within these antigens and, finally, inclusion of sets of peptide epitopes best suited for targeting the disease in question. The vast majority of malignancies are characterized by defects in apoptosis signalling, which is mediated by two group of apoptosis regulators: the BCL2 family (for example, BCL2, BCL-X sub(L) and MCL1) and the inhibitor of apoptosis proteins (IAP), such as survivin or melanoma IAP. Such apoptosis regulators are therefore crucial cellular factors contributing to the pathogenesis and progression of cancer. Consequently, they represent very attractive targets for the design of new anticancer drugs, such as antisense oligonucleotides and small-molecule BCL2 inhibitors. Notably, recently spontaneous cellular immune responses against these proteins have been identified as frequent features in cancer patients. Here, we summarize current knowledge of IAP and BCL2 family proteins as T-cell antigens, report the results of the first exploratory trials using these antigens for immunotherapy and discuss future opportunities, such as simultaneous targeting of several proteins or combination with conventional chemotherapy, because their expression in tumours is correlated with drug resistance and/or poor prognosis. Immunotherapy based on survivin, BCL2 or MCL1 might also compromise endothelial-cell viability and interfere with tumour angiogenesis. Targeting survivin or BCL2 family members in angiogenic immunotherapy might therefore deliver two distinct and potentially synergistic treatment modalities using a common procedure. In conclusion, a multi-epitope vaccine targeting regulators of apoptosis seems to be a promising, universal antitumour vaccine, which addresses both the tumour and the tumour stroma. Moreover, it synergistically boosts the effects of conventional cytotoxic therapies or radiation.

  4. Distinct but overlapping T helper epitopes in the 37-58 region of SSX-2

    M. Ayyoub, A. Merlo and C. S. Hesdorffer, et al.

    Clinical Immunology, Vol. 114, No. 1, Jan 2005, pp. 70-78.

    Because of their specific expression in tumors of different histological types, the products of the SSX genes are important candidate targets for development of cancer vaccines. We have previously identified two immunodominant SSX-2-derived T cell epitopes recognized by HLA-A2-restricted CD8 super(+) T cells (SSX-2 41-49) and HLA-DR11-restricted CD4 super(+) T cells (SSX-2 45-59), respectively. In this study, we report the identification of an HLA-DR3-restricted epitope mapping to the 37-51 region of SSX-2, overlapping both previously identified epitopes. As about one fifth of individuals from several major ethnic groups express HLA-DR3, the identification of this epitope significantly increases the percent of patients that are expected to mount specific CD4 super(+) T cell responses following vaccination with peptides in this region of SSX-2. Retrieval of multiple overlapping epitopes in a defined region of SSX-2 protein suggests the presence of a ''hot spot'' for T cell recognition that may prove sufficient for the induction of immune responses.

  5. Secretory production of biologically active rat interleukin-2 by Clostridium acetobutylicum DSM792 as a tool for anti-tumor treatment

    S. Barbe, L. Van Mellaert and J. Theys, et al.

    FEMS microbiology letters, Vol. 246, No. 1, 1 May 2005, pp. 67-73.

    The search for effective means of selectively delivering high therapeutic doses of anti-cancer agents to tumors has explored a variety of systems in the last decade. The ability of intravenously injected clostridial spores to infiltrate and thence selectively germinate in the hypoxic regions of solid tumors is exquisitely specific, making this system an interesting addition to the anti-cancer therapy arsenal. To increase the number of therapeutic proteins potentially useful for cancer treatment we have tested the possibility of Clostridium acetobutylicum to secrete rat interleukin-2 (rIL2). Therefore, rIL2 cDNA was placed under the control of the endo- beta -1,4-glucanase promoter and signal sequence of C. saccharobutylicum. Recombinant C. acetobutylicum containing the relevant construct secreted up to 800 mu gl super(-) super(1) biologically active rIL2. The obtained yield should be sufficient to provoke in vivo effects.

  6. Identification of Noncanonical Melanoma-Associated T Cell Epitopes for Cancer Immunotherapy

    Anne Bredenbeck, Florian O. Losch and Tumenjargal Sharav, et al.

    Journal of Immunology, Vol. 174, No. 11, 1 Jun 2005, pp. 6716-6724.

    The identification of tumor-associated T cell epitopes has contributed significantly to the understanding of the interrelationship of tumor and immune system and is instrumental in the development of therapeutic vaccines for the treatment of cancer. Most of the known epitopes have been identified with prediction algorithms that compute the potential capacity of a peptide to bind to HLA class I molecules. However, naturally expressed T cell epitopes need not necessarily be strong HLA binders. To overcome this limitation of the available prediction algorithms we established a strategy for the identification of T cell epitopes that include suboptimal HLA binders. To this end, an artificial neural network was developed that predicts HLA-binding peptides in protein sequences by taking the entire sequence context into consideration rather than computing the sum of the contribution of the individual amino acids. Using this algorithm, we predicted seven HLA A*0201-restricted potential T cell epitopes from known melanoma-associated Ags that do not conform to the canonical anchor motif for this HLA molecule. All seven epitopes were validated as T cell epitopes and three as naturally processed by melanoma tumor cells. T cells for four of the new epitopes were found at elevated frequencies in the peripheral blood of melanoma patients. Modification of the peptides to the canonical sequence motifs led to improved HLA binding and to improved capacity to stimulate T cells.

  7. Altered intracellular sorting signals do not influence the efficacy of genetic melanoma vaccines incorporating helper determinants in mice

    J. Brueck, J. Steitz, D. Strand and T. Tueting.

    Journal of Gene Medicine, Vol. 7, No. 5, May 2005, pp. 613-620.

    Background A genetic melanoma vaccine consisting of cDNA encoding the model self-antigen tyrosinase-related protein 2 (TRP2) fused in-frame to the immunogenic enhanced green fluorescent protein (EGFP) was able to break immune tolerance and stimulate CD8 super(+) T cells in vivo. In the present study we investigated whether alteration of the intracellular antigen localization as a result of the linkage with immune-enhancing helper proteins affects the resulting immune response. Methods Expression plasmids and recombinant adenoviruses were constructed encoding various fusion proteins with different intracellular sorting signals which direct the antigen to the cytosol, the endoplasmic reticulum or the endosomal compartments. Genetic immunization of C57BL/6 mice was performed with all constructs using particle-bombardment of the skin and injection of recombinant adenoviruses. The resulting immune response was analyzed using ELISPOT and tumor rejection assays. Results Induction of TRP2-specific CD8 super(+) T cells in vivo and autoimmune-mediated destruction of melanocytes in the bombarded area of the skin were observed with all constructs expressing fusion proteins between TRP2 and EGFP. Importantly, injections of the different recombinant adenoviruses all mediated protective immunity against transplanted B16 melanoma cells. Conclusions Altered intracellular sorting signals do not significantly influence the efficacy of genetic melanoma vaccines incorporating helper determinants in our model system. These results further support the concept that linkage of immunogenic helper sequences can be successfully applied for antigen-specific immunotherapy of melanoma and provide a scientific basis for the translation of this strategy in future clinical applications.

  8. Electroporation as a "Prime/Boost" Strategy for Naked DNA Vaccination against a Tumor Antigen

    Sarah Buchan, Eirik Groenevik, Iacob Mathiesen, Catherine A. King, Freda K. Stevenson and Jason Rice.

    Journal of Immunology, Vol. 174, No. 10, May 2005, pp. 6292-6298.

    We have developed novel DNA fusion vaccines encoding tumor Ags fused to pathogen-derived sequences. This strategy activates linked T cell help and, using fragment C of tetnus toxin, amplification of anti-tumor Ab, CD4 super(+), and CD8 super(+) T cell responses is achievable in mice. However, there is concern that simple DNA vaccine injection may produce inadequate responses in larger humans. To overcome this, we tested electroporation as a method to increase the transfection efficiency and immune responses by these tumor vaccines in vivo in mice. Using a DNA vaccine expressing the CTL epitope AH1 from colon carcinoma CT26, we confirmed that effective priming and tumor protection in mice are highly dependent on vaccine dose and volume. However, suboptimal vaccination was rendered effective by electroporation, priming higher levels of AH1-specific CD8 super(+) T cells able to protect mice from tumor growth. Electroporation during priming with our optimal vaccination protocol did not improve CD8 super(+) T cell responses. In contrast, electroporation during boosting strikingly improved vaccine performance. The prime/boost strategy was also effective if electroporation was used at both priming and boosting. For Ab induction, DNA vaccination is generally less effective than protein. However, prime/boost with naked DNA followed by electroporation dramatically increased Ab levels. Thus, the priming qualities of DNA fusion vaccines, integrated with the improved Ag expression offered by electroporation, can be combined in a novel homologous prime/boost approach, to generate superior antitumor immune responses. Therefore, boosting may not require viral vectors, but simply a physical change in delivery, facilitating application to the cancer clinic.

  9. Exosomes: A new delivery system for tumor antigens in cancer immunotherapy

    Jung-Ah Cho, Dong-jun Yeo and Hye-Youn Son, et al.

    International Journal of Cancer, Vol. 114, No. 4, 2005, pp. 613-622.

    Exosomes are small membrane vesicles that are released into the extracellular environment during fusion of multivesicular bodies with plasma membrane. Exosomes are secreted by various cell types including hematopoietic cells, normal epithelial cells and even some tumor cells. They are known to carry MHC class I, various costimulatory molecules and some tetraspanins. Recent studies have shown the potential of using native exosomes as immunologic stimulants. Here, we demonstrate a novel means of using exosomes engineered to express a specific tumor antigen to generate an immune response against tumors. We expressed a target tumor antigen, human MUC1 (hMUC1), in 2 MHC type-distinct mouse cell lines, CT26 and TA3HA. Analysis of exosomes purified from these cells revealed that exosomes contained the target MUC1 antigen on their surfaces as well as other well-described exosomal proteins, including Hsc70 and MHC class I molecules. In addition, both autologous and allogenic exosomes were able to stimulate the activation of immune cells and suppress hMUC1-expressing tumor growth in a MUC1-specific and dose-related manner. Therefore, these data suggest that exosomes can be engineered from tumor cell lines to deliver a target immunogen capable of inducing an effective immune response and that they may represent a new cell-free tumor vaccine.

  10. Immune-Reconstituted Influenza Virosome Containing CD40L Gene Enhances the Immunological and Protective Activity of a Carcinoembryonic Antigen Anticancer Vaccine

    Maria Grazia Cusi, Maria Teresa Del Vecchio and Chiara Terrosi, et al.

    Journal of Immunology, Vol. 174, No. 11, 1 Jun 2005, pp. 7210-7216.

    The correct interaction of a costimulatory molecule such as CD40L with its contrareceptor CD40 expressed on the membrane of professional APCs, provides transmembrane signaling that leads to APC activation. This process can be exploited to significantly improve the efficacy of cancer vaccines and the outcome of a possible cancer vaccine-induced, Ag-specific CTL response. Therefore, we investigated whether a novel intranasal delivery of immune-reconstituted influenza virosomes (IRIV), assembled with the CD40L gene (CD40L/IRIV), could be used to improve protective immunity and the Ag-specific CTL response against carcinoembryonic Ag (CEA) generated with a novel vaccine constituted of IRIV assembled with the CEA gene (CEA/IRIV). Our results suggest that CD40L/IRIV was able to augment CEA-specific CTL activity and CEA-specific protective immunity induced by CEA/IRIV most likely through the induction of a CTL response associated with a Th1 phenotype. In conclusion, we provide evidence that CD40L/IRIV, by acting through the CD40L/CD40 signaling pathway, acts as an immune-adjuvant that could increase the efficacy of a CEA-specific cancer vaccine, which could provide an efficacious new strategy for cancer therapy.

  11. Nitroaspirin corrects immune dysfunction in tumor-bearing hosts and promotes tumor eradication by cancer vaccination

    Carmela De Santo, Paolo Serafini and Ilaria Marigo, et al.

    Proceedings of the National Academy of Sciences, USA, Vol. 102, No. 11, 15 Mar 2005, pp. 4185-4190.

    Active suppression of tumor-specific T lymphocytes can limit the immune- mediated destruction of cancer cells. Of the various strategies used by tumors to counteract immune attacks, myeloid suppressors recruited by growing cancers are particularly efficient, often resulting in the induction of systemic T lymphocyte dysfunction. We have previously shown that the mechanism by which myeloid cells from tumor-bearing hosts block immune defense strategies involves two enzymes that metabolize L-arginine: arginase and nitric oxide (NO) synthase. NO-releasing aspirin is a classic aspirin molecule covalently linked to a NO donor group. NO aspirin does not possess direct antitumor activity. However, by interfering with the inhibitory enzymatic activities of myeloid cells, orally administered NO aspirin normalized the immune status of tumor-bearing hosts, increased the number and function of tumor-antigen-specific T lymphocytes, and enhanced the preventive and therapeutic effectiveness of the antitumor immunity elicited by cancer vaccination. Because cancer vaccines and NO aspirin are currently being investigated in independent phase I/II clinical trials, these findings offer a rationale to combine these treatments in subjects with advanced neoplastic diseases.

  12. Presentation of Endogenously Synthesized MHC Class II-Restricted Epitopes by MHC Class II Cancer Vaccines Is Independent of Transporter Associated with Ag Processing and the Proteasome

    Samudra K. Dissanayake, Natalia Tuera and Suzanne Ostrand-Rosenberg.

    Journal of Immunology, Vol. 174, No. 4, 15 Feb 2005, pp. 1811-1819.

    Cell-based vaccines consisting of invariant chain-negative tumor cells transfected with syngeneic MHC class II (MHC II) and costimulatory molecule genes are prophylactic and therapeutic agents for the treatment of murine primary and metastatic cancers. Vaccine efficacy is due to direct presentation of endogenously synthesized, MHC II-restricted tumor peptides to CD4 super(+) T cells. Because the vaccine cells lack invariant chain, we have hypothesized that, unlike professional APC, the peptide-binding groove of newly synthesized MHC II molecules may be accessible to peptides, allowing newly synthesized MHC II molecules to bind peptides that have been generated in the proteasome and transported into the endoplasmic reticulum via the TAP complex. To test this hypothesis, we have compared the Ag presentation activity of multiple clones of TAP-negative and TAP-positive tumor cells transfected with I-A super(k) genes and the model Ag hen egg white lysozyme targeted to the endoplasmic reticulum or cytoplasm. Absence of TAP does not diminish Ag presentation of three hen egg white lysozyme epitopes. Likewise, cells treated with proteasomal and autophagy inhibitors are as effective APC as untreated cells. In contrast, drugs that block endosome function significantly inhibit Ag presentation. Coculture experiments demonstrate that the vaccine cells do not release endogenously synthesized molecules that are subsequently endocytosed and processed in endosomal compartments. Collectively, these data indicate that vaccine cell presentation of MHC II-restricted endogenously synthesized epitopes occurs via a mechanism independent of the proteasome and TAP complex, and uses a pathway that overlaps with the classical endosomal pathway for presentation of exogenously synthesized molecules.

  13. A polytope DNA vaccine elicits multiple effector and memory CTL responses and protects against human papillomavirus 16 E7-expressing tumour

    T. Doan, K. Herd, I. Ramshaw, S. Thomson and R. W. Tindle.

    Cancer Immunology, Immunotherapy, Vol. 54, No. 2, Feb 2005, pp. 157-171.

    Vaccine-induced CD8 T cells directed to tumour-specific antigens are recognised as important components of protective and therapeutic immunity against tumours. Where tumour antigens have pathogenic potential or where immunogenic epitopes are lost from tumours, development of subunit vaccines consisting of multiple individual epitopes is an attractive alternative to immunising with whole tumour antigen. In the present study we investigate the efficacy of two DNA-based multiepitope ('polytope') vaccines containing murine (H-2 super(b)) and human (HLA-A*0201)-restricted epitopes of the E7 oncoprotein of human papillomavirus type 16, in eliciting tumour-protective cytotoxic T-lymphocyte (CTL) responses. We show that the first of these polytopes elicited powerful effector CTL responses (measured by IFN- gamma ELISpot) and long-lived memory CTL responses (measured by functional CTL assay and tetramers) in immunised mice. The responses could be boosted by immunisation with a recombinant vaccinia virus expressing the polytope. Responses induced by immunisation with polytope DNA alone partially protected against infection with recombinant vaccinia virus expressing the polytope. Complete protection was afforded against challenge with an E7-expressing tumour, and reduced growth of nascent tumours was observed. A second polytope differing in the exact composition and order of CTL epitopes, and lacking an inserted endoplasmic reticulum targeting sequence and T-helper epitope, induced much poorer CTL responses and failed to protect against tumour challenge. These observations indicate the validity of a DNA polytope vaccine approach to human papillomavirus E7-associated carcinoma, and underscore the importance of design in polytope vaccine construction.

  14. DNA Vaccines: Progress and Challenges

    John J. Donnelly, Britta Wahren and Margaret A. Liu.

    Journal of Immunology, Vol. 175, No. 2, Jul 2005, pp. 633-639.

    In the years following the publication of the initial in vivo demonstration of the ability of plasmid DNA to generate protective immune responses, DNA vaccines have entered into a variety of human clinical trials for vaccines against various infectious diseases and for therapies against cancer, and are in development for therapies against autoimmune diseases and allergy. They also have become a widely used laboratory tool for a variety of applications ranging from proteomics to understanding Ag presentation and cross-priming. Despite their rapid and widespread development and the commonplace usage of the term "DNA vaccines," however, the disappointing potency of the DNA vaccines in humans underscores the challenges encountered in the efforts to translate efficacy in preclinical models into clinical realities. This review will provide a brief background of DNA vaccines including the insights gained about the varied immunological mechanisms that play a role in their ability to generate immune responses.

  15. IL-4-Transfected Tumor Cell Vaccines Activate Tumor-Infiltrating Dendritic Cells and Promote Type-1 Immunity

    Junichi Eguchi, Naruo Kuwashima and Manabu Hatano, et al.

    Journal of Immunology, Vol. 174, No. 11, 1 Jun 2005, pp. 7194-7201.

    We previously demonstrated that IL-4 gene-transfected glioma cell vaccines induce effective therapeutic immunity in preclinical glioma models, and have initiated phase I trials of these vaccines in patients with malignant gliomas. To gain additional mechanistic insight into the efficacy of this approach, we have treated mice bearing the MCA205 (H-2 super(b)) or CMS-4 (H-2 super(d)) sarcomas. IL-12/23 p40 super(-/-) and IFN- gamma super(-/-) mice, which were able to reject the initial inoculation of IL-4 expressing tumors, failed to mount a sustained systemic response against parental (nontransfected) tumor cells. Paracrine production of IL-4 in vaccine sites promoted the accumulation and maturation of IL-12p70-secreting tumor-infiltrating dendritic cells (TIDCs). Adoptive transfer of TIDCs isolated from vaccinated wild-type, but not IL-12/23 p40 super(-/-), mice were capable of promoting tumor-specific CTL responses in syngeneic recipient animals. Interestingly, both STAT4 super(-/-) and STAT6 super(-/-) mice failed to reject IL-4-transfected tumors in concert with the reduced capacity of TIDCs to produce IL-12p70 and to promote specific antitumor CTL reactivity. These results suggest that vaccines consisting of tumor cells engineered to produce the type 2 cytokine, IL-4, critically depend on type 1 immunity for their observed therapeutic efficacy.

  16. CD4 T cells in tumor immunity

    M. Gerloni and M. Zanetti.

    Springer seminars in immunopathology, Vol. 27, No. 1, Jun 2005, pp. 37-48.

    T cell immunity is the key to protective immune responses against tumors. Traditionally, this function has been ascribed to CD8 T lymphocytes with cytotoxic activity, which are restricted by MHC class I molecules. In recent years the realization that CD4 T cells can also play a relevant role in protective anti-tumor responses has received growing attention. Here we will discuss the role of MHC class II-restricted T cells in response to, and in the regulation of, tumor antigens. Emphasis will be placed on four areas: (1) the role of CD4 T cell immunity in tumor protection in animal models and putative mode of action, (2) tumor antigens recognized by human CD4 T cells, (3) the cooperation between two CD4 T cells of different specificity as a new way to jump start the response against sub-immunogenic determinants of tumor antigens in a tolerant environment, and (4) the negative impact of regulatory CD4 T cells on anti-tumor T cell responses. By drawing attention to these four areas, it is our intention to provide the reader with a comprehensive view of issues of contemporary importance for this field, in the expectation that the information will help a better design of therapeutic cancer vaccines.

  17. Gene Therapy to Manipulate Effector T Cell Trafficking to Tumors for Immunotherapy

    Michael Gough, Marka Crittenden and Uma Thanarajasingam, et al.

    Journal of Immunology, Vol. 174, No. 9, 1 May 2005, pp. 5766-5773.

    Strategies that generate tumor Ag-specific effector cells do not necessarily cure established tumors. We hypothesized that the relative efficiency with which tumor-specific effector cells reach the tumor is critical for therapy. We demonstrate in this study that activated T cells respond to the chemokine CCL3, both in vitro and in vivo, and we further demonstrate that expression of CCL3 within tumors increases the effector T cell infiltrate in those tumors. Importantly, we show that adenoviral gene transfer to cause expression of CCL3 within B16ova tumors in vivo increases the efficacy of adoptive transfer of tumor-specific effector OT1 T cells. We additionally demonstrate that such therapies result in endogenous immune responses to tumor Ags that are capable of protecting animals against subsequent tumor challenge. Strategies that modify the "visibility" of tumors have the potential to significantly enhance the efficacy of both vaccine and adoptive transfer therapies currently in development.

  18. T-cell triggering by CD3-and CD28-binding molecules linked to a human virus-modified tumor cell vaccine

    C. Haas, M. Lulei, P. Fournier, A. Arnold and V. Schirrmacher.

    Vaccine, Vol. 23, No. 19, Mar 2005, pp. 2439-2453.

    The aim was to develop T cell costimulatory molecules that are broadly applicable to augment anti-tumor immune responses upon application of a virus- modified tumor vaccine to cancer patients. We generated recombinant bispecific single-chain antibodies with one specificity directed against the CD3 or the CD28 antigen on human T cells and the other against the viral target molecule hemagglutinin-neuraminidase (HN) of Newcastle Disease Virus (NDV). By re- directing unstimulated primary human T cells against HN-expressing NDV-infected tumor cells, the bispecific molecule bsHN-CD3 cross-linked effector and target cells and rapidly induced cytotoxicity at nanomolar concentrations. The bsHN- CD28 molecule exerted T cell co-stimulatory function. Maximal T cell activation was achieved with tumor cells infected by NDV and modified with both new stimulatory molecules. This was revealed by T cell proliferation, upregulation of CD69 and CD25 and by release of cytokines, interferons and chemokines. The new molecules combine high-effectivity with specificity and safety.

  19. Re-engineered CD40 receptor enables potent pharmacological activation of dendritic-cell cancer vaccines in vivo

    B. A. Hanks, J. Jiang and R. A. K. Singh, et al.

    Nature medicine, Vol. 11, No. 2, 2005, pp. 130-137.

  20. Re-engineered CD40 receptor enables potent pharmacological activation of dendritic-cell cancer vaccines in vivo

    B. A. Hanks, Jianghong Jiang and R. A. K. Singh, et al.

    Nature medicine, Vol. 11, No. 2, Feb 2005, pp. 130-137.

    Modest clinical outcomes of dendritic-cell (DC) vaccine trials call for the refinement of DC vaccine design. Although many potential antigens have been identified, development of methods to enhance antigen presentation by DCs has lagged. We have engineered a potent, drug-inducible CD40 (iCD40) receptor that permits temporally controlled, lymphoid-localized, DC-specific activation. iCD40 is comprised of a membrane-localized cytoplasmic domain of CD40 fused to drug-binding domains. This allows it to respond to a lipid-permeable, high-affinity dimerizer drug while circumventing ectodomain-dependent negative-feedback mechanisms. These modifications permit prolonged activation of iCD40-expressing DCs in vivo, resulting in more potent CD8 super(+) T-cell effector responses, including the eradication of previously established solid tumors, relative to activation of DCs ex vivo (P < 0.01), typical of most clinical DC protocols. In addition, iCD40-mediated DC activation exceeded that achieved by stimulating the full-length, endogenous CD40 receptor both in vitro and in vivo. Because iCD40 is insulated from the extracellular environment and can be activated within the context of an immunological synapse, iCD40-expressing DCs have a prolonged lifespan and should lead to more potent vaccines, perhaps even in immunecompromised patients.

  21. Cancer vaccines and immunotherapies: emerging perspectives

    R. A. Henderson, S. Mossman, N. Nairn and M. A. Cheever.

    Vaccine, Vol. 23, No. 17-18, Mar 2005, pp. 2359-2362.

    Research efforts over the last two decades studying immune responses to human carcinomas have demonstrated that antigens expressed by tumor cells can elicit specific cellular and humoral immune responses. Unfortunately, despite the observation that existent immune responses to these antigens are present in some patients with cancer, the tumors continue to progress. Thus, there has been considerable interest to augment these immune responses by immunization. Some of the clinical trials of the first cancer vaccines have provided evidence of clinical benefit thus encouraging the development of other vaccines. Challenges to development of such cancer vaccines include the identification and characterization of the antigen(s) to be targeted, the definition of the desired immune response to be elicited by the vaccine, and the choice of the appropriate vaccine delivery system.

  22. Preclinical evaluation of autologous dendritic cells transfected with mRNA or loaded with apoptotic cells for immunotherapy of high-risk neuroblastoma

    S. Jarnjak-Jankovic, R. D. Pettersen, S. Saeboee-Larssen, F. Wesenberg, M. R. K. Olafsen and G. Gaudernack.

    Cancer gene therapy, Vol. 12, No. 8, Aug 2005, pp. 699-707.

    Children with high-risk neuroblastoma (NB) have a poor clinical outcome. The purpose of the present study was to evaluate different strategies for immunotherapy of high-risk NB based on vaccination with antigen-loaded dendritic cells (DCs). DCs are professional antigen-presenting cells with the ability to induce antitumor T-cell responses. We have compared DCs either loaded with apoptotic tumor cells or fransfected with mRNA from the NB cell line HTB11 SK-N-SH, for their capacity to induce T-cell responses in vitro. Monocyte-derived DCs from healthy donors were loaded with tumor-derived antigens in the form of apoptotic cells or mRNA, matured and used to prime autologous T cells in vitro. After 1 week, T-cell responses against antigen-loaded DCs were measured by ELISPOT assay. DCs loaded with apoptotic NB cells or transfected with NB-cell mRNA were both able to efficiently activate autologous T cells. Both T cells of the CD8+ and CD4+ subset were activated. T cells activated by NB mRNA transfected DCs extensively crossreacted with DCs loaded with apoptotic NB cells and vice versa. The results indicate that loading of DCs with apoptotic NB cells or transfection with tumor mRNA represent promising strategies for development of individualized cancer vaccines/cancer gene therapy in treatment of NB.

  23. Comparison of antigen constructs and carrier molecules for augmenting the immunogenicity of the monosaccharide epithelial cancer antigen Tn

    E. Kagan, G. Ragupathi and S. S. Yi, et al.

    Cancer Immunology, Immunotherapy, Vol. 54, No. 5, May 2005, pp. 424-430.

    We have demonstrated previously that the optimal method for inducing an antibody response against defined cancer antigens is covalent conjugation of the antigen to keyhole limpet hemocyanin (KLH) and use of the potent saponin adjuvant QS-21. Single molecules of glycolipids (tetrasaccharides, pentasaccharides, or hexasaccharides) and MUC1 peptides (containing between one and five MUC1 tandem repeats) conjugated to KLH have proven sufficient for antibody recognition and vaccine construction. However, cancer specificity of monoclonal antibodies against the monosaccharide Tn and disaccharide sTn comes largely from recognition of clusters (c) of these molecules on the cell surface. Tn consists of a monosaccharide (GalNAc) O-linked to serine or threonine on epithelial cancer mucins which are uniquely rich in serines and threonines. We test here several Tn constructs: Tn monosaccharide, Tn(c) prepared on a triple threonine backbone, and Tn prepared on a partially or fully glycosylated MUC1 backbone. We determine that Tn(c) is more effective than Tn, and conjugation to KLH is more effective than conjugation to BSA or polystyrene beads for inducing ELISA reactivity against Tn, and FACS reactivity against Tn-positive tumor cells. Surprisingly, MUC1 glycosylated with Tn at three or five sites per 20 amino acid MUC1 tandem repeat and conjugated to KLH, induced the strongest antibody response against Tn and tumor cells expressing Tn, and had the additional advantage of inducing antibodies against MUC1.

  24. Modification of Professional Antigen-Presenting Cells with Small Interfering RNA In vivo to Enhance Cancer Vaccine Potency

    Tae Woo Kim, Jin-Hyup Lee and Liangmei He, et al.

    Cancer research, Vol. 65, No. 1, Jan 2005, pp. 309-316.

    RNA interference using small interfering RNA (siRNA) is an effective means of silencing gene expression in cells. Intradermal administration of nucleic acids via gene gun represents an efficient method for delivering nucleic acids to professional antigen-presenting cells in vivo. In this study, we show that the coadministration of DNA vaccines encoding human papillomavirus type 16 E7 with siRNA targeting key proapoptotic proteins Bak and Bax prolongs the lives of antigen-expressing dendritic cells in the draining lymph nodes, enhances antigen-specific CD8 super(+) T-cell responses, and elicits potent antitumor effects against an E7-expressing tumor model in vaccinated mice. Our data indicate that intradermal administration of siRNA to manipulate gene expression represents a plausible strategy for modification of the properties of professional antigen- presenting cells in vivo to enhance cancer vaccine potency.

  25. Rapid Assessment of Recognition Efficiency and Functional Capacity of Antigen-Specific T-Cell Responses

    H. E. Kohrt, C. -T Shu, T. B. Stuge, S. P. Holmes, J. Weber and P. P. Lee.

    Journal of Immunotherapy, Vol. 28, No. 4, Jul-Aug 2005, pp. 297-305.

    It is increasingly recognized that cells within an antigen-specific CD8 T-cell population may be diverse in recognition efficiency for target, which may significantly affect the overall efficacy of the response in clinical settings such as viral infections and cancer. CD8 T cells with seemingly identical antigen specificity, particularly those elicited by cancer vaccines, may be heterogeneous for sensitivity and recognition efficiency for the cognate peptide and functional state in vivo. Analysis of individual T-cell clones derived from an antigen-specific T-cell population would provide an accurate assessment of the overall response; however, this is time- and labor-intensive, preventing rapid and routine assessment of patient samples from clinical trials. By stimulating antigen-specific T cells that otherwise appear homogeneous on tetramer staining with graded amounts of cognate peptides, the authors show that individual cells downmodulate surface T-cell receptors (TCR) and thus lose tetramer reactivity with variable dynamics within the T-cell population. The dynamics of TCR downregulation represent an accurate assessment of an individual cell's antigen sensitivity, recognition efficiency, and relative functional state within an antigen-specific population and have direct correlation to killing capacity by chromium release as well as degranulation by CD107 mobilization. Furthermore, despite correlation of average T-cell function by all three techniques, TCR downregulation uncovered heterogeneity in T-cell responses after vaccination among patient samples directly ex vivo. When examined using this novel technique, antigen-specific T cells elicited by vaccination with heteroclitic peptides exhibited significantly different recognition efficiencies for the heteroclitic versus native peptides, translating into differences in functional responses. With advancing cancer vaccine trials, the capacity to detect and functionally characterize antigen-specific T-cell responses in detail is critical. Techniques, as presented here, that rapidly assess the overall antigen sensitivity, recognition efficiency, and functional status of patients' T-cell responses will guide future vaccine trials and immunotherapies.

  26. Immunotherapy for Pancreatic Cancer : Science Driving Clinical Progress

    Dan Laheru and Elizabeth M. Jaffee.

    Nature Reviews: Cancer, Vol. 5, No. 6, Jun 2005, pp. 459-467.

    The identification of key signalling pathways involved in immune-system regulation, along with the development of early pancreatic tumours in mouse models have provided new opportunities for pancreatic cancer treatment and prevention. Immunotherapy for pancreatic cancer is one approach that is at a crucial crossroads, as therapeutics that are designed to target pancreatic- cancer-associated antigens and regulatory signalling molecules are entering clinical trials. Pancreatic cancer represents a significant challenge, as the tumour cells are naturally resistant to current chemotherapy and radiation therapy. Mechanisms of immune escape both at the local and systemic level are recognized. Such mechanisms will probably need to be circumvented to fully develop an effective pancreatic cancer vaccine. So far, monoclonal antibodies to vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR) have been tested in combination with chemotherapy in patients with advanced pancreatic cancer. Several pancreatic vaccine approaches have been tested including peptide-based and gene-modified whole-cell vaccine approaches, both in patients with resected pancreatic cancer who are at risk for recurrence and in patients with advanced disease. New immunotherapy targets have been identified and the discovery of a relevant pancreatic cancer animal model should lead to more efficient and rapid testing and development of vaccine strategies. It is clear that the most effective strategy will require a combined approach incorporating the best targeted interventions taken from each respective modality.

  27. Surrogate Tumor Antigen Vaccination Induces Tumor-Specific Immunity and the Rejection of Spontaneous Metastases

    Jennifer D. Lewis, Michael H. Shearer, Ronald C. Kennedy and Robert K. Bright.

    Cancer research, Vol. 65, No. 7, 1 Apr 2005, pp. 2938-2946.

    The nonimmunogenic 4T1 murine mammary carcinoma model and a model surrogate tumor antigen (sTA) were employed to explore the possibility of inducing tumor-specific immunity through active immunization in the absence of defined tumor-associated antigens. Immunization of naive mice with protein-based sTA resulted in protection from s.c. challenge, with 4T1 modified to express the sTA (4T1.sTA), or from a sTA-expressing unrelated tumor cell line (mKSA). Immunization had no effect on parental 4T1 tumor growth or the formation of parental 4T1 spontaneous lung metastases. Mice that were sTA immunized and successfully rejected 4T1.sTA challenge also rejected a subsequent challenge in the contralateral flank with parental 4T1 and strikingly prevented the formation of spontaneous parental 4T1 lung metastases. The rejection of parental 4T1 seemed to be specific for and associated with unknown 4T1 tumor-associated antigens, because rejection of mKSA did not induce cross-protection against a challenge with parental 4T1. To evaluate the effect of this vaccine approach on established disease, mice were simultaneously challenged on day 0 with 4T1.sTA and parental 4T1 in contralateral flanks and then immunized on days 3, 10, 17, and 24 with sTA protein. Tumor growth and metastasis were delayed in four of five animals, and 20% (2 of 5) of the animals were tumor free at the completion of the experiment. Together, these data suggest that prior vaccination with a sTA followed by inoculation with poorly immunogenic tumor cells modified to express the sTA activates determinant spreading and the induction of systemic tumor immunity resulting in indigenous tumor rejection.

  28. Recombinant NY-ESO-1 Cancer Antigen: Production and Purification under cGMP Conditions

    R. Murphy, S. Green and G. Ritter, et al.

    Preparative Biochemistry and Biotechnology, Vol. 35, No. 2, 2005, pp. 119-134.

    The cancer-testis antigen, NY-ESO-1, has been engineered into a bacterial expression plasmid which incorporates a His sub(6)-tag. The plasmid was transfected into E. coli strain BL21 and Master and Working cell banks generated from this expression system. Three 15-litre fermentations were performed under cGMP (code of Good Manufacturing Practice) conditions and the crude NY-ESO-1 tagged protein isolated as solubilised inclusion bodies. A three-step cGMP chromatography process (immobilised metal affinity, anion exchange, and hydrophobic interaction) was utilised to purify the protein. The purified NY-ESO-1 is being used in early stage human cancer vaccine trials in Australia and the U.S.A.

  29. CpG Oligonucleotide Therapy Cures Subcutaneous and Orthotopic Tumors and Evokes Protective Immunity in Murine Bladder Cancer

    C. Ninalga, A. Loskog, M. Klevenfeldt, M. Essand and T. H. Totterman.

    Journal of Immunotherapy, Vol. 28, No. 1, Jan 2005, pp. 20-27.

    Bacillus Calmette-Guerin (BCG) instillation is standard immunotherapy for superficial bladder carcinoma. However, many patients become refractory to BCG, giving impetus to the development of alternative therapies. CpG oligodeoxynucleotide (ODN) therapy has been shown to promote TH1-oriented antitumor responses in various tumor models. To investigate its therapeutic effect in bladder cancer, we used different CpG ODNs to treat C57BL/6 mice bearing the subcutaneous murine bladder tumor MB49. CpG type B ODN 1668 was superior at inhibiting tumor growth, leading to complete regression of large tumors. More importantly, CpG ODN 1668 also regressed orthotopically growing MB49 tumors for the first time. Rechallenge of CpG ODN-cured mice with MB49 showed that a majority of the mice were protected long term, demonstrating that CpG ODN therapy evokes a memory response. Adenoviral vectors (Ad) encoding CD40L, tumor necrosis factor-related activation-induced cytokine, lymphotactin, interleukin (IL) 2, and IL-15 were also investigated. AdCD40L and AdIL-15 transduction could abolish MB49 tumorigenicity, and these vectors were combined with CpG ODN 1668 to investigate any enhanced effects. No such effects were seen. All groups of mice treated with CpG ODNs, alone or in combination with adenoviral vector, exhibited increased serum concentrations of IL-12, indicative of a TH1 response. Our results show that CpG ODN therapy cures established subcutaneous and orthotopic bladder cancer via a TH1-mediated response and provides long-lasting protective immunity.

  30. Breaking tolerance in cancer immunotherapy: time to ACT

    W. W. Overwijk.

    Current opinion in immunology, Vol. 17, No. 2, Apr 2005, pp. 187-194.

    The discovery of defined tumor antigens and their application in therapeutic cancer vaccines has not yet resulted in a successful therapy for cancer patients. Recent data suggest that this might be because most current clinical immunotherapeutic strategies rely on a tolerized tumor-reactive T-cell repertoire, resulting in a weak T-cell response that cannot induce tumor regression in the face of a multitude of normal and tumor-induced immunoregulatory mechanisms. New insights from animal models and clinical trials suggest a rationale for combination approaches in which the ineffective endogenous anti-tumor immune response is enhanced through a combination of adoptive cell transfer (ACT), specific vaccination and cytokine help for the reliable induction of a robust anti-tumor immune response and tumor regression.

  31. Measuring Melanoma-Specific Cytotoxic T Lymphocytes Elicited by Dendritic Cell Vaccines with a Tumor Inhibition Assay In Vitro

    S. Paczesny, H. Shi and H. Saito, et al.

    Journal of Immunotherapy, Vol. 28, No. 2, Mar 2005, pp. 148-157.

    Improving cancer vaccines depends on assays measuring elicited tumor-specific T-cell immunity. Cytotoxic effector cells are essential for tumor clearance and are commonly evaluated using 51Cr release from labeled target cells after a short (4 hours) incubation with T cells. The authors used a tumor inhibition assay (TIA) that assesses the capacity of cytotoxic T lymphocytes (CTLs) to control the survival/growth of EGFP-labeled tumor cell lines. TIA was validated using CD8+ T cells primed in vitro against melanoma and breast cancer cells. TIA was then used to assess the CTL function of cultured CD8+ T cells isolated from patients with metastatic melanoma who underwent vaccination with peptide-pulsed CD34+ HPCs-derived DCs. After the DC vaccination, T cells from six of eight patients yielded CTLs that could inhibit the survival/growth of melanoma cells. The results of TIA correlated with killing of tumor cells in a standard 4-hour 51Cr release assay, yet TIA allowed detection of CTL activities that appeared marginal in the 51Cr release assay. Thus, TIA might prove valuable for measuring spontaneous and induced antigen-specific cytotoxic T cells.

  32. Targeted Delivery of the ErbB2/HER2 Tumor Antigen to Professional APCs Results in Effective Antitumor Immunity

    Florian Rohrbach, Robert Weth, Mischo Kursar, Arjen Sloots, Hans-Willi Mittruecker and Winfried S. Wels.

    Journal of Immunology, Vol. 174, No. 9, 1 May 2005, pp. 5481-5489.

    Activation of T cells by professional APCs that present peptide epitopes of tumor-associated Ags is critical for the induction of cell-mediated immunity against tumors. To facilitate targeted delivery of the ErbB2 (HER2, neu) tumor Ag to APCs in vivo, we have generated chimeric proteins that contain the extracellular domain of CTLA-4 for binding to B7 molecules on the APC surface, which is genetically fused to a human ErbB2 fragment as an antigenic determinant. Bacterially expressed CTLA-4-ErbB2 fusion protein and a similar molecule harboring in addition the translocation domain of Pseudomonas exotoxin A as an endosome escape function displayed specific binding to B7-expressing cells, followed by protein internalization and intracellular degradation. Vaccination of BALB/c mice with the fusion proteins resulted in the induction of ErbB2-specific CD8 super(+) T cells and CTL-dependent protection from subsequent challenge with ErbB2-expressing but not ErbB2-negative murine renal carcinoma cells. In a therapeutic setting, injection of CTLA-4-ErbB2 protein vaccines caused rejection of established ErbB2-expressing tumors. Thereby, immunological memory was induced, leading to long-term systemic immunity and protection against rechallenge several months later. Our results demonstrate that these chimeric protein vaccines are effective tools for the induction of ErbB2-specific, T cell-mediated immunity.

  33. Induction of effective and antigen-specific antitumour immunity by a liposomal ErbB2/HER2 peptide-based vaccination construct

    A. Roth, F. Rohrbach, R. Weth, B. Frisch, F. Schuber and W. S. Wels.

    British journal of cancer, Vol. 92, No. 8, 25 Apr 2005, pp. 1421-1429.

    Efficient delivery of tumour-associated antigens to appropriate cellular compartments of antigen-presenting cells is of prime importance for the induction of potent, cell-mediated antitumour immune responses. We have designed novel multivalent liposomal constructs that co-deliver the p63-7l cytotoxic T Lymphocyte epitope derived from human ErbB2 (HER2), and HA307-319, a T-helper (Th) epitope derived from influenza haemagglutinin. Both peptides were conjugated to the surface of liposomes via a Pam sub(3)CSS anchor, a synthetic lipopeptide with potent adjuvant activity. In a murine model system, vaccination with these constructs completely protected BALB/c mice from subsequent s.c. challenge with ErbB2-expressing, but not ErbB2-negative, murine renal carcinoma (Renca) cells, indicating the induction of potent, antigen-specific immune responses. l.v. re-challenge of tumour-free animals 2 months after the first tumour cell inoculation did not result in the formation of lung tumour nodules, suggesting that long-lasting, systemic immunity had been induced. While still protecting the majority of vaccinated mice, a liposomal construct lacking the Th epitope was less effective than the diepitope construct, also correlating with a lower number of CD8 super(+) IFN- gamma super(+) T-cells identified upon ex vivo peptide restimulation of splenocytes from vaccinated animals. Importantly, in a therapeutic setting treatment with the liposomal vaccines resulted in cures in the majority of tumour-bearing mice and delayed tumour growth in the remaining ones. Our results demonstrate that liposomal constructs which combine Tc and Th peptide antigens and lipopeptide adjuvants can induce efficient, antigen-specific antitumour immunity, and represent promising synthetic delivery systems for the design of specific antitumour vaccines.

  34. Recombinant modified vaccinia Ankara primes functionally activated CTL specific for a melanoma tumor antigen epitope in melanoma patients with a high risk of disease recurrence

    Caroline L. Smith, P. Rod Dunbar and Fareed Mirza, et al.

    International Journal of Cancer, Vol. 113, No. 2, 20 Jan 2005, pp. 259-266.

    Recombinant plasmid DNA and attenuated poxviruses are under development as cancer and infectious disease vaccines. We present the results of a phase I clinical trial of recombinant plasmid DNA and modified vaccinia Ankara (MVA), both encoding 7 melanoma tumor antigen cytotoxic T lymphocyte (CTL) epitopes. HLA-A*0201-positive patients with surgically treated melanoma received either a "prime-boost" DNA/MVA or a homologous MVA-only regimen. Ex vivo tetramer analysis, performed at multiple time points, provided detailed kinetics of vaccine-driven CTL responses specific for the high-affinity melan- A[26[27[28[29[30[31[32[33[34[35 analogue epitope. Melan-A26-35-specific CTL were generated in 2/6 patients who received DNA/MVA (detectable only after the first MVA injection) and 4/7 patients who received MVA only. Ex vivo ELISPOT analysis and in vitro proliferation assays confirmed the effector function of these CTL. Responses were seen in smallpox-vaccinated as well as vaccinia-naive patients, as defined by anti-vaccinia antibody responses demonstrated by ELISA assay. The observations that 1) CTL responses were generated to only 1 of the recombinant epitopes and 2) that the magnitude of these responses (0.029-0.19% CD8 super(+) T cells) was below the levels usually seen in acute viral infections suggest that to ensure high numbers of CTL specific for multiple recombinant epitopes, a deeper understanding of the interplay between CTL responses specific for the viral vector and recombinant epitopes is required.

  35. Linked Foreign T-Cell Help Activates Self-Reactive CTL and Inhibits Tumor Growth

    Lucilla Steinaa, Peter Birk Rasmussen and Anne Marie Wegener, et al.

    Journal of Immunology, Vol. 175, No. 1, 1 Jul 2005, pp. 329-334.

    Transgenic mice expressing membrane-bound OVA under the rat insulin promoter, RIP-mOVA, has previously been suggested to display deletional tolerance toward the dominant CTL epitope, SIINFEKL, and provide an elegant model system to test the hypothesis that the lack of T cell help contributes to the tolerance. To understand how the CD8 tolerance is maintained in these mice, a set of neo-self-Ags, OVA, modified to contain a foreign Th peptide, were constructed and tested for their ability to induce CTL responses in RIP-mOVA mice. Immunization with these Th peptide-modified OVA molecules and not with the wild-type OVA induced self-reactive CTLs recognizing dominant CTL peptide, SIINFEKL. Importantly, immunization with the modified OVA constructs also prevented the growth of OVA-expressing tumors in transgenic mice. Since endogenous OVA Th peptides did not contribute toward breaking self CTL tolerance, these results also highlighted a very robust CD4 T cell tolerance toward OVA in RIP-mOVA mice that has not been previously described. These results therefore provide direct evidence that it is the tolerance in the CD4 Th cell compartment that helps maintain the CTL tolerance against self-Ag in these mice. Since the CTL tolerance can be broken or bypassed by foreign Th peptides inserted into a self Ag, potential of using this approach in generating effective therapeutic cancer vaccines is discussed.

  36. Anti-angiogenic therapy subsequent to adeno-associated-virus-mediated immunotherapy eradicates lymphomas that disseminate to the liver

    Xueying Sun, Geoffrey W. Krissansen and Peter W. C. Fung, et al.

    International Journal of Cancer, Vol. 113, No. 4, 2005, pp. 670-677.

    Liver cancer has a very poor prognosis and lacks effective therapy. We have previously demonstrated that intraportal injection of adeno-associated-viral (AAV) particles that express angiostatin lead to long-term expression of angiostatin capable of suppressing the outgrowth of EL-4 tumors in the liver. Here we combine AAV-mediated angiostatin therapy with immunotherapy by employing an AAV vector encoding the T-cell costimulator B7.1. Incubation of EL-4 cells with AAV-B7.1 viruses resulted in the rapid expression of B7.1 on the surface of 80% of EL-4 cells. Mice that were vaccinated with B7.1-engineered tumor cells rejected the tumor cells and resisted a secondary challenge with unmodified parental cells. Splenocytes from the vaccinated mice were highly cytotoxic towards parental EL-4 cells in vitro. However, the vaccinated mice failed to resist the challenge of a heavy burden of EL-4 cells. Intraportal injection of AAV particles that express angiostatin into mice that had been vaccinated 1 month earlier with B7.1-engineered tumor cells protected mice against the challenge of a heavy burden of EL-4 cells and eradicated tumors that had disseminated to the liver. The combinational therapy increased the survival rate of mice with advanced liver cancer. These encouraging results warrant investigation of the employment of anti-angiogenic therapy subsequent to cancer immunotherapy for targeting unresectable disseminated liver metastases.

  37. Modulation of antitumor responses by dendritic cells

    J. Vieweg and A. Jackson.

    Springer seminars in immunopathology, Vol. 26, No. 3, Jan 2005, pp. 329-341.

    The discovery that dendritic cells (DC) play a key role in regulating antitumor immunity has prompted considerable efforts in developing DC-based cancer vaccines for use in clinical oncology. Early translational trials using antigen-loaded DC have established clear evidence of vaccine safety, and demonstrated bioactivity by stimulating immunological and even clinical responses in selected subjects. Despite these encouraging results, the vaccine-induced immune responses achieved to date are not yet sufficient to attain a robust and durable therapeutic effect in the cancer patient. Therefore, further improvements are required to enhance vaccine potency and optimize the potential for clinical success. This article presents a set of emerging concepts that, together, form a framework for a multi-pronged approach that will further enhance the efficacy of DC-based vaccination by either directly improving DC-mediated T cell activation or by inhibiting mechanisms that suppress the induction of an effective antitumor response. The clinical translation of these concepts will result in new opportunities to successfully modulate immune responses in clinical settings.

  38. Vaccination of Apoptotic Fas Ligand-expressing Tumors Decreased Antitumor Responses by Enhanced Production of Immunosuppressive Cytokines

    A. Wada, Y. Tada and O. Shimozato, et al.

    Anticancer Research, Vol. 25, No. 1A, Jan-Feb 2005, pp. 299-304.

    Expression of Fas ligand (FasL) in tumors produced antitumor effects by generating both inflammation and T cell-mediated immunity, although the Fas/FasL interaction induces an apoptotic process of Fas-positive activated T cells. Our previous study, however, showed that immunization of mice with ultraviolet (UV)-treated FasL-expressing tumors rather induced immune suppression to the tumors, whereas mice rejected UV-untreated FasL-expressing tumors and developed protective immunity subsequently. Since dendritic cells (DCs) control tumor-specific immune responses in vivo, we examined a possible role of DCs in the immune suppression induced. Administration of DCs that were co-cultured with UV-treated FasL-expressing tumors did not influence the growth of parent tumors that were subsequently inoculated. Migration of immunocompetent cells into UV-treated FasL-expressing tumors was not significantly different from that into UV-untreated FasL-expressing tumors. However, production of immunosuppressive but not T helper type 1 cytokines was enhanced when UV-treated FasL-expressing tumors were administered. These data collectively suggest that the immune suppression induced by UV-treated FasL-expressing tumors was not attributable to tolerance of DCs, but due to cytokine-induced suppression of cell-mediated immunity.

  39. Understanding the response to immunotherapy in humans

    E. Wang, M. C. Panelli and F. M. Marincola.

    Springer seminars in immunopathology, Vol. 27, No. 1, Jun 2005, pp. 105-117.

    Whether the efforts of the last decade aimed at the development of vaccines against tumor-specific antigens encountered success or failure is a matter of expectations. On the bright side, we could optimistically observe that anti-cancer-vaccines stand as an outstanding example of the successful implementation of modern biotechnology tools for the development of biologically sound therapeutics. In particular, vaccines against melanoma (the prototype model of tumor immunology in humans) can reproducibly induce cytotoxic T cell (CTL) responses exquisitely specific for cancer cells. This achievement trespasses the specificity of any other anti-cancer therapy. The skeptics, on the other end, might point out that immunization only rarely leads to cancer regression, labeling, therefore, this approach is ineffective. In our opinion this judgment stems from the naive expectation that CTL induction is sufficient for an effective immune response. Here we propose that more needs to be understood about the mechanisms required for the induction of a therapeutically relevant immune response in humans. In particular, we will discuss the variables related to cancer heterogeneity, the weight of individual patients' polymorphism(s), the role of the T cell activation and differentiation and, finally, the complex relationship between immune and cancer cells within the tumor microenvironment.

  40. Hsp70-Like Protein 1 Fusion Protein Enhances Induction of Carcinoembryonic Antigen-Specific CD8 super(+) CTL Response by Dendritic Cell Vaccine

    Yanfeng Wu, Tao Wan and Xiangyang Zhou, et al.

    Cancer research, Vol. 65, No. 11, 1 Jun 2005, pp. 4947-4954.

    Heat shock proteins (HSP) have been revealed to interact with antigen-presenting cells and have potent adjuvant capability to induce antigen-specific CD8 super(+) CTL and Th1 responses. Our previous work shows how Hsp70-like protein 1 (Hsp70L1), as a new member of the Hsp70 subfamily, acts as potent Th1 adjuvant. Here, we report the efficient induction of tumor antigen-specific immune response by dendritic cells pulsed with recombinant fusion protein of Hsp70L1 and CEA sub(576-669) fragment of the carcinoembryonic antigen (CEA) containing CAP-1 (a HLA-A2-restricted CTL epitope). Fusion protein CEA sub(576-669)-Hsp70L1 can promote dendritic cell maturation and activate dendritic cells to produce cytokines, such as interleukin-12, interleukin-1 beta , and tumor necrosis factor- alpha , and chemokines, such as macrophage inflammatory protein-1 alpha , macrophage inflammatory protein-1 beta , and regulated on activation, normal T expressed and secreted, indicating the adjuvant ability of Hsp70L1 in the fusion protein. CEA-specific HLA-A2.1-restricted CD8 super(+) CTLs either from patients with CEA super(+)/HLA-A2.1 super(+) colon carcinoma or from splenocytes of immunized HLA-A2.1/K super(b) transgenic mice can be generated more efficiently after stimulations or immunizations with dendritic cells pulsed by CEA sub(576-669)-Hsp70L1 than with dendritic cells pulsed by CEA sub(576-669) alone, resulting in secreting more Th1 cytokine IFN- gamma and killing target cells more potently in an antigen-specific and HLA-A2.1-restricted manner. Adoptive transfer of splenocytes from transgenic mice immunized with CEA sub(576-669)-Hsp70L1-pulsed dendritic cells can inhibit tumor growth and prolong survival in nude mice bearing CEA super(+)/HLA-A2.1 super(+) human colon carcinoma more markedly. Therefore, Hsp70L1 has potent adjuvant effect in form of fusion protein, indicating that Hsp70L1 may be widely used as Th1 adjuvant to prepare antigenic fusion protein for the therapeutics of cancer or infectious diseases.

  41. MUC1 Peptide Vaccination in Patients with Advanced Pancreas or Biliary Tract Cancer

    Kotaro Yamamoto, Tomio Ueno and Toru Kawaoka, et al.

    Anticancer Research, Vol. 25, No. 5, Sep-Oct 2005, pp. 3575-3580.

    To evaluate the immunogenicity of MUC1 peptide vaccine in advanced pancreatic and bile duct cancers, a phase I clinical trial was conducted. A 100-mer MUC1 peptide consisting of the extracellular tandem repeat domain and incomplete Freund's adjuvant were subcutaneously administered to 6 pancreatic and 3 bile duct cancer patients at weeks 1, 3 and 5 and doses ranging from 300 to 3000 mu g. Circulating intracytoplasmic cytokine-positive CD4 super(+) T cells and anti-MUC1 IgG antibodies were measured before and after vaccination. There were no adverse events, except for mild reddening and swelling at the vaccination site. In 8 patients eligible for clinical evaluation, 7 had progressive disease and 1 stable disease with a tendency for increased circulating anti-MUC1 IgG antibody after vaccination. This phase I clinical trial revealed the safety of a vaccine containing 100-mer MUC1 peptides and incomplete Freund's adjuvant.

  42. Diverse CD8 super(+) T-Cell Responses to Renal Cell Carcinoma Antigens in Patients Treated with an Autologous Granulocyte-Macrophage Colony-Stimulating Factor Gene-Transduced Renal Tumor Cell Vaccine

    Xianzheng Zhou, Do Youn Jun and Amy Morck Thomas, et al.

    Cancer research, Vol. 65, No. 3, 1 Feb 2005, pp. 1079-1088.

    A phase I clinical trial with granulocyte-macrophage colony-stimulating factor tumor cell vaccines in patients with metastatic renal cell carcinoma (RCC) showed immune cell infiltration at vaccine sites and delayed-type hypersensitivity (DTH) responses to autologous tumor cells indicative of T-cell immunity. To further characterize RCC T-cell responses and identify relevant RCC-associated antigens, we did a detailed analysis of CD8 super(+) T-cell responses in two vaccinated RCC patients who generated the greatest magnitude of DTH response and also displayed a strong clinical response to vaccination (>90% reduction in metastatic tumor volume). Three separate CD8 super(+) T-cell lines (and subsequent derived clones) derived from patient 24 recognized distinct RCC- associated antigens. One recognized a shared HLA-A*0201-restricted antigen expressed by both renal cancer cells and normal kidney cells. This recognition pattern correlated with a positive DTH test to normal kidney cells despite no evidence of impairment of renal function by the patient's remaining kidney after vaccination. A second line recognized a shared HLA-C7-restricted antigen that was IFN-gamma inducible. A third line recognized a unique HLA-A*0101-restricted RCC antigen derived from a mutated KIAA1440 gene specific to the tumor. In addition, two independent CTL lines and three clones were also generated from patient 26 and they recognized autologous tumor cells restricted through HLA- A*0205, HLA-A/B/C, and HLA-B/C. These results show that paracrine granulocyte- macrophage colony-stimulating factor tumor vaccines may generate a diverse repertoire of tumor-reactive CD8 super(+) T-cell responses and emphasize the importance of polyvalency in the design of cancer immunotherapies.