Intensive pharmacological immunosuppression allows for repetitive liver gene transfer with recombinant adenovirus in nonhuman primates

Repeated administration of gene therapies is hampered by host immunity toward vectors and transgenes. Attempts to circumvent antivector immunity include pharmacological immunosuppression or alternating different vectors and vector serotypes with the same transgene. Our studies show that B-cell depletion with anti-CD20 monoclonal antibody and concomitant T-cell inhibition with clinically available drugs permits repeated liver gene transfer to a limited number of nonhuman primates with recombinant adenovirus. Adenoviral vector–mediated transfer of the herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene was visualized in vivo with a semiquantitative transgene-specific positron emission tomography (PET) technique, liver immunohistochemistry, and immunoblot for the reporter transgene in needle biopsies. Neutralizing antibody and T cell–mediated responses toward the viral capsids were sequentially monitored and found to be repressed by the drug combinations tested. Repeated liver transfer of the HSV1-tk reporter gene with the same recombinant adenoviral vector was achieved in macaques undergoing a clinically feasible immunosuppressive treatment that ablated humoral and cellular immune responses. This strategy allows measurable gene retransfer to the liver as late as 15 months following the first adenoviral exposure in a macaque, which has undergone a total of four treatments with the same adenoviral vector

Carcinoma-derived interleukin-8 disorients dendritic cell migration without impairing T-cell stimulation

BACKGROUND: Interleukin-8 (IL-8, CXCL8) is readily produced by human malignant cells. Dendritic cells (DC) both produce IL-8 and express the IL-8 functional receptors CXCR1 and CXCR2. Most human colon carcinomas produce IL-8. IL-8 importance in malignancies has been ascribed to angiogenesis promotion. PRINCIPAL FINDINGS: IL-8 effects on human monocyte-derived DC biology were explored upon DC exposure to recombinant IL-8 and with the help of an IL-8 neutralizing mAb. In vivo experiments were performed in immunodeficient mice xenografted with IL-8-producing human colon carcinomas and comparatively with cell lines that do not produce IL-8. Allogenic T lymphocyte stimulation by DC was explored under the influence of IL-8. DC and neutrophil chemotaxis were measured by transwell-migration assays. Sera from tumor-xenografted mice contained increasing concentrations of IL-8 as the tumors progress. IL-8 production by carcinoma cells can be modulated by low doses of cyclophosphamide at the transcription level. If human DC are injected into HT29 or CaCo2 xenografted tumors, DC are retained intratumorally in an IL-8-dependent fashion. However, IL-8 did not modify the ability of DC to stimulate T cells. Interestingly, pre-exposure of DC to IL-8 desensitizes such cells for IL-8-mediated in vitro or in vivo chemoattraction. Thereby DC become disoriented to subsequently follow IL-8 chemotactic gradients towards malignant or inflamed tissue. CONCLUSIONS: IL-8 as produced by carcinoma cells changes DC migration cues, without directly interfering with DC-mediated T-cell stimulation

Lysine 63 polyubiquitination in immunotherapy and in cancer-promoting inflammation

Covalent and reversible post-translational modifications of proteins are a common theme in signaling. Ubiquitin conjugation was originally described to target proteins to proteasomal degradation by ubiquitin polymerization involving lysine (K) 48 residues. Differently linked polymers of polyubiquitin have been found that modify proteins without targeting to proteasomal degradation. Instead this pathway creates docking sites for signaling scaffolds that are key to control the nuclear factor-kappaB (NF-kappaB) pathway. Indeed TRAF-2, TRAF-6, and TRAF-3 are E3 ubiquitin ligases that form K63-linked ubiquitin polymers. Therefore signaling via TNF family receptors, IL1R, IL-18R, T-cell receptor (TCR), and Toll-like receptors (TLR) use this type of post-translational modification. Specific enzymes exist (DUBs) that deactivate this system, degrading K63 polyubiquitin chains. Interestingly, mice deficient in these deubiquitinases develop autoimmunity and inflammation. In carcinogenesis, the K63 polyubiquitin pathway is possibly critical for inflammation-driven tumor promotion. The pathway is also critically involved in costimulation of tumor immunity/immunotherapy as well as in the biology of malignant cells themselves. The elements of this new signaling paradigm offer the opportunity for therapeutic exploitation and drug discovery

Lysine 63 polyubiquitination in immunotherapy and in cancer-promoting inflammation

Covalent and reversible post-translational modifications of proteins are a common theme in signaling. Ubiquitin conjugation was originally described to target proteins to proteasomal degradation by ubiquitin polymerization involving lysine (K) 48 residues. Differently linked polymers of polyubiquitin have been found that modify proteins without targeting to proteasomal degradation. Instead this pathway creates docking sites for signaling scaffolds that are key to control the nuclear factor-kappaB (NF-kappaB) pathway. Indeed TRAF-2, TRAF-6, and TRAF-3 are E3 ubiquitin ligases that form K63-linked ubiquitin polymers. Therefore signaling via TNF family receptors, IL1R, IL-18R, T-cell receptor (TCR), and Toll-like receptors (TLR) use this type of post-translational modification. Specific enzymes exist (DUBs) that deactivate this system, degrading K63 polyubiquitin chains. Interestingly, mice deficient in these deubiquitinases develop autoimmunity and inflammation. In carcinogenesis, the K63 polyubiquitin pathway is possibly critical for inflammation-driven tumor promotion. The pathway is also critically involved in costimulation of tumor immunity/immunotherapy as well as in the biology of malignant cells themselves. The elements of this new signaling paradigm offer the opportunity for therapeutic exploitation and drug discovery

Molecular pathways: hypoxia response in immune cells fighting or promoting cancer

Both malignant and stromal components in tumors are influenced by the physiologic conditions of the microenvironment. Hypoxia is a prominent feature of solid tumors as a result of defective vascularization and intense metabolic activity. The gene-expression control mechanisms that adapt tissues to hypoxia are exploited by tumors to promote angiogenesis and vasculogenesis. The functions of infiltrating immune cells (macrophages and lymphocytes) and other stromal components are also influenced by a limited O(2) supply. Hypoxia-inducible factors (HIF) are the main molecular transcriptional mediators in the hypoxia response. The degradation and activity of HIF-1α and HIF-2α are tightly controlled by the fine-tuned action of oxygen-sensing prolyl and asparaginyl hydroxylase enzymes. Recent evidence indicates that hypoxia can modulate the differentiation and function of T lymphocytes and myeloid cells, skewing their cytokine-production profiles and modifying the expression of costimulatory receptors. This conceivably includes tumor-infiltrating lymphocytes. Hypoxia not only directly affects tumor-infiltrating leukocytes but also exerts effects on tumor cells and vascular cells that indirectly cause selective chemokine-mediated recruitment of suppressive and proangiogenic T-cell subsets. This review focuses on changes induced by hypoxia in immune cells infiltrating solid malignancies. Such changes may either promote or fight cancer, and thus are important for immunotherapy

Molecular pathways: hypoxia response in immune cells fighting or promoting cancer

Both malignant and stromal components in tumors are influenced by the physiologic conditions of the microenvironment. Hypoxia is a prominent feature of solid tumors as a result of defective vascularization and intense metabolic activity. The gene-expression control mechanisms that adapt tissues to hypoxia are exploited by tumors to promote angiogenesis and vasculogenesis. The functions of infiltrating immune cells (macrophages and lymphocytes) and other stromal components are also influenced by a limited O(2) supply. Hypoxia-inducible factors (HIF) are the main molecular transcriptional mediators in the hypoxia response. The degradation and activity of HIF-1α and HIF-2α are tightly controlled by the fine-tuned action of oxygen-sensing prolyl and asparaginyl hydroxylase enzymes. Recent evidence indicates that hypoxia can modulate the differentiation and function of T lymphocytes and myeloid cells, skewing their cytokine-production profiles and modifying the expression of costimulatory receptors. This conceivably includes tumor-infiltrating lymphocytes. Hypoxia not only directly affects tumor-infiltrating leukocytes but also exerts effects on tumor cells and vascular cells that indirectly cause selective chemokine-mediated recruitment of suppressive and proangiogenic T-cell subsets. This review focuses on changes induced by hypoxia in immune cells infiltrating solid malignancies. Such changes may either promote or fight cancer, and thus are important for immunotherapy

Palettes of vaccines and immunostimulatory monoclonal antibodies for combination

Various monoclonal antibodies (mAb) target immune system molecules to enhance immunity by costimulating T cells (i.e., CD137, OX40, CD40, GITR) or interfering in coinhibitory signals (i.e., CTLA-4, PD-1). These powerful agents can be guided by cancer vaccines to enhance immunity against tumor but not self tissues. Clinically powerful therapeutic synergies are at hand

Delivery of immunostimulatory monoclonal antibodies by encapsulated hybridoma cells

Immunostimulatory monoclonal antibodies are immunoglobulins directed toward surface proteins of immune system cells that augment the immune response against cancer in a novel therapeutic fashion. Exogenous administration of the recombinant humanized immunoglobulins is being tested in clinical trials with agents of this kind directed at a variety of immune-controlling molecular targets. In this study, the encapsulation of antibody-producing hybridoma cells was tested in comparison with the systemic administration of monoclonal antibodies. Hybridomas producing anti-CD137 and anti-OX40 mAb were encapsulated in alginate to generate microcapsules containing viable cells that secrete antibody. Immobilized cells in vitro were able to release the rat immunoglobulin produced by the hybridomas into the supernatant. Microcapsules were implanted by injection into the subcutaneous tissue of mice and thereby provided a platform for viable secreting cells, which lasted for more than 1 week. The pharmacokinetic profile of the rat monoclonal antibodies following microcapsule implantation was similar to that attained following an intraperitoneal administration of the purified antibodies. The rat-mouse hybridoma cells did not engraft as tumors in immunocompetent mice, while they lethally xenografted in immunodeficient mice, if not microencapsulated. The antitumor therapeutic activity of the strategy was studied on established CT26 colon carcinomas resulting in complete tumor eradication in an elevated fraction of cases and strong tumor-specific CTL responses with either anti-CD137 or anti-OX40 producing hybridomas, thus offering proof of the concept. This form of administration permitted combinations of more than one immunostimulatory monoclonal antibody to exploit the synergistic effects such as those known to be displayed by anti-CD137 and anti-OX40 mAb

Delivery of immunostimulatory monoclonal antibodies by encapsulated hybridoma cells

Immunostimulatory monoclonal antibodies are immunoglobulins directed toward surface proteins of immune system cells that augment the immune response against cancer in a novel therapeutic fashion. Exogenous administration of the recombinant humanized immunoglobulins is being tested in clinical trials with agents of this kind directed at a variety of immune-controlling molecular targets. In this study, the encapsulation of antibody-producing hybridoma cells was tested in comparison with the systemic administration of monoclonal antibodies. Hybridomas producing anti-CD137 and anti-OX40 mAb were encapsulated in alginate to generate microcapsules containing viable cells that secrete antibody. Immobilized cells in vitro were able to release the rat immunoglobulin produced by the hybridomas into the supernatant. Microcapsules were implanted by injection into the subcutaneous tissue of mice and thereby provided a platform for viable secreting cells, which lasted for more than 1 week. The pharmacokinetic profile of the rat monoclonal antibodies following microcapsule implantation was similar to that attained following an intraperitoneal administration of the purified antibodies. The rat-mouse hybridoma cells did not engraft as tumors in immunocompetent mice, while they lethally xenografted in immunodeficient mice, if not microencapsulated. The antitumor therapeutic activity of the strategy was studied on established CT26 colon carcinomas resulting in complete tumor eradication in an elevated fraction of cases and strong tumor-specific CTL responses with either anti-CD137 or anti-OX40 producing hybridomas, thus offering proof of the concept. This form of administration permitted combinations of more than one immunostimulatory monoclonal antibody to exploit the synergistic effects such as those known to be displayed by anti-CD137 and anti-OX40 mAb