ABSTRACT
BACKGROUND: Wilson's disease (WD) is a rare condition resulting from autosomal recessive mutations in ATP7B, a copper transporter, manifesting with hepatic, neurological, and psychiatric symptoms. Timely diagnosis and appropriate treatment yield a positive prognosis, while delayed identification and/or insufficient therapy lead to a poor outcome. Our aim was to establish a prognostic method for WD by characterising biomarkers based on circulating microRNAs. METHODS: We conducted investigations across three cohorts: discovery, validation (comprising unrelated patients), and follow-up (revisiting the discovery cohort 3 years later). All groups were compared to age- and gender-matched controls. Plasma microRNAs were analysed via RNA sequencing in the discovery cohort and subsequently validated using quantitative PCR in all three cohorts. To assess disease progression, we examined the microRNA profile in Atp7b-/- mice, analysing serum samples from 6 to 44 weeks of age and liver samples at three time points: 20, 30, and 40 weeks of age. RESULTS: In patients, elevated levels of the signature microRNAs (miR-122-5p, miR-192-5p, and miR-885-5p) correlated with serum activities of aspartate transaminase, alanine aminotransferase and gamma-glutamyl transferase. In Atp7b-/- mice, levels of miR-122-5p and miR-192-5p (miR-885-5p lacking a murine orthologue) increased from 12 weeks of age in serum, while exhibiting fluctuations in the liver, possibly attributable to hepatocyte regenerative capacity post-injury and the release of hepatic microRNAs into the bloodstream. CONCLUSIONS: The upregulation of the signature miR-122-5p, miR-192-5p, and miR-885-5p in patients and their correlation with liver disease progression in WD mice support their potential as biomarkers of WD.
ABSTRACT
Wilson's disease (WD) is an inherited disorder of copper metabolism associated with mutations in ATP7B gene. We have shown that the administration of an adeno-associated vector (AAV) encoding a mini version of human ATP7B (VTX-801) provides long-term correction of copper metabolism in a murine WD model. In preparation of a future clinical trial, we have evaluated by positron emission tomography (PET) the value of 64Cu biodistribution, excretion pattern, and blood kinetics as pharmacodynamic biomarkers of VTX-801 effects. Six-week-old WD mice were injected intravenously with increasing doses of VTX-801 and 3 weeks or 3 months later with [64Cu]CuCl2. Untreated WD and wild-type (WT) mice were included as controls. Control WD mice showed increased hepatic 64Cu retention, reduced fecal excretion of the radiotracer, and altered 64Cu blood kinetics (BK) compared with WT mice. VTX-801 treatment in WD mice resulted in a significant reduction of hepatic 64Cu accumulation, the restoration of fecal 64Cu excretion, and the correction of 64Cu BK. This study showed that VTX-801 restores physiological copper metabolism in WD mice, confirming the mechanism of action of VTX-801, and demonstrated the translational potential of [64Cu]CuCl2-PET to explore VTX-801 pharmacodynamics in a minimally invasive and sensitive manner in WD patients.
ABSTRACT
Gene therapy with an adeno-associated vector (AAV) serotype 8 encoding the human ATPase copper-transporting beta polypeptide (ATP7B) complementary DNA (cDNA; AAV8-ATP7B) is able to provide long-term copper metabolism correction in 6-week-old male Wilson disease (WD) mice. However, the size of the genome (5.2 kilobases [kb]) surpasses the optimal packaging capacity of the vector, which resulted in low-yield production; in addition, further analyses in WD female mice and in animals with a more advanced disease revealed reduced therapeutic efficacy, as compared to younger males. To improve efficacy of the treatment, an optimized shorter AAV vector was generated, in which four out of six metal-binding domains (MBDs) were deleted from the ATP7B coding sequence, giving rise to the miniATP7B protein (Δ57-486-ATP7B). In contrast to AAV8-ATP7B, AAV8-miniATP7B could be produced at high titers and was able to restore copper homeostasis in 6- and 12-week-old male and female WD mice. In addition, a recently developed synthetic AAV vector, AAVAnc80, carrying the miniATP7B gene was similarly effective at preventing liver damage, restoring copper homeostasis, and improving survival 1 year after treatment. Transduction of approximately 20% of hepatocytes was sufficient to normalize copper homeostasis, suggesting that corrected hepatocytes are acting as a sink to eliminate excess of copper. Importantly, administration of AAVAnc80-miniATP7B was safe in healthy mice and did not result in copper deficiency. Conclusion: In summary, gene therapy using an optimized therapeutic cassette in different AAV systems provides long-term correction of copper metabolism regardless of sex or stage of disease in a clinically relevant WD mouse model. These results pave the way for the implementation of gene therapy in WD patients.
Subject(s)
Copper-Transporting ATPases/genetics , Copper/metabolism , Genetic Therapy/methods , Hepatolenticular Degeneration/therapy , Animals , Copper-Transporting ATPases/metabolism , Dependovirus , Disease Models, Animal , Female , Genetic Vectors , Hepatolenticular Degeneration/mortality , Homeostasis , Liver/metabolism , Male , Mice, Inbred C57BLSubject(s)
Hepatolenticular Degeneration/genetics , Hepatolenticular Degeneration/therapy , Targeted Gene Repair/methods , Animals , Copper/metabolism , Copper-Transporting ATPases/deficiency , Copper-Transporting ATPases/genetics , Copper-Transporting ATPases/metabolism , Dependovirus/genetics , Hepatolenticular Degeneration/metabolism , Humans , Liver/metabolism , Male , Mass Spectrometry/methods , Mice , Mice, KnockoutABSTRACT
BACKGROUND & AIMS: Wilson's disease (WD) is an autosomal recessively inherited copper storage disorder due to mutations in the ATP7B gene that causes hepatic and neurologic symptoms. Current treatments are based on lifelong copper chelating drugs and zinc salts, which may cause side effects and do not restore normal copper metabolism. In this work we assessed the efficacy of gene therapy to treat this condition. METHODS: We transduced the liver of the Atp7b(-/-) WD mouse model with an adeno-associated vector serotype 8 (AAV8) encoding the human ATP7B cDNA placed under the control of the liver-specific α1-antitrypsin promoter (AAV8-AAT-ATP7B). After vector administration we carried out periodic evaluation of parameters associated with copper metabolism and disease progression. The animals were sacrificed 6months after treatment to analyze copper storage and hepatic histology. RESULTS: We observed a dose-dependent therapeutic effect of AAV8-AAT-ATP7B manifested by the reduction of serum transaminases and urinary copper excretion, normalization of serum holoceruloplasmin, and restoration of physiological biliary copper excretion in response to copper overload. The liver of treated animals showed normalization of copper content and absence of histological alterations. CONCLUSIONS: Our data demonstrate that AAV8-AAT-ATP7B-mediated gene therapy provides long-term correction of copper metabolism in a clinically relevant animal model of WD providing support for future translational studies.
Subject(s)
Copper/metabolism , Genetic Therapy/methods , Genetic Vectors/administration & dosage , Hepatolenticular Degeneration , Liver , Adenosine Triphosphatases/genetics , Animals , Cation Transport Proteins/genetics , Copper-Transporting ATPases , Disease Models, Animal , Disease Progression , Gene Transfer Techniques , Hepatolenticular Degeneration/genetics , Hepatolenticular Degeneration/metabolism , Hepatolenticular Degeneration/therapy , Humans , Liver/metabolism , Liver/pathology , Mice , Peptide Fragments/genetics , Treatment Outcome , alpha 1-Antitrypsin/geneticsABSTRACT
Agonist anti-CD137 (4-1BB) mAbs enhance CD8-mediated antitumor immunity. Agonist anti-human CD137 mAbs binding to four distinct epitopes on the CD137 glycoprotein costimulated T cell activation irrespective of the engaged epitope or its interference with CD137L binding. CD137 perturbation with all these agonist mAbs resulted in Ag and Ab internalization toward an endosomal vesicular compartment. Internalization was observed in activated T lymphocytes from humans and mice, not only in culture but also in Ab-injected living animals. These in vivo experiments were carried out upon systemic i.v. injections with anti-CD137 mAbs and showed CD137 internalization in tumor-infiltrating lymphocytes and in activated human T cells transferred to immunodeficient mice. Efficient CD137 internalization required K63 polyubiquitination and endocytosed CD137-containing vesicles recruited TNFR-associated factor (TRAF) 2 and were decorated with K63 polyubiquitins. CD137 stimulation activates NF-κB through a K63-linked polyubiquitination-dependent route, and CD137-associated TRAF2 becomes K63 polyubiquitinated. Consistent with a role for TRAF2 in CD137 signaling, transgenic mice functionally deficient in TRAF2 showed delayed immunotherapeutic activity of anti-CD137 mAbs. As a whole, these findings advance our knowledge of the mechanisms of action of anti-CD137 immunostimulatory mAbs such as those currently undergoing clinical trials in cancer patients.
Subject(s)
Antibodies, Monoclonal/immunology , Lymphocyte Activation/immunology , Neoplasms, Experimental/immunology , T-Lymphocytes/immunology , Tumor Necrosis Factor Receptor Superfamily, Member 9/metabolism , Animals , Antibodies, Monoclonal/pharmacology , Blotting, Western , Cell Line , Endocytosis/drug effects , Endocytosis/immunology , Endosomes/drug effects , Endosomes/immunology , Endosomes/metabolism , Female , Humans , Immunoprecipitation , Immunotherapy/methods , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , NF-kappa B/immunology , NF-kappa B/metabolism , Neoplasms, Experimental/therapy , Polyubiquitin/immunology , Polyubiquitin/metabolism , Signal Transduction/drug effects , Signal Transduction/immunology , TNF Receptor-Associated Factor 2/immunology , TNF Receptor-Associated Factor 2/metabolism , Transfection , Tumor Necrosis Factor Receptor Superfamily, Member 9/immunologyABSTRACT
CD47, a "don't eat me" signal for phagocytic cells, is expressed on the surface of all human solid tumor cells. Analysis of patient tumor and matched adjacent normal (nontumor) tissue revealed that CD47 is overexpressed on cancer cells. CD47 mRNA expression levels correlated with a decreased probability of survival for multiple types of cancer. CD47 is a ligand for SIRPα, a protein expressed on macrophages and dendritic cells. In vitro, blockade of CD47 signaling using targeted monoclonal antibodies enabled macrophage phagocytosis of tumor cells that were otherwise protected. Administration of anti-CD47 antibodies inhibited tumor growth in orthotopic immunodeficient mouse xenotransplantation models established with patient tumor cells and increased the survival of the mice over time. Anti-CD47 antibody therapy initiated on larger tumors inhibited tumor growth and prevented or treated metastasis, but initiation of the therapy on smaller tumors was potentially curative. The safety and efficacy of targeting CD47 was further tested and validated in immune competent hosts using an orthotopic mouse breast cancer model. These results suggest all human solid tumor cells require CD47 expression to suppress phagocytic innate immune surveillance and elimination. These data, taken together with similar findings with other human neoplasms, show that CD47 is a commonly expressed molecule on all cancers, its function to block phagocytosis is known, and blockade of its function leads to tumor cell phagocytosis and elimination. CD47 is therefore a validated target for cancer therapies.
Subject(s)
Antigens, Differentiation/metabolism , CD47 Antigen/immunology , Neoplasms/immunology , RNA, Messenger/genetics , Receptors, Immunologic/metabolism , Antibodies/immunology , CD47 Antigen/genetics , Cell Division/immunology , Flow Cytometry , Humans , Neoplasms/pathology , Neoplasms/therapy , Phagocytosis/immunology , Prognosis , Survival AnalysisABSTRACT
Apolipoprotein A-I (Apo A-I) is a major component of high density lipoproteins (HDL) that transport cholesterol in circulation. We have constructed an expression plasmid encoding a chimeric molecule encompassing interleukin-15 (IL-15) and Apo A-I (pApo-hIL15) that was tested by hydrodynamic injections into mice and was co-administered with a plasmid encoding the sushi domain of IL-15Rα (pSushi) in order to enhance IL-15 trans-presentation and thereby bioactivity. The pharmacokinetics of the Apo A-I chimeric protein were much longer than non-stabilized IL-15 and its bioactivity was enhanced in combination with IL-15Rα Sushi. Importantly, the APO-IL-15 fusion protein was incorporated in part into circulating HDL. Liver gene transfer of these constructs increased NK and memory-phenotype CD8 lymphocyte numbers in peripheral blood, spleen and liver as a result of proliferation documented by CFSE dilution and BrdU incorporation. Moreover, the gene transfer procedure partly rescued the NK and memory T-cell deficiency observed in IL-15Rα(-/-) mice. pApo-hIL15+ pSushi gene transfer to the liver showed a modest therapeutic activity against subcutaneously transplanted MC38 colon carcinoma tumors, that was more evident when tumors were set up as liver metastases. The improved pharmacokinetic profile and the strong biological activity of APO-IL-15 fusion protein holds promise for further development in combination with other immunotherapies.
Subject(s)
Gene Transfer Techniques , Genetic Therapy , Immunotherapy , Interleukin-15/genetics , Interleukin-15/therapeutic use , Lipoproteins, HDL/blood , Liver/metabolism , Animals , Apolipoprotein A-I/blood , Blotting, Western , CD8-Positive T-Lymphocytes/immunology , Cell Count , Cell Line, Tumor , Cell Proliferation , Humans , Immunologic Memory , Injections, Subcutaneous , Interleukin-15/blood , Interleukin-15 Receptor alpha Subunit/chemistry , Interleukin-15 Receptor alpha Subunit/metabolism , Killer Cells, Natural/immunology , Mice , Mice, Inbred C57BL , Neoplasm Metastasis , Phenotype , Protein Structure, Tertiary , Recombinant Proteins/genetics , Recombinant Proteins/therapeutic use , Spleen/metabolismABSTRACT
Anti-CD137 mAb are capable of inducing tumor rejection in several syngeneic murine tumor models and are undergoing clinical trials for cancer. The anti-tumor effect involves co-stimulation of tumor-specific CD8(+) T cells. Whether antigen cross-presenting DC are required for the efficacy of anti-CD137 mAb treatment has never been examined. Here we show that the administration of anti-CD137 mAb eradicates EG7-OVA tumors by a strictly CD8beta(+) T-cell-dependent mechanism that correlates with increased CTL activity. Ex vivo analyses to determine the identity of the draining lymph node cell type responsible for tumor antigen cross-presentation revealed that CD11c(+) cells, most likely DC, are the main players in this tumor model. A minute number of tumor cells, revealed by the presence of OVA cDNA, reach tumor-draining lymph nodes. Direct antigen presentation by tumor cells themselves also participates in anti-OVA CTL induction. Using CD11c diphtheria toxin receptor-green fluorescent protein-->C57BL/6 BM chimeric mice, which allow for sustained ablation of DC with diphtheria toxin, we confirmed the involvement of DC in tumor antigen cross-presentation in CTL induction against OVA(257-264) epitope and in the antitumor efficacy induced by anti-CD137 mAb.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Dendritic Cells/immunology , Neoplasms/therapy , T-Lymphocytes, Cytotoxic/immunology , Tumor Necrosis Factor Receptor Superfamily, Member 9/agonists , Animals , Antigen Presentation/immunology , Antigens, Neoplasm/immunology , Cell Line, Tumor , Cross-Priming/immunology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neoplasms/immunology , Ovalbumin/immunology , Peptide Fragments/immunology , Tumor Necrosis Factor Receptor Superfamily, Member 9/immunologyABSTRACT
PURPOSE: Eradication of post-treatment residual myeloma cells is needed to prevent relapses, and immunostimulatory monoclonal antibodies (mAb) such as anti-CD137, CTLA-4, CD40, etc., which enhance the immune response against malignancies, represent a means of achieving this purpose. This study explores anti-CD137 mAbs for multiple myeloma treatment in preclinical models of the disease because they safely augment tumor immunity and are in clinical trials for other cancers. EXPERIMENTAL DESIGN: The antitumor effect of anti-CD137 mAb on mouse plasmacytomas derived from HOPC and NS0 cell lines was studied and compared with that of anti-CTLA-4, anti-CD40, and anti-ICAM-2 mAbs. The antitumor effect of anti-CD137 mAb was also examined in a mouse syngeneic disseminated myeloma (5TGM1) model, which more closely resembles human multiple myeloma. Depletions of specific cell populations and gene-targeted mice were used to unravel the requirements for tumor rejection. RESULTS: Agonistic mAb against CD137 and blocking anti-CTLA-4 mAb showed activity against i.p. HOPC tumors, resulting in extended survival of mice that also became immune to rechallenge. Anti-CD137 mAbs induced complete eradications of established s.c. NS0-derived tumors that were dependent on IFN-gamma, natural killer cells, and CD8(+) T lymphocytes. Natural killer cells accumulated in tumor draining lymph nodes and showed increased IFN-gamma production. Antitumor efficacy of anti-CD137 mAb was preserved in CD28-deficient mice despite the fact that CD28 signaling increases the expression of CD137 on CD8(+) T cells. Importantly, anti-CD137 mAb treatment significantly decreased systemic tumor burden in the disseminated 5TGM1 model. CONCLUSIONS: The immune-mediated antitumor activity of anti-CD137 mAb in mouse models holds promise for myeloma treatment in humans.
Subject(s)
Adjuvants, Immunologic/therapeutic use , Antibodies, Monoclonal/therapeutic use , Multiple Myeloma/drug therapy , Plasmacytoma/drug therapy , Tumor Necrosis Factor Receptor Superfamily, Member 9/immunology , Animals , Antigens, CD/immunology , CD40 Antigens/immunology , CTLA-4 Antigen , Disease Models, Animal , Female , Humans , Mice , Mice, Inbred BALB C , Plasmacytoma/immunology , Survival Analysis , Tumor BurdenABSTRACT
CD137 (also known as 4-1BB) is a surface co-stimulatory glycoprotein originally described as present on activated T lymphocytes. Artificial stimulation of this molecule with monoclonal antibodies or other agonist moieties therapeutically augments the cellular immune response against tumors, regardless of the absence of CD137 on tumor cells. These pharmacological agents, when administered systemically, surpass the immune effects of the membrane-bound natural ligand (CD137 or 4-1BB ligand), the activity of which is confined to cell-to-cell interactions. Greater affinity and broader distribution of the CD137 pharmacological agonists cause much more intense receptor crosslinking and stronger intracellular signals than the natural ligand. Target engagement on a variety of immune cell types such as T, natural killer and dendritic cells and on tumor vessels could switch on multiple mechanisms of action. As an agonist, anti-CD137 monoclonal antibody has entered Phase II clinical trials; elucidation of the mechanisms behind the antitumor efficacy requires further research in mice and patients to understand and rationally combine these new treatments.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Immunotherapy/methods , Tumor Necrosis Factor Receptor Superfamily, Member 9/metabolism , Animals , Antibodies, Monoclonal/immunology , Drug Delivery Systems , Humans , Neoplasms/immunology , Neoplasms/therapy , Signal Transduction , T-Lymphocytes/metabolism , Tumor Necrosis Factor Receptor Superfamily, Member 9/immunologyABSTRACT
Immunotherapy encompasses a variety of interventions and techniques with the common goal of eliciting tumor cell destructive immune responses. Colorectal carcinoma often presents as metastatic disease that impedes curative surgery. Novel strategies such as active immunization with dendritic cells (DCs), gene transfer of cytokines into tumor cells or administration of immunostimulatory monoclonal antibodies (such as anti-CD137 or anti-CTLA-4) have been assessed in preclinical studies and are at an early clinical development stage. Importantly, there is accumulating evidence that chemotherapy and immunotherapy can be combined in the treatment of some cases with colorectal cancer, with synergistic potentiation as a result of antigens cross-presented by dendritic cells and/or elimination of competitor or suppressive T lymphocyte populations (regulatory T-cells). However, genetic and epigenetic unstable carcinoma cells frequently evolve mechanisms of immunoevasion that are the result of either loss of antigen presentation, or an active expression of immunosuppressive substances. Some of these actively immunosuppressive mechanisms are inducible by cytokines that signify the arrival of an effector immune response. For example, induction of 2, 3 indoleamine dioxygenase (IDO) by IFNgamma in colorectal carcinoma cells. Combinational and balanced strategies fostering antigen presentation, T-cell costimulation and interference with immune regulatory mechanisms will probably take the stage in translational research in the treatment of colorectal carcinoma.
Subject(s)
Colorectal Neoplasms/immunology , Colorectal Neoplasms/therapy , Immunotherapy/methods , Antibodies, Monoclonal/therapeutic use , Colorectal Neoplasms/genetics , Genetic Therapy , Humans , Immunization , Tumor Escape/immunologyABSTRACT
There is compelling evidence for the role of natural killer (NK) cells in tumor immunosurveillance and their beneficial effects on many experimentally successful immunotherapy strategies. NK cells mediate cell contact-dependent cellular cytotoxicity and produce pro-inflammatory cytokines, but do not rearrange antigen receptors. Their activation depends on various germline-encoded receptors, including CD16, which mediates recognition of antibody-coated target cells. NK cytotoxicity is checked by a repertoire of inhibitory receptors that scan adequate expression of major histocompatibility complex class I molecules on the potential target cell. Functional cross-talk of NK and dendritic cells suggests a critical role for NK cells in the initiation and regulation of cellular immunity. Considerable knowledge on the molecular basis of NK recognition/activation contrasts with a lack of successful translational research on these matters. However, there is plenty of opportunity for targeted intervention of inhibitory/activatory surface receptors and for adoptive cell therapy with autologous or allogeneic NK cells.
Subject(s)
Cell Communication/immunology , Dendritic Cells/immunology , Immunotherapy, Adoptive , Killer Cells, Natural/immunology , Neoplasms/immunology , T-Lymphocytes/immunology , Animals , Cytotoxicity, Immunologic , Humans , Immunity, Cellular , Immunotherapy, Adoptive/methods , Killer Cells, Natural/metabolism , Killer Cells, Natural/transplantation , Ligands , Mice , Neoplasms/metabolism , Neoplasms/therapy , Receptors, Immunologic/immunology , Receptors, Immunologic/metabolism , Receptors, Natural Killer CellABSTRACT
Better understanding of the mechanisms that mediate spontaneous immune rejections ought to be important in the quest for improvements in immunotherapy of cancer. A set of intraperitoneal tumors of mesenchymal origin that had been chemically induced in ubiquitously expressing EGFP transgenic mice provided a model in which both T and NK cells were absolutely required for tumor rejection. Tumor cells were traceable because of being fluorescent and readily grafted in RAG1(-/-) immunodeficient mice, whereas they were rejected in a majority of syngeneic C57BL/6 and EGFP-transgenic mice. Tumor-cell clones with the highest EGFP expression tended to be rejected, but a direct involvement of EGFP as the antigen recognized for the immune rejections was ruled out. Rejections were absolutely dependent on NK cells as well as on CD4(+) and CD8(+) T lymphocytes according to selective depletion studies. Furthermore, CD8(+) and CD4(+) T lymphocytes as well as NK cells were detected in the inflammatory infiltrate that mediates tumor rejection along with some DC. The effects of IFN gamma, produced at the tumor site by T and NK lymphocytes, were only required at the malignant cell level and were necessary for tumor eradication. NK recognition of tumor cells was mediated by the NKG2D-activating receptor and blocking its function in vivo partially interfered with rejection. Therefore, complete rejection of these mesenchymal tumors requires a concerted set of activities including direct tumor-cell destruction and IFN gamma production that are mediated by both NK and T cells.
Subject(s)
Green Fluorescent Proteins/genetics , Interferon-gamma/metabolism , Killer Cells, Natural/immunology , Peritoneal Neoplasms/immunology , Receptors, Immunologic/metabolism , T-Lymphocytes/immunology , Animals , Cell Line, Tumor , Female , Flow Cytometry , Fluorescent Antibody Technique , Mesoderm/pathology , Mice , Mice, Inbred C57BL , Mice, Transgenic , NK Cell Lectin-Like Receptor Subfamily K , Neoplasms, Experimental/immunology , Neoplasms, Experimental/metabolism , Peritoneal Neoplasms/metabolism , Receptors, Natural Killer CellABSTRACT
Recombinant adenovirus administration gives rise to transgene-independent effects caused by the ability of the vector to activate innate immunity mechanisms. We show that recombinant adenoviruses encoding reporter genes trigger IFN-alpha and IFN-beta transcription from both plasmacytoid and myeloid mouse dendritic cells. Interestingly, IFN-beta and IFN-alpha5 are the predominant transcribed type I IFN genes both in vitro and in vivo. In human peripheral blood leukocytes type I IFNs are induced by adenoviral vectors, with a preponderance of IFN-beta together with IFN-alpha1 and IFN-alpha5 subtypes. Accordingly, functional type I IFN is readily detected in serum samples from human cancer patients who have been treated intratumorally with a recombinant adenovirus encoding thymidine kinase. Despite inducing functional IFN-alpha release in both mice and humans, gene transfer by recombinant adenoviruses is not interfered with by type I IFNs either in vitro or in vivo. Moreover, IFN-alpha does not impair replication of wild-type adenovirus. As a consequence, cancer gene therapy strategies with defective or replicative-competent adenoviruses are not expected to be hampered by the effect of the type I IFNs induced by the vector itself. However, type I IFN might modulate antitumor and antiadenoviral immune responses and thus influence the outcome of gene immunotherapy.
Subject(s)
Adenoviridae/genetics , Gene Expression/genetics , Interferon Type I/genetics , Transgenes/genetics , Animals , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/therapy , Cell Line, Tumor , Cell Survival/drug effects , Dendritic Cells/cytology , Dendritic Cells/metabolism , Female , Genetic Therapy/methods , Genetic Vectors/administration & dosage , Genetic Vectors/genetics , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HeLa Cells , Humans , Interferon Type I/pharmacology , Interferon-alpha/genetics , Interferon-alpha/pharmacology , Interferon-beta/genetics , Interferon-beta/pharmacology , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Liver Neoplasms/genetics , Liver Neoplasms/therapy , Mice , Mice, Inbred C57BL , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Thymidine Kinase/genetics , Transcription, Genetic/geneticsABSTRACT
Artificially enforced expression of CD80 (B7-1) and CD86 (B7-2) on tumor cells renders them more immunogenic by triggering the CD28 receptor on T cells. The enigma is that such B7s interact with much higher affinity with CTLA-4 (CD152), an inhibitory receptor expressed by activated T cells. We show that unmutated CD80 is spontaneously expressed at low levels by mouse colon carcinoma cell lines and other transplantable tumor cell lines of various tissue origins. Silencing of CD80 by interfering RNA led to loss of tumorigenicity of CT26 colon carcinoma in immunocompetent mice, but not in immunodeficient Rag-/- mice. CT26 tumor cells bind CTLA-4Ig, but much more faintly with a similar CD28Ig chimeric protein, thus providing an explanation for the dominant inhibitory effects on tumor immunity displayed by CD80 at that expression level. Interestingly, CD80-negative tumor cell lines such as MC38 colon carcinoma and B16 melanoma express CD80 at dim levels during in vivo growth in syngeneic mice. Therefore, low CD80 surface expression seems to give an advantage to cancer cells against the immune system. Our findings are similar with the inhibitory role described for the dim CD80 expression on immature dendritic cells, providing an explanation for the low levels of CD80 expression described in various human malignancies.
Subject(s)
Adenocarcinoma/immunology , B7-1 Antigen/immunology , Colonic Neoplasms/immunology , Adenocarcinoma/genetics , Adenocarcinoma/metabolism , Animals , Antigens, CD , Antigens, Differentiation/immunology , Antigens, Differentiation/metabolism , B7-1 Antigen/biosynthesis , B7-1 Antigen/genetics , Base Sequence , CTLA-4 Antigen , Cell Line, Tumor , Colonic Neoplasms/genetics , Colonic Neoplasms/metabolism , Female , Gene Silencing , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Molecular Sequence Data , TransfectionABSTRACT
In the course of a clinical trial consisting of intratumoral injections of dendritic cells (DCs) transfected to produce interleukin-12, the use of (111)In-labeled tracing doses of DCs showed that most DCs remained inside tumor tissue, instead of migrating out. In search for factors that could explain this retention, it was found that tumors from patients suffering hepatocellular carcinoma, colorectal or pancreatic cancer were producing IL-8 and that this chemokine attracted monocyte-derived dendritic cells that uniformly express both IL-8 receptors CXCR1 and CXCR2. Accordingly, neutralizing antihuman IL-8 monoclonal antibodies blocked the chemotactic attraction of DCs by recombinant IL-8, as well as by the serum of the patients or culture supernatants of human colorectal carcinomas. In addition, tissue culture supernatants of colon carcinoma cells inhibited DC migration induced by MIP-3beta in an IL-8-dependent fashion. IL-8 production in malignant tissue and the responsiveness of DCs to IL-8 are a likely explanation of the clinical images, which suggest retention of DCs inside human malignant lesions. Impairment of DC migration toward lymphoid tissue could be involved in cancer immune evasion.
Subject(s)
Cell Movement , Colonic Neoplasms/immunology , Colonic Neoplasms/therapy , Dendritic Cells/immunology , Interleukin-8/biosynthesis , Interleukin-8/immunology , Liver Neoplasms/immunology , Liver Neoplasms/therapy , Pancreatic Neoplasms/immunology , Pancreatic Neoplasms/therapy , Antibodies, Monoclonal , Chemotaxis , Humans , Immunotherapy , Interleukin-12/biosynthesis , TransfectionABSTRACT
Like paratroopers in special operations, dendritic cells (DCs) can be deployed behind the enemy borders of malignant tissue to ignite an antitumour immune response. 'Cross-priming T cell responses' is the code name for their mission, which consists of taking up antigen from transformed cells or their debris, migrating to lymphoid tissue ferrying the antigenic cargo, and meeting specific T cells. This must be accomplished in such an immunogenic manner that specific T lymphocytes would mount a robust enough response as to fully reject the malignancy. To improve their immunostimulating activity, local gene therapy can be very beneficial, either by transfecting DCs with genes enhancing their performance, or by preparing tumour tissue with pro-inflammatory mediators. In addition, endogenous DCs from the tumour host can be attracted into the malignant tissue following transfection of certain chemokine genes into tumour cells. On their side, tumour stroma and malignant cells set up a hostile immunosuppressive environment for artificially released or attracted DCs. This milieu is usually rich in transforming growth factor-beta, vascular endothelial growth factor, and IL-10, -6 and -8, among other substances that diminish DC performance. Several molecular strategies are being devised to interfere with the immunosuppressive actions of these substances and to further enhance the level of anticancer immunity achieved after artificial release of DCs intratumourally.