Targeted therapy against VEGF and mTOR pathways has been established as the standard-of-care for metastatic clear cell renal cell carcinoma (ccRCC); however, these treatments frequently fail and most ...patients become refractory requiring subsequent alternative therapeutic options. Therefore, development of innovative and effective treatments is imperative. About 80%-90% of ccRCC tumors express an inactive mutant form of the von Hippel-Lindau protein (pVHL), an E3 ubiquitin ligase that promotes target protein degradation. Strong genetic and experimental evidence supports the correlate that pVHL functional loss leads to the accumulation of the transcription factor hypoxia-inducible factor 2α (HIF2α) and that an overabundance of HIF2α functions as a tumorigenic driver of ccRCC. In this report, we describe an RNAi therapeutic for HIF2α that utilizes a targeting ligand that selectively binds to integrins αvβ3 and αvβ5 frequently overexpressed in ccRCC. We demonstrate that functional delivery of a HIF2α-specific RNAi trigger resulted in HIF2α gene silencing and subsequent tumor growth inhibition and degeneration in an established orthotopic ccRCC xenograft model.
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Internalization of hu14.18‐IL2 occurs through IL2 receptors on NK effectors and GD2 antigen on tumor targets, with slower tumor progression accounting for effective NK/tumor interactions.
The ...hu14.18‐IL2 (EMD 273063) IC, consisting of a GD2‐specific mAb genetically engineered to two molecules of IL‐2, is in clinical trials for treatment of GD2‐expressing tumors. Anti‐tumor activity of IC in vivo and in vitro involves NK cells. We studied the kinetics of retention of IC on the surface of human CD25+CD16– NK cell lines (NKL and RL12) and GD2+ M21 melanoma after IC binding to the cells via IL‐2R and GD2, respectively. For NK cells, ∼50% of IC was internalized by 3 h and ∼90% by 24 h of cell culture. The decrease of surface IC levels on NK cells correlated with the loss of their ability to bind to tumor cells and mediate antibody‐dependent cellular cytotoxicity in vitro. Unlike NK cells, M21 cells retained ∼70% of IC on the surface following 24 h of culture and maintained the ability to become conjugated and lysed by NK cells. When NKL cells were injected into M21‐bearing SCID mice, IT delivery of IC augmented NK cell migration into the tumor. These studies demonstrate that once IC binds to the tumor, it is present on the tumor surface for a prolonged time, inducing the recruitment of NK cells to the tumor site, followed by tumor cell killing.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Established s.c. NXS2 murine neuroblastoma tumors exhibited transient resolution after suboptimal therapy using the hu14.18-IL2
immunocytokine (IC). The hu14.18-IL2 IC is a fusion protein that has ...linked a molecule of interleukin 2 (IL-2) to the COOH
terminus of each of the IgG heavy chains on the humanized anti-GD 2 monoclonal antibody hu14.18. To induce more potent and longer lasting in vivo antitumor effects, we tested hu14.18-IL2 IC in a regimen combining it with constant infusion IL-2 in NXS2 tumor-bearing mice.
The addition of the constant infusion IL-2 augmented the antitumor response induced by treatment with the hu14.18-IL2 IC in
animals with experimentally induced hepatic metastases and in animals bearing localized s.c. tumors. The combined treatment
induced prolonged tumor eradication in most animals bearing s.c. tumors and involved both natural killer cells and T cells.
The enhanced ability of this combined treatment to prevent tumor recurrence was not observed when a larger dose of hu14.18-IL2
IC, similar in IL-2 content to the IC plus systemic IL-2 regimen, was tested as single-agent therapy. Animals showing prolonged
tumor eradication of established tumors after the combined hu14.18-IL2 plus IL-2 regimen exhibited a protective T-cell-dependent
antitumor memory response against NXS2 rechallenge.
Bispecific antibodies (bsAbs) that bind to cell surface antigens and to digoxigenin (Dig) were used for targeted small interfering RNA (siRNA) delivery. They are derivatives of immunoglobulins G ...(IgGs) that bind tumor antigens, such as Her2, IGF1-R, CD22, and LeY, with stabilized Dig-binding variable domains fused to the C-terminal ends of the heavy chains. siRNA that was digoxigeninylated at its 3′end was bound in a 2:1 ratio to the bsAbs. These bsAb–siRNA complexes delivered siRNAs specifically to cells that express the corresponding antigen as demonstrated by flow cytometry and confocal microscopy. The complexes internalized into endosomes and Dig-siRNAs separated from bsAbs, but Dig-siRNA was not released into the cytoplasm; bsAb-targeting alone was thus not sufficient for effective mRNA knockdown. This limitation was overcome by formulating the Dig-siRNA into nanoparticles consisting of dynamic polyconjugates (DPCs) or into lipid-based nanoparticles (LNPs). The resulting complexes enabled bsAb-targeted siRNA-specific messenger RNA (mRNA) knockdown with IC50 siRNA values in the low nanomolar range for a variety of bsAbs, siRNAs, and target cells. Furthermore, pilot studies in mice bearing tumor xenografts indicated mRNA knockdown in endothelial cells following systemic co-administration of bsAbs and siRNA formulated in LNPs that were targeted to the tumor vasculature.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Genetic immunization is an attractive approach to generate antibodies because native proteins are expressed in vivo with normal posttranscriptional modifications, avoiding time-consuming and costly ...antigen isolation or synthesis. Hydrodynamic tail or limb vein delivery of naked plasmid DNA expression vectors was used to induce antigen-specific antibodies in mice, rats, and rabbits. Both methods allowed the efficient generation of high-titer, antigen-specific antibodies with an overall success rate of Western detectable antibodies of 78% and 92%, respectively. High-titer antibodies were typically present after 3 hydrodynamic tail vein plasmid DNA deliveries, 5 weeks after the initial injection (i.e., prime). For hydrodynamic limb vein plasmid DNA delivery, two deliveries were sufficient to induce high-titer antibody levels. Tail vein delivery was less successful at generating antibodies directed against secreted proteins as compared with limb vein delivery. Material for screening was generated by transfection of the immunization vector into mammalian cell lines. The cell line (COS-7) that produced the highest level of antigen expression performed best in Western blot analysis screens. In summary, intravenous delivery of antigen-expressing plasmid DNA vectors is an effective genetic immunization method for the induction of antigen-specific antibodies in small and large research animals.
Tumor-associated antigens (TAA) are typically poorly immunogenic “self” antigens. An effective strategy to break tolerance and induce antitumor immunity is by genetic vaccination, employing the ...orthologous TAA-sequence from a different species. We recently developed a clinically relevant approach for intravascular hydrodynamic limb vein (HLV) delivery of nucleic acids to skeletal muscle. Using the human gp100 xenogeneic TAA in the murine B16 melanoma model, we show that genetic vaccination of mice by HLV plasmid DNA delivery was highly effective at breaking tolerance against the homologous murine gp100 (mgp100) TAA and induced prophylactic antitumor protection. HLV vaccination resulted in an anti-hgp100 humoral and cellular response, with 4–5% of CD8+ T cells being gp10025–33-epitope-specific. Vaccinated animals demonstrated in vivo cytolytic activity against human and mgp10025–33 peptide-pulsed targets. Antitumor immunity could be adoptively transferred by splenocytes from human gp100-vaccinated animals. Furthermore, a durable antitumor memory response was established as ∼3% of CD8+ T cells were gp10025–33 antigen-specific in mice 6 months after vaccination. Following a single HLV human gp100 DNA boost, this level increased to ∼17% and protected animals from subsequent B16 tumor rechallenge. Our results warrant further consideration of HLV as a clinically relevant method for cancer gene therapy.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Therapeutic treatment with hu14.18-IL-2 immunocytokine (IC) or Flt3-L (FL) protein is initially effective at resolving established intradermal NXS2 neuroblastoma tumors in mice. However, many treated ...animals develop recurrent disease. We previously found that tumors recurring following natural killer (NK) mediated IC treatment show augmented MHC class I expression, while the tumors that recurred following T cell dependent Flt3-L treatment exhibited decreased MHC class I expression. We hypothesized that this divergent MHC modulation on recurrent tumors was due to therapy-specific immunoediting. We further postulated that combining IC and Flt3-L treatments might decrease the likelihood of recurrent disease by preventing MHC modulation as a mechanism for immune escape. We now report that combinatorial treatment of FL plus hu14.18-IL-2 IC provides greater antitumor benefit than treatment with either alone, suppressing development of recurrent disease. We administered FL by gene therapy using a clinically relevant approach: hydrodynamic limb vein (HLV) delivery of DNA for transgene expression by myofibers. Delivery of FL DNA by HLV injection in mice resulted in systemic expression of >10 ng/ml of FL in blood at day 3, and promoted up to a fourfold and tenfold increase in splenic NK and dendritic cells (DCs), respectively. Furthermore, the combination of FL gene therapy plus suboptimal IC treatment induced a greater expansion in the absolute number of splenic NK and DCs than achieved by individual component treatments. Mice that received combined FL gene therapy plus IC exhibited complete and durable resolution of established NXS2 tumors, and demonstrated protection from subsequent rechallenge with NXS2 tumor.
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EMUNI, FZAB, GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UL, UM, UPUK, VKSCE, ZAGLJ
We recently developed a clinically relevant approach for intravascular hydrodynamic limb vein (HLV) delivery of nucleic acids that results in high-level transgene expression in myofibers. This novel ...delivery method is a very effective means to induce a high-titer antibody response against expressed transgene antigens following genetic immunization. Here, we examine the utility of HLV as a method to deliver a genetic cancer vaccine in mice.Using the human gp100 (hgp100) xenogeneic DNA cancer vaccine in the murine B16 melanoma model, we show that genetic vaccination of mice by HLV delivery was highly effective at breaking tolerance against the homologous murine gp100 tumor-associated-antigen and induced prophylactic antitumor protection against B16 tumor challenge. Mice were vaccinated by repeated (days 0, 14 and 28) injections of 50 μg of plasmid DNA encoding the hgp100 into a distal site of the hind limb saphenous vein in a volume of 1.0 ml at a rate of 8.0 ml/min. Tolerance was broken in hgp100-vaccinated mice as demonstrated by prophylactic antitumor protection and in vitro cytolytic activity against B16 and hgp100-transfected B16 (B16-hgp100) cells. Tetramer (hgp100 25-33 /H2Db) analysis of peripheral blood lymphocytes indicated that 3-5% of the CD8+ T cells were gp100-specific following HLV vaccination. Although HLV genetic vaccination elicited an anti-hgp100 antibody response able to recognize and bind appropriately (processed in B16 melanocytic cells) and aberrantly (processed in non-melanosomal COS-7 cells) processed hgp100, adoptive and passive transfer studies demonstrated that protection was cell-mediated. Furthermore, a durable antitumor memory response was evident in HLV vaccinated mice 6 months after tumor challenge. Survivor mice showed delayed tumor progression upon B16 rechallenge, with 3-4% of their CD8 + T cells still exhibiting gp100-specificity. Effective reactivation of antitumor protection was achieved in survivor mice by a single HLV hgp100 boost that resulted in expansion of gp100-specific CD8+ T cells (17%) and protection from tumor rechallenge.Our results indicate that genetic vaccination by HLV delivery is an effective method to break tolerance and induce T cell immunity capable of prophylactic antitumor protection. Most importantly, since HLV gene delivery is equally effective in small and large research animals (mice, rats, rabbits, dogs and monkeys), it represents a clinically promising alternative to existing genetic vaccination strategies.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
We evaluated recurrent NXS2 neuroblastoma tumors that developed following NK- or T-cell-mediated immunotherapy in tumor-bearing mice. Recurrent tumors developed following an NK-dependent antitumor ...response using a suboptimal dose of hu14.18-IL2, a humanized IL-2 immunocytokine targeted to the GD(2)-ganglioside. This treatment initially induced complete resolution of measurable tumor in the majority of mice, followed, however, by delayed tumor recurrence in some mice. These recurrent NXS2 tumors revealed markedly enhanced (> fivefold) MHC class I antigen expression when compared with NXS2 tumors growing in PBS-treated control mice. A similar level of enhanced MHC class I antigen-expression could be induced on NXS2 cells in vitro by culturing with interferon gamma, and was associated with reduced susceptibility to both NK-cell-mediated tumor cell lysis and antibody-dependent cellular cytotoxicity in vitro. In contrast, Flt3-ligand treatment of NXS2-bearing mice induced a protective T-cell-dependent antitumor memory response. Recurrent NXS2 tumors that developed following Flt3-L therapy revealed a decreased expression of MHC class I antigens. While NXS2 tumors are susceptible to in vivo destruction following either hu14.18-IL2 or Flt3-ligand immunotherapies, these results suggest that some tumor cells may be selected to survive and progress by expressing either higher or lower levels of MHC class I antigen in order to resist either NK- or T-cell-mediated antitumor responses, respectively.
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EMUNI, FZAB, GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UL, UM, UPUK, VKSCE, ZAGLJ
The huKS1/4-IL2 fusion protein, directed against the human epithelial cell adhesion molecule (huEpCAM) has been shown to induce a strong CD8+ T-cell-dependent, natural killer (NK) cell-independent, ...antitumor response in mice bearing the huEp-CAM-transfected CT26 colon cancer CT26-EpCAM. Here we investigate the effectiveness of huKS1/4-IL2 against CT26-Ep21.6, a subclone of CT26-EpCAM, expressing low levels of MHC class I. In vitro antibody-dependent cellular cytotoxicity (ADCC) assays in the presence of huKS1/4-IL2 demonstrate that murine NK cells from spleen and blood can kill CT26-Ep21.6 significantly better than they kill CT26-EpCAM. NK-mediated ADCC of CT26-EpCAM can be enhanced by blocking the murine NK cell-inhibitory receptor, Ly-49C. A potent in vivo antitumor effect was observed when BALB/c mice bearing experimental metastases of CT26-Ep21.6 were treated with huKS1/4-IL2. The depletion of NK cells during huKS1/4-IL2 treatment significantly reduced the antitumor effect against CT26-Ep21.6. Together our in vitro and in vivo data in the huEp-CAM-transfected CT26 models indicate that the amount of MHC class I expressed on the tumor target cell plays a critical role in the in vivo antitumor mechanism of huKS1/4-IL2 immunotherapy. A low MHC class I level favors NK cells as effectors, whereas a high level of MHC class I favors T cells as effectors. Given the heterogeneity of MHC class I expression seen in human tumors and the prevailing T-cell suppression in many cancer patients, the observation that huKS1/4-IL2 has the potential to effectively activate an NK cell-based antitumor response may be of potential clinical relevance.
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