Meriem Bourajjaj

Self-Assembling Peptide Epitopes as Novel Platform for Anticancer Vaccination

The aim of the present study was to improve the immunogenicity of peptide epitope vaccines using novel nanocarriers based on self-assembling materials. Several studies demonstrated that peptide antigens in nanoparticulate form induce stronger immune responses than their soluble forms. However, several issues such as poor loading and risk of inducing T cell anergy due to premature release of antigenic epitopes have challenged the clinical success of such systems. In the present study, we developed two vaccine delivery systems by appending a self-assembling peptide (Ac-AAVVLLLW-COOH) or a thermosensitive polymer poly(N-isopropylacrylamide (pNIPAm) to the N-terminus of different peptide antigens (OVA250–264, HPV-E743–57) to generate self-assembling peptide epitopes (SAPEs). The obtained results showed that the SAPEs were able to form nanostructures with a diameter from 20 to 200 nm. The SAPEs adjuvanted with CpG induced and expanded antigen-specific CD8+ T cells in mice. Furthermore, tumor-bearing mice vaccinated with SAPEs harboring the HPV E743–57 peptide showed a delayed tumor growth and an increased survival compared to sham-treated mice. In conclusion, self-assembling peptide based systems increase the immunogenicity of peptide epitope vaccines and therefore warrants further development toward clinical use.

Strong : In vivo antitumor responses induced by an antigen immobilized in nanogels via reducible bonds

Cancer vaccines are at present mostly based on tumor associated protein antigens but fail to elicit strong cell-mediated immunity in their free form. For protein-based vaccines, the main challenges to overcome are the delivery of sufficient proteins into the cytosol of dendritic cells (DCs) and processing by, and presentation through, the MHC class I pathway. Recently, we developed a cationic dextran nanogel in which a model antigen (ovalbumin, OVA) is reversibly conjugated via disulfide bonds to the nanogel network to enable redox-sensitive intracellular release. In the present study, it is demonstrated that these nanogels, with the bound OVA, were efficiently internalized by DCs and were capable of maturating them. On the other hand, when the antigen was just physically entrapped in the nanogels, OVA was prematurely released before the particles were taken up by cells. When combined with an adjuvant (polyinosinic-polycytidylic acid, poly(I:C)), nanogels with conjugated OVA induced a strong protective and curative effect against melanoma in vivo. In a prophylactic vaccination setting, 90% of the mice vaccinated with nanogels with conjugated OVA + poly(I:C) did not develop a tumor. Moreover, in a therapeutic model, 40% of the mice showed clearance of established tumors and survived for the duration of the experiment (80 days) while the remaining mice showed substantial delay in tumor progression. In conclusion, our results demonstrate that conjugation of antigens to nanogels via reducible covalent bonds for intracellular delivery is a promising strategy to induce effective antigen-specific immune responses against cancer.

Macrophage selective photodynamic therapy by meta-tetra(hydroxyphenyl)chlorin loaded polymeric micelles: A possible treatment for cardiovascular diseases

Abstract Selective elimination of macrophages by photodynamic therapy (PDT) is a new and promising therapeutic modality for the reduction of atherosclerotic plaques. m‐Tetra(hydroxyphenyl)chlorin (mTHPC, or Temoporfin) may be suitable as photosensitizer for this application, as it is currently used in the clinic for cancer PDT. In the present study, mTHPC was encapsulated in polymeric micelles based on benzyl‐poly(&egr;‐caprolactone)‐b‐methoxy poly(ethylene glycol) (Ben‐PCL‐mPEG) using a film hydration method, with loading capacity of 17%. Because of higher lipase activity in RAW264.7 macrophages than in C166 endothelial cells, the former cells degraded the polymers faster, resulting in faster photosensitizer release and higher in vitro photocytotoxicity of mTHPC‐loaded micelles in those macrophages. However, we observed release of mTHPC from the micelles in 30 min in blood plasma in vitro which explains the observed similar in vivo pharmacokinetics of the mTHPC micellar formulation and free mTHPC. Therefore, we could not translate the beneficial macrophage selectivity from in vitro to in vivo. Nevertheless, we observed accumulation of mTHPC in atherosclerotic lesions of mice aortas which is probably the result of binding to lipoproteins upon release from the micelles. Therefore, future experiments will be dedicated to increase the stability and thus allow accumulation of intact mTHPC‐loaded Ben‐PCL‐mPEG micelles to macrophages of atherosclerotic lesions. Graphical abstract Photosensitizer (PS) loaded polyester‐based micelles (big red dots) accumulate in atherosclerotic plaques of blood vessels (not in healthy endothelial). Micelles are degraded by high lipase activity in macrophages, causing PS release and selective photocytotoxicity towards the macrophages. Intervention of the inflammation is thus possible without harming healthy tissue. Figure. No Caption available.

Anginex lipoplexes for delivery of anti-angiogenic siRNA

Angiogenesis is one of the hallmarks of cancer which renders it an attractive target for therapy of malignancies. Tumor growth suppression can be achieved by inhibiting angiogenesis since it would deprive tumor cells of oxygen and vital nutrients. Activation of endothelial cells of tumor vasculature is the first step in angiogenesis which is mediated by various factors. One of the major triggers in this process is vascular endothelial growth factor (VEGF) which binds to VEGF receptors on endothelial cells of tumor vessels. This induces a series of signaling cascades leading to activation of cellular processes involved in angiogenesis, and therefore down-regulation of VEGF receptor-2 (VEGFR-2) expression seems a viable option to inhibit angiogenesis. In our investigations, this aim has been pursued by using siRNA interfering with the expression of VEGFR-2. Since the discovery of RNA interference (RNAi) as a gene regulation process, successful delivery of small non-coding RNA has presented itself as a major challenge. In the current study, we have characterized a galectin-1 targeted anginex-coupled lipoplex (Angiplex) containing siRNA against the gene of VEGFR-2 as an angiostatic therapeutic. Angiplex particles had a size of approximately 120 nm with a net negative charge and were stable in vitro. These particles were internalized in a specific manner by HUVECs compared to a non-targeted lipoplex system, and their uptake was higher than Lipofectamine 2000. Gene silencing efficiency of Angiplex was shown to be 61%.

Abstract A12: Identification of microRNA-based therapeutic candidates using a unique lentiviral microRNA overexpression library

microRNA (miRNA) genes transcribed by RNA polymerase II generate small noncoding miRNAs of 18 to 24 nucleotides after maturation process. The mature miRNAs and their associated isomirs specifically bind to different mRNA transcripts, resulting in down regulation of multiple genes within the cell in a highly multiplexed way. miRNA expression profiles differ between human cell types suggesting cell-specific impacts of each miRNA on the regulation of different biological processes. Comparison of miRNA profiles of tumor samples and adjacent normal tissues showed that some miRNAs are up- or down- regulated and suggested their implication during tumor progression. However, such a miRNA profiling approach is not sufficient to identify the respective role of each miRNA gene during the tumorigenesis. Here, to assess the individual role of each miRNA gene and its different isomirs in a specific cell environment, we have constructed a lentiviral miRNA expression library containing more than 1100 human known and novel miRNA precursors. The arrayed layout of our library allowed high-throughput screens with a large spectrum of functional read-outs using either normal or tumor cells. To exemplify this approach, the results of three different screens will be presented; i.e. identification of miRNAs that inhibit the BRAF pathway, miRNAs that inhibit tumor angiogenesis and miRNAs that stimulate the mesenchymal to epithelial transition. In addition, beyond this hit identification step, we will present detailed characterization of the role of the identified miRNAs in tumor progression by means of molecular and cellular functional assays. Combining our unique miRNA expression library with a functional screening platform has allowed the identification and the further characterization of several miRNAs able to significantly impact on tumor behavior supporting the therapeutic interest of some candidates. Citation Format: Paula I. van Noort, Negar Babae, Gerald W. Verhaegh, Willemijn M. Gommans, Francesco Cerisoli, Mark Verheul, Raymond M. Schiffelers, Arjan W. Griffioen, Jack A. Schalken, Eugene Berezikov, Edwin Cuppen, Roel Q. J. Schaapveld, Jos B. Poell, Gregoire P. Prevost, Meriem Bourajjaj, Suzanna Vidic, Judy R. van Beijnum, Rick J. van Haastert, Iman Schultz, Thijs de Gunt, Onno van Hooij. Identification of microRNA-based therapeutic candidates using a unique lentiviral microRNA overexpression library [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr A12.