High-Affinity DARPin Allows Targeting of MeV to Glioblastoma Multiforme in Combination with Protease Targeting without Loss of Potency.

Measles virus (MeV) is naturally cytolytic by extensive cell-to-cell fusion. Vaccine-derived MeV is toxic for cancer cells and is clinically tested as oncolytic virus. To combine the potential of MeV with enhanced safety, different targeting strategies have been described. We generated a receptor-targeted MeV by using receptor-blind viral attachment protein genetically fused to designed ankyrin repeat protein (DARPin) binding domains specific for the epidermal growth factor receptor (EGFR). To reduce on-target toxicity for EGFR+ healthy cells, we used an engineered viral fusion protein activatable by tumor-associated matrix metalloproteases (MMPs) for additional protease targeting. The dual-targeted virus replicated exclusively on EGFR+/MMP+ tumor cells but was safe on healthy EGFR+ target cells, primary human keratinocytes. Nevertheless, glioblastoma and other tumor cells were efficiently killed by all targeted viruses, although replication and oncolysis were slower for protease-targeted MeV. In vivo, efficacy of EGFR-targeted MeV was virtually unimpaired, whereas also dual-targeted MeV showed significant intra-tumoral spread and efficacy and could be armed with a prodrug convertase. The use of DARPin-domains resulted in potent EGFR-targeted MeV and for the first time effective dual retargeting of an oncolytic virus, further enhancing tumor selectivity. Together with powerful cell-toxic genes, the application as highly tumor-specific platform is promising.

Enhanced lysis by bispecific oncolytic measles viruses simultaneously using HER2/neu or EpCAM as target receptors.

To target oncolytic measles viruses (MV) to tumors, we exploit the binding specificity of designed ankyrin repeat proteins (DARPins). These DARPin-MVs have high tumor selectivity while maintaining excellent oncolytic potency. Stability, small size, and efficacy of DARPins allowed the generation of MVs simultaneously targeted to tumor marker HER2/neu and cancer stem cell (CSC) marker EpCAM. For optimization, the linker connecting both DARPins was varied in flexibility and length. Flexibility had no impact on fusion helper activity whereas length had. MVs with bispecific MV-H are genetically stable and revealed the desired double-target specificity. In vitro, the cytolytic activity of bispecific MVs was superior or comparable to mono-targeted viruses depending on the target cells. In vivo, therapeutic efficacy of the bispecific viruses was validated in an orthotopic ovarian carcinoma model revealing an effective reduction of tumor mass. Finally, the power of bispecific targeting was demonstrated on cocultures of different tumor cells thereby mimicking tumor heterogeneity in vitro, more closely reflecting real tumors. Here, bispecific excelled monospecific viruses in efficacy. DARPin-based targeting domains thus allow the generation of efficacious oncolytic viruses with double specificity, with the potential to handle intratumoral variation of antigen expression and to simultaneously target CSCs and the bulk tumor mass.

Lentiviral Protein Transfer Vectors Are an Efficient Vaccine Platform and Induce a Strong Antigen-Specific Cytotoxic T Cell Response.

UNLABELLED: To induce and trigger innate and adaptive immune responses, antigen-presenting cells (APCs) take up and process antigens. Retroviral particles are capable of transferring not only genetic information but also foreign cargo proteins when they are genetically fused to viral structural proteins. Here, we demonstrate the capacity of lentiviral protein transfer vectors (PTVs) for targeted antigen transfer directly into APCs and thereby induction of cytotoxic T cell responses. Targeting of lentiviral PTVs to APCs can be achieved analogously to gene transfer vectors by pseudotyping the particles with truncated wild-type measles virus (MV) glycoproteins (GPs), which use human SLAM (signaling lymphocyte activation molecule) as a main entry receptor. SLAM is expressed on stimulated lymphocytes and APCs, including dendritic cells. SLAM-targeted PTVs transferred the reporter protein green fluorescent protein (GFP) or Cre recombinase with strict receptor specificity into SLAM-expressing CHO and B cell lines, in contrast to broadly transducing vesicular stomatitis virus G protein (VSV-G) pseudotyped PTVs. Primary myeloid dendritic cells (mDCs) incubated with targeted or nontargeted ovalbumin (Ova)-transferring PTVs stimulated Ova-specific T lymphocytes, especially CD8(+) T cells. Administration of Ova-PTVs into SLAM-transgenic and control mice confirmed the observed predominant induction of antigen-specific CD8(+) T cells and demonstrated the capacity of protein transfer vectors as suitable vaccines for the induction of antigen-specific immune responses. IMPORTANCE: This study demonstrates the specificity and efficacy of antigen transfer by SLAM-targeted and nontargeted lentiviral protein transfer vectors into antigen-presenting cells to trigger antigen-specific immune responses in vitro and in vivo. The observed predominant activation of antigen-specific CD8(+) T cells indicates the suitability of SLAM-targeted and also nontargeted PTVs as a vaccine for the induction of cytotoxic immune responses. Since cytotoxic CD8(+) T lymphocytes are a mainstay of antitumoral immune responses, PTVs could be engineered for the transfer of specific tumor antigens provoking tailored antitumoral immunity. Therefore, PTVs can be used as safe and efficient alternatives to gene transfer vectors or live attenuated replicating vector platforms, avoiding genotoxicity or general toxicity in highly immunocompromised patients, respectively. Thereby, the potential for easy envelope exchange allows the circumventing of neutralizing antibodies, e.g., during repeated boost immunizations.

Triple therapy in chronic hepatitis C: initial series in a public health program in the South of Brazil.

BACKGROUND: Chronic hepatitis C has great impact on world's health. Current therapy for genotype 1 hepatitis C virus includes protease inhibitors boceprevir and telaprevir, associated to standard therapy - peginterferon alfa + ribavirin. There are no published data in Brazil on the results of this new therapy, and it is interesting an evaluation of what was accomplished up to this moment. Objectives To evaluate virologic response to triple therapy, as well as the safety profile and tolerability. METHOD: This study is a clinical series of patients receiving triple therapy for C hepatitis in a single center of a Public Health System of South Brasil. Out of the 121 patients that initiated the triple therapy, the first patients that finished the treatment and evaluated the sustained virological response (24 weeks after the end of treatment) were included. RESULTS: Twenty four genotype 1 chronic hepatitis C monoinfected patients were included. Nineteen (79.2%) patients had been previously treated. Thirteen (54.2%) patients were cirrhotic. Nineteen (79.2%) patients completed the planned therapy. By the end of the treatment, 14 (58.3%) out of 24 patients had undetectable viral load. Sustained virologic response occurred in 12 (50.0%) out of 24 patients, 07 (58.3%) in telaprevir group and 05 (41.7%) in boceprevir group. Out of 24 patients under triple therapy, 58% (n=14) presented anemia. CONCLUSIONS: In conclusion, despite the small number of patients treated with triple therapy evaluated in the current study, it possibly reflects the population under this therapy in real-life.

TRIPLE THERAPY IN CHRONIC HEPATITIS C: initial series in a public health program in the South of Brazil / Terapia tripla em pacientes com hepatite C: experiência inicial em um pólo de aplicação do Sul do Brasil

Background Chronic hepatitis C has great impact on world’s health. Current therapy for genotype 1 hepatitis C virus includes protease inhibitors boceprevir and telaprevir, associated to standard therapy - peginterferon alfa + ribavirin. There are no published data in Brazil on the results of this new therapy, and it is interesting an evaluation of what was accomplished up to this moment. Objectives To evaluate virologic response to triple therapy, as well as the safety profile and tolerability. Method This study is a clinical series of patients receiving triple therapy for C hepatitis in a single center of a Public Health System of South Brasil. Out of the 121 patients that initiated the triple therapy, the first patients that finished the treatment and evaluated the sustained virological response (24 weeks after the end of treatment) were included. Results Twenty four genotype 1 chronic hepatitis C monoinfected patients were included. Nineteen (79.2%) patients had been previously treated. Thirteen (54.2%) patients were cirrhotic. Nineteen (79.2%) patients completed the planned therapy. By the end of the treatment, 14 (58.3%) out of 24 patients had undetectable viral load. Sustained virologic response occurred in 12 (50.0%) out of 24 patients, 07 (58.3%) in telaprevir group and 05 (41.7%) in boceprevir group. Out of 24 patients under triple therapy, 58% (n=14) presented anemia. Conclusions In conclusion, despite the small number of patients treated with triple therapy evaluated in the current study, it possibly reflects the population under this therapy in real-life. .

Contexto A hepatite crônica pelo vírus C tem grande impacto na saúde mundial. A terapia atual do genótipo 1 inclui os inibidores de protease (IP) boceprevir e telaprevir, associados à terapia padrão - alfapeginterferona + ribavirina (PR). No Brasil ainda não há estudos publicados sobre os resultados dessa nova terapia, sendo de interesse uma avaliação do que foi realizado até o momento. Objetivos Avaliar a resposta virológica ao tratamento triplo, bem como o perfil de segurança e tolerabilidade. Métodos O estudo consta de série de casos dos pacientes em uso de terapia tripla para o tratamento da hepatite C em um polo de tratamento da Secretaria Estadual da Saúde do Estado do Rio Grande do Sul, Brasil. Dentre os 121 pacientes que estão em uso de terapia tripla (PR e IP) foram apresentados os dados referentes aos primeiros que finalizaram o tratamento e realizaram avaliação da resposta virológica sustentada na semana 24 pós-tratamento. Resultados Foram incluídos 24 pacientes monoinfectados por hepatite C crônica genótipo 1. Dezenove (79%) pacientes eram previamente experimentados. Treze (54,2%) pacientes apresentavam cirrose. Dezenove (79,2%) pacientes completaram o tratamento planejado. Ao final do tratamento, 14 (58,3%) dos 24 pacientes apresentaram carga viral indetectável. Resposta virológica sustentada ocorreu em 12 (50%) dos 24 pacientes, sendo 07 (58,3%) no grupo telaprevir e 05 (41,7%) no grupo boceprevir. Dos 24 pacientes submetidos à terapia tripla, 58% (n=14) apresentaram anemia. Conclusões Embora o presente estudo tenha avaliado um pequeno número de casos, possivelmente reflete a população submetida à terapia tripla na vida real, despida das restrições dos estudos de registro. .