@phdthesis{Monjezi2018,
  author    = {Monjezi, Razieh},
  title     = {Engineering of chimeric antigen receptor T cells with enhanced therapeutic index in cancer immunotherapy using non-viral gene transfer and genome editing},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-152521},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {The advances in genetic engineering have enabled us to confer T cells new desired functions or delete their specific undesired endogenous properties for improving their antitumor function. Due to their efficient gene delivery, viral vectors have been successfully used in T-cell engineering to provide gene transfer medicinal products for the treatment of human disease. One example is adoptive cell therapy with T cells that were genetically modified with gamma-retroviral and lentiviral (LV) delivery vectors to express a CD19-specific chimeric antigen receptor (CAR) for cancer treatment. This therapeutic approach has shown remarkable results against B-cell malignancies in pilot clinical trials. Consequently, there is a strong desire to make CAR T cell therapy scalable and globally available to patients. However, there are persistent concerns and limitations with the use of viral vectors for CAR T cell generation with regard to safety, cost and scale of vector production. In order to address these concerns, we aimed to improve non-viral gene transfer and genome editing tools as an effective, safe and broadly applicable alternative to viral delivery methods for T-cell engineering. In the first part of the study, we engineered CAR T cells through non-viral Sleeping Beauty (SB) transposition of CAR genes from minimalistic DNA vectors called minicircles rather than conventional SB plasmids. This novel approach dramatically increased stable gene transfer rate and cell viability and resulted in higher yield of CAR+ T cells without the need of long ex vivo expansion to generate therapeutic doses of CAR+ T cells. Importantly, CD19-CAR T cells modified by MC-based SB transposition were equally effective as LV transduced CD19-CAR T cells in vitro and in a murine xenograft model (NSG/Raji-ffLuc), where a single administration of CD8+ and CD4+ CAR T cells led to complete eradication of lymphoma and memory formation of CAR T cells after lymphoma clearance. To characterize the biosafety profile of the CAR T cell products, we did the most comprehensive genomic insertion site analysis performed so far in T cells modified with SB. The data showed a close-to-random integration profile of the SB transposon with a higher number of insertions in genomic safe harbors compared to LV integrants. We developed a droplet digital PCR assay that enables rapid determination of CAR copy numbers for clinical applications. In the second part of the study, we ablated expression of PD-1, a checkpoint and negative regulator of T cell function to improve the therapeutic index of CAR T cells. This was accomplished using non-viral CRISPR/Cas9 via pre-assemble Cas9 protein and in vitro-transcribed sgRNA (Cas9 RNP). Finally, we combined our developed Cas9 RNP tool with CAR transposition from MC vectors into a single-step protocol and successfully generated PD-1 knockout CAR+ T cells. Based on the promising results achieved from antibody-mediated PD-1 blockade in the treatment of hematological and solid tumors, we are confident that PD-1 knockout CAR T cells enhance the potency of CAR T cell therapies for treatment of cancers without the side effects of antibody-based therapies. In conclusion, we provide a novel platform for virus-free genetic engineering of CAR T cells that can be broadly applied in T-cell cancer therapy. The high level of gene transfer rate and efficient genome editing, superior safety profile as well as ease-of-handling and production of non-viral MC vectors and Cas9 RNP position our developed non-viral strategies to become preferred approaches in advanced cellular and gene-therapy.},
  subject      = {Krebs <Medizin>},
  language  = {en}
}
@phdthesis{Hotz2008,
  author    = {Hotz, Christian},
  title     = {Improvement of Salmonella vaccine strains for cancer immune therapy based on secretion or surface display of antigens},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-29548},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2008},
  abstract  = {Cancer immune therapy represents a promising alternative to conventional anti tumour therapy like radiation, surgical excision of the tumour or classical chemotherapy. The biggest advantage of cancer immune therapy is specificity, achieved by targeting tumour-associated antigens with the effector arms of the host immune system. This is believed to result in less adverse effects than standard therapy and reaches presumably also metastatic lesions at distant sites from the primary tumour. However, cancer immune therapy by vaccination against tumour antigens failed to translate into clinical success, yet. Furthermore, despite tremendous clinical efforts malignant disease still results in high mortalities giving rise to the need for novel vaccination-based therapies against cancer. An interesting approach in this respect is the use of bacteria like attenuated salmonellae as carriers for heterologous cancer antigens. In numerous preclinical studies Salmonella-based vaccines could elicit cell mediated immune responses of the CD4+ and CD8+ type against own and heterologous antigens which make them ideally suited for anti tumour therapy. Special delivery systems in Salmonella carriers like surface display or secretion of antigens were shown to be advantageous for the immunological outcome. This work focussed on developing novel Salmonella carriers for immune therapy against cancer. In a first project, TolC, a multifunctional outer membrane protein of E. coli was utilized as membrane anchor for 3 heterologous antigens. Respective TolC fusion proteins encoded on plasmids were analysed for expression, functionality and plasmid stability in different engineered Salmonella strains. The amount of membrane localized recombinant TolC was enhanced in tolC-deficient strains. Furthermore, fusion proteins were functional and plasmid stability was very high in vitro and in vivo. Disappointingly, neither specific CD4+/CD8+ T-cell responses against the model antigen ovalbumin nor CD8+ responses against the cancer antigen BRAFV600E were detectable in murine model systems. However, mice immunized with Salmonella strains displaying an immunodominant epitope of the cancer related prostate specific antigen (PSA) were partially protected from subsequent tumour challenge with a PSA expressing melanoma cell line. Tumour growth in mice immunized with the respective strain was significantly decelerated compared to controls, thus indicating that this surface display system confers protective immunity against tumours. In a second study, the approved typhoid vaccine strain Salmonella enterica serovar Typhi Ty21a (Ty21a) was improved for the hemolysin type I secretion system of E. coli. This secretion system is widely used for heterologous antigen delivery in live bacterial vaccines. It was demonstrated throughout this work that a mutation of rpoS in Ty21a correlated with decreased ability for hemolysin secretion compared to other Salmonella strains. Complementation with rpoS or the presumed downstream target of rpoS, rfaH resulted in enhanced expression and secretion of heterologous hemolysin in Ty21a. Presumably by raising the amount of free antigen, rfaHcomplemented Ty21a elicited higher antibody titres against heterologous hemolysin in immunized mice than controls and even rpoS-positive Ty21a. Therefore, rfaHcomplemented Ty21a could form the basis of a novel generation of vaccines for human use based on (cancer) antigen secretion.},
  subject      = {Impfstoff},
  language  = {en}
}