<p>  </p> <p>Adoptive cell transfer of chimeric antigen receptors (CAR) T cells has successfully targeted hematological malignancies in human patients. However, unpredicted side effects experienced after injection of the CAR T cells suggests the need for an optimal predictive preclinical animal model. Dogs have intact immune systems and develop solid tumors spontaneously with similar morphology and genetics to humans. I hypothesize that generating CAR T cells for dogs will closely mimic human patients' outcomes, thus providing new understandings of the safety of this immunotherapy. In addition to the dog as a preclinical model, we propose using a universal CAR T cell to overcome various tumor-related immunosuppressive challenges and control the killing of the target cells. To achieve this, we established methods for activating and expanding canine T cells to a clinically relevant scale. Then, we expressed a second-generation anti-FITC-8-41BB-ζ CAR T cell via lentiviral transduction. In the presence of the correct low-molecular-weight bispecific adapter, we showed <em>in-vitro</em> CAR-mediated function. Our results proved that it is feasible to generate functional canine anti-FITC-8-BB-ζ CAR T cells for therapy.</p>

<p>Approximately 50% of all cancer patients undergo radiotherapy (RT) as part of their treatment regimen. However, the development of genetic mutations can severely impair cell death arising from radiation-induced DNA damage, leading to cancer recurrence and poor disease prognosis after treatment. Photodynamic therapy (PDT) offers an alternative approach to induce localized cancer cell death by damaging the cell and organelle membranes instead of relying on DNA damage. Yet, its clinical application is typically limited to surface-level lesions due to the poor tissue-penetration properties of visible light photons, which are required as an activation source.</p><p>Herein, we report the usage of calcium tungstate nanoparticles (CWO NPs) as energy transducers for potentiating PDT using X-ray photons from RT as the activation source. CWO NPs undergo “radioluminescence” wherein they can absorb incident high energy X-ray photons and emit lower energy UV-A and blue photons. Therefore, by intratumorally administering NPs, visible light photons can be generated <i>in situ</i> during RT. Since X-ray photons can penetrate tissue more efficiently than visible light photons, this strategy addresses the limitations of both RT and PDT.</p><p>Firstly, we demonstrate the compatibility of CWO NPs as energy transducers for activating two different photosensitizers: bilirubin (BR) and protoporphyrin IX (PPIX). In the case of bilirubin, we conjugated it with poly(ethylene glycol) (PEG) to form amphiphilic chains that self-assembled to encapsulate CWO NPs. For PPIX, CWO NPs were formulated by encapsulating them with poly(ethylene glycol-b-D,L-lactic acid) block copolymer (PEG-PLA/CWO NPs), while systemically delivering PPIX through its hydrophilic prodrug 5-aminolevulinic acid (ALA). In both scenarios, mechanistic studies revealed that X-ray irradiated CWO NPs generated sufficient blue light photons <i>in situ </i>to activate photosensitizers. This yielded significant improvement in cell-killing effects compared to RT alone, as demonstrated by clonogenic assays conducted in radio-resistant 4T1 and HN31 cell lines. The inherent non-toxicity of both formulations was also demonstrated through MTT assays. <i>In vivo</i> efficacy studies using intratumorally administered NPs demonstrated a significant improvement in tumor growth control and mouse survival compared to conventional RT treatments.</p><p>These studies highlight the potential of RT-PDT in achieving enhanced local tumor control. However, a notable limitation of this approach is its inability to effectively treat metastatic lesions. To address this challenge, recent research has explored the combination of RT-PDT with immune checkpoint inhibition, particularly targeting indoleamine-2,3-dioxygenase (IDO) to induce systemic abscopal responses. To investigate this idea, we conducted efficacy studies in mice upon simultaneous treatment with Epacadostat, a small molecule IDO inhibitor. Although some improvement in tumor control and survival was observed across two separate studies, these results did not reach statistical significance. Consequently, further optimization of treatment schedules and immune checkpoint inhibitor delivery is necessary to obtain a more conclusive understanding of the compatibility of these treatment modalities.</p><p>Next, computed tomography (CT) imaging studies revealed that the current formulation of PEG-PLA/CWO NPs exhibits limited spreading in collagen-dense tumors like 4T1 when administered intratumorally. To overcome this, a modified formulation was developed by surface-functionalization with collagenase (Col-PEG-PLA/CWO NPs) to degrade collagen within tumors. The results suggest approximately 2.4× improvement in intratumoral spreading volume relative to non-functionalized NPs. In the context of RT-PDT, this could imply significantly improved illumination of the tumor volume.</p><p>Lastly, one limitation of the current platform design is the requirement of intratumoral administration to deliver NPs. When administered systemically, less than 1% of NPs passively accumulate in the tumor. To address this, NPs were loaded into chimeric antigen receptor-functionalized neutrophils (CAR-neutrophils) differentiated from human pluripotent stem cells. Specifically, the receptors were modified with chlorotoxin peptide which is capable of selectively targeting glioblastomas. The results presented in this study demonstrate the optimal conditions for uptake of NPs by CAR-neutrophils. Furthermore, purification steps to separate NP-loaded CAR-neutrophils from unloaded NPs are described.</p><p>In summary, these studies describe the development and biological evaluation of two distinct NP platforms for RT-PDT. However, a few key limitations currently hinder the clinical translation of these technologies, including the inability to treat metastases, poor intratumoral spreading, and the need for intratumoral injections. Preliminary solutions have been identified for each of these challenges, providing a foundation for future investigations.</p>

No description available.

Lower-grade glioma (LGG) is a group of tumors arising from the cells of the central nervous system. Although various therapy interventions are used, the prognosis remains different. Novel biomarkers are needed for the prognosis of disease and novel therapeutic strategies in LGG. The procollagen-lysine, 2-oxoglutarate 5-dioxygenase (PLOD) family contains three members and is related to multiple cancers, yet it was not investigated in LGG. Data from the Chinese Glioma Genome Atlas (CGGA) and The Cancer Genome Atlas (TCGA) cohorts were used to analyze the role of PLOD in LGG. As the PLOD family is involved in processes, such as tumor formation and cancer metastasis, we focused on its relationship to the tumor microenvironment (TME) in LGG. A high expression of the PLOD family relates to poor prognosis and high infiltration of immune cells within the TME. The expression level of the PLOD family might become a novel biomarker for prognosis and is a potential target for individual treatment decisions in LGG.

Background: Treatment options for poorly differentiated (PDTC) and anaplastic (ATC) thyroid carcinoma are unsatisfactory and prognosis is generally poor. Lenvatinib (LEN), a multi-tyrosine kinase inhibitor targeting fibroblast growth factor receptors (FGFR) 1-4 is approved for advanced radioiodine refractory thyroid carcinoma, but response to single agent is poor in ATC. Recent reports of combining LEN with PD-1 inhibitor pembrolizumab (PEM) are promising. Materials and Methods: Primary ATC (n=93) and PDTC (n=47) tissue samples diagnosed 1997-2019 at five German tertiary care centers were assessed for PD-L1 expression by immunohistochemistry using Tumor Proportion Score (TPS). FGFR 1-4 mRNA was quantified in 31 ATC and 14 PDTC with RNAscope in-situ hybridization. Normal thyroid tissue (NT) and papillary thyroid carcinoma (PTC) served as controls. Disease specific survival (DSS) was the primary outcome variable. Results: PD-L1 TPS≥50% was observed in 42% of ATC and 26% of PDTC specimens. Mean PD-L1 expression was significantly higher in ATC (TPS 30%) than in PDTC (5%; p<0.01) and NT (0%, p<0.001). 53% of PDTC samples had PD-L1 expression ≤5%. FGFR mRNA expression was generally low in all samples but combined FGFR1-4 expression was significantly higher in PDTC and ATC compared to NT (each p<0.001). No impact of PD-L1 and FGFR 1-4 expression was observed on DSS. Conclusion: High tumoral expression of PD-L1 in a large proportion of ATCs and a subgroup of PDTCs provides a rationale for immune checkpoint inhibition. FGFR expression is low thyroid tumor cells. The clinically observed synergism of PEM with LEN may be caused by immune modulation.

Anti-CD19 CAR-T cell immunotherapy is a hopeful treatment option for patients with B cell lymphomas, however it copes with partly severe adverse effects like neurotoxicity. Single-cell resolved molecular data sets in combination with clinical parametrization allow for comprehensive characterization of cellular subpopulations, their transcriptomic states, and their relation to the adverse effects. We here present a re-analysis of single-cell RNA sequencing data of 24 patients comprising more than 130,000 cells with focus on cellular states and their association to immune cell related neurotoxicity. For this, we developed a single-cell data portraying workflow to disentangle the transcriptional state space with single-cell resolution and its analysis in terms of modularly-composed cellular programs. We demonstrated capabilities of single-cell data portraying to disentangle transcriptional states using intuitive visualization, functional mining, molecular cell stratification, and variability analyses. Our analysis revealed that the T cell composition of the patient’s infusion product as well as the spectrum of their transcriptional states of cells derived from patients with low ICANS grade do not markedly differ from those of cells from high ICANS patients, while the relative abundancies, particularly that of cycling cells, of LAG3-mediated exhaustion and of CAR positive cells, vary. Our study provides molecular details of the transcriptomic landscape with possible impact to overcome neurotoxicity.

No description available.

A major obstacle in the implementation of adoptive cell therapy (ACT) for solid tumors is CD8+ T cell quantity and functional quality. In order to address this issue, the ACT field has directed considerable effort toward the generation of less-differentiated memory T cells (Tm), which demonstrate superior effector function and engraftment over effector T cells. An obstacle in using Tm for ACT is their requirement for in vivo activation before full effector function can be acquired. We sought to determine if a rhabdovirus expressing a defined tumor antigen (i.e. a rhabdoviral oncolytic vaccine) could activate adoptively-transferred Tm in vivo and eliminate established tumors. We used ex vivo cultured DUC18 TCR-transgenic Tm combined with a rhabdoviral oncolytic vaccine to target established CMS5 fibrosarcomas in both balb/c and NRG mice, and we compared the efficacy of the combination treatment versus monotherapies. Our data demonstrate that the rhabdoviral oncolytic vaccine was capable of expanding adoptively-transferred Tm in order to eliminate established tumors. Furthermore, synergy between ACT and oncolytic vaccination was required for optimal therapeutic outcome. Interestingly, we observed a population of endogenous, tumor-primed lymphocytes which appeared to be required for complete tumor elimination and subsequent memory formation. This was in contrast to the current consensus in the ACT field which is that endogenous lymphocytes are detrimental to therapeutic outcome, thus necessitating lymphodepletion prior to the commencement of therapy. Our data suggest that endogenous lymphocytes may be a beneficial cell population which is overlooked by current approaches to ACT. / Thesis / Master of Science (MSc) / Current approaches to the T cell therapy of cancer are hindered by poor cell quality. It is simple to grow higher quality T cells, but it is difficult to grow very large numbers of them. Furthermore, higher quality T cells need a signal in order to “switch on” before they can start killing cancer cells. Here, we use a cancer-targeting virus as a signal for these cells to activate, grow to very large numbers in the patient, and destroy their tumor. Our vaccine also switches on other immune cells in the patient, which help guarantee the destruction of the tumor. The significance of this work is that it will improve T cell therapy for cancer by opening the possibility of using higher-quality T cells which are much better at killing cancer than the currently used type of T cells.

Immunotherapy, by enhancing the endogenous anti-tumor immune responses, is showing promising results for the treatment of numerous cancers refractory to conventional therapies. However, its effectiveness for advanced castration-resistant prostate cancer remains unsatisfactory and new therapeutic strategies need to be developed. To this end, mathematical modeling provides a quantitative framework for testing in silico the efficacy of new treatments and combination therapies, as well as understanding unknown biological mechanisms. In this dissertation we present two mathematical models of prostate cancer immunotherapy defined as systems of ordinary differential equations. The first work, introduced in Chapter 2, provides a mathematical model of prostate cancer immunotherapy which has been calibrated using data from pre-clinical experiments in mice. This model describes the evolution of prostate cancer, key components of the immune system, and seven treatments. Numerous combination therapies were evaluated considering both the degree of tumor inhibition and the predicted synergistic effects, integrated into a decision tree. Our simulations predicted cancer vaccine combined with immune checkpoint blockade as the most effective dual-drug combination immunotherapy for subjects treated with androgen-deprivation therapy that developed resistance. Overall, this model serves as a computational framework to support drug development, by generating hypotheses that can be tested experimentally in pre-clinical models. The Chapter 3 is devoted to the description of a human prostate cancer mathematical model. The potential effect of immunotherapies on castration-resistant form has been analyzed. In particular, the model includes the dendritic vaccine sipuleucel-T, the only currently available immunotherapy option for advanced prostate cancer, and the ipilimumab, a drug targeting the cytotoxic T-lymphocyte antigen 4 , exposed on the CTLs membrane, currently under Phase II clinical trial. From a mathematical analysis of a simplified model, it seems likely that, under continuous administration of ipilimumab, the system lies in a bistable situation where both the no-tumor equilibrium and the high-tumor equilibrium are attractive. The schedule of periodic treatments could then determine the outcome, and mathematical models could help in deciding an optimal schedule.

Gastric cancer is a leading cause of cancer death worldwide. Systemic treatment comprising chemotherapy and targeted therapy is the standard of care in advanced/metastatic gastric cancer. Comprehensive molecular characterization of gastric adenocarcinomas by the TCGA Consortium and ACRG has resulted in the definition of distinct molecular subtypes. These efforts have in parallel built a basis for the development of novel molecularly stratified treatment approaches. Based on this molecular characterization, an increasing number of specific genomic alterations can potentially serve as treatment targets. Consequently, the development of promising compounds is ongoing. In this review, key molecular alterations in gastric and gastroesophageal junction cancers will be addressed. Finally, the current status of the translation of targeted therapy towards clinical applications will be reviewed.