<p> Vaccination is arguably the most effective tool at our disposal to prevent the morbidity and mortality associated with infectious disease. However, there are currently several infectious diseases, notably HIV, malaria and tuberculosis, for which we do not posses effective vaccines. Further complicating matters, traditional methods to construct vaccines for these diseases have been unsuccessful. Advances in our understanding of adaptive immunity have demonstrated that vaccines for these diseases likely rely upon potent T cell immunity to be effective. Recombinant adenovirus (rAd) vectors have shown great promise as vaccination platforms since they are easily constructed, stable, well-tolerated and elicit robust T cell responses. The robust activity of rAd vectors based on the human serotype 5 virus (rHuAd5) in murine and simian models merits futher investigation as a prototypic T cell vaccine. To this end, we have undertaken a comprehensive evaluation of T cell immunity following rAd vaccination. Our previous observations determined that the CD8+ T cell response produced by rHuAd5 vaccines displayed a prolonged effector phase that was associated with long-lived antigen presentation. We have further investigated the mechanisms underlying the maintenance of this memory population. Our results have revealed that the memory phenotype is not due to continual recruitment of naive CD8+ T cells. Rather, the sustained effector phenotype appears to depend upon prolonged expression of the antigen-encoding transgene from the rHuAd5 vector. Interestingly, transgene expression was only required for 60 days after which point the memory population stabilized. Further investigation of the relationship between antigen structure and the CD8+ T cell response revealed that antigens which traffic through the ER produce a CD8+ T cell response that expands more rapidly and displays a more pronounced contraction phase than antigens which are produced within the cytosol. While the exact mechanism underlying this phenomenon is not known, we suspect that pathways related to ER stress may be involved. Despite the more dramatic contraction phase associated with antigens that traffic through the ER, the memory phenotype was unchanged. Interestingly, the CD4+ T cell response was not influenced by antigen structure and displays a sharp contraction phase regardless of whether the antigen traffics through the ER or is produced in the cytosol. We further investigated the relationship between CD4+ T cell help and CD8+ T cell immunity produced by rHuAd5. Based on the partially-exhausted phenotype of the CD8+ T cells produced by rHuAd5 (diminished TNF-a production and little IL-2 production), we suspected that inadequate CD4+ T cell help may have been responsible. However, removal of CD4+ T cells did not further impair the CD8+ T cell response produced by rHuAd5. Rather, a lack of CD4+ T cell help only impacted the magnitude of the primary CD8+ T cell response generated by rHuAd5; the functionality of the CD8+ T cell population, including the ability to proliferate following secondary stimulation, were not affected by the absence of CD4+ T cells. Thus, although CD8+ T cell expansion following immunization with rHuAd5 is dependent upon the availability of CD4+ T cell help, the memory functions of the CD8+ T cell population appears to be independent of CD4+ T cell help. Finally, we compared the magnitude of the CD8+ T cell response produced by rHuAd5 and recombinant vaccinia virus. Our results demonstrated that the functionality of the early T cell response produced by both vectors were identical. However, the primary transgene-specific CD8+ T cell responses produced by rHuAd5 were significantly larger than rVV because the vector specific responses were negligible in the case of rAd but very strong following rVV inoculation. This research has contributed to our understanding of T cell immunity following rAd immunization and will assist in the construction and implementation of future vaccines. </p> / Thesis / Doctor of Philosophy (PhD)

京都大学 / 新制・論文博士 / 博士(農学) / 乙第13497号 / 論農博第2901号 / 新制||農||1093(附属図書館) / 学位論文||R4||N5403(農学部図書室) / (主査)教授 谷 史人, 教授 佐々木 努, 教授 保川 清 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

INTRODUCTION: Multiple myeloma (MM) is a disease that results in the production of ineffective immunoglobulins and monoclonal proteins in the blood and urine, leading to insufficient organ function or death. Currently, there is a 5-year survival rate of 47% for patients diagnosed with MM, with a proportion of patients ultimately succumbing to the disease. The current standard of care for MM includes toxic combinations of chemotherapy. The evolution of chimeric antigen receptor (CAR) T-cell therapy for hematologic cancers such as lymphoma, leukemia, and now myeloma has provided another effective treatment option for patients who have relapsed after standard treatments for MM. Idecabtagene Vicleucel (ide-cel), was approved in March 2021 for patients with relapsed and refractory MM. While CAR T-cell treatment appears to be far less toxic than standard chemotherapy, this therapy comes with its own associated toxicities, mainly cytokine release syndrome (CRS) and neurotoxicity (NT). In clinical trials, ide-cel demonstrated to be an effective treatment in some patients, leading to the FDA approval for patients who have exhausted multiple other lines of therapy. Currently, it is unclear why patients respond differently to CAR T-cell treatment and why some patients present with more severe toxicity than others. Therefore, this study aims to examine patient factors such as demographics, age, and treatment history to determine if such characteristics may influence the CAR T-cell response; also, we assess the efficacy of ide-cel in a real-world experience outside of a clinical trial. METHODS: In this study, 14 patients’ medical records were reviewed after receiving commercial CAR T-cell therapy between August 2021 and January 2022. Eligible patients for the therapy were determined by strict inclusion criteria, including having a confirmed diagnosis of MM and exhausting at least four prior lines of therapy, as well as exclusion criteria, such as excluding individuals who have received CAR T-cells prior in a clinical trial setting. Approximately one month before preparation lymphodepletion chemotherapy, eligible patients underwent leukapheresis and had their blood sent to a laboratory to extract T-cells and genetically modify them to express the CAR for reinfusion. On 3 and 5 days prior to CAR T-cell infusion, patients underwent lymphodepletion using fludarabine and cyclophosphamide. Patients remained in the hospital for approximately one week following infusion, pending adverse reactions. After discharge, patients returned to the hospital for routine follow-ups. Data analysis was then performed on collected clinical readouts such as: prior treatments, bone marrow biopsies, response rates, laboratory values from blood samples, and pre- and post-infusion scans of various tissues within the body. RESULTS: At a median follow-up time of 15 weeks, six patients (43%) achieved a complete response (CR), three patients demonstrated a partial response (PR, 21%), and four patients showed disease progression (PD, 28%). Post-infusion scans were not available for one subject (7%) as they were still in the hospital. These results are similar to the phase I and phase II trials in which 45% and 33% of patients demonstrated a CR post-infusion, respectively. As for associated toxicities, 10 patients (71%) experienced CRS and one patient (7%) presented with ICANS. All patients that achieved a CR experienced ide-cel related toxicities, compared with only 38% of those with less favorable or unknown outcomes, which indicates that systemic immune system activation which causes CRS may be required to achieve a CR but CRS is not always linked with a CR outcome. There were 28 different chemotherapy regimens used as the standard of care treatment prior to ide-cel therapy. We assessed the most recent chemotherapeutic regimen in each patient to assess whether there is an association with most recent treatment and response. Of the six patients that achieved a CR to ide-cel, all were previously treated with RVD or CyBorD regimens, compared to the four patients who had disease progression who were mainly treated with salvage DCEP chemotherapy. Four patients (29%) received DCEP as their final chemotherapy regimen, and 3 of these 4 (75%) demonstrated progressive disease after ide-cel. Two patients received Belantamab-Mafodotin prior to ide-cel treatment, with one patient presenting with disease progression and the other patient achieving CR. 71% of patients experienced CRS following ide-cel infusion, which is resembles the phase II trial of ide-cel in which 84% of patients demonstrated CRS. In this study, only 7% of patients experienced neurological toxicity, which is comparable to the 18% of patients that demonstrated to have ICANS in the phase II study. CONCLUSIONS: We found similar performance of the ide-cel CAR-T therapy in the real world setting as in the clinical trial. Also, the complete responses were achieved by subjects with an array of characteristics, including varying recent chemotherapeutic treatments, IgG, IgA, and light-chain only subtypes of MM, and diverse demographics and other characteristics. The characteristic that demonstrated the most predictability and somewhat unique to subjects with CR was the associated toxicities from ide-cel. Development of these associated toxicities may attest that substantial immune activation, of CAR T-cells and other immune cells, leads to the efficacy of the product in eliminating cancer cells. Further analysis will need to be completed as more individuals enroll in this study to be able to determine if there are significant associations between demographics and prior lines of treatment with response to ide-cel CAR-T therapy. Lastly, future studies should assess the immune cell effector functions that are generated in CR patients that will help to specify the association between ide-cel activation, experienced associated toxicities, and its efficacy.

CAR T-cellbehandling är en immunterapi som har visat lovande resultat vid behandling av cancer. Trots det riktade immunsvaret som kan uppnås, betonar komplexiteten i tillverkningsprocessen och behandlingsproceduren det utrymme somm finns för förbättringar. Omprogrammerade T-celler har illustrerat en hög persistens hos patienter, som utsätter dem för risken för systemisk toxicitet. In-vivo transienta CAR T-celler som använder självförstärkande mRNA leverade genom affinitetsproteinbelagda LNP, föreslås som ett standardiserat alternativ som möjligör dosering av terapin vid behov.  Med hjälp av fagdisplay utfördes ett urval av affibody molekyler mot de tre immunonkologiska målproteinerna CD5, CD8 och CD19, i fyra cykler. Monoklonal fag-ELISA och DNA-sekvensering identifierade sju förmodade kandidater mot CD5, en förmodad kandidat mot CD8 och tre mot CD19. SPR analys visade specifik binding från CD5 kandidaterna, medan binding till målprotein inte kunde påvisas för CD8- och CD19 kandidaterna. De identifierade CD5-bindarna kan konjugeras till LNP för T-cell inriktad leverans av själv-amplififerande mRNA, med genetisk kod för en valfri CAR. / Chimeric antigen receptor (CAR) T-cell therapy is an immunotherapy which has shown promising results in treating patients suffering from oncological malignancies. Despite the targeted immune response that can be achieved, elaborate manufacturing and procedure processes emphasise room for improvement. Engineered T-cells have illustrated a high persistence in patients, exposing them to the risk of systemic toxicity. In-vivo transient CAR T-cells using self-amplifying mRNA by delivery through affinity protein coated lipid nanoparticles (LNP) is proposed as a standardised and reversible alternative, allowing for dosing when needed. Using phage display technology, selection of affibody molecules toward the three immune oncology proteins CD5, CD8 and CD19 was performed in four cycles. Monoclonal phage enzyme-linked immunosorbent assay (ELISA) and DNA sequencing identified seven putative candidates toward CD5, one putative candidate was isolated toward CD8, and three toward CD19. Surface plasmon resonance analysis (SPR) showed specific target binding of the CD5 candidate binders, while target binding could not be demonstrated for the CD8 and CD19 candidates. The identified CD5 binders could be conjugated to LNP for T-cell targeted delivery of self-amplifying mRNA encoding any CAR of interest.

T cells play an important role in adaptive immune system against viral infections, while premature aging and dysfunction of T cells induced by unrepaired DNA damages are always non-negligible snags during the long-term of fighting with chronic viral infections, such as Hepatitis B virus (HBV), Hepatitis C virus (HCV) or Human Immunodeficiency Virus (HIV) infection. In this dissertation, we investigated the role of topological DNA damage in reprogramming telomeric DNA damage responses (DDR), mitochondrial metabolisms, and T cell functions using CD4+ T cells derived from individuals with chronic viral infections or healthy subjects treated with topoisomerase inhibitors. The healthy human T cells were treated with camptothecin (CPT) for mitochondrial topoisomerases I (Top1mt) or ICRF-193 or etoposide (ETP) for topoisomerases IIα (Top2α) as models. We found a significant suppression of Top2α and Top1mt protein levels and enzymatic activity in CD4+ T cells in chronically HCV/HIV-infected patients compared to age and gender-matched healthy subjects, along with an accumulation of the topoisomerase cleavage complex (Topcc) in genomic DNA as well as mitochondrial DNA (mtDNA). Mechanistically, topoisomerase inhibition in healthy CD4+ T cells caused topological DNA damage, telomere attrition, mitochondrial metabolic disorder and T cell apoptosis or dysfunction via inducing Topcc accumulation, PARP1 cleavage and failure in DNA repair, thus recapitulating T cell dysregulation in the setting of chronic viral infections. In addition, T cells from virally infected subjects with lower topoisomerase levels were vulnerable to the inhibitor-induced cell apoptosis, indicating an important role for Top2α and Top1mt in preventing DNA topological disruption and cell death. These results demonstrate that accumulation of Topcc and topoisomerase deficiency lead to unrepaired DNA damage and render virally infected patients’ T cells prone to senescence and apoptosis, thus contributing to mitochondrial metabolic disturbance or dysfunction in CD4+ T cell during chronic HCV or HIV infection. This study reveals a novel mechanism by which topoisomerase deficiency promotes telomeric DNA or mtDNA damage and premature T cell aging, and provides a new therapeutic target for restoring the DNA topologic machinery protecting T cells from unwanted DNA damage and to maintain immune competence.

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