Indiana University-Purdue University Indianapolis (IUPUI) / Alternative splicing is a regulatory mechanism that generates multiple mRNA transcripts from a single gene, allowing significant expansion in proteome diversity. Disruption of splicing mechanisms has a large impact on the transcriptome and is a significant driver of complex diseases by producing condition-specific transcripts. Recent studies have reported that mis-spliced RNA transcripts can be another major source of neoantigens directly associated with immune responses. Particularly, aberrant peptides derived from unspliced introns can be presented by the major histocompatibility complex (MHC) class I molecules on the cell surface and elicit immunogenicity. In this dissertation, we first developed an integrated computational pipeline for identifying IR-induced neoantigens (IR-neoAg) from RNA sequencing (RNA-Seq) data. Our workflow also included a random forest classifier for prioritizing the neoepitopes with the highest likelihood to induce a T cell response. Second, we analyzed IR neoantigen using RNA-Seq data for multiple myeloma patients from the MMRF study. Our results suggested that the IR-neoAg load could serve as a prognosis biomarker, and immunosuppression in the myeloma microenvironment might offset the increasing neoantigen load effect. Thirdly, we demonstrated that high IR-neoAg predicts better overall survival in TCGA pancreatic cancer patients. Moreover, our results indicated the IR-neoAg load might be useful in identifying pancreatic cancer patients who might benefit from immune checkpoint blockade (ICB) therapy. Finally, we explored the association of IR-induced neo-peptides with neurodegeneration disease pathology and susceptibility. In conclusion, we presented a state-of-art computational solution for identifying IR-neoAgs, which might aid neoantigen-based vaccine development and the prediction of patient immunotherapy responses. Our studies provide remarkable insights into the roles of alternative splicing in complex diseases by directly mediating immune responses. / 2023-08-16

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Introduction: Mortality in cancer has declined during recent years due to more efficient cancer treatments. Some of the novel immunotherapies against cancer under development has a risk of lacking regulatory guidance for the approval, such as neoantigen-based therapeutic vaccines. Aim: The purpose of the degree project is to evaluate the potential process of approval of neoantigen based vaccines from a regulatory perspective. Methods: The method used to gather data in the study is through qualitative interviews, regulatory documents, guidelines and data from current trials. Results: The results indicate that during the preclinical phase, neoantigen vaccines can receive warrants of a case-by-case approach leading to a more optimized development pathway. During clinical trials, the trial design could consist of master protocols, singlearm trials, comparative trials and adaptive trials where the control group may use the standard of care or historical controls. The indication in the trials should be cancer types with a high tumor mutational burden, low mortality rate, and high medical need. The biomarkers should evaluate immune and tumor response. Endpoints in early trials should evaluate safety and efficacy, and the tumor and immune response in pivotal trials. Neoantigen vaccines can use several incentives during the development and can be approved through conventional or conditional approval. Finally, the study suggests that it is yet too early for the agencies to have established any guidelines. However, advices from regulatory agencies can guide developers towards regulatory approval. Conclusion: This study shows that different study designs and various incentives can be used for the regulatory approval of individualized neoantigen based vaccines. Further guidelines need to be developed to enhance future development.

Cancer is a global health issue and is estimated to be the second leading cause of death worldwide. Within cancer treatment, it has become attractive to introduce precision medicine to harness the body’s own immune system to fight cancer. Personalised peptide cancer vaccine can activate the immune system and elicit an immune response by using the patient’s own blood and tumour cells.   The aim of this study was to gather information to better understand the production of individual-specific peptides and the challenges when producing these peptides with focus from a GMP perspective. The methodology for conducting this study and retrieve information for the result was gathered by three different data bases; EMA, European Commission and FDA, and by qualitative semi-structured interviews.   The findings show that these personalised peptides should be manufactured by using solid-phase peptide synthesis, cleavage from the resin, HPLC purification, and final salt exchange. The traditional GMP requirements must be used as a basis to build a pragmatic program. In terms of delivery time, the peptides can be delivered within a few weeks. The main challenges will be to manufacture successful peptides since the peptide sequences vary from patient to patient and due to that fact have different production efficacy, deliver within the set timeframe, and have flexibility in the manufacture process.   Multiple guidelines need to be followed in order to set up a process that consistently delivers the intended product. When producing oncology drugs on-demand it is essential to keep the timeline since the patients are severely ill. However, further studies are needed to determine how the manufacture of personalised peptides could be performed to harmonise with regulatory guidelines.

Background Cancer immunotherapy has yielded many successes. Yet to some hard-to-treat brain tumors, such as glioblastoma multiforme (GBM) and diffuse intrinsic pontine glioma (DIPG), it still lacks substantial improvement. Neoantigens resulting from mutations in malignant cells are the key targets for employing adoptive cell therapies. A novel therapeutical strategy may be developed based on the identification of T cell receptors (TCRs) targeting specific neoantigens. Methods Previous work had been done to provide essential materials, including candidate neoantigen peptides, human leukocyte antigen (HLA) genotypes, and peripheral blood mononuclear cell (PBMCs) from patients and healthy donors (HDs). Autologous antigen-presenting cells (APCs) and T cells were isolated from PBMCs for in vitro assays. The activation of T cells against peptides was evaluated by the upregulation of 41BB utilizing flow cytometry (FACS). The cell populations with positive signals were sorted through FACS for TCR sequencing directly or after rapid cell expansion. Results T cells and APCs from 12 HDs were isolated. T cells from 10 HDs were analyzed after in vitro stimulation. T cells from HD30 showed reactions to several public neoantigens; while T cells from HD49 and HD53 showed reactions also to private neoantigens restricted in GBM patient C6. Conclusion The upregulation of 41BB indicated the activation of T cells and the existence of reactive TCRs against either public or private neoantigens in some HDs. Those reactive TCRs and their encoding sequences were the fundamentals of future works. Due to practical reasons, TCR sequencing cannot be done within this project. In future works, wildtype peptides will be included to further validate the results, ensuring identified TCRs recognize neoantigens specifically. Furthermore, the identified TCRs will be cloned and transferred to freshly isolated T cells to confirm their functionality. Keywords Cancer immunotherapy, brain cancer, neoantigen, MHC/HLA, TCR

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