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Employing engineered exosomes for combating colon cancer and engineering CD38 as an optimized drug carrier

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Employing engineered exosomes for combating colon cancer and engineering CD38 as an optimized drug carrier

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Content Employing Engineered Exosomes for Combating Colon Cancer and Engineering CD38 as an Optimized Drug Carrier By Yuanteng Zhao A Thesis Presented to the FACULTY OF THE USC SCHOOL OF PHARMACY UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree MASTER OF SCIENCE (Molecular Pharmacology and Toxicology) December 2023 Copyright 2023 Yuanteng Zhao i Table of Contents List of Tables..................................................................................................................................ii List of Figur es...............................................................................................................................iii Abstr act....................................................................................................................................1&22 Chapter 1: Employing Engineered Exosomes for Combating Colon Cancer 1.1 Abstract..........................................................................................................................1 1.2 Origin and cellular function of exosome.......................................................................2 1.3 The Role of Protein and Antibody on Exosome Surface...............................................3 1.4 Exosome’s Cargo and its Functions in Cancer Biology................................................5 1.5 Exosome as a Tool in Tumor Treatment........................................................................7 1.6 Development and Application of Genetically Engineered Multifunctional Exosomes.............................................................................................................................9 1.7 The Design Rationale of GEMINI-Exosome...............................................................11 1.8 Exosome Surface Display Design................................................................................12 1.9 Production and Purification of Exosomes....................................................................15 1.10 Future Experiment Design.........................................................................................20 Chapter 2:Engineering CD38 as an Optimized Drug Carrier.................................................22 2.1 Abstract .......................................................................................................................22 2.2 Introduction of CD38...................................................................................................23 2.3 Study of CD38 through NAD+ analogues..........................................................,........24 2.4 CD38 as a Drug carrier through NAD+ analogues......................................................25 2.5 Conjugation of multiple CD38 to Antibodies Decreased the Pharmacokinetics Performance.......................................................................................................................27 2.6 Aim and Hypothesis ....................................................................................................28 2.7.Experimental Design ...................................................................................................28 2.8 In silico secondary structure analysis..........................................................................33 2.9 In silico tertiary structure analysis...............................................................................34 Refer ences ....................................................................................................................................49 ii List of Tables Table 1: Secondary Structure and Solvent Accessibility Prediction.............................................36 Table 2: Protein Disorder and Flexibility Prediction.....................................................................38 Table 3: Phyre-generated alpha helix and beta sheet distribution.................................................39 Table 4: Phyre-generated catalytic pocket view............................................................................43 Table 5:Phyre-generated tertiary structure prediction...................................................................45 iii List of Figures Fig 1: Mechanism of GEMINI-Exos. ..........................................................................................17 Fig 2: Molecular Interaction of Gemini-Exos. ..............................................................................17 Fig 3a: Western blot of anti-CD9 and anti-HA for purifiedUE and PCO exosomes.....................19 Fig 3b: Western blot of anti-CD9 and anti-HA for cotransfected (GEMINI) exosomes...............19 Fig 4: Coomassie-stained SDS-Page gels of purified truncated CD38s and wildtype CD38........31 Fig 5: Wildtype and truncated CD38’s NAD cyclase catalyzing activity measured by NGD assay...............................................................................................................................................33 1 Chapter I: Employing Engineered Exosomes for Combating Colon Cancer 1.1 Abstract: Exosomes are nano-sized vesicles secreted by cells, containing a variety of molecules such as proteins, RNA, and lipids. These vesicles play a crucial role in intercellular communication and have gained significant attention for their regenerative capabilities. Exosome therapy involves isolating and purifying exosomes from specific cell sources, such as cancer cells and dendritic cells, and administering them to target tissues or organs to elicit therapeutic activities. These exosomes act as messengers, delivering bioactive molecules and genetic materials to recipient cells and modulating their behavior and promoting tissue repair. By harnessing the immune regulating properties of exosomes, scientists and clinicians are exploring their potential applications in oncology. Exciting research findings and clinical trials are paving the way for exosome therapy to become a revolutionary approach in regenerative medicine. However, clinical trials failed because of insufficient response. Genetically engineered exosomes emerged as a promising option due to their effectiveness and multi-functionality for immunotherapy. Colon cancer is the second most prevalent form of cancer worldwide and is associated with significant morbidity and mortality. Colon cancer typically arises from abnormal growths called polyps, which can be detected and removed during routine screenings, reducing the risk of developing cancer. The primary therapeutic approaches for colon cancer include surgery, chemotherapy, radiation therapy, targeted therapy, and immunotherapy. Surgery is the most common treatment for colon cancer and involves the removal of the tumor and nearby lymph nodes. If the cancer has spread to surrounding tissues or organs, drug therapies are required, though advanced states patients have lower response rate to the therapy. In addition, chemotherapy causes intolerable side effects, which calls for the need for safer and more 2 effective immunotherapies Several antibodies and checkpoint inhibitors have been approved by FDA to be utilized as first-line therapy, but the overall response rate is less than 20%. The suboptimal response rate could be attributed to the fact that eliciting immunological response from a single pathway is insufficient. Since genetically-engineered exosomes enable creating a multifunctional tool in a single molecular entity, we proposed to apply such exosomes to colon cancer theray, which can trigger multiple pathways simultaneously in a site-specific manner. In this chapter, the rationale design, generation, and preliminary characterization of genetically engineered exosomes are included. 1.2 Origin and cellular function of exosome Exosome is a type of cell-secreted extracellular vesicles, which plays important roles in the body's physiological process. Exosomes are secreted by almost all types of cells, and they enter diverse categories of biofluid including blood, urine, gastric acid, ascitic fluid, bile etc. (Doyle & Wang, 2019) The size of the exosome usually ranges from 30 - 200 nm, while other members in the extracellular vesicle family such as microvesicles range from 100-1000 nm and apoptotic bodies are usually greater than 1000 nm. (Gurung et al., 2021) Similar to the cell membrane and other organelles, exosomes’ surface is composed of phospholipid bilayers with diverse type of protein, antibody and lipid. Different cell origins lead to distinct surface characteristics of the exosomes, which not only enhance their surface structure stability but also endow the exosome with their unique ability in cell signaling and communication. (Wang et al., 2020) The cellular communication can in turn promote the biogenesis of exosome, suggesting that exosomes are critical information transit stations for the pathological process. (De Toro et al., 2015) Within the vesicle, the exosome contains DNA, protein, enzymes, mRNA, miRNA, lncRNA and cRNA (J. Dai et al., 2020), which could be delivered to the target cell’s cytosol by membrane fusion or 3 endocytosis. The cargo-like internal structure and the lipid bilayer of the exosome enables its potential as a therapeutic agent delivery vehicle. Exosomes’ formation of different origins follows a similar mechanism, which starts at the endosomal pathway. After endocytosis, the plasma membrane invaginates to form a cup-like structure through an early secretory endosomal pathway. The early endosome then develops into intraluminal vesicles (ILV) after content is loaded by Golgi and endoplasmic reticulum (ER). ESCRT (endosomal sorting complexes required for transport) then facilitate the acidification of the ILV to form the late endosome. The late endosome is also referred to multivesicular bodies (MVBs), which subsequently release the exosome to extracellular spaces after fusing with the plasma membrane through the exocytosis process. 1.3 The Role of Protein and Antibody on Exosome Surface The endogenous marker and protein are critical regulators for the production, transportation, membrane fusion and communication of exosomes. The endosomal destination heavily depends on the ESCRT, whose member ESCRT-0 is responsible for binding to the cargo with the ubiquitination tag to avoid the lysosomal pathway(Ruivo et al., 2017). ESCRT-I and ESCRT-II facilitate the budding of the payload, while ESCRT-III nips the exosome off the MVBs.(Schoneberg et al., 2017) ALIX and TSG101 also participate in the ESCRT pathway by identifying the ubiquitinated protein and arranging them on the target exosome. (Juan & Furthauer, 2018) Rab GTPase protein modulates the exosomes’ intracellular destination assignment by regulating the MVB trafficking to lysosome or to the recycling center. Rab27a and Rab27b plays a role in the process of exosome attaching to the plasma membrane. One of the most notable families of protein on exosomes are tetraspanins, including CD9, CD63 and CD81, which also contribute to the formation of the exosome by sorting proteins into the exosomes. 4 CD9 also aids the exosomal content delivery by assisting the fusing of the exosomal membrane with the target cell membrane.Heat shock protein (HSP) family proteins are populated in the exosomes to promote the biogenesis process. HSP protein on exosome are also highly correlated with the tumor environment; HSP70’s expression is upregulated on the tumor cell secreted exosome compared to the normal cells(Chanteloup et al., 2020). HSP90 regulates the exosome release and promote the tumor cell growth under the hypoxia conditions.(Sager et al., 2022) CD47 on the surface releases the “don’t eat me” signal to help exosome evade from the immune surveillance and the clearance in the circulation. (Belhadj et al., 2020) CD63 also installs the latent membrane protein 1 (LMP1) on the exosome to aid the escape from the lysosomal pathways. (Hurwitz et al., 2017) The surface lipid layer protein docking and the encapsulated cargo feature of exosomes appoint them the role in immune regulation, cell to cell communication, cell proliferation and differentiation and environment regulation. Exosomes secreted by dendritic cells carry major histocompatibility complexes (MHC) class I and II proteins bound with antigenic peptide, which could subsequently stimulate T cells, indicating their role in inducing the innate and adaptive immune response. (Li et al., 2022) B-cell derived exosomes could also present the allergen peptide to stimulate the allergen-specific T-cells to stimulate the TH2 cytokine release (Admyre et al., 2007). In addition, exosomes also present the co-stimulatory molecule and adhesion molecule to the target cells to induce the generation of CD8 + and CD4 + T cells related immune response. (J. Dai et al., 2020) During the T cell activation process, the exosome could serve as a direct antigen presenter or a vehicle for transferring antigen to antigen presenting cells (APC). Alternatively, the tumor antigen presented on the exosome surface could also promote tumor 5 angiogenesis and metastasis through the cohort effect of the surface pro-angiogenic biomolecules. (Olejarz et al., 2020) 1.4 Exosome’s Cargo and its Functions in Cancer Biology A variety of RNA is encapsulated in the exosomes for different purposes, with some of the functions uncharacterized. mRNA, microRNA (miRNA) and small interfering RNA target at protein synthesis, and protein translation regulation. tRNA, snRNA, Y RNA and vault RNA in exosomes have not been well-characterized, but they are predicted to prevent oligomerization and autophagy. Some populations of exosomes contain RNA fragments which are caused by methylation and uridylylation, and their function remains unexplored. Of all types of RNA, miRNA stands for the most prominent type in the exosome. miRNA containing exosomes secreted by epithelial cells is reported to promote tumorigenesis, invasion and cell proliferation; breast cancer cells secreted exosomes with miRNA to increase drug resistance.(O'Brien et al., 2020) The cargo-carrying feature of the exosome endorses its important role in cancer biology. Tumor microenvironment facilitates the modifications in gene expression profiles. These alterations stimulate cancer cells to adapt to new environments by enabling them to infiltrate the neighboring tissue, penetrate to lymphatic or blood vessels and establish in distant organs, and cultivate resilience against cytotoxic medications. Cancer-associated fibroblasts (CAFs) are essential parts of the tumor microenvironment, and normal fibroblasts (NF) engagement with cancer cells significantly influences their initiation and stimulation. (Melillo, 2007) NFs suppress tumor growth, while CAF promotes tumor cell invasions and expansions. miRNAs in the exosome could convert NFs into CAFs, but the communication mechanisms remain largely unknown. Tumor cells transport a large number of exosomes containing various angiogenic 6 molecules, which promote angiogenesis systematically. The tumor-derived exosome can transport within tumor tissues or between tumor and endothelial system. (Ahmadi & Rezaie, 2020) Malignant mesothelioma (MM) produces exosomes containing cargo that promote upregulation of microtubule formation molecules and tumor invasion. Exosomes produced by myeloma cells could elicit metastatic site formation in bone marrow and initiate angiogenesis in mouse models. (Wang et al., 2016) In addition, brain cancer with highly vascularized characteristics demonstrated high expression levels of angiogenic biomolecules in both cellular and mouse models. (Lang et al., 2017)In the case of leukemia, tumor cell utilizes the paracrine exosome for the formation of initiation niche and suppression of hematopoiesis. Exosome trafficking alters the generations of IL-8, which assist cancer cells to evade from the chemo cytotoxicity. VEGF- or VEGFR-carrying exosomes stimulate the glycolysis in endothelial cells, which further strengthens the survivability from chemotherapy. T-cell and endothelial cells' function is also altered by internalization of exosomes by reprogramming the cellular activity with exosomal cargo content. Exosomes enter the immune and endothelial cells through endocytosis, and after completing their tasks in a cell, they will transfer to other cells through tubule networks. The transferring process suggests that exosomes facilitate cell to cell communication to promote angiogenesis. Pro-angiogenic ligands on the exosome surface interact with receptors on the cancer cell to regulate the cellular signaling pathways to drive angiogenesis pathways. Exosomes originate from different types of cancer cells that differ greatly in their surface protein type, whereas they facilitate cancer cell migration through a variety of pathways. Matrix metalloproteinases (MMPs) family protein, HAX-1, VEGF, Interleukin-6/8, VEGF and etc on cancer-sourced exosome exert angiogenesis, mobility-enhancing and proliferation in different combination's synergistic effect. (Wang et al., 2016) The construction of pre-metastatic 7 niche is highly correlated with epithelial-mesenchymal transition mediated by cancer-produced exosomes with angiogenic Rac-1 and PAK-2. (Gopal et al., 2016)The cancer cells produced exosomes contain diverse cargo and surface protein that promote the development of the cancer environment. 1.5 Exosome as a Tool in Tumor Treatment Despite that tumor-released exosomes (TEX) played an important role in tumor progression, they could be also utilized as potential therapeutic agents by eliciting anti-tumor immunity. Tumor cells secreted exosomes containing tumor-specific antigen, immunostimulatory and immunosuppressive molecules that may interact with immune cells including T cells, dendritic cells and NK cells. (Xu et al., 2020) Exosomes produced by cancer cells present antigen to dendritic cells to induce tumor-antigen-specific cytotoxic T cell (CTL) response and to increase CD8+ T cell population. Rab27 on tumor exosomes upregulates the major histocompatibility complex class molecules (MHC) and other costimulatory molecules to promote the development of dendritic cells. The immune-stimulation effect of TEXs encourages the development of TEX- based cancer vaccines by incubation of TEXs with dendritic cells isolated from cancer patients, and TEXs-induced immune reaction is stronger than tumor cell lysate induced immune reaction. Nevertheless, TEXs are also reported to suppress the expansion of CD8+ T cells but increase the expansion of CD4+ T cells. The immunosuppressive components including Tumor necrosis factor(TNF)-related apoptosis inducing ligand (TRAIL) and Programmed death-ligand 1 (PD-L1) could lead to immune escape for various cancer cells. TEXs possess a set of characteristics that allow them to either dampen or enhance the immune response, most likely influenced by the surrounding conditions where they are released. (Xu et al., 2020) As the result, though several TEXs-based vaccines have been developed and been tested in animal experiments and in vitro, 8 they could not enter clinical trials due to the ambiguous effect in the immune system and poor disease outcome. Exosomes derived from dendritic cells (DEX) play an important role in immune modulation by priming CTL and inhibiting tumor growth. Tumor-peptide activated dendritic cells secreted exosomes that also contain MHC-peptide complexes. After internalization into cells through the endosomal pathway, they assist the proliferation and activation of the T cells population.(Xia et al., 2022) In mice with melanoma, DEX increases the accumulation of T cells at the tumor location, and it also suppresses the number of Treg and increases the number of CD4+ and CD8+ T cells. DEXs demonstrate a lower efficiency in stimulating T cells compared to dendritic cells, and several studies showed that pre-processing by dendritic cells is the major pathway in presenting antigen to effector cells. (Santos & Almeida, 2021)Unlike TEXs, DEXs do not carry significant population of immunosuppressive molecule or angiogenic molecule that can result in the worsening of the disease. With the encouraging preclinical results, three DEXs vaccines have entered clinical trial I with one entered clinical trial I for treating cancers. DEXs exhibited limited efficacy in eliciting antigen-specific immune responses, and only a minor portion of patients were observed to show slowed disease progress. In a few cases, DEXs were able to reverse the metastasis, but an insufficient number of patients met the criteria of progression-free survival, which caused failure of these clinical trials. Nevertheless, all DEXs vaccines are welltolerated with no significant side effects, signifying future feasibility in developing other exosome-based treatments. (Xia et al., 2022) To enhance the treatment efficacy of TEXs and DEXs, engineering of exosomal cargo contents or surface protein have been attempted and demonstrated improved performance in vitro and in vivo. By loading breast cancer cells generated exosome with CDK4 siRNA, the expression of 9 CDK4 will decrease through inhibition of the synthesis of CDK4 mRNA. Expression of IL-18 molecule on exosome surface could serve as the adjuvant for exosome vaccine by acting as immunostimulatory cytokines for T cells. IL-18 promotes the secretion of Th1 and TNF-α to enhance the cytotoxic T cell effect in killing tumor cells, and it also acts as chemoattractant for dendritic cells and T cells for lymphocyte maturation. Conjugation of KLA, a short cationic amphipathic peptide to disrupt mitochondria membrane, on the exosome surface can induce cancer cell apoptosis. Exosome modified by SEA, superantigen staphylococcal enterotoxin， will increase the TEXs efficiency in increasing the anti-tumor T-cell response. These modifications showed certain efficacy in vitro and in vivo, but they have not been validated in clinical trials. 1.6 Development and Application of Genetically Engineered Multifunctional Exosomes It has been almost 10 years since the last exosome vaccine was tested in clinical trials, signifying limitations of current types of DEXs and TEXs in treating cancers. TEXs were notorious for their immunosuppressive effects, while DEX could not raise sufficient antigen-specific T-cell responses. With the goal of overcoming these disadvantages, a novel type of exosome named as synthetic multivalent antibodies retargeted exosomes (SMART-Exos) was developed(Cheng et al., 2018) . SMART-Exos displays fusion antibodies targeting both tumor surface-specific antigen and CD3 on T cells by genetic engineering. SMART-Exos facilitate the accumulation and activation by anti-CD3 with accurate targeting efficiency at the tumor site by tumor specific antigen. SMART-Exos demonstrated excellent antibody displaying and binding ability to both T- cell and cancer cells through flow cytometry. Confocal microscopy showed SMART-Exos's capability in cross-linking of Jurkat(immortalized T cells) and cancer cells. The cytotoxicity was validated in both cellular assays and mice models. For cellular assays, solely applying anti-tumor 10 SMART-Exos or anti-CD3 SMART-Exos do not exert any cytotoxicity even at concentration more than 100ng/mL concentration, while the fusion SMART-exos’s EC50 are around 10ng/mL. In mice planted with breast cancer cells, the fusion SMART-Exos were able to completely inhibit tumor growth, and the harvested T cells are characterized by an increased percentage of CD3+/CD45+ T cells. No significant tumor regrowth was observed in the treatment group after treatment was stopped. Furthermore, the exosome produced by HEK 293, an immortalized human embryonic kidney cell line, does not carry immunosuppressive ligands as TEXs do. The application of SMART-Exos has been tested effective on both EGFR+ or HER2+ breast cancer cells, which indicates the potential broader application for more types of cancers with variable surface antigen. These groundbreaking studies unveil a novel achievement: the successful deployment of two unique types of monoclonal antibodies on the surface of exosomes. This controlled and precise approach effectively triggers anti-tumor immunity, marking a significant advancement in the field. (Cheng et al., 2018; Shi et al., 2020) With the encouraging outcomes from SMART-Exos, more functions have been developed to extrapolate the full potential of genetically engineered exosomes. Following the development of SMART-Exos, it was demonstrated that the genetically infused functionally tailored exosomes (GIFTed-Exos) could efficiently deliver multi types of proteins to the disease site. Structurally, GIFTED-Exos contains transmembrane protein CD9 due to its high abundance in the exosome, and the protein of interest is genetically fused to CD9 in order to be displayed on the exosomal surface with high expression level. To enable exosome-mediated intracellular delivery, a photocleavable protein could be inserted between CD9 and the protein payload. After the exosome fused with cells, the photocleavable part would be cleaved by UV 405 nm irradiation to release the protein cargo intracellularly. For protein payloads that don't need to be released but 11 only functionally displayed, they could be directly fused to the CD9. GIFTed-Exos showed outstanding capability in displaying CD70 or glucocorticoid ‐ induced tumor necrosis factor receptor family ‐ related ligand (GITRL). Both GITRL-GIFTed-Exos and CD70-Gifted-Exos were able to induce T-cell stimulatory activities by increasing the secretion of IL-2 or IFN-γ secretion level. For delivery of soluble protein to cytoplasm of target cells, fluorescence protein mCherry, apoptosis-inducing protein–apoptin and catalase for antioxidizing activity were tested. mCherry demonstrated successful intracellular delivery with its fluorescence characteristics under confocal microscopy. The CD9-PhoCl-apoptin construct delivers apoptin to HELA cell lines to induce apoptosis but not HEK293 cell lines after violet light treatment due to their endogenous high level of apoptin, signifying that delivery of apoptin to target cancer cell could induce apoptosis. In addition, CD9-PhoCl-catalase was co-expressed with ApoA-I, the ligand of high-density lipoprotein scavenger receptor class B type I (SR-BI), for facilitating the accurate delivery to the hepatocytes or liver. The CD9-PhoCl-catalase exosome selectively binds to SR- BI+ HepG2 cells to deliver antioxidant enzymes, thus catalyzing the breakdown of H2O2 to alleviate liver damage. The delivery accuracy and the enzyme activity were evaluated in vitro and in vivo, and the CD9-PhoCl-catalase exosome protects the liver against acute damage by CCl4 in the mice model. The GIFTed-Exos delivers a high concentration of protein cargo with the assistance of highly expressed CD9, and the endosomal pathway of the exosome and the photocleavable constructs enables the intracellular delivery. (Cheng et al., 2021) 1.7 The Design Rationale of GEMINI-Exosome After validating the feasibility of GIFTed-Exosome, the next question is how to integrate the immune checkpoint inhibitors into the exosome therapy. Under normal circumstances, the immune surveillance function of the body can recognize and eliminate malignant cells. However, 12 tumor cells employ various methods to evade immune system surveillance, ultimately leading to tumor initiation and progression. Cell-mediated immunity mediated by T lymphocytes is the primary mechanism of anti-tumor immunity. In addition to expressing certain specific antigens recognizable by the immune system, tumor cells can also express multiple immune inhibitory ligands, such as Programmed Death-Ligand 1–PD-L1. PD-L1 binds to the PD-1–inhibitory receptor Programmed Death-1 expressed on T cells, leading to the loss of T cells' ability to attack cancer cells. The principle of the anti-tumor action of PD-(L)1 inhibitors, or checkpoint inhibitors, is to block the binding of PD-1 and PD-L1, thereby restoring the immune cytotoxic function of the body against tumor cells. So far, the FDA has approved seven immune checkpoint inhibitors targeting the PD-1/PD-L1 pathway: four PD-1 monoclonal antibodies and three PD-L1 monoclonal antibodies. Currently, there are more than 5000 clinical trials testing PD-1/PD-L1 monoclonal antibodies as monotherapy or in combination with other therapies, including targeted therapy, chemotherapy, and radiation therapy. (29) The wide application of PD-1/PDL-1 in clinical trials indicates their groundbreaking efficacy in treating cancers. Despite the fact the number of PD-1/PD-L1 combination therapy clinical trials is constantly increasing, the average planned patient enrollment for single-therapy trials has been consistently decreasing, with a reduction of nearly 90% compared to 2014. Such reduction suggests the huge clinical need for combination therapy in order to boost the therapy efficacy. A part of patients didn’t benefit from the checkpoint inhibitor therapy because of the low response rate or the developed drug resistance mechanism. Combination therapy helps enhance the overall treatment effectiveness. To further boost immune response, additional T cell activation machinery OX-40 is added to the exosome design. OX40 is a type I transmembrane glycoprotein primarily expressed 13 constitutively by regulatory T cells and inducible by effector T cells. The ligand for OX40 is OX40L, which is mainly expressed on antigen-presenting cells APCs, NK cells, T cells. (Croft et al., 2009) A portion of patients who received PD-1/PD-L1 therapy did not benefit from immune checkpoint blockade (ICB) as they developed primary resistance.(Lei et al., 2020) Furthermore, the effectiveness of ICB is also limited by acquired resistance, ultimately leading to disease progression. (Pathak et al., 2020) The immune activation elicited by the interaction between antigen MHC/peptide complexes and T cell receptors is a prerequisite for T cell activation, but it is not sufficient to initiate T cell responses alone. Further immune signal strengthening through costimulatory molecules is crucial for optimal T cell priming, expansion, and differentiation. Costimulatory molecules can be primarily classified into two major groups: the immunoglobulin superfamily (IgSF) and the tumor necrosis factor receptor superfamily (TNFRSF). The TNFRSF consists of CD27, OX40, and glucocorticoid-induced tumor necrosis factor receptor (TNFR)- related proteins. Unlike standard ICB therapies that target tumor surface receptors and inhibit anti-tumor immune responses of T cells, drugs targeting OX40 can exert their effects by directly activating and modulating immune responses. Therefore, OX40 has been widely tested as the drug target for tumor immunotherapy. Notably, co-administration of OX40 activator and PD-L1 blocker increases the population of CD4+ and CD8+ T cells in mice tumor models. Comparable populations of CD8+ T cells expressing NK cell receptors can also be observed in the bloodstream of cancer patients who respond positively to immunotherapy. (van der Sluis et al., 2023) The result potentially validated the importance of adding OX40-stimulatory group to the ICB treatment. Given that co-administration of antibodies to OX40 to PD-L1 might improve the treatment outcome, we envisioned applying the exosome scaffold for the combination therapy. To further 14 increase the targeting efficiency and the immune activation effectiveness, we also integrated the function SMART-Exos of co-targeting tumor surface antigen and immune activation into the design. Contrary to immunotherapeutic agents that are defined at the single molecule level, such as immune checkpoint inhibitors and bispecific antibodies, exosomes have the potential to exhibit multifunctional presentation of immunomodulatory proteins on their spherical surface. This characteristic enhances their ability to bind with higher avidity to target receptors or ligands on both immune and cancer cells, facilitating the formation of immunological junctions. The resulting exosomes display PD-1, OX-40L, anti-CD3 and anti-EGFR. Of these functional moieties, PD-1 blocks the PD-L1 on the tumor cell surface to avoid immune escape; OX-40L interacts with OX-40 expressed on T cells to further stimulate T-cells, anti-EGFR enables accurate delivery and the formation of immune synapse between T cells and cancer cells; anti- CD3 bind to CD3/TCR complex to activate of cytotoxic T cells to selectively kill the EGFR- expressing tumor cells. With targeting of multi-immunological pathways, it may lead to heightened activation of the immune system. To achieve high yield with minimum number of immune suppression ligands, the exosomes were produced by Expi293F cells. With its unique multivalent presentation characteristics, the resulting exosome is named as Genetically Engineered Multifunctional Immune-Modulating Exosomes (GEMINI-Exos). Previously, GEMINI-Exos has been applied to the treatment of EGFR-positive triple negative breast cancer. GEMINI-Exos successfully redirects cancer cells to the site of cancer and kills cancer cells through eliciting T-cell immunity both in vitro and in vivo. To fully explore the potential of GEMINI-Exos, we propose to extend its application to EGFR-positive colon cancer cell lines, because a significant portion of colorectal cancer patient's tumor samples have EGFR binding sites.(Pabla et al., 2015) 15 1.8 Exosome Surface Display Design To balance among structural stability, activity, manufacturing efficiency of the four different proteins concurrently presented on a single exosome, we opted to introduce and co-express two fusion protein constructs within exosome-producing cells. By employing the the design of SMART-Exos and GIFTed-Exos, αCD3 scFv and αEGFR scFv could be effectively docked on exosome surfaces through tandem fusion with the transmembrane domain (TMD) of the human platelet-derived growth factor receptor (PDGFR). An HA epitope tag was placed at the terminal of the αCD3-αEGFR-PDGFR TMD fusion. PD-1 and OX40L fall into the categories of type I and type II membrane proteins, respectively. To display both structures on the surface in a single-chain fusion format with correct orientations, we endeavored to genetically engineer fulllength PD-1 and OX40L to the N- and C-terminus of CD9, respectively. Considering the substantial abundance and intracellular location of CD9 within exosome membranes, CD9-based fusions were anticipated to facilitate the display of transmembrane proteins on exosome surfaces and enriched presentation of antibodies. The designed PD-1-CD9-OX40L fusion construct encompasses an HA tag at the beginning, a 6×His tag at the end, and flexible (GGGGS)2 linkers before and after the fused CD9 domain. 1.9 Production and Purification of Exosomes The molecular cloning of GEMINI-Exos was conducted as the previously publication described. (Cheng et al., 2022) Expi293F cells were acquired from Thermo Fisher Scientific and sustained in BalanCD HEK293 medium containing 4 mM L-glutamine with agitation at a rate of 125 rpm min −1 at 37°C in 8% CO2. Expi293F cells were cultivated to P3-P6 at a concentration of 2.5 million cells/mL for transfection purposes. Transfection-grade plasmids for the sequence-verified expression constructs were purified from using ZymoPURE II plasmid kits (ZYMO Research, 16 Irvine, CA) and transiently transfected into Expi293F cells using PEI MAX 40K (Polysciences, PA) in accordance with the manufacturer's instructions. Cell culture supernatants were collected on day 3 and day 6 after transfection via centrifugation and stored at −80°C. Exosomes were isolated from cell culture supernatants of Expi293F cells through differential centrifugation and ultracentrifugation. Initially, cell cultures were centrifuged at 100×g for 10 minutes to eliminate Expi293F cells, and subsequently centrifuged at 4000×g for 30 minutes to eliminate deceased cells and cell debris using a Heraeus Megafuge 40R refrigerated centrifuge equipped with a TX- 750 swinging bucket rotor (Thermo Fisher Scientific). The collected supernatant was then subjected to centrifugation at 14,000×g for 60 minutes using a J2-21 floor model centrifuge with a JA-17 fixed-angle aluminum rotor (Beckman Coulter, Indianapolis, IN) to eliminate large vesicles. Clarified supernatants were subsequently centrifuged in a Type 70 Ti rotor by an Optima L-80 XP ultracentrifuge (Beckman Instruments) at 60,000 rpm (371,000 ×g) for 1.5 hours to pellet exosomes. All centrifugation procedures were carried out at 4°C. The resultant exosome pellets were subjected to two washes with PBS, resuspended in PBS, and then filtered using 0.2μm syringe filters. Protein concentrations of the purified exosomes were measured using Bradford assays by adhering to the manufacturer's instructions. The average yield for the final purified exosome product was around 2.0 mg from every liter for collected medium. 17 Figure 1. Mechanism of GEMINI-Exos. Adapted from (Cheng et al., 2022) Figure 2. Molecular Interaction of Gemini-Exos. Adapted from (Cheng et al., 2022) Ten ug of purified exosomes quantified by Bradford Assay (Thermo Fisher Scientifics) was lysed in NuPAGE LDS sample buffer (Thermo Fisher Scientific) with or without 10 mM dithiothreitol and boiled at 95C for 5 min. The lysates were then ran on 4% to 20% ExpressPlus- PAGE gels(GeneScript, NJ) for 50 mins under 150 V, followed by transferring to PVDF immunoblots membranes (Bio-Rad Laboratories, Inc, CA), blocked with 5% non-fat milk (Bio- 18 Rad, CA) in PBS with 0.5% Tween20 (PBST) and probed with corresponding antibodies. Anti- HA antibody was purchased from Thermo Fisher Scientific; anti-CD9 was purchased from Cell Signaling Technology. Anti-HA and anti-CD9 primary antibody were detected by goat anti- mouse horseradish peroxidase (purchased from Thermo Fisher) and goat anti-rabbit horseradish peroxidase (purchased from Thermo Fisher). For control purpose, native exosomes with no transfection, UE exosome (transfected with antiCD3-antiEGFR), PCO exosome (transfected with PD-1-CD9-OX40L), and co-transfection exosome were purified. Lane Number Anti-CD9 blot Anti-HA blot 1 UE UE 2 UE UE 3 PCO PCO 4 PCO PCO 5 UE UE 6 UE UE 7 UE UE Anti-CD9 Anti-HA 19 Figure 3a. Western blot of anti-CD9 and anti-HA for purifiedUE and PCO exosomes Anti HA Anti-CD9 Co-transfection Figure 3b. Western blot of anti-CD9 and anti-HA for cotransfected (GEMINI) exosomes. 1. 2. 3. 4. 5. 6. 7 1. 2. 3. 4. 5. 6. 7 20 The native, UE, PCO, and co-transfection exosome all yield approximately 1 mg from 240 mL of cell culture supernatant. The yield of the exosome production is negatively related to the time of storage in the -80°C freezer, which suggests that exosome might degrade after long-term freezer storage. The successful purification of each exosome construct was confirmed through western blot analysis. Specifically, the successful purification of the UE construct was validated by the presence of a positive signal from the anti-HA antibody. Similarly, the purification of the PCO constructs was confirmed by the positive signal obtained from the anti-CD9 and anti-HA antibody. The UE constructs have a weak anti-CD9 signal, which be explained by the fact that exosome naturally carries the CD9 molecule. The PCO exosome has a significant higher signal of anti-CD9 due to the transfected construct also contains CD9-derived fusion. Furthermore, the successful purification of co-transfection exosomes was demonstrated by the presence of positive signals on both the anti-HA and anti-CD9 western blots. Anti-HA western blot visualized UE as a clean single band blot and PCO as a triple band with smears, which might be explained by the long-term freezer storage and protein degradation. In preparation of following in vitro and in vivo experiments, 10 mg blank exosome, 20 mg UE exosome, 20 mg PCO exosome, and 10 mg co-transfection exosomes. 1.10 Future Experiment Design Due to time constraints, future experiments have been planned but have not yet been conducted. Three colorectal cancer cell lines SW480, HT29, HCT 116 with varied expression levels of EGFR will be selected. To validate the binding affinity of EGFR, flow cytometry will be performed for UE, blank, PCO, and co-transfection exosomes using the above mentioned three cell lines. UE and co-transfection (GEMINI) exosomes are expected to have strong binding affinity to high EGFR-expressing cell lines. OX40, PD-L1, and PD-L2 will serve as the coating 21 antigens for ELISA to validate the binding affinity of PD1 and OX40L displayed on the exosome surface. The GEMINI-exosome might have higher binding affinity due to the PD1/PD-L1 interactions. Binding to PD-L1, PD-L2 and OX40 will be analyzed using PD-L1/PD-L2 constitutively upregulated cancer cells and OX40+ T cells. The stimulatory capability for T cells will be measured by quantifying the amounts of secreted interferon (IFN)-gamma and interleukin (IL)-2 cytokines after incubation of human peripheral blood mononuclear cells (PBMCs) with cancer cells and each type of exosome. Native exosomes and PCO should have minimum level of stimulation, UE type should have enhanced level of simulation, while the GEMINI-Exos should have the highest stimulation capability. Next, the MTT assay will be conducted to evaluate the cytotoxicity of each exosome type by incubating cancer cells/human PBMC and exosomes together. If binding affinity, T-cell activation capability and cytotoxicity experiment yield positive results, GEMINI-Exosome’s antitumor activity will be evaluated in colorectal tumor in mice models. Specifically, colon cancer with high levels of EGFR and PD-L1/L2 will be implanted into the flank of NSG mice with n=5 in each group. A total of six groups are planned for the animal experiment, including: PBS, native exosome, UE exosome, PCO exosome, UE+PCO exosome and GEMINI exosomes in order to understand the key factor affecting the treatment efficacy. The tumor volume will be used as the primary scale to determine the efficacy. At the endpoint of the experiment, tumor tissue will be harvested to analyze CD8 +CD45 + T cell percentage, CD4+CD25+ percentage, CD8+ T cell percentage in order to learn the effect of exosome on the regulation of T cell. The blood will also be harvested to measure alanine aminotransferase activity and creatine level in order to evaluate liver and renal damage. 22 Chapter II: Engineering CD38 as an Optimized Drug Carrier 2.1 Abstract: CD38 is a transmembrane glycoprotein and highly expressed on the surface of various cells, including immune cells and certain cancer cells. It possesses enzymatic activity and is involved in the metabolism of nicotinamide adenine dinucleotide (NAD+ ) into Cyclic ADP-ribose (cADP) and nicotinamide. Several NAD+ analogues have been developed in order to study CD38’s structural characteristics and catalytic mechanism. Particularly, 2’-Cl-araNAD+ analogue features a clickable azide group and covalently inhibits CD38, enabling facile conjugation of cytotoxic payloads. Through genetic engineering, CD38 is fused to antibodies for tumor surface antigens, so 2’-Cl-araNAD+-payload could be easily conjugated to the antibody through a single-step enzymatic reaction in a site-specific manner. The CD38 has been proved to be a good candidate of drug carrier for site-specific delivery in preclinical study. To enhance the therapeutic effects, increased numbers of CD38 could be conjugated to a single antibody in order to increase drug-to-antibody ratio, and such strategy was proved to be successful. Nevertheless, an antibody fused to multiple CD38 molecules is suffered from low stability and short half-life issues. The stability issue might be a result of increased molecular weight and steric hindrance. To resolve this problem, we propose to develop a truncated version of CD38 while maintaining a similar level of enzymatic activity through rational design. Through C-terminal truncation of 22 amino acids, we were able to obtain a new version of CD38, whose activity is comparable to that of wildtype CD38. With the truncated version of CD38, we expect to see improved overall antibody stability. 23 2.2 Introduction of CD38 Mammalian CD38 is a type II transmembrane glycoprotein. CD38 is expressed on both cellular membranes, cytoplasm and intracellular compartment. It catalyzes nicotinamide adenine dinucleotide (NAD+) to form the cyclic ADP ribose (cADPR), which is an essential coenzyme involved in cellular energy production and redox reactions. (Shrimp et al., 2014)cADPR induces calcium release at lower cytosolic concentrations of Ca 2+ , so the cADPR also acts as a second messenger mobilizer.(Lee et al., 2022) Besides cADPR, CD38 also catalyzes NAD+ to form ADPR, Nicotinic acid adenine dinucleotide phosphat (NAADP), and more metabolites for more cellular events. CD38 is highly expressed on normal cells including T cells, NK cells, B cells, and dendritic cells and malignant cells including lymphocytic leukemia, prostate cancer, and multiple myeloma. (Dwivedi et al., 2021; Konen et al., 2020)The high expression of CD38 on cancer cells is usually a negative prognostic marker for cancer progression.(Durig et al., 2002) Due to its enzymatic activity and distribution in multi types of cells, CD38 also widely participates in cell signaling and immune regulation. For immune regulations, it participates in cell-cell interactions and immune cell activation. In T cells, CD38 activation can cause T cell proliferation and cytokine production.(Piedra-Quintero et al., 2020) It is also involved in the differentiation of Tregs, which play a role in maintaining immune capability and tolerance. Increased CD38 expression on B cells is associated with high antibody production and immune response regulation. Additionally, CD38 expression on NK cells contributes to NK cell cytotoxicity and antitumor activity. CD38 has been implicated in inflammation and autoimmune disorders. Escalated CD38 expression and activity have been observed in inflammatory conditions, such as rheumatoid arthritis, multiple sclerosis, and asthma.(Cole et al., 2018) CD38- generated metabolites, particularly cADPR, can modulate immune cell function and 24 inflammatory responses. Given that CD38 is highly expressed on the surface of malignant cells, CD38-targeting antibodies, such as daratumumab, have been developed and approved by FDA for the treatment of multiple myeloma. These antibodies can induce direct cell death, antibody- dependent cellular cytotoxicity, and antibody-dependent cellular phagocytosis of CD38- expressing cancer cells. (van de Donk, 2018) 2.3 Study of CD38 through NAD+ analogues In addition to CD38’s pathological and physiological role, CD38’s enzymatic role has drawn more research attention for determining their mechanism of action. Studies have been conducted to determine the crystal structure of CD38 when it reacts with NAD+ . The reaction of NAD+ and CD38 is transient, so it is almost impossible to capture the catalytic state crystal structure. Instead, the generated crystal structure often used inactive enzymes or in dimer or trimer state, which might cause misinterpretation of true CD38 structure. To improve the understanding towards CD38’s structure and catalytic activity, NAD+ analogues have been synthesized and tested. Previous studies showed that 2′-F-arabinose nicotinamide mononucleotide (2′-F-araNMN) and 2′-F-araNAD+ covalently inhibits CD38 by irreversibly constructing a stable arabinosyl-ester bond with the glutamate 226 residue in catalytic pocket by MALDI-PSD identification. The F- araNAD+ targets CD38 in a highly efficient and specific manner to fully inhibit CD38’s activity on the cellular surface.(Baum et al., 2021) A NAD + mimic, 4’thioribose NAD + (S-NAD +), that is resistant to enzymatic cleavage by CD38 helped solved the high-resolution X-ray crystal structure. (Z. F. Dai et al., 2020)The complex of S-NAD+ and CD38 demonstrated important molecular interaction between CD38 and its substrate as well as CD38’s catalytic residues. Interestingly, the CD38 bind NAD+ do not largely interact with the enzyme residue but mainly contact with surrounding solvent. The pyrophosphate diester bond present in NAD+ structure 25 exhibited remarkable plasma durability and prompt payload release after being internalized into cells for ADCs. Thus, we anticipate that the combination of NAD+ analogues together with CD38 could serve as potential drug delivery vehicles. 2′-Cl–substituted NAD+ was shown to exhibit higher stability compared to 2′-F-araNAD, providing an improved tool for drug delivery. To enable the drug carrying through easy click chemistry conjugation, a novel 2′-Cl-araNAD+ analog with an azido group modification at N6 of adenine (2′-Cl-araNAD+ -N3) was synthesized. CD38 genetically fused to a tumor antigen directing antibody could then react with the resulting 2′-Cl-araNAD+-payload to create Antibody -Drug-Conjugate(ADC). The majority of the existing ADCs being used or developed are created through indiscriminate conjugation to cysteine or lysine residues on the antibody or protein surface. This unselective process leads to the formation of heterogeneous ADCs with diverse drug-to-antibody ratios (DARs). Variable DARs could lead to different pharmacological activities, which is unfavorable for product quality consistency and clinical results outcome. ADCs that undergo site-specific conjugation exhibit enhanced performance and safety characteristics compared to the heterogeneous ADCs. Previous studies have reported that synthetic or non-natural amino acids, carbohydrates, and enzymes facilitate ligation to enable site-specific conjugation. However, the conjugation methods often involve multiple intricate steps or lengthy reaction times due to inefficient chemical reactions. While genetic fusions of peptide motifs or engineered enzymes enable more efficient production of site- specific ADCs, and the introduction of mutations or sequences derived from non-human sources may result in significant immunogenicity concerns. The reaction between CD38 and 2′-Cl- araNAD+_payload is a single-step highly efficient enzymatic reaction with minimum exogenous immunogenicity moiety, due to NAD+ is also a naturally occurring structure. 2.4 CD38 as a Drug carrier through NAD+ analogues 26 To selectively target HER2+ breast cancer cells, a cytotoxic payload tubulin inhibitor monomethyl auristatin F(MMAF) was chosen. For a more specific design, the extracellular domain of native human CD38 was genetically fused to the C-terminus of the anti-HER2 immunoglobulin G (IgG) to facilitate the conjugation of the payloads of interest, forming ADPribosyl cyclase ADC (ARC-ADC). Fusion of the CD38 enzymatic domain to the IgG was facilitated through a flexible glycine-glycine-serine (GGS) linker. By mammalian cell transient expression in human embryonic kidney-293 (HEK293) cells, the designated constructs were produced and purified. Similar to conjugated CD38, the fused constructs were then combined with CD38 inhibitor 2'-Cl-araNAD+ covalently, which could rapidly bind with CD38 through a stable arabinosyl-ester bond with the catalytic E226) residue. The successful conjugation was examined by CD38 activity assay using nicotinamide guanine dinucleotide (NGD+)-based fluorescence assays. CD38 catalyzes NGD+ to release of nicotinamide and cGDPR, whose production could be measured by an activation wavelength of 300 nm and fluorescent wavelength of 410 nm. Successful conjugation of CD38 C-fusion IgG is characterized by complete inhibition of CD38 activity. Mass spectrometry also detected the exact mass increase after incubation of CD38 C-fusion IgG with 2′-Cl-araNAD+–MMAF, determining that the DAR was 2 as expected from the design. The purified anti-HER2 ARC-ADC was then tested for its binding capability with both HER2 positive and HER2 negative cell lines; it showed only selective binding towards HER2 positive cell lines, indicating the accurate delivery selectivity and minor toxicity to the normal tissues. By testing with multiple cell lines with differential HER2+ expression, the anti-HER2 ARC-ADC’s cytotoxic activity was most prominent in high HER2 expression cell lines but less significant in low HER2 expression cell lines. More importantly, anti-HER2 ARC-ADC demonstrated powerful antitumor efficacy in mouse 27 xenograft models. IRDye-labeled anti-HER2 ARC-ADC showed high accumulation in HER2 tumor sites with very low signals in heart, lung, liver, spleen and kidney. Compared with phosphate-buffered saline (PBS) treated groups, anti-HER2 ARC-ADC effectively reduced tumor size and extended the survival time. The good treatment outcome proved that CD38 and 2′-Cl-araNAD+_payload could act as a promising combination for drug delivery. 2.5 Conjugation of multiple CD38 to Antibodies Decreased the Pharmacokinetics Performance Encouraged by this finding, an increased number of CD38 is being genetically fused to the antibody that recognizes tumor surface antigen, by ligating the C-terminus of the light chain with CD38 enzymatic domain. Together with the CD38 on the C-terminus of IgG, a total of 4 CD38 can be conjugated on a single antibody targeting (human C-type lectin-like molecule-1) hCLL-1 positive acute myeloid leukemia, making the final DAR of 4. The higher number DAR positively correlates to higher cellular cytotoxicity for hCLL-1 positive leukemia cells. In animal experiments, higher DAR and higher dose of drug more effectively control the tumor development, indicating the positive relationship between efficacy and number of drugs that can be delivered by a single antibody. While a higher drug-to-antibody ratio (DAR) is generally more beneficial for antitumor effectiveness, it should be noted that an antibody with a DAR of 4 has a shorter half-life. Considering that larger antibodies typically exhibit longer circulation in the plasma, the shorter half-life observed in DAR-4 antibodies suggests that an increased CD38 count is associated with instability. We hypothesized that the significant size of CD38(30 kDa) is the main factor that affects the antibody’s overall stability in circulation and production yield. When the number of CD38 for ARC-ADCs increases, it may cause drug delivery issues in terms of tissue penetration and immunogenicity. 28 2.6 Aim and Hypothesis Aim: To address pharmacokinetics issues of conjugating multiple CD38, we designed five types of truncated CD38 based on structure analysis. The engineered CD38 variants were attempted for expression and purification from mammalian cells, followed by characterization of their enzymatic activity. Hypothesis: Structure-guided protein engineering may result in truncated CD38 with sufficient catalytic activity for drug conjugation. 2.7 Experimental Design By using the software Pymol to visualize the CD38 crystal structure, we located most important structural regions for the thermodynamic stability and activity, including the catalytic pocket, disulfide bonds, beta sheets and alpha helices. When designing the truncation sites, we avoid the region next to the catalytic pocket to maintain its structural integrity and capability to react with NAD+. Previous study in engineered cytosolic CD38 indicates that to maintain CD38’s enzymatic activity, the engineered CD38 enzyme requires the formation of six disulfide linkages within the cytosol. The most critical disulfide bond is Cys-254–Cys-275 to maintain the catalytic pocket’s activity. (Zhao et al., 2011) The clipping of the structure is usually involved with a full length of alpha helix, beta sheet or disulfide bond in order to prevent the residue structure’s instability. Truncation has been tested from both C-terminus and N-terminus of the structure in order to find the best truncation site. With such modification logic, the truncated structures were generated, which are named as 58-300 (C terminal truncation, length 255 aa), 45-278 (N terminal truncation, length 246 aa), 95-300(C terminal truncation, length 219aa), 45-246(N terminal truncation, length 214aa), 54-300 (C terminal truncation, length 259aa). The nomenclature is based on the sequence of the CD38. The wild type of CD38 has a length of 300 amino acids with 29 the initial 44 amino acids at C-terminus have no activity, and the wildtype sequence we adopted here refers to the amino acid from 45-300. For the convenience of comparison, the discussion below is based on the normalized full length CD38 position from 45-300, not the labeling position starting from the first amino acid. To make the truncation without affecting the catalytic activity, the optimal truncated structure will mimic the natural structure, especially the crucial parts including the strands. Therefore, we hypothesized that the truncated structure that simulates most characteristics of the wildtype structure will have the best activity, while the low similarity to the wildtype structure could result in failure for protein production and activity. Of all the truncated versions of CD38, only 45-278 could be purified and tested with NAD+ cyclase activity validated by NGD assay. The full length or the truncated sequence of CD38 was genetically engineered to a pFUSE back bone for expression in HEK 293 cell lines. A 6XHis tag was added to the C terminus for purification and downstream in vitro validation. The polymerase chain reaction (PCR) using Pfx polymerase (Thermo Fisher Scientific) was used to generate PCR encoding CD38s.Each DNA fragments for each fusion protein were visualized by bands on agarose gel and so that they can be excised and purified with DNA extraction kits (Zymo Research, CA). Cleaned DNA fragments were excised by DNA restriction enzymes (New England Biolabs, MA), followed by ligation into empty pFUSE vector backbone by DNA T4 ligase (New England Biolabs, MA). The ligated products were used to transform into DH10B E. coli electrocompetent cells. Colonies that were resistant on zeocin agarose plate were identified as positive colonies, which were further picked for DNA sequencing (Genewiz, NJ) for correct sequencing confirmation. Expi293F cells were acquired from Thermo Fisher Scientific and sustained in BalanCD HEK293 medium containing 4 mM L-glutamine with agitation at a rate of 125 rpm min−1 at 37°C in 8% 30 CO2. Expi293F cells were cultivated to P3-P6 at a concentration of 2.5 million cells/mL for transfection purposes. Transfection-grade plasmids for the sequence-verified expression constructs were purified from E.Coli culture using ZymoPURE II plasmid kits (ZYMO Research, Irvine, CA) and transiently transfected into Expi293F cells using PEI MAX 40K (Polysciences, PA) in accordance with the manufacturer's instructions. Cell culture supernatants were collected on day 3 and day 6 after transfection via 4000g x 30mins centrifugation. Cell culture media was first dialyzed against storage buffer at 4°C (25 mM Hepes and 250 mM NaCl, pH 7.5) by 100 folds dilution for overnight before purification by Ni-NTA resins (Thermo Fisher Scientific, MA). Dialyzed media were washed through the Ni-NTA resins in a gravity flow column for two times, followed by washing with 15 column volumes of wash buffer which was consisted of (20 mM tris-HCl (pH 8.0), 200 mM NaCl, and 30 mM imidazole). Protein was then eluted in 15 column volumes of elution buffer which was consisted of (20 mM tris-HCl (pH 8.0), 200 mM NaCl, and 400 mM imidazole) and were dialyzed into PBS buffer for 10000 folds overnight at 4°C. On the second day, recombinant CD38-His6 was concentrated with 10-kDa cutoff concentrator (MilliporeSigma, MA). The purified proteins were examined by SDS-PAGE stained with Coomassie blue. Protein concentrations were determined using a NanoDrop 2000C spectrophotometer (Thermo Fisher Scientific, MA). 31 Figure 4. Coomassie-stained SDS-Page gels of purified truncated CD38s and wildtype CD38. Lane number Lane description 1 45-246 30mM Imidazole Wash 2 45-246 400mM Imidazole Elution 3 58-300 30mM Imidazole Wash 4 58-300 400mM Imidazole Elution 5 95-300 30mM Imidazole Wash 1 2 3 4 5 6 7 8 9 10 32 6 95-300 400mM Imidazole Elution 7 45-278 400mM Imidazole Elution 8 Wildtype 400mM Imidazole Elution 9 54-300 30mM Imidazole Wash 10 54-300 400mM Imidazole Elution Enzymatic activities of Wildtype CD38 and 45-278 CD38 NGD+ (Sigma-Aldrich, MO) was adopted as the reactant to determine ADP-ribosyl cyclase activity of purified CD38 and 45-278 truncated version of. CD38 catalyze the reaction from NGD+ to (Cyclic guanosine diphosphate–ribose) cGDPR and nicotinamide. cGDPR formed from CD38 cyclase activity was characterized by fluorescence emission at 410 nm (excitation at 300 nm), and the slope of emission curve reflects the rate of the reaction. Reactions were conducted by mixing of two different types of purified CD38 of two different concentrations (50 nM and 100 nM) into assay wells with 100 μM NGD+ in PBS buffer, followed by monitoring the reactions using excitation wavelength of 300nM and fluorescence wavelength at 410 nm for 5 min using a Synergy H1 plate reader (BioTek, VT). 33 Figure 5. Wildtype and truncated CD38’s NAD cyclase catalyzing activity measured by NGD assay. 2.8 In silico secondary structure analysis To explore the reason behind the successful truncation while maintaining good activity, we used protein secondary structure prediction and tertiary structure prediction software for analysis. For the secondary structure prediction, PredictProtein from Rostlab, Technische Universität München, Germany was used. The sequence of the truncated CD38s were used as input, and the software used pre-identified long regions with no regular secondary structure (NORS) for detecting protein disorder and flexibility regions. Disordered regions are characterized by a significant abundance of two types of short amino acid patterns: a proline-rich pattern and linear motifs. The polyproline-II (PPII) helix represents a widely observed helical structure motif that adopts an extended conformation and is prominently present in molecular recognition features (MoRF) within unstructured regions. It has been estimated that approximately 85% of the linear motifs cataloged in the Eukaryotic Linear Motif (ELM) database are situated within disordered Fluorescence Intensity Time 34 regions. The lab developed a type of methodology called MD (Meta-Disorder predictor), which is an amalgamation of multiple independent methods that effectively captures diverse forms of disorder with enhanced performance, while still maintaining the ability to differentiate between different types of disorder. By integrating the outputs from various prediction methods along with sequence profiles and valuable features such as predicted solvent accessibility and secondary structure, MD achieves improved accuracy in identifying and characterizing disorder. The table below summarizes the disordered secondary region. The predicted results are generally aligned with the experimental data, since 45-278 has the highest similarity to the wildtype structure. The distribution of alpha helix and strands are highly correlated with the structure stability and catalytic activity, since the location of these critical structures could significantly affect the shape of the catalytic pocket, the catalytic activity for cyclizing the NAD+. After the truncation of a certain part of the protein, the whole protein could be affected significantly due to the overall charge, disulfide bond location and the relative hydrogen bond location has shifted. During the protein expression process, the unstable structure could lead to protein folding process failure, leading to inability for purification. The truncation could also affect the solvent accessibility for the catalytic pocket, while previous studies has reported that CD38’s solvent accessibility sensitively shifted for unbound state and the active state when catalyzing NAD+. (Boittier et al., 2020) Particularly, we compared the predicted helix, strand, solvent accessibility, protein disorder, and flexibility. For the helix structure distribution, the wildtype has 7 fragments of helix structure distribution. For C-terminal truncated CD38 58-300 and 54-300, they have a similar pattern of alpha helix sequence, except for a few short additional alpha helices. The other N-terminal truncated CD38 95-300, an alpha helix misformed which could lead to incorrect folding. For the C-terminal 35 truncated type 45-246, a C-terminal alpha helix disappeared due to truncation. The feasible 45- 278 is featured with all alpha helix as the wildtype protein does, except for Y83 that can cause a short helical structure. The truncated CD38s have a more similar distribution of strand structures to wild type CD38. 58- 300 formed a short strand at the beginning; 54-300 and 95-300 maintains similar pattern of strand; 45-246 and 45-278 lost partial strand structure due to the truncation. According to the predicted data, the percentage of protein disorder positively correlates to the amount of protein truncated. The lightly truncated constructs (within 20 aa), 58-300&54- 300&45-278 have a similar size of disordered region. When the region of truncation exceeded 45, the constructs 45-246 and 95-300 both gained a large region of disorder. The relative B-value or PROFbva, predicts flexible and rigid residues in proteins. The rigidity and flexibility of amino acid sequence in the catalytic pocket are correlated enzymatic activity, since the protein will experience shape change during the activated state and the non-binding state. The excessively flexible amino acid sequence leads to instability of the structure, while excessively rigid amino acid sequence will limit the conformation change during the activation state. Nevertheless, the PROFbva method calculates the relative B value mainly from the primary structure of the protein rather than the secondary structure, which makes the prediction less accurate. According to the predicted B-value, for all truncated types of CD38, the flexibility next to the catalytic pocket is well retained. The high rigidity areas for the wild type CD38 are short and widely distributed through the whole sequence. After N-terminal truncation, the length of each rigidity fragment increases, and the rigidity area becomes less distributed. The rigidity characteristics of N-terminal truncation were less affected. 36 By summarizing the information obtained from secondary structure prediction analysis and comparing with the only feasible isotype 45-278, some vague conclusions could be drawn: 1st, the integral of all current helical structures are crucial for the protein structure, but the increased region of the helix or false location of the helix could exert a negative effect. 2nd, loss of partial C-terminal strand structure seems to not affect the expression and activity. Given that 54-300 retains the similar strand pattern as the wild type but 58-300 does not, the strand structure is sensitive to more than 10aa truncation. 3rd, large size of terminal truncation (>45 aa) will significantly increase the disordered region.4th, even short C-terminal truncation (<20 aa) could affect the overall rigidity of the CD38 structure. To further validate the reliability of the predicted result, secondary structures of the truncated CD38 were also evaluated with Phyre. The question mark below indicated the low confidence rate in prediction. The prediction is mainly based on the most similar sequence matched found in the library. Accuracy is only reached if there is a substantial number of diverse sequence homologous detectable in the sequence database. If the sequence has very few homologues, the sequence drastically drops. Prediction given by Phyre and Predictprotein do not match exactly, mainly because of the difference in the algorithm difference. Though the predicted results are different, it is important to note that removing a large trunk of peptide chain will have a greater impact on the alpha helix and beta sheet distribution, according to the analysis for both prediction methods. Moreover, the feasible structure 48-278 has an exact match of all helix and sheet structure compared to the wildtype structure except for the last piece of short sheet structure, which suggests that high similarity to the wildtype structure will enhance the successful rate for truncating the CD38. Table 1. Secondary Structure and Solvent Accessibility Prediction. 37 CD38 Secondary Structure and Solvent Accessibility Prediction Wildtype 58-300 54-300 45-246 95-300 38 45-278 Table 2. Protein Disorder and Flexibility Prediction. CD38 Secondary structure Protein Disorder and Flexibility Prediction Wild type 58-300 39 54-300 45-246 95-300 45-278 Table 3. Phyre-generated alpha helix and beta sheet distribution. CD38 Alpha helix and beta sheet distribution 40 Wild type 58-300 54-300 41 45-246 95-300 45-278 2.9 In silico tertiary structure analysis For a more intuitive understanding of the truncated structure by taking in the consideration of disorder caused by truncated structure, tertiary structure models were also generated by Phyre2. 42 Phyre 2 generated the truncated structure by protein sequence matching, so that model could adopt the most similar homologous structures from the library. From the homologous structure and analysis of the secondary structure, Phyre 2 constructed a hidden Markov model (HMM), which represents the information extracted from ‘sequence’ of observations over time. Next, it creats three-dimensional representations of protein by comparing the protein HMM with the HMMs of library structures. By incorporating insertions and deletions through the utilization of a library of loop structures, a fitting method known as cyclic coordinate descent, and a collection of empirical energy factors would be generated. Finally, the system would optimize the selection of rotamers for each amino acid sidechain by utilizing a rotamer library developed by Roland Dunbrack's laboratory, along with our own efficient graph-based method (R3) implementation. The objective is to model the side chains while minimizing the occurrence of steric clashes. (34) The tertiary generated figure predicted the conformation change after truncation. Rainbow color scheme was used to label protein from N-terminal to C-terminal. (From purple to red). For reference, 2EF1, crystal structure of the extracellular domain of human CD38, was retrieved from PDB bank. 2EF1 showed high similarity with the predicted wildtype structure, validating the predicting ability of the Phyre 2. Compared to the 58-300, 54-300 structure and wildtype both have an additional short strand at the green region despite that nothing in the green region was truncated. The wildtype structure has 4 beta sheets arranged side by side next to the catalytic pocket, but one beta sheet was missing from 45-246, which may explain the unsuccessful purification. For 95-300, one major alpha helix was missing next to the alpha helix, and the missing alpha helix shifted the shape of the catalytic pocket. 54-300 and 45-278 are both equipped with an essential structure next to the catalytic pocket. However, when viewed from the top of the catalytic pocket, the modification made in 54-300 has a stronger impact on the beta 43 sheet alignment directions. Though 45-278 lost a piece of alpha helix at C-terminal, it has a smaller impact for the catalytic pocket configuration. Table 4. Phyre-generated catalytic pocket view. Catalytic pocket view Wildtype 44 54-300 45-278 Summarizing the information obtained from secondary structure prediction and tertiary structure prediction, both the active and inactive truncated CD38s shed light for understanding the key structure for maintaining the catalytic pocket structure’s integrity. From currently available experimental data, truncation of the C-terminal 22 amino acids is a feasible method. In contrast, even 9 amino acids N-terminal truncation could lead to inactivation of CD38. The ultimate goal 45 of this project is to minimize the CD38 structure so that the final antibody structure could be more stable. The construct of 45-278 successfully reduced the molecular weight of each CD38 by 2.5 kDa, and it might enhance the thermodynamics stability of the CD38 conjugated antibody by reducing the possible steric hindrance. In spite of that, a more truncated version of CD38 might still be needed to achieve longer antibody half-life. Given the fact that N-terminal truncation is not a viable path, truncation from the non-terminal parts together with the current 45-278 truncation might be a more optimal option. To facilitate the truncation process with high efficiency, algorithm-based design could be employed to better visualize the impact of truncation on the catalytic pocket. Table 5. Phyre-generated tertiary structure prediction. CD38 Tertiary Structure Wildtype 46 PDB 6EDR 58-300 47 54-300 45-246 48 95-300 45-278 49 References Admyre, C., Bohle, B., Johansson, S. M., Focke-Tejkl, M., Valenta, R., Scheynius, A., & Gabrielsson, S. (2007). B cell-derived exosomes can present allergen peptides and activate allergen-specific T cells to proliferate and produce T(H)2-like cytokines. Journal of Allergy and Clinical Immunology, 120(6), 1418-1424. https://doi.org/10.1016/j.jaci.2007.06.040 Ahmadi, M., & Rezaie, J. (2020). Tumor cells derived-exosomes as angiogenenic agents: possible therapeutic implications. 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Abstract (if available)

Abstract Exosomes are nano-sized vesicles secreted by cells, containing a variety of molecules such as proteins, RNA, and lipids. Genetically engineered exosomes emerged as a promising option due to their effectiveness and multi-functionality for immunotherapy. Colon cancer is the second most prevalent form of cancer worldwide and is associated with significant morbidity and mortality. Surgery, chemotherapy and antibodies demonstrated limited benefits. Since genetically-engineered exosomes enable creating a multifunctional tool in a single molecular entity, we proposed to apply such exosomes to colon cancer therapy, which can trigger multiple pathways simultaneously in a site-specific manner.

CD38 is a transmembrane glycoprotein, and highly expressed on the surface of various cells. . It possesses enzymatic activity and is involved in the metabolism of nicotinamide adenine dinucleotide (NAD+) into Cyclic ADP-ribose (cADP) and nicotinamide. 2’-Cl-araNAD+ analogue features a clickable azide group and covalently inhibits CD38, enabling facile conjugation of cytotoxic payloads. The CD38 has been proved to be a good candidate of drug carrier for site-specific delivery in preclinical study. To enhance the therapeutic effects, increased numbers of CD38 could be conjugated to a single antibody in order to increase drug-to-antibody ratio, and such strategy was proved to be successful. Nevertheless, an antibody fused to multiple CD38 molecules is suffered from low stability and short half-life issues. To resolve this problem, we propose to develop a truncated version of CD38 while maintaining a similar level of enzymatic activity through rational design.

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Creator Zhao, Yuanteng (author)

Core Title Employing engineered exosomes for combating colon cancer and engineering CD38 as an optimized drug carrier

School School of Pharmacy

Degree Master of Science

Degree Program Molecular Pharmacology and Toxicology

Degree Conferral Date 2023-12

Publication Date 12/04/2023

Defense Date 12/01/2023

Publisher Los Angeles, California (original), University of Southern California (original), University of Southern California. Libraries (digital)

Tag ADC,exosome,immunotherapy,OAI-PMH Harvest,protein engineering

Format theses (aat)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Zhang, Yong (committee chair), Duncan, Roger (committee member), Okamoto, Curtis (committee member)

Creator Email 619818454@qq.com,yzhao714@usc.edu

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