Immunotherapy based on chimeric antigen receptor (CAR)-expressing T lymphocytes has proven to be highly successful in the treatment of acute lymphoblastic leukemia (ALL), leading to development of CAR-based immunotherapies for other hematologic malignancies. Currently, efforts are underway to refine T cell modifications to make patient treatment more effective. Each time, this modification then needs to be empirically validated in in vitro experiments. We decided to study the effect of the cytokine IL-21 on the antitumor function of CD19-specific CAR T cells using in vitro assays. A construct that co-expressed IL-21 under the control of the inducible NFAT promoter together with CARs against CD19 was introduced into T cells. In a series of experiments, the properties of these cells were compared after coculture with tumor B cell lines and CLL cells obtained from patients. The results showed that CAR T cells that express IL-21 proliferate and activate better, even after repeated stimulation with leukemia cells. In addition to CARs specific against the CD19 molecule, we also investigated CARs specific against the CLL1 molecule, which has been described in the literature as one of the promising targets for the treatment of AML. We prepared CAR T cells against CLL1 producing IL-21. For this purpose, we...

CAR-T-Zell-Therapie ist eine vielversprechende neuartige Behandlungsform für Patienten mit aggressiven B-Zell Non-Hodgkin-Lymphomen (B-NHL). In dieser Arbeit wurde die anti-CXCR5 CAR-T-Zell-Therapie als Alternative zur anti-CD19 CAR-T-Zell-Therapie für die Behandlung von reifen B-NHLs untersucht. CXCR5 ist ein B-Zell-homing Rezeptor, der von reifen B Zellen und follikulären T-Helferzellen (TFH Zellen) exprimiert wird. TFH Zellen wurden als tumor-unterstützend in chronisch lymphatischer Leukämie (CLL) und im follikulären Lymphom (FL) beschrieben. Dieses Expressionsmuster erlaubt es, auf einzigartige Weise zeitgleich die malignen Zellen und die tumorunterstützende Mikroumgebung mithilfe von CAR-T-Zell-Therapie gerichtet gegen einen Chemokinrezeptor anzugreifen. Die wichtigsten Ergebnisse dieser Arbeit waren, dass (1) die anti-CXCR5 CAR T-Zellen zielgerichtet CXCR5 positive reife B-NHL Zelllinien und Patientenproben in vitro eliminierten und eine starke anti-Tumor Reaktivität in einem immundefizienten Xenotransplantationsmausmodell zeigten, (2) die anti-CXCR5 CAR T-Zellen zielgerichtet die tumorunterstützenden TFH Zellen in CLL und FL Patientenproben in vitro erkannten und dass (3) CXCR5 ein sicheres Expressionsprofil zeigte. CXCR5 war stark und häufig auf B-NHL exprimiert und die Expression auf gesundem Gewebe war auf lymphoide Zellen beschränkt. Zusammenfassend lässt sich sagen, dass die anti-CXCR5 CAR-T-Zell-Therapie eine neue Behandlungsmöglichkeit für Patienten mit reifen B-NHL darstellt, indem durch die anti-CXCR5 CAR-T Zellen sowohl der Tumor als auch ein Anteil der tumorunterstützende Mikroumgebung eliminiert werden. Im zweiten Teil der Arbeit wurde das Eμ-Tcl1 murine CLL Lymphommodell genutzt um die Auswirkung der Lymphomentwicklung auf die CXCR5+ T Zellen zu untersuchen. Mittels RNA-Einzelzell-Sequenzierung konnte ein profunder Einfluss des Lymphomwachstums auf das T Zell-Kompartiment der Mäuse, denen Eμ-Tcl1 Zellen gespritzt wurden, gezeigt werden. / CAR T cell therapy is a promising new treatment option for patients suffering from aggressive B non-Hodgkin lymphomas (NHLs). In CAR T cell therapy, patient-derived T cells are genetically modified to express a chimeric receptor commonly directed towards a surface antigen expressed by neoplastic cells. In this thesis, anti-CXCR5 CAR T cell therapy was investigated as an alternative to anti-CD19 CAR T cell therapy for the treatment of mature B-NHLs. CXCR5 is a B cell homing receptor expressed by mature B cells and follicular helper T (TFH) cells. TFH cells were described to support the tumor cells in chronic lymphocytic leukemia (CLL) and follicular lymphoma (FL). This expression pattern allows simultaneous targeting of the malignant cells and the tumor-supporting microenvironment by CAR T cell therapy against a chemokine receptor in an unprecedented manner. Main findings included that (1) anti-CXCR5 CAR T cells targeted specifically CXCR5 expressing mature B-NHL cell lines and patient samples in vitro and showed strong in vivo anti-tumor reactivity in an immunodeficient xenograft mouse model, (2) anti-CXCR5 CAR T cells targeted tumor-supportive TFH cells derived from CLL and FL patient samples in vitro and (3) CXCR5 showed a safe expression profile. CXCR5 was strongly and frequently expressed by B-NHLs and its expression on healthy tissue was restricted to lymphoid cells. In summary, anti-CXCR5 CAR T cell therapy presents a novel treatment option for patients suffering from mature B-NHLs by eliminating the tumor and part of the tumor-supportive microenvironment. The second part of the project, the Eμ-Tcl1 murine lymphoma model, which mimics human CLL, was used to study the impact of lymphomagenesis on CXCR5+ T cells. Using single cell RNA sequencing, a profound influence of lymphoma growth on the T cell compartment in Eμ-Tcl1 tumor-challenged mice could be shown.

Liver metastases are the most common cause of death in colorectal cancer patients. The standard of care and potential for cure for colorectal liver metastases is resection, but often times disease it too extensive for this treatment. Over the years, cancer research has made way for advances in treating progressive disease through immunotherapy. By genetically modifying an individual’s immune system using virally transduced chimeric antigen receptor T cells (CAR-T), patients are better able to receive exquisitely specific T cells to target specific tumors. Furthermore, selective delivery strategies may enhance efficacy while limiting detrimental, systemic adverse effects. Not only this, CAR-Ts have also lead to complete remission in some liquid tumors while maintaining the potential for remission in solid tumors as well. This literature review takes readers through the emergence of the different generations of CAR-T and the various studies including clinical trials that have demonstrated the safety and efficacy of CAR-T. The second portion of this paper will outline the design for a phase II clinical trial using intrahepatic CAR-T therapy in addition to selective internal radiation therapy (SIRT) for refractory CEA+ colorectal liver metastases. Benefits and limitations of using these therapies are further discussed.

Olika typer av blodcancer är några av de vanligast förekommande cancerformerna i världen. Ju tidigare sjukdomen upptäcks och beroende på vilken typ av blodcancer någon drabbats av ser prognosen i många fall god ut då dagens behandlingsmetoder under flera år effektiviserats. Dock finns det fall där patienter inte svarar på traditionella terapier som cytostatika och stålning eller gånger där sjukdomen kommer tillbaka i aggressivare former. I dessa fall krävs andra metoder för att försöka behandla och bota cancern.   En metod som på senare tid fått mycket uppmärksamhet i forskarvärlden är användandet av kroppens egna immunceller som behandlingsform mot blodcancer. Genom att rena fram en patients egna T-celler från ett blodprov, kan dessa sedan genetiskt modifieras till att känna igen specifika tumörassocierade antigener (TAA) som bara vissa typer av cancerceller uttrycker. Metoden går ut på att T-cellerna får chimära antigen-receptorer (CAR), som uttrycks på cellytan, där CAR-T cellerna sedan injiceras tillbaka till patienten. CAR-T cellerna känner igen cancercellerna och attackerar sedan, med målet att patienten efter behandlingen inte ska ha några cancerceller kvar i kroppen.   Kliniska försök gjorda på patienter med olika typer av blodcancer har visat lovande resultat, särskilt gällande patienter som fått återfall av blodcancertypen Akut Lymfatisk Leukemi (ALL). De som fått delta i studierna har haft mycket dåliga prognoser och har innan blivit behandlade med de konventionella behandlingsterapierna, men utan eller med mycket dåligt resultat. Förhoppningar som väckts från dessa forskningsresultat har lett till diskussion att CAR-T celler kan komma att förändra cancervården och i framtiden kanske vara en lika vanlig behandlingsmetod som strålning eller kemoterapi. Dock kvarstår många problem som forskarna måste lyckas lösa innan CAR-T celler kan räknas som konventionell. Bland annat finns stora risker att patienter vid behandling kan drabbas av cytokinfrisläppningssyndrom (CRS, eng. cytokine release syndrome), där immunförsvaret kan attackera kroppens egna organ som i värsta fall kan leda till döden. Cancercellerna kan även komma tillbaka efterbehandling, då de har utvecklat en resistens mot CAR-T cellerna. Metoder för att undvika dessa toxiska responser och göra T-cellerna mer effektiva är bara några av de problem som kvarstår.

INTRODUCTION: Chimeric antigen receptor (CAR) T-cell therapy is a new treatment for hematologic malignancies including aggressive B-cell non-Hodgkin’s lymphoma (NHL). Although it has provided an effective treatment option for patients who have few options, CAR T-cell therapy does have many associated toxicities. Prolonged cytopenias are one of the lesser understood toxicities that can affect upwards of 40% of patients. METHODS: In this retrospective study, we reviewed 106 patients who received commercial CAR T-cell therapy between November 2017 and September 2019. Prolonged cytopenias were defined as having absolute neutrophil count (ANC) <1000/mm3, platelets (PLT) <50,000/mm3, and/or hemoglobin (Hgb) <10 g/dL at least once after 30 days post-CAR T-cell infusion. Furthermore, if only one incidence of cytopenia was recorded 30 days post infusion, we required that the patient had to have received either a transfusion or granulocyte-colony stimulating factor (GCSF) after the date of the recorded cytopenic value to be considered a part of the cytopenic cohort. RESULTS: 22 patients met the criteria of having prolonged cytopenias. 64% of the cytopenic cohort had >1 type of prolonged cytopenias. Anemia was the most prevalent affecting 72% of cytopenic patients. The length of time from diagnosis of aggressive B-cell NHL to date of CAR T-cell infusion was found to be positively correlated with an increased risk of developing prolonged cytopenias following CAR T-cell therapy. Additional risk factors associated with an increased risk of delayed cytopenias by univariate analysis included neutropenia on the day of infusion (day 0), a high C-reactive protein (CRP) before lymphodepletion and on day 0, day 0 PLT count, and Hgb before lymphodepletion and on day 0. On multivariate analysis, only high CRP before lymphodepletion was associated with an increased risk of prolonged cytopenias while high ferritin and PLT values on day 0 were associated with not developing prolonged cytopenias. There was no statistical difference between the cytopenic and non-cytopenic cohorts in rates of progression free survival (PFS) and overall survival (OS). Also, no difference was seen in rates or severity of other toxicities between cohorts. 41% of the cytopenic cohort experienced infectious complications post-infusion with one patient dying from their infectious complications. However, there was no association with incidence of infection and prolonged cytopenias when compared to the incidence of infection in the non-cytopenic cohort. CONCLUSIONS: A longer time from diagnosis of aggressive B-cell NHL to time of CAR T-cell infusion was associated with prolonged cytopenias while the number of lines of prior chemotherapy and rate of prior high dose chemotherapy with an autologous stem cell transplant (HD-ASCT) were not associated. It would be valuable to confirm this association and why it is associated since the other two factors were not. We lacked bone marrow biopsies before CAR T-cell infusion and did not have bone marrow biopsies for many patients after CAR T-cell infusion. It would be beneficial to collect data regarding bone marrow biopsies from these time points to highlight any changes that could be related to CAR T-cell therapy. Cytogenetic information of individual patient’s diseases would be worth analyzing to help determine if there are biological factors associated with prolonged cytopenias in response to CAR T-cell therapy. Additional studies should investigate the laboratory values we found to have associations with either cohort to help identify possible predictive values providers could use to identify patients at higher risk of having prolonged cytopenias. There is also a need to see if specific prior chemotherapy regimens increase a patient’s risk of having prolonged cytopenias. Overall, since prolonged cytopenias after CAR T-cell infusions have not been heavily investigated, further investigation is needed to better understand the predictive factors and identify possible mechanisms of prolonged cytopenias seen in CAR T-cell patients.

abstract: The success of genetically-modified T-cells in treating hematological malignancies has accelerated the research timeline for Chimeric Antigen Receptor-T (CAR-T) cell therapy. Since there are only two approved products (Kymriah and Yescarta), the process knowledge is limited. This leads to a low efficiency at manufacturing stage with serious challenges corresponding to high cost and scalability. In addition, the individualized nature of the therapy limits inventory and creates a high risk of product loss due to supply chain failure. The sector needs a new manufacturing paradigm capable of quickly responding to individualized demands while considering complex system dynamics. The research formulates the problem of Chimeric Antigen Receptor-T (CAR-T) manufacturing design, understanding the performance for large scale production of personalized therapies. The solution looks to develop a simulation environment for bio-manufacturing systems with single-use equipment. The result is BioMan: a discrete-event simulation model that considers the role of therapy's individualized nature, type of processing and quality-management policies on process yield and time, while dealing with the available resource constraints simultaneously. The tool will be useful to understand the impact of varying factor inputs on Chimeric Antigen Receptor-T (CAR-T) cell manufacturing and will eventually facilitate the decision-maker to finalize the right strategies achieving better processing, high resource utilization, and less failure rates. / Dissertation/Thesis / Masters Thesis Industrial Engineering 2020

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22882号 / 医博第4676号 / 新制||医||1047(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙折 晃史, 教授 濵﨑 洋子, 教授 羽賀 博典 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Most Adoptive Cell Therapies (ACT), including CAR T cell therapies, suffer failure because of the severe side effects due to loss-of-control of the therapeutic cells once they are inside the patient’s body, suggesting that novel strategies must be developed for a better in vivo control of these engineered cells. In the meantime, CAR T cell therapies targeting solid tumors have not experienced the remarkable success achieved with hematopoietic cancers, mainly due to continuous tumor antigen exposure and a suppressive tumor microenvironment. Here we designed a private passageway fusion receptor, which is composed of a ligand binding domain and a glycosylphosphatidylinositol (GPI) anchoring domain, to be expressed and localized to the surface of CAR T cells independently to the classical CAR T construct. These ligand binding domains preserve high binding affinity towards their cognate ligands and are only expressed on the CAR T cells that have been transduced. Therefore, cytotoxic drugs or immunosuppressants linked to the corresponding targeting ligands are shown to be specifically delivered to these fusion receptor positive CAR T cells for lowering the activity of the over-activated CAR T cells. On the other hand, we discovered that a potent TLR7 agonist is able to enhance the lysis effect of the exhausted CAR T cells in a co-culture model. Serial releasable and non-releasable targeted TLR7 agonists were prepared and tested. Based on these data, we suggest that our secret passageway fusion receptor platform provides a better control of the activity of CAR T cells using the corresponding targeting ligand-payload conjugates in a dose dependent manner and function as a doorway for the delivery of instructions to CAR T cells for versatile purposes.

T cells engineered with chimeric antigen receptors (CAR-T) have had breakthrough successes in cancer immunotherapy with FDA approvals. Still, the high cost of personalized CAR-T cell therapy hinders the accessibility from the public as a therapeutic option, and the lack of uniquely defined cancer-specific antigens brings the risk of life-threatening on-target, off-tumor toxicity. Changing the immune cell therapy platform to Natural Killer cells (NK cells) can be an option to develop allogenic off-the-shelf cell therapy. To reduce the on-target, off-tumor toxicity, developing an inhibitory CAR (iCAR) for cell therapy is necessary for precise logic computation and implementation of iCAR can broaden the spectrum of treatable cancers. This dissertation optimized the primary NK cell expansion platform with autologous PBMC and set robust virus transduction using pseudotyped retrovirus on the primary NK cells to use the NK cells as the cell therapy platform. I engineered iCAR candidates to suppress in response to a “safety antigen” on healthy cells, first in T cells and validated in NK cells, showing its efficacy against various versions of activating CAR (aCAR) drove killing of targets expressing activating antigen, while an inhibitory CAR (iCAR) suppressed cytotoxicity against targets expressing a safety antigen. Therapeutic immune cells equipped with the right pair of aCAR and iCAR platform will improve safety for currently validated cancer antigens and enable new therapies for previously unaddressed indications. / 2024-05-23T00:00:00Z

This thesis addresses multiple approaches to investigating mechanisms of immune linked disease. There are four projects outlined below which describe the work of these investigations. First, educating students about techniques to study disease and therapies is an important area of research. Flow cytometry is a common technique in immunology and its versatility and high throughput abilities can be applied to many fields. While it is very useful, flow cytometry is a complex technique that requires training to operate and understand, and there are very few reports about administering effective training. This thesis outlines the first report of a full semester university course about flow cytometry. Students who completed the course reported increased confidence in their skill levels in conceptual, technical and analytical areas. Second, in the fight against cancer, immunotherapies may provide the necessary adaptability to successfully combat many cancer types. By strengthening and educating the immune system, clinicians can help patients fight cancer without resorting to harmful chemotherapeutics, or immunotherapies can be used in tandem with current treatments. Chimeric antigen receptor (CAR) T cells and checkpoint blockade are two of the most successful immunotherapies. CAR T cells combine the extraordinary binding ability of an antibody with T cell signaling molecules via genetic engineering, for a faster and more efficient cancer killing version of the patient's own T cells. These have been remarkably successful, but results depend on the specific signaling co-receptors that are included in the design. Increased understanding of co-receptor function could help in making CAR T cell design more specific, and enable CAR T cells to be effective against more types of cancers. Metabolic function is crucial in understanding T cell therapeutics because T cells need to use energy efficiently enough to compete with ravenous cancer cells. This thesis outlines an ongoing investigation into a co-receptor's effect on CAR T cell metabolism, suggesting that co-receptors can alter CAR T cell metabolism by increasing maximal respiration. Third, CD5 is a negative regulatory co-receptor on T cells that can modulate T cell activation. Related inhibitory co-receptors (PD-1 and CTLA-4) are currently being effectively blocked as checkpoint therapies to reactivate T cells towards cancerous cells. This thesis outlines ongoing work investigating CD5's impact on cellular metabolism. We have found that T cells without CD5 are hypermetabolic as compared to normal naïve T cells. CD5 deficient T cells also have higher maximal respiration, higher basal respiration and higher glycolytic capacity. These differences are also present transiently after non-specific activation. Thus, CD5 significantly regulates the ability of a T cell to use energy, suggesting that CD5 may be a good target for creating more efficient T cell immunotherapies. Fourth, in a separate project, this thesis examines environmental causes of disease. Asthma and allergies are common and growing problems in children and adults. Evaporative cooling can be a less expensive alternative to central cooling, but its effects on allergens and other bioaerosols in the home remains unclear. This project examines the relationship between evaporative cooling and bioaerosols (dust mites, bacterial endotoxin, and fungal β-(1→3)-D-glucans) in low income homes in Utah. We report significantly higher levels of these bioaerosols, particularly fungi in homes with evaporative cooling after adjusting for home-specific factors.