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

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

Cancer immunotherapy has recently made remarkable clinical progress. Adoptive transfer of T-cells engineered with a chimeric antigen receptor (CAR) against CD19 has been successful in treatment of B-cell leukemia. Patient’s T-cells are isolated, activated, transduced with a vector encoding the CAR molecule and then expanded before being transferred back to the patient. However some obstacles restrict its success in solid tumors. This thesis explores different aspects to improve CAR T-cells therapy of cancer. Ex vivo expanded T-cells are usually sensitive to the harsh tumor microenvironment after reinfusion. We developed a novel expansion method for T-cells, named AEP, by using irradiated and preactivated allo-sensitized allogeneic lymphocytes (ASALs) and allogeneic mature dendritic cells (DCs). AEP-expanded T-cells exhibited better survival and cytotoxic efficacy under oxidative and immunosuppressive stress, compared to T-cells expanded with established procedures. Integrating retro/lentivirus (RV/LV) used for CAR expressions randomly integrate in the T-cell genome and has the potential risk of causing insertional mutagenesis. We developed a non-integrating lentiviral (NILV) vector containing a scaffold matrix attachment region (S/MAR) element (NILV-S/MAR) for T-cells transduction. NILV-S/MAR-engineered CAR T-cells display similar cytotoxicity to LV-engineered CAR T-cells with undetectable level of insertional event, which makes them safer than CAR T-cells used in the clinic today. CD19-CAR T-cells have so far been successful for B-cell leukemia but less successful for B-cell lymphomas, which present semi-solid structure with an immunosuppressive microenvironment. We have developed CAR T-cells armed with H. pylorineutrophil-activating protein (HP-NAP). HP-NAP is a major virulence factor and plays important role in T-helper type 1 (Th1) polarizing. NAP-CAR T-cells showed the ability to mature DCs, attract innate immune cells and increase secretion of Th1 cytokines and chemokines, which presumably leads to better CAR T-cell therapy for B-cell lymphoma. Allogeneic-DCs (alloDCs) were used to further alter tumor microenvironment. The premise relies on initiation of an allo-reactive immune response for cytokine and chemokines secretion, as well as stimulation of T-cell response by bringing in tumor-associated antigen. We demonstrated that alloDCs promote migration and activation of immune cells and prolong the survival of tumor-bearing mice by attracting T-cells to tumors and reverse the immune suppressive tumor microenvironment.

In the last decade, six CAR T cell therapies against hematological malignancies have been approved for commercial manufacturing and several clinical trials are underway. This has led to extensive preclinical research focused on optimizing individual manufacturing steps of adoptive T cell therapeutics. Ex vivo expansion of T cells is one of the crucial manufacturing steps, as is necessary to obtain clinically required cell numbers for infusion. However, ex vivo expansion is also a complex step as it involves multiple different variables including culture medium, serum and cytokine supplementation, activation reagent and mode of genetic modification. Consequently, our understanding of changes in T cells during ex vivo expansion and the impact of expansion conditions on the final product; and thus the outcome of the therapy remain mostly elusive. Therefore, this project was designed to understand the changes in T cells at different stages of ex vivo expansion compared to freshly isolated T cells with a focus on understanding the ongoing transcriptional changes. The T cells were isolated from healthy blood donor buffy coats using FABian®-Cell Isolation System based on Fab-TACS technology. T cells were cultured for 7 days in X-VIVO 15 media (supplemented with 5% human serum and 50 IU/ml IL-2) with activation for initial 3 days using anti-CD3/CD28 TranAct. The T cell kinetics during ex vivo expansion was characterized based on cell activation, differentiation and proliferation. For whole transcriptome sequencing, Total RNA was harvested from 6 different time points, freshly isolated cells (0 hr), and cells cultured for 4, 12, 24, 72 and 168 hr (7 days). The RNA sequencing libraries were prepared using “Illumina TruSeq Stranded Total RNA library prep' workflow and whole transcriptome sequencing was performed on Illumina Novaseq 6000. Further, changes in T cell trafficking capabilities, cell size and cell cycle progression were studied in freshly isolated T cells and cultured cells. The changes in T cell trafficking was studied by analyzing the changes in VLA4 mediated T cell adhesion to VCAM1 coated surface under increasing shear stress. The cell size and volume of freshly isolated T cells and cultured cells were analyzed using multisizer instrument. Additionally, an in vitro model was developed to simulate the behavior of cultured T cells upon re-infusion into the blood and changes in cell cycle was analyzed. The components of in vitro reconstituted blood model were pooled human AB serum, erythrocyte concentrates and cultured T cells. The absolute lymphocyte count in buffy coats and total number of T cells isolated per buffy coat were in range compared to cell isolation and enrichment through standard leukapheresis. Thus suggesting that healthy donor-derived buffy coats and enrichment of T cells using Fab-TACS technology can be a suitable starting material and cell enrichment device respectively. The T cell growth kinetics was analyzed based on surface expression of specific markers, which also closely resembled their gene expression. The T cell kinetics observed during ex vivo expansion was similar to T cell kinetics observed in several preclinical CAR T cell expansion studies. The T cell proliferation in terms of increase in cell numbers and gene ontology (GO) terms related to DNA replication and cell division were significantly enriched only after 3 days of ex vivo expansion. The final cell numbers after 7 days of ex vivo expansion were approx. 1.0E+9 T cells, which was well above the clinically required infusion dosage of currently approved CAR T cell therapies. Taken together, the ex vivo expansion protocol followed in this study generates T cells in range required for clinical infusion dose and the growth kinetics of T cells observed were in line with the commercial expansion protocol. Hierarchical clustering of genes based on their expression over time identified 29 different gene-clusters which followed the pattern of mono-, bi- and triphasic modulation. The gene-clusters 11 and 18 were significantly enriched with T cell immune function related GO terms. The GO analysis of differentially expressed genes identified enrichment several bioprocesses, signaling pathways and T cell immune functions including commonly known activation, differentiation and proliferation. The ex vivo expansion of T cells was associated with early (i.e. upto 24 hr time point) enrichment of several GO terms associated with cytokine production such as IL-1, IL-2, IL-5, IL-6, IL-10, IL-13, IL-17, TNF and IFN-γ. The Janus kinase-signal transducer and activator of transcription (JAK-STAT) and Mitogen-activated protein kinase (MAPK) signaling cascades related GO terms were enriched as a result of autocrine signaling mediated by cytokines released during ex vivo expansion. These data demonstrate that cytokine release in T cells is activated during ex vivo manufacturing, and should be considered during future optimization. The gene expression analysis of commonly known exhaustion markers revealed two different patterns of expression. The CTLA4, TIGIT, TBX21 and BATF was upregulated at early time points. Whereas, the expression of TIM3, LAG3 and CX3CR1 was upregulated at later time points. The early expression of exhaustion markers can be attributed to immune check point function to prevent over-activation, and later expression of exhaustion markers may contribute to inhibitory function. In vitro investigation of ex vivo expanded T cells exhibited stronger VLA4 mediated adhesion to VCAM1 coated surface compared to freshly isolated cells under increasing shear stress. This was in contrast to the downregulation of alpha 4/beta 1 integrin gene expression during ex vivo expansion. The cell size analysis revealed cultured T cells were larger in terms of both size and volume at the end of 7-day culture period with doubled cell volume compared to freshly isolated T cells. These results taken together, suggest that increased adhesion capacity and increased cell size, after T cell expansion may be associated with accumulation of T cells in lungs upon infusion. The freshly isolated T cells that closely represent the T cells circulating in peripheral blood were arrested in G0/G1 phase. However, during ex vivo expansion T cells entered cell cycle, and T cells were found to be predominantly in S+G2/M phase on day 3, 5 and 7. Surprisingly, the cultured T cells were still in cell cycle even after 48 h of incubation in reconstituted blood in vitro. This suggests that a prolonged resting phase of ex vivo expanded T cells for more 48 hr before infusion into the patients can be advantageous in minimizing the risks associated with T cell therapy. In conclusion, this study has revealed a number of novel insights into transcriptional regulation and signaling processes occurring during culture expansion. In the study, the different patterns of transcriptional regulation and enrichment of various associated bio-processes and signaling pathways during ex vivo expansion were explored. In addition, an in-depth analysis of genes related to T cell activation and differentiation, adhesion and migration, and exhaustion markers was performed. The protein-protein interaction analysis and transcriptional factor enrichment analysis provide valuable data for further in silico investigations of transcriptional changes in T cells during ex vivo expansion. Additionally, this study provides a comprehensive overview of long non-coding RNAs at different stages of ex vivo expansion of T cells, thus providing a resource for novel understanding of impact of lncRNAs on T cells during ex vivo expansion for adoptive T cell therapies. The complete data of 48 transcriptomes derived from 8 donors over 6 time points is reposited (GEO: GSE250311) to a publicly available database and will allow exploration for future studies which aim at the characterization of alterations in expanded T cells for therapy, and optimization of conditions for their future use in patients.

Das Glykoprotein A33 (GPA33) ist ein aussichtsreiches Antigen für eine zielgerichtete Therapie, da es vorwiegend in Darmepithelien und Darmkrebs vorkommt. Klinische Studien zu GPA33-gerichteten Antikörpertherapien haben aber festgelegte Ansprechraten nicht erreicht. Zur Untersuchung der geringen Wirksamkeit und Verbesserung der Therapien habe ich GPA33-Expression in Darmkrebs untersucht, mit GPA33-Regulation interferiert und eine zelluläre GPA33-gerichtete Therapiealternative entwickelt. Mithilfe von Immunhistochemie und Immunfluoreszenz in zwei Kolorektalkarzinom-Kohorten entdeckte ich Heterogenität in der GPA33-Expression. Ich fand eine inverse Korrelation zwischen hoher GPA33-Expression und Metastasierung sowie einen konsistenten GPA33-Expressionsphänotyp im Primärtumor und Fernmetastasen. GPA33-positive Zellen waren zentral lokalisiert und hatten geringe WNT-Aktivität. GPA33-negative Zellen an der invasiven Front wiesen erhöhte WNT-Aktivität auf. Zur Untersuchung der Verbindung zwischen Zellsignalwegen und GPA33-Expression griff ich in onkogene Signalwege und Transkriptionsfaktoren ein. Meine Ergebnisse zeigten eine CDX1-abhängige transkriptionelle Regulierung der GPA33-Expression und eine inverse Korrelation mit dem WNT-Signalweg in vitro. Die Herunterregulierung der WNT-Aktivität erhöhte GPA33-Expression in vitro und in GPA33-negativen Tumorzellsubpopulationen in Xenotransplantaten. Schließlich entwickelte ich GPA33-CAR-T-Zellen und untersuchte ihre Anti-Tumor-Aktivität. GPA33-CAR T-Zellen wurden als Reaktion auf GPA33-positive Darmkrebszellen aktiviert und reduzierten das Tumorwachstum in Mäusen. Zusammenfassend identifiziert diese Studie intratumorale Heterogenität als Ursache für die geringe Wirksamkeit GPA33-gerichteter Therapie und zeigt, dass diese Therapie durch WNT-Inhibition verbessert werden könnte. Zudem liefere ich präklinische Beweise für den Einsatz einer GPA33-gerichteten Immuntherapie gegen Darmkrebs. / The cell surface Glycoprotein A33 (GPA33) is a promising antigen for targeted therapy, predominantly expressed in intestinal epithelia and colorectal cancer. Prior clinical trials with GPA33-targeted antibodies have not achieved desired response rates. To investigate low efficacy and improve therapies, I explored GPA33 expression, interfered with GPA33-regulatory mechanisms, and developed a cellular GPA33-targeted therapy alternative. Using immunohistochemistry and immunofluorescence on two colorectal cancer cohorts, I discovered heterogeneity in GPA33 expression. I found an inverse correlation of high GPA33 expression with progressive disease and consistent GPA33 expression in primary tumor and distant metastasis. GPA33-positive cells were centrally localized with low WNT activity, whereas GPA33-negative cells were at the invasive front with elevated WNT signaling. To understand the functional link between cell signaling networks and GPA33 expression, I interfered with oncogenic signaling pathways and transcription factors. My findings revealed CDX1-dependent transcriptional regulation of GPA33 and an inverse correlation with WNT signaling in vitro. Downregulation of WNT activity increased GPA33 expression in vitro and in GPA33-negative tumor cell subpopulations in subcutaneous xenograft models. Finally, I developed GPA33-CAR T cells and assessed their anti-tumor activity using flow cytometry, ELISA, live-cell imaging, and adoptive transfer. GPA33-CAR T cells were activated in response to GPA33-positive colorectal cancer cells and reduced xenograft growth in tumor-bearing mice. In conclusion, this study identifies intratumoral heterogeneity as a potential cause for low treatment efficacy and demonstrates that GPA33-targeted therapy could be enhanced by simultaneous WNT inhibition to boost GPA33 expression in colorectal cancer cells. Additionally, I provide preclinical evidence supporting the use of cellular immunotherapy to target GPA33 in colorectal cancer.

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.

No description available.