archives@tulane.edu / Background: Human Immunodeficiency Virus-1 (HIV-1) has killed over 35 million and infects 1.8 million new people each year. Antiretroviral therapy (ART), although effective controlling plasma viremia and transmission, does not purge latent or persistent reservoirs necessary to eliminate infection, and must be maintained for life. It is thus imperative to discover therapeutics that provide both lifetime suppression of viral loads and depletion of viral reservoirs. Methods: To harness the immunosurveillance capacity of highly functional and persistent CMV-specific adaptive response, rhesus PBMCs were stimulated with rhCMV peptide pools (IE1, IE2, and pp65) to expand rhCMV-specific T cells. These cells were then genetically modified with retroviral vectors expressing a CD4 extracellular domain linked to T cell intracellular signaling domains that instruct CTL activity, converting them into HIV-specific effector cells. Vectors combine CD4 targeting with an maC46 fusion inhibitor to protect against viral entry. In a reversal of the critical step in the HIV viral lifecycle whereby virus targets new CD4+ host cells using its Env glycoprotein, these genetic modifications redirect host immune responses to target and kill Env expressing infected cells. We hypothesize that continuous stimulation of CD4-CAR T cells through their rhCMV-specific TCR will maintain activated T effector memory CTL capable of targeting HIV infected cells. Results: We find that autologous rhPBMCs can be expanded ex vivo with rhCMV peptides up to therapeutically relevant numbers for adoptive transfer. This rhCMV-specific T cell expansion enriches cells in a phenotype consistent with T effector memory differentiation. Following genetic modification and adoptive transfer, cells reach peak expansion at seven days post infusion into ART suppressed or unsuppressed SHIV infected Rhesus Macaques. We observe these cells capable of persisting in vivo for at least 2 years following reinfusion. Furthermore, these cells are maintained in vivo in an effector memory phenotype throughout the duration they were analyzed. Despite this, SHIV plasma viral loads remain unchanged. Conclusion: These studies establish use of rhCMV-specific T cells as an effective way to produce persistent genetically modified cells targeting SHIV. Future studies will need to further increase in vivo expansion, protection, and CTL activity as viral loads remain detectable. / 1 / Nathan Michel Johnson

INTRODUCTION: Multiple Myeloma (MM) is a plasma cell malignancy that causes improper production of immunoglobulins and elevated levels of monoclonal protein. Resulting morbidity is a conglomeration of symptoms due to organ failure, lytic bone disease, and hematological insufficiencies. The American Cancer Society estimates more than 35,000 patients will be diagnosed with multiple myeloma in the United States in 2023. Current therapeutic regimen hinge on the idea of myeloma as a chronic disease that cannot be entirely cured and toxic chemotherapies with long-term treatment cycles are the standard of care. The need for a one-time therapy that is both safe and efficacious and with potentially curative action has led to the development of anti-BCMA CAR T-cell infusions. The overwhelming success of this novel therapy in MM has been demonstrated in clinical trials, but the need for data surrounding the long-term toxicities post-CAR T-cell treatment in a real-world population of MM patients still exists. Common expected adverse events that have been identified in clinical trials include cytokine release syndrome, neurotoxic events, hematological toxicities, and infections associated with immunosuppression. This study was formed to elucidate the long-term adverse events associated with anti-BCMA CAR T-cell therapy in a real-world patient population. METHODS: A total of 54 patients who received a CAR T-cell infusion for their relapsed and refractory multiple myeloma were studied in a retrospective analysis at Dana-Farber Cancer Institute. Data were collected prior, during, and after infusion to gauge treatment performance and toxic side effects. Analyses of collected data, including complete blood counts, serum protein electrophoresis, fluorescence in-situ hybridization (FISH) data from bone marrow biopsy, and imaging were performed. RESULTS: Patients were followed for a mean average of 165 days (range 29-462) post-infusion. Patients either received CiltaCel (n = 7) or IdeCel (n = 47). Grade 3 or greater cytopenia occurred in 48% of patients at some point following infusion and the median time to first onset was 30 days (10-189). Forty-six patients (85%) achieved a partial response or better as their best response to therapy. During inpatient infusion, 76% of patients experienced grade 1 or 2 cytokine release syndrome (CRS) and 8% experienced grade 1 or 2 immune effector cell-associated neurotoxicity syndrome (ICANS). A total of 12 patients (22%) developed infections after infusion with respiratory infections being the most frequent (17%). Nine patients were also evaluated on a closer scale for their experience with prolonged cytopenia, but no significant commonalities were found. DISCUSSION: The analysis of this study found this patient population to have a considerably less frequent incidence of high grade cytopenia as compared to clinical trial data. However, 92% of patients developed grade 1-3 anemia and 77% developed any grade thrombocytopenia, both figures are greater than those presented in the KarMMa-2 clinical trial study for ide-cel. Patients who developed severe cytopenia were able to recover absolute neutrophil counts (ANC) over the course of their follow-up appointments which is an important aspect in the prevention and avoidance of serious infection. This same recovery was not observed in platelet or hemoglobin counts. Additionally, 15 patients were reported to still have high-grade cytopenia at 30—60-days post infusion, but this number drops to only 5 patients for the 60—90-day timeframe, this steep drop is indicative of an early onset of severe cytopenia that may not carry on as the patient progresses further from their infusion date. Compared to the KarMMa-2 study which reported an infection incidence of 69%, observations from this current study suggest this real-world patient population remained healthier after infusion in terms of infection with only 23% of patients developing post-infusion infection. Instances of CRS and ICANS were comparable to data evaluated in clinical trials. Finally, treatment responses did not significantly differ between the population of patients who developed grade 3 or greater cytopenia and those patients who did not. More data is required to determine the risk-benefit profile of early intervention with CAR T-cell therapy as directly compared to the current standard of care. This study is an encouraging insight into the performance of real-world RRMM patients that should assure patients and clinicians of the safety and uncompromising efficacy of anti-BCMA therapy as a treatment option for multiple myeloma.

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

Immunotherapy is a promising tool in the fight against cancer and aims to recruit patients own immune systems to seek out and destroy malignant cells. Options such as oncolytic viruses (OVs), autologous tumour vaccines and chimeric antigen receptors have shown clinical success to date, yet there remain significant hurdles to overcome. Here, we demonstrate a novel vaccine combining irrCell priming and infected cell boosting dramatically improves the tumour-specific CTL response against CT26 tumours and can be further enhanced using additional immunogenic factors (armed OVs, adjuvants). We also developed a novel fluorescence-based high-throughput screening platform to identify compounds that sensitize resistant solid tumours to killing by CAR-T cells, which ultimately revealed cardiac glycosides as putative tumour sensitizers. Overall, this thesis identifies several novel enhancers of the anticancer immune response, including a heterologous irr:ICV vaccine regimen and the potential ability to identify molecules to overcome resistance to CAR-T therapy.

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

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

Glioblastoma (GBM) is the most frequent and lethal malignant brain tumor in adults. It emerges with an incidence of 3.2 per 100.000 in the US and 3.91 in Europe. Today, standard treatment after diagnosis consists of surgical removal of tumor tissue, followed by radiation therapy and adjuvant chemotherapy using temozolomide. Even after this rigorous therapy, patients show a median overall survival of only 15.6 months or 20.5 months when the tumor is additionally treated with so-called tumor treating fields. GBM is characterized by molecular heterogeneity within the same patient but also between different patients, which impedes development of novel therapeutics. During the last decades various immunotherapies including (multi-epitope) peptide vaccines, oncolytic viruses or immune checkpoint inhibitors against GBM were tested in small clinical studies, but failed to show a benefit in large studies. A novel kind of immunotherapies that showed great success in hematological tumors so far, is based on chimeric antigen receptors (CAR). These synthetic receptors can be introduced into immune cells to retarget their function towards tumor cells, independently of the major histocompatibility complex (MHC) that is often down regulated by tumors for immune evasion. A large hurdle for treatment of GBM using immunotherapies such as CAR-T cells, is antigen heterogeneity that limits the effect of therapies against single targets and renders the need for discovery of novel targets to enable treatment of a wide variety of patients with high success. Analyzing publicly available data and performing RT-qPCR experiments with RNA isolated from GBM tissue of a local cohort of patients, overexpression of two candidate GBM antigens, namely TMEM158 and PTPRZ1 were observed. Overexpression of both antigens in GBM in comparison to normal brain tissue and low-grade gliomas (only TMEM158) was revealed. In addition, a negative correlation between expression and patient survival was detected, as well as a correlation between TMEM158 and CD44 expression, the latter being a marker for GBM stem cells and the mesenchymal GBM subtype. Induction of chemoresistance by TMEM158 seems likely for GBM, since this was already discovered for several other tumor entities. Protein expression of TMEM158 was confirmed by Western blot analysis of different GBM cell lines. Since cell surface expression of a target protein is a prerequisite for targeting by a CAR-therapy, the expression of TMEM158 on cells from GBM cell lines was analyzed by flow cytometry. For this analysis a fluorescence-labeled peptide, based on sequence information of a known naturally occurring TMEM158 ligand (BINP) was designed. Binding to T98G and U-87 MG was observed, while only very low binding to the neuroblastoma cell line SH-SY5Y was seen in flow cytometry. Partial knockdown of TMEM158 was achieved using DsiRNAs, followed by Western blot (antibody staining) and flow cytometry (peptide staining), confirming the specificity of binding detectable by both methods. A recombinant fusion protein, consisting of the extracellular part of TMEM158 and a human Fc-antibody fragment was produced in 293T cells by transient transfection of an expression vector. The expected size of the protein produced was confirmed by Western blot. Furthermore, binding of the BINP-peptide to the recombinant protein was analyzed and compared to a scrambled BINP-peptide. In these experiments specific binding of the BINP-peptide was observed, also indicating the functionality of the recombinant protein. Next, CAR-constructs were designed using the original sequence information from BINP as binding domain and additional variants with amino acid exchanges at different positions. Significant cytotoxicity of all BINP-CAR-T cells was observed against T98G, which showed highest binding of BINP when analyzed by flow cytometry. A BINP-CAR version in which phenylalanine 11 was exchanged with alanine (BINP-F11A-CAR) showed significantly higher cytotoxicity against T98G than the BINP-CAR containing the original BINP sequence (BINP-WT-CAR). Against the U-87 MG cell line, only a version of the BINP-CAR containing an RGD- (arginine-glycine-aspartic acid) motif showed significant cytotoxicity. RGD-motifs are known to bind integrins like αVβ3, which was abundantly present on this cell line, as it was confirmed by flow cytometry within this work. Using this BINP-RGD-CAR version, targeting of both antigens at the same time seems possible. No significant cytotoxicity of the different CAR versions was observed against the TMEM158- and αVβ3-low cell line SH-SY5Y. In conclusion, overexpression of TMEM158 and PTPRZ1 and their negative influence on survival of patients, as found in recent literature, was confirmed for glioblastoma. Significantly higher expression of TMEM158 in GBM in comparison to low-grade gliomas as well as the correlation with CD44 hint at an association of TMEM158 with the aggressive phenotype of GBM. For all of these reasons, targeting of TMEM158 appears to be very feasible. Cytotoxicity of the produced BINP-CAR-T cells, which are the first CAR-T cells targeting TMEM158 so far, was demonstrated against GBM cells. Additional to cytotoxicity of the CAR-T cells, other in vitro assays and in vivo models should be utilized to determine more aspects of CAR-T cell function, in the future. For example, proliferation, cytokine release, invasion of tumor tissue, and inactivation of CAR-T cells by the tumor milieu should be quantified. To estimate how many patients could benefit from a therapy against it, percentage of patients and distribution within the tumors should be determined.

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.

T cells are a cornerstone of the mammalian adaptive immune system. A range of T-cell subsets exist that can orchestrate the overall immune response to pathogens or cancers, either by directly killing infected cells or licensing other cells to do so. Dysregulation of this important process can result in immunodeficiency or autoimmunity. Although T cells have been studied extensively over many decades, the detailed mechanisms underlying T-cell activation remain to be fully resolved. This thesis describes the development of new optogenetic approaches for the modulation of T-cell signalling dynamics and the interrogation of key events in T-cell activation to help investigate this question. Optogenetics is a rapidly emerging technique whereby light can be used to control the spatial and temporal activation, or inactivation of signalling pathways at unprecedented resolution. The methods described in this work utilise the blue light-responsive LOV2 photo-domain from the common oat A. Sativa, which is the foundation of the both the ‘LOVTRAP’ and ‘TULIPs’ optogenetic toolkits. T-cell antigen receptor (TCR) microclusters arise early during the interaction between T cells and antigen presenting cells (APCs). These TCR signalling platforms contain the proteins necessary for sustained T-cell activation, yet the processes underlying their formation and dissociation are still not fully characterised as they have been difficult to investigate with current chemical and genetic manipulations of T cells. Using two optogenetics systems combining either LOVTRAP or TULIPs and the microcluster- scaffolding protein LAT (Linker for the Activation of T cells), it was possible to modulate early T-cell signalling events and measure functional outputs in real-time. Unfortunately, the biological limitations of these LAT-based systems meant that they could not be used to quantitatively investigate microcluster formation. However, in an alternative approach, a drug-inducible, light-controllable chimeric antigen receptor was successfully developed that yielded important new insights into the rapid rate of signal decay within the TCR signalling pathway and the temporal dynamics of T-cell activation over several timescales. T cell-dependent bispecific antibodies (TDBs) are a new class of immuno-therapeutics that can specifically direct a T-cell response towards tumours, by crosslinking the TCR complex to a surface- expressed target on the cancerous cells. However, their mechanism of action has not been studied in detail. The close apposition of the T cell and target cells driven by the TDB interaction can result in the steric exclusion of phosphatases, such as CD45, away from the TCR at the TDB-generated cell-cell interface due to their large, rigid extracellular domains. Using the myeloma-expressed antigen, FcRH5, it was found that membrane-proximal epitopes of FcRH5 drive more robust TCR clustering and increased CD45 exclusion than membrane distal epitopes, which strongly correlated with effective killing of the target cell. These findings have important implications for therapeutic design and implementation of TDBs.