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

Glioblastoma (GBM) remains the most aggressive primary brain tumor in adults. Since 2005, Standard of Care (SoC) consists of surgical resection followed by radiation and adjuvant chemotherapy with temozolomide. Treatment failure is attributed to intratumoral heterogeneity with populations capable of mechanisms to repair damaged DNA. Given the lack of progress to improve patient outcomes, the current work encompasses how multi-omic approaches can be utilized to uncover novel biology in GBM and develop precision medicines to exploit these cancer specific phenomena. Using patient derived GBM samples I first used the surface marker CD133 to interrogate glioblastoma stem cells, a subpopulation of cells identified to withstand conventional therapies and lead to tumor relapse. I used a genome-wide CRISPR-Cas9 library to conduct an unbiased loss-of-function phenotypic screen to identify regulators of CD133. I then validated SOX2 as a direct transcription factor to PROM1 encoding CD133. These findings further show the untapped potential of CRISPR to uncover novel biology to directly apply to broader fields of stem cells and cancer biology. Next, I combed GBM data sets at transcriptomic and proteomic levels to identify understudied proteins as potential targets for immunotherapies. Glycoprotein nonmetastatic melanoma protein B (GPNMB) has previously been identified as a clinically relevant target in GBM and shown to be active in the tumor immune microenvironment. I found GPNMB to be upregulated in recurrent GBM and macrophage populations which can be exploited in a more comprehensive manner to treat GBM. Through a series of models, I elucidated how GPNMB influences GBM biology, its effectiveness as a target for Chimeric Antigen Receptor T-cells, and how it can be paired with CD133 therapies to provide better coverage of tumor cells. Together, these studies highlight how advances in pre-clinical models and technologies can be leveraged to develop new therapies in a rational manner. / Thesis / Doctor of Science (PhD) / Glioblastoma (GBM) remains an aggressive and incurable brain cancer despite decades of intense research. Treatment failure is due to the untargeted approaches currently undertaken in the clinic. The current work uses multiples methods to interrogate how GBM grows and develops over time. Using GBM samples from consenting patients, I investigated an important population of the tumor using a surface marker CD133 and CRISPR to study which genes influenced it. I then successfully validated SOX2 as a direct regulator of CD133 expression. Next, I combed multiple data sets for a target to kill GBM cells without harming healthy tissue in patients. I found Glycoprotein Non-Metastatic Melanoma Protein B (GPNMB) to be exploitable and used several experimental methods to investigate its role in GBM progression. Finally, we used a novel immunotherapy to eliminate cells which express GPNMB. Together, these findings could apply to the broader field of stem cell biology and be used for a more targeted method to eliminate the cancer entirely.

Adoptive immunotherapy using chimeric antigen receptor (CAR)-modified T cells is an effective treatment for hematological malignancies that are refractory to conventional chemotherapy. To address a wider variety of cancer entities, there is a need to identify and characterize additional target antigens for CAR-T cell therapy. The two members of the receptor tyrosine kinase-like orphan receptor family, ROR1 and ROR2, have been found to be overexpressed on cancer cells and to correlate with aggressive cancer phenotypes. Recently, ROR1-specific CAR-T cells have entered testing in phase I clinical trials, encouraging us to assess the suitability of ROR2 as a novel target for CAR-T cell therapy. To study the therapeutic potential of targeting ROR2 in solid and hematological malignancies, we selected two representative cancer entities with high unmet medical need: renal cell carcinoma and multiple myeloma. Our data show that ROR2 is commonly expressed on primary samples and cell lines of clear cell renal cell carcinoma and multiple myeloma. To study the efficacy of ROR2-specific CAR T cell therapy, we designed two CAR constructs with 10-fold binding affinity differences for the same epitope of ROR2. We found both cell products to exhibit antigen-specific anti-tumor reactivity in vitro, including tumor cell lysis, secretion of the effector cytokines interleukin-2 (IL-2) and interferon-gamma (IFNγ), and T cell proliferation. In vivo studies revealed ROR2 specific CAR-T cells to confer durable responses, significant survival benefits and long-term persistence of CAR-expressing T cells. Overall, there was a trend towards more potent anti-tumor efficacy upon treatment with T cells that expressed the CAR with higher affinity for ROR2, both in vitro and in vivo. We performed a preclinical safety and toxicology assessment comprising analyses of ROR2 expression in healthy human and murine tissues, cross-reactivity, and adoptive T cell transfer in immunodeficient mice. We found ROR2 expression to be conserved in mice, and low-level expression was detectable in the male and female reproductive system as well as parts of the gastrointestinal tract. CAR-T cells targeting human ROR2 were found to elicit similarly potent reactivity upon recognition of murine ROR2. In vivo analyses showed transient tissue-specific enrichment and activation of ROR2-specific CAR-T cells in organs with high blood circulation, such as lung, liver, or spleen, without evidence for clinical toxicity or tissue damage as determined by histological analyses. Furthermore, we humanized the CAR binding domain of ROR2-specific CAR-T cells to mitigate the risk of adverse immune reactions and concomitant CAR-T cell rejection. Functional analyses confirmed that humanized CARs retained their specificity and functionality against ROR2-positive tumor cells in vitro. In summary, we show that ROR2 is a prevalent target in RCC and MM, which can be addressed effectively with ROR2-specific CAR-T cells in preclinical models. Our preliminary toxicity studies suggest a favorable safety profile for ROR2-specific CAR-T cells. These findings support the potential to develop ROR2-specific CAR-T cells clinically to obtain cell products with broad utility. / Adoptive Immuntherapie mit T-Zellen, die chimäre Antigenrezeptoren (CAR) exprimieren, ist ein effektiver Behandlungsansatz für Chemotherapie-resistente Blutkrebserkrankungen. Die Übertragung dieses Konzepts auf weitere Krebsarten erfordert die Identifikation und Charakterisierung neuer Zielstrukturen für die CAR-T Zelltherapie. ROR1 und ROR2, die beiden Mitglieder der Familie der Rezeptortyrosinkinase-ähnlichen Orphan-Rezeptoren, werden auf einer Vielzahl von Tumoren überexprimiert und korrelieren mit einer schlechten Prognose und höherer Krebs-Invasivität. Kürzlich konnte ROR1 als Zielstruktur für die CAR-T Zelltherapie bestätigt werden und die Effektivität und Sicherheit ROR1 spezifischer CAR-T Zellen wird derzeit im Rahmen klinischer Phase-I Studien näher untersucht. Aus diesem Grund waren wir daran interessiert, das therapeutische Potenzial ROR2-spezifischer Zelltherapie zu untersuchen. Als Modellsysteme hierfür wählten wir das Nierenzellkarzinom und das Multiple Myelom als repräsentative hämatologische und solide Krebserkrankungen mit hohem medizinischem Bedarf aus. Unsere Daten zeigen, dass ROR2 häufig auf Zelllinien und primären Tumorproben des klarzelligen Nierenzellkarzinoms und des Multiplen Myeloms vorkommt. Um die Effektivität ROR2-spezifischer CAR-T Zellen zu untersuchen, wurden zwei CAR Konstrukte mit zehnfach unterschiedlichen Bindungsaffinitäten für dasselbe Epitop von ROR2 hergestellt. Beide Zellprodukte zeigten hohe, antigen-spezifische Antitumor-Reaktivität in vitro – insbesondere im Hinblick auf Tumorzell-Lyse, Sekretion der Zytokine Interleukin-2 (IL-2) und Interferon gamma (IFNγ) und T-Zell Proliferation. In vivo beobachteten wir langanhaltende Antitumor-Effektivität durch ROR2-spezifische CAR-T Zellen, sowie signifikante Überlebensvorteile und langfristige T-Zell Persistenz. Außerdem beobachteten wir, sowohl in vitro als auch in vivo, einen Trend zu stärkerer Antitumor-Effektivität von T-Zellen, die den CAR mit höherer Affinität für ROR2 exprimierten. Im Rahmen einer präklinischen Toxikologie-Studie analysierten wir die Expression von ROR2 im gesunden Gewebe, die Kreuz-Reaktivität ROR2-spezifischer CAR-T Zellen und deren Sicherheit durch adoptiven T-Zell Transfer in immun-defiziente Mäuse. Unsere Daten zeigen, dass ROR2 in H. sapiens und M. musculus gleichermaßen exprimiert wird und ROR2 Expression war insbesondere in den weiblichen und männlichen Reproduktionsorganen und Teilen des Gastrointestinaltrakts detektierbar. Wir konnten außerdem zeigen, dass CAR-T Zellen, die menschliches ROR2 erkennen, vergleichbare Antitumor-Reaktivität gegen Zellen, die murines ROR2 exprimieren, auslösen. Unsere in vivo Analysen zeigten temporäre Anreicherung und Aktivierung ROR2-spezifischer CAR-T Zellen in gut durchbluteten Geweben, wie Lunge, Leber und Milz, in der Abwesenheit klinischer Anzeichen für Toxizität oder histologisch nachweisbarer Gewebsschädigungen. Um die Risiken immunologischer Nebenwirkungen und die damit einhergehende Abstoßung ROR2-spezifischer CAR-T Zellen zu reduzieren, humanisierten wir die CAR Bindedomäne. Unsere Daten zeigen, dass humanisierte ROR2-spezifische CAR-T Zellen vergleichbare Spezifität und Funktionalität gegen ROR2-positive Tumorzellen in vitro aufweisen. Insgesamt zeigen unsere Daten, dass ROR2 eine häufig auftretende Zielstruktur auf der Oberfläche von RCC und MM Zellen ist und diese in präklinischen Modellen effektiv mittels ROR2-spezifischer CAR-T Zellen adressiert werden kann. Unsere vorläufigen Toxizitätsdaten deuten darauf hin, dass ROR2-spezifische CAR-T Zellen ein vorteilhaftes Sicherheitsprofil aufweisen. Alles in allem unterstützen diese Daten das Potenzial der klinischen Entwicklung ROR2-spezifischer CAR-T Zellen als Zellprodukte mit breit gefächerter Anwendbarkeit.

Glioblastoma is a deadly brain cancer with discouraging standard of care. New methods like convection enhanced delivery and chimeric antigen receptor T cells (CAR-T) are promising treatments that can be translated to glioblastoma. In this study, CAR-T cell flow through a hydrogel was explored in the context of in-vitro convection enhanced delivery. A culture method to create large spheroids mimicking tumors from preexisting glioblastoma stem cell lines was fabricated, a convection enhanced delivery system for in-vitro testing was designed, and characterization of the CAR-T cells using the in-vitro system took place. The spheroid culture method was successfully optimized to produce spheroids large enough to act as a sufficient tumor in little time, the in-vitro set-up successfully administered treatment, and CAR-T cells were found to increase their velocities through a medium as their injection velocity increased. It was discovered that the density of the spheroid plays a crucial role in treatment delivery, often times driving how treatment will move through the spheroid. This system can be used in the future studies to test the killing potential of CAR-T cells to a tumor in-vitro. / Master of Science / Glioblastoma is a deadly brain cancer with current treatments that are discouraging at best. New methods must be utilized to aid in patient recovery. Chimeric antigen receptor T-Cells (CAR-T) are a promising treatment that can be used in glioblastoma. In this study, CAR-T cell behavior is defined in the context of in-vitro convection enhanced delivery. A large spheroid, or sphere of cells, mimicking a tumor was created, a convection enhanced delivery system set-up for in-vitro testing was designed, and characterization of CAR-T cell behavior using the in-vitro system took place. The spheroids were successfully cultured to act as a sufficient tumor, the in-vitro set-up successfully administered treatment, and CAR-T cells were found to increase their velocities in a gel as their injection velocity increases. It was discovered that the density of the spheroid plays a crucial role in treatment delivery, often times driving how treatment will move through the spheroid. This system can be used in the future studies to test the killing potential of CAR-T cells to a tumor in-vitro.

The current field of cancer treatment is undergoing a revolution. The influx of novel therapies derived from basic research on the immune system has shifted the landscape of modern medicine. Immunotherapy seeks to use the body’s own immune system as a medium to terminate neoplastic cells. This is performed by manipulating the immune system into either targeting cancer antigens or breaking down barriers towards T cell infiltration. The former mechanism uses CAR-T cells as an instrument to target specific cancer neo-antigens. CAR-T cells begin as T cells derived from a patient’s immune system. These cells are removed from the body and engineered to express a chimeric antigen receptor (CAR) through a process of viral transduction. This CAR allows the T cell to recognize and bind to a specific antigen of interest. In most cases, the antigen is present on cancer cells. The T cells, now expressing the CAR receptor, are transplanted back into the body of the patient and proceed to target cancer cells. This therapy has been used in hematological malignancies to great effect. Applying CAR-T cells to solid tumors is an ongoing process, but has been difficult to establish due to the immunosuppressive aspects of the tumor microenvironment. As such, combining CAR-T cells with traditional anti-cancer therapies has been proven to be efficacious in treating patients with solid tumors. In general, immunosuppression is a large problem in the treatment of cancer. Cancer cells and the tumor microenvironment express receptors that downregulate tumor-targeting actions of the immune system. The discovery of the programmed cell death protein 1 (PD1) allowed researchers to create novel antibodies that inhibit immunosuppression. PD1 located on T cells, binds to PDL1 on cancer and stromal cells. This interaction induces exhaustion and anergy in infiltrating T cells, thereby prevent T cells from targeting cancer cells. As such, the newly approved checkpoint blockade antibodies, Nivolumab and Pembrolizumab, block this interaction and allow T cells to carry out their targeting function. CAR-T cells and checkpoint blockade have both seen immense success in clinical trials and are currently being used the clinic. Nonetheless, development of these therapies for different types of cancers is an ongoing process and one that will require immense effort on behalf of the medical and pharmaceutical establishment

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.

CAR-T cell immunotherapy is a highly efficacious treatment for CD19-positive hematological malignancies, however, some patients are non-responsive for reasons that are not well understood. Clinical efficacy has been correlated with long-term persistence, a propensity that can be predicted by the differentiation state of transplanted cells. Despite this, decades-old methods for expanding T cells have not been updated to prevent the deleterious effects of excessive differentiation in CAR-T cells. Uncoupling proliferation and differentiation is a long-held goal in the field of immunotherapy with both cytokines and pharmacological approaches being implemented to dissociate these parallel processes. Histone methyltransferases rewire transcriptional programs in T cells and simultaneously regulate multitudes of genes, making them attractive targets for modifying the proliferation-differentiation axis. Despite this, only a handful of studies have examined their role in regulating the transcriptional programs of human CD8+ T cells. MLL4 (encoded by KMT2B) belongs to the six-member group of MLL histone methyltransferases. MLL1, a paralog of MLL4, has been implicated in regulating the maintenance of IL-4 and GATA-3 expression in TH2 CD4 memory T cell populations, however the function of MLL4 in human CD8+ T cells is unknown. We report a critical role for MLL4 in the proliferation and differentiation of CD8+ T cells. CRISPR-Cas9-editing of MLL4 uncoupled the processes of proliferation and differentiation, increasing proliferation but maintaining central memory T cell (TCM)-like populations, allowing for the production of increased numbers of TCM-like CD62L+CD45RO+ cells. Pharmacologically inhibiting the MLL4-Menin complex with MI-2 during T cell expansion enriched the frequency of minimally differentiated TCM-like CD8+ T cells. TCM-associated CD62L, CCR7, CD122 and CD127 surface markers were upregulated and early memory-associated transcription factor TCF7, LEF1, EOMES, and FOXP1 transcripts were increased. CD8+ CAR-T cells expanded in the presence of MI-2 responded earlier, while improving both tumor burden and survival in a NSG xenograft model of human leukemia. This finding has important translational impact in improving the persistence and proliferative capacity of CD8+ CAR-T cells. / Infectious Disease & Immunity

Advances in our understanding of the molecular events leading to cancer have facilitated the development of next-generation targeted therapies. Among the most promising new approaches is immuno-oncology, where therapeutic agents engage the immune system to fight cancer. One exciting strategy therein is the adoptive transfer of ex vivo cultivated tumor-specific T lymphocytes into a cancer patient. Tumor-specific T cells can be produced by engineering a patient’s own T cells with synthetic receptors (e.g. chimeric antigen receptors (CARs)) designed to redirect T cell cytotoxicity against a tumor target. CAR-engineered T cells (CAR-T cells) were expected to be a non-toxic cellular therapy which would seek out and specifically eliminate disseminated tumors. The clinical experience supports the promise of CAR-T cell therapy (striking efficacy has been observed in the treatment of hematological malignancies), while highlighting areas for improvement; CAR-T cell use has been associated with a host of toxicities and robust clinical efficacy has yet to be replicated in solid tumors. This thesis uses pre-clinical models to describe previously unappreciated aspects of CAR-T cell-associated toxicity and novel synthetic receptor strategies, including: i. The capacity of NKG2D-based CAR-T cells to mediate toxicity. ii. The utility of designed ankyrin repeat proteins as CAR antigen-binding domains. iii. The discovery that variables intrinsic to human CAR-T cell products contribute to toxicity. iv. A novel synthetic receptor capable of redirecting T cell specificity against a tumor target – the T cell antigen coupler (TAC). Unlike equivalent CAR-T cells, TAC-T cells are capable of mediating efficacy against a solid tumor in the absence of toxicity. We anticipate that these results will contribute towards the development of next-generation synthetic receptor-engineered T cell products that can deliver upon the promise of safe, systemic cancer therapeutics. / Thesis / Doctor of Philosophy (PhD) / The human immune system has the unique capacity to “seek and destroy” tumor cells throughout the body. A novel class of drugs, immuno-oncology agents, harness this ability to fight cancer. Within this class is a new cellular drug where genetic engineering is used to create killer immune cells (called T cells) capable of recognizing and eliminating tumors. Two of these cellular drugs have recently received FDA approval, supporting the feasibility of this approach. However, further research is needed to improve the safety of engineered-T cells and increase the number of patients whom can benefit from their use. This thesis uses laboratory investigations to better understand the side-effects associated with anti-cancer engineered-T cells and evaluate new engineering strategies. We anticipate that these results will contribute towards the development of next-generation engineered-T cell drugs which retain the ability to function systemically against cancer but offer an enhanced safety profile.

Breast cancer is one of the most common cancer types in women and its metastases cause most patient deaths in the advanced stage of this disease. 1,2,3 Unfortunately, metastases develop drug resistance to chemotherapy and impaired T lymphocyte infiltration into metastatic lesions by compressing blood vessels. 4,5,10,11,12 Although losartan decompressed vessels and increased the presence of T lymphocytes in the metastatic lesions, T-cells were not effective at eliminating tumors.10 In this thesis, we generated chimeric antigen receptor constructs that have specificity against epithelial cell adhesion molecule (EpCAM). After optimizing a retroviral transfection/transduction system, we successfully generated EpCAM CAR T-cells and tested their efficacy against tumor spheroids. We noticed a dramatic reduction of spheroids' area and spheroids' diameter after 36 hours of treatment and observed spheroids’ destruction and tumor cell elimination after 96 hours of treatment, compared to non-specific stimulated T-cells treatment on tumor spheroids. EpCAM CAR T-cells have been shown to be effective against cancer in vitro; therefore, injection of EpCAM CAR T-cells into mice with breast cancer will be conducted to determine whether losartan is able to improve infiltration. We expect that the use of losartan will improve the number of infiltrated CAR-T-cells and their efficacy against breast tumors.

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...