In addition to defending against invasive microbes, T cells of the mammalian adaptive immune system also provide crucial protection against cancerous cells that spontaneously emerge from healthy host tissue. Cancer cells, in turn, co-opt many of the regulatory signaling pathways used by the body to prevent excessive T cell proliferation and activation. The tumor microenvironment (TMI) is frequently a highly immunosuppressive environment, and much research has been focused on identifying new targets that can enhance T cell antitumor immunity. Novel observations from my lab has found monoamine oxidase A (MAO-A) plays an important role in maintaining this immunosuppressive TMI. In Chapter 2, I observe that MAO-A functions as a “checkpoint” molecule that regulates CD8+ T cell antitumor immunity through modulating metabolization of serotonin, a neurotransmitter previously unassociated with the immune response. In Chapter 3, I observe that MAO-A also indirectly affect T cell antitumor immunity by promoting tumor associated macrophage polarization towards an immunosuppressive phenotype through signaling with oxidative stress. These two chapters provide exciting preclinical data and clinical correlation for repurposing clinical approved MAO-A inhibitors for cancer immunotherapy. Allogeneic hematopoietic stem cell transplantation is another approach to treating cancer, in particular hematological malignancies, and relies on donor T cell recognition of minor histocompatibility antigens to target residual cancer cells. However, its application is severely limited due to the risk of graft versus host disease (GvHD). Higher invariant natural killer T (iNKT) cell load following transplantation has been clinically associated with reduced GvHD without loss of the beneficial graft versus leukemia/lymphoma (GvL) effect. However, the accessibly of iNKT cells remains a major hurdle to their research and potential clinical application. In Chapter 4, I provide two protocols for generating large amounts of mouse and human iNKT cells in vivo through TCR engineering of hematopoietic stem cells (HSC-engineered iNKT) and a murine intermediary. In Chapter 5, a protocol to generate human HSC-engineered iNKT cells in vitro is introduced, and the resultant HSC-engineered iNKT are found capable of ameliorating xeno-GvHD without loss of GvL effect when added to the allograft, providing exciting preclinical data highlighting the possibly of using HSC-engineered iNKT cells as additives to the allograft to reduce GvHD risk and improve patient outcomes.

Adoptive cell therapy, especially chimeric antigen receptor (CAR) therapy, has revolutionized cancer immunotherapy, showing remarkable results in the treatment of chronic viral infections and malignancies. Currently, there are six US Food and Drug Administration (FDA) approved adoptive CAR-engineered T (CAR-T) cell products for treating B cell lymphoma (BCL) or multiple myeloma (MM) patients: Yescarta (Kite Pharma, 2017), Kymriah (Novartis, 2017), Tecartus (Kite Pharma, 2020), Breyanzi (Bristol Myers Squibb, 2021), Abecma (Bristol Myers Squibb, 2021), and Carvykti (Legend, 2022).Despite the impressive outcomes, the current FDA-approved cell therapies fall into the autologous category: T cells obtained from the patient via leukapheresis and reinjected back into the same patient. This approach is high-cost, time-consuming, and patient-selective. Some patients may not have enough time waiting for the manufacturing to be completed. Therefore, new approaches that allow rapid production of large-scale and ready-to-use therapeutic cells are in great demand. The work presented in this thesis aims to provide alternative platforms for off- the-shelf cancer immunotherapy. By differentiating genetically engineered stem cells (i.e., ii hematopoietic stem cells and pluripotent stem cells), we developed multiple in vitro culture systems to robustly generate large-scale CAR-armed invariant nature killer T (NKT) cells suitable for allogeneic cell therapy. Chapter 1 introduces the background of iNKT cells, the-state-of-the-arts of stem cell engineering, and the previous study of engineering stem cell into iNKT cells. Chapter 2 reports a successful platform that utilizing artificial thymic organoid (ATO) culture methods to generate large-scale CAR-armed iNKT (CAR-iNKT) cells for cancer immunotherapy. The HSC-engineered iNKT cells closely resembled endogenous iNKT cells and exhibited potent antitumor efficacy and high safety in the xenograft multiple myeloma mouse model. Chapter 3 reports a feeder-free in vitro culture system to differentiate engineered stem cells into CAR-eNKT cells. The novel platform is robust and compatible for delivering different CAR cargos and additional molecules. The independence of murine derived feeder cells eliminates the potential risk of cross-species contamination during manufacturing. Chapter 4 switch the focus from previous chapters and starts to develop pluripotent stem cells (PSCs) derived CAR-iNKT cells for cancer immunotherapy. The unlimited expansion capability, capacity for clonal selection and ease of genetic engineering of PSCs allow them to be an ideal candidate providing unlimited source for off-the-shelf allogeneic cell therapies. Chapter 5 summarizes the thesis, discusses the challenge, and provides future perspectives for developing stem cell and iNKT-based off-the-shelf cancer immunotherapy. It is believed that more fundamental studies will be critical to further develop the next generation immunotherapy.

Cancer immunotherapy has become the new medicine for cancer patients providing curative hopes. The adoptive cell transfer of engineered T cells has demonstrated promising clinical response rates, especially for chimeric antigen receptor (CAR) therapies and T cell receptor (TCR) engineered therapies. This dissertation aims to extend the findings of previous engineered T cell therapies focusing on TCR-engineered T cells. Two TCRs are studied here: NY-ESO-1-specific TCR (esoTCR) and invariant natural killer T (iNKT) cell TCR. Towards the “off-the-shelf” T cell immunotherapy, we demonstrated the efforts in developing the TCR toolbox, the ex vivo allogeneic T cell generation platform, combination engineering methods for next-generation allogeneic T cell product, and nanomaterials for T cell activation and expansion.

NY-ESO-1 attracts wide attention for developing targeted cancer therapies for its broad aberrant expression across tumor types and strong ability to elicit immune responses. Due to the MHC restriction nature of TCR, a toolbox of esoTCRs with various NY-ESO-1 epitope specificity and MHC restriction is in great need to expand the patient pool and prevent tumor evasion through the loss of MHC heterozygosity. Chapter 2 of this dissertation will present the work on isolation and characterization of NY-ESO-1-specific TCRs restricted on various MHCs.

Most current engineered T cell therapies, including CAR and TCR therapies, fall under autologous cell therapy. The autologous approach has demonstrated its feasibility and effectiveness. The personalized nature of autologous therapies has also greatly limited the further extended use of engineered T cells in the clinic. In Chapters 3 and 4, we established a novel ex vivo HSC-based TCR-engineered T cell generation platform for allogeneic “off-the-shelf” T cell therapies for cancer. In two separate works, we demonstrated the use of esoTCR and iNKT TCR in this platform. The combinational uses of CAR, CRISPR-Cas9 gene editing, and other enhancement genes were also explored in these two chapters.

T cell activation and expansion is an essential step required for all T cell-based immunotherapies. The developmental path of T cell activating methods starts from the simple addition of anti-CD3 antibodies to magnetic antibody-conjugated beads that are commercially available nowadays. Striving to mimic the natural cell-cell interaction and immunological synapse relating to T cell activation, in Chapter 5, we present a novel nanomaterial-based method for ultrahigh T cell activation and expansion.

Collectively, the work described here advances the field of T cell therapies by enriching the toolbox of TCRs restricted on various MHC, establishing an ex vivo HSC-based TCR-engineered T cell generation platform which provides new allogeneic T cell sources towards the “off-the-shelf” T cell therapies for cancer patients, and providing a new nanomaterial-based method for ultrahigh T cell activation and expansion.

Adoptive cellular therapy using T cells programmed with chimeric antigen receptor (CAR) to target tumor cells has demonstrated tremendous efficacy in numerous pre-clinical and clinical trials. However, current FDA approved therapies are autologous, T cells collected from a patient will be manufactured and used to treat that single patient, making this type of therapy costly, labor intensive, and difficult to efficiently deliver to all patients in need1. Allogeneic, or “off-the-shelf” CAR-Ts could offer a potential solution to some of these hurdles. In most studies CARs are transduced into conventional αβ T cells, while the potential of introducing CARs into other cell types has yet received relatively little attention.In human, 1-5% of circulating T cells express γδ TCR. γδ TCR-CD3 complex are responsible for antigen stimulation of γδ T cells and the downstream signaling cascade for T cell activation. Unlike αβ TCR, γδ TCR directly recognizes tumor in the non-MHC restricted fashion, which makes γδ T cells a better CAR carrier for allogeneic cellular therapy because they do not cause graft-versus-host-disease (GvHD). In chapter 2, we discussed the unique features of different subsets of human γδ T cells and their antitumor properties. We also summarized the past and the ongoing pre-clinical studies and clinical trials utilizing γδ T cell-based cancer immunotherapy. Human Vγ9Vδ2 T cells are the predominant γδ T cell population in peripheral blood. They bind to butyrophilin-3A1/2A1 (BTN3A1/2A1) complex on various tumors, and can be selectively expanded in vivo and ex vivo by Zoledronate (ZOL), a clinically approved aminobisphosphonate drug for treatment of osteoporosis and cancer. Despite some positive results from clinical trials for cancer treatments using this subset of T cells, many patients’ γδ T cells failed to respond to ZOL and did not show robust cytotoxicity against tumors. The more recent studies have suggested that not all Vγ9Vδ2 T cells are identical, and there is a huge variation between people. In chapter 3 of this dissertation, we present a new screening strategy to select and expand peripheral blood mononuclear cell (PBMC)-derived Vγ9Vδ2 T cells that are more powerful against tumors. In addition, engineering of these γδ T cells with CAR and interleukin 15 (IL-15) transgenes to enhance the efficacy and persistence of the cells is shown in this chapter. The current method of generating Vγ9Vδ2 T cells for adoptive cell therapy involves either in vitro or in vivo expansion of PBMC-derived γδ T cells using aminobisphosphonates, such as ZOL. However, this method generates highly variable yields of γδ T cells depending on PBMC donors; and most importantly, such a γδ T cell product will likely contain conventional αβ T cells and thereby incurring GvHD risk in patients receiving these allogeneic cell products. A novel method that can reliably generate a homogenous monoclonal population of γδ T cells at large quantities with a feeder-free differentiation system is thus pivotal to developing an off-the-shelf γδ T cell therapy. In chapter 4, we demonstrate a novel feeder free-serum free in vitro culture system to generate Vγ9Vδ2 T cells from hematopoietic stem cells (HSCs). This approach provides an alternative strategy to generate γδ T cells, and overcomes the challenges of using PBMC-derived γδ T cells for cancer treatments. In the last chapter, we summarize our works and point out important future directions that may advance the field of γδ T cell‐based cancer immunotherapy.