The Combined Treatment Efficacy of Anti-CD123 CAR T cells with Azacitidine for the Treatment of Acute Myeloid Leukaemia

“Joint Degree Program between Adelaide Medical School, The University of Adelaide and Medizinischen Universitätsklinik, der Albert-Ludwigs-Universität Freiburg Im Breisgau Freiburg Im Breisgau, Deutschland”Chimeric antigen receptor (CAR) T cells have yielded impressive remission rates in treatment-refractory B cell malignancies (B-ALL and B-lymphomas) by targeting CD19, resulting in CAR T cell therapies entering into clinical practice. However, the utility of CAR T cells for acute myeloid leukaemia (AML) remains a challenge. CAR T cells against AML-associated antigens are typically hampered by cytotoxic effects against normal haematopoietic progenitor cells and by CAR T cell exhaustion. Current clinical trials using CAR T cells that target various antigens in AML have resulted in either transient leukaemia cell clearance, or complete clearance of leukaemia at the expense of severe on-target off-tumour toxicities. In this thesis, third-generation anti-CD123 CAR T cells were developed with a humanised binding moiety for CD123 incorporating two intracellular signalling domains. The CAR developed in this project demonstrated strong anti- AML activity without elimination of the healthy haematopoietic system or epithelial tissue damage in mouse xenograft models. However, a sustained and long-term tumour eradication was not observed in the mice. In the clinical setting, this would mean that patients have suboptimal responses to the CAR T cells and may relapse. The AML microenvironment is immunosuppressive by employing a variety of mechanisms to escape the host immune surveillance, which may hamper the efficacy of CAR T cell therapy. To improve the long-term efficacy of the CAR T cells, combination therapy with DNA methyltransferase inhibitors, such as azacitidine (AZA), was explored. AZA has previously been shown to upregulate the expression of leukaemia-associated antigens on AML thereby inducing more effective T cell responses. AZA was therefore combined with CD123 CAR T cells and evaluated in AML xenograft models. Priming of AML cells with AZA increased the expression of the target antigen, CD123, on the cell surface. CD123 CAR T cells were more effective at eliminating AML cells in vivo and induced long term eradication. Interestingly, the combined treatment strategy induced a CTLA-4negative CD123 CAR T cell population. Functionally, these CTLA-4negative CD123 CAR T cells exhibited superior cytotoxicity against AML cells with sustained tumour necrosis factor (TNF) production and higher proliferative capacity compared to CTLA-4positive CD123 CAR T cells. Furthermore, AML xenograft mice treated with CTLA-4negative CD123 CAR T cells survived longer than CTLA-4positive CD123 CAR T cell treated mice, and demonstrated recall immunity in secondary AML xenograft recipients. Mechanistically, when AML cells were primed with AZA, the CAR T cells demonstrated increased intracellular retention of CTLA-4 and reduced extracellular expression upon exposure to the AML cells. The decreased expression of extracellular CTLA-4 was associated with decreased numbers of regulatory CAR T cells. Normally, high extracellular CTLA-4 expression prevents the phosphorylation of Lck and Zap70; intracellular molecules required for effective T cell induction and function. In this case, a higher phosphorylation level of these molecules was observed in the CAR T cells exposed to AML cells previously primed with AZA compared to without priming. The findings in this thesis project indicate that AZA increases the target antigen, CD123, on AML cells, allowing enhanced recognition and elimination by cytotoxic CTLA-4negative CD123 CAR T cells. These novel findings pave the way for a clinical trial combining AZA and CD123 CAR T cells for AML treatment.Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 202

mTORC1 plays an important role in osteoblastic regulation of B-lymphopoiesis

Skeletal osteoblasts are important regulators of B-lymphopoiesis, serving as a rich source of factors such as CXCL12 and IL-7 which are crucial for B-cell development. Recent studies from our laboratory and others have shown that deletion of Rptor, a unique component of the mTORC1 nutrient-sensing complex, early in the osteoblast lineage development results in defective bone development in mice. In this study, we now demonstrate that mTORC1 signalling in pre-osteoblasts is required for normal B-lymphocyte development in mice. Targeted deletion of Rptor in osterix-expressing pre-osteoblasts (Rptor; ob; -/-; ) leads to a significant reduction in the number of B-cells in the bone marrow, peripheral blood and spleen at 4 and 12 weeks of age. Rptor; ob; -/-; mice also exhibit a significant reduction in pre-B and immature B-cells in the BM, indicative of a block in B-cell development from the pro-B to pre-B cell stage. Circulating levels of IL-7 and CXCL12 are also significantly reduced in Rptor; ob; -/-; mice. Importantly, whilst Rptor-deficient osteoblasts are unable to support HSC differentiation to B-cells in co-culture, this can be rescued by the addition of exogenous IL-7 and CXCL12. Collectively, these findings demonstrate that mTORC1 plays an important role in extrinsic osteoblastic regulation of B-cell development

mTORC1 plays an important role in osteoblastic regulation of B-lymphopoiesis

Skeletal osteoblasts are important regulators of B-lymphopoiesis, serving as a rich source of factors such as CXCL12 and IL-7 which are crucial for B-cell development. Recent studies from our laboratory and others have shown that deletion of Rptor, a unique component of the mTORC1 nutrient-sensing complex, early in the osteoblast lineage development results in defective bone development in mice. In this study, we now demonstrate that mTORC1 signalling in pre-osteoblasts is required for normal B-lymphocyte development in mice. Targeted deletion of Rptor in osterix-expressing pre-osteoblasts (Rptor; ob; -/-; ) leads to a significant reduction in the number of B-cells in the bone marrow, peripheral blood and spleen at 4 and 12 weeks of age. Rptor; ob; -/-; mice also exhibit a significant reduction in pre-B and immature B-cells in the BM, indicative of a block in B-cell development from the pro-B to pre-B cell stage. Circulating levels of IL-7 and CXCL12 are also significantly reduced in Rptor; ob; -/-; mice. Importantly, whilst Rptor-deficient osteoblasts are unable to support HSC differentiation to B-cells in co-culture, this can be rescued by the addition of exogenous IL-7 and CXCL12. Collectively, these findings demonstrate that mTORC1 plays an important role in extrinsic osteoblastic regulation of B-cell development

ROCK1/2 signaling contributes to corticosteroid-refractory acute graft-versus-host disease

Patients with corticosteroid-refractory acute graft-versus-host disease (aGVHD) have a low one-year survival rate. Identification and validation of novel targetable kinases in patients who experience corticosteroid-refractory-aGVHD may help improve outcomes. Kinase-specific proteomics of leukocytes from patients with corticosteroid-refractory-GVHD identified rho kinase type 1 (ROCK1) as the most significantly upregulated kinase. ROCK1/2 inhibition improved survival and histological GVHD severity in mice and was synergistic with JAK1/2 inhibition, without compromising graft-versus-leukemia-effects. ROCK1/2-inhibition in macrophages or dendritic cells prior to transfer reduced GVHD severity. Mechanistically, ROCK1/2 inhibition or ROCK1 knockdown interfered with CD80, CD86, MHC-II expression and IL-6, IL-1β, iNOS and TNF production in myeloid cells. This was accompanied by impaired T cell activation by dendritic cells and inhibition of cytoskeletal rearrangements, thereby reducing macrophage and DC migration. NF-κB signaling was reduced in myeloid cells following ROCK1/2 inhibition. In conclusion, ROCK1/2 inhibition interferes with immune activation at multiple levels and reduces acute GVHD while maintaining GVL-effects, including in corticosteroid-refractory settings

Oncolytic virotherapy with chimeric VSV-NDV synergistically supports RIG-I-dependent checkpoint inhibitor immunotherapy

Unraveling the complexities of the tumor microenvironment (TME) and its correlation with responsiveness to immunotherapy has become a main focus in overcoming resistance to such treatments. Targeting tumor-intrinsic retinoic acid-inducible gene-I (RIG-I), a sensor for viral RNA, was shown to transform the TME from an immunogenically “cold” state to an inflamed, “hot” lesion, which we demonstrated previously to be a crucial mediator of the efficacy of immune checkpoint inhibition with anti-cytotoxic T lymphocyte-associated protein 4 (CTLA-4). In this study, we focus on the chimeric oncolytic virus vesicular stomatitis virus (VSV)-Newcastle disease virus (NDV), comprised of genetic components of VSV and NDV, and we investigate its utility to support tumor-intrinsic RIG-I-dependent therapy with anti-CTLA-4. Overall, we demonstrate that treatment with VSV-NDV efficiently delays tumor growth and significantly prolongs survival in a murine model of malignant melanoma, which was further enhanced in combination with anti-CTLA-4. Although the direct oncolytic and pro-inflammatory effects of VSV-NDV therapy were independent of RIG-I activation, the synergism with anti-CTLA-4 therapy and associated activation of tumor-specific T cells was critically dependent on active RIG-I signaling in tumor cells. This work highlights the therapeutic value of utilizing an immune-stimulatory oncolytic virus to sensitize tumors to immune checkpoint inhibition

Demethylating therapy increases anti-CD123 CAR T cell cytotoxicity against acute myeloid leukemia

Successful treatment of acute myeloid leukemia (AML) with chimeric antigen receptor (CAR) T cells is hampered by toxicity on normal hematopoietic progenitor cells and low CAR T cell persistence. Here, we develop third-generation anti-CD123 CAR T cells with a humanized CSL362-based ScFv and a CD28-OX40-CD3ζ intracellular signaling domain. This CAR demonstrates anti-AML activity without affecting the healthy hematopoietic system, or causing epithelial tissue damage in a xenograft model. CD123 expression on leukemia cells increases upon 5′-Azacitidine (AZA) treatment. AZA treatment of leukemia-bearing mice causes an increase in CTLA-4negative anti-CD123 CAR T cell numbers following infusion. Functionally, the CTLA-4negative anti-CD123 CAR T cells exhibit superior cytotoxicity against AML cells, accompanied by higher TNFα production and enhanced downstream phosphorylation of key T cell activation molecules. Our findings indicate that AZA increases the immunogenicity of AML cells, enhancing recognition and elimination of malignant cells by highly efficient CTLA-4negative anti-CD123 CAR T cells