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Establishing canine osteosarcoma as a solid tumor model for the evaluation of B7-H3 CAR T cell therapy

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dc.contributor.authorCao, Jennifer, author
dc.contributor.authorDow, Steven, advisor
dc.contributor.authorAvery, Anne, committee member
dc.contributor.authorSchenkel, Alan, committee member
dc.contributor.authorThamm, Douglas, committee member
dc.date.accessioned2023-08-28T10:29:06Z
dc.date.available2024-08-28T10:27:54Z
dc.date.issued2023
dc.description.abstractOsteosarcoma (OS) is a highly aggressive primary bone cancer that mainly affects children and young adults. OS is the third most common childhood cancer, after lymphoma and brain tumors. Major advances in the 1980's in neoadjuvant chemotherapy has increased 5-year survival rates in OS from 30% to 70%. Unfortunately, for patients that do not respond to standard therapy or that have metastatic disease the 5-year survival rate is still 20% with no major improvements in the last 4 decades. Approximately 15-20% of patients have metastatic lesions at the time of diagnosis and 25-30% of all OS patients will develop metastatic disease. For this subset of patients advances in treatment options are desperately needed. OS also occurs in high rates in large breed dogs with an estimated 10,000 cases in dogs per year in the United States compared to 1,000 cases per year in humans. The dog has been a well-established translational model for OS due to the similar clinical presentation, cell origin, histological features, and disease progression between canine and human OS. Development of chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment for advanced and relapsed B-cell lymphomas and leukemias. CAR T cell targeting of B cell marker CD19 has shown up to 90% complete remission in patients with advanced B cell leukemia. However, efforts to expand CAR T cell therapy to solid tumor types have not seen the same clinical success as with blood cancers. Major barriers unique to solid tumor CAR T cell therapy are A) selection of tumor associated antigen target, B) CAR T cell trafficking to tumor sites from the circulation and C) immune suppressor cells within the tumor microenvironment (TME). To develop more effective CAR T cell therapies against solid tumor, we utilized canine OS as a translational animal model. To establish canine osteosarcoma as a platform for evaluating B7-H3 CAR T cell therapy, first we validated B7-H3 as an antigen target in canine OS. We found differential expression of B7-H3 with high levels of B7-H3 expression on OS cell lines and FFPE biopsies, whereas normal canine tissues were B7-H3 negative or low. Next, we optimized generation of canine B7-H3 CAR T cells from whole blood isolated from tumor bearing dogs to maximize both T cell expansion and CAR transduction efficiency. We also found that the addition of cytokines IL-7 and IL-15 minimize CAR T cell exhaustion due to ex vivo activation and expansion. We next determined that canine B7-H3 CAR T cells exerted antigen specific killing and cytokine activity against B7-H3+ canine OS cell lines. To address issues with CAR T cell trafficking we evaluated the addition of chemokine receptor CXCR2 to B7-H3 CAR T cells. To assess the utility of the B7-H3-CXCR2 CAR we determined that canine OS cell lines secreted high levels of ligand chemokine CXCL8 at baseline. To further evaluate functionality, we evaluated the two CAR constructs in a mouse xenograft model of canine OS. We found that the B7-H3-CXCR2 CAR construct had significantly greater anti-tumoral activity than the single B7-H3 CAR construct in inhibiting tumor growth and achieving complete tumor elimination. Studies were also designed to determine if modifying the TME with combination drugs losartan and propranolol improved CAR T cell activity. This is based on recent successful studies with losartan and propranolol in dogs with OS and glioma. We found that combination losartan and propranolol decreased the population of mouse CD11b+Ly6Chigh tumor associated macrophages (TAMs) to xenografted canine OS tumors. In vitro assays showed that immune suppressive macrophages enhance B7-H3-CXCR2 CAR T cell function when co-cultured, likely through CAR activation by macrophage B7-H3 expression. Collectively, the results from these studies pave the way for assessing B7-H3-CXCR2 CAR T cells in dogs with metastatic OS. Clinical outcomes in spontaneous OS in dogs will likely give more clinically relevant results serving as a platform for evaluating new CAR T cell therapies and combination therapies with TME modification, radiation, or checkpoint blockade. Success of this work can provide a new adoptive cell immunotherapy treatment option to patients both canine and human with metastatic osteosarcoma. Additionally, B7-H3-CXCR2 CAR T cells can be applied to other B7-H3 positive CXCL8 secreting tumor types.
dc.format.mediumborn digital
dc.format.mediumdoctoral dissertations
dc.identifierCao_colostate_0053A_17959.pdf
dc.identifier.urihttps://hdl.handle.net/10217/236962
dc.languageEnglish
dc.language.isoeng
dc.publisherColorado State University. Libraries
dc.relation.ispartof2020-
dc.rightsCopyright and other restrictions may apply. User is responsible for compliance with all applicable laws. For information about copyright law, please see https://libguides.colostate.edu/copyright.
dc.rights.accessEmbargo expires: 08/28/2024.
dc.subjectCAR T cell
dc.subjectimmunotherapy
dc.subjecttranslational
dc.subjectchimeric antigen receptor
dc.subjectcanine
dc.subjectosteosarcoma
dc.titleEstablishing canine osteosarcoma as a solid tumor model for the evaluation of B7-H3 CAR T cell therapy
dc.typeText
dcterms.embargo.expires2024-08-28
dcterms.embargo.terms2024-08-28
dcterms.rights.dplaThis Item is protected by copyright and/or related rights (https://rightsstatements.org/vocab/InC/1.0/). You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s).
thesis.degree.disciplineMicrobiology, Immunology, and Pathology
thesis.degree.grantorColorado State University
thesis.degree.levelDoctoral
thesis.degree.nameDoctor of Philosophy (Ph.D.)

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