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CD19-targeting CAR T Cells for Treatment of B Cell Malignancies: From Bench to Bedside
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical Immunology.
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Immunotherapy for cancer is a young research field progressing at high speed. The first chimera of an antibody and a signaling chain was designed by Zelig Eshhar and was later further developed to enhance existing T cell therapy by combining a single-chain fragment of an antibody with the CD3 zeta chain of the TCR complex. T cells expressing these chimeric antigen receptors (CARs) could recognize and specifically kill tumor cells. However the T cells, lacked in persistence and tumor rejection did not occur. Thus, the CAR constructs have been improved by providing the T cell with costimulatory signals promoting activation. The focus of this thesis has been to evaluate second and third generation αCD19-CAR T cells for the treatment of B cell leukemia and lymphoma.

B cell tumors commonly upregulate anti-apoptotic proteins such as Bcl-2, which generates therapy resistance. In the first paper a second generation (2G) αCD19-CD28-CAR T cell was combined with the Bcl-2 family inhibitor ABT-737. ABT-737 sensitized tumor cells to CAR T cell therapy and may be an interesting clinical combination treatment. In paper II, the phenotype and function of a third generation (3G) αCD19-CD28-4-1BB-CAR T cell were evaluated. B cell-stimulated CAR T cells showed increased proliferation and an antigen-driven accumulation of CAR+ T cells. 3G CAR T cells had equal cytotoxic capacity, similar lineage, memory and exhaustion profile phenotype compared to 2G CARs. However, 3G CAR T cells proliferated better and had increased activation of intracellular signaling pathways compared to 2G CAR T cells. In paper III, αCD19-CD28-4-1BB-CAR T cells were used to stimulate immature dendritic cells leading to an upregulation of maturation markers on co-cultured dendritic cells. Hence, CAR T cells may not only directly kill the tumor cells, but may induce bystander immunity that indirectly aids tumor control. This thesis also include supplementary information about the development and implementation of protocols for GMP production of CAR T cell batches for a phase I/IIa clinical trial currently ongoing for patients with refractory B cell leukemia and lymphoma. So far, two patients have safely been treated on the lowest dose.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. , 72 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1031
Keyword [en]
chimeric antigen receptors, CAR T cells, T cell therapy, immunotherapy, CD19, Bcl-2 family inhibitors, B cell malignancies
National Category
Immunology
Research subject
Clinical Immunology
Identifiers
URN: urn:nbn:se:uu:diva-232638ISBN: 978-91-554-9047-8 (print)OAI: oai:DiVA.org:uu-232638DiVA: diva2:749422
Public defence
2014-11-21, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2014-10-27 Created: 2014-09-22 Last updated: 2015-01-23
List of papers
1. Combining CAR T cells and the Bcl-2 family apoptosis inhibitor ABT-737 for treating B-cell malignancy
Open this publication in new window or tab >>Combining CAR T cells and the Bcl-2 family apoptosis inhibitor ABT-737 for treating B-cell malignancy
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2013 (English)In: Cancer Gene Therapy, ISSN 0929-1903, E-ISSN 1476-5500, Vol. 20, no 7, 386-393 p.Article in journal (Refereed) Published
Abstract [en]

B-cell malignancies upregulate the B-cell lymphoma 2 (Bcl-2) family inhibitors of the intrinsic apoptosis pathway, making them therapy resistant. However, small-molecule inhibitors of Bcl-2 family members such as ABT-737 restore a functional apoptosis pathway in cancer cells, and its oral analog ABT-263 (Navitoclax) has entered clinical trials. Gene engineered chimeric antigen receptor (CAR) T cells also show promise in B-cell malignancy, and as they induce apoptosis via the extrinsic pathway, we hypothesized that small-molecule inhibitors of the Bcl-2 family may potentiate the efficacy of CAR T cells by engaging both apoptosis pathways. CAR T cells targeting CD19 were generated from healthy donors as well as from pre-B-ALL (precursor-B acute lymphoblastic leukemia) patients and tested together with ABT-737 to evaluate apoptosis induction in five B-cell tumor cell lines. The CAR T cells were effective even if the cell lines exhibited different apoptosis resistance profiles, as shown by analyzing the expression of apoptosis inhibitors by PCR and western blot. When combining T-cell and ABT-737 therapy simultaneously, or with ABT-737 as a presensitizer, tumor cell apoptosis was significantly increased. In conclusion, the apoptosis inducer ABT-737 enhanced the efficacy of CAR T cells and could be an interesting drug candidate to potentiate T-cell therapy.

Keyword
ABT-737, BH3 mimetics, engineered T cells, CAR, Bcl-2, pre-B-ALL
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-204980 (URN)10.1038/cgt.2013.35 (DOI)000321951700002 ()
Available from: 2013-08-16 Created: 2013-08-13 Last updated: 2017-12-06Bibliographically approved
2. CARs expressing CD28 and 4-1BB demonstrate increased intracellular signaling activity and proliferative capacity while maintaining a memory phenotype upon repeated antigen stimulation
Open this publication in new window or tab >>CARs expressing CD28 and 4-1BB demonstrate increased intracellular signaling activity and proliferative capacity while maintaining a memory phenotype upon repeated antigen stimulation
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(English)Manuscript (preprint) (Other academic)
National Category
Immunology
Identifiers
urn:nbn:se:uu:diva-232643 (URN)
Available from: 2014-09-22 Created: 2014-09-22 Last updated: 2015-01-23
3. CAR T Cells Induce DC Maturation via a Cell Contact-dependent Process and Enhance Their Ability to Stimulate T Cell Responses
Open this publication in new window or tab >>CAR T Cells Induce DC Maturation via a Cell Contact-dependent Process and Enhance Their Ability to Stimulate T Cell Responses
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(English)Manuscript (preprint) (Other academic)
National Category
Immunology
Identifiers
urn:nbn:se:uu:diva-232644 (URN)
Available from: 2014-09-22 Created: 2014-09-22 Last updated: 2015-01-23
4. Third Generation CD28/4-1BB CAR T Cells for Refractory CD19+ B Cell Malignancy: Generation of a GMP Protocol an Evaluation of Clinical Grade Batches
Open this publication in new window or tab >>Third Generation CD28/4-1BB CAR T Cells for Refractory CD19+ B Cell Malignancy: Generation of a GMP Protocol an Evaluation of Clinical Grade Batches
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(English)Manuscript (preprint) (Other academic)
Abstract
National Category
Immunology
Identifiers
urn:nbn:se:uu:diva-232646 (URN)
Available from: 2014-09-22 Created: 2014-09-22 Last updated: 2017-03-24

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