Development of approaches for immunotherapy by chimeric antigen receptor modified hematopoietic stem cell transfer

Persistent Link:
http://hdl.handle.net/10150/193512
Title:
Development of approaches for immunotherapy by chimeric antigen receptor modified hematopoietic stem cell transfer
Author:
Badowski, Michael Steven
Issue Date:
2009
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
Cancer is an uncontrolled growth of the body's own cells. While cancer rates increase with age, this disease afflicts both young and old. Traditional cancer therapy has had three major facets: 1) chemotherapy, which can non-specifically damage healthy tissue, 2) radiation, which can make some types of cancer more likely in the future, and 3) surgery, which can be physically traumatic and is not effective in removing unseen microtumors or circulating metastases. Immunotherapy, by its very nature, is drastically different. Immunotherapy seeks to employ cells or molecules from the immune system, in their original or a modified form, to augment, assist or replace missing elements of the native functioning immune system. Our immunotherapeutic approach has been to develop novel chimeric antigen receptors (CAR) and deliver the engineered transgene into hematopoietic stem cells (HSC). We have developed a novel single chain TCR (scTCR) in which the TCR V-alpha and V-beta segments are joined by a flexible linker. In addition to our scTCR we developed a single chain antibody molecule (scFv) to increase avidity to the tumor antigen and avoid the potential limitation of MHC restriction. Our lab has previously developed a signaling cassette based on the CD3 zeta chain, CD28 and p56Lck proteins which are prominent in the T-cell signaling pathway. The single chain specificities are linked to the signaling cassette that we have shown to function in T-cells. With specificity and signaling coupled, the chimeric antigen receptor can be transduced into hematopoietic stem cells (HSC) via a lentivirus vector. This adoptive immunotherapy can potentially eliminate malignant cells or supplement traditional therapies by providing engineered specificity and a useful method to transfer and expand tumor specific T-cells. We show in this study that the CAR can be delivered effectively to HSC and that the introduced transgene is expressed in multiple cell lineages. We also have developed a novel method of increasing lentiviral transduction efficiency. Both transduced fraction of cells and overall expression can be increased by proper timing and coordination of cell growth, cell cycle phase, vector addition and treatment with heat shock.
Type:
text; Electronic Dissertation
Keywords:
CAR; chimeric antigen receptor; heat shock; lentivirus; scFv; stem cell
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Microbiology & Immunology; Graduate College
Degree Grantor:
University of Arizona
Advisor:
Harris, David T
Committee Chair:
Harris, David T

Full metadata record

DC FieldValue Language
dc.language.isoENen_US
dc.titleDevelopment of approaches for immunotherapy by chimeric antigen receptor modified hematopoietic stem cell transferen_US
dc.creatorBadowski, Michael Stevenen_US
dc.contributor.authorBadowski, Michael Stevenen_US
dc.date.issued2009en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractCancer is an uncontrolled growth of the body's own cells. While cancer rates increase with age, this disease afflicts both young and old. Traditional cancer therapy has had three major facets: 1) chemotherapy, which can non-specifically damage healthy tissue, 2) radiation, which can make some types of cancer more likely in the future, and 3) surgery, which can be physically traumatic and is not effective in removing unseen microtumors or circulating metastases. Immunotherapy, by its very nature, is drastically different. Immunotherapy seeks to employ cells or molecules from the immune system, in their original or a modified form, to augment, assist or replace missing elements of the native functioning immune system. Our immunotherapeutic approach has been to develop novel chimeric antigen receptors (CAR) and deliver the engineered transgene into hematopoietic stem cells (HSC). We have developed a novel single chain TCR (scTCR) in which the TCR V-alpha and V-beta segments are joined by a flexible linker. In addition to our scTCR we developed a single chain antibody molecule (scFv) to increase avidity to the tumor antigen and avoid the potential limitation of MHC restriction. Our lab has previously developed a signaling cassette based on the CD3 zeta chain, CD28 and p56Lck proteins which are prominent in the T-cell signaling pathway. The single chain specificities are linked to the signaling cassette that we have shown to function in T-cells. With specificity and signaling coupled, the chimeric antigen receptor can be transduced into hematopoietic stem cells (HSC) via a lentivirus vector. This adoptive immunotherapy can potentially eliminate malignant cells or supplement traditional therapies by providing engineered specificity and a useful method to transfer and expand tumor specific T-cells. We show in this study that the CAR can be delivered effectively to HSC and that the introduced transgene is expressed in multiple cell lineages. We also have developed a novel method of increasing lentiviral transduction efficiency. Both transduced fraction of cells and overall expression can be increased by proper timing and coordination of cell growth, cell cycle phase, vector addition and treatment with heat shock.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
dc.subjectCARen_US
dc.subjectchimeric antigen receptoren_US
dc.subjectheat shocken_US
dc.subjectlentivirusen_US
dc.subjectscFven_US
dc.subjectstem cellen_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineMicrobiology & Immunologyen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorHarris, David Ten_US
dc.contributor.chairHarris, David Ten_US
dc.contributor.committeememberAblin, Richarden_US
dc.contributor.committeememberSchluter, Samuel Fen_US
dc.contributor.committeememberBernstein, Harrisen_US
dc.identifier.proquest10817en_US
dc.identifier.oclc659753674en_US
All Items in UA Campus Repository are protected by copyright, with all rights reserved, unless otherwise indicated.