Engineering VHH-based Chimeric Antigen Receptor (CAR) T Cell Therapy for Solid Tumor Treatment
Engineering VHH-based Chimeric Antigen Receptor (CAR) T Cell Therapy for Solid Tumor Treatment

Author: Yushu Joy Xie

Publisher:

Published: 2019

Total Pages: 154

ISBN-13:

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Chimeric antigen receptor (CAR) T cells are a promising cancer therapeutic, as they can specifically redirect the cytotoxic function of a T cell to a chosen target of interest. CAR T cells have been successful in clinical trials against hematological cancers, but have experienced low efficacy against solid tumors for a number of reasons, including a paucity of tumor-specific antigens to target and a highly immunosuppressive solid tumor microenvironment. In chapter 2 of this thesis, we develop a strategy to target multiple solid tumor types through markers in their microenvironment. The use of single domain antibody (VHH)-based CAR T cells that recognize these markers circumvents the need for tumor-specific targets. Chapter 3 will describe methods to overcome the immunosuppressive microenvironment of solid tumors. Here, we have developed VHH-secreting CAR T cells that can modulate additional aspects of the tumor microenvironment, including the engagement of the innate immune system through secretion of a VHH against an inhibitor of phagocytosis. We show that this strategy of VHH-secretion by CAR T cells can lead to significant benefits in outcome. We also demonstrate that delivery of therapeutics by CAR T cells can improve the safety profile of the therapeutic. Chapter 4 of this thesis explores strategies to increase the targeting capacity of CAR T cells by building logic-gated CARs. Finally, chapter 5 will describe work in imaging CAR T cells specifically, longitudinally, and non-invasively through PET imaging. Our results demonstrate the flexibility of VHHs in CAR T cell engineering and the potential of VHH-based CAR T cells to target the tumor microenvironment, modulate the tumor microenvironment, and treat solid tumors.

Engineering Chimeric Antigen Receptors to Overcome the Immunosuppressive Solid Tumor Microenvironment
Engineering Chimeric Antigen Receptors to Overcome the Immunosuppressive Solid Tumor Microenvironment

Author: Andrew J Hou

Publisher:

Published: 2022

Total Pages: 118

ISBN-13:

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Adoptive T-cell therapy is a cancer treatment strategy where T cells from a cancer patient are harvested, modified ex vivo to target tumor cells, and subsequently reinfused back into the patient's body. Although remarkably successful against blood-based B-cell malignancies, efficacy has been limited against solid tumors, in large part due to the immunosuppressive tumor microenvironment (TME). Among the many inhibitory factors in the TME, transforming growth factor-beta (TGF-[Beta]) plays a prominent role in suppressing anti-tumor immunity through both direct inhibition of T-cell cytotoxicity, as well as recruitment and polarization of immunosuppressive cell types such as myeloid-derived suppressor cells and regulatory T cells. We therefore hypothesized that T-cell function in the solid TME could be potentiated by pairing tumor-targeting CARs with TGF-[Beta] CARs that program T-cell activation, rather than inhibition, in the presence of TGF-[Beta]. Wefirst verified that TGF-[Beta] CAR expression is neither counterproductive to cytotoxic T-cell function, nor does it pose a significant risk of toxicity. Pairing TGF-[Beta] CARs with tumor-specific TCRs or CARs did not significantly enhance therapeutic outcomes of adoptive T-cell transfer in preclinical models of melanoma and prostate cancer, warranting further engineering efforts. In models of glioblastoma, however, single-chain bispecific CAR-T cells targeting TGF-[Beta] and tumor antigen were not only more resistant to tumor-mediated dysfunction, but also remodeled the immune-cell composition of the tumor microenvironment to potentiate anti-tumor immunity.

Engineering Bispecific Chimeric Antigen Receptors to Improve the Efficacy of Adoptive T-cell Therapy
Engineering Bispecific Chimeric Antigen Receptors to Improve the Efficacy of Adoptive T-cell Therapy

Author: Eugenia Zah

Publisher:

Published: 2018

Total Pages: 136

ISBN-13:

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The recent FDA approval of CD19 chimeric antigen receptor (CAR) adoptive T-cell therapy for B-cell leukemias serves to highlight CAR-T cell therapy as a promising treatment approach for refractory cancers. More recently, adoptive transfer of T cells expressing CARs targeting B-cell maturation antigen (BCMA) has had numerous successes in clinical trial with 80-100% of multiple myeloma patients responding to treatment. However, CAR-T-cell therapy still faces several limitations including tumor antigen escape, a circumstance where tumor cells downregulate their surface antigen to avoid detection by CAR-T cells, and T-cell inhibition by cytokines such as transforming growth factor (TGF)- in the solid tumor environment. These factors can significantly limit the efficacy of CAR-T-cell therapy. To overcome antigen escape, we designed single-chain bispecific CARs (OR-gate CARs) capable of signaling in the presence of two antigens instead of one. Using rational design principles, we constructed and evaluated CD19-OR-CD20 CARs that are able to prevent tumor antigen escape by CD19- leukemia. We further demonstrate that unlike single-input CD19 CARs, CD19-OR-CD20 CARs also prevent the emergence of spontaneous CD19-downregulated tumors in vivo. In a second study, we describe the rapid design and characterization of BCMA-OR-CS1 CARs and demonstrate that BCMA-OR-CS1 CARs can be rationally engineered to prevent antigen escape by BCMA- as well as CS1- myeloma cells. Finally, we explore the utility of the TGF- CAR, a receptor capable of rewiring inhibitory TGF- signaling to an activating response, in improving CD20 CAR function in TGF- -rich environments. We evaluated three different bispecific targeting strategies, OR-gate CAR, DualCAR (co-expressing two receptors in one cells), and CARpool (pooling two different CAR-T cells), and demonstrate that TGF- CAR-T cells are able to shield neighboring CD20 CAR-T cells from the inhibitory effects of TGF- .

Chimeric Antigen Receptor T-Cell Therapies for Cancer E-Book
Chimeric Antigen Receptor T-Cell Therapies for Cancer E-Book

Author: Daniel W. Lee

Publisher: Elsevier Health Sciences

Published: 2019-11-30

Total Pages: 246

ISBN-13: 0323755976

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From patient referral to post-therapy management, Chimeric Antigen Receptor (CAR) T-Cell Therapies for Cancer: A Practical Guide presents a comprehensive view of CAR modified T-cells in a concise and practical format. Providing authoritative guidance on the implementation and management of CAR T-cell therapy from Drs. Daniel W. Lee and Nirali N. Shah, this clinical resource keeps you up to date on the latest developments in this rapidly evolving area. Covers all clinical aspects, including patient referral, toxicities management, comorbidities, bridging therapy, post-CAR monitoring, and multidisciplinary approaches to supportive care. Includes key topics on associated toxicities such as predictive biomarkers, infections, and multidisciplinary approaches to supportive care. Presents current knowledge on FDA approved CAR T-cell products as well as developments on the horizon. Editors and authors represent leading investigators in academia and worldwide pioneers of CAR therapy.

Molecular and Cellular Engineering to Guide CAR T Cell Therapy Through the Immunosuppressive Tumor Microenvironment
Molecular and Cellular Engineering to Guide CAR T Cell Therapy Through the Immunosuppressive Tumor Microenvironment

Author: Chi-Wei Man

Publisher:

Published: 2022

Total Pages: 0

ISBN-13:

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Chimeric Antigen Receptor (CAR) T cell therapy is a revolutionary treatment option for cancer therapy, demonstrating widespread clinical success in treating hematological malignancies such as acute lymphoblastic leukemia and certain lymphomas. Despite its widespread success treating hematological malignancies, CAR T cells still struggle to treat solid tumors. One reason for this is the immunosuppressive tumor microenvironment. Expressed in certain tumors, Programmed Death-Ligand 1 (PD-L1) actively suppresses T cell activation and function. To both neutralize this immunoinhibitory effect and eliminate tumor cells, I used yeast display mediated directed evolution to engineer PDbody, derived from the monobody scaffold, to bind to PD-L1. I then employed PDbody as a SynNotch-gated CAR receptor to eliminate a triple-negative breast cancer model in vitro and slow tumor growth in vivo. CAR T cell therapy can also fail when tumors do not homogenously express the CAR target antigen. To combat this problem, I developed heat-inducible Cis-activated CAR (CisCAR) to allow CAR T cells to self-present their target antigen. I then used CisCAR to eliminate antigen-negative leukemic and breast cancer cells in vitro, demonstrating the universal applicability of this treatment strategy. Overall, this dissertation presents new methods that enhance CAR T cell therapy, enabling them to more effectively target a wider range of diseases.

CAR-T Cell Therapies for Non-Hematopoietic Malignancies: Taking Off The Training Wheels
CAR-T Cell Therapies for Non-Hematopoietic Malignancies: Taking Off The Training Wheels

Author: Avery Dexter Posey, Jr.

Publisher: Frontiers Media SA

Published: 2020-04-24

Total Pages: 164

ISBN-13: 2889636879

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Chimeric antigen receptor (CAR) T cell therapies for leukemia (e.g. tisagenlecleucel) and lymphoma (e.g. axicabtagene ciloleucel) have recently received regulatory approval in the United States. Phase I/II trials have demonstrated complete remission of refractory or relapsed tumors in 50% - 94% patients. However, the clinical successes of engineered T cells for the treatment of solid malignancies have thus far been few and far between. Furthermore, several instances of severe and lethal toxicities have arisen due to on-target, off-tumor recognition of antigen by T cell products. Recent advances in phase I trials for solid tumors, as well as in pre-clinical models, have revealed several variables that will be important to consider for the successful use of CAR-T cells in treating solid tumors. These variables include (i) regional versus systemic delivery; (ii) scFv versus ligand interactions; (iii) antigen loss versus escape; (iv) epitope spreading and (v) checkpoint expression on immune cells or tumor cells. Also, there remains outstanding mechanistic questions related to why differences exist in the persistence and tonic signaling of second-generation CD28 versus 4-1BB co-stimulated CAR-T cells. In addition, we are now learning the roles of lympho-depleting regimens (and associated toxicities) in modifying the persistence of engineered T cell therapies. A more comprehensive view of CAR-T cell strategies and important advances, both of pre-clinical and clinical evaluations, in solid tumors is necessary to drive these therapies forward.

Genome Engineering to Expand Applications of Human T-cell Immunotherapy
Genome Engineering to Expand Applications of Human T-cell Immunotherapy

Author: Alexandra E. Grier

Publisher:

Published: 2017

Total Pages: 102

ISBN-13:

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Adoptive T-cell therapy, particularly chimeric antigen receptor (CAR) therapy, is a revolutionary and quickly-evolving means of treating cancer patients who can no longer be helped by standard therapies. In multiple clinical trials, including our own at Seattle Children’s Hospital, CD19 CAR therapy for B-cell leukemia and lymphoma has achieved a complete remission rate of >90%. Unfortunately, in its present form, CAR therapy has had limited success against solid tumors. It is also not currently an option for patients who lack sufficient numbers of their own T-cells due to their disease or prior treatments. Thus, genome engineering strategies to overcome these limitations could be of great benefit to patients. We chose a two-pronged approach to achieve this goal: knock-out of the endogenous TCR and multiplex knock-out of the T-cell inhibitory checkpoints PD-1, Tim3, Lag3, and TIGIT. Knocking out these inhibitory checkpoint proteins specifically in the CAR T-cells will maintain the synergistic effects recently seen in combination monoclonal antibody therapy without the serious, sometimes fatal, immune-mediated side effects seen with systemic antibody therapy. To this end, we first developed a linear mRNA expression vector with a long, encoded poly(A) tail to allow transient delivery of nucleases such as TALENs or CRISPR to primary human cells in a consistent, clinically applicable, and scalable fashion. We then used IVT mRNA made from this vector to deliver a TALEN pair targeting the TCR locus to CD19 CAR T-cells, and demonstrated that removal of the endogenous TCR does not hinder CAR T-cell function in vitro or in vivo in a murine xenograft tumor model. Knockout of the endogenous TCR will facilitate production of an allogeneic CAR T-cell product to be used as a bridge to HSCT in patients who cannot receive autologous CAR therapy. Removal of the endogenous TCR will also add a measure of safety when creating CAR T-cells lacking inhibitory checkpoint proteins by preventing GvHD while retaining anti-tumor effects. These technologies and methods may allow a wider variety of patients to benefit from the recent advances in CAR T-cell therapy.