Engineering chimeric antigen receptor - modified T cells to target the tissue resident HIV reservoir
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Type
ThesisThesis type
Masters by ResearchAuthor/s
York, JarrodAbstract
A landmark study employing in vitro models of interactions between HIV infection of DCs, macrophages and memory T cells cocultured with pDCs, demonstrates that the production of IFNα by pDCs significantly increased HIV infection of resting memory T cells by inducing reactivation ...
See moreA landmark study employing in vitro models of interactions between HIV infection of DCs, macrophages and memory T cells cocultured with pDCs, demonstrates that the production of IFNα by pDCs significantly increased HIV infection of resting memory T cells by inducing reactivation without viral spread (Tong et al. 2021). Reactivated latently infected memory T cells (p24+) expressed gp120 env antigen on the cell membrane after pDC interaction, in addition to high levels of the early activation and tissue resident marker, CD69. Although HIV reactivation can be achieved by IFNα (Tong et al. 2021), the conventional cytotoxic T lymphocyte response to the HIV reservoir is limited by several mechanisms including mutational and non-mutational escape by sequestration of the virus in sites where access by CD8 T cells is limited; HIV-specific CD8 T cell exhaustion and apoptosis; and HIV nef downregulation of MHC-I antigen presentation thus preventing viral clearance. Therefore, the aim of this project was to explore an approach that may potentially enhance an immunotherapy strategy targeting the latent tissue reservoirs with anti-HIV CAR T cells following IFNα treatment and latent HIV reactivation. This is a novel “kick and kill” strategy which can be translated into clinical adoptive cell therapies. The CAR designs in the current research plan will utilise HIV bnAb (broadly neutralising antibody) scFvs of VRC07-523LS and PGT121 for universal targeting of the CD4 binding site on gp120 and specific targeting of the V3 loop of CCR5 tropic HIV gp120, respectively. CD69 expression on tissue resident cells will be targeted in a novel manner, utilising its specific ligand, galectin 1. Based on our collaborators established protocols (Bishop et al. 2018), electroporation with the piggyBac transposon system was used to generate bnAb or galectin-1 based single antigen recognition domain CD8+ CAR T cells with three different spacer regions derived from human CD8α chain, IgG 1 hinge-CH2-CH3 mutated to ablate FcγR binding and prevent non-specific activation of CAR T cells (Hudecek et al. 2015), or truncated human IgG 1 hinge. Following a 22-day tEGFR (truncated epidermal growth factor receptor)-Cetuximab and cytokine mediated expansion all CAR T cells were analysed by flow cytometry for memory phenotypes and co-expression of exhaustion markers. CAR T cell function was investigated via expression of effector cytokines and cytotoxic activity following co-culture with target cells (HIVBal infected Sup-T1-CCR5 or CD69 expressing Jurkat cells). Sup-T1-CCR5 cells are derived from human T cell lymphoma and have been engineered to express high levels of CCR5. Our CAR T cell expansion protocol was shown to produce favourable memory phenotype populations (TCM and TEM), without co-expression of exhaustion markers, for all CAR design variants. Our results demonstrate six anti-HIV bnAb variant CAR T cells with highly specific activation and potent cytotoxic activity when co-cultured with HIVBal-infected Sup-T1-CCR5 cells as detected by the intracellular expression of effector cytokines and reduction of p24+ cells. Furthermore, novel galectin-1 CAR T cells targeting CD69+ Jurkat cells demonstrated functional specificity by effector cytokine expression and killing. Following functional validation of CAR variants incorporating different spacer regions we found that CAR T cells designed with human CD8α chains were the most efficient in expressing cytokines and killing their target cell. Based on the data presented in this thesis, we propose a split tandem duo CAR T cells with a CD8α spacer capable of targeting both gp120 and CD69. In conclusion, this project has demonstrated the feasability of a novel cell therapy targeting markers of the HIV latent reservoir. Our CAR constructs are highly effective and will form an ideal platform to develop combination strategies to develop more precise T cell therapies for use against cells harbouring HIV. Increasing precision is paramount as markers such as CD69 are ubiquitous (Sathaliyawala et al. 2013) and inadvertent damage to healthy cells needs to be avoided. Towards this we are designing novel dual targeting CARs for future studies. We expect these kinds of novel therapeutics combined with other approaches will eliminate latent HIV reservoirs and contribute towards a HIV cure.
See less
See moreA landmark study employing in vitro models of interactions between HIV infection of DCs, macrophages and memory T cells cocultured with pDCs, demonstrates that the production of IFNα by pDCs significantly increased HIV infection of resting memory T cells by inducing reactivation without viral spread (Tong et al. 2021). Reactivated latently infected memory T cells (p24+) expressed gp120 env antigen on the cell membrane after pDC interaction, in addition to high levels of the early activation and tissue resident marker, CD69. Although HIV reactivation can be achieved by IFNα (Tong et al. 2021), the conventional cytotoxic T lymphocyte response to the HIV reservoir is limited by several mechanisms including mutational and non-mutational escape by sequestration of the virus in sites where access by CD8 T cells is limited; HIV-specific CD8 T cell exhaustion and apoptosis; and HIV nef downregulation of MHC-I antigen presentation thus preventing viral clearance. Therefore, the aim of this project was to explore an approach that may potentially enhance an immunotherapy strategy targeting the latent tissue reservoirs with anti-HIV CAR T cells following IFNα treatment and latent HIV reactivation. This is a novel “kick and kill” strategy which can be translated into clinical adoptive cell therapies. The CAR designs in the current research plan will utilise HIV bnAb (broadly neutralising antibody) scFvs of VRC07-523LS and PGT121 for universal targeting of the CD4 binding site on gp120 and specific targeting of the V3 loop of CCR5 tropic HIV gp120, respectively. CD69 expression on tissue resident cells will be targeted in a novel manner, utilising its specific ligand, galectin 1. Based on our collaborators established protocols (Bishop et al. 2018), electroporation with the piggyBac transposon system was used to generate bnAb or galectin-1 based single antigen recognition domain CD8+ CAR T cells with three different spacer regions derived from human CD8α chain, IgG 1 hinge-CH2-CH3 mutated to ablate FcγR binding and prevent non-specific activation of CAR T cells (Hudecek et al. 2015), or truncated human IgG 1 hinge. Following a 22-day tEGFR (truncated epidermal growth factor receptor)-Cetuximab and cytokine mediated expansion all CAR T cells were analysed by flow cytometry for memory phenotypes and co-expression of exhaustion markers. CAR T cell function was investigated via expression of effector cytokines and cytotoxic activity following co-culture with target cells (HIVBal infected Sup-T1-CCR5 or CD69 expressing Jurkat cells). Sup-T1-CCR5 cells are derived from human T cell lymphoma and have been engineered to express high levels of CCR5. Our CAR T cell expansion protocol was shown to produce favourable memory phenotype populations (TCM and TEM), without co-expression of exhaustion markers, for all CAR design variants. Our results demonstrate six anti-HIV bnAb variant CAR T cells with highly specific activation and potent cytotoxic activity when co-cultured with HIVBal-infected Sup-T1-CCR5 cells as detected by the intracellular expression of effector cytokines and reduction of p24+ cells. Furthermore, novel galectin-1 CAR T cells targeting CD69+ Jurkat cells demonstrated functional specificity by effector cytokine expression and killing. Following functional validation of CAR variants incorporating different spacer regions we found that CAR T cells designed with human CD8α chains were the most efficient in expressing cytokines and killing their target cell. Based on the data presented in this thesis, we propose a split tandem duo CAR T cells with a CD8α spacer capable of targeting both gp120 and CD69. In conclusion, this project has demonstrated the feasability of a novel cell therapy targeting markers of the HIV latent reservoir. Our CAR constructs are highly effective and will form an ideal platform to develop combination strategies to develop more precise T cell therapies for use against cells harbouring HIV. Increasing precision is paramount as markers such as CD69 are ubiquitous (Sathaliyawala et al. 2013) and inadvertent damage to healthy cells needs to be avoided. Towards this we are designing novel dual targeting CARs for future studies. We expect these kinds of novel therapeutics combined with other approaches will eliminate latent HIV reservoirs and contribute towards a HIV cure.
See less
Date
2021Rights statement
The author retains copyright of this thesis. It may only be used for the purposes of research and study. It must not be used for any other purposes and may not be transmitted or shared with others without prior permission.Faculty/School
Faculty of Medicine and Health, Westmead Clinical SchoolAwarding institution
The University of SydneyShare