Targeting of B-cell malignancy using novel parallel chimeric antigen receptor (pCAR) engineered T-cells

Student thesis: Doctoral ThesisDoctor of Philosophy


Second generation chimeric antigen receptors (CARs) containing a CD28 or 4-1BB costimulatory endodomain have shown remarkable efficacy against hematological malignancies. However, treatment failure and recurrence of disease after CAR T-cell therapy are considerable obstacles to overcome. Here, parallel (p)CARs in which a second-generation (CD28+CD3z) CAR is co-expressed with a 4-1BB containing chimeric co-stimulatory receptor (CCR), were engineered. The hypothesis underlying pCAR design is that optimal dual-costimulation requires positioning of endodomains in trans and proximal to the membrane which will, in turn, improve CAR T-cell efficacy. To investigate this premise, pCARs in which targeting moieties engage CD19, CD20 and CD22 were designed. In order to dual target CD19, the avidity of FMC63 scFv was altered using single-site mutagenesis thus creating a panel of second-generation CARs with a spectrum of binding avidities. These novel CARs demonstrated that an optimal window of avidity, lower than that of FMC63, improves T-cell responses in vitro and in vivo. Furthermore, using in vitro pre-clinical models, we demonstrated that CD19 pCARs showed greater cytotoxic responses, sustained cytokine release, increased proliferation and reduced expression of exhaustion markers upon repeated antigen exposure. In vivo CD19 pCAR T-cells showed significantly enhanced disease control and extended survival in a NALM-6 xenograft model, when compared to second-generation CAR T-cells. However, pCARs dual-targeting CD20 and CD19 or CD22 and CD19 did not show improved functionality in vitro or in vivo, respectively. The data shown here warrant further investigation and development of future pCARs to uncover the mechanisms behind their functionality.
Date of Award1 Jul 2021
Original languageEnglish
Awarding Institution
  • King's College London
SupervisorJohn Maher (Supervisor) & Richard Dillon (Supervisor)

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