Optimising endosialin targeting CAR-T cells
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Embargo End Date
2029-02-04
ICR Authors
Authors
Dinesh Kumar, M
Document Type
Thesis or Dissertation
Date
2026-02-04
Date Accepted
Abstract
Despite the recent success of CAR-T cell therapy in haematological malignancies, the efficacy of CAR-T cells against solid tumours is constrained by the immunosuppressive tumour microenvironment. Endosialin is a transmembrane glycoprotein that is minimally expressed in healthy adult tissues but is notably upregulated on tumour pericytes and perivascular CAFs in solid tumours, making it a promising target in the tumour microenvironment. Our laboratory had previously generated mouse anti-endosialin E3K CAR-T cells, derived from a rat mAb 3K2L that recognises both mouse and human endosialin, and validated its activity in multiple mouse tumour models. My PhD project aimed to engineer a humanised and deimmunised form of the E3K CAR construct, suitable for clinical application.During the humanisation and deimmunisation process, the E3K scFv sequence was closely examined, revealing a potential N-linked glycosylation site on the variable heavy chain region at <78-NQS-80>. After determining that this site was inactive, the serine residue was substituted with leucine. Next, the E3K CAR construct was modified for insertion into a lentiviral vector and optimised for lentiviral transduction of human T cells (renamed to Kozak E3K CAR). In silico design of 16 humanised and deimmunised Variants of Kozak E3K CAR scFv was carried out, and their binding was assessed using human and mouse endosialin-Fc fusion proteins. NFAT Jurkat cells were used to evaluate downstream signalling of the humanised Variants, and the four most active Variants were further evaluated for cytotoxic activity against target cells. Variant 16 demonstrated the highest cytotoxic activity across multiple donor T cells and against both human and mouse endosialin-positive target cells.In conclusion, this PhD research has developed a humanised, deimmunised endosialin-targeting CAR construct that can be advanced to subsequent stages of therapeutic development, aiding the creation of a viable clinical treatment.
Citation
2026
DOI
Source Title
Publisher
Institute of Cancer Research (University Of London)
ISSN
eISSN
Collections
Research Team
Molecular Cell Biology