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Conversion of anti-tissue factor antibody sequences to chimeric antigen receptor and bi-specific T-cell engager
S C Saunderson1, J C Halpin1,2, G M Y Tan1,3
1Department of Microbiology and Immunology, University of Otago, Dunedin, 9016, New Zealand.
Background:
The efficacy of antibody-targeted therapy of solid cancers is limited by the lack of consistent tumour-associated antigen expression. However, tumour-associated antigens shared with non-malignant cells may still be targeted using conditionally activated-antibodies, or by chimeric antigen receptor (CAR) T cells or CAR NK cells activated either by the tumour microenvironment or following 'unlocking' via multiple antigen-recognition. In this study, we have focused on tissue factor (TF; CD142), a type I membrane protein present on a range of solid tumours as a basis for future development of conditionally-activated BiTE or CAR T cells. TF is frequently upregulated on multiple solid tumours providing a selective advantage for growth, immune evasion and metastasis, as well as contributing to the pathology of thrombosis via the extrinsic coagulation pathway.
Methods:
Two well-characterised anti-TF monoclonal antibodies (mAb) were cloned into expression or transposon vectors to produce single chain (scFv) BiTE for assessment as CAR and CD28-CD3-based CAR or CD3-based BiTE. The affinities of both scFv formats for TF were determined by surface plasmon resonance. Jurkat cell line-based assays were used to confirm the activity of the BiTE or CAR constructs.
Results:
The anti-TF mAb hATR-5 and TF8-5G9 mAb were shown to maintain their nanomolar affinities following conversion into a single chain (scFv) format and could be utilised as CD28-CD3-based CAR or CD3-based BiTE format.
Conclusion:
Because of the broad expression of TF on a range of solid cancers, anti-TF antibody formats provide a useful addition for the development of conditionally activated biologics for antibody and cellular-based therapy.
Insights
Targeting tissue factor (TF) with antibody formats offers a promising strategy for solid cancer therapy. These anti-TF biologics can be conditionally activated for improved treatment efficacy.
Area of Science:
- Oncology
- Immunotherapy
- Biotechnology
Background:
- Antibody-targeted cancer therapy faces challenges due to inconsistent tumor antigen expression.
- Conditionally activated antibodies or chimeric antigen receptor (CAR) T/NK cells can overcome this by targeting shared antigens like tissue factor (TF).
- TF is upregulated on various solid tumors, promoting tumor growth, immune evasion, metastasis, and thrombosis.
Purpose of the Study:
- To develop and assess antibody formats targeting tissue factor (TF) for conditional cancer therapy.
- To engineer BiTE and CAR constructs using anti-TF monoclonal antibodies (mAbs).
Main Methods:
- Two anti-TF mAbs (hATR-5 and TF8-5G9) were converted into single-chain variable fragment (scFv) formats.
- Constructs were evaluated as CAR and BiTE (CD28-CD3 or CD3-based) using expression/transposon vectors.
- Surface plasmon resonance determined scFv affinities; Jurkat cell assays confirmed construct activity.
Main Results:
- Anti-TF scFv formats (hATR-5 and TF8-5G9) retained nanomolar affinities for TF.
- These scFv formats were successfully utilized in CD28-CD3-based CAR and CD3-based BiTE constructs.
Conclusions:
- Broad TF expression across solid cancers makes anti-TF antibody formats valuable for therapy.
- These formats are suitable for developing conditionally activated biologics for antibody and cellular therapies.
