Bispecific light T-cell engagers for gene-based immunotherapy of epidermal growth factor receptor (EGFR)-positive malignancies
- 412 Downloads
The recruitment of T-cells by bispecific antibodies secreted from adoptively transferred, gene-modified autologous cells has shown satisfactory results in preclinical cancer models. Even so, the approach’s translation into the clinic will require incremental improvements to its efficacy and reduction of its toxicity. Here, we characterized a tandem T-cell recruiting bispecific antibody intended to benefit gene-based immunotherapy approaches, which we call the light T-cell engager (LiTE), consisting of an EGFR-specific single-domain VHH antibody fused to a CD3-specific scFv. We generated two LiTEs with the anti-EGFR VHH and the anti-CD3 scFv arranged in both possible orders. Both constructs were well expressed in mammalian cells as highly homogenous monomers in solution with molecular weights of 43 and 41 kDa, respectively. In situ secreted LiTEs bound the cognate antigens of both parental antibodies and triggered the specific cytolysis of EGFR-expressing cancer cells without inducing T-cell activation and cytotoxicity spontaneously or against EGFR-negative cells. Light T-cell engagers are, therefore, suitable for future applications in gene-based immunotherapy approaches.
KeywordsCancer immunotherapy Bispecific antibody T-cell recruitment EGFR
Bispecific killer-cell engager
Light T-cell engager
Size exclusion chromatography with multiangle light scattering
T-cell recruiting bsAb
Single-domain antibodies from camelid heavy-chain-only immunoglobulins
Luis Alvarez-Vallina was involved in the study conception and design. Kasper Mølgaard, Seandean L. Harwood, Marta Compte, Nekane Merino, Jaume Bonet, Ana Alvarez-Cienfuegos, Kasper Mikkelsen, Natalia Nuñez-Prado, Ana Alvarez-Méndez, Laura Sanz, and Francisco J. Blanco were involved in acquisition, analysis, and interpretation of data. Kasper Mølgaard, Seandean L. Harwood, and Luis Alvarez-Vallina drafted the manuscript, and all the authors were involved in critical revision of the manuscript.
Luis Alvarez-Vallina was supported by grants from the Danish Council for Independent Research, Medical Sciences (DFF-6110-00533) and the Novo Nordisk Foundation (NNF14OC0011019). Jaume Bonet was supported by the ‘EPFL Fellows’ fellowship program co-funded by Marie Skłodowska-Curie, Horizon 2020 Grant agreement no. 665667. Francisco J. Blanco thanks the Spanish Ministry of Economy and Competitiveness (MINECO) for support through grant CTQ2017-83810-R and Severo Ochoa Excellence Accreditation (SEV-2016-0644). Laura Sanz was supported by grants from the Fondo de Investigación Sanitaria/Instituto de Salud Carlos III (PI13/00090), co-funded by European Regional Development FEDER funds, and the Comunidad de Madrid (S2010/BMD-2312).
Compliance with ethical standards
Conflict of interest
The authors declare no conflict of interest related to this work.
All procedures involving human blood products were in accordance with the ethical standards of the Aarhus University Hospital Ethical Committee and with the 1964 Helsinki declaration and its later amendments. Human peripheral blood mononuclear cells were isolated from fresh peripheral blood of anonymized healthy volunteer donors.
Blood donors were recruited to donate blood by standard phlebotomy. The investigational nature of the studies in which their blood would be used, and the risks and discomforts of the donation process were carefully explained to the donors, and a signed informed consent document was obtained.
- 5.Klinger M, Brandl C, Zugmaier G et al (2012) Immunopharmacologic response of patients with B-lineage acute lymphoblastic leukemia to continuous infusion of T cell-engaging CD19/CD3-bispecific BiTE antibody blinatumomab. Blood 119:6226–6233. https://doi.org/10.1182/blood-2012-01-400515 CrossRefPubMedGoogle Scholar
- 6.Blanco B, Holliger P, Vile RG et al (2003) Induction of human T lymphocyte cytotoxicity and inhibition of tumor growth by tumor-specific diabody-based molecules secreted from gene-modified bystander cells. J Immunol 171:1070–1077. https://doi.org/10.4049/jimmunol.171.2.1070 CrossRefPubMedGoogle Scholar
- 12.Harwood SL, Alvarez-Cienfuegos A, Nuñez-Prado N et al (2017) ATTACK, a novel bispecific T cell-recruiting antibody with trivalent EGFR binding and monovalent CD3 binding for cancer immunotherapy. Oncoimmunology 7:e1377874. https://doi.org/10.1080/2162402X.2017.1377874 CrossRefPubMedPubMedCentralGoogle Scholar
- 13.Alvarez-Cienfuegos A, Nuñez-Prado N, Compte N et al et al (2016) Intramolecular trimerization, a novel strategy for making multispecific antibodies with controlled orientation of the antigen binding domains. Sci Rep 6:28643. https://doi.org/10.1038/srep28643 CrossRefPubMedPubMedCentralGoogle Scholar
- 22.Muyldermans S (2013) Nanobodies: natural single-domain antibodies. Annu Rev Biochem 82:775–797. https://doi.org/10.1146/annurev-biochem-063011-092449 CrossRefPubMedGoogle Scholar
- 34.Baker JH, Lindquist KE, Huxham LA et al (2008) Direct visualization of heterogeneous extravascular distribution of trastuzumab in human epidermal growth factor receptor type 2 overexpressing xenografts. Clin Cancer Res 14:2171–2179. https://doi.org/10.1158/1078-0432.CCR-07-4465 CrossRefPubMedGoogle Scholar