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Bispecific light T-cell engagers for gene-based immunotherapy of epidermal growth factor receptor (EGFR)-positive malignancies

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Abstract

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.

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Abbreviations

BiKE:

Bispecific killer-cell engager

bsAb:

Bispecific antibody

Fc:

Fragment crystallizable

LiTE:

Light T-cell engager

Luc:

Luciferase

SEC-MALS:

Size exclusion chromatography with multiangle light scattering

T-bsAbs:

T-cell recruiting bsAb

VHH :

Single-domain antibodies from camelid heavy-chain-only immunoglobulins

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Funding

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).

Author information

Authors and Affiliations

  1. Immunotherapy and Cell Engineering Laboratory, Department of Engineering, Aarhus University, Gustav Wieds Vej 10, 8000 C, Aarhus, Denmark

    Kasper Mølgaard, Seandean L. Harwood, Kasper Mikkelsen, Natalia Nuñez-Prado & Luis Alvarez-Vallina

  2. Molecular Immunology Unit, Hospital Universitario Puerta de Hierro Majadahonda, Manuel de Falla 1, 28222, Madrid, Spain

    Marta Compte, Ana Alvarez-Cienfuegos & Laura Sanz

  3. CIC bioGUNE, Parque Tecnológico de Bizkaia 800, 48160, Derio, Spain

    Nekane Merino & Francisco J. Blanco

  4. Laboratory of Protein Design and Immunoengineering, École Polytechnique Fédérale de Lausanne, Station 19, 1015, Lausanne, Switzerland

    Jaume Bonet

  5. Universidad Complutense de Madrid, Plaza de Ramón y Cajal s/n, 28040, Madrid, Spain

    Ana Alvarez-Mendez

  6. IKERBASQUE, Basque Foundation for Science, María Díaz de Haro 3, 48013, Bilbao, Spain

    Francisco J. Blanco

Authors
  1. Kasper Mølgaard
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  2. Seandean L. Harwood
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  9. Ana Alvarez-Mendez
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  11. Francisco J. Blanco
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  12. Luis Alvarez-Vallina
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Contributions

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.

Corresponding author

Correspondence to Luis Alvarez-Vallina.

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The authors declare no conflict of interest related to this work.

Ethical approval

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.

Informed consent

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.

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Mølgaard, K., Harwood, S.L., Compte, M. et al. Bispecific light T-cell engagers for gene-based immunotherapy of epidermal growth factor receptor (EGFR)-positive malignancies. Cancer Immunol Immunother 67, 1251–1260 (2018). https://doi.org/10.1007/s00262-018-2181-5

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