Advertisement

Cancer Immunology, Immunotherapy

, Volume 67, Issue 8, pp 1251–1260 | Cite as

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

  • Kasper Mølgaard
  • Seandean L. Harwood
  • Marta Compte
  • Nekane Merino
  • Jaume Bonet
  • Ana Alvarez-Cienfuegos
  • Kasper Mikkelsen
  • Natalia Nuñez-Prado
  • Ana Alvarez-Mendez
  • Laura Sanz
  • Francisco J. Blanco
  • Luis Alvarez-Vallina
Original Article
  • 412 Downloads

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.

Keywords

Cancer immunotherapy Bispecific antibody T-cell recruitment EGFR 

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

Notes

Author 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.

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).

Compliance with ethical standards

Conflict of interest

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.

Supplementary material

262_2018_2181_MOESM1_ESM.pdf (1.3 mb)
Supplementary material 1 (PDF 1381 KB)

References

  1. 1.
    Kontermann RE (2005) Recombinant bispecific antibodies for cancer therapy. Acta Pharmacol Sin 26:1–9.  https://doi.org/10.1111/j.1745-7254.2005.00008.x CrossRefPubMedGoogle Scholar
  2. 2.
    Nuñez-Prado N, Compte M, Harwood S et al (2015) The coming of age of engineered multivalent antibodies. Drug Discov Today 20:588–594.  https://doi.org/10.1016/j.drudis.2015.02.013 CrossRefPubMedGoogle Scholar
  3. 3.
    Kontermann RE, Brinkmann U (2015) Bispecific antibodies. Drug Discov Today 20:838–847.  https://doi.org/10.1016/j.drudis.2015.02.008 CrossRefPubMedGoogle Scholar
  4. 4.
    Löffler A, Kufer P, Lutterbüse R et al (2000) A recombinant bispecific single-chain antibody, CD19 × CD3, induces rapid and high lymphoma-directed cytotoxicity by unstimulated T lymphocytes. Blood 95:2098–2103PubMedGoogle Scholar
  5. 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. 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
  7. 7.
    Compte M, Blanco B, Serrano F et al (2007) Inhibition of tumor growth in vivo by in situ secretion of bispecific anti-CEA × anti-CD3 diabodies from lentivirally transduced human lymphocytes. Cancer Gene Ther 14:380–388.  https://doi.org/10.1038/sj.cgt.7701021 CrossRefPubMedGoogle Scholar
  8. 8.
    Compte M, Cuesta AM, Sánchez-Martín D et al (2009) Tumor immunotherapy using gene-modified human mesenchymal stem cells loaded into synthetic extracellular matrix scaffolds. Stem Cells 27:753–760.  https://doi.org/10.1634/stemcells.2008-0831 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Compte M, Alonso-Camino V, Santos-Valle P et al (2010) Factory neovessels: engineered human blood vessels secreting therapeutic proteins as a new drug delivery system. Gene Ther 17:745–751.  https://doi.org/10.1038/gt.2010.33 CrossRefPubMedGoogle Scholar
  10. 10.
    Mølgaard K, Compte M, Nuñez-Prado N et al (2017) Balanced secretion of anti-CEA × anti-CD3 diabody chains using the 2A self-cleaving peptide maximizes diabody assembly and tumor-specific cytotoxicity. Gene Ther 24:208–214.  https://doi.org/10.1038/gt.2017.3 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Compte M, Alvarez-Cienfuegos A, Nuñez-Prado N et al (2014) Functional comparison of single-chain and two-chain anti-CD3-based bispecific antibodies in gene immunotherapy applications. Oncoimmunology 3:e28810.  https://doi.org/10.4161/onci.28810 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 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. 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
  14. 14.
    Webb B, Sali A (2016) Comparative protein structure modeling using MODELLER. Curr Protoc Bioinform 54:5.6.1–5.6.37.  https://doi.org/10.1002/cpbi.3 CrossRefGoogle Scholar
  15. 15.
    Blanco-Toribio A, Sainz-Pastor N, Alvarez-Cienfuegos A et al (2013) Generation and characterization of monospecific and bispecific hexavalent trimerbodies. MAbs 5:70–79.  https://doi.org/10.4161/mabs.22698 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Kim JH, Song DH, Youn SJ et al (2016) Crystal structures of mono- and bi-specific diabodies and reduction of their structural flexibility by introduction of disulfide bridges at the Fv interface. Sci Rep 6:34515.  https://doi.org/10.1038/srep34515 CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Camacho C, Coulouris G, Avagyan V et al (2009) BLAST+: architecture and applications. BMC Bioinform 10:421.  https://doi.org/10.1186/1471-2105-10-421 CrossRefGoogle Scholar
  18. 18.
    Boehm MK, Corper AL, Wan T et al (2000) Crystal structure of the anti-(carcinoembryonic antigen) single-chain Fv antibody MFE-23 and a model for antigen binding based on intermolecular contacts. Biochem J 346:519–528CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Schmitz KR, Bagchi A, Roovers RC et al (2013) Structural evaluation of EGFR inhibition mechanisms for nanobodies/VHH domains. Structure 21:1214–1224.  https://doi.org/10.1016/j.str.2013.05.008 CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Kung P, Goldstein G, Reinherz EL et al (1979) Monoclonal antibodies defining distinctive human T cell surface antigens. Science 206:347–349CrossRefPubMedGoogle Scholar
  21. 21.
    Huehls AM, Coupet TA, Sentman CL (2015) Bispecific T-cell engagers for cancer immunotherapy. Immunol Cell Biol 93:290–296.  https://doi.org/10.1038/icb.2014.93 CrossRefPubMedGoogle Scholar
  22. 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
  23. 23.
    Els Conrath K, Lauwereys M, Wyns L et al (2001) Camel single-domain antibodies as modular building units in bispecific and bivalent antibody constructs. J Biol Chem 276:7346–7350.  https://doi.org/10.1074/jbc.M007734200 CrossRefPubMedGoogle Scholar
  24. 24.
    Li L, He P, Zhou C et al (2015) A novel bispecific antibody, S-Fab, induces potent cancer cell killing. J Immunother 38:350–356.  https://doi.org/10.1097/CJI.0000000000000099 CrossRefPubMedGoogle Scholar
  25. 25.
    Li A, Xing J, Li L et al (2016) A single-domain antibody-linked Fab bispecific antibody Her2-S-Fab has potent cytotoxicity against Her2-expressing tumor cells. AMB Express 6:32.  https://doi.org/10.1186/s13568-016-0201-4 CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Wang XB, Zhao BF, Zhao Q et al (2004) A new recombinant single chain trispecific antibody recruits T lymphocytes to kill CEA (carcinoma embryonic antigen) positive tumor cells in vitro efficiently. J Biochem 135:555–565.  https://doi.org/10.1093/jb/mvh065 CrossRefPubMedGoogle Scholar
  27. 27.
    Alvarez-Vallina L (2001) Genetic approaches for antigen-selective cell therapy. Curr Gene Ther 1:385–397.  https://doi.org/10.2174/1566523013348418 CrossRefGoogle Scholar
  28. 28.
    Sanz L, Blanco B, Alvarez-Vallina L (2004) Antibodies and gene therapy: teaching old ‘magic bullets’ new tricks. Trends Immunol 25:85–91.  https://doi.org/10.1016/j.it.2003.12.001 CrossRefPubMedGoogle Scholar
  29. 29.
    Iwahori K, Kakarla S, Velasquez MP et al (2015) Engager T cells: a new class of antigen-specific T cells that redirect bystander T cells. Mol Ther 23:171–178.  https://doi.org/10.1038/mt.2014.156 CrossRefPubMedGoogle Scholar
  30. 30.
    Velasquez MP, Torres D, Iwahori K et al (2016) T cells expressing CD19-specific engager molecules for the immunotherapy of CD19-positive malignancies. Sci Rep 6:27130.  https://doi.org/10.1038/srep27130 CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Bonifant CL, Szoor A, Torres D et al (2016) CD123-engager T cells as a novel immunotherapeutic for acute myeloid leukemia. Mol Ther 24:1615–1626.  https://doi.org/10.1038/mt.2016.116 CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Liu X, Barrett DM, Jiang S et al (2016) Improved anti-leukemia activities of adoptively transferred T cells expressing bispecific T-cell engager in mice. Blood Cancer J 6:e430.  https://doi.org/10.1038/bcj.2016.38 CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Stadler CR, Bähr-Mahmud H, Celik L et al (2017) Elimination of large tumors in mice by mRNA-encoded bispecific antibodies. Nat Med 23:815–817.  https://doi.org/10.1038/nm.4356 CrossRefPubMedGoogle Scholar
  34. 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
  35. 35.
    Frankel SR, Baeuerle PA (2013) Targeting T cells to tumor cells using bispecific antibodies. Curr Opin Chem Biol 17:385–392.  https://doi.org/10.1016/j.cbpa.2013.03.029 CrossRefPubMedGoogle Scholar
  36. 36.
    Rezvani K, Rouce RH (2015) The application of Natural Killer cell immunotherapy for the treatment of cancer. Front Immunol 6:578.  https://doi.org/10.3389/fimmu.2015.00578 CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Yano S, Kondo K, Yamaguchi M et al (2003) Distribution and function of EGFR in human tissue and the effect of EGFR tyrosine kinase inhibition. Anticancer Res 23:3639–3650PubMedGoogle Scholar
  38. 38.
    Ballestrero A, Garuti A, Cirmena G et al (2012) Patient-tailored treatments with anti-EGFR monoclonal antibodies in advanced colorectal cancer: KRAS and beyond. Curr Cancer Drug Targets 12:316–328.  https://doi.org/10.2174/156800912800190956 CrossRefPubMedGoogle Scholar
  39. 39.
    Lutterbuese R, Raum T, Kischel R et al (2010)) T cell-engaging BiTE antibodies specific for EGFR potently eliminate KRAS- and BRAF-mutated colorectal cancer cells. Proc Natl Acad Sci U S A 107:12605–12610.  https://doi.org/10.1073/pnas.1000976107 CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Sanchez-Martin D, Sørensen MD, Lykkemark S et al (2015) Selection strategies for anticancer antibody discovery: searching off the beaten path. Trends Biotechnol 33:292–301.  https://doi.org/10.1016/j.tibtech.2015.02.008 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Immunotherapy and Cell Engineering Laboratory, Department of EngineeringAarhus UniversityAarhusDenmark
  2. 2.Molecular Immunology UnitHospital Universitario Puerta de Hierro MajadahondaMadridSpain
  3. 3.CIC bioGUNEDerioSpain
  4. 4.Laboratory of Protein Design and ImmunoengineeringÉcole Polytechnique Fédérale de LausanneLausanneSwitzerland
  5. 5.Universidad Complutense de MadridMadridSpain
  6. 6.IKERBASQUE, Basque Foundation for ScienceBilbaoSpain

Personalised recommendations