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Lunch & Poster Display session

34P - Development of LAG-3 nanobodies as potent cancer imaging tracers


12 Dec 2019


Lunch & Poster Display session


Quentin Lecocq


Annals of Oncology (2019) 30 (suppl_11): xi1-xi11. 10.1093/annonc/mdz447


Q. Lecocq1, R.M. Awad1, K. Zeven1, Y. De Vlaeminck1, W. de Mey1, P. Debie2, J. Puttemans2, C. Goyvaerts1, G. Raes2, M. Keyaerts3, N. Devoogdt2, K. Breckpot1

Author affiliations

  • 1 Laboratory For Molecular And Cellular Therapy, VUB, 1090 - Jette/BE
  • 2 In Vivo Cellular And Molecular Imaging Lab, VUB, 1090 - Jette/BE
  • 3 Nucg, UZ Brussels, 1090 - Jette/BE


Abstract 34P


Immune checkpoint blockade revolutionized anti-cancer therapy but unfortunately not all patients can benefit from it. The development of innovative and efficacious diagnostic methods that can guide treatment decisions is warranted. Nanobodies (Nbs) are small antigen-binding moieties that efficiently penetrate cell-cell interfaces in tumors and generate high contrast in noninvasive imaging, making them prime candidates for development of novel imaging tracers. In this study, we generated and characterized Nbs as tools for nuclear imaging of the immune checkpoint LAG-3.


Nanobody generation was initiated by immunization of llamas with recombinant LAG-3. Periplasmic extracts were generated and analyzed for their binding to mouse LAG-3 using ELISA and flow cytometry. Selected Nbs were cloned in a vector for high yield production and purified in order to analyze their affinity using SPR. A total of 9 high affinity Nbs were subsequently evaluated for their labeling efficiency with Technetium-99m (99mTc), after which the biodistribution of 99mTc labeled Nbs was assessed in mice. Additionally, we evaluated the noninvasive detection of LAG-3 expressed by immune cells in the tumor environment of murine colon adenocarcinoma (MC38) using 99mTc-Nb3132.


Nine high affinity Nbs were selected to evaluate their potential as noninvasive imaging tracers. Subsequently, Nb3132 was reported as the most potent tracer for noninvasive imaging of the immune checkpoint LAG-3 (1). SPECT/CT of MC38 tumor-bearing mice 1h after injection of 99mTc-Nb3132 presented specific uptake in spleen, thymus, lymph nodes as well as at the tumor site. This uptake pattern coincided with the presence of LAG-3 expressed on immune cells in these organs, as determined by flow cytometry and immunohistochemistry. No specific uptake was observed when injecting mice with a radiolabeled control Nb.


Nb3132 showed excellent in vivo imaging capacities to specifically identify LAG-3 expressing immune cells within the tumor site. These findings support the predictive potential of Nb3132 to noninvasively detect LAG-3 by nuclear imaging and to guide treatment decisions in future, improving the shortcomings of using full-sized antibody formats as diagnostic tracers.

Clinical trial identification

Editorial acknowledgement

Legal entity responsible for the study

Geert Raes.


Kom op tegen kanker and FWO flanders.


All authors have declared no conflicts of interest.

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