Evaluating a human PD-L1 (huPD-L1) specific single domain antibody (sdAb) as a theranostic.

Immune checkpoint therapy exemplified by blockade of the PD-1:PD-L1 pathway represents a promising cancer treatment. Nonetheless, a large number of patients and cancer types do not respond well to the therapy.

We generated human PD-L1 (huPD-L1) binding sdAbs and evaluated them as a tool to perform noninvasive immune imaging of huPD-L1pos tumors. In addition, we used them to block the interaction between huPD-1:huPD-L1 as such enhancing the de novo activation of antigen-specific T cells and augmenting their functionality when interacting with tumor cells.


HuPD-L1 binding sdAbs were generated through immunization of alpaca’s with human and mouse PD-L1. The resulting sdAbs were characterized, evaluating binding on recombinant huPD-L1 by ELISA and on huPD-L1-expressing cells by flow cytometry, and determining their affinity and blocking capacity using surface plasmon resonance. Selected high affinity sdAbs were labeled with Technetium-99m (99mTc) and used for SPECT/CT imaging of naïve mice or mice bearing either MCF7 breast cancer cells or 624-mel melanoma cells that were lentivirally engineered to express huPD-L1. Ex vivo analysis was performed to assess the 99mTc-anti-huPD-L1 sdAb uptake (γ-counting) and PD-L1 expression (flow cytometry). Furthermore, these tumor cell lines or PD-L1 positive dendritic cells (DCs) were co-cultured with human CD8+ T cells, either or not in combination with anti-PD-L1 mAbs or sdAbs, whereafter we evaluated the amount of antigen-specific T cells and their functionality.


Out of 42 sdAbs, one sequence family, represented by sdAbs K1, K2, K3 and K4, was shown to have high affinity for hu-PD-L1. K2 was selected as lead compound for further evaluation of huPD-L1 imaging in xenograft cancer models. SPECT/CT and dissection analysis in immunodeficient mice bearing huPD-L1-modified or nonmodified MCF7 or 624-mel cells showed high uptake in both huPD-L1pos tumors, which correlated with huPD-L1 expression levels determined by flow cytometry.


We furthermore showed that adding Nb K2 to immature antigen (gp100 or melA) presenting dendritic or tumor cells, co-cultured with T cells, restores T cell receptor (TCR) triggering and leads to a higher number of antigen specific T cells, better proliferation and higher IFN-g secretion.


These data show that sdAb K2 holds promise to noninvasively assess PD-L1 expression levels in tumors and that it is able to block the interaction between PD-1 and PD-L1 leading to enhanced T-cell activity.



Broos K(1)
Lecocq Q(1)
Bridoux J(2)
Raes G(3, 4)
Xavier C(2)
Keyaerts M(2, 5)
Devoogdt N(2)
Breckpot K(1)


Laboratory of Molecular and Cellular therapy, Vrije Universiteit Brussel, Laarbeeklaan 103/E, B-1090 Brussels, Belgium(1)
In Vivo Cellular and Molecular Imaging, Vrije Universiteit Brussel, Laarbeeklaan 103/E, B-1090 Brussels, Belgium (2)
Cellular and Molecular Immunology, Vrije Universiteit Brussel, Pleinlaan 2, B-1000 Brussels, Belgium(3)
Myeloid Cell Immunology Lab, VIB Inflammation Research Center, Pleinlaan 2, B-1000 Brussels, Belgium (4)
Nuclear Medicine Department, UZ Brussel, Laarbeeklaan 101, B-1090 Brussels, Belgium (5)

Presenting author

Katrijn Broos, PhD student, Vrije Universiteit Brussel
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