Evaluation of single domain antibodies as nuclear tracers for imaging of the immune checkpoint receptor human lymphocyte activation gene-3 in cancer

Abstract

Recent advancements in the field of immune-oncology have led to a significant increase in life expectancy of patients with diverse forms of cancer, such as hematologic malignancies, melanoma and lung cancer. Unfortunately, these encouraging results are not observed in the majority of patients, who remain unresponsive and/or encounter adverse events. Currently, researchers are collecting more insight into the cellular and molecular mechanisms that underlie these variable responses. As an example, the human lymphocyte activation gene-3 (huLAG-3), an inhibitory immune checkpoint receptor, is increasingly studied as a therapeutic target in immune-oncology. Noninvasive molecular imaging of the immune checkpoint programmed death protein-1 (PD-1) or its ligand PD-L1 has shown its value as a strategy to guide and monitor PD-1/PD-L1-targeted immune checkpoint therapy. Yet, radiotracers that allow dynamic, whole body imaging of huLAG-3 expression are not yet described. We here developed single-domain antibodies (sdAbs) that bind huLAG-3 and showed that these sdAbs can image huLAG-3 in tumors, therefore representing promising tools for further development into clinically applicable radiotracers.

Keywords: Immune checkpoint; Immunotherapy; Lymphocyte activation gene-3; Molecular imaging; Nanobody; Single-domain antibody.

Fig. 1

SPR analysis of anti-huLAG-3 sdAbs. A The table shows the code, sequence family and affinity of the sixteen selected sdAbs as determined by SPR on immobilized huLAG-3-Fc. B RaPID plot illustrating the association (k_a) and dissociation (k_d) values for each of the sdAbs, determined via affinity analysis using the biacore T100 instrument. Each sdAb, color-coded for family-homology, is indicated as a dot on the two-dimensional graph so that pairings with identical affinity (K_D) values are located along iso-affinity diagonals

Fig. 2

Binding characteristics of selected anti-LAG-3 sdAbs on huLAG-3 expressing 2D3 cells. A The table shows the code, sequence family and the binding of the sdAb to 2D3 cells versus 2D3-huLAG-3 cells, calculated using the MFI determined in flow cytometry. B Histogram showing binding of anti-huLAG-3 sdAbs on 2D3 (black line) or 2D3-huLAG-3 (red line) cells, measured using flow cytometry (n = 2)

Fig. 3

Biodistribution of selected anti-huLAG-3 sdAbs in healthy mice. A The table shows the code, sequence family, potential of labeling with ^99mTc, and uptake of the tracer in the liver. The successful or unsuccessful labeling of sdAbs with ^99mTc is depicted with ✓ or ✗ respectively. B SPECT/CT images generated 1 h after injection of ^99mTc-labeled sdAbs in healthy C57BL/6 mice (n = 3). K = kidney, L = liver, H = heart, I = intestines, B = bladder

Fig. 4

Expression of huLAG-3 and its binding by selected sdAbs on lentivirally engineered TC-1 cells. A Representative flow cytometry results, showing staining of TC-1 cells (black line) or TC-1-huLAG-3 cells (blue line) with an antibody specific for huLAG-3. B The MFI of TC-1 cells was used to determine the fold increase in MFI when these sdAbs bound to TC-1-huLAG-3 cells (n = 2)

Fig. 5

Visualization of huLAG-3 in mice bearing TC-1 and TC-1-huLAG-3 tumors using SPECT/CT imaging. A Schematic representation of the study design. B TC-1 and TC-1-huLAG-3 cells were injected subcutaneously at opposite hind legs of NU(NCr)Foxn1nu mice and allowed to grow tumors up till day 10. The graph shows the tumor volume in function of time as mean ± SD (n = 3). c Images of the SPECT/CT scans performed to evaluate the utility of selected ^99mTc-labeled sdAbs to detect huLAG-3 expressed in tumors. The arrows indicate the tumors on the images (n = 3). MIP = Maximum Intensity Projection

Fig. 6

SdAb-mediated SPECT/CT imaging in TC-1-huLAG-3 tumor bearing mice shows that sdAb 3187 generates high signal-to-background ratios enabling detection of huLAG-3 in the tumor. A Results of ex vivo γ-counting analysis of uptake in isolated organs (expressed in %IA/g and as mean ± SD (n = 3)). B The graph shows the ratio in uptake in the TC-1-huLAG-3 positive tumor compared to the indicated organs. C The table shows the statistical results of comparing TC-1-huLAG-3 positive tumor/organ ratio values between control sdAb and huLAG-3 targeting sdAbs, confirming significant differences for sdAbs 3187 and 3202