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Department of Radiology, Molecular Imaging Innovations Institute (MI3), Weill Cornell Medicine, New York, NY 10065, USADepartment of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
Lymphocyte activation gene 3 (LAG-3) is a negative immune checkpoint and a key regulator of immune homeostasis with multiple biological activities related to T-cell functions. Fibrinogen-like protein 1 (FGL1) is a major LAG-3 functional ligand that is upregulated in various human cancers. LAG-3 positive T cells bind FGL1 expressed by cancer cells, which inhibits T-cell activation and cytokine secretion via indirect blocking of T cell receptor (TCR) signaling. High expression of LAG-3 and FGL1 in patients with solid tumors is associated with drug resistance and decreased survival in response to FDA-approved immune checkpoint inhibitors. Therefore, targeting the LAG-3/FGL1 pathway represents a promising therapeutic strategy to maximize the number of patients benefiting from checkpoint blockade therapy. However, there are no small molecules in existence that target LAG-3/FGL1 interaction. Herein, we report a time-resolved fluorescence resonance energy transfer (TR-FRET) assay to evaluate the ability of small molecules to inhibit LAG-3/FGL1 interaction. We further demonstrate the implementation of the developed assay in screening chemical libraries of small molecules from the NCI Diversity Set VII, FDA-approved drugs, and a focused library of NF-κB modulators. This work will pave the way for drug discovery efforts focused on therapeutic targeting of LAG-3/FGL1 interaction using small molecules.
Immune checkpoint blockade (ICB) has revolutionized the therapy landscape of several types of cancers, providing unprecedented survival in a subset of patients [
The history and advances in cancer immunotherapy: understanding the characteristics of tumor-infiltrating immune cells and their therapeutic implications.
]. This is exemplified by the clinical success of monoclonal antibodies (mAbs) targeting immune checkpoints, such as Programmed Cell Death Ligand 1 (PD-1) and Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) [
Current landscape and future of dual anti-CTLA4 and PD-1/PD-L1 blockade immunotherapy in cancer; lessons learned from clinical trials with melanoma and non-small cell lung cancer (NSCLC).
]. ICB resistance could be explained by malignant cell-intrinsic mechanisms as well as extrinsic factors from the tumor microenvironment (TME). These factors include the upregulation of other immune checkpoints, such as Lymphocyte activation gene 3 (LAG-3), T-cell immunoglobulin mucin domain 3 (TIM-3), and V-domain Ig suppressor of T-cell activation (VISTA) [
]. Therefore, remarkable research efforts are currently directed towards therapeutic targeting of these alternative immune checkpoints (e.g., LAG-3 and VISTA) in order to maximize the number of cancer patients benefiting from ICB.
LAG-3 is a cell surface inhibitory receptor and a key regulator of immune homeostasis with multiple biological activities related to T-cell functions [
]. In a large clinical trial called RELATIVITY-047, the combination of anit-LAG-3 and anti-PD-1 mAbs achieved median progression-free survival of 10.1 months in metastatic melanoma patients, as compared with 4.6 months with anti-PD-1 mAb alone [
]. The combination of relatlimab (anti-LAG-3 mAb) and nivolumab (anti-PD-1 mAb) has been recently approved by the U.S. Food and Drug Administration (FDA) as a combination therapy for metastatic melanoma patients [
]. In addition to melanoma, LAG-3 represents a promising target for non-small cell lung cancer (NSCLC) as it is highly detected in tumor-infiltrating lymphocytes (TILs) of NSCLC patients [
Expression analysis and significance of PD-1, LAG-3, and TIM-3 in human non–small cell lung cancer using spatially resolved and multiparametric single-cell analysis.
]. Elevated LAG-3 expression in patients with advanced NSCLC treated with PD-1 inhibitors was significantly associated with shorter progression-free survival [
Expression analysis and significance of PD-1, LAG-3, and TIM-3 in human non–small cell lung cancer using spatially resolved and multiparametric single-cell analysis.
]. We further quantified the binding affinity of FGL1 to LAG-3 using surface plasmon resonance (Fig. S1). The equilibrium dissociation constant (KD) value of 63.4 nM (Fig. S1) for FGL-1 to LAG-3 is in close agreement with the quality control assessment for KD provided by the commercial vendor. In addition, the KD value is close to the concentrations selected by us for the TR-FRET assay based on 2D titration (as detailed below), which maximizes the sensitivity for the inhibitors. LAG-3 positive T cells bind to ligands, such as FGL1 expressed by cancer cells, which inhibits activation and cytokine secretion via indirect blocking of T cell receptor (TCR) signaling [
]. These results indicate that the LAG-3/FGL1 interaction represents a mechanism of tumor evasion in NSCLC patients. Relatlimab, the first FDA-approved anti-LAG-3 mAb, has demonstrated ability to block LAG-3/FGL1 interaction and reverse T-cell inhibition induced by LAG-3/FGL1 signaling in co-culture assays [
Given the complexity of the TME, the combination of mAbs targeting immune checkpoints with small molecule agonists/inhibitors has emerged as a powerful approach to maximize the number of patients benefiting from ICB and prevent tumor recurrence [
]. Multiple clinical trials are currently underway to evaluate the efficacy of anti-LAG-3 mAbs in combination with anti-PD-1 mAbs as therapies for several solid tumors. However, there are currently no small molecule LAG-3 inhibitors in existence, which entails a major limitation for the development of effective combination of therapeutic modalities for cancer patients. Therefore, the discovery of small molecule LAG-3 inhibitors would enable the development of combination treatments that have potential for clinical translation as efficient immunotherapies for NSCLC as well as multiple solid tumors. To tackle this problem, we developed a screening assay that will enable the identification of small molecules that target the interaction of LAG-3 with FGL1. In comparison to mAbs, small molecules possess oral bioavailability and enhanced tumor penetration [
]. Remarkably, small molecules are more amenable to pharmacokinetic optimization, which allows adopting flexible dosage regimens that may enable avoiding immune-related adverse events associated with mAbs. These benefits offered by the introduction of small molecules as immune checkpoint inhibitors would be significant as the field moves toward synergistic combination therapies that are tailored to target multiple receptors and enhance overall response rates of cancer immunotherapy approaches. Notably, LAG-3 and FGL1 have been reported to be expressed in the cytoplasm of NSCLC cells [
]. Unlike mAbs, small molecules with cell-penetrating ability have the potential to modulate intracellular LAG-3/FGL1 interaction.
Methods
Our workflow for the TR-FRET assay for LAG-3/FGL1 interaction is illustrated in Fig. 1. For the TR-FRET assay, we obtained human His-tagged FGL1 at the N-terminus from Acro Biosystems (Cat. No. FG1-H52Hy). We procured LAG-3 extracellular domain (ECD) expressed with the Fc region of human IgG1 from Sino Biological (Cat No.16498-H02H). Both terbium (Tb) cryptate gold labeled anti-His mAb and XL665 labeled anti-human mAb were obtained from Cisbio (part of PerkinElmer). Optimization and validation experiments of the TR-FRET assay are displayed in Fig. S2-S4. TR-FRET measurements were done on Tecan Infinite M1000 Pro. (Donor Read-620): excitation wavelength 340 nm/20 nm, emission wavelength 620 nm/10nm, (Acceptor Read-665): excitation wavelength 340 nm/20 nm, emission wavelength 665 nm/10nm; both with 100 flashes/well, 500 ms integration time, 60 ms lag time). The assay mixture (18 μL) was immediately mixed prior to the assay to a final concentration of: LAG-3 (100 nM), FGL1 (100 nM), Tb cryptate labeled anti-His mAb (1 nM), and XL665 labeled anti-human Ab (10 nM). Stock solutions of tested compounds in DMSO (0.1%) and phosphate-buffered solution (PBS, pH 7.4) from the chemical library were applied to assay plates (medium binding white plates, Greiner #784075) at a final concentration (50 μM, n=3). The assay mixture (18 μL) was added to the plated compounds (2 μL) and incubated for 2 h at room temperature. The buffer used for both proteins and fluorophores was PBS, pH 7.4. Measurements were performed as described above, and TR-FRET signals were calculated as a ratio as follows: (intensity of 665 nm)/(intensity of 620 nm) × 100. All assay plates contained control wells (n=3) that include DMSO (0.1%) and wells that include all assay components except for Tag 2 (n=3). Percent inhibition was calculated on a scale of 0% (i.e., activity with DMSO vehicle only) to 100% (1 μM relatlimab) using full column of controls on each plate. The interquartile mean of control wells was used to calculate Z’ factor. It is noteworthy to mention that our established workflow includes a single incubation time, which is advantageous in comparison to the commercially available TR-FRET assay for LAG-3/FGL1 from BPS Bioscience (Cat# 79739-1), which entails two separate incubation times.
Fig. 1Our established workflow for LAG-3/FGL1 TR-FRET assay.
Hit identification: (1) We identified hits as compounds that result in 50% inhibition of the TR-FRET signal. (2) Fluorescent attenuators were identified using an additional readout of donor channel fluorescence data. Changes in donor fluorescence that mirror or contribute to overall activity (ratio of FRET/donor) indicate assay interference, and these compounds were not identified as hits.
Results and discussion
In continuation to our efforts focused on the development of screening assays for the identification of small molecule inhibitors of negative immune checkpoints,[
]. we established a TR-FRET assay for LAG-3/FGL1 interaction (Fig. 1). TR-FRET uses two fluorophores, termed a donor and an acceptor dye, that transfer energy when in close proximity to each other. In TR-FRET protein: protein interaction assay, one protein is labeled (directly or indirectly) with the donor, and the other protein is labeled (directly or indirectly) with the acceptor [
]. When the two proteins interact, the donor molecule is brought within proximity of the acceptor molecule. Excitation of the donor will result in signal generation proportional to the binding of proteins. Agents that block the interaction between both proteins result in attenuated TR-FRET signal. Aiming to identify the best acceptor-donor pairs for the LAG-3/FGL1 interaction, we explored a different LAG-3: FGL1 cross-titration with different tags versus multiple combinations of donor and acceptor reagents. We then miniaturized the assay volumes for use in 384-well format. We also optimized the assay buffer, plate type, order of reagent addition, and incubation time. The largest signal ratio was detected upon using the Tb cryptate gold labeled anti-His Ab as the donor and XL665 labeled anti-human Ab as the acceptor (Fig. 2) with a ratio of 1:10. We have selected the concentrations of both LAG-3 and FGL-1 in our assay based on the outcome of 2D titration of LAG-3 and FGL-1 (Table 1). The signal to background (S/B) ratio of the LAG-3/FGL1 TR-FRET assay was 10.7. Optimized conditions for maximizing the signal ratio involved final concentrations of 100 nM for both human Fc-tagged LAG-3 and His-tagged FGL1.
Fig. 2Schematic illustration of TR-FRET assay developed to identify small molecules that inhibit LAG-3/FGL1 interaction.
Table 12D titration of LAG-3 and FGL1 in the presence of Tb cryptate labeled anti-His mAb (1 nM), and XL665 labeled anti-human Ab (10 nM) and the corresponding signal-to-background ratio from the TR-FRET assay.
Titration of XL665-labeled LAG-3 to Tb-labeled FGL1 illustrated the FRET efficiency based on hyperbolic dependence of TR-FRET ratio on the acceptor concentration (Fig. 3A). Compounds that can block LAG-3/FGL1 interaction would consequently result in attenuated TR-FRET signal. Remarkably, a concentration-dependent decrease in the TR-FRET ratio was detected in the presence of varying concentrations of relatlimab, an anti-LAG-3 mAb (Fig. 3B). Dose-dependent FRET screening for relatlimab revealed a half-maximal inhibitory concentration (IC50) value of 0.39 ± 0.02 nM (Fig. 3B). It is noteworthy to mention that evaluation of relatlimab for LAG-3/FGL1 inhibition using a commercially available enzyme-linked immunosorbent assay (ELISA)-based LAG-3/FGL1 inhibitor screening assay from BPS Bioscience (Catalog # 79789) indicated an IC50 value of 0.51 ± 0.04 nM. Moreover, we have verified the ability of untagged LAG-3 (Fig. 3C) and FGL-1 (Fig. 3D) to induce dose-dependent reduction in the TR-FRET signal.
Fig. 3(A) Changes in TR-FRET ratio upon variation of the acceptor to donor ratio. (B) Dose-response curves of relatlimab binding in LAG-3 TR-FRET assay. Dose-dependent reduction in the TR-FRET signal of the LAG-3/FGL-1 upon incubation with increasing concentrations of untagged LAG-3 (C) and FGL-1 (D). Error bars represent standard deviation (n = 3).
The close agreement between the outcome of the ELISA-based LAG-3/FGL1 inhibition assay and the TR-FRET assay for relatlimab demonstrates the efficiency of the TR-FRET assay in screening inhibitors of LAG-3/FGL1 interaction. The potential utility of the assay for high-throughput screening (HTS) has been demonstrated with a mean Z’ factor of 0.74 ± 0.05, revealing a high-quality assay for HTS. Z’ factor corresponds to the ratio of data signal variability (standard deviation) to dynamic range (i.e., change in TR-FRET signal for positive and negative controls) [
]. Z’ factor of the LAG-3/FGL1 TR-FRET assay was calculated based on three independent runs. Moreover, DMSO tolerance screening revealed that the TR-FRET signal was stable and robust up to 2.0% (v/v) DMSO.
Following the optimization of the assay, we performed a pilot screen using a subset of the National Cancer Institute (NCI) Diversity Set VII chemical library (Fig. S5A) as well as subset of the MedChemExpress library (Cat. No. HY-L022) of FDA approved drugs (Fig. S5B). Both libraries were screened at 50 μM final concentration. As shown in Fig. S5A and S5B, this screening resulted in the identification of 10 hits from the NCI library as well as 5 hits from the MedChemExpress library with inhibition >50% of the TR-FRET signal. As this library's purpose was primarily for demonstration of the HTS assays utility, hits from this library were inspected chemically and it was decided not to pursue them further due to low likelihood for medicinal chemistry tractability.
We next implemented our developed TR-FRET assay in screening a focused chemical library from APExBIO. The library (Cat. No. L1044P) is composed of a unique collection of 73 modulators of nuclear factor kappa B (NF-κB) for NF-κB signaling pathway research. Numerous studies indicate that dysregulated NF-κB activity causes inflammation-related diseases as well as cancers, and NF-κB has been long proposed as the potential target for therapy of diseases [
]. Notably, recent reports have indicated the potential of the implementation of dual targeted strategies to modulate NF-κB signaling as well as immune checkpoints for improved immunotherapeutic outcomes in patients with cancer [
]. As shown in Fig. 4A, screening of the APExBIO-focused library at a single concentration (50 μM) resulted in the identification of three hits with >50% inhibition of the TR-FRET signal. Screening of the APExBIO-focused library demonstrated excellent reproducibility as shown in Fig. 4B. These three hits were ZLN005, pracinostat, and piceatannol. We identified piceatannol as a false hit based on demonstrated quenching effect of the TR-FRET signal by its polyphenolic structure. The chemical structures of ZLN005 and pracinostat along with their % inhibition of TR-FRET at 50 μM are depicted in Fig. 4C. Dose-dependent screening of both compounds is displayed in Fig. 4D and S6. Using our established protocol, the IC50 values of ZLN005 and pracinostat in Fig. 4D and S6 are 28.42 ± 1.26 μM and 46.28 ± 3.06 μM, respectively. Changing the order of addition of the TR-FRET assay by adding the inhibitors before the complex is formed resulted in minimal effects on the IC50 values of these two compounds (27.19 ± 1.03 μM and 47.95 ± 2.49 μM, for ZLN005 and pracinostat, respectively). Moreover, the purity assessment (1HNMR and LC-MS) of both compounds is disclosed in Fig. S7-S10. ELISA-based LAG-3/FGL1 inhibition for ZLN005 and pracinostat using the kit from BPS Bioscience (Catalog # 79789) resulted in IC50 values of 36.02 ± 2.42 μM and 61.34 ± 4.31 μM, respectively. Notably, the identified compounds share a benzimidazole core, which might represent a promising scaffold in future drug discovery efforts focused on the development of small molecule inhibitors of LAG-3/FGL1 interaction. It is noteworthy to mention that although our chemical library included other compounds with benzimidazole core, none of them advanced as a hit based on our criteria detailed above.
Fig. 4(A) Scatterplot showing single-dose inhibition data screened at 50 μM from the APExBIO-focused library. (B) Correlation plot of the APExBIO set duplicate runs in the TR-FRET LAG-3/FGL1 assay. (C) Chemical structures of ZLN005 and pracinostat along with their % inhibition of TR-FRET signal at 50 μM. (D) Dose-response curve of ZLN005 in TR-FRET assay of LAG-3/FGL1. Error bars represent standard deviation (n = 3).
In summary, we are introducing in this study a TR-FRET assay for HTS of chemical libraries for LAG-3/FGL1 inhibition. This developed assay will be critical in the identification of first-in-class small molecule inhibitors of LAG-3/FGL1 interaction, which will set the stage for further preclinical evaluation of these compounds in animal models of immuno-oncology. Small molecule inhibitors of LAG-3/FGL1 interaction can be evaluated in combination treatments to maximize the efficacy of FDA-approved anti-PD-1 mAbs in preclinical models of melanoma and NSCLC.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
The history and advances in cancer immunotherapy: understanding the characteristics of tumor-infiltrating immune cells and their therapeutic implications.
Current landscape and future of dual anti-CTLA4 and PD-1/PD-L1 blockade immunotherapy in cancer; lessons learned from clinical trials with melanoma and non-small cell lung cancer (NSCLC).
Expression analysis and significance of PD-1, LAG-3, and TIM-3 in human non–small cell lung cancer using spatially resolved and multiparametric single-cell analysis.
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