Small molecules to minimize adverse effects in immunotherapies
Antibody therapies have proved quite efficient in immuno-oncology, particularly in targeting immune checkpoints on tumor-infiltrated T cells, most famously the PD-1/PD-L1 axis. However, immune-related adverse effects have represented a significant drawback to these therapies. In an effort to minimize these deleterious effects, another category of compounds is focusing a lot of interest: small molecules. Indeed, these drugs are known for their shorter half-lives, higher rate of clearance, and diffusion-based distribution.1 They also easily penetrate membranes and can thus offer alternative mechanisms of action to monoclonal antibodies, which mostly block extracellular protein–protein interactions. In addition, they are mainly administered orally, and can be produced at a lower cost than antibodies.2
The first small-molecule immuno-oncology drug, TLR7-targeting Imiquimod, was approved in 2004 by the FDA for the treatment of basal cell carcinoma.3 Since then, numerous small molecules are being developed and tested in preclinical settings and clinical trials to improve immune checkpoint blockade.2 One of these promising PD-L1-targeting compounds was tested in genOway’s optimized preclinical models4 and proved efficient in inhibiting tumor growth.5
Mechanism of action of human PD-L1-targeting small molecule CCX559
A recent report described another small molecule PD-L1 inhibitor inducing anti-tumor immunity—CCX559.6 In this study, CCX559 was shown to potently and selectively inhibit human PD-L1 binding to PD-1 and CD80, while enhancing TCR signaling in vitro.
Figure 1: A–B) hPD-L1 membrane staining of MC38-hPD-L1 cells upon treatment with CCX559 or DMSO; C–D) Corresponding DAPI staining; E–F) Intracellular hPD-L1 staining of MC38-hPD-L1 cells. PD-L1 was observed in intracellular vesicles only in CCX559-treated cells.
Other PD-L1-targeting small molecule inhibitors were described to induce its dimerization and internalization. Similarly, PD-L1 dimerization was observed upon treatment with CCX559 in vitro, preventing its interaction with PD-1. The cellular distribution of hPD-L1 was assessed by immunofluorescence staining in MC38-hPD-L1 cells from genOway: MC38 cancer cells invalidated for mouse PD-L1 and expressing human PD-L1. Upon CCX559 treatment, hPD-L1 staining was reduced at the membrane (Figure 1A–D), while increased in intracellular vesicles, demonstrating potent internalization (Figure 1E–F). Similar treatment with anti-human PD-L1 monoclonal antibody MEDI4736 did not impact human PD-L1 localization (not shown).
These data show that CCX559 does not directly compete with PD-1 to bind PD-L1, as MEDI4736 and other PD-L1 monoclonal antibodies do, but, rather, prevents PD-L1/PD-1 binding through dimerization of PD-L1. In addition, CCX559 induces PD-L1 internalization, further preventing its interaction with PD-1.
Efficient tumor growth inhibition and tissue distribution of CCX559 in vivo
To assess CCX559 potency as an anti-tumor agent, the small molecule was tested in two murine models. One of the models consisted of the injection of MC38-hPD-L1 in immunocompetent mice. When administered orally once per day after tumor formation, CCX559 significantly reduced tumor growth (Figure 2A).
Figure 2: A) CCX559 dose-dependently suppressed MC38-hPD-L1 tumor growth in C57BL/6 mice. B) Plasma CCX559 concentrations were measured 24 hours after the last dose (MC38-hPD-L1 tumors in C57BL/6 mice). C) CCX559 accumulated in MC38-hPD-L1 tumors in human PD-L1 KI mice. D) Tumor, plasma, and tissue drug levels were measured on days 1 and 5 post-dosing (MC38-hPD-L1 tumors in hPD-L1 KI mice).
This effect was dose-dependent, and similar to the one observed with MEDI4736. CCX559 plasma levels were measured and showed consistent and dose-dependent drug exposure throughout the treatment period (Figure 2B). Ex vivo staining for cell membrane human PD-L1 on tumor cells demonstrated target engagement in tumors (not shown). MC38-hPD-L1 cells were also injected in human PD-L1 Knockin mice to assess the small molecule’s distribution and clearance. Interestingly, CCX559 was shown to accumulate preferentially in tumors one day after treatment (Figure 2C), and appeared almost completely cleared in all organs five days post-administration (Figure 2D). Complete clearance was observed by day 12 post-dose (not shown).
CCX559 thereby represents a promising alternative to known PD-L1-targeting therapies, considering its potency to inhibit tumor growth, preferential on-target (tumors) accumulation, and rapid clearance in vivo. Last, but not least, this molecule is now in clinical development with a Phase 1 study in subjects with solid tumors.
Of note, the MC38-hPD-L1 cell line used in this publication, as well as a similar and optimized human PD-L1 Knockin mouse model as the one mentioned here, were generated by and are available off-the-shelf at genOway, a designer and provider of numerous physiologically relevant preclinical models in multiple research areas, including immuno-oncology, metabolism, cardiovascular diseases, and neuroscience.
Adams, J. L., Smothers, J., Srinivasan, R. & Hoos, A. Big opportunities for small molecules in immuno-oncology. Nat Rev Drug Discov14, 603-622 (2015).
van der Zanden, S. Y., Luimstra, J. J., Neefjes, J., Borst, J. & Ovaa, H. Opportunities for Small Molecules in Cancer Immunotherapy. Trends Immunol 41, 493-511 (2020).
Geisse, J. et al. Imiquimod 5% cream for the treatment of superficial basal cell carcinoma: results from two phase III, randomized, vehicle-controlled studies. J Am Acad Dermatol 50, 722-733 (2004).
Acurcio, R. C. et al. Therapeutic targeting of PD-1/PD-L1 blockade by novel small-molecule inhibitors recruits cytotoxic T cells into solid tumor microenvironment. J Immunother Cancer 10 (2022).
Sullivan, K. M. C. et al. CCX559 is a potent, orally-administered small molecule PD-L1 inhibitor that induces anti-tumor immunity. PLoS One 18, e0286724 (2023).
The hPD-1 mouse enables the in vivo efficacy assessment and profiling of immuno-oncology agents targeting the human immune checkpoint PD-1 in fully immunocompetent mice.
The MC38-hPD-L1-LZ clonal cell line expresses high levels of human PD-L1 and forms solid tumors in vivo. An optimized version of a luciferase-ZsGreen (LZ) fusion protein is also stably expressed for in vivo imaging and ex vivo tracking.