(T1230-10-59) Discovery of Anti-LAG-3 Peptides for Cancer Immunotherapy

Purpose: Although targeting CTLA-4 and PD-1/PD-L1 have significantly enhanced the outcomes of patients with cancer, a large group of patients have shown to develop resistance mechanisms to these therapies after a period of treatment. In a recent study, lymphocyte activation gene-3 (LAG-3) has been found to be highly expressed on T cells upon treatment with monoclonal antibody (mAb) targeting PD-1/PD-L1 pathway. This potentially develops resistance against anti-PD-1/PD-L1 treatment. FGL1 has been identified to be the major inhibitory ligand for LAG-3 that is responsible for the T-cells inhibitory function. When LAG-3 and FGL1 bind together, they send an “off” signal to the T cell that help keep the immune system from destroying the tumor cells. Blocking the interaction between LAG-3 receptor and its partner protein, FGL1, can enhance the T cells’ antitumor activity and boost the immune response against various cancers. However, several anti-LAG-3 antibodies are examined in several clinical trials to treat several cancers. Despite their specificity and affinity, antibody-based checkpoint inhibitors possess poor tumor permeability and high manufacturing cost. Here we aim to discover a low-molecular-weight peptide inhibitors to block the LAG-3/FGL1 interaction, which may have a great deal of promise in treating cancer and advancing the patients’ clinical results.

Methods: In this study, an innovative biopanning technique developed in our lab was used to discover small peptide-based anti-LAG-3 inhibitors. The peptides were synthesized by the solid-phase peptide synthesis using PurePep^TM Chorus synthesizer (Gyros Protein Technologies, Tucson, AZ). The molecular weights and purity were confirmed by mass spectrometry and HPLC, respectively. The binding affinity of the discovered peptides were evaluated using appropriate assays. Anti- tumor activity of the peptides was examined in five-week-old male and female Balb/c mice bearing murine colorectal cancer cell line CT26. The mice were randomly divided into five different groups (8 mice/group, 50% female, 50% male). Mice were inoculated subcutaneously with 1x10⁶ CT26 cells. When tumors became palpable (size 50-100 mm³), the animals were injected with multiple intraperitoneal injections of the tested peptides. The tumor size was measured with a digital caliper.

Results: We discovered several anti-LAG-3 inhibitor peptides that can block the LAG-3/FGL1 interaction. Among them, the CMA16 peptide inhibitor shows the highest blocking efficacy of the LAG-3/FGL1 interaction. The dimerization of CMA16 increased the affinity to LAG-3. CMA16 peptide and its dimeric counterpart significantly inhibit tumor growth of CT26 tumor-bearing mice and demonstrate comparable activity to anti-PD-L1 peptide inhibitor that previously developed in our laboratory. The combination therapy of CMA16 dimer and anti-PD-L1 peptide significantly inhibits the tumor growth compared to monotherapies.

Conclusion: This study demonstrated the benefit of using phage display to discover low-molecular-weight peptide inhibitors. CMA16 and its dimeric counterpart blocked the LAG-3/FGL1 interaction and showed a significant antitumor activity in a mouse CT26 tumor model. The combination of LAG-3 and PD-L1 peptide inhibitors further improved the antitumor response. The result of this study supports further development of CMA16 for cancer immunotherapy.

References: 1. Kraman, M., et al., FS118, a Bispecific Antibody Targeting LAG-3 and PD-L1, Enhances T-Cell Activation Resulting in Potent Antitumor Activity. Clin Cancer Res, 2020. 26(13): p. 3333-3344.
2. Liu, H., et al., Discovery of low-molecular weight anti-PD-L1 peptides for cancer immunotherapy. J Immunother Cancer, 2019. 7(1): p. 270.
3. Qian, W., et al., Fibrinogen-like protein 1 (FGL1): the next immune checkpoint target. Journal of Hematology & Oncology, 2021. 14(1): p. 147.
4. Wang, J., et al., Fibrinogen-like Protein 1 Is a Major Immune Inhibitory Ligand of LAG-3. Cell, 2019. 176(1-2): p. 334-347.e12.

Acknowledgments: This work was supported by the National Institutes of Health (NIH) [grant number R01CA231009].