(T1330-04-23) Discovery of Anti-Transforming Growth Factor β Receptor II (TGFβRII) Cyclic Peptides

Purpose: Transforming Growth Factor β (TGFβ) is a cytokine over-expressed in most cancers, including colon cancer and breast cancer. It plays a crucial role in regulating cancer pathogenesis. TGFβ diminishes the activation and cytotoxic function of effector CD8⁺ T cells and induces the differentiation of regulatory T cells (Tregs), thereby promoting immunosuppression. Moreover, it promotes metastasis and invasion by facilitating the epithelial-to-mesenchymal transition (EMT) process. TGFβ also remodels the tumor microenvironment by promoting angiogenesis, peritumoral collagen deposition, and cancer-associated fibroblast activation, leading to tumor fibrosis. These combined effects result in poor tumor infiltration by immune cells, leading to "cold" tumors. Furthermore, these effects of TGFβ hinder the efficacy and promote resistance to immunotherapies and chemotherapies. Blocking the TGFβ pathway has been shown to revert immune exclusion and enhances the infiltration of CD8⁺ T cells and natural killer cells into tumors. Binding of TGFβ ligands to the TGFβRII receptor is an essential step in initiating the TGFβ signaling pathway. However, progress in developing antagonists for blocking the TGFβRII has been limited. This study investigates the use of low molecular weight cyclic peptides to block the TGFβ/TGFβRII interaction. Low molecular weight peptides have several advantages over antibodies and small molecules, such as high selectivity, low immunogenicity, improved tumor penetration due to their small size, and ease of production.

Methods: Our laboratory utilized an innovative phage display biopanning procedure to discover small cyclic anti-TGFβRII peptides using an in-house generated cyclic peptide library. The peptides were synthesized via solid phase peptide synthesis using Fmoc-based chemistry and the cyclization via disulfide bond formation on a PurePepTM Chorus peptide synthesizer. The peptides’ mass and purity were confirmed using LC/MS. The peptides' binding affinity, and IC₅₀ were determined using competitive ELISA assays and Surface Plasmon Resonance (SPR). We also determined the serum stability of the peptides. The antitumor activity of the best peptide was examined in an MC38 subcutaneous murine colon cancer model. Once tumors reached 50-100 mm³, the mice were randomized into four groups (8 mice per group, 4 males, 4 females) for treatments. The tumor sizes were measured using a digital caliper.

Results: Through phage display biopanning, we identified anti-TGFβRII peptides that can block the interaction between TGFβ1 and TGFβRII. We optimized the peptides using alanine scanning to improve the blocking efficiency. The CMCP5 R3 peptide showed the highest blocking efficiency with an IC₅₀ of 300 nM using an ELISA assay and 630 nM using competitive SPR. Serum stability tests showed high metabolic stability with a half-life of 2.35 hours in human serum. Preliminary in-vitro studies indicated that it can neutralize the TGFβ1-induced proliferation of cancer-associated fibroblasts. The CMCP5 R3 peptide demonstrated significant antitumor activity in MC8-bearing mice and exhibited better activity than the anti-PDL1 peptide previously developed in our laboratory.

Conclusion: In this study, we utilized phage display biopanning to identify and optimize low molecular weight cyclic peptide inhibitors. The CMCP5 R3 peptide successfully inhibited the TGFβ/TGFβRII interaction and exhibited significant antitumor activity in a mouse colon cancer MC38 model, which is poorly responsive to anti-PDL1 peptide inhibitors. These results further support the use of anti-TGFβRII peptide as therapeutic agents for modulating the tumor microenvironment and treating TGFβRII-dependent cancers.

References: 1. Fetse, J. et al. Discovery of Cyclic Peptide Inhibitors Targeting PD-L1 for Cancer Immunotherapy. J. Med. Chem. 65, 12002–12013 (2022).
2. Liu, H. et al. Discovery of low-molecular weight anti-PD-L1 peptides for cancer immunotherapy. J. Immunother. Cancer 7, 270 (2019).
3. Colak, S. & ten Dijke, P. Targeting TGF-β Signaling in Cancer. Trends in Cancer 3, 56–71 (2017).
4. Derynck, R., Turley, S. J. & Akhurst, R. J. TGFβ biology in cancer progression and immunotherapy. Nature Reviews Clinical Oncology vol. 18 9–34 (2021).
5. Zhong, Z. et al. Anti–transforming growth factor β receptor II antibody has therapeutic efficacy against primary tumor growth and metastasis through multieffects on cancer, stroma, and immune cells. Clinical Cancer Research 16, 1191–1205 (2010).

Acknowledgements: This work was supported by the National Institutes of Health (NIH) [grant number R01CA231009 and R01AA021510].