*3.3. Potential Diagnostic and Therapeutic Applications of CgA-Derived Peptides That Interact with Integrins in Cancer*

The integrin αvβ6 is overexpressed by several types of cancer cells, such as head and neck squamous cell carcinoma, pancreatic ductal adenocarcinoma, breast, colon, liver, and ovarian cancers, and others [73,74,79–83]. This integrin modulates cancer cell invasion, inhibits apoptosis, and, importantly, is involved in the maturation of TGFβ1, a potent immunosuppressive cytokine. Increased expression levels of αvβ6 are prognostic indicators of poor survival in patients with various types of tumors [79,82,84–86], and various ligands of this integrin coupled to tumor imaging agents are currently being tested in cancer patients for tumor imaging purposes [87–91]; thus, the development of CgA-derived peptides capable of recognizing this integrin in tumors is of great experimental and clinical interest. Following this line of thought, experimental work has been carried out to obtain new peptides with higher affinity for αvβ6-integrin, starting from CgA39-63 as a lead compound. The model of CgA39-63/αvβ6 interactions, obtained by NMR and computational studies, allowed to predict that restoring the canonical RGDLXXL motif by replacing the glutamate (E) residue in the RGDERIL site of CgA39-63 with a leucine (L) may increase its affinity for αvβ6. Intriguingly, the replacement of E46 with L not only increased, as expected, the binding affinity for αvβ6, but, unexpectedly, also that for the integrin αvβ8 (Table 1); thus, the E46L replacement converted CgA39-63 into a bi-selective ligand of both αvβ6 and αvβ8 integrins (Ki: 1.6 ± 0.3 nM and 8.5 ± 3.7 nM, respectively) integrins [71]. Chemical "stapling" of the α-helix of the E46L-CgA39-63 mutant, by side-chain-to-side-chain cross linking with a triazole-bridge, further increased the affinity for both αvβ6 and αvβ8 (Ki: 0.6 ± 0.1 nM and 3.2 ± 1.2 nM, respectively) by stabilizing the α-helix [71]. Notably, the αvβ8 integrin represents another cell-surface receptor expressed by various carcinoma

cells [92–94]; thus, the mutated/chemically stapled peptide (called peptide **5a**) represents a strong bi-selective ligand for these integrins, which can be potentially exploited as a tumor-homing ligand for delivering imaging and anticancer compounds to αvβ6/αvβ8 single- or double-positive tumors, such as oral and skin squamous cell carcinoma [95] (see Figure 2 for a schematic representation of this concept). This hypothesis is supported by the results of a very recent study aimed at investigating the tumor-homing properties of compounds consisting of peptide **5a** coupled with IRDye 800 CW (a near-infrared fluorescent dye) or with 18F-NOTA (a label for positron emission tomography) [96]. This study showed that both conjugates can bind αvβ6 and αvβ8 with an affinity similar to that of the free peptide and that they can selectively recognize various αvβ6/αvβ8 single- or double-positive cancer cells, including cells from melanoma, pancreatic carcinoma, oral mucosa, prostate, and bladder cancer. Furthermore, biodistribution studies, performed with these conjugates in mice bearing orthotopic or subcutaneous αvβ6-positive pancreatic tumors, showed high target-specific uptake of fluorescence- and radio-labeled peptide by tumors [96]. Tumor-specific uptake of the fluorescent conjugate was also observed in mice bearing αvβ8-positive prostate tumors [96], confirming the hypothesis that peptide **5a** can home to αvβ6- and/or αvβ8-positive tumors.

**Figure 2.** Use of the CgA-derived peptide **5a** (stapled) for delivering imaging or therapeutic compounds to αvβ6/αvβ8 single- or double-positive tumors. The peptide **5a**, derived from the region 38–63 of human CgA (originally published in [96]) is characterized by the sequence CFETLRGDLRILSILRX1QNLX2KELQ, where X1 and X2 are propargylglycine and azidolysine residues, respectively, which form a triazole bridge after a click chemistry reaction, thereby increasing the α-helix stability. Peptide **5a** can be exploited for delivering radioactive or fluorescent imaging compounds to αvβ6/αvβ8 single- or double-positive tumors or for developing new therapeutic tumor-homing agents. The image in the right panel shows the radiotracer uptake in a mouse bearing a pancreatic tumor implanted subcutaneously (arrow), as assessed by PET/CT scan (originally published in [96]).

Remarkably, both αvβ6 and αvβ8 integrins (which are upregulated in many tumors and, in the case of αvβ8, also in tumor infiltrating Treg cells) can activate the latency associated peptide/TGFβ complex, through interactions of integrins with the RGD sites of the complex [73,83,92]. These interactions can lead to the local activation of TGFβ in the tumor microenvironment, a potent immunosuppressive mechanism that may contribute to tumor progression. Interestingly, in vitro studies have shown that peptide **5a** can inhibit the integrin-mediated TGFβ activation [96]. Thus, in principle, the peptide **5a** can be used not only as a ligand for delivering imaging or anticancer agents to αvβ6/αvβ8 single- or double-positive tumors, but also as a tumor-homing inhibitor of these TGFβ activators.

Finally, considering the role of αvβ6/αvβ8-mediated TGFβ activation in fibrosis [97] the dual targeting capability of peptide **5a** might be also exploited in the development of anti-fibrotic drugs. This is another hypothesis that deserves to be investigated.

### **4. Conclusions**

The results obtained so far suggest that integrins (particularly the integrin αvβ6) and neuropilin-1 are important receptors that mediate relevant pathophysiological functions of CgA and its fragments in angiogenesis, wound healing, and tumor growth. Experimental evidence indicates that these interactions may also represent important targets for cancer imaging and therapy. Although further work is necessary to clarify the receptor mechanisms of CgA and its fragments in the regulation of cardiovascular homeostasis, metabolism, and tumor growth, the results obtained so far highlight the complexity of the "CgA system", which consists of a multitude of CgA-derived peptides and various receptors. The complexity of this system is even higher if we consider that full-length CgA and some of its fragments show biphasic dose-response curves in angiogenesis assays, as well as in cardio-regulatory and tumor pre-clinical models, likely because of the activation of counterregulatory mechanisms at higher doses. These mechanisms are not clearly understood and, therefore, their full elucidation remains a challenge. A third level of complexity is related to the fact that CgA undergoes differential post-translational modifications in different cells and tissues, such as glycosylation, sulfation, and phosphorylation. As most of the studies carried out so far on the biological functions of CgA have been performed with recombinant or synthetic peptides lacking these modifications, the impact of these structural modifications on proteolytic cleavage, fragment generation, receptor recognition, and biological activity, remains to be investigated.

**Author Contributions:** A.C., G.A. and F.C. performed literature research, interpreted data, prepared figures, and wrote and approved the submitted manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Associazione Italiana per la Ricerca sul Cancro (AIRC) under IG 2019–ID. 23470 project–P.I. Angelo Corti and by Fondazione AIRC 5 per Mille 2019 (ID 22737) program, P.I. MC Bonini, Group Leader A. Corti.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** A.C. and F.C. are inventor of a patent on CgA-derived peptides and their use in cancer imaging.
