Review. 26 July 2024. The MOE Key Laboratory of Spectrochemical Analysis and Instrumentation, Department of Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China. Author to whom correspondence should be addressed.
The landscape of cancer therapy has gained major impetus through the development of materials capable of selectively targeting cancer cells while sparing normal cells. Synthetic peptides are appealing as scaffolds for the creation of such materials. They are small in size, amenable to chemical synthesis and functionalization, and possess diverse chemical and structural space for modulating targeting properties. Here, we review some fundamental insights into the design, discovery, and evolution of peptide-based targeting agents, with a particular focus on two types of cancer cell targets: unique/overexpressed surface receptors and abnormal physiological properties. We highlight the cutting-edge strategies from the literature of the last two decades that demonstrate innovative approaches to constructing receptor-specific cyclic binders and stimulus-responsive targeting materials. Additionally, we discuss potential future directions for advancing this field, with the aim of pushing the frontiers of targeted cancer therapy forward.
Cancer remains one of the leading causes of death worldwide, with 20 million new cases expected annually by 2025. This underscores the need for highly effective cancer therapies [1]. Surgery, radiation therapy, chemotherapy, and combinations of these therapies dominate clinical practice. However, they often result in severe side effects and unintended toxicity due to their non-selective action against normal cells and tissues [2,3,4]. Immunotherapy is a transformative approach in this field by deploying our body’s own immune system as a personalized medicine to fight against cancer [5,6]. This can be achieved by targeting inhibitory pathways with immune checkpoint inhibitors, or activating pathways with chimeric antigen receptor immune cells or cell engagers. Compared to chemotherapy or radiation, immunotherapy typically has better action specificity with fewer side effects. In some cases, immunotherapy can induce durable responses, contrasting with conventional therapies where cancer may recur more frequently. Clearly, in almost all cancer therapies, it is critical to ensure therapeutic activity against cancer cells while sparing normal cells from harm. Paul Ehrlich’s “magic bullet” concept in the 1890s laid the foundation for targeted therapies by emphasizing the importance of selective-targeting capabilities [7]. Developing molecules or materials that specifically target cancer cells has thus become a highly rewarding endeavor. Cancer cells have a large number of unique or overexpressed surface receptors compared to normal cells. These proteins serve as prime molecular targets for the design or discovery of receptor-specific binding molecules. These binders can facilitate targeted cargo delivery, enable immune checkpoint inhibition, and recruit immune and effector cells, thereby advancing both cancer therapy and diagnostics. In addition, cancer cells exhibit aberrant physiological properties such as overexpressed enzymes, elevated redox potentials, and acidic pH conditions. Effectively targeting these features, as opposed to the receptor-based approaches, is also critical for distinguishing cancer cells from normal cells in therapeutic interventions.
Monoclonal antibodies (mAbs) are widely used because of their exceptional binding specificity and high affinity for cell-surface receptors, making them valuable for generating cell-targeting binders. However, their biological production is costly and can lead to variability in potency between batches. The large size of mAbs may also pose challenges in vivo, such as inadequate pharmacokinetics and limited tissue penetration [8,9,10,11]. In contrast, nucleic acid aptamers—a short, single strand of DNA or RNA—function like chemical antibodies. Like antibodies, aptamers can specifically recognize molecular targets based on defined nucleotide sequences and conformations. The SELEX random library technique has been used to generate numerous high-affinity aptamers that selectively bind to various cellular targets [12,13,14]. Aptamer-based antagonists and aptamer–drug/toxin conjugates have been developed for cancer treatment, as reviewed in detail elsewhere [15,16,17].
Here, we highlight peptides as promising candidates for cancer cell-targeting agents. Compared to mAbs, peptides offer distinct advantages: they are small in size, amenable to chemical synthesis and functionalization, exhibit minimal batch-to-batch variability, possess low immunogenicity, and have an extended shelf life [18,19,20]. Peptides also offer greater chemical diversity for modulating targeting properties compared to nucleic acid aptamers, thanks to the wide range of natural and unnatural amino acids available. Their diverse secondary and higher order structures further expand the scope for tailoring chemical distribution and folding or assembly behaviors. As shown in recent reviews, peptides have been increasingly explored for cancer treatments and diagnostics [20,21,22,23,24]. Previous reviews have systematically discussed their therapeutic effects in different cell types and ways to improve their in vivo efficacy. However, limited attention has been paid to the approaches for molecular design and the search for cancer cell-targeting peptides. Practical methods to correlate peptide sequence-dependent folding and assembly behaviors with cancer-related physiological properties remain elusive [25,26,27]. This mini-review illustrates selected studies to show cutting-edge strategies in the discovery and design of peptide-based binders and dynamic peptide materials, and their receptors and physiological signals, respectively. By leveraging the rich molecular codes encoded in peptides, this review promises to reliably and predictably design targeting agents, advancing targeted cancer therapy.
Cancer cells possess oncogenic aberrations that promote abnormal proliferation, migration, and the evasion of immune surveillance [28,29]. In particular, unique/overexpressed membrane receptors have become prime targets for distinguishing cancer cells from their normal counterparts [20,30,31,32]. Early efforts in developing receptor-specific binding molecules centered on structure-guided design by exploring natural proteins that engage with target receptors. For example, integrins play a central role in cell adhesion to the extracellular matrix (ECM), facilitating cellular motility and invasion [33]. The well-known RGD peptide was originally derived from the sequence of fibronectin, an abundant ECM protein [34,35]. RGD has been identified as a key interacting motif with integrin heterodimers, such as α 5 β 1, α V β 3, α V β 6, and α II β 3. Consequently, a variety of synthetic RGD peptides and their derivatives have been constructed as potent binders for integrin-overexpressed cancer cells in malignancies, such as melanoma, glioblastoma, and breast, prostate, and ovarian malignancies [36,37,38]. This approach is time-consuming and highly dependent on the availability of high-resolution structural information on the protein-receptor complex. In contrast, techniques such as phage display [39], and mRNA display [40] enable the screening of large numbers of random peptides against nearly any given molecular target. For example, the overexpression of human epidermal growth factor receptor 2 (HER2) on cancer cells triggers receptor homodimerization and clustering. This activates downstream MAPK and PI3K pathways to drive cell proliferation, growth, and anti-apoptosis [41]. Quinn and coworkers used a random 6-amino-acid peptide bacteriophage display library to find the HER2-specific peptide (KCCYSL) with a dissociation constant (K D) in the range of hundreds of micromolar [42]. In a separate study, Sioud and colleagues used a phage display biopanning technique to identify a HER2 binder (LTVSPWY) with a remarkable K D value in the nanomolar range [43].
Cyclic peptides dominate in cell-related molecular binders based on the literature of the last two decades [44,45]. Studies suggest that cyclization of linear RGD peptides can significantly increase their binding affinity to α V β 3 receptors from several micromolar to nanomolar levels [46,47]. The increase is attributed to reduced chain flexibility, which facilitates peptide–target engagements by minimizing entropic loss during binding. In addition, cyclization of many linear peptide-based binders has resulted in improved proteolytic resistance during in vivo cancer cell targeting. This is due to constrained backbone conformations that impede access to protease catalytic sites. Clearly, cyclization has emerged as a facile route to enhancing the targeting performance of peptide-based binders.
Numerous bioactive cyclic peptides found in nature maintain their three-dimensional structures through intramolecular disulfide bonds. Wu and coworkers were inspired to develop a series of cysteine-rich motifs, such as CXC and CPPC, to direct peptide cyclization. These motifs enable the programmable formation of intramolecular disulfide bonds, leading to single-, bi-, and multi-cyclic architectures with predictable topologies (Figure 1A) [48,49]. In addition to the disulfide-based approach, various chemical conjugation and crosslinking strategies have been developed, allowing the creation of cyclic scaffolds with exceptional efficiency [50]. The screening library (such as phage [39] and mRNA display [40]) has been extensively used to search for unnatural cyclic binders that target given molecular entities. For example, Heinis, Winter and coworkers presented a strategy using phage display to screen and isolate cyclic binders for various molecular targets [45,51]. Cysteine-containing peptides presented on phage tips were cyclized using thiol-reactive chemical linkers, resulting in redox-stable conformations as opposed to disulfide-bridged cyclic structures (Figure 1B) [52]. Suga and coworkers established the random nonstandard peptide integrated discovery (RaPID) platform to generate the library of thioether-closed macrocyclic peptides containing non-proteinogenic amino acids (Figure 1C) [53,54]. This platform integrates genetic code reprogramming using a flexible in vitro translation (FIT) system with mRNA display, greatly expanding the repertoire of cyclic binders [55]. An increasing number of cyclic peptides with nanomolar and even picomolar binding affinities have been generated for a wide range of targets, including cancer cell-surface receptors.
Peptide-based binders facilitate cancer therapy by guiding the delivery of chemotherapeutics, radioisotopes, and other cytotoxic agents. They also serve to inhibit and antagonize cell-surface receptors as well as intracellular proteins [56,57,58]. Among them, many binder-based drugs and peptide–drug/toxin conjugates have been approved or are under clinical evaluation (Table 1). For instance, romidepsin [59], a bicyclic peptide isolated from natural fermentation products, has received FDA approval for the treatment of cutaneous T-cell lymphoma. This prodrug features an intrapeptide disulfide bond that is reduced within the cell. The active peptide form is then released to specifically target and inhibit histone deacetylase enzymes. Another notable advancement is 177 Lu DOA-TATE [60], the first FDA-approved radiopharmaceutical for the treatment of gastroenteropancreatic neuroendocrine tumors. The molecule conjugates a radionuclide to octreotide, and a cyclic peptide and somatostatin analogue. The octreotide selectively delivers ionizing radiation to cancer cells bearing overexpressed somatostatin receptors. Similarly, 177 Lu-AB-3PRGD is in a Phase I trial to determine its effectiveness in various advanced solid tumors (NCT06375564) [61]. This conjugate employs a dimeric RGD peptide with 3 PEG 4 linkers for high-avidity multivalent targeting of α V β 3 on cancer cells. In addition to these targeting peptides derived from natural protein sources, screened synthetic binders are also being explored in preclinical or clinical trials. A notable example is the bicycle toxin conjugate (BTC) BT8009, which combines a nectin-4 targeting bicyclic peptide with the cytotoxin monomethyl auristatin E [62]. This conjugate has shown promising anticancer activity in patients with advanced or metastatic malignancies, including urothelial cancer (NCT04561362, NCT06225596). Comprehensive lists of binder-based therapeutics in clinical trials and approved for marketing have been presented elsewhere [63,64]. Concurrent with the rapid development of cancer immunotherapy, peptide-based binders have also been harnessed as cell engagers to orchestrate cancer elimination using immune effector cells. For example, Wang and coauthors designed a bispecific triblock peptide AKMGEGGWGANDY-GNNQQNY-RGD to facilitate interactions between T cells and cancer cells [65]. The first and third blocks of the peptide selectively targeted CD3 on T cells and integrins on MCF-7 cells, respectively. Upon RGD-integrin interaction, the fibril-forming sequence (the second block) drove the clustering of the cell-surface peptides, activating T cells and culminating in the cytolysis of cancer cells.
Figure 1. (A) Schematics of disulfide pairing of CPIEC motifs and the oxidation of peptides containing two CPIEC motifs [49]. Reprinted (adapted) with permission from 49. Copyright 2023 American Chemical Society. (B) Phage selection of bicyclic peptides using a thiol-reactive chemical linker [52]. Reprinted (adapted) with permission from 52. Copyright 2017 American Chemical Society. (C) mRNA selection of cyclic peptides on the RaPID platform [54]. Reprinted (adapted) with permission from 54. Copyright 2019 American Chemical Society.
