Yuanming Qi

Peptide-based cancer therapy: Opportunity and challenge

Cancer is one of the leading causes of death worldwide. Conventional cancer therapies mainly focus on mass cell killing without high specificity and often cause severe side effects and toxicities. Peptides are a novel class of anticancer agents that could specifically target cancer cells with lower toxicity to normal tissues, which will offer new opportunities for cancer prevention and treatment. Anticancer peptides face several therapeutic challenges. In this review, we present the sources and mechanisms of anticancer peptides and further discuss modification strategies to improve the anticancer effects of bioactive peptides.

Blocking of the PD‐1/PD‐L1 Interaction by a D‐Peptide Antagonist for Cancer Immunotherapy

Blockade of the protein-protein interaction between the transmembrane protein programmed cell death protein 1 (PD-1) and its ligand PD-L1 has emerged as a promising immunotherapy for treating cancers. Using the technology of mirror-image phage display, we developed the first hydrolysis-resistant D-peptide antagonists to target the PD-1/PD-L1 pathway. The optimized compound (D) PPA-1 could bind PD-L1 at an affinity of 0.51 μM in vitro. A blockade assay at the cellular level and tumor-bearing mice experiments indicated that (D) PPA-1 could also effectively disrupt the PD-1/PD-L1 interaction in vivo. Thus D-peptide antagonists may provide novel low-molecular-weight drug candidates for cancer immunotherapy.

Anti-tumor effects of a novel chimeric peptide on S180 and H22 xenografts bearing nude mice.

In recent years, many endogenous peptides have been identified by screening combinatory phage display peptide library, which play important roles in the process of angiogenesis. A heptapeptide, ATWLPPR, binds specifically to NRP-1 and selectively inhibits VEGF165 binding to VEGFR-2. Another heptapeptide, NLLMAAS, blocks both Ang-1 and Ang-2 binding to Tie-2 in a dose-dependent manner. In the present study, we aimed to connect ATWLPPR (V1) with NLLMAAS (V2) via a flexible linker, Ala-Ala, to reconstruct a novel peptide ATWLPPRAANLLMAAS (V3). We firstly investigated the anti-tumor and anti-angiogenic effects of peptide V3 on sarcoma S180 and hepatoma H22 bearing BALB/c nude mice. Mice were continuously subcutaneously administrated with normal saline, V1 (320microg/kg/d), V2 (320microg/kg/d), V1+V2 (320microg/kg/d), and V3 (160, 320 and 480microg/kg/d), for 7 days. Treatment with peptide V3 could significantly reduce the tumor weight and volume. Pathological examination showed that the tumors treated with peptide V3 had a larger region of necrosis than that of peptide V1, V2, and V1+V2 at the same dose. A significant decrease of microvessel density (MVD) in a dose-dependent manner was observed in each group of peptide V3. The results of pathological examination on normal tissue, lung, heart, liver, spleen, kidney and white blood cells showed that peptide V3 might have no significant toxicity. In conclusion, our results demonstrated that peptide V3 could be more effective on inhibiting tumor growth and angiogenesis than that of V1, V2, and V1+V2. Peptide V3 could be considered as a novel chimeric peptide with potent anti-tumor activity.

Identification of novel T cell epitopes from efflux pumps of Mycobacterium tuberculosis

Cytotoxic T lymphocytes (CTLs) play an important role in the immunity of Mycobacterium tuberculosis (Mtb) infection. In the present study, the identification of novel CTL epitopes from efflux pumps, Rv1258c and Rv1410c, was reported. Candidate native peptides and their analogues were predicted with prediction programs. Rv1410c-p510 (TLAPQVEPL) and Rv1410c-p510-1Y9V (YLAPQVEPV) showed potent binding affinity and stability towards HLA-A*0201 molecule. In enzyme-linked immunospot (ELISPOT) assay, the CTLs induced from peripheral blood mononuclear cells (PBMCs) by these peptides could release interferon-γ (IFN-γ) in at least one healthy donor (HLA-A*02(+), PPD(+)). In cytotoxicity assay in vitro and in vivo, the CTLs induced by Rv1410c-p510-1Y9V could specifically lyse peptide-loaded T2 cells. This is the first report to identify CTL epitopes from the efflux pumps of Mtb. The novel epitope identified could serve as candidate to the multivalent peptide vaccine against drug-resistant M. tuberculosis.

Identification of a novel cytotoxic T lymphocyte epitope from CFP21, a secreted protein of Mycobacterium tuberculosis.

CFP21 is a major secreted protein of Mycobacterium tuberculosis (Mtb) which is considered as a promising antigen for immunotherapy. To identify CFP21-derived HLA-A*0201 restricted epitopes, a series of native peptides and their analogues were predicted with prediction programs and synthesized. The native peptide, p134 (AVADHVAAV), and its analogues, p134-1Y2L and p134-1Y2L9L, showed potent binding affinity and stability to HLA-A*0201 molecule. In ELISPOT assay, the cytotoxic T lymphocytes (CTLs) induced by these peptides could release IFN-γ. In cytotoxicity assay, the CTLs induced by p134 and p134-1Y2L9L could specifically lyse peptide-loaded T2 cells. In these two assays, the native peptide, p134, showed the most potent activity. Our results indicated that p134 could be a novel epitope which could serve as a good candidate to develop peptide vaccines against M. tuberculosis.

Identification of a new broad-spectrum CD8+ T cell epitope from over-expressed antigen COX-2 in esophageal carcinoma.

Cyclooxygenase-2 (COX-2) has been found to be over-expressed in esophageal carcinoma (EC) and it could be considered as a potential tumor-associated antigen (TAA). In the present study, six candidate peptides from COX-2 were firstly predicted and synthesized. Among them, P(479) had the highest affinity and stability toward both HLA-A *0201 and HLA-A *03 molecules and it could significantly promote the IFN-gamma release. The cytotoxic T lymphocytes (CTLs) induced by P(479) could specifically lyse COX-2-expressed EC cell lines, EC-1 (HLA-A3 supertype) and EC-9706 (HLA-A2 supertype). These results suggested that P(479) as a novel broad-spectrum T cell epitope would be very useful in immunotherapy against esophageal carcinoma.

Antitumor activity of novel chimeric peptides derived from cyclinD/CDK4 and the protein transduction domain 4

CyclinD1/CDK4 and cyclinD3/CDK4 complexes are key regulators of the cell progression and therefore constitute promising targets for the design of anticancer agents. In the present study, the key peptide motifs were selected from these two complexes. Chimeric peptides with these peptides conjugated to the protein transduction domain 4 (PTD4) were designed and synthesized. The chimeric peptides, PTD4-D1, PTD4-D3, PTD4-K4 exhibited significant anti-proliferation effects on cancer cell lines. These peptides could compete with the cyclinD/CDK4 complex and induce the G1/S phase arrest and apoptosis of cancer cells. In the tumor challenge experiment, these peptides showed potent antitumor effects with no significant side effects. Our results suggested that these peptides could be served as novel leading compounds with potent antitumor activity.

Antioxidant and free radical-scavenging activity of the extracellular death factor in Escherichia coli

The extracellular death factor (EDF) is a newly discovered linear pentapeptide with the sequence of H-Asn-Asn-Trp-Asn-Asn-OH (NNWNN). It could act as a quorum-sensing molecule and participate in the mazEF-mediated cell death in Escherichia coli. In the present study, we firstly studied the antioxidant and free radical-scavenging activities of EDF. EDF could scavenge hydroxyl radicals in vitro and protect protein, lipid and DNA from being damaged by hydroxyl radicals. Our results indicated that this extracellular death factor might have dual effects during the programmed cell death process of Escherichia coli.

A novel peptide targeting Clec9a on dendritic cell for cancer immunotherapy

Dendritic cells (DCs) are professional antigen-presenting cells with antigen recognition molecules on the surface. Clec9a is selectively expressed on mouse CD8a+ DCs and CD103+ DCs subsets, which are functionally similar to human BDCA3+ DCs. It is reported that Clec9a is responsible for the antigen cross-presentation of these DC subsets. In the present study, by using phage display technique, we discovered a novel peptide WH, which can selectively bind to mouse Flt3L induced Clec9a+ DCs or Clec9a over-expressed HEK-293T cells. Furthermore, by using computer-aided docking model and mutation assay, we observed that Asp248 and Trp250 are two key residues for Clec9a to bind with peptide WH. When coupled with OVA257-264 epitope, peptide WH can significantly enhance the ability of Clec9a+ DCs to activate OVA-specific CD8+ T cells, which elicit strong ability to secret IFN-γ, express perforin and granzyme B mRNA. In B16-OVA lung metastasis mouse model, WH-OVA257-264 fusion peptide can also enhance the activation of CD8+ T cells and decrease the lung metastasis loci. All these results suggested that peptide WH could be considered as an antigen delivery carrier targeting Clec9a+ DCs for cancer immunotherapy.

In vitro and in vivo identification of a novel cytotoxic T lymphocyte epitope from Rv3425 of Mycobacterium tuberculosis

The identification of novel cytotoxic T lymphocyte (CTL) epitopes is important to analysis of the involvement of CD8+ T cells in Mycobacterium tuberculosis infection as well as to the development of peptide vaccines. In this study, a novel CTL epitope from region of difference 11 encoded antigen Rv3425 was identified. Epitopes were predicted by the reversal immunology approach. Rv3425‐p118 (LIASNVAGV) was identified as having relatively strong binding affinity and stability towards the HLA‐A*0201 molecule. Peripheral blood mononuclear cells pulsed by this peptide were able to release interferon‐γ in healthy donors (HLA‐A*02+ purified protein derivative+). In cytotoxicity assays in vitro and in vivo, Rv3425‐p118 induced CTLs to specifically lyse the target cells. Therefore, this epitope could provide a subunit component for designing vaccines against Mycobacterium tuberculosis.