The biologically functional identification of a novel TIM3-binding peptide P26 in vitro and in vivo

Purpose Recent studies have shown that TIM3 plays an important role in T-cell failure, which is closely related to the resistance to anti-programmed cell death protein 1 (PD-1) treatment. However, there have been no reports on the application of peptide blockers to TIM3. In this study, we endeavored to identify the in vitro and in vivo anti-tumor activities of a TIM3-targeting peptide screened from the phage peptide library. Methods Phage display peptide library technology, surface plasmon resonance, flow cytometry, and mixed lymphocyte reaction were utilized to screen and demonstrate the bioactivities of P26, a TIM3-targeting peptide. Meanwhile, tumor growth assay was performed to evaluate the anti-tumor effect of P26. Results In terms of affinity, we demonstrated that P26 specifically binds to TIM3 at the cellular and molecular levels, which therefore blocks the interaction between TIM3 and Galectin-9 (Gal-9) and competes with Gal-9 to bind TIM3. Additionally, P26 significantly increases T-cell activity and elevates IFN-γ and IL-2 levels in a dose-dependent manner. Notably, P26 also counteracts Gal-9-mediated T-cell suppression. More importantly, P26 can inhibit growth of MC38-hPD-L1 tumor in mice. Conclusions P26, as a novel TIM3-binding peptide, has the ideal bioactivity connecting to TIM3 and the potential prospect of application in immunotherapy as an alternative or adjuvant to existing agents.

Screening and Identification of a Phage Display Derived Peptide That Specifically Binds to the CD44 Protein Region Encoded by Variable Exons

CD44, especially the isoforms with variable exons (CD44v), is a promising biomarker for the detection of cancer. To develop a CD44v-specific probe, we screened a 7-mer phage peptide library against the CD44v3-v10 protein using an improved subtractive method. The consensus sequences with the highest frequency (designated CV-1) emerged after four rounds of panning. The binding affinity and specificity of the CV-1 phage and the synthesized peptide for the region of CD44 encoded by the variable exons were confirmed using enzyme-linked immunosorbent assay and competitive inhibition assays. Furthermore, the binding of the CV-1 probe to gastric cancer cells and tissues was validated using immunofluorescence and immunohistochemistry assays. CV-1 sensitively and specifically bound to CD44v on cancer cells and tissues. Thus, CV-1 has the potential to serve as a promising probe for cancer molecular imaging and target therapy.

Role of molecular mimicry of hepatitis C virus protein with platelet GPIIIa in hepatitis C–related immunologic thrombocytopenia

Patients with HIV-1 immune-related thrombocytopenia (HIV-1–ITP) have a unique Ab against platelet GPIIIa49-66 capable of inducing oxidative platelet fragmentation in the absence of complement. HIV-1–seropositive drug abusers are more prone to develop immune thrombocytopenia than non–drug abusers and have a higher coinfection with hepatitis C virus (HCV) than non–drug abusers (90% vs 30%). Molecular mimicry was sought by screening a phage peptide library with anti–GPIIIa49-66 antibody as bait for peptides sharing homology sequences with HCV. Several phage peptide clones had 70% homology with HCV protein. Sera from dually infected thrombocytopenic patients with HCV and HIV-ITP reacted strongly with 4 nonconserved peptides from HCV core envelope 1. Reactivity correlated inversely with platelet count (r2 = 0.7, P < .01). Ab raised against peptide PHC09 in GPIIIa−/− mice induced thrombocytopenia in wild-type mice. Affinity-purified IgG against PHC09 induced oxidative platelet fragmentation in vitro. Drug abusers dually infected with HCV and HIV-1 had a greater incidence and severity of thrombocytopenia as well as titer of anti–GPIIIa49-66/PHC09 Ab. NZB/W F1 mice injected with recombinant core envelope 1 developed Ab versus PHC09 and significantly decreased their platelet count (P < .001). Thus, HCV core envelope 1 can induce thrombocytopenia by molecular mimicry with GPIIIa49-66.

Antimicrobial Activity of Novel Dendrimeric Peptides Obtained by Phage Display Selection and Rational Modification

ABSTRACT A large 10-mer phage peptide library was panned against whole Escherichia coli cells, and an antimicrobial peptide (QEKIRVRLSA) was selected. The peptide was synthesized in monomeric and dendrimeric tetrabranched form (multiple antigen peptide [MAP]), which generally allows a dramatic increase of peptide stability to peptidases and proteases. The antibacterial activity of the dendrimeric peptide against E. coli was much higher than that of the monomeric form. Modification of the original sequence, by residue substitution or sequence shortening, produced three different MAPs, M4 (QAKIRVRLSA), M5 (KIRVRLSA), and M6 (QKKIRVRLSA) with enhanced stability to natural degradation and antimicrobial activity against a large panel of gram-negative bacteria. The MICs of the most potent peptide, M6, were as low as 4 to 8 μg/ml against recent clinical isolates of multidrug-resistant Pseudomonas aeruginosa and members of the Enterobacteriaceae. The same dendrimeric peptides showed high stability to blood proteases, low hemolytic activity, and low cytotoxic effects on eukaryotic cells, making them promising candidates for the development of new antibacterial drugs.