Cystine knot peptides can be successfully radiolabeled with (177)Lu for potential therapeutic applications. Knottin 2.5F labeled with (177)Lu exhibits favorable distribution in murine U87MG xenograft model; thus, it is a promising agent for radionuclide therapy of integrin-positive tumors.

Recently, a truncated form of the agouti-related protein (AgRP), a 4-kDa cystine-knot peptide of human origin, was used as a scaffold to engineer mutants that bound to αvβ3 integrin with high affinity and specificity. In this study, we evaluated the potential of engineered integrin-binding AgRP peptides for use as cancer imaging agents in living subjects. Methods Engineered AgRP peptides were prepared by solid-phase peptide synthesis and were folded in vitro and purified by reversed-phase high-performance liquid chromatography. Competition assays were used to measure the relative binding affinities of engineered AgRP peptides for integrin receptors expressed on the surface of U87MG glioblastoma cells. The highest-affinity mutant, AgRP clone 7C, was site-specifically conjugated with 1,4,7,10-tetraazacyclododecane-N,N′,N′′N′′′-tetraacetic acid (DOTA). The resulting bioconjugate, DOTA-AgRP-7C, was radiolabeled with 64Cu for biodistribution analysis and small-animal PET studies in mice bearing U87MG tumor xenografts. In addition to serum stability, the in vivo metabolic stability of 64Cu-DOTA-AgRP-7C was assessed after injection and probe recovery from mouse kidney, liver, tumor, and urine. Results AgRP-7C and DOTAAgRP-7C bound with high affinity to integrin receptors expressed on U87MG cells (half maximal inhibitory concentration values, 20 ± 4 and 14 ± 2 nM, respectively). DOTA-AgRP-7C was labeled with 64Cu with high radiochemical purity (>99%). In biodistribution and small-animal PET studies, 64Cu-DOTA-AgRP-7C displayed rapid blood clearance, good tumor uptake and retention (2.70 ± 0.93 percentage injected dose per gram [%ID/g] and 2.37 ± 1.04 %ID/g at 2 and 24 h, respectively), and high tumor-to-background tissue ratios. The integrin-binding specificity of 64Cu-DOTA-AgRP-7C was confirmed in vitro and in vivobyshowing thata large molar excessofthe unlabeled peptidomimetic c(RGDyK) could block probe binding and tumor uptake. Serum stability and in vivo metabolite assays demonstrated that engineered AgRP peptides are sufficiently stable for in vivo molecular imaging applications. Conclusion A radiolabeled version of the engineered AgRP peptide 7C showed promise as a PET agent for tumors that express the αvβ3 integrin. Collectively, these results validate AgRP-based cystine-knot peptides for use in vivo as molecular imaging agents and provide support for the general use of AgRP as a scaffold to develop targeting peptides, and hence diagnostics, against other tumor receptors.

Human hepatocellular carcinoma (HCC) is considered difficult to cure because it is resistant to radio-and chemotherapy and has a high recurrence rate after curative liver resection. Epidermal growth factor receptor variant III (EGFRvIII) has been reported to express in HCC tissues and cell lines. This article describes the efficacy of an anti-EGFRvIII monoclonal antibody (mAb CH12) in the treatment of HCC xenografts with EGFRvIII expression and the underlying mechanism of EGFRvIII as an oncogene in HCC. The results demonstrated that CH12 bound preferentially to EGFRvIII with a dissociation constant (K d ) of 1.346 nm/liter. In addition, CH12 induces strong antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity in Huh7-EGFRvIII (with exogenous expression of EGFRvIII) and SMMC-7721 (with endogenous expression of EGFRvIII) cells. Notably, CH12 significantly inhibited the growth of Huh7-EGFRvIII and SMMC-7721 xenografts in vivo with a growth inhibition ratio much higher than C225, a U. S. Food and Drug Administration-approved anti-EGFR antibody. Treatment of the two HCC xenografts with CH12 significantly suppressed tumor proliferation and angiogenesis. Mechanistically, in vivo treatment with CH12 reduced the phosphorylation of constitutively active EGFRvIII, Akt, and ERK. Down-regulation of the apoptotic protectors Bcl-x L , Bcl-2, and the cell cycle regulator cyclin D1, as well as up-regulation of the cell-cycle inhibitor p27, were also observed after in vivo CH12 treatment. Collectively, these results indicate that the monoclonal antibody CH12 is a promising therapeutic agent for HCC with EGFRvIII expression. Hepatocellular carcinoma (HCC)3 is the fifth most common cancer and the third most common cause of cancer-related death in the world (1). The cancer is usually diagnosed at a stage when the disease is already advanced and incurable. Surgery is the most effective treatment for HCC. However, tumor recurrence after a curative liver resection is high (2, 3). Adjuvant chemotherapy has not significantly improved survival of HCC patients (3). Sorafenib is the only targeted therapy approved by the U. S. Food and Drug Administration to treat HCC. Although sorafenib showed good tolerance in the studied populations, most patients in clinical practice suffer from underlying liver cirrhosis with impaired metabolic function and experience dose-limiting toxicities with the need to reduce the overall dose of sorafenib subsequently (4).The epidermal growth factor receptor (EGFR) has been successfully targeted for cancer therapy (5). EGFR expression in HCC has been reported (6). EGFR expression has been suggested to contribute to the aggressive growth characteristics of HCC tumors (7-9). EGFR overexpression has been demonstrated to be positively correlated with early tumor recurrence and a negative prognostic factor in poorly differentiated HCCs (6, 10). Hence, EGFR represent a promising target for developing innovative HCC treatment strategies. However, despite the clinical success of several EGFR-targ...

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Pullorum disease caused by Salmonella Pullorum remains an important disease for the poultry industry due to high morbidity and mortality in many countries. Phage therapy is becoming an alternative strategy to control multidrug-resistant Salmonella infections in young chicks. However, how bacteriophages affect the growth performance of chicks infected with S. Pullorum remains poorly understood. Herein, we assessed the therapeutic efficacy of Salmonella phage CKT1 against hypervirulent arthritis - causing S. Pullorum. The results showed that single phage treatment after hypervirulent S. Pullorum infection significantly improved body weight loss of chicks. Compared with enlarged liver and spleen in only Salmonella challenged group, phage administration substantially reduced the liver/body and spleen/body weight ratios, bacterial loads in organs and the degree of hepatic sinusoidal dilatation and congestion. Moreover, phage CKT1 can enter the organs of chicks and stay for at least 3 d in liver and spleen, and promote higher serum levels of IL-6 production within 6 d postinfection, indicating phage-induced bacterial lysis may be involved in inflammatory immune response to S. Pullorum infection. Analysis of the microbiome of gastrointestinal tract of chicks demonstrated that Salmonella challenge significantly reduced the relative abundances of Lachnoclostridium and Blautia, resulting in remarkably increased Escherichia-Shigella and Klebsiella becoming the predominant bacterial taxa. In contrast, the use of phage CKT1 significantly reduced Escherichia-Shigella and Klebsiella populations in intestine, permitting the proliferation of beneficial microbiota in Firmicutes including Lachnoclostridium, Ruminococcus, Lactobacillus , and Pseudoflavonifractor . In addition, phage alone treatments did not affect the normal gut microbiota structure of chicks, and phage therapy on Salmonella infected chicks increased bacteria species richness in the cecum. These results suggest that Salmonella phage CKT1 could improve growth performance of chicks challenged with S. Pullorum by normalizing the abnormal intestinal microbiome.