A DNA vaccine expressing an optimized secreted FAPα induces enhanced anti-tumor activity by altering the tumor microenvironment in a murine model of breast cancer

Cancer-associated fibroblasts (CAFs), major components of the tumor microenvironment (TME), promote tumor growth and metastasis and inhibit the anti-tumor immune response. We previously constructed a DNA vaccine expressing human FAPα, which is highly expressed by CAFs, to target these cells in the TME, and observed limited anti-tumor effects in the 4T1 breast cancer model. When the treatment time was delayed until tumor nodes formed, the anti-tumor effect of the vaccine completely disappeared. In this study, to improve the safety and efficacy, we constructed a new FAPα-targeted vaccine containing only the extracellular domain of human FAPα with a tissue plasminogen activator signal sequence for enhanced antigen secretion and immunogenicity. The number of CAFs was more effectively reduced by CD8⁺ T cells induced by the new vaccine. This resulted in decreases in CCL2 and CXCL12 expression, leading to a significant decrease in the ratio of myeloid-derived suppressor cells in the TME. Moreover, when mice were treated after the establishment of tumors, the vaccine could still delay tumor growth. To facilitate the future application of the vaccine in clinical trials, we further optimized the gene codons and reduced the homology between the vaccine and the original sequence, which may be convenient for evaluating the vaccine distribution in the human body. These results indicated that the new FAPα-targeted vaccine expressing an optimized secreted human FAPα induced enhanced anti-tumor activity by reducing the number of FAPα⁺ CAFs and enhancing the recruitment of effector T cells in the 4T1 tumor model mice.     

Increased stem cell somatic mutation in the non-neoplastic colorectal mucosa of patients with familial adenomatous polyposis

Colorectal tumorigenesis in familial adenomatous polyposis (FAP) results from somatic mutation of either the normal APC allele or another growth control gene in epithelial cells bearing a germline APC defect. The rate at which tumors develop is therefore dependent on the somatic mutation frequency; it is not known whether this is normal or elevated in FAP. We aimed to quantify stem cell somatic mutation in FAP, comparing it with hereditary nonpolyposis colorectal cancer (HNPCC) and Crohn's disease (CD). Stem cell somatic mutation frequency was studied in 47 FAP patients, 5 HNPCC patients, and 13 CD patients, all younger than 49 years, by quantifying crypt-restricted loss of O-acetyltransferase activity in sections of morphologically normal colonic mucosa from individuals heterozygous for this monogenically inherited polymorphism. Median stem cell somatic mutation frequency was significantly higher in FAP than HNPCC (4.2 × 10⁻⁴v 1.4 × 10⁻⁴, Mann-Whitney U, P < .02). The level in CD (4.0 × 10⁻⁴) was similar to FAR Mutated crypts occurred in groups more frequently in FAP (22%) than HNPCC (12%) or CD (10%), suggesting an increase in stem cell division associated with crypt fission in FAP. We conclude that stem cell somatic mutation frequency is raised in non-neoplastic colorectal mucosa in FAR This is probably related to increased stem cell proliferation and contributes to the high rate of tumor formation in this condition.     

Immunohistochemical characteristics of duodenal adenomas in familial adenomatous polyposis with special reference to cell kinetics

The duodenum is the second most frequent site of cancer in patients with familial adenomatous polyposis (FAP). The main objective of this study was to evaluate the cell kinetics in duodenal and ampullary adenomas in FAP. The endoscopic and biopsy findings of duodenal adenomas in 22 FAP subjects and 18 non-FAP subjects were compared. Adenomas in FAP included 15 ampullary adenomas and 17 nonampullary adenomas. The cell kinetics was evaluated by immunohistochemistry for Ki-67, p53, bcl-2, and cyclooxygenase 2 (COX2), and the apoptotic index (AI) as determined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) method. Any correlations between the indices for cell kinetics and the endoscopic findings were identified. All 50 adenomas were histologically verified to be tubular adenoma with low-grade dysplasia. Neither the expression of Ki-67, p53, bcl-2, and COX2 nor the AI differed substantially between FAP and non-FAP subjects. In patients with FAP, duodenal adenoma tended to have a higher Ki-67-labeling index than the ampullary adenoma (54.3 +/- 11.3 versus 46.8 +/- 12.7; .05 < P < .1). In addition, the Ki-67-labeling index in endoscopically normal or slightly enlarged ampullary adenoma was significantly higher than that in markedly enlarged ampullary adenoma (51.8 +/- 11.4 versus 39.4 +/- 11.3; P < .05). Duodenal adenoma in FAP subjects was not found to have a higher proliferative activity or a smaller degree of apoptosis compared with those in non-FAP subjects. The smaller proliferative activity in larger ampullary adenoma may thus be related to the static nature of ampullary adenoma in FAP.     

5′-Cytosine DNA-methyltransferase mRNA levels in hereditary colon carcinoma

 DNA methylation plays an important part in the regulation of gene expression. Alterations in DNA methylation in tumours have been reported and have been used to generate hypotheses about mutagenesis and silencing of tumour suppressor genes. However, the underlying mechanism is still poorly understood, and conflicting data on the levels of overexpression of 5′-cytosine DNA methyltransferase in sporadic colon carcinoma have been published. We used a competitive RT-PCR assay for quantification of mRNA of 5′-cytosine DNA methyltransferase in colon biopsies obtained from patients with hereditary colon carcinoma syndromes and compared the results with those obtained in a control group. No significant difference was found between the flat mucosa of FAP patients and the mucosa of the control group. In FAP and HNPCC patients, the 5′-cytosine DNA methyltransferase mRNA levels of adenomas were significantly higher (P<0.05) than of flat mucosa in the same group, but both showed great variability from patient to patient. Our findings suggest that the mRNA levels of methyltransferase cannot be used as predictive marker for screening in families affected by hereditary colon carcinoma. Received: 20 July 1998 / Accepted: 21 September 1998     

An ultra-high-affinity small organic ligand of fibroblast activation protein for tumor-targeting applications

We describe the development of OncoFAP, an ultra-high-affinity ligand of fibroblast activation protein (FAP) for targeting applications with pan-tumoral potential. OncoFAP binds to human FAP with affinity in the subnanomolar concentration range and cross-reacts with the murine isoform of the protein. We generated various fluorescent and radiolabeled derivatives of OncoFAP in order to study biodistribution properties and tumor-targeting performance in preclinical models. Fluorescent derivatives selectively localized in FAP-positive tumors implanted in nude mice with a rapid and homogeneous penetration within the neoplastic tissue. Quantitative in vivo biodistribution studies with a lutetium-177–labeled derivative of OncoFAP revealed a preferential localization in tumors at doses of up to 1,000 nmol/kg. More than 30% of the injected dose had already accumulated in 1 g of tumor 10 min after intravenous injection and persisted for at least 3 h with excellent tumor-to-organ ratios. OncoFAP also served as a modular component for the generation of nonradioactive therapeutic products. A fluorescein conjugate mediated a potent and FAP-dependent tumor cell killing activity in combination with chimeric antigen receptor (CAR) T cells specific to fluorescein. Similarly, a conjugate of OncoFAP with the monomethyl auristatin E-based Vedotin payload was well tolerated and cured tumor-bearing mice in combination with a clinical-stage antibody-interleukin-2 fusion. Collectively, these data support the development of OncoFAP-based products for tumor-targeting applications in patients with cancer.

Small organic ligands which selectively bind with high affinity to tumor-associated antigens are increasingly applied as targeting delivery vehicles of small payloads such as radionuclides (1, 2), drugs (3–5), and fluorophores (6, 7) to tumor sites. In principle, the use of small ligands for targeting applications offers several advantages compared to intact immunoglobulins, including superior penetration of solid neoplastic lesions (8), lower immunogenicity (9), and a reduced cost of goods (10). Low molecular weight compounds may reach their target in vivo in a matter of seconds, thanks to rapid extravasation after intravenous administration (8). A strikingly selective accumulation of small ligands in neoplastic masses has been demonstrated for a small number of targets including somatostatin receptor type 2 (SSTR-2) (11), prostate-specific membrane antigen (PSMA) (12), and carbonic anhydrase IX (CAIX) (13), for which high-affinity small organic ligands are available. Those ligands are typically specific for defined tumor entities, such as neuroendocrine tumors (11), prostate cancer (3), and clear cell renal cell carcinoma (2).¹⁷⁷Lu-DOTATATE (Lutathera), a small-molecule product targeting SSTR-2, has been approved based on phase III data in which a clinically meaningful 82% reduction in the risk of disease progression or death was demonstrated in patients with gastroenteropancreatic neuroendocrine tumors (GEP-NETs) (14). Similar data are expected from the currently ongoing phase III VISION trial for ¹⁷⁷Lu-PSMA-617 (clinical trial no. {"type":"clinical-trial","attrs":{"text":"NCT03511664","term_id":"NCT03511664"}}NCT03511664), a radiolabeled small molecule that binds with high affinity to PSMA and that enables targeted beta particle therapy in metastatic castration-resistant prostate cancer patients (15). PHC-102, a ^99mTc-labeled small-molecule derivative targeting CAIX, exhibited favorable uptake in primary and metastatic lesions in patients with renal cell carcinoma (RCC) (2). In light of the promising performance of small organic ligands, it would be desirable to discover and develop small molecules with a broader tumor-targeting potential, therefore covering multiple cancer types.Fibroblast activation protein (FAP) is a type II integral membrane serine protease which is abundantly expressed in the stroma of more than 90% of the epithelial cancers, including malignant breast, colorectal, skin, prostate, and pancreatic cancers (16, 17), while exhibiting a restricted expression in normal adult tissues (18, 19). Haberkorn and coworkers (1, 20, 21) have recently described a series of FAP ligands capable of selective accumulation in FAP-positive tumors in mice and in patients. One of these products (named FAPI-04) showed impressive tumor to background ratios at early time points (i.e., few hours after administration) in a broad range of different cancer types in patients. More than 28 tumor types including breast, lung, pancreatic, head and neck, esophagus, and colorectal cancer presented a remarkably high uptake of a FAP-targeted small molecule labeled with gallium-68 (1, 20, 21). For this reason, FAP has recently been dubbed as “the next billion-dollar target for theranostic products” (22).Here, we describe how the chemical modification of a quinoline moiety in position 8 led to the discovery of OncoFAP, a small organic FAP ligand with a dissociation constant in the subnanomolar concentration range. OncoFAP exhibited a strikingly selective and efficient tumor-targeting performance when equipped with various types of payloads, including radionuclides, fluorophores, and cytotoxic drugs. The targeting delivery of radionuclides to solid tumors is rapidly gaining in popularity, as it may open theranostic opportunities, associated with the use of gallium-68 for positron emission tomography (PET) imaging and of lutetium-177 for therapeutic applications (23). The delivery of fluorescein to tumors enables the conditional activation of chimeric antigen receptor (CAR) T cells, which display a potent biocidal activity only in the presence of fluorescein-labeled adaptor molecules specific to a tumor antigen (24, 25). Finally, small-molecule–drug conjugates (SMDCs) promise to represent a valid alternative to antibody–drug conjugates for cancer therapy, with better tumor penetration and a lower cost of goods (8, 26, 27).     

Probiotics in functional bowel disorders

Functional bowel disorders (FBDs) are the most common gastrointestinal (GI) disorders seen by gastroenterologists and primary care physicians. The disorders affect patients functioning and quality of life (QOL) and are associated with significant healthcare burden. The current theory regarding the development of FBDs suggests brain-gut axis dysfunctions associated abnormal GI motility and sensation. Recent data suggest that alterations in the intestinal microbiota may have a role in the pathogenesis of FBDs; or at least have the potential to affect intestinal functions that are thought to be relevant to the development of functional GI symptoms. This has led to growing interest of healthcare providers and patients in targeting the intestinal microbiota for the treatment of FBDs. In this article we discuss the potential role probiotic interventions in the treatment of FBDs. We review the evidence from pre-clinical and clinical studies and discuss the current recommendations for the use of probiotics for FBDs in clinical practice.     

Safety assessment for octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)-propionate (CAS Reg. No. 2082-79-3) from use in food contact applications

Octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate (CAS Reg. No. 2082-79-3), currently marketed as Irganox 1076 (I-76), is a sterically hindered phenolic antioxidant used in a variety of organic substrates, including those used in the manufacture of food contact articles. In 2012, the US Food and Drug Administration (USFDA), Office of Food Additive Safety (OFAS), initiated a post-market re-evaluation of the food contact applications of I-76. This project aimed to ensure that current dietary exposures from the use of I-76 in food contact articles are accurately captured and the safety assessment considered all relevant and available toxicological information. To accomplish these aims, the USFDA reviewed the available toxicological studies and chemistry information on food contact applications of I-76. Based on this in-depth analysis, a NOAEL of 64 mg/kg-bw/d (female rats) from a chronic rat study and a cumulative estimated dietary intake (CEDI) of 4.5 mg/p/d, was used to calculate a margin of exposure (MOE) of ∼850. We concluded that the previous and current exposure levels provide an adequate margin of safety (MOS) and remain protective of human health for the regulated uses.     

Glutathione-S-transferase P1-1 protects aberrant crypt foci from apoptosis induced by deoxycholic acid

BACKGROUND & AIMS: Aberrant crypt foci, precursors of colonic adenoma, are frequently positive for glutathione-S-transferase P1-1. Because deoxycholic acid is an apoptosis-inducing xenobiotic in the colon, we examined the possibility that aberrant crypt foci, through the cytoprotecting function of glutathione-S-transferase P1-1, resist deoxycholic acid-induced apoptosis, thereby surviving to become adenomas and subsequently cancer. METHODS: Glutathione-S-transferase P1-1 or cyclooxygenase-2 expression and the percentage of apoptotic cells in aberrant crypt foci were examined by immunohistochemistry and by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling, respectively. Glutathione-S-transferase P1-1 was transfected into colon cancer cells (M7609) and human lung fibroblasts, and deoxycholic acid-induced apoptosis was evaluated by a dye-uptake assay and flow cytometry. Binding of deoxycholic acid to glutathione-S-transferase P1-1 was analyzed by circular dichroism and immunoprecipitation. Caspase activities were determined by colorimetric protease assay, and sulindac binding to glutathione-S-transferase P1-1 was determined by inhibition assay of glutathione-S-transferase P1-1 activity. RESULTS: Aberrant crypt foci showed positive immunostaining for glutathione-S-transferase P1-1 but negative staining for cyclooxygenase-2. The percentage of apoptotic cells in aberrant crypt foci was significantly lower than in healthy epithelium, and the difference became more apparent with deoxycholic acid treatment. The impaired sensitivity of aberrant crypt foci to deoxycholic acid was restored by the glutathione-S-transferase P1-1-specific inhibitor gamma-glutamyl-S-(benzyl)cysteinyl-R-phenylglycine diethylester. By transfection of glutathione-S-transferase P1-1, M7609 cells became more resistant to deoxycholic acid-induced apoptosis than mock transfectants. Direct binding of glutathione-S-transferase P1-1 to deoxycholic acid was proven by circular dichroism and by immunoprecipitation. The aberrant crypt foci in adenoma patients treated with sulindac, which was shown to bind to glutathione-S-transferase P1-1, underwent apoptosis in 4 days and mostly regressed in 2-3 months. CONCLUSIONS: Glutathione-S-transferase P1-1 protects aberrant crypt foci from deoxycholic acid-induced apoptosis and may play a pivotal role in early colon carcinogenesis.