TAG-72 Research Articles

Improved targeting of an anti-TAG-72 antibody drug conjugate for the treatment of ovarian cancer.

IntroductionOvarian cancer has only a 17% 5‐year survival rate in patients diagnosed with late stage disease. Tumor‐associated glycoprotein‐72 (TAG72), expressed in 88% of all stages of ovarian cancer, is an excellent candidate for antibody‐targeted therapy, as it is not expressed in normal human adult tissues, except in the secretory endometrium.MethodsUsing the clinically relevant anti‐TAG72 murine monoclonal antibody CC49, we evaluated antibody drug conjugates (ADCs) incorporating the highly potent, synthetic antimitotic agent monomethylauristatin E (MMAE). MMAE was conjugated to CC49 via reduced disulfides in the hinge region, using three different types of linker chemistry, vinylsulfone (VS‐MMAE), bromoacetamido (Br‐MMAE), and maleimido (mal‐MMAE).ResultsThe drug antibody ratios (DARs) of the three ADCs were 2.3 for VS‐MMAE, 10 for Br‐MMAE, and 9.5 for mal‐MMAE. All three ADCs exhibited excellent tumor to blood ratios on PET imaging, but the absolute uptake of CC49‐mal‐MMAE (3.3%ID/g) was low compared to CC49‐Br‐MMAE (6.43%ID/g), at 142 hours. Blood clearance at 43 hours was 38% for intact CC49, about 24% for both CC49‐VS‐MMAE and CC49‐Br‐MMAE, and 7% for CC49‐mal‐MMAE. CC49‐VS‐MMAE was not further studied due to its low DAR, while CC49‐mal‐MMAE was ineffective in the OVCAR3 xenograft likely due to its rapid blood clearance. In contrast, CC49‐Br‐MMAE treated mice exhibited an average of a 15.6 day tumor growth delay and a 40% increase in survival vs controls with four doses of 7.5 or 15 mg/kg of CC49‐Br‐MMAE.ConclusionWe conclude that CC49‐Br‐MMAE with a high DAR and stable linker performs well in a difficult to treat solid tumor model.

Humanized Anti–Tumor-Associated Glycoprotein–72 for Submillimeter Near-Infrared Detection of Colon Cancer in Metastatic Mouse Models

BackgroundTumor-associated glycoprotein (TAG)–72 is a pancarcinoma antigen that is overexpressed in greater than 80% of colorectal adenocarcinomas. CC49 is a TAG-72–specific antibody. The aim of the present study was to demonstrate selective imaging of colon tumors and metastases with the humanized TAG-72 antibody (anti-huCC49) conjugated to a near-infrared fluorophore in orthotopic mouse models. MethodsAnti-huCC49 was conjugated to near-infrared dye IR800CW. Mouse imaging was performed with the Pearl Trilogy Small Animal and FLARE Imaging Systems. Subcutaneous mouse models of colon cancer cell line LS174T were used to determine the optimal dose of administration and timing of imaging. Orthotopic mouse models of LS174T were established by surgical orthotopic implantation of LS174T tumors onto the serosa of the cecum. Peritoneal carcinomatosis models were established by injection of LS174T cells into the peritoneum of nude mice. Mice were administered anti-huCC49–IR800 via tail vein injection. Mice were euthanized 72 h later and imaged after laparotomy. ResultsSubcutaneous LS174T xenografts demonstrated optimal tumor detection 72 h after administration with 50 μg anti-huCC49-IR800CW. Tumors were visualized with fluorescence imaging with a mean tumor-to-liver ratio of 7.39 (standard deviation: 2.76). In the orthotopic model, metastases smaller than 1 mm were fluorescently visualized that were invisible with bright light. ConclusionsAnti-huCC49–IR800CW provides sensitive and specific imaging of colon cancer and metastases at a submillimeter resolution in metastatic nude mice models. This provides a promising near-infrared probe for the imaging of colon cancer and metastases for preoperative diagnosis and fluorescence-guided surgery.

Aptamer-Functionalized Drug Nanocarrier Improves Hepatocellular Carcinoma toward Normal by Targeting Neoplastic Hepatocytes

Site-specific delivery of chemotherapeutics specifically to neoplastic hepatocytes without affecting normal hepatocytes should be a focus for potential therapeutic management of hepatocellular carcinoma (HCC). The aptamer TLS 9a with phosphorothioate backbone modifications (L5) has not been explored so far for preferential delivery of therapeutics in neoplastic hepatocytes to induce apoptosis. Thus, the objective of the present investigation was to compare the therapeutic potential of L5-functionalized drug nanocarrier (PTX-NPL5) with those of the other experimental drug nanocarriers functionalized by previously reported HCC cell-targeting aptamers and non-aptamer ligands, such as galactosamine and apotransferrin. A myriad of well-defined investigations such as cell cycle analysis, TUNEL (terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling) assay, and studies related to apoptosis, histopathology, and immunoblotting substantiated that PTX-NPL5 had the highest potency among the different ligand-attached experimental formulations in inducing selective apoptosis in neoplastic hepatocytes via a mitochondrial-dependent apoptotic pathway. PTX-NPL5 did not produce any notable toxic effects in healthy hepatocytes, thus unveiling a new and a safer option in targeted therapy for HCC. Molecular modeling study identified two cell-surface biomarker proteins (tumor-associated glycoprotein 72 [TAG-72] and heat shock protein 70 [HSP70]) responsible for ligand-receptor interaction of L5 and preferential internalization of PTX-NPL5 via clathrin-mediated endocytosis in neoplastic hepatocytes. The potential of PTX-NPL5 has provided enough impetus for its rapid translation from the pre-clinical to clinical domain to establish itself as a targeted therapeutic to significantly prolong survival in HCC.

Humanized Fluorescent Tumor-associated Glycoprotein-72 Antibody Selectively Labels Colon-cancer Liver Metastases in Orthotopic Mouse Models.

Fluorescence imaging has been shown to improve intra-operative detection of liver metastasis. The present study aimed to determine whether humanized anti-TAG-72 antibody (huCC49) conjugated to a near-infrared dye provides selective labeling of colorectal-cancer liver metastasis in orthotopic mouse models. Humanized anti-TAG-72 (huCC49) was conjugated to IRDye800CW (huCC49-IR800). Orthotopic liver-metastasis nude-mouse models (n=5) were established with the human colon-cancer LS174T cell-line. Three weeks later, mice were administered huCC49-IR800 and intra-vital imaging was performed 48 h later. The mean tumor-to-liver ratio (TLR) was calculated. Intra-vital imaging demonstrated clear tumor margins with minimal liver fluorescence 48 h after administration of 50 μg huCC49-IR800 with mean TLR=7.53 (SD±2.76). Anti-TAG-72 monoclonal antibody conjugated to IRDye800 provides distinct and bright labeling of colorectal tumors in orthotopic nude-mouse models of liver metastasis. TAG-72 may be a useful target for intra-operative imaging of colorectal cancer liver metastasis in the clinic.

First clinical study of a pegylated diabody 124I-labeled PEG-AVP0458 in patients with tumor-associated glycoprotein 72 positive cancers

Through protein engineering and a novel pegylation strategy, a diabody specific to tumor-associated glycoprotein 72 (TAG-72) (PEG-AVP0458) has been created to optimize pharmacokinetics and bioavailability to tumor. We report the preclinical and clinical translation of PEG-AVP0458 to a first-in-human clinical trial of a diabody.Methods: Clinical translation followed characterization of PEG-AVP0458 drug product and preclinical biodistribution and imaging assessments of Iodine-124 trace labeled PEG-AVP0458 (124I-PEG-AVP0458). The primary study objective of the first-in-human study was the safety of a single protein dose of 1.0 or 10 mg/m2 124I-PEG-AVP0458 in patients with TAG-72 positive relapsed/ metastatic prostate or ovarian cancer. Secondary study objectives were evaluation of the biodistribution, tumor uptake, pharmacokinetics and immunogenicity. Patients were infused with a single-dose of 124I labeled PEG-AVP0458 (3-5 mCi (111-185 MBq) for positron emission tomography (PET) imaging, performed sequentially over a one-week period. Safety, pharmacokinetics, biodistribution, and immunogenicity were assessed up to 28 days after infusion.Results: PEG-AVP0458 was radiolabeled with 124I and shown to retain high TAG-72 affinity and excellent targeting of TAG-72 positive xenografts by biodistribution analysis and PET imaging. In the first-in-human trial, no adverse events or toxicity attributable to 124I-PEG-AVP0458 were observed. Imaging was evaluable in 5 patients, with rapid and highly specific targeting of tumor and minimal normal organ uptake, leading to high tumor:blood ratios. Serum concentration values of 124I-PEG-AVP0458 showed consistent values between patients, and there was no significant difference in T½α and T½β between dose levels with mean (± SD) results of T½α = 5.10 ± 4.58 hours, T½β = 46.19 ± 13.06 hours.Conclusions: These data demonstrates the safety and feasibility of using pegylated diabodies for selective tumor imaging and potential delivery of therapeutic payloads in cancer patients.

Abstract 2395: A bispecific T- cell engager antibody (BiTE) against TAG-72/ CD3 for targeted cancer immunotherapy

Abstract Background: Immunotherapies involving cytotoxic T lymphocytes (CTL’s) have been central to cancer immunotherapy. Bi-specific T cell engager antibody (BiTE) therapy has emerged as an effective immunotherapy by redirecting cytotoxic T cells against cell surface protein on tumor cells. Our BiTE antibody targets human tumor associated glycoprotein 72 (TAG-72). TAG-72 is a pan-carcinoma marker expressed on the surfaces of many cancer tissues including breast, prostate, ovary, endometrium, stomach, esophagus, and pancreas. It is not expressed in normal tissues making it an ideal target therapy for cancer. Our BiTE antibody can bind CD3 to activate the T-cell’s while simultaneously binding TAG-72 on the surface of the cancer cell leading to perforin induced cancer cell death. The in vitro and in vivo effects of TAG-72/ CD3 BiTE antibody on different cancer cells are presented here. Methods: We studied in vitro and in vivo effects of TAG-72/ CD3 BiTE antibody on different cancer cells; breast (DOX resistant MCF-7, SKBR3, MDA-MB-175 VII), ovarian (OVCAR-3), endometrium (KLE) and colon (Ls174T). In vitro cytotoxicity assay was performed by flow cytometry and immunofluorescence staining using live/ dead cell labelling fluorescent dyes. The cells were co-cultured with naïve human CD8 T cells with/ without BiTE antibody (1 µg, 5 µg and 10 µg) for different time intervals and assayed. In vivo study was carried out in NSG mice implanted with MCF-7 breast cancer cells + naïve human CD8 T cells and treated with a total of 4 doses of BiTE antibody (200µg/ dose). Results: Different tumor cells showed varying (%) of TAG-72 expression determined by flow cytometry, viz; DOX (55%), SKBR3 (16%), MDA 175 VII (8%), OVCAR-3 (27%), KLE (27%), Ls174T (30%). The BiTE antibody also showed dose dependent binding to these cells. Flow cytometry analysis showed that addition of the BiTE antibody to naïve CD8 T-cells and incubation for 4 days significantly increases tumor cell death as indicated by the Annexin V/ PI+ve signal from DOX (52.4% vs. 41.4% CD8 alone), SKBR-3 (47.9% vs. 30% CD8 alone), MDA 175 VII (23% vs 11% CD8 alone), OVCAR-3 (31.1% vs. 23.8% CD8 alone), KLE (27% vs 13% CD8 alone) and Ls174T (54% vs 29% CD8 alone) respectively (p<0.05). Further, a significant level of IFN-γ secreted by activated T-cells was only detected in the bispecific T-cell engager Ab + CD8 conditions. In vivo data showed good tumor regression at day 45 with tumors measuring (8 mm2) in group treated with BiTE antibody as compared to untreated group (31 mm2) (p<0.01). Conclusion: Our in vitro and in vivo data indicates that the TAG-72/ CD3 BiTE antibody can effectively redirect cytotoxic T cells against different cancer cells expressing TAG-72 glycoprotein and may be effective in the treatment of breast, ovarian, endometrium and colon cancers. Citation Format: Fatema Khambati, Hatem Soliman. A bispecific T- cell engager antibody (BiTE) against TAG-72/ CD3 for targeted cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2395.

CURRENT UPDATES ON DIAGNOSTIC BIOMARKERS OF HEPATOCELLULAR CARCINOMA

Hepatocellular carcinoma (HCC) is a liver malignancy, which is a cause of several deaths related to cancer worldwide. In early stages, curative treatment such as surgical resection, liver transplant and local ablation can improve the patient’s survival. However, the disease is often diagnosed in an advanced stage; moreover, some available therapies are restricted to palliative care and local treatment. Early diagnosis of HCC and adequate therapy are crucial to increasing survival as well as to improve the patient’s quality of life. Therefore, many researchers have been investigating biomarkers such as alpha-fetoprotein (AFP), glypican-3 (GPC3), des-γ-carboxyprothrombin, gamma-glutamyl transferase (GGT), serum α-L-fucosidase (AFU), carbonyl reductase 2, golgi phosphoprotein 2, transforming growth factor-β1 (TGF-β1), vascular endothelial growth factor (VEGF), tumor- specific growth factor (TSGF), epidermal growth factor receptor family (EGFR), hepatocyte growth factor/scatter factor, fibroblast growth factor (FGF), circulating nucleic acids (mRNAs), gamma-glutamyl transferase mRNA (GGT mRNA), insulin-like growth factor II (IGF-II) mRNA, albumin mRNA, circulating micro RNAs, heat shock protein (HSP), Golgi protein 73 (GP73), squamous cell carcinoma antigen (SCCA), tumor-associated glycoprotein 72 (TAG-72), zinc-α2-glycoprotein (ZAG), cytokeratin 19, osteopontin, midkine (MDK), gankyrin, annexin A2, soluble urokinase plasminogen activator receptor (suPAR), AXL, thioredoxins (TRXs), cluster of differentiation 147 (CD147) and microRNAs, which can regulate important pathways in carcinogensis, tumor angiogenesis and progression. So, they can be considered as possible markers of progression in HCC and therapeutic targets for this type of cancer. In this review, we discuss the recent advances related to diagnostic biomarkers, clinical aspects and outcome in hepatocellular carcinoma.

Glycosylation markers in cancer.

Alteration of glycosylation, a hallmark of cancer, results in the production of tumor-associated glycans or glycoproteins. These molecules are subsequently secreted or membrane-shed into the blood stream and thus serve as tumor-associated markers. Increased glycosylation in cancer is triggered by overexpression of glycoproteins that carry certain specific glycans, increase or decrease of nucleotide sugar donors and altered expression of glycosyltransferase and glycosidase enzymes. In this chapter, the biochemistry and function of glycoprotein, glycan and enzyme markers are reviewed. These glycosylation markers, applicable for detection and monitoring of cancer, include CA19-9, CA125, CEA, PSA and AFP. Because of their specific affinity to distinct sugar moieties, lectins are useful for developing assays to detect these tumor associated glycans and glycoproteins in clinical samples. As such, various enzyme-linked lectin assays (ELLA) have been developed for diagnosis, monitoring and prognosis. Because glycosylation changes occur early in cancer, the detection of tumor associated glycosylation markers using lectin based assays is an effective strategy to improve diagnosis and treatment resulting better outcomes clinically.

Serum tumor-associated glycoprotein 72, a helpful predictor of lymph nodes invasion in esophagogastric junction adenocarcinoma

Disruption of cell-cell junction and adhesion to vessels are crucial steps in tumor metastasis. Tumor-associated glycoprotein 72 (TAG-72) is a crucial membrane mucin in gastroesophageal mucosa for microenvironment contact with cells. Thus, the TAG-72 value may be an indicator of the malignant involvement of lymph nodes in esophagogastric junction adenocarcinoma (EGAC) patients. Of the 183 patients suspected as gastroesophageal neoplasms, 129 were subsequently diagnosed as EGAC, and 54 were subsequently diagnosed as benign gastroesophageal diseases by imageological or/and histological examination. After we obtained preoperative serum TAG-72 values, the relationship between serum TAG-72 and lymphatic metastasis status, extent of invaded lymph nodes and clinical stage was tested using Spearman correlation analysis and χ2 tests. Compared with those in patients who suffered either benign gastroesophageal diseases or preinvasive carcinoma, the median serum TAG-72 values were statistically higher in EGAC patients with positive lymph nodes (Kruskal-Wallis test; P < 0.001). Serum TAG-72 values were significantly correlated with Lymph Node Ratio (LNR) (Spearman correlation; P < 0.001). Using corresponding ROC (95% CI = 0.621–0.783, P < 0.0001), serum TAG-72 values with an optimal cut-off (2.2 kU/mL) showed a sensitivity of 0.632 and a specificity of 0.690 for predicting malignant lymph node involvement in EGAC. These results suggest that the serum TAG-72 value is a clinically helpful predictor of lymph nodes invasion in resectable EGAC.

Effective Targeting of TAG72+ Peritoneal Ovarian Tumors via Regional Delivery of CAR-Engineered T Cells.

Impressive clinical efficacy of chimeric antigen receptor (CAR)-engineered T cell therapy for hematological malignancies have prompted significant efforts in achieving similar responses in solid tumors. The lack of truly restricted and uniform expression of tumor-associated antigens, as well as limited T cell persistence and/or tumor trafficking pose major challenges for successful translation of CAR T cell therapy in solid tumors. Recent studies have demonstrated that aberrantly glycosylated cell surface proteins on tumor cells are amenable CAR targets. Tumor-associated glycoprotein 72 (TAG72) antigen is the sialyl-Tn found on multiple O-glycoproteins expressed at high levels on the surface of several cancer types, including ovarian cancer. Here, we developed a humanized TAG72-specific CAR containing a 4-1BB intracellular co-stimulatory signaling domain (TAG72-BBζ). TAG72-BBζ CAR T cells showed potent antigen-dependent cytotoxicity and cytokine production against multiple TAG72+ ovarian cancer cell lines and patient-derived ovarian cancer ascites. Using in vivo xenograft models of peritoneal ovarian tumors, regional intraperitoneal delivery of TAG72-BBζ CAR T cells significantly reduced tumor growth, extended overall survival of mice, and was further improved with repeat infusions of CAR T cells. However, reduced TAG72 expression was observed in early recurring tumors, which coincided with a lack of T cell persistence. Taken together, we demonstrate efficacy with TAG72-CAR T cells in ovarian cancer, warranting further investigations as a CAR T cell therapeutic strategy for this disease.

The prognostic role of tumor associated glycoprotein 72 (TAG-72) in stage II and III colorectal adenocarcinoma

Tumor associated glycoprotein 72 (TAG-72) is a membrane-bound glycoprotein complex that is overexpressed in many adenocarcinomas. Recently, monoclonal antibody targeting TAG72, minretumomab, have been introduced as a potential therapeutic target in colorectal cancers (CRC) as well as breast and lung cancers. However, the detailed expression profile of TAG72 and its prognostic effect in CRC are not clear yet. We investigated the relationship between tumor associated glycoprotein 72 (TAG-72) expression and clinicopathologic characteristics in CRC using 3E8 antibody, a fully humanized antibody with the highest affinity to TAG-72. Immunohistochemical staining for TAG-72 was performed in 578 CRC patients, and the results were analyzed using a modified Remmele scoring system (score: 0–12). Of the 578 patients, 144 (24.9%) composed the TAG-72 overexpression (TAG-72high) group. TAG-72high was significantly associated with microsatellite stable tumor (P = .002), lymphatic invasion (P = .001), venous invasion (P = .005), and high pN status (P < .001). In survival analyses, TAG-72high group showed shorter disease-free survival in univariate analysis (P = .001), and TAG-72high was found to be an independent prognostic factor in multivariate analysis (P = .028), in addition to TNM stage. In conclusion, TAG-72 is thought to be the factors involved in the progression of CRC and may be considered as one of the potential therapeutic target.

Linker engineering in anti-TAG-72 antibody fragments optimizes biophysical properties, serum half-life, and high-specificity tumor imaging

Antibody (Ab) fragments have great clinical potential as cancer therapeutics and diagnostics. Their small size allows for fast clearance from blood, low immunoreactivity, better tumor penetration, and simpler engineering and production. The smallest fragment derived from a full-length IgG that retains binding to its antigen, the single-chain variable fragment (scFV), is engineered by fusing the variable light and variable heavy domains with a peptide linker. Along with switching the domain orientation, altering the length and amino acid sequence of the linker can significantly affect scFV binding, stability, quaternary structure, and other biophysical properties. Comprehensive studies of these attributes in a single scaffold have not been reported, making design and optimization of Ab fragments challenging. Here, we constructed libraries of 3E8, an Ab specific to tumor-associated glycoprotein 72 (TAG-72), a mucinous glycoprotein overexpressed in 80% of adenocarcinomas. We cloned, expressed, and characterized scFVs, diabodies, and higher-order multimer constructs with varying linker compositions, linker lengths, and domain orientations. These constructs dramatically differed in their oligomeric states and stabilities, not only because of linker and orientation but also related to the purification method. For example, protein L-purified constructs tended to have broader distributions and higher oligomeric states than has been reported previously. From this library, we selected an optimal construct, 3E8.G4S, for biodistribution and pharmacokinetic studies and in vivo xenograft mouse PET imaging. These studies revealed significant tumor targeting of 3E8.G4S with a tumor-to-background ratio of 29:1. These analyses validated 3E8.G4S as a fast, accurate, and specific tumor-imaging agent.

Tumor-Derived Microvesicles Modulate Antigen Cross-Processing via Reactive Oxygen Species-Mediated Alkalinization of Phagosomal Compartment in Dendritic Cells

Dendritic cells (DCs) are the only antigen-presenting cells able to prime naïve T cells and cross-prime antigen-specific CD8+ T cells. Their functionality is a requirement for the induction and maintenance of long-lasting cancer immunity. Albeit intensively investigated, the in vivo mechanisms underlying efficient antigen cross-processing and presentation are not fully understood. Several pieces of evidence indicate that antigen transfer to DCs mediated by microvesicles (MVs) enhances antigen immunogenicity. This mechanism is also relevant for cross-presentation of those tumor-associated glycoproteins such as MUC1 that are blocked in HLA class II compartment when internalized by DCs as soluble molecules. Here, we present pieces of evidence that the internalization of tumor-derived MVs modulates antigen-processing machinery of DCs. Employing MVs derived from ovarian cancer ascites fluid and established tumor cell lines, we show that MV uptake modifies DC phagosomal microenvironment, triggering reactive oxygen species (ROS) accumulation and early alkalinization. Indeed, tumor MVs carry radical species and the MV uptake by DCs counteracts the chemically mediated acidification of the phagosomal compartment. Further pieces of evidence suggest that efficacious antigen cross-priming of the MUC1 antigen carried by the tumor MVs results from the early signaling induced by MV internalization and the function of the antigen-processing machinery of DCs. These results strongly support the hypothesis that tumor-derived MVs impact antigen immunogenicity by tuning the antigen-processing machinery of DCs, besides being carrier of tumor antigens. Furthermore, these findings have important implications for the exploitation of MVs as antigenic cell-free immunogen for DC-based therapeutic strategies.

Assessment of tumor characteristics based on glycoform analysis of membrane-tethered MUC1

Clinical tissue specimens are useful for pathological diagnosis, which is, in some cases, supported by visualization of biomolecule localization. In general, diagnostic specificity in molecular pathology is increased by the acquisition of a probe to distinguish the modification of isomers. Although glycosylation is one of the candidate modifications in a protein, comparative glycan analysis of disease-associated proteins derived from a single tissue section is still challenging because of the lack of analytical sensitivity. Here we demonstrate a possible method for differential glycoform analysis of an endogenous tumor-associated glycoprotein MUC1 by an antibody-overlay lectin microarray. Tissue sections (5 μm thick) of patients with cholangiocarcinoma (CCA; n=21) and pancreatic ductal adenocarcinoma (PDAC; n=50) were stained with an anti-MUC1 antibody MY.1E12 that was established as a monoclonal antibody recognizing an MUC1 glycosylation isoform with a sialyl-core 1 structure (NeuAcα2-3galactosyl β1-3-N-acetylgalactosamine). MY.1E12-positive tissue areas (2.5 mm2) were selectively dissected with a laser capture microdissection procedure. The membrane MUC1 was enriched by immunoprecipitation with MY.1E12 and subjected to lectin microarray analysis. Even though the reactivities of MY.1E12 between CCA and PDAC were similar, the lectin-binding patterns varied. We found Maackia amurensis leukoagglutinin and pokeweed lectin distinguished MY.1E12-reactive MUC1 of CCA from that of PDAC. Moreover, MUC1 with M. amurensis hemagglutinin (MAH) reactivity potentially reflected the degree of malignancy. These results were confirmed with MAH-MY.1E12 double fluorescent immunostaining. These glycan changes on MUC1 were detected with high sensitivity owing to the cluster effect of immobilized lectins on a tandem repeat peptide antigen covered with highly dense glycosylation such as mucin. Our approach provides the information to investigate novel glycodynamics in biology, for example, glycoalteration, as well as diseases related to not only MUC1 but also other membrane proteins.

Phytochemical remedies: a key strategy towards reversing the aggressive murine colon cancer

Colon cancer accounts for about 500,000 death cases worldwide annually and represents approximately 6.5% of cancers in Egypt. This study employed a murine colon cancer model to identify the mechanism of the therapeutic value of gallic acid or ellagic acid against colon carcinogenesis in vivo. Fifty adult male rats were distributed into five groups. Group (1) was set as a negative control group. Groups (2), (3), (4), and (5) were intrarectally injected with N-methylnitrosourea to induce colon cancer. Group (2) was kept untreated, whereas group (3) and (4) were orally administered gallic acid and ellagic acid, respectively. Group (5) received intraperitoneal injection of 5-fluorouracil. The administration of gallic acid, ellagic acid or 5-fluorouracil harmonized the hazardous effect of N-methylnitrosourea in colon cancer-afflicted rats. This was evidenced by the significant depletion in both of serum tumor associated glycoprotein-72 (TAG72) and galectin-3 (GAL3) levels as well as the significant downregulation in the expression level of frizzled-8 receptor (FRZ-8) gene and significant amplification of adenomatous polyposis coli gene expression level in the colon tissues of all treated groups. Intriguingly, such treatments resulted in a notable improvement in the pathological features of colon tissue sections of N-methylnitrosourea-challenged rats. Generally speaking, the current investigation emphasizes the favorable impact of gallic acid and ellagic acid in monitoring the progression of colon cancer in vivo. We suggest that the mitigation of Wnt signaling pathway may be the probable mechanism by which these compounds can offer their therapeutic actions against colon cancer.

Sialic Acid-Targeted Biointerface Materials and Bio-Applications.

Sialic acids (SAs) are typically found as terminal monosaccharides attached to cell surface glycoconjugates, which play crucial roles in various biological processes, and aberrant sialylation is closely associated with many diseases, particularly cancers. As SAs are overexpressed in tumor-associated glycoproteins, the recognition and specific binding of SA are crucial for monitoring, analyzing and controlling cancer cells, which would have a considerable impact on diagnostic and therapeutic application. However, both effective and selective recognition of SA on the cancer cell surface remains challenging. In recent years, SA-targeted biointerface materials have attracted great attention in various bio-applications, including cancer detection and imaging, drug delivery for cancer therapy and sialylated glycopeptide separation or enrichment. This review provides an overview of recent advances in SA-targeted biointerface materials and related bio-applications.

In Vivo Pretargeted Imaging of HER2 and TAG-72 Expression Using the HaloTag Enzyme.

A novel pretargeted SPECT imaging strategy based on the HaloTag enzyme has been evaluated for the first time in a living system. To determine the efficacy of this approach, two clinically relevant cancer biomarkers, HER2 and TAG-72, were selected to represent models of internalizing and noninternalizing antigens, respectively. In MDA-MB-231/H2N (HER2-expressing) and LS174T (TAG-72-expressing) xenograft tumors in mice, pretargeting experiments were performed in which HaloTag-conjugated derivatives of the antibodies trastuzumab (anti-HER2) or CC49 (anti-TAG-72) were utilized as primary agents, and the small molecule HaloTag ligands 111In-HTL-1, -2, and -3 were evaluated as secondary agents. While this approach was not sufficiently sensitive to detect the internalizing HER2 antigen, pretargeting experiments involving the most optimal secondary agent, 111In-HTL-3, were successful in detecting the noninternalizing antigen TAG-72 and provided high-contrast SPECT images at 4 and 24 h postinjection.

A Fas Ligand (FasL)-Fused Humanized Antibody Against Tumor-Associated Glycoprotein 72 Selectively Exhibits the Cytotoxic Effect Against Oral Cancer Cells with a Low FasL/Fas Ratio.

Altered expression of the Fas ligand (FasL)/Fas ratio exhibits a direct impact on the prognosis of cancer patients, and its impairment in cancer cells may lead to apoptosis resistance. Thus, the development of effective therapies targeting the FasL/Fas system may play an important role in the fight against cancer. In this study, we evaluated whether a fusion protein (hcc49scFv-FasL) comprising of the cytotoxicity domain of the FasL fused to a humanized antibody (CC49) against tumor-associated glycoprotein 72, which is expressed on oral squamous cell carcinoma (OSCC), can selectively kill OSCC cells with different FasL/Fas ratios. In clinical samples, the significantly low FasL and high Fas transcripts were observed in tumors compared with normal tissues. A lower FasL/Fas ratio was correlated with a worse prognosis of OSCC patients and higher proliferative and invasive abilities of OSCC cells. The hcc49scFv-FasL showed a selective cytotoxic effect on OSCC cells (Cal-27 and SAS) but not on normal oral keratinocytes cells (HOK) through apoptosis induction. Moreover, SAS cells harboring a lower FasL/Fas ratio than Cal-27 were more sensitive to the cytotoxic effect of hcc49scFv-FasL. Unlike wild-type FasL, hcc49scFv-FasL was not cleaved by matrix metalloproteinases and did not induce nonapoptotic signaling in SAS cells. In vivo, we found that hcc49scFv-FasL drastically reduced the formation of lymph node metastasis and decreased primary tumor growth in SAS orthotopic and subcutaneous xenograft tumor models. Collectively, our data indicate that a tumor-targeting antibody fused to the FasL can be a powerful tool for OSCC treatment, especially in populations with a low FasL/Fas ratio. Mol Cancer Ther; 16(6); 1102-13. ©2017 AACR.

Review of the Third Domain Receptor Binding Fragment of Alphafetoprotein (AFP): Plausible Binding of AFP to Lysophospholipid Receptor Targets.

Alpha-fetoprotein (AFP) is a 69 kD fetal- and tumor-associated single-chain glycoprotein belonging to the albuminoid gene family. AFP functions as a carrier/transport molecule as well as a growth regulator and has been utilized as a clinical biomarker for both fetal defects and cancer growth. Lysophospholipids (LPLs) are plasma membrane-derived bioactive lipid signaling mediators composed of a small molecular weight single acyl carbon chain (palmitic, oleic acid) attached to a polar headgroup; they range in molecular mass from 250-750 daltons. The LPLs consist of either sphingosine-1-phosphate or lysophosphatidic acid, and mostly their choline, ethanolamine, serine or inositol derivatives. They are present only in vertebrates. These bioactive paracrine lipid mediators are ubiquitously distributed in tissues and are released from many different cell types (platelets, macrophages, monocytes, etc.) involved in developmental, physiological, and pathological processes. The LPLs bind to four different classes of G-protein coupled receptors described herein which transduce a multiple of cell effects encompassing activities such as morphogenesis, neural development, angiogenesis, and carcinogenesis. The identification of potential binding sites of LPL receptors on the AFP third domain receptor binding fragment was derived by computer modeling analysis. It is conceivable, but not proven, that AFP might bind not only to the LPL receptors, but also to LPLs themselves since AFP binds medium and long chain fatty acids. It is proposed that some of the activities ascribed to AFP in the past might be due in part to the presence of bound LPLs and/or their receptors.

Pretargeting with bispecific fusion proteins facilitates delivery of nanoparticles to tumor cells with distinct surface antigens

Tumor heterogeneity, which describes the genetically and phenotypically distinct subpopulations of tumor cells present within the same tumor or patient, presents a major challenge to targeted delivery of diagnostic and/or therapeutic agents. An ideal targeting strategy should deliver a given nanocarrier to the full diversity of cancer cells, which is difficult to achieve with conventional ligand-conjugated nanoparticles. We evaluated pretargeting (i.e., multistep targeting) as a strategy to facilitate nanoparticle delivery to multiple target cells by measuring the uptake of biotinylated nanoparticles by lymphoma cells with distinct surface antigens pretreated with different bispecific streptavidin-scFv fusion proteins. Fusion proteins targeting CD20 or tumor-associated glycoprotein 72 (TAG-72) mediated the specific in vitro uptake of 100nm biotin-functionalized nanoparticles by Raji and Jurkat lymphoma cells (CD20-positive and TAG-72-positive cells, respectively). Greater uptake was observed for pretargeted nanoparticles with increasing amounts of surface biotin, with 6- to 18-fold higher uptake vs. non-biotinylated nanoparticle and fusion protein controls. Fully biotin-modified particles remained resistant to cultured macrophage cell uptake, although they were still quickly cleared from systemic circulation in vivo (t1/2<1h). For single Raji tumor-bearing mice, pretargeting with CD20-specific FP significantly increased nanoparticle tumor targeting. In mice bearing both Raji and Jurkat tumors, pretargeting with both fusion proteins markedly increased nanoparticle targeting to both tumor types, compared to animals dosed with nanoparticles alone. These in vitro and in vivo observations support further evaluations of pretargeting fusion protein cocktails as a strategy to enhance nanoparticle delivery to a diverse array of molecularly distinct target cells.