Biomolecular Recognition of the Glycan Neoantigen CA19-9 by Distinct Antibodies.

Glycans decorate the cell surface, secreted glycoproteins and glycolipids, and altered glycans are often found in cancers. Despite their high diagnostic and therapeutic potential, however, glycans are polar and flexible molecules that are quite challenging for the development and design of high-affinity binding antibodies. To understand the mechanisms by which glycan neoantigens are specifically recognized by antibodies, we analyze the biomolecular recognition of the tumor-associated carbohydrate antigen CA19-9 by two distinct antibodies using X-ray crystallography. Despite the potential plasticity of glycans and the very different antigen-binding surfaces presented by the antibodies, both structures reveal an essentially identical extended CA19-9 conformer, suggesting that this conformer's stability selects the antibodies. Starting from the bound structure of one of the antibodies, we use the AbLIFT computational algorithm to design a variant with seven core mutations in the variable domain's light-heavy chain interface that exhibits tenfold improved affinity for CA19-9. The results reveal strategies used by antibodies to specifically recognize glycan antigens and show how automated antibody-optimization methods may be used to enhance the clinical potential of existing antibodies.

Efficacy of irreversible electroporation ablation combined with natural killer cells in treating locally advanced pancreatic cancer.

PURPOSE: To explore the efficacy and safety of irreversible electroporation (IRE) ablation combined with natural killer (NK) cells in the treatment of locally advanced pancreatic cancer (LAPC). METHODS: A total of 92 LAPC patients treated in our hospital from January 2016 to January 2017 were enrolled, and there were 46 cases of percutaneous IRE as IRE group, and 46 cases of IRE combined NK cell therapy as IRE-NK group. The clinical information of all the patients was collected, and the short-term efficacy, changes in the serum immunological indicators after treatment, carbohydrate antigen 19-9 (CA19-9) level, and incidence of adverse reactions were compared between the two groups of patients. Besides, the overall survival (OS) and disease-free survival (DFS) of patients were followed up and recorded. RESULTS: On 1 month after treatment, all the patients underwent efficacy assessment, which showed the overall response rate of patients in IRE-NK group was significantly superior to that in IRE group. One and 7 days after operation, the level of CA19-9 was obviously raised in the two groups, with a statistically significant difference, and it declined 30 days postoperatively. Seven and 30 days after operation IRE-NK group had a notably lower level of CA19-9 than IRE group. After treatment, all the patients exhibited considerably higher lymphocyte count and notably enhanced lymphocyte function, and all the indicators in IRE-NK group were higher than those in IRE group. Besides, the levels of serum interleukin (IL)-2, TNF-ß and IFN-γ in IRE-NK group were remarkably higher than those in IRE group, whereas there were no statistically significant differences in the levels of IL-4, IL-6 and IL-10 between the two groups. All the patients were followed up for 6-29 months, and there were no statistically significant differences in the DFS and OS between IRE group and IRE-NK group. CONCLUSION: IRE ablation combined with NK cells has excellent efficacy in treating LAPC, and they can exert a synergistic treatment effect to enhance the immune function of patients and reduce CA19-9 expression, with tolerable adverse reactions.

Label-assisted chemical adsorption triggered conversion of electroactivity of sensing interface to achieve the Ag/AgCl process for ultrasensitive detection of CA 19-9.

Efficient strategies in enhancing sensitivity are pivotal to ultrasensitive detection of tumor markers. In this work, based on the strategy of label-assisted chemical adsorption triggered conversion of electroactivity of sensing interface, a Ag/AgCl process was achieved to enhance sensitivity of the constructed sandwich-type amperometric immunosensor for ultrasensitive detection of carbohydrate antigen 19-9 (CA19-9). Briefly, polydopamine-Ag nanoparticles (PDA-Ag NPs), as signal precursor, combined with labeling antibody were served as labels and graphene oxide-melamine (GO-MA) substrate with chemical absorption capacity was applied as smart sensing interface. After successfully incubating labels, there was primitively no current response due to the poor conductivity between labels and electrode. However, in the presence of H2O2, Ag NPs from labels can be etched into Ag ions, which were adsorbed by GO-MA to form GO-MA-Ag as electroactive substrate. Then, the substrate exhibited a sharp and stable electrochemistry peak of solid-state Ag/AgCl process in the buffer containing KCl. The sensitivity toward detection of CA19-9 was notably enhanced based on the appearance of sharp peak. Under optimum conditions, the designed immunosensor demonstrated a wide working range from 0.0001 to 100 U mL-1 and an ultralow detection limit 0.032 mU mL-1. Thus, utilizing this strategy to construct immunosensor was highly promising in clinical diagnosis for ultrasensitive detection of tumor makers.

Retooling a Blood-Based Biomarker: Phase I Assessment of the High-Affinity CA19-9 Antibody HuMab-5B1 for Immuno-PET Imaging of Pancreatic Cancer.

PURPOSE: In patients with cancer who have an abnormal biomarker finding, the source of the biomarker in the bloodstream must be located for confirmation of diagnosis, staging, and therapy planning. We evaluated if immuno-PET with the radiolabeled high-affinity antibody HuMab-5B1 (MVT-2163), binding to the cancer antigen CA19-9, can identify the source of elevated biomarkers in patients with pancreatic cancer. PATIENTS AND METHODS: In this phase I dose-escalating study, 12 patients with CA19-9-positive metastatic malignancies were injected with MVT-2163. Within 7 days, all patients underwent a total of four whole-body PET/CT scans. A diagnostic CT scan was performed prior to injection of MVT-2163 to correlate findings on MVT-2163 PET/CT. RESULTS: Immuno-PET with MVT-2163 was safe and visualized known primary tumors and metastases with high contrast. In addition, radiotracer uptake was not only observed in metastases known from conventional CT, but also seen in subcentimeter lymph nodes located in typical metastatic sites of pancreatic cancer, which were not abnormal on routine clinical imaging studies. A significant fraction of the patients demonstrated very high and, over time, increased uptake of MVT-2163 in tumor tissue, suggesting that HuMab-5B1 labeled with beta-emitting radioisotopes may have the potential to deliver therapeutic doses of radiation to cancer cells. CONCLUSIONS: Our study shows that the tumor antigen CA19-9 secreted to the circulation can be used for sensitive detection of primary tumors and metastatic disease by immuno-PET. This significantly broadens the number of molecular targets that can be used for PET imaging and offers new opportunities for noninvasive characterization of tumors in patients.

Antibodies targeting sialyl Lewis A mediate tumor clearance through distinct effector pathways.

Sialyl Lewis A (sLeA, also known as CA19-9), a tetrasaccharide selectively and highly expressed on advanced adenocarcinomas including colon, stomach, and pancreatic cancers, has long been considered as an attractive target for active and passive vaccination. While progress in antibodies targeting tumor-associated protein antigens resulted in an impressive array of therapeutics for cancer treatment, similar progress in exploiting tumor-associated carbohydrate antigens, such as sLeA, has been hampered by the lack of a detailed understanding of the singular characteristics of these antigens. We have addressed this issue by analyzing antibodies derived from patients immunized with an sLeA/KLH vaccine. These antibodies were engineered to mediate tumor clearance in vivo in preclinical models through Fc-FcγR interactions. However, in contrast to protein antigens in which hFcγRIIIA engagement was both necessary and sufficient to mediate tumor clearance in both preclinical and clinical settings, a similar selective dependence was not seen for anti-sLeA antibodies. Thus, re-engineering the Fc portion of sLeA-targeting antibodies to broadly enhance their affinity for activating FcγRs led to an enhanced therapeutic effect. These findings will facilitate the development of more efficient anticancer therapies and further advance this promising class of therapeutic antibodies into clinical use.

A ratiometric electrochemical sensor for multiplex detection of cancer biomarkers using bismuth as an internal reference and metal sulfide nanoparticles as signal tags.

Ratiometric electrochemical sensors can provide a relatively accurate analysis of target analytes due to their self-calibration function. Herein, we report a simple ratiometric strategy for achieving the electrochemical detection of Cd(ii), Hg(ii), Pb(ii) and Zn(ii), as well as multiple cancer biomarkers by using metal sulfide nanoparticles as signal tags. A conductive polymer film of poly(2-amino terephthalic acid) (ATA) was electrochemically produced on a glassy carbon electrode (GCE) and doped with carbon nanotubes (CNTs) and mercaptosuccinic acid (MSA). Using Bi(iii) as an enhancer and internal reference in anodic stripping voltammetry, the MSA-CNT-ATA/GCE exhibited sensitive and distinguishable voltammetric responses to Cd(ii), Hg(ii), Pb(ii) and Zn(ii), with detection limits of 0.13, 0.49, 0.16 and 0.089 µg L-1, respectively. By using CdS, HgS, PbS and ZnS labeled secondary antibodies as the signal tags, alpha-fetoprotein, carbohydrate antigen 19-9, carbohydrate antigen 125, and carcinoembryonic antigen were determined simultaneously according to the amounts of metal sulfide in the sandwich-type complexes, with detection limits of 0.11 pg mL-1, 0.68 mU mL-1, 1.4 mU mL-1 and 0.23 pg mL-1, respectively. This ratiometric approach has a wide scope in the electrochemical detection of heavy metal ions as well as immunoassays with metal ions serving as signal tags.

The glycan CA19-9 promotes pancreatitis and pancreatic cancer in mice.

Glycosylation alterations are indicative of tissue inflammation and neoplasia, but whether these alterations contribute to disease pathogenesis is largely unknown. To study the role of glycan changes in pancreatic disease, we inducibly expressed human fucosyltransferase 3 and ß1,3-galactosyltransferase 5 in mice, reconstituting the glycan sialyl-Lewisa, also known as carbohydrate antigen 19-9 (CA19-9). Notably, CA19-9 expression in mice resulted in rapid and severe pancreatitis with hyperactivation of epidermal growth factor receptor (EGFR) signaling. Mechanistically, CA19-9 modification of the matricellular protein fibulin-3 increased its interaction with EGFR, and blockade of fibulin-3, EGFR ligands, or CA19-9 prevented EGFR hyperactivation in organoids. CA19-9-mediated pancreatitis was reversible and could be suppressed with CA19-9 antibodies. CA19-9 also cooperated with the KrasG12D oncogene to produce aggressive pancreatic cancer. These findings implicate CA19-9 in the etiology of pancreatitis and pancreatic cancer and nominate CA19-9 as a therapeutic target.

Glycoprotein CA19.9-specific monoclonal antibodies recognize sialic acid-independent glycotope.

A repertoire of monoclonal antibodies was generated by immunization of mice with cancer-associated glycoprotein CA19.9, and two of them were selected as optimal capture and detecting counterparts for sandwich test system for detection of CA19.9. Fine epitope specificity of the antibodies was determined using printed glycan array, enzyme-linked immunosorbent assay, and inhibitory enzyme-linked immunosorbent assay. Unexpectedly, both immunoglobulins did not bind key epitope of CA19.9 glycoprotein, tetrasaccharide SiaLeA, as well as its defucosylated form sialyl LeC (known as CA-50 epitope). The antibodies were found to have different glycan-binding profiles; however, they recognized similar glycotopes with common motif Galß1-3GlcNAcß (LeC), thus resembling specificity of human natural cancer-associated anti-LeC antibodies. We propose that cancer-specific glycopeptide epitope includes Galß1-3GlcNAcß fragment of a glycoprotein O-chain in combination with proximal hydrophobic amino acid(s) of the polypeptide chain.

Improving theranostics in pancreatic cancer.

BACKGROUND: Pancreatic cancer is the fourth most deadly cancer in the United States, and is expected to be the second most deadly by 2030. The major difficulty in treating pancreatic cancer is the late onset of symptoms. Generally, patients show metastatic disease by the time of diagnosis, with a survival rate of 5% beyond 5 years. In patients without metastatic disease, surgical resection increases 5 year survival rate to 25%. The remaining 75% succumb to undetected metastases. Clearly, improvements to both detection, surgical intervention, and therapeutic strategies will be needed to improve patient outcome in pancreatic cancer. METHODS: Recent literature has been surveyed and atomic models of new therapeutic approaches were generated. RESULTS AND CONCLUSIONS: Here, we focus on the recent progress employing monoclonal antibodies (mAbs) to target pancreatic cancer associated markers, and more specifically on recent chemical and protein engineering efforts to improve the homogeneity, stability, and administration of mAbs to precisely deliver imaging agents and cytotoxins to sites of disease.

Bifunctional 4MBA mediated recyclable SERS-based immunoassay induced by photocatalytic activity of TiO2 nanotube arrays.

We first report here a novel recyclable surface-enhanced Raman scattering (SERS)-based immunoassay via the photocatalytic ability of anatase titania nanotube (TiO2-NT) arrays. In this immunoassay, an immune probe was realized by immobilizing anti-CA19-9 onto Ag@SiO2@Ag three core-shell nanoparticles (TCSNPs), which showed a much higher SERS activity than bare Ag NPs with an enhancement ratio of 1.75. Then, the vertically oriented TiO2-NT immune substrate was synthesized by ultra-fast anodic oxidation of flexible titanium foils and functionalised with 4-mercaptobenzoic acid (4MBA) molecules to link them with anti-CA19-9. The immunoassay using the above immune probe and the substrate exhibited a wide linear range from 1000 to 0.5 U mL(-1) and a low detection limit of 0.5 U mL(-1) for CA19-9 due to the excellent SERS performance of Ag@SiO2@Ag TCSNPs. More importantly, the linkage between TiO2-NTs and 4MBA was destroyed by catalyzing 4MBA into 4-sulfobenzoate upon UV irradiation in O2-saturated water. The target antigen and the immune probe were simultaneously removed leading to a recyclable immunoassay and a detection limit of 5 U mL(-1) was achieved after six cycles. The simplicity and versatility of this strategy may bridge the technology gap between academia and practical detection, which makes it promising for clinical SERS-based immunoassay.

Site-specifically labeled CA19.9-targeted immunoconjugates for the PET, NIRF, and multimodal PET/NIRF imaging of pancreatic cancer.

Molecular imaging agents for preoperative positron emission tomography (PET) and near-infrared fluorescent (NIRF)-guided delineation of surgical margins could greatly enhance the diagnosis, staging, and resection of pancreatic cancer. PET and NIRF optical imaging offer complementary clinical applications, enabling the noninvasive whole-body imaging to localize disease and identification of tumor margins during surgery, respectively. We report the development of PET, NIRF, and dual-modal (PET/NIRF) imaging agents, using 5B1, a fully human monoclonal antibody that targets CA19.9, a well-established pancreatic cancer biomarker. Desferrioxamine (DFO) and/or a NIRF dye (FL) were conjugated to the heavy-chain glycans of 5B1, using a robust and reproducible site-specific (ss) labeling methodology to generate three constructs ((ss)DFO-5B1, (ss)FL-5B1, and (ss)dual-5B1) in which the immunoreactivity was not affected by the conjugation of either label. Each construct was evaluated in a s.c. xenograft model, using CA19.9-positive (BxPC3) and -negative (MIAPaCa-2) human pancreatic cancer cell lines. Each construct showed exceptional uptake and contrast in antigen-positive tumors with negligible nonspecific uptake in antigen-negative tumors. Additionally, the dual-modal construct was evaluated in an orthotopic murine pancreatic cancer model, using the human pancreatic cancer cell line, Suit-2. The (ss)dual-5B1 demonstrated a remarkable capacity to delineate metastases and to map the sentinel lymph nodes via tandem PET-computed tomography (PET/CT) and NIRF imaging. Fluorescence microscopy, histopathology, and autoradiography were performed on representative sections of excised tumors to visualize the distribution of the constructs within the tumors. These imaging tools have tremendous potential for further preclinical research and for clinical translation.

Toward Curative Fluorescence-Guided Surgery of Pancreatic Cancer.

BACKGROUND/AIMS: Negative surgical margins are critical to prevent recurrence in cancer surgery. We review the use of fluorophore-labeled monoclonal antibodies to aid in cancer visualization in orthotopic nude mouse models of human pancreatic cancer in order to achieve negative margins in fluorescence-guided surgery (FGS). METHODOLOGY: Anti-CEA or anti-CA 19-9 antibodies were conjugated with fluorophores of visible and near-infrared wavelengths. Orthotopic primary and metastatic human pancreatic tumors in nude mouse models were readily visualized with fluorescence imaging after administration of fluorophore conjugated anti-CEA or anti-CA 19-9. RESULTS: The fluorescence signal was detectable 30 minutes after systemic antibody delivery and remained present for two weeks, with minimal in vivo photobleaching after exposure to standard operating room lighting. There was greatly improved ability to resect labeled tumor tissue using FGS. CONCLUSIONS: Fluorophore-labeled anti-CEA or anti-CA 19-9 antibodies enable enhanced visualization of tumors for FGS of pancreatic cancer when CEA or CA 19-9 expression is present. The choice of fluorophore significantly affects the signal intensity in the labeled tumor. The technologies described herein have the potential to change the paradigm of surgical oncology to engender significantly improved outcomes.

[Preparation and characterization of the monoclonal antibodies against human CA19-9 and establishment of a sandwich CLIA system].

OBJECTIVE: To prepare the monoclonal antibodies (mAbs) against human carbohydrate antigen 19-9 (CA19-9) and establish a double-antibody sandwich chemiluminescent immunoassay (CLIA) detection system for CA19-9 in the human serum. METHODS: BALB/c mice were immunized with human CA19-9 antigen. The mAbs were obtained by hybridoma technique. The purity, titer, specificity and pairing of the mAbs were characterized and the sandwich CLIA system was established. The system was evaluated in its accuracy, limit of detection, linearity and repetitiveness after optimization of coating buffer, coating concentration and pipetting mode, and the serum sample was tested with it. RESULTS: Four mAbs named #1-1, #2-1, #3-1 and #4-1 were obtained against human CA19-9. The titers of the anti-CA19-9 mAbs were above 10(-8). The mAbs had nearly no cross-reaction with CA125, CA15-3 and CA72-4. The double-antibody sandwich CLIA system was established by #3-1 mAb and #2-1 mAb-HRP. After optimization, its property was detection range of 0-1 000 U/mL, limit detection of 2.0 U/mL, linear correlation coefficient of 0.9999. The results which were contrasted with Roche test showed that: Kappa>0.75, r(correlation coefficient)>0.9. CONCLUSION: The mAbs against human CA9-9 have been prepared and a sandwich CLIA system for detecting CA19-9 in the human serum has been established successfully.

Metastatic recurrence in a pancreatic cancer patient derived orthotopic xenograft (PDOX) nude mouse model is inhibited by neoadjuvant chemotherapy in combination with fluorescence-guided surgery with an anti-CA 19-9-conjugated fluorophore.

The aim of this study is to determine the efficacy of neoadjuvant chemotherapy (NAC) with gemcitabine (GEM) in combination with fluorescence-guided surgery (FGS) on a pancreatic cancer patient derived orthotopic xenograft (PDOX) model. A PDOX model was established from a CA19-9-positive, CEA-negative tumor from a patient who had undergone a pancreaticoduodenectomy for pancreatic adenocarcinoma. Mice were randomized to 4 groups: bright light surgery (BLS) only; BLS+NAC; FGS only; and FGS+NAC. An anti-CA19-9 or anti-CEA antibody conjugated to DyLight 650 was administered intravenously via the tail vein of mice with the pancreatic cancer PDOX 24 hours before surgery. The PDOX was brightly labeled with fluorophore-conjugated anti-CA19-9, but not with a fluorophore-conjugated anti-CEA antibody. FGS was performed using the fluorophore-conjugated anti-CA19-9 antibody. FGS had no benefit over BLS to prevent metastatic recurrence. NAC in combination with BLS did not convey an advantage over BLS to prevent metastatic recurrence. However, FGS+NAC significantly reduced the metastatic recurrence frequency to one of 8 mice, compared to FGS only after which metastasis recurred in 6 out of 8 mice, and BLS+NAC with metastatic recurrence in 7 out of 8 mice (p = 0.041). Thus NAC in combination with FGS can reduce or even eliminate metastatic recurrence of pancreatic cancer sensitive to NAC. The present study further emphasizes the power of the PDOX model which enables metastasis to occur and thereby identify the efficacy of NAC in combination with FGS on metastatic recurrence.

Hemothorax with a high carbohydrate antigen 19-9 level caused by a bronchogenic cyst.

A 58-year-old man presented with right-sided chest pain. Radiography and computed tomography showed a pleural effusion in the right chest and a mass in the right hilum. Thoracentesis showed a hemothorax. The carbohydrate antigen (CA) 19-9 level in the pleural effusion was very high, requiring differentiation from malignancy. Positron emission tomography showed no significant fluorodeoxy glucose (FDG) accumulation. Magnetic resonance imaging revealed a cystic lesion. The tumor was resected for both a diagnosis and treatment. A pathological examination demonstrated a bronchogenic cyst. An immunohistochemical study suggested that the cyst was the source of the hemothorax and the high CA19-9 level.

Amperometric carbohydrate antigen 19-9 immunosensor based on three dimensional ordered macroporous magnetic Au film coupling direct electrochemistry of horseradish peroxidase.

A sandwich-type electrochemical immunosensor for the detection of carbohydrate antigen 19-9 (CA 19-9) antigen based on the immobilization of primary antibody (Ab1) on three dimensional ordered macroporous magnetic (3DOMM) electrode, and the direct electrochemistry of horseradish peroxidase (HRP) that was used as both the label of secondary antibody (Ab2) and the blocking reagent. The 3DOMM electrode was fabricated by introducing core-shell Au-SiO2@Fe3O4 nanospheres onto the surface of three dimensional ordered macroporous (3DOM) Au electrode via the application of an external magnet. Au nanoparticles functionalized SBA-15 (Au@SBA-15) was conjugated to the HRP labeled secondary antibody (HRP-Ab2) through the Au-SH or Au-NH3(+) interaction, and HRP was also used as the block reagent. The formation of antigen-antibody complex made the combination of Au@SBA-15 and 3DOMM exhibit remarkable synergistic effects for accelerating direct electron transfer (DET) between HRP and the electrode. Under the optimal conditions, the DET current signal increased proportionally to CA 19-9 concentration in the range of 0.05 to 15.65 U mL(-1) with a detection limit of 0.01 U mL(-1). Moreover, the immunosensor showed high selectivity, good stability, satisfactory reproducibility and regeneration. Importantly, the developed method was used to assay clinical serum specimens, achieving a good relation with those obtained from the commercialized electrochemiluminescent method.

Label-free detection of hepatocellular carcinoma markers based on photoluminescence of antibody-conjugated ZnO arrays.

Hepatocellular carcinoma (HCC) is a malignant disease that is prevalent all around the world, especially in Asia. The combined detection of four common HCC markers, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), carbohydrate antigen 125 (CA125) and 19-9 (CA19-9), can significantly improve the accuracy of early screening and diagnosis of this disease, which is very important for its effective treatment in a curable stage. In this article, hierarchical ZnO column arrays with core-shell structure were prepared, and specific antibodies of HCC markers were successfully conjugated onto ZnO arrays via the carbodiimide chemistry. The photoluminescence (PL) intensity of antibody-ZnO increased after HCC markers were bound. In the range of 0.5-15 ng/mL for AFP or CEA (or 0.5-15 U/mL for CA125 or CA19-9), the apparent linear relations between the PL enhancements and the concentrations of HCC markers offered simple standard curve for HCC detection in serum samples, indicating that the PL-enhanced antibody-ZnO arrays could be utilized in early clinical screening. A preliminary mechanism of PL intensity enhancement can be established based on this work.

An engineered anti-CA19-9 cys-diabody for positron emission tomography imaging of pancreatic cancer and targeting of polymerized liposomal nanoparticles.

BACKGROUND: Antibody-based therapeutics is a rapidly growing field. Small engineered antibody fragments demonstrate similar antigen affinity compared with the parental antibody but have a shorter serum half-life and possess the ability to be conjugated to nanoparticles. The goal of this study was to engineer an anti-carbohydrate antigen 19-9 (CA19-9) cys-diabody fragment in hopes of targeting nanoparticles to pancreatic cancer. METHODS: The anti-CA19-9 cys-diabody was created by engineering a C-terminal cysteine residue into the DNA single-chain Fv construct of the anti-CA19-9 diabody and expressed in NS0 cells. Maleimide chemistry was used to conjugate the cys-diabody to polymerized liposomal nanoparticles (PLNs) through the cysteine residues. Flow cytometry was used to evaluate targeting of cys-diabody and cys-diabody-PLN conjugate to human pancreatic cancer cell lines. The cys-diabody was radiolabeled with a positron emitter ((124)I) and evaluated in a mouse model of CA19-9-positive and CA19-9-negative xenografts with micro-positron emission tomography/micro-computed tomography at successive time intervals after injection. Percentage of injected dose per gram of radioactivity was measured in blood and tumor to provide objective confirmation of the micro-positron emission tomographic images. RESULTS: Tumor xenograft imaging of the anti-CA19-9 cys-diabody demonstrated an average tumor-to-blood ratio of 3.0 and positive-to-negative tumor ratio of 7.4. Successful conjugation of the cys-diabody to PLNs was indicated by flow cytometry showing specific binding of cys-diabody-PLN conjugate to human pancreatic cancer cells in vitro. CONCLUSIONS: Our results show that the anti-CA19-9 cys-diabody targets pancreatic cancer providing specific molecular imaging in tumor xenograft models. Furthermore, the cys-diabody-PLN conjugate demonstrates target-specific binding of human pancreatic cancer cells with the potential to deliver targeted treatment.

Diverse monoclonal antibodies against the CA 19-9 antigen show variation in binding specificity with consequences for clinical interpretation.

The CA 19-9 antigen is currently the best individual marker for the detection of pancreatic cancer. In order to optimize the CA 19-9 assay and to develop approaches to further improve cancer detection, it is important to understand the specificity differences between CA 19-9 antibodies and the consequential affect on biomarker performance. Antibody arrays enabled multiplexed comparisons between five different CA 19-9 antibodies used in the analysis of plasma samples from pancreatic cancer patients and controls. Major differences were observed between antibodies in their detection of particular patient samples. Glycan array analysis revealed that certain antibodies were highly specific for the canonical CA 19-9 epitope, sialyl-Lewis A, while others bound sialyl-Lewis A in addition to a related structure called sialyl-Lewis C and modification with Nue5Gc. In a much larger patient cohort, we confirmed the binding of sialyl-Lewis C glycan by one of the antibodies and showed that the broader specificity led to the detection of an increased number of cancer patients without increasing detection of pancreatitis patient samples. This work demonstrates that variation between antibody specificity for cancer-associated glycans can have significant implications for biomarker performance and highlights the value of characterizing and detecting the range of glycan structures that are elevated in cancer.

Anti-CA19-9 diabody as a PET imaging probe for pancreas cancer.

BACKGROUND: Intact antibodies are poor imaging agents due to a long serum half-life (10-20 d) preventing adequate contrast between the tumor and surrounding blood. Smaller engineered antibody fragments overcome this problem by exhibiting shorter serum half-lives (4-20 h).The diabody (55 kDa) is the smallest antibody fragment, which retains the bivalency of the intact antibody. Our goal was to develop and characterize the anti-CA19-9 diabody fragment and determine its ability to provide antigen specific imaging of pancreas cancer. METHODS: The diabody DNA construct was created by isolation of the variable region genes of the intact anti-CA19-9 antibody. Diabody expression was carried out in NS0 cells and purified using HPLC from supernatant. Specific antigen binding was confirmed with flow cytometry and immunofluorescence. Radiolabeled diabody was injected into mice harboring an antigen positive xenograft (BxPC3 or Capan-2) and a negative xenograft (MiaPaca-2). MicroCT and MicroPET were performed at successive time intervals after injection. Radioactivity was measured in blood and tumor to provide objective confirmation of the microPET images. RESULTS: Immunofluorescence and flow cytometry showed specific binding of the anti-CA19-9 diabody. Pancreas xenograft imaging of BxPC3/MiaPaca-2 and Capan-2/MiaPaca-2 models with the anti-CA19-9 diabody demonstrated an average tumor:blood ratio of 5.0 and 2.0, respectively, and an average positive:negative tumor ratio of 11 and 6, respectively. With respect to the tumor:blood ratio, these data indicate five times and two times more radioactivity in the tumor than in the blood yielding adequate contrast between tumor tissue and background (i.e., blood) to create the representative microPET images. CONCLUSIONS: We successfully engineered a functional diabody against CA19-9, a tumor antigen present on the vast majority of pancreas cancers. Additionally, we demonstrate high contrast antigen specific microPET imaging of pancreas cancer in xenograft models.