Mapping PAM4 (clivatuzumab), a monoclonal antibody in clinical trials for early detection and therapy of pancreatic ductal adenocarcinoma, to MUC5AC mucin.

BACKGROUND: PAM4, an antibody that has high specificity for pancreatic ductal adenocarcinoma (PDAC), compared to normal pancreas, benign lesions of the pancreas, and cancers originating from other tissues, is being investigated as a biomarker for early detection, as well as antibody-targeted imaging and therapy. Therefore, the identity of the antigen bound by this monoclonal antibody (MAb) can provide information leading to improved use of the antibody. Prior results suggested the antigen is a mucin-type glycoprotein rich in cysteine disulfide bridges that provide stable conformation for the PAM4-epitope. METHODS: Indirect and sandwich enzyme immunoassays (EIA) were performed to compare and contrast the reactivity of PAM4 with several anti-mucin antibodies having known reactivity to specific mucin species (e.g., MUC1, MUC4, MUC5AC, etc.). Studies designed to block reactivity of PAM4 with its specific antigen also were performed. RESULTS: We demonstrate that MAbs 2-11 M1 and 45 M1, each reactive with MUC5AC, are able to provide signal in a heterologous sandwich immunoassay where PAM4 is the capture antibody. Further, we identify MAbs 21 M1, 62 M1, and 463 M1, each reactive with MUC5AC, as inhibiting the reaction of PAM4 with its specific epitope. MAbs directed to MUC1, MUC3, MUC4, MUC16 and CEACAM6 are not reactive with PAM4-captured antigen, nor are they able to block the reaction of PAM4 with its antigen. CONCLUSIONS: These data implicate MUC5AC as a specific mucin species to which PAM4 is reactive. Furthermore, this realization may allow for the improvement of the current PAM4 serum-based immunoassay for detection of early-stage PDAC by the application of anti-MUC5AC MAbs as probes in this sandwich EIA.

PAM4 enzyme immunoassay alone and in combination with CA 19-9 for the detection of pancreatic adenocarcinoma.

BACKGROUND: The monoclonal antibody PAM4 has high specificity for pancreatic ductal adenocarcinoma (PDAC), as well as its precursor lesions, but has not been found to be reactive with normal and benign pancreatic tissues. The objective of the current study was to evaluate a PAM4-based serum enzyme immunoassay alone and in combination with the carbohydrate antigen (CA) 19-9 assay for the detection of PDAC, with particular attention to early stage disease. METHODS: Sera from patients with confirmed PDAC (N = 298), other cancers (N = 99), benign disease of the pancreas (N = 120), and healthy adults (N = 79) were evaluated by a specific enzyme immunoassay for the concentration of PAM4 and CA 19-9 antigen levels by blinded analyses. All tests for statistical significance were 2-sided. RESULTS: The overall sensitivity for PAM4 detection of PDAC was 76%, with 64% of patients with stage I disease also identified. The detection rate was considerably higher (85%) for patients with advanced disease. The assay demonstrated high specificity compared with benign pancreatic disease (85%), with a positive likelihood ratio of 4.93. CA 19-9 provided an overall sensitivity of 77%, and was positive in 58% of patients with stage I disease; however, the specificity was significantly lower for CA 19-9 (68%), with a positive likelihood ratio of 2.85 (P = .026 compared with PAM4). It is important to note that a combined PAM4 and CA 19-9 biomarker serum assay demonstrated an improved sensitivity (84%) for the overall detection of PDAC without a significant loss of specificity (82%) compared with either arm alone. CONCLUSIONS: The PAM4 enzyme immunoassay identified approximately two-thirds of patients with stage I PDAC with high discriminatory power with respect to benign, nonneoplastic pancreatic disease. These results provide a rationale for testing patient groups considered to be at high risk for PDAC with a combined PAM4 and CA 19-9 biomarker serum assay for the detection of early stage PDAC.

Synergistic interaction between sphingomyelin and gemcitabine potentiates ceramide-mediated apoptosis in pancreatic cancer.

We have examined the mechanism by which sphingomyelin (SM) enhances chemotherapy in human pancreatic cancer cells, focusing on the correlation between ceramide metabolism and apoptosis. Dose response curves for gemcitabine in the absence or presence of 0.2 mg/mL SM provided IC(50) values of 78.3 +/- 13.7 and 13.0 +/- 3.0 nmol/L, respectively. The cytotoxic effect of the combined treatment was synergistic (combination index = 0.36). Using annexin-V staining, the percentage of apoptotic cells was 3.6 +/- 2.6% for the untreated cells, 6.5 +/- 3.8% for the 0.2 mg/mL SM-treated cells, and 19.9 +/- 12.9% for the 100 nmol/L gemcitabine-treated cells, but increased significantly to 42.1 +/- 12.7% with the combined treatment (P < 0.001, compared with gemcitabine-treated group). The percentage of cells losing mitochondrial membrane potential followed a similar trend. The ceramide content of untreated and gemcitabine-treated cells was not significantly different (0.46 +/- 0.29 and 0.59 +/- 0.34 pmol ceramide/nmole PO(4)). However, when 0.2 mg/mL SM was added, ceramide levels were 1.09 +/- 0.42 and 1.58 +/- 0.55 pmol ceramide/nmol PO(4), for the SM alone and SM with gemcitabine-treated cells, respectively (P = 0.038). Acidic SMase was activated by exposure to gemcitabine but not SM, whereas the activities of neutral SMase and glycosylceramide synthase did not change with either gemcitabine or SM. The data are consistent with gemcitabine-induced activation of acidic SMase and indicate that the addition of SM can yield increased production of ceramide, mitochondrial depolarization, apoptosis, and cell death. Because SM by itself is relatively nontoxic, addition of this lipid to agents that induce apoptosis may prove useful to enhance apoptosis and increase cytotoxicity in cancer cells.

Improved iodine radiolabels for monoclonal antibody therapy.

A major disadvantage of (131)iodine (I)-labeled monoclonal antibodies (MAbs) for radioimmunotherapy has been the rapid diffusion of iodotyrosine from target cells after internalization and catabolism of the radioiodinated MAbs. We recently reported that a radioiodinated, diethylenetriaminepentaacetic acid-appended peptide, designated immunomedics' residualizing peptide 1 (IMP-R1), was a residualizing iodine label that overcame many of the limitations that had impeded the development of residualizing iodine for clinical use. To determine the factors governing the therapeutic index of the labeled MAb, as well as the factors required for production of radioiodinated MAb in high yield and with high specific activity, variations in the peptide structure of IMP-R1 were evaluated. A series of radioiodinated, diethylenetriaminepentaacetic acid-appended peptide moieties (IMP-R1 through IMP-R8) that differed in overall hydrophilicity and charge were compared. Radioiodinations of the peptides followed by conjugations to disulfide-reduced RS7 (an anti-epithelial glycoprotein-1 MAb) furnished radioimmunoconjugates in good overall incorporations, with immunoreactivities comparable to that of directly radioiodinated RS7. Specific activities of up to 8 mCi/mg and yields > 80% have been achieved. In vitro processing experiments showed marked increases in radioiodine retention with all of the adducts; radioiodine retention at 45 h was up to 86% greater in cells than with directly iodinated RS7. Each of the (125)I-peptide-RS7 conjugates was compared with (131)I-RS7 (labeled by the chloramine-T method) in paired-label biodistribution studies in nude mice bearing human lung tumor xenografts. All of the residualizing substrates exhibited significantly enhanced retention in tumor in comparison to directly radioiodinated RS7, but the nontarget uptakes differed significantly among the residualizing labels. The best labels were IMP-R4 and IMP-R8, showing superior tumor-to-non-tumor ratios by virtue of high tumor uptake and retention and low normal organ uptake, as well as superior radiochemical properties. The therapeutic efficacy of (131)I-IMP-R4-RS7 was compared with that of conventionally (131)I-labeled RS7 and (90)yttrium-RS7 in the nude mice lung cancer model. The therapeutic efficacy of (131)I-IMP-R4-RS7 and (90)yttrium-RS7 were equivalent, and both agents yielded significantly improved control of tumor growth compared with conventional (131)I-labeled RS7.

Identification of a Mu-9 (anti-colon-specific antigen-p)-reactive peptide having homology to CA125 (MUC16).

Mu-9, an anti-colon specific antigen-p (CSAp) monoclonal antibody (MAb), has shown excellent gastrointestinal cancer targeting in both pre-clinical and clinical trials. With the recent development of the humanized version of this MAb, Mu-9 will receive further attention as a potential therapeutic agent for colorectal cancer. Hence, we have undertaken studies to examine the nature of the CSAp antigen and the structure of the Mu-9 epitope. M13 phage displaying random 12-mer peptides were used to isolate a peptide that binds both murine and humanized Mu-9 MAbs. The peptide, Mu-9-p16, was synthesized and found to inhibit binding of Mu-9 to CSAp with a Ka for the humanized antibody of 4.28 x 10(-7) +/- 0.91 x 10(-7) M. Control peptides did not bind Mu-9, nor did they inhibit the Mu-9-CSAp interaction. Three overlapping peptides were synthesized and used to demonstrate that the last six residues were sufficient to inhibit the Mu-9-CSAp interaction. A search of GenBank revealed that the peptide sequence IHPRP, was also present within CA125, a very high molecular weight ovarian cancer-associated antigen. The sequence is present outside of the known antigenic regions identified by the Oc125, M-11 and Ov197 anti-CA125 antibodies. To demonstrate that CSAp and CA125 may be the same protein, a heterologous sandwich enzyme immunoassay was developed with anti-CA125 MAbs used as capture reagents and Mu-9 as probe. By use of this ELISA system, we were able to specifically identify CSAp. In conclusion, our results indicate that the Mu-9-p16 peptide isolated through our screen identifies a peptide epitope shared by CSAp and CA125 and suggest that these proteins are related.

Differentiation of pancreatic ductal adenocarcinoma from chronic pancreatitis by PAM4 immunohistochemistry.

CONTEXT: PAM4 is a monoclonal antibody that shows high specificity for pancreatic ductal adenocarcinoma (PDAC) and its neoplastic precursor lesions. A PAM4-based serum immunoassay is able to detect 71% of early-stage patients and 91% with advanced disease. However, approximately 20% of patients diagnosed with chronic pancreatitis (CP) are also positive for circulating PAM4 antigen. The specificity of the PAM4 antibody is critical to the interpretation of the serum-based and immunohistochemical assays for detection of PDAC. OBJECTIVE: To determine whether PAM4 can differentiate PDAC from nonneoplastic lesions of the pancreas. DESIGN: Tissue microarrays of PDAC (N = 43) and surgical specimens from CP (N = 32) and benign cystic lesions (N = 19) were evaluated for expression of the PAM4 biomarker, MUC1, MUC4, CEACAM5/6, and CA19-9. RESULTS: PAM4 and monoclonal antibodies (MAbs) to MUC1, MUC4, CEACAM5/6, and CA19-9 were each reactive with the majority of PDAC cases; however, PAM4 was the only monoclonal antibody not to react with adjacent, nonneoplastic parenchyma. Although PAM4 labeled 19% (6 of 32) of CP specimens, reactivity was restricted to pancreatic intraepithelial neoplasia associated with CP; inflamed tissues were negative in all cases. In contrast, MUC1, MUC4, CEACAM5/6, and CA19-9 were detected in 90%, 78%, 97%, and 100% of CP, respectively, with reactivity also present in nonneoplastic inflamed tissue. CONCLUSIONS: PAM4 was the only monoclonal antibody able to differentiate PDAC (and pancreatic intraepithelial neoplasia precursor lesions) from benign, nonneoplastic tissues of the pancreas. These results suggest the use of PAM4 for evaluation of tissue specimens, and support its role as an immunoassay for detection of PDAC.

Detection of early-stage pancreatic adenocarcinoma.

BACKGROUND: Pancreatic adenocarcinoma is an almost universally lethal disease, in large part, due to our inability to detect early-stage disease. Monoclonal antibody PAM4 is reactive with a unique biomarker expressed by >85% of pancreatic adenocarcinomas. In this report, we examined the ability of a PAM4-based immunoassay to detect early-stage disease. MATERIALS AND METHODS: The PAM4-based immunoassay was used to quantitate antigen in the serum of healthy volunteers (n = 19), patients with known pancreatic adenocarcinoma (n = 68), and patients with a primary diagnosis of chronic pancreatitis (n = 29). RESULTS: Sensitivity for detection of pancreatic adenocarcinoma was 82%, with a false-positive rate of 5% for healthy controls. Patients with advanced disease had significantly higher antigen levels than those with early-stage disease (P < 0.01), with a diagnostic sensitivity of 91%, 86%, and 62% for stage 3/stage 4 advanced disease, stage 2, and stage 1, respectively. We also evaluated chronic pancreatitis sera, finding 38% positive for antigen; however, this was discordant with immunohistochemical findings that suggest the PAM4 antigen is not produced by inflamed pancreatic tissue. Furthermore, several of the serum-positive pancreatitis patients, for whom tissue specimens were available for pathologic interpretation, had evidence of neoplastic precursor lesions. CONCLUSIONS: These results suggest the use of the PAM4 serum assay to detect early-stage pancreatic adenocarcinoma and that positive levels of PAM4 antigen are not derived from inflamed pancreatic tissues but rather may provide evidence of subclinical pancreatic neoplasia. EFFECT: The ability to detect pancreatic adenocarcinoma at an early stage could provide for early therapeutic intervention with potentially improved patient outcomes.