Identification of PAM4 (clivatuzumab)-reactive epitope on MUC5AC: a promising biomarker and therapeutic target for pancreatic cancer.

PAM4 is a monoclonal antibody showing high specificity for pancreatic ductal adenocarcinoma (PDAC). Humanized PAM4 labeled with 90Y in combination with low-dose gemcitabine has shown promising therapeutic activity, and is being evaluated in a phase III clinical trial. Prior efforts have suggested that PAM4 potentially reacts with MUC5AC, a secretory mucin expressed de novo in early pancreatic neoplasia and retained throughout disease progression. In present study, we provide further evidence validating MUC5AC as the PAM4 antigen, and locate PAM4-reactive epitope within the N-terminal cysteine-rich subdomain 2 (Cys2), thus differentiating PAM4 from most anti-MUC5AC antibodies known to-date. Specifically, we show (i) PAM4-antigen and MUC5AC were co-localized in multiple human cancer cell lines, including Capan-1, BxPC-3, and CFPAC-1; (ii) MUC5AC-specific siRNA prominently reduced the expression of both MUC5AC and PAM4-antigen in CFPAC-1 cells; (iii) PAM4 preferentially binds to the void-volume fractions from Sepharose-CL2B chromatography of Capan-1 culture supernatants, which were revealed by Western blot to display the ladder pattern characteristic of oligomeric MUC5AC; and (iv) the N-terminal Cys2 within several recombinant MUC5AC fragments is essential for binding to PAM4. These findings shed light on the mechanism of PAM4-based diagnosis and treatment for pancreatic cancer, and guide further exploration of its clinical utility.

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.

Diagnostic and therapeutic implications of a novel immunohistochemical panel detecting duodenal mucosal invasion by pancreatic ductal adenocarcinoma.

BACKGROUND: We investigated a series of pancreaticoduodenectomy and duodenal biopsies with a panel of immunohistochemical markers to identify duodenal mucosal invasion by pancreatic ductal adenocarcinoma (PDAC), including markers of poor prognosis and targets of promising novel immunotherapies. MATERIALS AND METHODS: Eighteen consecutive pancreaticoduodenectomy specimens with duodenal mucosal invasion by PDAC were examined for expression of MUC1, MUC4, MUC5AC, MUC6, mesothelin, MUC2, CDX2, and DPC4 on formalin-fixed, paraffin-embedded sections of duodenal-ampullary-pancreatic junctions. Expression of all but MUC6 was also assessed in duodenal biopsies from 12 patients with duodenal mucosal invasion by PDAC. RESULTS: The duodenal mucosa expressed MUC1 (crypts), MUC2 (goblet cells), MUC6 (Brunner glands), CDX2, and DPC4. PDACs in the duodenal mucosa from the resection (n=16-18) and biopsy (n=12) specimens were marked as follows: MUC1 100% (30/30), MUC4 83% (24/29), MUC5AC 83% (25/30), mesothelin 82% (23/28), MUC2 7% (2/30), and CDX2 36% (10/28). Loss of DPC4 expression was seen in 16 of 29 (55%) cases. Reactive mucosa adjacent to PDAC expressed MUC4, MUC5AC and mesothelin in 65% (17/26), 19% (5/27), and 19% (5/26) of cases, respectively. While MUC5AC and mesothelin had high diagnostic accuracy for detection of PDAC, MUC2, CDX2 and DPC4 expression demonstrated negative correlation with PDAC, with absent expression being highly specific for PDAC. CONCLUSION: Immunohistochemical labeling for PDAC biomarkers may aid the diagnosis of PDAC in duodenal biopsy, especially in situations where diagnosis of a pancreatic mass is challenging.

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.

Milatuzumab-SN-38 conjugates for the treatment of CD74+ cancers.

CD74 is an attractive target for antibody-drug conjugates (ADC), because it internalizes and recycles after antibody binding. CD74 mostly is associated with hematologic tumors but is expressed also in solid cancers. Therefore, ADCs of the humanized anti-CD74 antibody, milatuzumab, were examined for the therapy of CD74-expressing solid tumors. Milatuzumab-doxorubicin and two milatuzumab-SN-38 conjugates with cleavable linkers, differing in their stability in serum and how they release SN-38 in the lysosome, were prepared. CD74 expression was determined by flow cytometry and immunohistology. In vitro cytotoxicity and in vivo therapeutic studies were conducted in the human cancer cell lines A-375 (melanoma), HuH-7 and Hep-G2 (hepatoma), Capan-1 (pancreatic), NCI-N87 (gastric), and Raji Burkitt lymphoma. The milatuzumab-SN-38 ADC was compared with SN-38 ADCs prepared with anti-Trop-2 and anti-CEACAM6 antibodies in xenografts expressing their target antigens. Milatuzumab-doxorubicin was most effective in the lymphoma model, whereas in A-375 and Capan-1 solid tumors, only milatuzumab-SN-38 showed a therapeutic benefit. Despite much lower surface expression of CD74 than Trop-2 or CEACAM6, milatuzumab-SN-38 had similar efficacy in Capan-1 as anti-Trop-2-SN-38, but in NCI-N87, anti-CEACAM6 and anti-Trop-2 conjugates were superior. Studies in two hepatoma lines at a single dose level showed significant benefit over saline controls but not against an irrelevant immunoglobulin G conjugate. CD74 is a suitable target for ADCs in some solid tumor xenografts, with efficacy largely influenced by uniformity of CD74 expression and with SN-38 conjugates providing the best therapeutic responses; SN-38 conjugates were preferable in solid cancers, whereas doxorubicin ADC was better in lymphoma tested.

Horizontal transmission of malignancy: in-vivo fusion of human lymphomas with hamster stroma produces tumors retaining human genes and lymphoid pathology.

We report the in-vivo fusion of two Hodgkin lymphomas with golden hamster cheek pouch cells, resulting in serially-transplanted (over 5-6 years) GW-532 and GW-584 heterosynkaryon tumor cells displaying both human and hamster DNA (by FISH), lymphoma-like morphology, aggressive metastasis, and retention of 7 human genes (CD74, CXCR4, CD19, CD20, CD71, CD79b, and VIM) out of 24 tested by PCR. The prevalence of B-cell restricted genes (CD19, CD20, and CD79b) suggests that this uniform population may be the clonal initiating (malignant) cells of Hodgkin lymphoma, despite their not showing translation to their respective proteins by immunohistochemical analysis. This is believed to be the first report of in-vivo cell-cell fusion of human lymphoma and rodent host cells, and may be a method to disclose genes regulating both organoid and metastasis signatures, suggesting that the horizontal transfer of tumor DNA to adjacent stromal cells may be implicated in tumor heterogeneity and progression. The B-cell gene signature of the hybrid xenografts suggests that Hodgkin lymphoma, or its initiating cells, is a B-cell malignancy.

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.

Prevention of acute graft-versus-host disease in a xenogeneic SCID mouse model by the humanized anti-CD74 antagonistic antibody milatuzumab.

Prevention and treatment of graft-versus-host disease (GVHD) remains a major challenge, given that current T-cell depletion and mainstay immunosuppressive therapies compromise preexisting T-cell immunity, often leading to severe infections and disease relapse. Thus, there is a critical need for novel anti-GVHD agents that can spare protective T-cell memory. Here we show that milatuzumab (hLL1), a humanized anti-CD74 antagonist monoclonal antibody, can moderately reduce the numbers of CD74-expressing B cells and myeloid dendritic cells, but has no effect on the survival of T cells that are CD74(-). Consequently, milatuzumab inhibits allogeneic T-cell proliferation in mixed leukocyte reactions. In a human/mouse xenogeneic SCID mouse model in which GVHD is induced and mediated by engrafted human CD4(+) T cells and dendritic cells, milatuzumab effectively prevents the onset and manifestations of acute GVHD, suppresses serum levels of human IFN-γ and IL-5, eliminates the infiltration of human lymphocytes into GVHD target organs (ie, lung, liver, and spleen), and significantly promotes survival (90% versus 20% for controls; P = .0012). Importantly, exposure to milatuzumab does not affect the number of cytomegalovirus-specific, IFN-γ-producing human CD8(+) T cells in allogeneic mixed leukocyte reactions. These encouraging results warrant further exploration of milatuzumab as a possible new therapeutic agent for GVHD.

Fractionated radioimmunotherapy with (90) Y-clivatuzumab tetraxetan and low-dose gemcitabine is active in advanced pancreatic cancer: A phase 1 trial.

BACKGROUND: It has been demonstrated that the humanized clivatuzumab tetraxetan (hPAM4) antibody targets pancreatic ductal carcinoma selectively. After a trial of radioimmunotherapy that determined the maximum tolerated dose of single-dose yttrium-90-labeled hPAM4 ((90) Y-hPAM4) and produced objective responses in patients with advanced pancreatic ductal carcinoma, the authors studied fractionated radioimmunotherapy combined with low-dose gemcitabine in this disease. METHODS: Thirty-eight previously untreated patients (33 patients with stage IV disease and 5 patients with stage III disease) received gemcitabine 200 mg/m(2) weekly for 4 weeks with (90) Y-hPAM4 given weekly in Weeks 2, 3, and 4 (cycle 1), and the same cycle was repeated in 13 patients (cycles 2-4). In the first part of the study, 19 patients received escalating weekly (90) Y doses of 6.5 mCi/m(2) , 9.0 mCi/m(2) , 12.0 mCi/m(2) , and 15.0 mCi/m(2) . In the second portion, 19 additional patients received weekly doses of 9.0 mCi/m(2) or 12.0 mCi/m(2) . RESULTS: Grade 3/4 thrombocytopenia or neutropenia (according to version 3.0 of the National Cancer Institute's Common Terminology Criteria for Adverse Events) developed in 28 of 38 patients after cycle 1 and in all retreated patients; no grade >3 nonhematologic toxicities occurred. Fractionated dosing of cycle 1 allowed almost twice the radiation dose compared with single-dose radioimmunotherapy. The maximum tolerated dose of (90) Y-hPAM4 was 12.0 mCi/m(2) weekly for 3 weeks for cycle 1, with ≤9.0 mCi/m(2) weekly for 3 weeks for subsequent cycles, and that dose will be used in future trials. Six patients (16%) had partial responses according to computed tomography-based Response Evaluation Criteria in Solid Tumors, and 16 patients (42%) had stabilization as their best response (58% disease control). The median overall survival was 7.7 months for all 38 patients, including 11.8 months for those who received repeated cycles (46% [6 of 13 patients] ≥1 year), with improved efficacy at the higher radioimmunotherapy doses. CONCLUSIONS: Fractionated radioimmunotherapy with (90) Y-hPAM4 and low-dose gemcitabine demonstrated promising therapeutic activity and manageable myelosuppression in patients with advanced pancreatic ductal carcinoma.

Evaluation of a non-viral vaccine in smallpox-vaccinated individuals and immunized HLA-transgenic mice.

The current poxvirus vaccine is associated with rare, but serious adverse events. Therefore, we investigated a non-replicating approach to vaccine design. Peptides encoding potential HLA-binding motifs were derived from the orthopoxvirus genes, D8L, A27L, and C12L (the IL-18-binding protein [vIL18BP105]), all of which are preserved among poxviruses that infect humans, and which may be a target of host immunity. The peptides were tested with poxvirus-vaccinated human PBMC and serum for eliciting memory responses, as well as with splenocytes and serum from peptide-immunized, human HLA-DR04 transgenic (HLA tg) mice. vIL18BP105 induced 5-fold proliferation of vaccinated-donor PBMC over non-vaccinated (P<0.001), including IL-2-producing CD8+ cells. Serum IgG recognizing vIL18BP105 was detected (P<0.002 vs non-vaccinated) by ELISA. Viral peptides were conjugated to the HLA-targeting mAb, L243, for immunization of HLA tg mice. Splenocytes from vIL18BP105-L243-immunized mice proliferated upon exposure to vIL18BP105 (P<0.001). Proliferating splenocytes were interferon-γ-producing CD4(+)CD45RA(neg). vIL18BP105-L243-immunized mice generated IgG more rapidly than free-peptide-immunized mice. Peptide-specific antibody was also detected when different L243-peptide conjugates were combined. vIL18BP, by eliciting human memory responses, is a viable antigen for inclusion in a virus-free vaccine. The immunogenicity of peptides was boosted by conjugation to L243, whether administered alone or combined.

Horizontal transmission and retention of malignancy, as well as functional human genes, after spontaneous fusion of human glioblastoma and hamster host cells in vivo.

Cell fusion in vitro has been used to study cancer, gene mapping and regulation, and the production of antibodies via hybridomas. However, in-vivo heterosynkaryon formation by cell-cell fusion has received less attention. This investigation describes the spontaneous fusion of a human glioblastoma with normal hamster cells after xenogeneic transplantation, resulting in malignant cells that express both human and hamster genes and gene products, and retention of glioblastoma traits with an enhanced ability to metastasize. Three of 7 human genes found showed translation of their proteins during serial propagation in vivo or in vitro for years; namely, CD74, CXCR4 and PLAGL2, each implicated with malignancy or glioblastoma. This supports the thesis that genetic hybridization of cancer and normal cells can transmit malignancy and also, as first described herein, regulatory genes involved in the tumor's organotypic morphology. Evidence also is increasing that even cell-free human cancer DNA can induce malignancy and transfer genetic information to normal cells. Hence, we posit that the transfer of genetic information between tumor and stromal cells, whether by cell-cell fusion or other mechanisms, is implicated in the progression of malignancy, and may further define the crosstalk between cancer cells and their stromal neighbors.

Treatment of advanced pancreatic carcinoma with 90Y-Clivatuzumab Tetraxetan: a phase I single-dose escalation trial.

PURPOSE: Humanized antibody hPAM4 specifically binds a mucin glycoprotein expressed in pancreatic adenocarcinomas. This phase I study evaluated a single dose of (90)Y-clivatuzumab tetraxetan ((90)Y-labeled hPAM4) in patients with advanced pancreatic cancer. EXPERIMENTAL DESIGN: Twenty-one patients (4 stage III; 17 stage IV) received (111)In-hPAM4 for imaging and serum sampling before (90)Y-hPAM4. Study procedures evaluated adverse events, safety laboratories, computed tomography (CT) scans, biomarkers, pharmacokinetics, radiation dosimetry, and immunogenicity (HAHA). RESULTS: (111)In-hPAM4 showed normal biodistribution with radiation dose estimates to red marrow and solid organs acceptable for radioimmunotherapy and with tumor targeting in 12 patients. One patient withdrew before (90)Y-hPAM4; otherwise, 20 patients received (90)Y doses of 15 (n = 7), 20 (n = 9), and 25 mCi/m(2) (n = 4). Treatment was well tolerated; the only significant drug-related toxicities were (NCI CTC v.3) grade 3 to 4 neutropenia and thrombocytopenia increasing with (90)Y dose. There were no bleeding events or serious infections, and most cytopenias recovered to grade 1 within 12 weeks. Three patients at 25 mCi/m(2) encountered dose-limiting toxicity with grade 4 cytopenias more than 7 days, establishing 20 mCi/m(2) as the maximal tolerated (90)Y dose. Two patients developed HAHA of uncertain clinical significance. Most patients progressed rapidly and with CA19-9 levels increasing within 1 month of therapy, but 7 remained progression-free by CT for 1.5 to 5.6 months, including 3 achieving transient partial responses (32%-52% tumor diameter shrinkage). CONCLUSION: (90)Y-Clivatuzumab tetraxetan was well tolerated with manageable hematologic toxicity at the maximal tolerated (90)Y dose, and is a potential new therapeutic for advanced pancreatic cancer.

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.

Enhanced expression of CD74 in gastrointestinal cancers and benign tissues.

CD74, a transmembrane glycoprotein that associates with MHC II, is an important chaperone that regulates antigen presentation for immune response. In addition, CD74 is the receptor for macrophage migration-inhibitory factor which, when bound to CD74, initiates survival pathways and cell proliferation. Formalin fixed, paraffin embedded clinical specimens were evaluated by immunohistochemical procedures for expression of CD74. Overall, expression of CD74 within gastrointestinal carcinomas showed a statistically greater expression than in the normal tissue counterparts (P<0.001 or better). CD74 expression was observed in 95% of pancreatic carcinomas with the majority of cases presenting a mostly intense, diffuse labeling pattern. The results suggested a trend towards greater expression within the higher grade carcinomas (P=0.06). Colorectal and gastric carcinomas gave similar results with 60% and 86%, respectively, positive for CD74 with an intense, diffuse staining pattern. We hypothesized that precursor lesions would express levels of CD74 as high, or higher, than their respective carcinomas, since activation of survival pathways would be of particular importance at the early stages of neoplastic development. For PanIN lesions there was greater expression of CD74 within higher grade, PanIN-3 lesions, whereas the colonic adenomas showed no such trend, but overall, a higher frequency and intensity of CD74 labeling than was observed within the colon carcinomas. These findings are supportive of a role for CD74 in the development and maintenance of gastrointestinal neo-plasia, and provide a rationale for development of therapeutic agents that are able to block CD74 function, specifically within the tumor cell.

Pretargeted radioimmunotherapy of pancreatic cancer xenografts: TF10-90Y-IMP-288 alone and combined with gemcitabine.

UNLABELLED: Pancreatic cancer is a silent disease that most commonly presents in an already metastatic form. Current treatment options provide little survival benefit. Radiolabeled PAM4 IgG, a monoclonal antibody that recognizes a unique epitope associated with a mucin found almost exclusively in pancreatic cancer, has shown encouraging therapeutic effects in animal models and in early clinical testing ((90)Y-humanized PAM4 IgG, (90)Y-clivatuzumab tetraxetan). The studies reported herein examine a new pretargeting procedure for delivering therapeutic radionuclides. METHODS: We prepared a humanized, recombinant tri-Fab bispecific monoclonal antibody (bsmAb) (TF10) using specificity for targeting pancreatic cancer of PAM4 and another Fab binding to a hapten (histamine-succinyl-glycine [HSG]) and tested this in a pretargeting setting with a (90)Y-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid-di-HSG-peptide (pretargeted radioimmunotherapy [PT-RAIT]). Nude mice bearing established Capan-1 human pancreatic cancer xenografts were given TF10 and then received the (90)Y peptide as a single bolus dose 19 h later, or the therapy cycle was fractionated weekly. Other studies examined different combinations with gemcitabine. RESULTS: PT-RAIT of 18.5 MBq ( approximately 50% of its maximum tolerated dose [MTD]) was as effective as the MTD of (90)Y-PAM4 IgG (5.55 MBq). Three monthly doses of 9.25 MBq of PT-RAIT combined with a monthly cycle of gemcitabine (3 weekly, 6-mg doses) significantly enhanced survival, compared with PT-RAIT alone. Adding gemcitabine as a radiosensitizer to 9.25 MBq of PT-RAIT enhanced objective responses. Weekly fractionation of the PT-RAIT, as compared with a single treatment, improved responses. CONCLUSION: PAM4-based PT-RAIT with (90)Y hapten peptide is an effective treatment for pancreatic cancer, with less toxicity than (90)Y-PAM4 IgG, in this model. Combinations with gemcitabine and dose fractionation of the PT-RAIT enhanced therapeutic responses.

Ceramide regulates gemcitabine-induced senescence and apoptosis in human pancreatic cancer cell lines.

Bioactive sphingolipids are potent intracellular signaling molecules having profound effects on cell death, growth, and differentiation. Pharmacologic manipulation of sphingolipid levels could have a significant effect on the induction of apoptosis by anticancer agents, and thus, improve treatment efficacy. We observed that gemcitabine cannot completely kill AsPc1 and Panc1 human pancreatic cancer cells in culture; even at high concentrations of gemcitabine, 30% to 40% of the cells remain viable. By adding sphingomyelin to the culture medium, gemcitabine-induced cell death increased synergistically to >90%. Panc1 cells that survived high concentrations of gemcitabine had an increase in beta-galactosidase activity, a marker of senescence. The inclusion of sphingomyelin with gemcitabine reduced beta-galactosidase activity, as compared with cells treated with gemcitabine alone. Expression of p21(waf1/cip1) in both cell lines exposed to sphingomyelin, gemcitabine, and gemcitabine + sphingomyelin varied relative to the untreated group. C(8)-ceramide induced both cell death and senescence in a dose-dependent manner. These results indicate that gemcitabine induces senescence in pancreatic cancer cells and that sphingomyelin-enhanced chemosensitivity is achieved through reducing the induction of senescence by redirecting the cell to enter the apoptotic pathway. Ceramide levels seem to be critical to this decision, with cell cycle progression being uninhibited at low ceramide levels, senescence induced at moderate levels, and apoptosis initiated at high levels. Our results provide further evidence that targeting the sphingolipid metabolism is a means of enhancing the efficacy of chemotherapeutic agents.

A novel bispecific, trivalent antibody construct for targeting pancreatic carcinoma.

Preclinical and clinical studies have demonstrated the application of radiolabeled mAb-PAM4 for nuclear imaging and radioimmunotherapy of pancreatic carcinoma. We have now examined the ability of a novel PAM4-based, bispecific monoclonal antibody (mAb) construct, TF10, to pretarget a radiolabeled peptide for improved imaging and therapy. TF10 is a humanized, bispecific mAb, divalent for mAb-PAM4 and monovalent for mAb-679, reactive against the histamine-succinyl-glycine hapten. Biodistribution studies and nuclear imaging of the radiolabeled TF10 and/or TF10-pretargeted hapten-peptide (IMP-288) were conducted in nude mice bearing CaPan1 human pancreatic cancer xenografts. (125)I-TF10 cleared rapidly from the blood, with levels decreasing to <1% injected dose per gram (ID/g) by 16 hours. Tumor uptake was 3.47 +/- 0.66% ID/g at this time point with no accumulation in any normal tissue. To show the utility of the pretargeting approach, (111)In-IMP-288 was administered 16 hours after TF10. At 3 hours postadministration of radiolabeled peptide, imaging showed intense uptake within the tumors and no evidence of accretion in any normal tissue. No targeting was observed in animals given only the (111)In-peptide. Tumor uptake of the TF10-pretargeted (111)In-IMP-288 was 24.3 +/- 1.7% ID/g, whereas for (111)In-IMP-288 alone it was only 0.12 +/- 0.002% ID/g at 16 hours. Tumor/blood ratios were significantly greater for the pretargeting group ( approximately 1,000:1 at 3 hours) compared with (111)In-PAM4-IgG ( approximately 5:1 at 24 hours; P < 0.0003). Radiation dose estimates suggested that TF10/(90)Y-peptide pretargeting would provide a greater antitumor effect than (90)Y-PAM4-IgG. Thus, the results suggest that TF10 pretargeting may provide improved imaging for early detection, diagnosis, and treatment of pancreatic cancer as compared with directly radiolabeled PAM4-IgG.

PAM4-reactive MUC1 is a biomarker for early pancreatic adenocarcinoma.

PURPOSE: The anti-MUC1 monoclonal antibody (MAb), PAM4, has a high specificity for pancreatic adenocarcinoma compared with other cancers, normal tissues, or pancreatitis. In order to assess its role in early pancreatic cancer development, we examined the expression of the PAM4-reactive MUC1 in the noninvasive precursor lesions, pancreatic intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasia (IPMN). EXPERIMENTAL DESIGN: Tissue microarrays prepared from formalin-fixed, paraffin-embedded specimens were assessed by immunohistology for expression of the PAM4-reactive, non-variable number of tandem repeats (VNTR), MUC1 epitope, and the VNTR epitope bound by the MA5 MAb. RESULTS: The PAM4-reactive MUC1 epitope was not detected in normal pancreas but was expressed in 87% (48 of 55) of invasive pancreatic adenocarcinomas, including early stage 1 disease: PAM4 labeled 94% (44 of 47) of the earliest PanIN lesions, PanIN-1A and 1B, along with 91% (10 of 11) of PanIN-2, 40% (2 of 5) of PanIN-3, and 86% (31 of 36) of intraductal papillary mucinous neoplasia lesions. A mostly diffuse pattern of labeling was observed. A second, unrelated, anti-MUC1 MAb, MA5, showed considerably less sensitivity with early PanIN-1 lesions; only 61% (25 of 41) were positive and the labeling did not differentiate normal pancreas from PanINs. CONCLUSIONS: The results suggest that expression of the PAM4-reactive antigen may represent an early event in the development of invasive pancreatic adenocarcinoma, and is unrelated to the VNTR peptide core epitopes of MUC1. Detection of this biomarker using immunohistology, in vitro immunoassays, and in vivo antibody-based imaging may provide new opportunities for the early detection and improved diagnosis of pancreatic cancer.

Sphingolipid targets in cancer therapy.

Considerable progress has been made recently in our understanding of the role of ceramide in the induction of apoptotic cell death. Ceramide is produced by cancer cells in response to exposure to radiation and most chemotherapeutics and is an intracellular second messenger that activates enzymes, leading to apoptosis. Because of its central role in apoptosis, pharmacologic manipulation of intracellular ceramide levels should result in attenuation or enhancement of drug resistance. This may be achieved through direct application of sphingolipids or by the inhibition/activation of the enzymes that either produce or use ceramide. In addition, attention should be given to the subcellular location of ceramide generation, because this has been shown to affect the biological activity of sphingolipids. This review summarizes the sphingolipid biosynthetic pathway, as it relates to the identification of important targets for drug discovery, and the development of novel agents capable of enhancing chemotherapy.

New MUC1 serum immunoassay differentiates pancreatic cancer from pancreatitis.

PURPOSE: To evaluate a new immunoassay for identification and quantitation of MUC1 in the sera of patients with pancreatic cancer or pancreatitis. The sensitivity and specificity of the assay are examined and compared to results from a CA19-9 immunoassay. METHODS: An in vitro enzyme immunoassay was established with monoclonal antibody PAM4 as the capture reagent, and a polyclonal anti-MUC1 antibody as the probe. Patient sera were obtained from healthy, adult patients with acute and chronic pancreatitis, and those with pancreatic and other forms of cancer, and were measured for PAM4-reactive MUC1. RESULTS: At a cutoff of 10.2 units/mL, 41 (77%) of 53 pancreatic cancer patients, none of the healthy individuals (n = 43), and only four (5%) of 87 patients with pancreatitis were positive above this value. Among nonpancreatic cancers investigated, colorectal cancers gave the highest percentage of positives (14%; five of 36). Overall, the sensitivity and specificity of the immunoassay for pancreatic cancer were 77% and 95%, respectively. Receiver operator characteristic analyses for discrimination of pancreatic cancer from pancreatitis provided an area under the curve of 0.89 (95% CI, 0.82 to 0.93), with a specificity of 95.4% and a positive likelihood ratio of 16.8. A direct pair-wise comparison of PAM4 and CA19-9 immunoassays for discrimination of pancreatic cancer and pancreatitis resulted in a significant difference (P < .003), with the PAM4 immunoassay demonstrating superior sensitivity and specificity. CONCLUSION The high sensitivity and specificity observed suggest that the PAM4-based immunoassay of circulating MUC1 may be useful in the diagnosis of pancreatic cancer.