Loop-mediated isothermal amplification test for detection of Neisseria gonorrhoeae in urine samples and tolerance of the assay to the presence of urea.

A loop-mediated isothermal amplification (LAMP) assay for open reading frame 1 (ORF1) of the glutamine synthetase gene of Neisseria gonorrhoeae was able to tolerate urea concentrations of ≤ 1.8 M, compared with a PCR assay that was functional at concentrations of <100 mM. The LAMP assay was as sensitive as the PCR assay while being faster and simpler to perform.

Circulating tumor cells: application as a biomarker for molecular characterization and predictor of survival in an all-comer solid tumor phase I clinical study.

PURPOSE: Clinical development of cancer drugs has a low success rate. Prognostic and predictive biomarkers using minimally invasive approaches hold promise for increasing the probability of success by enabling disease characterization, patient selection and early detection of drug treatment effect. Enumeration and molecular characterization of circulating tumor cells (CTC) may address some of these needs, and thus were evaluated for utility in a Phase I solid tumor clinical study. EXPERIMENTAL DESIGN: Blood samples for CTC analysis were obtained from 24 cancer patients in a multi-center all-comer Phase I study of MEDI-575, a novel anti-PDGFRα antibody. Samples were taken at screening and analyzed for enumeration of CTC using the CellSearch(®) platform and for molecular characterization using a novel quantitative RT-PCR assay. RESULTS: Fifty-nine percent of the patients showed at least 1 CTC per 7.5 ml of blood at baseline. Progression-free survival (PFS) and overall survival (OS) of patients with 0 CTCs at baseline were longer than PFS and Os for patients with 1-3 and >3 CTCs (8.8 versus 1.4 and 1.3 months PFS, P = 0.02; 9.0 vs 7.4 and 3.5 months OS, P = 0.20, respectively). Patients with 0 CTC showed a greater percentage of stable disease than the other 2 groups with 1-3 and >3 CTCs (57% vs 29% and 0%). The multimarker qRT-PCR method detected CTC in 40% of the patients, and 80% of these patients were positive for pre-selected drug target genes. CONCLUSION: CTC enumeration of patients in an all-comer study is feasible and may allow for patient stratification for PFS and Os to evaluate the clinical response of investigational agents. Gene expression profiling of isolated CTC may provide a means for molecular characterization of selected tumor targets.

The hematopoietic-specific beta1-tubulin is naturally resistant to 2-methoxyestradiol and protects patients from drug-induced myelosuppression.

Taxanes and other microtubule-targeting drugs (MTDs) represent one of the most effective classes of cancer chemotherapeutics. However, ultimately their utility is limited due to drug-induced myelosuppression. Here we identify 2-Methoxyestradiol (2ME2) as the first MTD able to specifically target tumor cells while sparing the bone marrow from dose-limiting, life-threatening toxicities. Following drug selection with 2ME2, epithelial cancer cells acquired a tubulin mutation at Vbeta236I that impaired the 2ME2-tubulin interaction and rendered cells resistant to 2ME2. We further show that the hematopoietic-specific Hbeta1 tubulin isotype naturally encodes Ibeta236 and is insensitive to 2ME2. Systemic administration of 2ME2 in C57BL6 mice revealed that there was no effect on bone marrow microtubules, in contrast to the taxane or Vinca alkaloid induced toxicities. Similar results were obtained upon drug treatment of human bone marrow and CD34-positive stem/progenitor cells. Herein, we describe the first isotype-targeted chemotherapeutic, setting a new paradigm for the entire class of MTDs, and providing a model that could allow the design of new tubulin inhibitors devoid of myelosuppression. The ability to design a drug with minimal side-effects would significantly augment the chances of clinical success by allowing the use of a truly therapeutic dose rather than the maximally tolerated.

Significant antitumor activity in vivo following treatment with the microtubule agent ENMD-1198.

Clinical studies using the microtubule-targeting agent 2-methoxyestradiol (2ME2; Panzem) in cancer patients show that treatment is associated with clinical benefit, including prolonged stable disease, complete and partial responses, and an excellent safety profile. Studies have shown that 2ME2 is metabolized by conjugation at positions 3 and 17 and oxidation at position 17. To define structure-activity relationships for these positions of 2ME2 and to generate metabolically stable analogues with improved anti-tubulin properties, a series of analogues was generated and three lead analogues were selected, ENMD-1198, ENMD-1200, and ENMD-1237. These molecules showed improved metabolic stability with >65% remaining after 2-h incubation with hepatocytes. Pharmacokinetic studies showed that oral administration of the compounds resulted in increased plasma levels compared with 2ME2. All three analogues bind the colchicine binding site of tubulin, induce G(2)-M cell cycle arrest and apoptosis, and reduce hypoxia-inducible factor-1alpha levels. ENMD-1198 and ENMD-1200 showed improved in vitro antiproliferative activities. Significant reductions in tumor volumes compared with vehicle-treated mice were observed in an orthotopic breast carcinoma (MDA-MB-231) xenograft model following daily oral treatment with all compounds (ANOVA, P < 0.05). Significantly improved median survival time was observed with ENMD-1198 and ENMD-1237 (200 mg/kg/d) in a Lewis lung carcinoma metastatic model (P < 0.05). In both tumor models, the high-dose group of ENMD-1198 showed antitumor activity equivalent to that of cyclophosphamide. ENMD-1198 was selected as the lead molecule in this analogue series and is currently in a phase I clinical trial in patients with refractory solid tumors.

Selective high-affinity ligand antibody mimics for cancer diagnosis and therapy: initial application to lymphoma/leukemia.

PURPOSE: More than two decades of research and clinical trials have shown radioimmunotherapy to be a promising approach for treating various forms of cancer. Lym-1 antibody, which binds selectively to HLA-DR10 on malignant B-cell lymphocytes, has proved to be effective in delivering radionuclides to non-Hodgkin's lymphoma and leukemia. Using a new approach to create small synthetic molecules that mimic the targeting properties of the Lym-1 antibody, a prototype, selective high-affinity ligand (SHAL), has been developed to bind to a unique region located within the Lym-1 epitope on HLA-DR10. EXPERIMENTAL DESIGN: Computer docking methods were used to predict two sets of small molecules that bind to neighboring cavities on the beta subunit of HLA-DR10 surrounding a critical amino acid in the epitope, and the ligands were confirmed to bind to the protein by nuclear magnetic resonance spectroscopy. Pairs of these molecules were then chemically linked together to produce a series of bidentate and bisbidentate SHALs. RESULTS: These SHALs bind with nanomolar to picomolar K(d)'s only to cell lines expressing HLA-DR10. Analyses of biopsy sections obtained from patients also confirmed that SHAL bound to both small and large cell non-Hodgkin's lymphomas mimicking the selectivity of Lym-1. CONCLUSIONS: These results show that synthetic molecules less than 1/50th the mass of an antibody can be designed to exhibit strong binding to subtle structural features on cell surface proteins similar to those recognized by antibodies. This approach offers great potential for developing small molecule therapeutics that target other types of cancer and disease.

Characterization of MUC1 glycoprotein on prostate cancer for selection of targeting molecules.

MUC1 glycoprotein that is overexpressed in aberrant forms in epithelial cancers has been used for diagnosis, staging and therapy. As normal prostate and prostate cancer tissues express MUC1, it represents a potential target, but MUC1 epitopes specific to prostate cancer have not been well characterized. In order to assess MUC1 epitopes in prostate cancer, and their correlation with Gleason grades, binding of 7 well-characterized anti-MUC1 monoclonal antibodies (MAbs) (BrE-3, SM3, BC2, EMA, B27.29, HMFG-1 and NCL MUC1 core), were studied on a prostate tissue microarray. This microarray contained 197 prostate tissue cores representing: i) normal/benign prostate; ii) prostatic intraepithelial neoplasia and Gleason grades 1 and 2; and iii) Gleason grades 3-5. These MAbs bind the MUC1 extracellular domain, but have variable sensitivity to MUC1 glycosylation. To further characterize the effect of glycosylation on their binding, MAb reactivities with unglycosylated MUC1 core peptide and breast and prostate cancer cell lysates were compared. These studies demonstrated strong binding of BrE-3, BC2 and EMA to the peptide core and recognition by BrE-3, SM3, BC2 and EMA of hypoglycosylated MUC1. The results for the microarray indicated that higher Gleason grades were associated with markedly increased cellular staining by MAbs that preferentially recognize less glycosylated MUC1 (BrE-3, p<0.001; SM3, p<0.004; EMA, p=0.009; and BC2, p<0.001). Staining by MAbs that bind preferentially to hyperglycosylated MUC1 (B27.29, p=0.33; HMFG-1, p=0.89; and NCL MUC1 core, p=0.96) did not correlate with Gleason grade. These results demonstrated that hypoglycosylated MUC1 expression increased with Gleason grade, thus supporting the targeting of hypoglycosylated MUC1 epitopes in prostate cancer for more specific imaging and therapy applications.

Enhancement of the therapeutic index: from nonmyeloablative and myeloablative toward pretargeted radioimmunotherapy for metastatic prostate cancer.

PURPOSE: New strategies that target selected molecular characteristics and result in an effective therapeutic index are needed for metastatic, hormone-refractory prostate cancer. EXPERIMENTAL DESIGN: A series of preclinical and clinical studies were designed to increase the therapeutic index of targeted radiation therapy for prostate cancer. (111)In/90Y-monoclonal antibody (mAb), m170, which targets aberrant sugars on abnormal MUC1, was evaluated in androgen-independent prostate cancer patients to determine the maximum tolerated dose and efficacy of nonmyeloablative radioimmunotherapy and myeloablative combined modality radioimmunotherapy with paclitaxel. To enhance the tumor to liver therapeutic index, a cathepsin degradable mAb linkage ((111)In/90Y-peptide-m170) was used in the myeloablative combined modality radioimmunotherapy protocol. For tumor to marrow therapeutic index improvement in future studies, anti-MUC1 scFvs modules were developed for pretargeted radioimmunotherapy. Anti-MUC1 and anti-DOTA scFvs were conjugated to polyethylene glycol scaffolds tested on DU145 prostate cancer cells and prostate tissue arrays, along with mAbs against MUC1 epitopes. RESULTS: The nonmyeloablative maximum tolerated dose of 90Y-m170 was 0.74 GBq/m2 for patients with not more than 10% axial skeleton involvement. Metastatic prostate cancer was targeted in all 17 patients; mean radiation dose was 10.5 Gy/GBq and pain response occurred in 7 of 13 patients reporting pain. Myeloablative combined modality radioimmunotherapy with 0.4 GBq/m2 of 90Y-peptide-m170 and paclitaxel showed therapeutic effects in 4 of 6 patients and 30% less radiation to the liver per unit of activity. Neutropenia was dose limiting without marrow support and patient eligibility was a major limitation to dose escalation. Hypoglycosylated MUC1 epitopes were shown to be abundant in prostate cancer and to increase with disease grade. Anti-MUC1 scFvs binding to prostate cancer tissue and live cells were developed into di-scFv binding modules. CONCLUSIONS: The therapeutic index enhancement for prostate radioimmunotherapy was achieved in clinical studies by the addition of cathepsin cleavable linkers to 90Y-conjugated mAbs and the use of paclitaxel. However, the need for marrow support in myeloablative combined modality radioimmunotherapy restricted eligible patients. Therefore, modular pretargeted radioimmunotherapy, aiming at improving the tumor to marrow therapeutic index, is being developed.

Production of soluble ScFvs with C-terminal-free thiol for site-specific conjugation or stable dimeric ScFvs on demand.

ScFv recombinant antibody fragments can provide specific tumor binding modules for targeting drugs. In the process of building multimeric tumor targeting pharmaceuticals, a prerequisite is the conservation of functional scFv antigen binding domains, thereby excluding scFv random conjugation to a carrier molecule or to another scFv. The pCANTAB 5E phage display/expression vector was genetically engineered to express any scFv gene as scFv with an additional C-terminal cysteine (scFv-Cys) such that the specific conjugation site is removed from the binding domain. Selected scFvs derived from an anti-MUC-1 scFv phage library were expressed in pCANTAB 5E and its modified version pCANTAB 5E Cys vectors, and compared for key characteristics. Production yields of scFv and scFv-Cys in shaker flask and biofermentor were compared. In the absence of a reducing agent, stable dimers (covalent scFv homodimers (scFv-Cys)2) were the major form of scFv-Cys. These diabodies provided substantial signal enhancement for immunohistochemical staining of tissues. In the presence of a reducing agent, scFv-Cys molecules remained monomeric, with the free SH available for conjugation to a PEG(maleimide)2 scaffold to form immunoreactive PEG(scFv)2 bioconjugates. ScFv expression from pCANTAB 5E Cys allowed for the production of soluble scFv-Cys protein from E.coli, either as stable scFv-Cys or (scFv-Cys)2. ScFv-Cys can be used for conjugation to PEG to form bivalent PEG (scFv-Cys)2 molecules or used as (scFv-Cys)2 for increased sensitivity in IHC.

Selection and characterization of anti-MUC-1 scFvs intended for targeted therapy.

PURPOSE: The selection and characterization of anti-MUC-1 single-chain antibody fragments (scFv) is a first step toward the construction of new anticancer molecules designed for optimal blood clearance and tumor penetration. The mucin MUC-1 was chosen as an antigen because it is abundantly expressed on epithelial cancers in an aberrantly glycosylated form, making it structurally and antigenically distinct from MUC-1 expressed on normal cells. EXPERIMENTAL DESIGN: A previously constructed anti-MUC-1 phage display library from hyperimmunized mice, with 5 x 10(5) calculated variants, was screened for the selection of anti-MUC-1 scFvs. Selection criteria were high binding to a MUC-1 peptide containing 4 tandem repeats of 20 amino acids and to MUC-1-positive MCF-7 (human breast cancer) cell lysates in ELISA. RESULTS: Six anti-MUC-1 scFv clones were selected and characterized. Nucleotide sequencing showed that four of them were full length scFv genes (variable heavy chain + variable light chain), whereas the remaining two contained either a variable heavy chain or a variable light chain alone. Their binding affinities (K(a)) range between 8 x 10(7) and 10(9) M(-1). Immunohistopathology demonstrated reactivity with breast cancer cells (MCF-7 and BT20) and human breast biopsy tissue. Molecular modeling revealed high structural similarity of the anti-MUC-1 scFvs with the X-ray-determined structure of the anti-CEA scFv (MFE-23). CONCLUSIONS: In vitro antigen binding was demonstrated for the selected anti-MUC-1 scFvs. The binding affinities of these scFvs are in a promising range for efficient in vivo antigen binding. These anti-MUC-1 scFvs will be evaluated as antigen-binding modules in new multifunctional agents for the detection and therapy of cancer.

Effect of molecular size of pegylated peptide on the pharmacokinetics and tumor targeting in lymphoma-bearing mice.

PURPOSE: Rapid blood and body clearances have hampered effective tumor targeting by small molecules. We used branched poly(ethylene glycol) (pegylated) polymers (M(r) 40,000, M(r) 70,000, M(r) 100,000, and M(r) 150,000) conjugated to tumor-specific and control peptides to assess the effect of both molecular weight and tumor specificity on pharmacokinetics and biodistribution. EXPERIMENTAL DESIGN: Pegylated specific lymphoma-binding peptide and control peptide (containing stereoisomers of proline and aspartate) were synthesized, radiolabeled with (111)In, fractionated by size, and injected into Raji lymphoma-bearing athymic mice (4-6 mice/group). Pharmacokinetics were followed for 2 days to evaluate effects of specificity and molecular size on blood clearance, body clearance, and biodistribution. RESULTS: As molecular size increased, blood and body clearances decreased (P < 0.001). The effect of molecular size on blood clearance was not altered by ligand binding specificity (P = 0.21), with t(1/2) ranging from 5.4 h (M(r) 40,000) to 17.7 h (M(r) 150,000). However, ligand specificity did alter body clearance, with pegylated control peptides clearing the body more slowly than pegylated specific peptides [P = 0.03; range, 19.1-91.3 h (specific peptides) versus 23.6-115.7 h (control peptides)]. At 24 h, there was more uptake of specific versus control pegylated peptides in tumor, liver, and marrow, but there was less uptake in kidneys, with a more pronounced difference for the higher molecular weight peptides (P < 0.01). CONCLUSIONS: These results demonstrate that the pharmacokinetics and biodistribution of peptides and resultant uptake in tumor and normal tissues can be altered by both molecular size and ligand specificity, with molecular size affecting pharmacokinetics and organ uptake in a predictable manner.

Cilengitide targeting of alpha(v)beta(3) integrin receptor synergizes with radioimmunotherapy to increase efficacy and apoptosis in breast cancer xenografts.

Although metastatic breast cancer is responsive to radioimmunotherapy (RIT), a systemic targeted radiation modality, complete and permanent remissions are not typical with single-modality treatment. Antiangiogenic agents, which target normal, proliferating endothelial cells, have the potential to provide relatively nontoxic continuous inhibition of tumor growth by blocking new blood vessel growth and may synergize with RIT to increase efficacy. This study was designed to determine whether, and how, the cyclic Arg-Gly-Asp peptide Cilengitide (EMD 121974), which targets the alpha(v)beta(3) integrin receptor expressed on neovasculature, could increase systemic RIT efficacy of therapy in a human breast cancer tumor model having mutant p53 and expressing bcl-2. HBT 3477 breast cancer tumor response in nude mice was compared between groups of untreated mice (n = 24), Cilengitide-treated mice (n = 18), RIT (200-260 mu Ci (90)Y-labeled 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA)-peptide ChL6; n = 46), and combined modality RIT (CMRIT) using RIT and six doses of Cilengitide (250 microg/dose; n = 41). Tumor size, survival, body weight, and blood counts were monitored for efficacy and toxicity of therapy. To clarify the mechanism of synergistic effect, tumors were evaluated at selected time points through 6 days for apoptosis, proliferation, and microvessel density. Cilengitide alone did not alter tumor growth when compared with untreated mice, but CMRIT with Cilengitide increased efficacy of treatment, with the cure rate for mice that received 260 mu Ci RIT increasing from 15 to 53% (P = 0.011). Lower-dose RIT (200 mu Ci) combined with Cilengitide resulted in less increase in cures (36 compared with 25% for RIT alone; P = 0.514). Combined analysis for high- and low-dose groups demonstrated increased efficacy of CMRIT (P = 0.020). Analysis of tumors from CMRIT mice indicated significantly increased apoptosis of tumor and endothelial cells 5 days after RIT compared with tumors from mice given RIT alone. Proliferation was decreased in CMRIT tumors compared with RIT tumors at 6 days (ANOVA, P < 0.05). Microvessel density in tumors from RIT and CMRIT mice was not different. No increased toxicity attributable to Cilengitide was observed based upon pooled blood sample and no statistical increase in mortality. In conclusion, CMRIT, combining Cilengitide and RIT, significantly increased the efficacy of therapy and increased apoptosis compared with single-modality therapy with either agent, in an aggressive, well-studied breast cancer model. The enhanced therapeutic synergy is of particular note, having been achieved without additional toxicity.

Combined modality radioimmunotherapy. Promise and peril.

BACKGROUND: Single-agent radioimmunotherapy (RIT), although potentially useful for slowing solid tumor growth, has not been effective in curing aggressive tumors, such as breast cancer. These cancers typically have p53 mutations and are less susceptible to apoptosis, the apparent mechanism of cell death from low dose-rate radiation. Thus, synergistic or combined modality radioimmunotherapy (CMRIT) agents are needed to increase radiosensitivity for therapeutic enhancement without additive toxicity. METHODS: To assess synergy in CMRIT in a breast cancer xenograft model, we evaluated RGD peptide EMD 121974, an inhibitor of alpha(v)beta(3) integrin; paclitaxel, an antimicrotubule; IMC-C225, a monoclonal antibody to epidermal growth factor receptor (EGFR); and bcl-2 antisense oligonucleotide G3139. Groups of mice received (90)Y-DOTA-ChL6 in combination with each agent. Tumor size, survival, and blood counts were monitored for efficacy and toxicity. Immunopathologic evaluation of apoptosis was performed at selected time points after RIT and RIT + RGD CMRIT. RESULTS: CMRIT with RGD peptide increased apoptosis and resulted in 57% cures, compared with 0% cures with RIT alone. CMRIT with paclitaxel after RIT increased cures to 88%, compared with 25% cures with RIT before paclitaxel administration. CMRIT with IMC-C225 resulted in up to 20% cures if given before RIT. A time-dependent increase in toxicity was observed with IMC-C225 after RIT. CMRIT with bcl-2 antisense G3139 resulted in no cures and an increased rate of regrowth compared with RIT alone. CONCLUSIONS: Some combined modality therapies resulted in higher numbers of cures, while others decreased cures and responses and increased toxicity compared with RIT alone. These results support the potential for CMRIT but illustrate the complexity of predicting the efficacy and toxicity and the importance of the relationship between dose and sequence of administration.