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PubMedDate not supplied

In vitro antitumor activity of 2'-deoxy-5-fluorouridine-monoclonal antibody conjugates.

5-Fluorouracil (5-FU) is an anticancer drug used in patients for the treatment of gastric and breast cancer and used either alone or in combination with methotrexate is one of the few drugs with some effect on colon cancer. 2'-Deoxy-5-fluorouridine (5-FUdr) (1) is an analogue based on 5-FU and can be covalently linked to a murine anti-Ly-2.1 monoclonal antibody (mAb) with the active ester derivative of 2'-deoxy-5-fluoro-3'-O-(carboxypropanoyl)uridine (5-FUdr-succ) (4). Such immunoconjugates can contain up to 42 residues of drug, although the most antibody activity was retained when substitution ratios were between 10 and 25 molecules of drug to mAb. In a cytotoxicity assay, 50% inhibition of [3H]deoxyuridine incorporation (IC50) with a murine Ly-2.1+ve thymoma cell line was 6 nM for 5-FUdr-anti-Ly-2.1, which is 12-fold more than that for free 5-FUdr (IC50 = 0.51 nM) but similar to that of 5-FUdr-succ (IC50 = 5.2 nM). The 5-FUdr-monoclonal antibody conjugates (5-FUdr-mAb) were 100-fold more active on the Ly-2.1+ve E3 cell line than on the Ly-2.1-ve BW5147 OU- cell line. The high in vitro activity and specificity of 5-FUdr-MoAb conjugates indicates that potent in vivo activity of these conjugates should be expected.

Animals↗
PubMedDate not supplied

Labeling monoclonal antibodies with 90yttrium- and 111indium-DOTA chelates: a simple and efficient method.

Yttrium-90 and indium-111 have been attached to a monoclonal antibody with a bifunctional chelating agent (DOTA-peptide). Using the unique features of this DOTA-peptide and its complexes with trivalent yttrium and indium, the bifunctional chelating agent was prelabeled with either radiometal and then conjugated to chimeric monoclonal antibody L6. Both radiolabeling procedures and yield are suitable for the practical preparation of radiopharmaceuticals. Biodistribution studies in tumor-bearing mice showed that, e.g., on day 3 after intravenous injection of a 90Y immunoconjugate, liver uptake was 5.4 +/- 1.5% ID/g, bone uptake 2.0 +/- 0.5% ID/g, and tumor uptake 18.0 +/- 8.0% ID/g.

Animals↗
PubMedDate not supplied

Three-dimensional model of the BR96 monoclonal antibody variable fragment.

Molecular modeling was used to build a three-dimensional model of the variable regions of the tumor-reactive monoclonal antibody BR96. An immunoconjugate of this antibody with the anticancer drug doxorubicin is currently in a phase I clinical trial for the treatment of solid tumors. A model structure of the BR96 variable fragment was generated to guide site-specific mutagenesis experiments and further improve the affinity of the antibody. The model displayed a distinct groove-type binding site which contained a significant number of aromatic residues. The dimensions and nature of the proposed binding site were consistent with the binding of the Ley tetrasaccharide which was found to bind to BR96. On the basis of the model, some BR96 residues are proposed to be crucial for antigen binding. BR96 and its complex with the Ley determinant have recently been crystallized, and structure determination is currently underway. Therefore, the detailed prediction of the BR96 combining site will soon be assessed, as a "blind test", based on crystallographic data.

Amino Acid Sequence↗
PubMedDate not supplied

Synthesis and in vitro testing of J591 antibody-dendrimer conjugates for targeted prostate cancer therapy.

Targeted therapeutics using antibodies are an attractive option over conventional cancer chemotherapeutics due to their potential to deliver a therapeutic specifically to cancer tissue without damaging normal tissue. However, there are known problems with immunoconjugates such as decreased immunoreactivity and poor solubility. Using dendrimers as carriers for these agents has the potential to resolve these issues. We synthesized J591 anti-PSMA (prostate specific membrane antigen) antibody dendrimer conjugates containing fluorophores on the dendrimer. The in vitro studies of these conjugates show that they specifically bind to cells expressing PSMA. Confocal microscopy experiments document the binding and internalization of these conjugates. This research encourages the further study of antibody-dendrimer-drug conjugates for use in targeted therapeutics.

Antibodies, Monoclonal↗
PubMedDate not supplied

Improved characteristics of a human beta-glucuronidase-antibody conjugate after deglycosylation for use in antibody-directed enzyme prodrug therapy.

Antibody-directed enzyme prodrug therapy (ADEPT) aims at the specific activation of relatively nontoxic prodrugs into active drugs at the tumor site. One of the enzymes described to be useful in ADEPT is human beta-glucuronidase (GUS), which is expected to have low immunogenicity in patients. A major obstacle for the use of GUS, however, is its rapid glycan-specific hepatic clearance. The carbohydrates of GUS have been modified by subsequent treatment with NaIO4 and NaBH4 to improve its retention in the circulation. The modification of GUS did not decrease the enzyme activity. In vitro it was demonstrated that a conjugate prepared with a pancarcinoma specific monoclonal antibody (mAb) 323/A3 and the modified enzyme (mGUS), when bound to tumor cells, was capable of complete prodrug activation. In vivo, the 323/A3-mGUS conjugate was cleared faster from the circulation of BALB/c mice (t1/2 = 9 h) than mAb 323/A3 (t1/2 = 32 h), but it was retained in the circulation much longer than an immunoconjugate prepared with native GUS (t1/2 = 24 min). In nude mice bearing subcutaneous OVCAR-3 tumors the distribution of 323/A3-mGUS was qualitatively comparable to that of mAb 323/A3. The 323/A3-mGUS conjugate showed specific localization in the tumor but to a lesser extent than mAb 323/A3 (2.7% vs 6.4% injected dose per gram at 1 day after iv injection). A favorable tumor-to-blood ratio of > 2 was observed for the conjugate at 7 days after administration, which is necessary for tumor-specific prodrug activation.

Animals↗
PubMedDate not supplied

New coupling reagents for the preparation of disulfide cross-linked conjugates with increased stability.

To improve the in vivo stability of disulfide-linked immunotoxins (ITs), a series of sterically hindered cross-linking reagents were designed and synthesized. These ligands are characterized by a thioimidate group linked to an S-acetyl thiol or a substituted aryldithio group. To select the reagent of choice, several aryldithio thioimidates, substituted with a methyl or a phenyl group adjacent to the disulfide, were analyzed in thiol-disulfide exchange reactions. Also analyzed were the following: (i) the stability and solubility of the linkers in aqueous solution, (ii) the rate of protein derivatization, and (iii) the steric hindrance due to methyl or phenyl group substituents toward cleavage of the disulfide bond by glutathione. Ethyl S-acetyl 3-mercaptobutyrothioimidate (M-AMPT) was chosen as reagent to prepare two types of stable disulfide-containing AR-3-gelonin conjugates (IT2 and IT3). IT2 was prepared by a 3-(4-carboxamidophenyldithio)propionthioimidate (CDPT)-derivatized antibody coupled to the M-AMPT-derivatized gelonin to afford a conjugate characterized by the presence of a methyl group adjacent to the sulfide bond. In the IT3 conjugate, an M-AMPT-derivatized toxin was coupled to the antibody thiolated with M-AMPT and then activated with Ellman's reagent (DNTB). The in vitro and in vivo stabilities of the three immunoconjugates were assayed, respectively, (i) by adding an excess of glutathione and monitoring protein release and (ii) by studying their pharmacokinetic behaviors. The specificity and cytotoxicity of all ITs were analyzed on target and unrelated cell lines, and no significant differences in activity were observed. IT3, consisting of a symmetrical dimethyl-substituted disulfide bond, was substantially more stable in vivo (t1/2 beta = 88.3 h) than the corresponding IT2, characterized by a disulfide-protected monomethyl substituent bond (t1/2 beta = 60.2 h) compared to the unhindered conjugate IT1 (t1/2 beta = 27.9 h). This family of cross-linking reagents therefore offers advantages, such as minimal perturbation of the protein structure and controlled reactivity due to the thioimidate moiety, as well as the capacity to yield immunotoxins possessing substantial stability in vivo.

Animals↗
PubMedDate not supplied

Quantitative analysis of derivatized proteins prepared with pyridyl disulfide-containing cross-linkers by high-performance liquid chromatography.

Determination of the introduced moieties into derivatized proteins is an essential step in the preparation and quality control of chemically defined immunoconjugates. For the derivatized proteins using pyridyl disulfide-containing cross-linkers such as N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and 4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyldithio)tolu ene (SMPT), the derivatization degree (ratio of pyridyl disulfide moieties to protein) has been traditionally determined by measuring the absorbance of both the derivatized protein and 2-thiopyridone (2-TP) released from the dithiothreitol (DTT) treatment (spectrophotometric method). This method, however, causes several problems including false high and low determinations of the protein and 2-TP, respectively, low selectivity, poor reproducibility, and relatively large amounts of sample consumption. A quantitative determination method of the derivatization ratios using bovine serum albumin derivatized with SPDP and SMPT as the model system has been developed. The concentration of protein and 2-TP released from the DTT treatment of derivatized proteins was determined directly without consideration of different reagents used and their concentrations. The present HPLC method was proved to be better in terms of accuracy, selectivity, and reproducibility with micro sample consumption. Moreover, this HPLC method can be directly applied to all derivatized proteins prepared with pyridyl disulfide-containing cross-linkers.

Animals↗
PubMed1999

Development of monoclonal ELISAs for azinphos-methyl. 1. Hapten synthesis and antibody production.

The development of monoclonal antibody-based enzyme-linked immunosorbent assays for azinphos-methyl is described. A panel of haptens was synthesized for immunoconjugate preparation, and a series of haptens for heterologous, coating or tracer, conjugates was also prepared. Hapten synthesis was based on a strategy in which only a fragment of the whole target molecule was present (fragmentary haptens). From immunized mice, a set of monoclonal antibodies was obtained and ELISA sensitivities were assayed in different formats. Affinities estimated as I(50) values in the low nanomolar range for azinphos-methyl and phosmet were observed for several monoclonal antibodies in the conjugate-coated format and in the antibody-coated format under nonoptimized assay conditions.

Animals↗
PubMed1989

Boron neutron capture therapy: linkage of a boronated macromolecule to monoclonal antibodies directed against tumor-associated antigens.

Two new protein-binding polyhedral boron derivatives, isocyanatoundecahydro-closo-dodecaborate(2-) (1) and isocyanato(trimethylamino)octahydro-closo-decaborate(1 -) (2), were synthesized. These anionic isocyanates have long hydrolysis half-lives at pH 7 and react readily with primary or secondary aliphatic amines resulting in spontaneous urea linkage. Utilizing 1, 1100 boron atoms (7.3% boron by weight) were incorporated per molecule of a polyclonal antibody directed against human thymocytes (anti-thymocyte globulin) without denaturation. However, immunoreactivity of the conjugates was lost. Reaction of 1 and 2 with polylysine yielded boronated macromolecules containing 21-28% boron by weight (up to 2000 boron atoms per molecule). Polylysine boronated with 2 was successfully linked to antibody molecules employing the heterobifunctional linking molecules N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and m-maleimidobenzoyl sulfosuccinimide ester (sulfo-MBS). Separation of the conjugated antibody from the free boronated macromolecules and unconjugated antibody molecules has been achieved by gel filtration on a Sephacryl S-300 column. By linking boronated polylysine to antibodies, greater than 10(3) boron atoms were incorporated with the attachment of this species to one or more sites on the antibody molecule. The resulting immunoconjugates contained greater than 10(3) boron atoms per molecule, retained their immunoreactivity, and potentially might be useful for the selective delivery of large numbers of boron atoms to tumor cells.

Animals↗
PubMed2004

Synthesis of taxoids with improved cytotoxicity and solubility for use in tumor-specific delivery.

To develop effective taxane-antibody immunoconjugates, we have prepared a series of modified taxanes that have both improved toxicity and solubility in aqueous systems as compared to paclitaxel (1a). These taxanes have been modified at either the C-10 or C-7 position and were found to be very cytotoxic against both normal and multi-drug-resistant (MDR) cells, as well as up to 30 times more soluble than paclitaxel in various buffer systems.

Antineoplastic Agents↗
PubMed2002

Modified immunoenriched (32)P-HPLC assay for the detection of O(4)-ethylthymidine in human biomonitoring studies.

Increased excretion of ethylated DNA bases has been reported in the urine of cigarette smokers. To study DNA ethylation in the target organs of smokers, an immunoenriched (32)P-postlabeling assay for O(4)-ethylthymidine (O(4)-etT) was developed. O(4)-etT-3'-monophosphate (O(4)-etT-3'P) was synthesized, purified, and characterized by LC-MS, ESI-MS, and NMR. DNA was enzymatically digested to 2'-deoxynucleoside-3'-monophosphate followed by immunoprecipitation of O(4)-etT-3'P using specific monoclonal antibodies. The immunoconjugate was washed by filtration, and O(4)-etT-3'P was recovered by ethanol treatment. The enriched O(4)-etT-3'P was labeled with [gamma-(32)P]ATP in the presence of T4-polynucleotide kinase at pH 6.8 to yield its 5'-labeled monophosphate and was subsequently resolved on RP-HPLC and detected with online detection of radioactivity. Adduct recovery was >80%, and the detection limit was approximately 500 amol. To further validate the method, O(4)-etT levels were determined in calf thymus DNA treated with N-ethyl-N-nitrosourea, and a dose-dependent formation of O(4)-etT was observed. Furthermore, O(4)-etT was found to be present in the cells obtained from the lower respiratory tract by sputum induction of two out of four smokers but not in three nonsmokers. O(4)-etT is a poorly repaired promutagenic DNA lesion; thus, it could be of potential use for biomonitoring smoking-related DNA damage. Our improved assay was found to be sufficiently sensitive and specific to detect O(4)-etT in surrogate cells from cigarette smoke exposed humans.

Carcinogens↗
PubMed2000

Efficient heterodimerization of recombinant bi- and trispecific antibodies.

Bispecific antibodies (BsAb) are promising therapeutic tools in tomorrow's medicine. Expression systems favoring efficient heterodimerization of intermediate-sized bispecific antibodies will significantly improve existing production methods. By C-terminal fusion of scFv molecules to the Fd- and the L-chains efficient heterodimerization in mammalian cells was obtained and a novel intermediate sized, disulfide stabilized BsAb could be efficiently produced. This type of antibody derivative easily allows for the production of trispecific antibodies, BsAb with bivalent binding for one antigen, or immunoconjugates.

Antibodies, Bispecific↗
PubMed1999

New therapeutic targets for rheumatoid arthritis.

New insights into the pathogenesis of rheumatoid arthritis (RA) and consequently new targets of therapy are covered in a broad overview fashion. Short-term significant beneficial effect on RA disease activity has been established in a small but rapidly growing number of double-blind placebo-controlled trials now including recombinant human IL-1 receptor antagonist, chimeric (mouse/human) monoclonal antibodies (mAb) against TNF alpha (cA2), humanised (human/mouse) anti-TNF alpha mAb (CDP571) and recombinant human TNF-receptor-Fc fusion protein (TNFR:Fc). Placebo-controlled trials of anti-T cells agents such as chimeric anti-CD4 mAb (cM-T412) and anti-CD5 immunoconjugate, did not demonstrate clinical benefit. A placebo-controlled study of the anti-T cell derived cytokine IL-2 (DAB486IL-2) showed only modes clinical improvement. Other anti-T cell approaches such as autologous T cell vaccination and induction of tolerance by oral type II collagen have been unsuccessful. The one controlled trial with an anti-inflammatory cytokine, recombinant human IFN-gamma, showed modest clinical benefits. Controlled trials with IL-4 and IL-10 and with anti-adhesion molecules are awaited.

Antibodies, Monoclonal↗
PubMed2000

Tumor specific activation of the VEGF/KDR angiogenic pathway in a subset of locally advanced squamous cell head and neck carcinomas.

Vascular endothelial growth factor (VEGF) and its receptors, Flt-1 and flk-1(KDR), constitute an important angiogenic pathway which, under hypoxic conditions, is up-regulated in many solid tumours. We used the monoclonal antibody 11B5, specific for recognizing VEGF expression and the 'VEGF/flk-1(KDR) complex' on tumour endothelium, to assess free VEGF protein expression and VEGF/receptor activated microvessel density (aMVD) in a series of 104 inoperable locally advanced squamous cell carcinomas of the head and neck, treated with chemo-radiotherapy. High VEGF expression in cancer cells was strongly associated with high VEGF/receptor expression in the vasculature. The high VEGF expression and the aMVD were not associated with the standard microvessel density (sMVD), as assessed with the monoclonal antibody anti-CD31 and, were not detected in normal tissue. An increased sMVD, however, was significantly related with the expression thymidine phosphorylase (TP), and also with the nuclear accumulation of the oncoprotein p53, but neither p53 nor TP was associated with VEGF expression by cancer cells or VEGF/receptor complex aMVD. In 35% of cancer cases examined, more than 20% of the microvessels assessed with anti-CD31 also expressed the VEGF/KDR complex. The vasculature of the normal head and neck mucosa did not express the VEGF/KDR complex. There was no association between VEGF expression or VEGF/receptor complex aMVD and response to chemo-radiotherapy or patient's survival. It is concluded that activation of the angiogenic pathway VEGF/flk-1(KDR) is tumor specific in a subgroup of locally advanced squamous cell carcinomas of the head and neck. Selective destruction of this type of vasculature, using immunoconjugates directed against the VEGF/receptor complex, may prove therapeutically useful for patients with a high tumoral VEGF/flk-1(KDR) activated microvessel fraction.

Adult↗
PubMed1995

Diffusion of an anti-transferrin receptor antibody in cultured murine melanoma cell layers.

PURPOSE: The purpose of the study was to develop a cell culture system representing a layer of cells in a solid tumor, and to use the system to study factors affecting the diffusion of a binding monoclonal antibody. METHODS: A mouse melanoma cell line, B16F10, was grown on Matrigel coated Transwell inserts. The diffusion of a binding monoclonal antibody which recognizes the murine transferrin receptor (a-TfR) was investigated, and compared to that of an isotype-matched, nonbinding control. RESULTS: At a cell density of 6.4 x 10(5) cells/cm2, 37 degrees C and a donor antibody concentration of 10 nM, the permeability of the a-TfR antibody was approximately half that of the control. Blocking experiments, in which the cell layer was pretreated with excess a-TfR, resulted in equal permeabilities for a-TfR and control antibodies, suggesting that the difference in permeabilities observed in the absence of blocking is due to a-TfR binding. Experiments at 4 degrees C and in the presence of metabolic inhibitors also equalized the permeation rates of the two antibodies, indicating that internalization of the antibody/antigen complex is important in retarding the permeation of the binding antibody. CONCLUSIONS: In this system, the diffusion of the a-TfR antibody is retarded by binding and internalization processes. The results have implications for the use of immunoconjugates in cancer chemotherapy, and for the delivery of other binding macromolecules to tissue sites of action.

Animals↗
PubMed2002

Apparent seroprevalence of Salmonella spp. in harp seals in the Greenland Sea as determined by enzyme-linked immunosorbent assay.

An indirect ELISA was developed as a possible tool for surveillance of the seroprevalence of Salmonella spp. in harp seals. This species is hunted for human consumption and thus transmission of disease to humans cannot be excluded. To cover a broad spectrum of serogroups, a mixture of the lipopolysaccharides (LPS) of S. typhimurium and S. choleraesuis was used as the antigen in this pilot study. Chicken anti-harp-seal immunoglobulin horseradish peroxidase conjugate served as the immunoconjugate. Sera from four captive harp seals, which were Salmonella culture-negative and had no clinical or historical evidence of salmonellosis, were used as negative controls. After immunization with an inactivated S. typhimurium vaccine, further sera from these seals were used as positive controls, as no serum from naturally infected animals was available. Serum samples from 93 harp seals caught in the Greenland sea in 1999 were examined, and anti-Salmonella antibodies were found in the samples from two individuals (seroprevalence 2.2%). Although Salmonella has been isolated from other pinniped species, this is the first documentation of Salmonella-seropositive harp seals. This study contributes to the evaluation of the importance of salmonellosis in arctic marine mammals and thus to the prevention of potential outbreaks of this important zoonosis.

Animals↗
PubMed1993

Radioimmunolocalisation in breast cancer using the gene product of c-erbB2 as the target antigen.

Lymph node status is still the single most important prognostic factor in breast cancer. Axillary surgery remains the only reliable means of providing this information. This pilot study evaluates using a highly specific radiolabelled monoclonal antibody to provide equivalent information by a non-invasive technique. After optimisation of labelling conditions, our first antibody, ICR12 (against the gene product of c-erbB-2) was evaluated in a mouse model system. Twenty-four hours post i.v. injection the mice were killed and their organs, blood and tumours harvested for counting. Tumour localisation was four times greater than that into normal tissues, reaching 20% injected dose per gram of tumour. Eight patients have had this Tc99m-ICR12. Patient selection was by immunocytochemical staining of fine needle aspirates from the patient's own breast cancer. After intravenous administration of the immunoconjugate, tomographic images were obtained at 24 h. These results were compared to the subsequent histopathological examinations. Three patients acted as normal controls, one patient was negative due to inappropriate sampling, and two patients had strong membrane staining and provided excellent tumour localisation to both breast primary and regional node metastases. A further two patients only had moderate antigen expression on staining and did not localise well. The good performance of this radiolabelled antibody with patients that strongly stain for the antigen encourages the development of this system as both a method of staging breast cancer and a potential means of immunotherapy in this subgroup of patients.

Adult↗
PubMed1993

pH in human tumour xenografts: effect of intravenous administration of glucose.

pH frequency distributions of tumours grown s.c. from 30 human tumour xenograft lines in rnu/rnu rats were analysed with the use of H+ ion-sensitive semi-microelectrodes prior to and following stimulation of tumour cell glycolysis by i.v. infusion of glucose. At normoglycemia, the average pH of the tumours investigated was 6.83 (range, 6.72-7.01; n = 268). Without exception, all xenografts responded to the temporary increase in plasma glucose concentration (PGC) from 6 +/- 1 to 30 +/- 3 mM by an accumulation of acidic metabolites, as indicated by a pH reduction to an average value of 6.43 (range, 6.12-6.78; n = 292). This pH value corresponds to a ten-fold increase in H+ ion activity in tumour tissue as compared to arterial blood. Tumour pH approached minimum values at 2-4 h after the onset of glucose administration and could be maintained at acidic levels for 24 h by controlled glucose infusion. Irrespective of pH variations between tumours grown from individual xenograft lines, there was no major difference in pH response to glucose between the four main histopathological tumour entities investigated, i.e. breast, lung and gastrointestinal carcinomas, and sarcomas. In tumours from several xenograft lines, an increase in blood glucose to only 2.5-times the normal value (14 mM) was sufficient to reduce the mean pH to 6.4. Glucose-induced acidosis was tumour-specific. The pH frequency distributions in liver, kidney and skeletal muscle of tumour-bearing rnu/rnu rats were only marginally sensitive to hyperglycemia (average pH, 6.97 vs normal value of 7.14). Tumour-selective activation of pH-sensitive anti-cancer agents, e.g. alkylating drugs, acid-labile prodrugs or pH-sensitive immunoconjugates may thus be feasible in a wide variety of human cancers.

Animals↗