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1.
A novel bifunctional maleimido CHX-A' DTPA chelator 5 was developed and conjugated to the monoclonal antibody trastuzumab (Herceptin) and subsequently radiolabeled with (111)In. The resulting (111)In labeled immunoconjugate 2 was demonstrated to bind to SKOV-3 ovarian cancer cells comparably to an isothiocyanato CHX-A' DTPA modified native trastuzumab, 1. Through efficient thiol-maleimide chemistry, antibodies, peptides or other targeting vectors can now be modified with an established radioactive metal chelating agent CHX-A' DTPA for imaging and/or therapies of cancer. 相似文献
2.
B J Clarke H C C?té D E Cool I Clark-Lewis H Saito R A Pixley R W Colman R T MacGillivray 《The Journal of biological chemistry》1989,264(19):11497-11502
We have localized the binding epitope(s) of two murine monoclonal antibodies (B7C9 and P5-2-1) that were shown previously to inhibit the activation of human coagulation factor XII by negatively charged surfaces. A factor XII cDNA expression library in lambda gt11 was screened with antibody B7C9, and 16 immunoreactive bacteriophage were isolated. Fusion proteins from each of the recombinant phage were reactive with both monoclonal antibodies. Two of the phage cDNA inserts were found to code for amino acid residues -6-+31 and +1-+47 of factor XII, respectively, thereby defining the limits of the antigenic peptide to amino acids +1-+31. Each of the remaining 14 recombinant phage contained longer factor XII cDNA inserts that included sequences coding for the amino-terminal 31 amino acid residues. These results were confirmed by direct binding of antibody B7C9 to synthetic peptides containing amino acids 1-14 and 1-28 of factor XII. Further experiments with a set of nested peptides also indicated that amino acid residues 1-4 were essential but not sufficient for binding of B7C9 to the peptides. Hydrophobicity analysis of the amino-terminal region of plasma factor XII revealed a highly hydrophilic region between amino acid residues 5 and 15 that contained positively charged lysine residues at positions 8, 11, and 13. We conclude that a major epitope(s) recognized by monoclonal antibodies B7C9 and P5-2-1 is present in the amino-terminal 28 amino acids of factor XII. It is proposed that binding of these antibodies to factor XII blocks interaction of the positively charged region between residues 5 and 15 with negatively charged surfaces, thereby inhibiting activation. 相似文献
3.
Identification of antigenically important domains in the glycoproteins of Sindbis virus by analysis of antibody escape variants. 总被引:9,自引:7,他引:2
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To study important epitopes on glycoprotein E2 of Sindbis virus, eight variants selected to be singly or multiply resistant to six neutralizing monoclonal antibodies reactive against E2, as well as four revertants which had regained sensitivity to neutralization, were sequenced throughout the E2 region. To study antigenic determinants in glycoprotein E1, four variants selected for resistance to a neutralizing monoclonal antibody reactive with E1 were sequenced throughout the E2 and E1 regions. All of the salient changes in E2 occurred within a relatively small region between amino acids 181 and 216, a domain that encompasses a glycosylation site at residue 196 and that is rich in charged amino acids. Almost all variants had a change in charge, suggesting that the charged nature of this domain is important for interaction with antibodies. Variants independently isolated for resistance to the same antibody were usually altered in the same amino acid, and reversion to sensitivity occurred at the sites of the original mutations, but did not always restore the parental amino acid. The characteristics of this region suggest that this domain is found on the surface of E2 and constitutes a prominent antigenic domain that interacts directly with neutralizing antibodies. Previous studies have shown that this domain is also important for penetration of cells and for virulence of the virus. Resistance to the single E1-specific neutralizing monoclonal antibody resulted from changes of Gly-132 of E1 to either Arg or Glu. Analogous to the findings with E2, these changes result in a change in charge and are found near a glycosylation site at residue 139. This domain of E1 may therefore be found near the 181 to 216 domain of E2 on the surface of the E1-E2 heterodimer; together, they could form a domain important in virus penetration and neutralization. 相似文献
4.
An important criterion in design of acylation agents for the radioiodination of internalizing monoclonal antibodies (mAbs) is to maximize the retention of radioiodine in the tumor following mAb intracellular processing. We have previously shown that labeling methods that generate positively charged catabolites have enhanced tumor retention. Herein we have extended this strategy to investigate the potential utility of labeling internalizing mAbs with an acylation agent that yielded labeled catabolites that would be negatively charged at lysosomal pH. The negatively charged acylation agent, N-succinimidyl 3-[(131)I]iodo-4-phosphonomethylbenzoate ([(131)I]SIPMB), was prepared from its tin precursor, N-succinimidyl 4-di-tert-butylphosphonomethyl-3-trimethylstannylbenzoate (tBu-SPMTB), in 40% radiochemical yield. The free acid, 3-[(131)I]iodo-4-phosphonomethylbenzoic acid ([(131)I]IPMBA), was also prepared from the corresponding precursor, 4-di-tert-butylphosphonomethyl-3-trimethylstannylbenzoic acid (tBu-PMTBA), in 80% radiochemical yield. The rapidly internalizing mAb L8A4 was conjugated to [(131)I]SIPMB in 25-40% yield with preservation of its immunoreactivity. Internalization and processing in the U87DeltaEGFR glioma cell line was studied in a paired label format with L8A4 labeled with (125)I using the Iodogen method. Retention of initially bound radioactivity in these cells at 24 h from [(131)I]SIPMB-labeled mAb was approximately 6-fold higher than that for directly labeled mAb. Catabolite analysis demonstrated that this difference reflected an order of magnitude higher retention of low molecular weight species in these cells. The [(131)I]SIPMB-L8A4 conjugate was intact over the first 2 h; thereafter, lysine-[(131)I]SIPMB was the predominant catabolite. In contrast, L8A4 labeled using Iodogen rapidly gave rise to mono-[(125)I]iodotyrosine within 2 h, which then cleared rapidly from the cells. These results suggest that SIPMB could be a potent candidate for labeling internalizing mAbs and warrant further study. 相似文献
5.
《International journal of radiation applications and instrumentation. Part B, Nuclear medicine and biology》1991,18(3):275-280
Thrombus immunoscintigraphy with radiolabeled monoclonal antibodies are presently undergoing intense clinical evaluations. Reports on clinical trials of radiolabeled antifibrins are very encouraging and results of antiplatelet antibody evaluations are forthcoming. Animal studies with antiplatelet antibodies indicate that a diagnosis can be made within the critical “lytic window” of 4–6 h, and thus the imaging procedure may be used as an adjunct to thrombolytic therapy, i.e. screening of patients.We now report on a potentially new application of monoclonal antibodies, immunoimaging for monitoring thrombolysis. In vitro studies were performed with “standardized clots” incubated with 99mTc 50H.19 and re-incubated with streptokinase (SK), urokinase (UK) or recombinant tissue plasminogen activator (rt-PA). The decrease in clot-bound 99mTc 50H.19 activity after SK, UK or rt-PA incubation was proportional to the decrease in clot weight (r = 0.90–0.98). The direct effects of these thrombolytic agents on the labeled antibody and the possible interference of aspirin, warfarin and heparin in thrombus immunoimaging were also investigated. Aspirin, heparin and warfarin did not interfere with clot-binding of 99mTc 50H.19. Thrombolytic agents did not affect the stability of the radiolabel or immunoreactivity of 50H.19. These results indicate that 99mTc 50H.19 is a promising agent that may enable monitoring thrombolysis in addition to thrombus immunoimaging. 相似文献
6.
《International journal of radiation applications and instrumentation. Part B, Nuclear medicine and biology》1990,17(2):247-254
A monoclonal antibody to carcinoembryonic antigen showing minimal cross-reactivity with blood cells and normal tissues was derivatized with benzylisothiocyanate derivatives of EDTA and DTPA. Seven chelators per immunoglobulin could be incorporated without loss of immunoreactivity. The resulting conjugates, labeled with indium-111, showed low liver uptake in animals. A cold kit, comprising the DTPA conjugate at a molarity of antibody bound chelator exceeding 1 x 10−4M, gave radiochemical yields of indium labeled antibody of ⩾95% and was stable for 1 yr. 相似文献
7.
《International journal of radiation applications and instrumentation. Part B, Nuclear medicine and biology》1988,15(6):717-720
Our laboratory investigated the use of a rapid miniaturized chromatography system, ITLC-SG with 0.9% NaCl, to assess the radiochemical purity of 111In labeled monoclonal antibodies (MoAbs). Radiochemical analysis was performed on numerous 111In labeled antibody preparations with labeling efficiencies ranging from 40 to greater than 95% and the results compared to those obtained with size exclusion high performance liquid chromatography (HPLC). The chromatographic procedure involved challenging radiolabeled antibodies with 0.05 M DTPA to chelate unbound and/or non-specific bound 111In, spotting on miniaturized instant thin layer-silica gel chromatography strips, developing in 0.9% NaCl, and counting appropriate segments for radioactivity. Results of the study demonstrated that the miniaturized chromatography procedure was rapid, taking less than 4 min to complete, and accurate in assessing the amount of unbound or non-specific bound 111In in 111In labeled monoclonal antibodies, when compared to size exclusion HPLC. 相似文献
8.
Reactive antigenic epitopes on presumed autoantigens of biologic interest have been examined by many researchers. The central third complementarity-determining region (CDR3) residues of a human monoclonal anti-proteinase 3 (PR3) antibody contained many negatively charged aspartic acid residues, perhaps contributing to its reactivity with positively charged PR3 regions. Examination of four other human monoclonal anti-PR3 antibodies shows a number of negatively charged residues within their CDR3 regions. Mapping of segments of linear PR3-epitopes reacting with anti-neutrophil cytoplasmic antibodies (ANCA) demonstrated a preliminary estimate of structures contributing to antigenic determinants. T-cell epitopes on PR3 are reported in studies of chronic myeloid leukemia. These T-cell epitopes appear to be human leukocyte antigen (HLA) A2.1 restricted. 相似文献
9.
Reactive antigenic epitopes on presumed autoantigens of biologic interest have been examined by many researchers. The central third complementarity-determining region (CDR3) residues of a human monoclonal anti-proteinase 3 (PR3) antibody contained many negatively charged aspartic acid residues, perhaps contributing to its reactivity with positively charged PR3 regions. Examination of four other human monoclonal anti-PR3 antibodies shows a number of negatively charged residues within their CDR3 regions. Mapping of segments of linear PR3-epitopes reacting with anti-neutrophil cytoplasmic antibodies (ANCA) demonstrated a preliminary estimate of structures contributing to antigenic determinants. T-cell epitopes on PR3 are reported in studies of chronic myeloid leukemia. These T-cell epitopes appear to be human leukocyte antigen (HLA) A2.1 restricted. 相似文献
10.
X Wen Q P Wu Y Lu Z Fan C Charnsangavej S Wallace D Chow C Li 《Bioconjugate chemistry》2001,12(4):545-553
Several biological barriers, including significant liver uptake, limit the clinical application of radiolabeled antibodies in radioimmunoscintigraphy. Here, a general approach is described for radiolabeling of monoclonal antibodies conjugated with poly(ethylene glycol) (PEG). This strategy is demonstrated with C225, a monoclonal antibody directed against epidermal growth factor (EGF) receptor. We synthesized a heterofunctional PEG with one end attached to a radiometal chelator, diethylenetriaminepentaacetic acid (DTPA), and the other end to a protected thiol group, S-acetylthioacetate. After a deprotection step, the resulting DTPA-PEG-SH was conjugated to maleimide-activated C225 to yield DTPA-PEG-C225 conjugate. Characterization of DTPA-PEG-C225 with immunoprecipitation and Western blot analysis revealed that the conjugate was biologically active in binding to the EGF receptor in A431 cells. Competitive EGF receptor binding assay in MDA-MB-468 cells showed that DTPA-PEG-C225, with up to 60% of the amino groups in C225 substituted, retained 66% of C225's binding affinity. Moreover, DTPA-PEG-C225 with increasing degrees of NH(2) substitution from 20% to 70% retained the activity of C225 to induce apoptosis in DiFi cells. More importantly, DTPA-PEG-C225 demonstrated less nonspecific interaction than DTPA-C225. Pharmacokinetic analysis using (111)In-labeled compounds revealed narrower steady-state distribution of (111)In-DTPA-PEG-C225 than (111)In-DTPA-C225, probably due to reduced nonspecific binding of PEG-modified antibody to tissues. The terminal half-life (t(1/2,)(gamma)) of (111)In-DTPA-PEG-C225, 21.1 h, was shorter than that of (111)In-DTPA-C225, 52.9 h. These data suggest that (111)In-DTPA-PEG-C225 may provide better imaging characteristics than (111)In-DTPA-C225, and that using PEG as a linker between the monoclonal antibody and DTPA may be a promising strategy in optimizing the imaging characteristics of immunoscintigraphic agents. 相似文献
11.
Solid-phase peptide quantitation assay using labeled monoclonal antibody and glutaraldehyde fixation
P G Kasprzyk F Cuttitta I Avis Y Nakanishi A Treston H Wong J H Walsh J L Mulshine 《Analytical biochemistry》1988,174(1):224-234
A solid-phase radioimmunoassay utilizing iodinated peptide-specific monoclonal antibody as a detection system instead of labeled peptide has been developed. Regional specific monoclonal antibodies to either gastrin-releasing peptide or gastrin were used as models to validate the general application of our modified assay. Conditions for radioactive labeling of the monoclonal antibody were determined to minimize oxidant damage, which compromises the sensitivity of other reported peptide quantitation assays. Pretreatment of 96-well polyvinyl chloride test plates with a 5% glutaraldehyde solution resulted in consistent retention of sufficient target peptide on the solid-phase matrix to allow precise quantitation. This quantitative method is completed within 1 h of peptide solid phasing. Pretreatment of assay plates with glutaraldehyde increased binding of target peptide and maximized antibody binding by optimizing antigen presentation. The hypothesis that glutaraldehyde affects both peptide binding to the plate and orientation of the peptide was confirmed by analysis of several peptide analogs. These studies indicate that peptide binding was mediated through a free amino group leaving the carboxy-terminal portion of the target peptide accessible for antibody binding. It was observed that the length of the peptide also affects the amount of monoclonal antibody that will bind. Under the optimal conditions, results from quantitation of gastrin-releasing peptide in relevant samples agree well with those from previously reported techniques. Thus, we report here a modified microplate assay which may be generally applied for the rapid and sensitive quantitation of peptide hormones. 相似文献
12.
The 528 murine monoclonal antibody (MAb) to the human epidermal growth factor receptor (EGFR) was sequentially cationized with hexamethylenediamine and conjugated with diethylenetriaminepentaacetic acid (DTPA) as a potential antibody radiopharmaceutical for imaging EGFR-expressing cancer. The cationized 528 MAb was characterized with isoelectric focusing and electrophoresis, and an immunoradiometric assay, which showed the affinity of the 528 MAb for the human EGFR was retained following cationization. The native or cationized 528 MAb, labeled with (111)In, was injected intravenously in scid mice bearing human U87 flank tumors, which express the EGFR, and tumor imaging was performed with both external detection in live animals and with whole body autoradiography. However, the tumor signal was not increased with the cationized MAb, relative to the native MAb, and this was due to a serum inhibition phenomenon that was confirmed by a pharmacokinetics analysis in control mice. In an attempt to block the serum inhibition, the cationized 528 MAb was pegylated with 2000 Da poly(ethylene glycol), and the cationized/pegylated MAb was conjugated with DTPA and labeled with (111)In. However, a pharmacokinetics analysis showed the pegylation did not reverse the serum inhibition of the cationic charge on the MAb. These studies describe methods for reformulating monoclonal antibodies to develop improved radiopharmaceuticals, but show that radiolabeling a cationized MAb with DTPA produces a serum neutralization of the initial cationization modification. 相似文献
13.
《International journal of radiation applications and instrumentation. Part B, Nuclear medicine and biology》1990,17(3):321-329
A new approach for covalent coupling diethylenetriaminepentaacetic acid (DTPA) molecules to a partially reduced monoclonal antibody utilizes a malemide modified copolymer of hydroxyethyl methylacrylate and methyl methacrylate (DTPA copolymer) prepared by the group transfer polymerization (GTP) method. An average of 6 DTPA molecules were incorporated per mol maleimeide DTPA copolymer and 1.5 mol maleimide DTPA copolymer per mol antibody. Maleimide DTPA copolymer modified antibody was intramolecularly cross-linked, reduced immunoactivity and had a high in vivo liver uptake. 相似文献
14.
Squaric monoamide monoester as a new class of reactive immunization hapten for catalytic antibodies 总被引:1,自引:0,他引:1
Xu Y Yamamoto N Ruiz DI Kubitz DS Janda KD 《Bioorganic & medicinal chemistry letters》2005,15(19):4304-4307
A squaric monoester monoamide motif was employed as an effective reactive immunogen for the discovery of monoclonal antibodies with reactive residue(s) in their combining sites. Two antibodies, 2D4 and 3C8, were uncovered that enhance paraoxon hydrolysis over background. Kinetic analysis of these antibodies was performed and interestingly both undergo a single turnover event due to covalent modification within the antibody combining site. Because antibodies 2D4 and 3C8 result in covalent attachment and thus inactivation of paraoxon, they could be useful probes for investigating paraoxon intoxication. 相似文献
15.
《International journal of radiation applications and instrumentation. Part B, Nuclear medicine and biology》1991,18(7):667-676
The direct labeling of antibodies and antibody fragments to form a highly stable bond between technetium and the sulfide groups of proteins is now well established. To optimize this reaction, the antibody protein must have sufficient reactive sulfides available to accept that technetium metal ions that are formed by the reduction of pertechnetate in the presence of a weak complexing agent. The reactive sulfide groups are provided by first reducing a small fraction of the disulfide bridges in the antibody protein or by starting with Fab′ fragments, which already have reactive sulfide groups. When the antibody protein has been appropriately reduced, and the reactive sulfide groups protected by a metal ion with a lower binding affinity than technetium, such as tin or zinc, very high labeling yields of high-affinity-bonded 99mTc can be achieved. This can be accomplished without loss of immunoreactivity, measured as either affinity or immunoreactive fraction.Side reactions can produce radiochemical impurities such as low-affinity, bound 99mTc; 99mTc colloids; 99mTc peptides or antibody aggregates; or 99mTc-complexes. Also, pertechnetate ions may be an impurity if the sodium pertechnetate solution added to the reduced antibodies is not completely reduced. The specifics of minimizing these side reactions have not been extensively discussed in the prior literature; however, it is clear that appropriate reduction of the protein prior to labeling and complete removal of the reducing agent, particularly if it contains reactive sulfide groups or is toxic, are critical.One- or two-step 99mTc-labeling kits for preparing 99mTc-labeled antibody or antibody fragments are rapidly being introduced for use in clinical nuclear medicine studies. These direct labeling methods employ a common sequence of chemical reactions, although the reducing agents for both the antibody and the [99mTc]pertechnetate may vary. Different 99mTc transfer agents may be used, but all transfer agents have the common feature of quickly forming weak to moderately strong complexes with reduced technetium. Most use Sn(II) to reduce the pertechnetate, although other reducing agents can be used. 相似文献
16.
Diethylenetriaminepentaacetic acid (DTPA), a strong chelating agent, was covalently linked to murine monoclonal anti-HLA IgG1 antibody (H-1) with the use of poly(L-lysine) (Mr 14,000) as a multivalent, intermediary carrier, via thiol-disulfide exchange reaction. The conjugates contained up to 42.5 mol DTPA per mol antibody, and retained over 90% of their antibody activity in vitro. The conjugates incorporated gadolinium (Gd) through an exchange reaction with Gd-EDTA, used to prevent colloid formation and nonspecific binding of the free metal. The IgG-poly(L-lysine)-DTPA-Gd had a greater effect per mol on proton relaxation rates than DTPA-Gd itself. Use of poly(L-lysine) as an intermediary carrier for attachment of chelating agents to IgG thus offers great potential for achieving high-specific-activity conjugates, particularly for use as biologically specific contrast agents in nuclear magnetic resonance imaging. 相似文献
17.
The FAD-dependent enzyme cyclohexanone oxygenase carries out biological Baeyer-Villiger oxidations of ketones and aldehydes with O2 as cosubstrate. Sulfides are enzymically oxygenated to sulfoxides but thiolactones appear to be enzymically processed to acyl sulfoxides which are reactive acylating and then sulfenylating agents, inducing mechanism-based inactivation. 相似文献
18.
The objective of this study was to develop an acylation agent for the radioiodination of monoclonal antibodies that would maximize retention of the label in tumor cells following receptor- or antigen-mediated internalization. The strategy taken was to add a polar substituent to the labeled aromatic ring to impede transport of labeled catabolites across lysosomal and cell membranes after antibody degradation. Preparation of unlabeled N-succinimidyl 4-guanidinomethyl-3-iodobenzoate (SGMIB) was achieved in six steps from 3-iodo-4-methylbenzoic acid. Preparation of 4-guanidinomethyl-3-[131I]iodobenzoic acid from the silicon precursor, 4-(N1,N2-bis-tert-butyloxycarbonyl)guanidinomethyl-3-trimethylsilylbenzoic acid proceeded in less than 5% radiochemical yield. A more successful approach was to prepare [131I]SGMIB directly from the tin precursor, N-succinimidyl 4-(N1,N2-bis-tert-butyloxycarbonyl)guanidinomethyl-3-trimethylstannylbenzoate, which was achieved in 60-65% radiochemical yield. A rapidly internalizing anti-epidermal growth factor receptor variant III antibody L8A4 was labeled using [131I]SGMIB in 65% conjugation efficiency and with preservation of immunoreactivity. Paired-label in vitro internalization assays demonstrated that the amount of radioactivity retained in cells after internalization for L8A4 labeled with [131I]SGMIB was 3-4-fold higher than that for L8A4 labeled with 125I using either Iodogen or [125I]SIPC. Catabolite assays documented that the increased retention of radioiodine in tumor cells for antibody labeled using [131I]SGMIB was due to positively charged, low molecular weight species. These results suggest that [131I]SGMIB warrants further evaluation as a reagent for labeling internalizing antibodies. 相似文献
19.
Recombinant monoclonal antibodies (Ab's) have widespread application as research tools, diagnostic reagents and as biotherapeutics. Whilst studying the cellular molecular switch protein m‐ras, a recombinant monoclonal antibody to m‐ras was generated for use as a research tool. Antibody genes from a single rabbit B cell secreting IgG to an m‐ras specific peptide sequence were expressed in mammalian cells, and monoclonal rabbit IgG binding was characterized by ELISA and peptide array blotting. Although the monoclonal Ab was selected for specificity to m‐ras peptide, it also bound to both recombinant full‐length m‐ras and h‐ras proteins. The cross‐reactive binding of the monoclonal Ab to h‐ras was defined by peptide array blot revealing that the Ab showed preference for peptide sequences containing multiple positively charged amino acid residues. These data reinforce the concept of antibody multispecificity through multiple interactions of the Ab paratope with diverse polypeptides. They also emphasize the importance of immunogen and Ab selection processes when generating recombinant monoclonal Ab's. 相似文献
20.
A V Hospattankar S W Law K Lackner H B Brewer 《Biochemical and biophysical research communications》1986,139(3):1078-1085
The human liver apoB-100 gene cloned in the lambda gt-11 expression vector expresses fusion proteins reacting with apoB antibodies. A fusion protein induced from a apoB-lambda gt-11 clone reacted with apoB-100 monoclonal antibodies known to block the binding of LDL to the LDL receptor. The fusion protein contains an amino acid sequence domain enriched in positively charged residues which is complementary to the negatively charged amino acids present in the consensus LDL receptor binding domain. This sequence of apoB-100 is proposed as a binding domain for the interaction with the LDL receptor. Comparison of derived amino acid sequences from the entire structure of apoB-100 molecule revealed several similar domains enriched in positively charged amino acids. A consensus sequence of the potential LDL binding domain was identified which contained positively charged amino acids at positions 1, 5 and 8 and a loop of 8-11 amino acids followed by two adjacent positively charged amino acids. These results are interpreted as indicating that there are several potential LDL receptor binding domains in apoB-100. 相似文献