Pretargeted peptide imaging and therapy.
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Animals↗
Biomedical subjects
C F Meares
Publications and source records attributed to C F Meares.
67Cu-2IT-BAT-Lym-1 pharmacokinetics, radiation dosimetry, toxicity and tumor regression in patients with lymphoma.
UNLABELLED: Lym-1, a monoclonal antibody that preferentially targets malignant lymphocytes, has induced therapeutic responses and prolonged survival in patients with non-Hodgkin's lymphoma when labeled with 1311. Radiometal-labeled antibodies provide higher tumor radiation doses than corresponding 1311 antibodies. 67Cu has an exceptional combination of properties desirable for radioimmunotherapy, including gamma and beta emissions for imaging and therapy, respectively, a biocompatible half-time and absence of pathways contributing to myelotoxicity. The radioimmunoconjugate, 67Cu-21T-BAT-Lym-1, has been shown to be efficacious in nude mice bearing human Burkitt's lymphoma (Raji) xenografts. Based on these results, a clinical study of the pharmacokinetics and dosimetry of 67Cu-21T-BAT-Lym-1 in patients with lymphoma was initiated. METHODS: Eleven patients with advanced stage 3 or 4 lymphoma were given a preload dose of unmodified Lym-1, then an imaging dose of 126-533 MBq (3.4-14.4 mCi) 67Cu-21T-BAT-Lym-1. Total Lym-1 ranged from 25 to 70 mg dependent on the specific activity of the radioimmunoconjugate and was infused at a rate of 0.5-1 mg/min. Imaging, physical examination, including caliper measurement of superficial tumors, and analysis of blood, urine and fecal samples were performed for a period of 6-13 d after infusion to assess pharmacokinetics, radiation dosimetry, toxicity and tumor regression. RESULTS: In 7 patients, in whom superficial tumors had been accurately measured, tumors regressed from 18% to 75% (mean 48%) within several days of 67Cu-21T-BAT-Lym-1 infusion. The uptake and biological half-time of 67Cu-21T-BAT-Lym-1 in tumors were greater than those of normal tissues, except the mean liver half-time exceeded the mean tumor half-time. The mean tumor-to-marrow radiation ratio was 32:1, tumor-to-total body was 24:1 and tumor-to-liver was 1.5:1. Images were of very good quality; tumors and normal organs were readily identified. Mild and transient Lym-1 toxicity occurred in 6 patients; 1 patient developed a human antimouse antibody. There were no significant changes in blood counts or serum chemistries indicative of radiation toxicity. CONCLUSION: Because of the long residence time of 67Cu-21T-BAT-Lym-1 in tumors, high therapeutic ratios were achieved and, remarkably, numerous tumor regressions were observed after imaging doses. The results indicate considerable therapeutic potential for 67Cu-21T-BAT-Lym-1.
Adult↗
67Cu-versus 131I-labeled Lym-1 antibody: comparative pharmacokinetics and dosimetry in patients with non-Hodgkin's lymphoma.
Antilymphoma mouse monoclonal antibody (MoAb) Lym-1, labeled with 67Cu or 131I, has demonstrated promising results in radioimmunotherapy (RIT) for lymphoma. Although 131I has played a central role in RIT thus far, some properties of 67Cu are preferable. A subset of our patients received both 67Cu- and 131I-labeled Lym-1, allowing a comparative evaluation of the two radiopharmaceuticals administered to a matched population of patients. Four patients with B-lymphocytic non-Hodgkin's lymphoma that had progressed despite standard therapy entered trials of 67Cu- and 131I-labeled Lym-1, which were injected 3-26 days apart. Lym-1 was conjugated to 6-[p-(bromoacetamido)benzyl]-1,4,7,11-tetraazacyclotetradecane-N,N ',N",N'"-tetraacetic acid (BAT) via 2-iminothiolane (2IT) and radiolabeled with 67Cu to prepare 67Cu-2IT-BAT-Lym-1; 131I-Lym-1 was preparred by the chloramine-T reaction. Planar imaging was used to quantitate 67Cu-2IT-BAT-Lym-1 or 131I-Lym-1 in organs and tumors daily for 3 days or longer. 67Cu-2IT-BAT-Lym-1 exhibited higher peak concentration in 92% (12 of 13) of tumors and a longer biological half-time in every tumor than 131I-Lym-1. The mean tumor concentration (%ID/g) of 67Cu-2IT-BAT-Lym-1 was 1.7, 2.2, and 2.8 times that of 131I-Lym-1 at 0, 24, and 48 h after injection, respectively. The mean biological half-times of 67Cu-2IT-BAT-Lym-1 and 131I-Lym-1 in tumor were 8.8 and 2.3 days, respectively. Consequently, the mean tumor radiation dose delivered by 67Cu-2IT-BAT-Lym-1 was twice that of 131I-Lym-1, 2.8 (range 0.8-6.7), and 1.4 (range 0.4-35) Gy/GBq, respectively. 67Cu-2IT-BAT-Lym-1 delivered a lower marrow radiation dose than 131I-Lym-1; hence, the tumor:marrow therapeutic indices were 29 and 9.7, respectively. Radiation doses from 67Cu-2IT-BAT-Lym-1 and 131I-Lym-1 to normal tissues were similar except for liver, which received a higher dose from 67Cu-2IT-BAT-Lym-1. Images obtained with 67Cu-2IT-BAT-Lym-1 were superior. Radiation dosimetry data for 67Cu-2IT-BAT-Lym-1 and 131I-Lym-1 agreed with corresponding data from the larger populations of patients from which the matched population for the current study was drawn. In conclusion, 67Cu-2IT-BAT-Lym-1 given to non-Hodgkin's lymphoma patients in close temporal proximity to 131I-Lym-1 exhibited greater uptake and longer retention in tumor, resulting in higher radiation dose and therapeutic index than 131I-Lym-1. These as well as other factors suggest that 67Cu-2IT-BAT-Lym-1 may be superior to 131I-Lym-1 for RIT.
Adult↗
Mapping the promoter DNA sites proximal to conserved regions of sigma 70 in an Escherichia coli RNA polymerase-lacUV5 open promoter complex.
Base-specific interactions between promoter DNA and Escherichia coli RNA polymerase are regulated by a sigma (sigma) protein during transcription initiation. To map spatial relations between evolutionarily conserved regions of the primary sigma (sigma 70) and each DNA strand along the lacUV5 promoter in the transcriptionally active "open" complex, we have used a cysteine-tethered cutting reagent to cleave DNA strands. The chemical nuclease FeBABE [iron (S)-1-(p-bromoacetamidobenzyl)ethylenediaminetetraacetate] was conjugated to single-cysteine mutants of sigma 70 at sites 132C, 376C, 396C, 422C, 496C, 517C, or 581C. After formation of open promoter complexes between lacUV5 DNA and RNA polymerase holoenzymes carrying conjugated sigma 70 subunits, we observed promoter DNA cleavage spanning at least 60 bases, between positions -48 and +12. The results show that sigma 70 region 2.1, otherwise implicated in core enzyme binding, is proximal to the nontemplate strand of lacUV5 DNA between the -10 promoter element and positions as far downstream of the transcription start site as +12. Conserved region 3.2 of sigma 70 is proximal to the template strand near the +1 transcription start site, and region 3.1 is positioned between the lacUV5-10 and -35 promoter elements. We propose a model for the orientation of sigma 70 and DNA in the open complex.
Amino Acid Sequence↗
Mapping the sigma70 subunit contact sites on Escherichia coli RNA polymerase with a sigma70-conjugated chemical protease.
The core enzyme of Escherichia coli RNA polymerase acquires essential promoter recognition and transcription initiation activities by binding one of several sigma subunits. To characterize the proximity between sigma70, the major sigma for transcription of the growth-related genes, and the core enzyme subunits (alpha2 beta beta'), we analyzed the protein-cutting patterns produced by a set of covalently tethered FeEDTA probes [FeBABE: Fe (S)-1-(p-bromoacetamidobenzyl)EDTA]. The probes were positioned in or near conserved regions of sigma70 by using seven mutants, each carrying a single cysteine residue at position 132, 376, 396, 422, 496, 517, or 581. Each FeBABE-conjugated sigma70 was bound to the core enzyme, which led to cleavage of nearby sites on the beta and beta' subunits (but not alpha). Unlike the results of random cleavage [Greiner, D. P., Hughes, K. A., Gunasekera, A. H. & Meares, C. F. (1996) Proc. Natl. Acad. Sci. USA 93, 71-75], the cut sites from different probe-modified sigma70 proteins are clustered in distinct regions of the subunits. On the beta subunit, cleavage is observed in two regions, one between residues 383 and 554, including the conserved C and Rif regions; and the other between 854 and 1022, including conserved region G, regions of ppGpp sensitivity, and one of the segments forming the catalytic center of RNA polymerase. On the beta' subunit, the cleavage was identified within the sequence 228-461, including beta' conserved regions C and D (which comprise part of the catalytic center).
Bacterial Proteins↗
Dimeric association of Escherichia coli RNA polymerase alpha subunits, studied by cleavage of single-cysteine alpha subunits conjugated to iron-(S)-1-[p-(bromoacetamido)benzyl]ethylenediaminetetraacetate.
Proximity relationships between the two associated monomers of the Escherichia coli RNA polymerase alpha subunit were studied using a set of four mutant alpha subunits, each with a single Cys residue at one of the naturally occurring positions (54, 131, 176, and 269). These mutant alpha subunits were conjugated with the cutting reagent iron-(S)-1-[p-(bromoacetamido)benzyl]ethylenediaminetetraacetate (Fe-BABE), and the peptide backbone was cleaved at locations near the modified Cys. Analysis of the cleavage sites identified segments within approximately 12 A of the conjugation site. These results show that, for intermolecular cutting, segments of the subunit assembly domain (N-terminal domain) of one subunit and the linker region between N- and C-terminal domains of the other subunit are near each other, and the N-terminal domains of both subunits are in close proximity to one another. Intramolecular cutting however, was observed only within an individual N- or C-terminal domain.
Alkaline Phosphatase↗
New anti-Cu-TETA and anti-Y-DOTA monoclonal antibodies for potential use in the pre-targeted delivery of radiopharmaceuticals to tumor.
Monoclonal antibodies were raised against yttrium(III)-1, 4, 7, 10-tetraazacyclododecane-N,N',N''N'''--tetraacetic acid (Y-DOTA) and copper(II)-1, 4, 8, 11-tetraazacyclotetradecane-N,N',N'',N'''-tetraacetic acid (Cu-TETA). Four hybridomas with high Y-DOTA binding activity and one hybridoma with Cu-TETA activity were selected. MAbs were purified from mouse ascites by Protein A affinity chromatography and characterized. Affinity constants were determined by equilibrium dialysis and the highest affinity Y-DOTA MAb (K(aff) = 1.9 x 10(8) M(-1)) was further characterized by competitive ELISA. Gd-DOTA competed as well as Y-DOTA, whereas In-DOTA required 740x higher concentrations for 50% inhibition of this Y-DOTA MAb binding to human serum albumin-Y-DOTA-coated microtiter plates. These anti-metal chelate MAbs have potential use as vehicles for the pretargeted delivery of radiometal chelates to tumors.
Animals↗
Yttrium-90-DOTA-peptide-chimeric L6 radioimmunoconjugate: efficacy and toxicity in mice bearing p53 mutant human breast cancer xenografts.
UNLABELLED: The novel radioimmunoconjugate, 90Y-DOTA-peptide-chimeric L6 (ChL6), was designed to reduce radiation to critical normal tissues with an exceptionally stable 90Y chelate moiety and a biodegradable linker. Human breast cancer tumors (HBT 3477) in mice were treated with 90Y-DOTA-peptide-ChL6 to examine the effects of increasing dose on the therapeutic efficacy and toxicity of this new agent. METHODS: Groups of athymic mice bearing HBT 3477 xenografts received 4.1- to 14.1-MBq doses of 90Y-DOTA-peptide-ChL6 intravenously. The lethal dose (LD)(50/30), general well-being (weight loss), hematotoxicity and therapeutic efficacy were studied. RESULTS: The LD(50/30) was 12.8 MBq, which corresponded to doses of 17.9 and 50.9 Gy to the total body and tumor (200 mm3), respectively. Deaths were associated with hematotoxicity; no deaths occurred at doses of 9.6 MBq or less. At sublethal doses, the rate of tumor response (cures +/- complete responses + partial responses) increased with increasing dose: 4.1 MBq, 27%; 5.9 MBq, 41%; 8.5 MBq, 69%; and 9.6 MBq, 79% (maximum tolerated dose, MTD). In mice receiving doses of 4.1-9.6 MBq, 6 of 74 (8%) of tumors were cured. Increasing the 90Y dose led to smaller tumor size at nadir and longer tumor regrowth delay but no increase in cure. Although the HBT 3477 p53 gene was found to be mutant resulting in p53 protein not binding DNA breaks, tumors at MTD demonstrated evidence of apoptosis. CONCLUSION: In the human breast cancer athymic mouse model, 90Y-DOTA-peptide-ChL6 had a high therapeutic index and LD(50/30) leading to a 79% response rate at the MTD. The evidence of apoptosis as a mechanism for this tumor response in p53 mutant breast cancer warrants further studies because these observations are relevant to the treatment of lethal breast cancer.
Adenocarcinoma↗
Expression of recombinant Lym-1 single-chain Fv in Escherichia coli.
Lym-1 single-chain Fv (sFv) can be used for targeted radiodiagnosis and therapy of B-lymphocytic malignancies. Lym-1 sFv was constructed and expressed as a glutathione S-transferase fusion protein, using a (G4S)3 linker connecting the C-terminus of the VH domain and the N-terminus of the VL domain of Lym-1. Six histidine residues and an E Tag epitope were introduced at the C-terminus of the sFv. Lym-1 sFv was purified with glutathione-Sepharose 4B affinity chromatography followed by digestion with thrombin. Lym-1 sFv of 28 kDa was confirmed by Western blotting with anti-(E Tag) monoclonal antibody. An antigen binding assay of Lym-1 and a CD study indicated that it is functionally active.
Amino Acid Sequence↗
Biological properties of biotin-chelate conjugates for pretargeted diagnosis and therapy with the avidin/biotin system.
UNLABELLED: Three-step pretargeting increases target-to-background ratios in radioimmunodetection and can potentially decrease harmful radiation to normal tissues in radioimmunotherapy. We studied four biotin-chelate conjugates (BCCs) for use in the avidin/biotin pretargeting system. METHODS: Pharmacokinetics and biodistribution were studied in normal BALB/c (IAk-negative), normal C3H (IAk-positive) and LS174T tumor-bearing BALB/c severe combined immunodeficient mice. Streptavidin alone and antibody-streptavidin conjugates [monoclonal antibody (MAb) 10-3.6 anti-IAk IgG2a] were used. Indium-111- or 88Y-BCCs were given alone intravenously; they were mixed with streptavidin or MAb-streptavidin conjugate and given intravenously; or streptavidin and MAb-streptavidin conjugate were pretargeted, and 2-3, 5 and 21 hr later, BCCs were injected intravenously. Samples were taken 2-3 hr after intravenous injection of labeled BCCs. RESULTS: Three of the four BCCs were rapidly excreted by the kidneys, with <2.5%/g in any organ or tumor at 2-3 hr. Gut excretion eliminated biotinyl-(S)-1-p-aminobenzylethylenediaminetetraacetic acid (EDTA) for use in pretargeting. Ninety percent of BCCs were bound to circulating pretargeted streptavidin at 1-6 hr, and approximately 15% were bound to pretargeted streptavidin at 24 hr. Kidney uptakes were: preformed streptavidin-BCC given intravenously, approximately 80%/g (24 hr); streptavidin pretargeted for 2-3 hr, approximately 60%/g; and streptavidin pretargeted for 5-21 hr, approximately 10%-20%/g. Kidney uptake was dose-dependent: 0.2, 0.67 and 1.0 nmol of streptavidin pretargeted for 21 hr showed increasing concentrations (24 hr). Uptake of monoclonal anti-IAk-streptavidin-BCC complex into spleen (70% +/- 10%/g; p < 0.05) and lymph nodes (10% +/- 3.5%/g; p < 0.01) was higher in IAk-positive C3H mice than it was in IAk-negative control BALB/c mice, and it was much higher than that in streptavidin controls. No significant target uptake was seen with anti-IAk MAb-streptavidin pretargeted for 3 or 20 hr. Kidney uptake approximately 20%/g, which was lower than that of streptavidin alone. CONCLUSION: Three biotinyl chelates bind the diagnostic and therapeutic radiometals 111In and 88Y (and, by analogy, 90Y) with the required in vivo stability and physiological properties for pretargeted diagnosis and therapy. Kidney uptake of streptavidin was decreased by conjugation to MAb. Failure of anti IAk MAb-streptavidin conjugate to bind BCC after pretargeting may be due to rapid internalization of MAb-streptavidin-IAk complex by the lymphocyte or to endogenous biotin. Either or both of these would make streptavidin unavailable to subsequent BCCs.
Animals↗
Comparison of 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA)-peptide-ChL6, a novel immunoconjugate with catabolizable linker, to 2-iminothiolane-2-[p-(bromoacetamido)benzyl]-DOTA-ChL6 in breast cancer xenografts.
Radioimmunotherapy using 131I-ChL6 antibody has shown promise in patients with breast cancer. To enhance this potential, a novel ChL6 immunoconjugate that is catabolizable and tightly binds 90Y and (111)In was developed. The immunoconjugate, 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA)-peptide-ChL6, consists of the macrocyclic chelator DOTA linked to ChL6 by a peptide that is preferentially catabolized in the liver. The pharmacokinetic and dosimetric properties of the radioimmunoconjugates (RICs) (111)In- and 90Y-DOTA-peptide-ChL6 and (111)In- and 90Y-2-iminothiolane (2-IT)-2-[p-(bromoacetamido)benzyl]-DOTA-ChL6 were compared in athymic mice bearing HBT3477 human breast cancer xenografts. Each of the RICs was stable in vivo and concentrated well in the xenografts. Liver concentration, cumulative radioactivity (activity over time), and radiation dose of the DOTA-peptide-ChL6 RICs were one-third to one-half of those of the corresponding 2-IT-2-[p(bromoacetamido)benzyl]-DOTA-ChL6 RICs. Indium-111 RICs were imperfect tracers for corresponding 90Y RICs, although their pharmacokinetics and radiation dosimetries were similar. The results of this study were consistent with previously published in vitro data, which indicated that the peptide linker of DOTA-peptide-ChL6 was catabolized by cathepsin B. The cumulative activities and radiation doses to the liver of DOTA-peptide-ChL6 RICs were one-half of those of corresponding RICs with the 2-IT linker. Preliminary data from pilot studies in patients with breast cancer are in accord with these observations. These novel DOTA-peptide RICs seem to have excellent clinical potential for radioimmunotherapy associated with marrow transplantation, for which liver radiation is likely to be dose limiting for 90Y.
Animals↗
Optimized conditions for chelation of yttrium-90-DOTA immunoconjugates.
UNLABELLED: Radioimmunotherapy (RIT) with 90Y-labeled immunoconjugates has shown promise in clinical trials. The macrocyclic chelating agent 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) binds 90Y with extraordinary stability, minimizing the toxicity of 90Y-DOTA immunoconjugates arising from loss of 90Y to bone. However, reported 90Y-DOTA immunoconjugate product yields have been typically only < or =50%. Improved yields are needed for RIT with 90Y-DOTA immunoconjugates to be practical. METHODS: (S) 2-[p-(bromoacetamido)benzyl]-DOTA (BAD) was conjugated to the monoclonal antibody Lym-1 via 2-iminothiolane (2IT). The immunoconjugate product, 2IT-BAD-Lym-1, was labeled in excess yttrium in various buffers over a range of concentrations and pH. Kinetic studies were performed in selected buffers to estimate radiolabeling reaction times under prospective radiopharmacy labeling conditions. The effect of temperature on reaction kinetics was examined. Optimal radiolabeling conditions were identified and used in eight radiolabeling experiments with 2IT-BAD-Lym-1 and a second immunoconjugate, DOTA-peptide-chimeric L6, with 248-492 MBq (6.7-13.3 mCi) of 90Y. RESULTS: Ammonium acetate buffer (0.5 M) was associated with the highest uptake of yttrium. On the basis of kinetic data, the time required to chelate 94% of 90Y (four half-times) under prospective radiopharmacy labeling conditions in 0.5 M ammonium acetate was 17-148 min at pH 6.5, but it was only 1-10 min at pH 7.5. Raising the reaction temperature from 25 degrees C to 37 degrees C markedly increased the chelation rate. Optimal radiolabeling conditions were identified as: 30-min reaction time, 0.5 M ammonium acetate buffer, pH 7-7.5 and 37 degrees C. In eight labeling experiments under optimal conditions, a mean product yield (+/- s.d.) of 91%+/-8% was achieved, comparable to iodination yields. The specific activity of final products was 74-130 MBq (2.0-3.5 mCi) of 90Y per mg of monoclonal antibody. The immunoreactivity of 90Y-labeled immunoconjugates was 100%+/-11%. CONCLUSION: The optimization of 90Y-DOTA chelation conditions represents an important advance in 90Y RIT because it facilitates the dependable and cost-effective preparation of 90Y-DOTA pharmaceuticals.
Buffers↗
Pretargeting: general principles; October 10-12, 1996.
BACKGROUND: Human imaging studies show that maximum human tumor concentrations of monoclonal antibody (MoAb) are achieved in 1 day, but several days are required for background reduction and sensitive radioimmunoscintigraphy of tumors. With therapeutic radionuclides such as yttrium-90 (90Y), this long biologic half-life imposes a high radiation burden on sensitive normal tissues from the large amount of unlocalized radioactivity. Normal tissue toxicity, especially to the bone marrow, has been the major limiting factor in the application of radioimmunotherapy to solid tumors. Pretargeting techniques provide an alternative method with which to obtain high selective tumor uptake of 90Y with simultaneous minimization of nontarget tissue background. METHODS: Current MoAb techniques employ either the biotin/avidin or the hapten/antibody system. DNA/DNA and prodrug/enzyme systems also have been used and many other ligand/receptor systems are possible. All the methods depend on a long circulating conjugate to obtain high target uptake with a diffusible, rapidly excreted effector molecule. RESULTS: Fast in, slow out tumor kinetics, ideal for therapy, have been achieved. In one mouse tumor system the biologic half-life of a bivalent 88Y JANUS tetraazacyclodidecanetetraacetic acid hapten, measured over 5 days, was approximately 24 hours. The therapeutic ratio, obtained from the integrated tumor and blood concentrations over 5 days, was approximately 20/1 compared with 2-3/1 with directly labeled MoAb. The total injected dose remaining in the mouse at 24 hours was 5.5%, 23% of which was in the tumor. CONCLUSIONS: These result suggest that it may be possible to deliver tumoricidal radiation doses with 90Y using pretargeting techniques without severe normal bone marrow irradiation.
Animals↗
Positioning of two alpha subunit carboxy-terminal domains of RNA polymerase at promoters by two transcription factors.
Interactions between the cAMP receptor protein (CRP) and the carboxy-terminal regulatory domain (CTD) of Escherichia coli RNA polymerase alpha subunit were analyzed at promoters carrying tandem DNA sites for CRP binding using a chemical nuclease covalently attached to alpha. Each CRP dimer was found to direct the positioning of one of the two alpha subunit CTDs. Thus, the function of RNA polymerase may be subject to regulation through protein-protein interactions between the two alpha subunits and two different species of transcription factors.
Amino Acid Substitution↗
Synergy of Taxol and radioimmunotherapy with yttrium-90-labeled chimeric L6 antibody: efficacy and toxicity in breast cancer xenografts.
Synergistic multimodality therapy is needed for breast cancer. Breast cancer frequently has p53 mutations that result in cells less likely to undergo apoptosis when exposed to DNA damaging therapies. Taxol (paclitaxel) is more effective in the presence of mutant p53. (90)Y-labeled DOTA-peptide-ChL6 ((90)Y-ChL6, where ChL6 is chimeric L6 antibody and DOTA is 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid) is a novel radioimmunoconjugate for targeting radiation to cancer. It has a stable metal chelator and a peptide linker that can be catabolized by hepatic lysozymes. This study was designed to assess potential synergism between Taxol and (90)Y-ChL6 in a highly anaplastic breast cancer model, HBT 3477. There was no tumor response in mice receiving ChL6 or Taxol alone. In mice receiving (90)Y-ChL6 alone, 79% (15 of 19) of tumors responded although none were cured. If Taxol was administered 24-72 hours before (90)Y-ChL6, again, 79% (23 of 29) of tumors responded but 21% were cured. When Taxol was administered 6 or 24 hours after (90)Y-ChL6, 100% (46 of 46) of tumors responded and 48% were cured. Taxol given with (90)Y-ChL6 did not substantially increase toxicity. Enhancement of the therapeutic effect when Taxol was added to (90)Y-ChL6 therapy for HBT 3477 xenografts was striking. The synergistic therapeutic effect of Taxol with (90)Y-ChL6 may relate to the p53 mutant status and BCL2 expression in HBT 3477 cells, observations that increase the likelihood that the results of this study are relevant to therapy for breast cancer in patients. In conclusion, Taxol seemed to be synergistic with (90)Y-ChL6 in this human breast cancer model. Up to 50% of these anaplastic breast cancer xenografts were cured by combined modality therapy.
Animals↗
The two alpha subunits of Escherichia coli RNA polymerase are asymmetrically arranged and contact different halves of the DNA upstream element.
RNA polymerase core enzyme of Escherichia coli is composed of two alpha subunits and one each of the beta and beta' subunits. The C-terminal domain of the RNA polymerase alpha subunit plays a key role in molecular communications with class I transcription factors and upstream (UP) elements of promoter DNA, using the same protein surface. To identify possible differences in the functional roles of the two alpha subunits, we have developed a reconstitution method for hybrid RNA polymerases containing two distinct alpha subunit derivatives in a defined orientation ("oriented alpha-heterodimer"). The binding sites of two alpha C-terminal domains on the UP element DNA were determined by hydroxyl radical-based DNA cleavage mediated by (p-bromoacetamidobenzyl)-EDTA x Fe, which was bound at Cys-269 on the UP recognition surface of one or both alpha subunits. The results clearly indicated that the two alpha subunits bind in tandem to two helix turns of the rrnBP1 UP element, and that the beta'-associated alpha subunit is bound to the promoter-distal region.
Binding Sites↗
Efficacy and toxicity of 67Cu-2IT-BAT-Lym-1 radioimmunoconjugate in mice implanted with human Burkitt's lymphoma (Raji).
Radioimmunotherapy has shown promising results for treatment of radiosensitive malignancies such as lymphoma. Positive responses have been reported in patients with non-Hodgkin's lymphoma treated with 131I-radiolabeled Lym-1, a mouse anti-lymphoma monoclonal antibody. In this study, the efficacy of 67Cu-radiolabeled Lym-1 was examined. Nude mice bearing human Burkitt's lymphoma (Raji) tumors (20-524 mm3) were treated with 12.4, 14.8, 18.5, and 23.3 MBq of 67Cu-2IT-BAT-Lym-1. Tumor size was measured to assess efficacy, and mouse weight, blood counts, and mortality were monitored to assess toxicity. In mice treated with 12.4, 14.8, and 18.5 MBq of 67Cu-2IT-BAT-Lym-1, 50% (9 of 18), 42% (5 of 12), and 50% (3 of 6) of tumors achieved remission or cure; 33% of tumors were cured overall; and significant regrowth delay was observed. The 23.3 MBq dose group did not yield meaningful efficacy data because of high mortality. In control groups receiving 14.8 and 18.5 MBq of the isotype-matched nonspecific monoclonal antibody radioimmunoconjugate, 67Cu-2IT-BAT-L6, 0% (0 of 15) and 17% (2 of 12) of tumors achieved a response; hence, targeted delivery of radiation was the dominant antitumor mechanism of 67Cu-2IT-BAT-Lym-1. LD50/30 for mice treated with 67Cu-2IT-BAT-Lym-1 and -L6 were 21.6 and 20.6 MBq, respectively. In conclusion, 67Cu-2IT-BAT-Lym-1 provided a therapeutic and frequently curative dose of radiation to tumored mice with modest toxicity.
Animals↗
Radiolytic protein surface mapping.
We report that high energy beta particles may function as a means for mapping the surface of a protein. Comparable to Fe-EDTA in the presence of ascorbate and peroxide, 90Y-EDTA alone can break polypeptide backbone bonds on the surface of E. coli RNA polymerase. The two methods give very similar fragmentation patterns, although some unique fragments are produced by each. Radiolytic footprinting may prove useful for mapping proteins inside living cells, since beta-radiolysis produces reactive species up to approximately 1 cm away from the emitting 90Y.
Beta Particles↗