A tetravalent single-chain antibody-streptavidin fusion protein for pretargeted lymphoma therapy.

Single-chain Fv antibody fragments from the CD20-specific murine monoclonal antibody B9E9 were genetically engineered as streptavidin fusions [single-chain Fv-streptavidin (scFvSA) fusion protein] for use in pretargeted radioimmunotherapy. The scFvSA constructs were expressed as soluble, tetrameric species in the periplasm of Escherichia coli. Expression levels were affected by the order of the variable regions and the length and composition of the single-chain Fv linker. The best expressor was obtained with the variable regions in the heavy chain-light chain configuration separated by a 25-mer Gly4Ser linker. This construct produced 250-300 mg of soluble, tetrameric fusion protein per liter of fermentor culture. The fusion protein (Mr 173,600) was purified from crude lysates by iminobiotin affinity chromatography with an overall yield of about 50% and was analyzed for functionality both in vitro and in vivo. Immunoreactivity of the scFvSA fusion protein and its nanomolar affinity to CD20-positive Ramos cells were comparable with the B9E9 monoclonal antibody. The fusion protein had a biotin dissociation rate identical to recombinant streptavidin and bound an average of 3.6 biotins/molecule of a possible 4 biotins/molecule. Labeled fusion protein cleared from the blood of BALB/c mice with a P half-life of about 16 h. In nude mice bearing Ramos xenografts, the fusion protein demonstrated sufficient tumor localization of functional streptavidin to enable efficient, tumor-specific targeting of a subsequently administered radionuclide-chelate/biotin molecule. These results suggest that large quantities of functional scFvSA can be produced for clinical testing as a therapy for non-Hodgkin's lymphoma.

Molecular modeling and preclinical evaluation of the humanized NR-LU-13 antibody.

A mouse-human chimeric monoclonal antibody (chNR-LU-13), specific for the EGP40 pancarcinoma antigen, was humanized through three-dimensional molecular modeling. Humanization of the chNR-LU-13 antibody is expected to enhance its use for patients undergoing immunotherapy. On the basis of the observed amino acid sequence identity, chNR-LU-13 complementary determining regions (CDRs) of the V(L) and V(H) regions were grafted onto the human anti-DNA-associated idiotype immunoglobulin clone, R3.5H5G'CL. Ten amino acids residues within the humanized framework were back-mutated to their corresponding chNR-LU-13 sequence, because they were predicted to disrupt the canonical classification of the CDRs or were within 5 A of a CDR. Synthesis of the V(L) and V(H) regions was accomplished by recursive PCR, and the dual-chain expression vector p451.C4 was positioned under control of the CMV(P+E). We observed by competitive ELISA that the recombinant humanized NR-LU-13 (huNR-LU-13) IgG1 antibody exhibited an indistinguishable immunoreactivity profile when compared with the murine monoclonal antibody (muNR-LU-10). The huNR-LU-13 antibody was effective in mediating both antibody-dependent cellular cytotoxicity and complement-mediated cytotoxicity when assayed against either the breast carcinoma cell line, MCF-7, or the colon adenocarcinoma cell line, SW1222. Biodistribution studies using i.v. coinjected 131I-muNR-LU-10 and 125I-huNR-LU-13 confirmed that the huNR-LU-13 specifically targets to the tumor in athymic BALB/c mice bearing the SW1222 human tumor xenograft. Humanization of the chNR-LU-13 antibody is expected to eliminate an undesired human antimouse antibody response, allowing for repeated i.v. administration into humans.

Phase II trial of yttrium-90-DOTA-biotin pretargeted by NR-LU-10 antibody/streptavidin in patients with metastatic colon cancer.

A Phase II study of yttrium-90-tetra-azacyclododecanetetra-acetic acid-biotin (90Y-DOTA-biotin) pretargeted by NR-LU-10 antibody/streptavidin (SA) was performed. The primary objectives of the study were to evaluate the efficacy and safety of this therapy in patients with metastatic colon cancer. Twenty-five patients were treated with a single dose of 110 mCi/m2 (mean administered dose, 106.5 +/- 10.3 mCi/m2) of 90Y-DOTA-biotin. There were three components of the therapy. Patients first received NR-LU-10/SA on day 1. A clearing agent (biotin-galactose-human serum albumin) was administered approximately 48 h after the NR-LU-10/SA to remove residual circulating unbound NR-LU-10/SA. Lastly, 24 h after administration of clearing agent, patients received biotin-DOTA-labeled with 110 mCi/m2 90Y. All three components of the therapy were administered i.v. Both hematological and nonhematological toxicities were observed. Diarrhea was the most frequent grade 4 nonhematological toxicity (16%; with 16% grade 3 diarrhea). Hematological toxicity was less severe with 8% grade 3 and 8% grade 4 neutropenia and 8% grade 3 and 16% grade 4 thrombocytopenia. The overall response rate was 8%. Two partial responders had freedom from progression of 16 weeks. Four patients (16%) had stable disease with freedom from progression of 10-20 weeks. Despite the relatively disappointing results of this study in terms of therapeutic efficacy and toxicity, proof of principle was obtained for the pretargeting approach. In addition, valuable new information was obtained about normal tissue tolerance to low-dose-rate irradiation that will help to provide useful guidelines for future study designs.

Clinical optimization of pretargeted radioimmunotherapy with antibody-streptavidin conjugate and 90Y-DOTA-biotin.

UNLABELLED: Pretargeted radioimmunotherapy (PRIT) was evaluated using an antibody-streptavidin conjugate, followed by a biotin-galactose-human serum albumin clearing agent and 90Y-dodecane tetraacetic acid (DOTA)-biotin as the final step for therapy. The objective was to develop a clinical protocol that could show an improved tumor-to-red marrow therapeutic ratio compared with conventional radioimmunotherapy (RIT) and at the same time preserve the efficiency of tumor targeting. METHOD: Forty-three patients with adenocarcinomas reactive to NR-LU-10 murine monoclonal antibody received the 3 components. Doses and timing parameters were varied to develop an optimized schema. In some patients, the conjugate was radiolabeled with 186Re as an imaging tracer to assess biodistribution of the conjugate and effectiveness of the clearing agent. 111In-DOTA-biotin was coinjected with 90Y-DOTA-biotin for quantitative imaging. Safety, biodistribution, pharmacokinetics, dosimetry, and antiglobulin formation were evaluated. RESULTS: The optimal schema was defined as a conjugate dose of 125 microg/mL plasma volume followed at 48 h by a clearing agent in a 10:1 molar ratio of clearing agent to serum conjugate. The therapeutic third step was 0.5 mg radiobiotin administered 24 h later. No significant adverse events were observed after administration of any of the components. The mean tumor-to-marrow absorbed dose ratio when using the optimized PRIT schema was 63:1, compared with a 6:1 ratio reported previously for conventional RIT. Antiglobulin to murine antibody and to streptavidin developed in most patients. CONCLUSION: This initial study confirmed that the PRIT approach is safe and feasible and achieved a higher therapeutic ratio than that achieved with conventional RIT using the same antibody.

Long-term cognitive and emotional impact of genetic testing for carriers of cystic fibrosis: the effects of test result and gender.

The cognitive and emotional responses to genetic testing for carriers of cystic fibrosis (CF) of 241 female and 36 male carriers and a matched sample who had received a negative screening result were compared 3 years after testing. The main predictor of responses to testing was the type of result received. Gender differences in response to screening were also found: Women were more likely to feel relieved and less likely than men to feel indifferent, regardless of test results. There was an interaction between test results and gender for feeling happy and healthy about test results. The greater impact of testing on women may reflect gender differences in appraisal or in coping with the threat of being a carrier for a genetic disorder.

Structural characterization of the major covalent adduct formed in vitro between acetaminophen and bovine serum albumin.

The structure of the covalent adduct formed in vitro between [14C]-acetaminophen ([14C]APAP) and bovine serum albumin (BSA) has been investigated with the aid of new analytical methodology. The APAP-BSA adduct, isolated from mouse liver microsomal incubations to which the radiolabeled drug and BSA had been added, was cleaved using a combination of specific (cyanogen bromide) and non-specific (acid hydrolysis) procedures, following which the mixture of amino acids obtained was derivatized, in aqueous solution, with ethyl chloroformate. The resulting ethoxycarbonyl derivatives were recovered by extraction into ethylacetate, methylated and subjected to profile analysis using both reverse-phase and normal-phase HPLC techniques. In each HPLC step, one major radioactive amino acid adduct was detected and was identified by mass spectrometry as the derivative of 3-cystein-S-yl-4-hydroxyaniline. Based on this finding, and with a knowledge of the behavior under acidic hydrolysis conditions of the 3-cysteinyl conjugate of APAP, it could be concluded that the major APAP-BSA adduct is one in which the drug is bound, via a thioether linkage at the C-3 position, to a sulfhydryl group on the protein. Furthermore, it could be established that this -SH function almost certainly is that associated with the cys-34 residue of BSA.

Covalent binding of acetaminophen to mouse hemoglobin. Identification of major and minor adducts formed in vivo and implications for the nature of the arylating metabolites.

When hepatotoxic doses of [ring-U-14C]acetaminophen ([ring-U-14C]APAP) were administered to mice, radioactivity became bound irreversibly to hemoglobin as well as to proteins in the liver and kidney. The covalent binding to hemoglobin was dose-dependent, and in phenobarbital-pretreated mice occurred to the extent of approximately 8% of the corresponding binding to liver proteins. Degradation of the modified globin by acid hydrolysis yielded 3-cystein-S-yl-4-hydroxyacetanilide as the major radioactive product, accounting for approximately 70% of protein-bound drug residues. This finding is consistent with the view that the majority of covalent binding of APAP to proteins is mediated by N-acetyl-p-benzoquinone imine (NAPQI), a reactive metabolite which preferentially arylates cysteinyl thiol residues. However, after administration of [acetyl-3H]APAP to mice, it was found that approximately 20% of the drug bound to hemoglobin had lost the N-acetyl side-chain, indicating the existence of a second type of APAP-protein adduct. One minor component of the globin hydrolysate was identified as S-(2,5-dihydroxyphenyl)-cysteine, which most likely arises from binding to hemoglobin of p-benzoquinone, a hydrolysis product of NAPQI. The two adducts reported represent the first identified examples of arylating drugs binding to hemoglobin. Experiments on the influence of different cytochrome P-450 inducing agents on the ratio of drug bound to hemoglobin versus hepatic proteins suggested that the reactive metabolites of APAP are formed in the liver and migrate to the erythrocyte, rather than being produced by hemoglobin-catalyzed oxidation of APAP. These findings imply that the reactive metabolites of APAP escape from hepatocytes in some latent forms, which then participate in the arylation of protein thiols in red blood cells and, possibly, at other remote sites.

Preclinical evaluation of a humanized NR-LU-10 antibody-streptavidin fusion protein for pretargeted cancer therapy.

A humanized single chain Fv antibody fragment specific to the EGP40 antigen was genetically engineered as a streptavidin fusion (scFvSA) for use in pretargeted radioimmunotherapy. The scFvSA construct was expressed as a soluble, tetrameric species in the Escherichia coli periplasm at 110-140 mg/liter. The fusion protein was purified from crude lysates by iminobiotin affinity chromatography with an overall yield of 50-60%. Characterization of the purified protein by SDS-PAGE, light scattering, and size exclusion chromatography demonstrated that the fusion protein was tetrameric with a molecular weight of approximately 172,000. Competitive immunoreactivity assays showed a two-fold greater binding to the antigen than the comparable whole antibody. The purified protein had a biotin disassociation rate identical to recombinant streptavidin and bound an average of three of four possible biotins per molecule. The radiolabeled fusion protein showed a faster blood clearance rate in normal mice than the corresponding whole antibody-streptavidin chemical conjugate. Tumor-specific targeting of a subsequently administered radionuclidechelate/biotin molecule was demonstrated in nude mice bearing SW1222 human colon carcinoma xenografts. A single dose of 800 microCi of 90Y-DOTA-biotin produced cures in mice with established subcutaneous human small cell lung or colon cancer xenografts.

Pretargeted radioimmunotherapy (PRIT) for treatment of non-Hodgkin's lymphoma (NHL): initial phase I/II study results.

Pretargeted radioimmunotherapy (PRIT) was investigated in patients with non-Hodgkin's lymphoma (NHL). The PRIT approach used in this study is a multi-step delivery system in which an antibody is used to target streptavidin to a tumor associated antigen receptor, and subsequently biotin is then used to target 90Y radioisotope to the tumor localized streptavidin. A chimeric, IgG1, anti-CD20 antibody, designated C2B8 or Rituximab, was conjugated to streptavidin (SA) and administered to patients with NHL. Thirty-four hours later, a clearing agent, synthetic biotin-N-acetyl-galactosamine, was administered to remove non-localized conjugate from the circulation. Finally, a DOTA-biotin ligand, labeled with 111In for imaging and/or 90Y for therapy was administered. Ten patients with relapsed or refractory NHL were studied. In three patients, the C2B8/SA conjugate was radiolabeled with a trace amount of 186Re in order to assess pharmacokinetics and biodistribution using gamma camera imaging. Seven patients received 30 or 50 mCi/m2 90Y DOTA-biotin. Re-186 C2B8/SA images confirmed that the conjugate localized to known tumor sites and that the clearing agent removed > 95% of the conjugate from the circulation. Radiolabeled biotin localized well to tumor. Unbound radiobiotin was rapidly excreted from the whole body and normal organs. The mean tumor dose calculated was 29 +/- 23 cGy/mCi 90Y and the average whole body dose was 0.76 +/- 0.3 cGy/mCi 90Y, resulting in a mean tumor to whole body dose ratio of 38:1. Only grade I/II non-hematologic toxicity was observed. Hematologic toxicity was also not severe; i.e., five of the seven patients who received 30 or 50 mCi/m2 of 90Y-DOTA-biotin experienced only transient grade III (but no grade IV) hematologic toxicity. Although six of ten patients developed humoral immune responses to the streptavidin, these were delayed and transient and hence may not preclude retreatment. Six of seven patients who received 30 or 50mCi/m2 90Y achieved objective tumor regression, including three complete and one partial response. The estimate of tumor to whole body dose ratio (38:1) achieved with PRIT in these NHL patients is higher than has been achieved in other studies using conventional RIT. Toxicity was mild and tumor response encouraging. PRIT clearly deserves additional study in patients with NHL.

The personality and psycho-sexual development of boys with 47 XXY chromosome constitution.

Twelve XXY boys identified at birth in a population screening study were followed up at a mean age of 16.7 yr and compared with a control group of chromosomally normal boys matched for age and social class. Assessment of personality and psycho-sexual development showed the XXY boys to be more timid and less self-confident and to have problems in relating to their peer group than the controls. They also showed some evidence of delayed or impaired development of sexual interest. In general, the differences were not marked. Possible causes for these differences and potential methods of preventing them are discussed.

Balanced rearrangements of the autosomes: results of a longitudinal study of a newborn survey population.

Thirty-six infants were identified by cytogenetic screening at birth as having balanced rearrangements of their autosomes, and 30 of them took part in a longitudinal study of their development, together with four of their affected sibs. With the exception of one child with a de novo reciprocal translocation who died, all children attended normal schools. Congenital malformations and short stature were present in only one child who had a de novo reciprocal translocation. The IQ scores of the 10 children with de novo translocations were significantly lower than those of the 20 children with familial translocations, but there were children in the de novo group of above average intelligence. Children with familial reciprocal translocations had significantly higher IQ scores than both the Robertsonian translocations and the controls, but the numbers in each category were small and a variety of different chromosomes were involved.

Klinefelter's syndrome in adolescence.

Twelve boys with Klinefelter's syndrome (47,XXY) identified by sex chromatin screening at birth were examined at between ages 16 and 18 years, together with 12 controls matched for social class and birth order from the same newborn population. Physical examination, psychometric assessment, personality, and degree of psychosexual development were assessed without knowledge of the karyotype. Anthropometry showed increased leg length and decreased head circumference in the XXY boys. Gynaecomastia was present in 4 boys, and testicular volume was reduced in the majority but one boy had normal sized testes. On the Wechsler intelligence scale there was a significant reduction in verbal score but not in either performance or full-scale score compared with the controls. Although appreciable differences were found in growth, personality, intelligence test scores, and psychosexual development, these were of small degree.

Mechanistic studies on the metabolic activation of acetaminophen in vivo.

Three analogues of acetaminophen (APAP), labeled at specific positions with either oxygen-18 or deuterium, were administered by ip injection to male BALB/c mice at the moderately hepatotoxic dose of 200 mg kg-1 in order to probe the mechanism by which APAP undergoes metabolic activation in vivo. The thioether conjugates of APAP present in bile, urine, and feces, which are believed to derive from the electrophilic intermediate N-acetyl-p-benzoquinone imine (NAPQI), were isolated following aqueous-phase derivatization, separated by HPLC, and converted to a common volatile derivative for analysis by GC-MS. The observed labeling patterns of these conjugates indicated that APAP undergoes metabolism to NAPQI by a process that does not involve the generation of a free oxygenated intermediate, but which more likely entails the sequential removal of two electrons from the substrate. On the basis of these findings, an integrated metabolic scheme is proposed which invokes initial cytochrome P-450 mediated generation of a caged oxygen-centered APAP radical species. Subsequent reactions of this intermediate may account for the formation of all known oxidative metabolites of APAP.

Psychological impact of population-based carrier testing for cystic fibrosis: 3-year follow-up. UK Cystic Fibrosis Follow-Up Study Group.

BACKGROUND: The objective of this study was to show the long-term psychological effects of population-based screening for cystic fibrosis. METHODS: The sample comprised all carriers (n = 435) and, for each carrier, two matched screen-negative individuals (n = 870) detected during screening programmes for cystic fibrosis in the general population and in antenatal populations carried out a median of 3 years earlier in six UK centres. Questionnaires were sent to all eligible participants, with reminders sent to non-responders. The main endpoints were understanding of test results, degree of anxiety, perceptions of health, and reproductive intentions, and behaviour. FINDINGS: 746 (62%) of 1201 questionnaires were returned. Recall of the meaning of test results was accurate in 225 (80%) of 280 carriers but only 200 (43%) of 466 screen-negative individuals. 46 (16%) of 280 proven carriers believed that their result meant that they were only likely, rather than definitely, to be a carrier; 232 (50%) of 466 of those with a screen-negative result erroneously believed that the result meant that they were definitely not carriers. There was no significant difference between carriers and screen-negative individuals in degree of general anxiety, although 16% of carriers reported feeling worried about their test results. Carriers had a poorer perception of their current health than did non-carriers, even though they had been told that carrier status confers no disadvantages to their own health. There were no differences between carriers and screen-negative individuals in reproductive intentions or behaviour. INTERPRETATION: We have shown that in the long-term, retention of the meaning of test results from cystic fibrosis screening is poor. Further research is needed to improve the performance of test-related counselling programmes to ensure that the main objectives of these programmes, to provide information on carrier status and to allow informed reproductive decisions, are met.

Prenatal screening for cystic fibrosis carriers: does the method of testing affect the longer-term understanding and reproductive behaviour of women?

A comparative study of women who underwent prenatal cystic fibrosis (CF) carrier screening by either the 'two-step method' or the 'couple method' was carried out 2-4 years after testing. Recall of the screening test and test result, understanding of the implications of the test result, and reproductive intentions and behaviour were compared. Women screened by the two-step method were significantly better informed on the genetic implications of the test result and the significance of being a single gene carrier than their couple screen counterparts. Regardless of the method of screening, a majority of those who had received a negative test result erroneously believed that they were definitely not a carrier. However, women who intended having further children were significantly more likely to understand correctly that a negative test result meant that they were unlikely to be a CF carrier. The method of testing had no influence on reproductive intentions or behaviour. Differences in emphasis, content and presentation of pre-screening information and counselling between the two methods of screening are identified. Reasons for variation in the long-term understanding between women screened by the two methods are discussed.

Identification of the major covalent adduct formed in vitro and in vivo between acetaminophen and mouse liver proteins.

Improved analytical methodology has been developed for the structural characterization of covalently bound drug-protein adducts and has been applied to an investigation of the conjugates formed in vivo and in vitro between [14C]acetaminophen and mouse liver proteins. The major adduct released by acid hydrolysis of hepatic protein samples, which accounted for approximately 70% of the bound radioactivity in vivo and in vitro, was identified as 3-cystein-S-yl-4-hydroxyaniline, a derivative whose structure reflects the predominance of acetaminophen thioether adducts in drug-modified proteins. It is concluded that the reactive, electrophilic metabolite of acetaminophen, which most likely is N-acetyl-p-benzoquinoneimine, binds with a high degree of selectivity to cysteinyl thiol groups on protein, formally in a Michael-type addition reaction. Cysteine residues thus represent primary target sites for arylation by the reactive metabolite of acetaminophen, and proteins rich in free thiols may be especially vulnerable to damage by this toxic intermediate.

The microsomal metabolism and site of covalent binding to protein of 3'-hydroxyacetanilide, a nonhepatotoxic positional isomer of acetaminophen.

Incubations of 3'-hydroxyacetanilide (3HAA) with hepatic microsomal preparations from phenobarbital-pretreated mice led to the formation of three products of aromatic hydroxylation, viz. 2',5'-, 3',4'-, and 2',3'-dihydroxyacetanilide, which were identified by GC/MS techniques and quantified by GLC analysis. NADPH-dependent covalent binding of radioactivity from [14C]3HAA to microsomal protein took place at almost four times the rate at which [14C]acetaminophen became irreversibly bound to protein under the same experimental conditions. This binding was inhibited by the addition to incubation media of ascorbate, glutathione, and the soluble proteins bovine serum albumin and bovine alpha s1-casein, but not by superoxide dismutase. Radioactivity from [14C]3HAA also became covalently bound to the added soluble proteins, the extent of which was greatest when the proteins contained a high content of free -SH groups. From an analysis of the effect of ascorbate and glutathione on both the covalent binding of 14C to protein and the production of the noncovalently bound products of 3HAA metabolism, it is concluded that reactive intermediates most likely derive from further oxidation of the primary 3HAA metabolites to electrophilic semiquinone and/or quinone species. Sulfhydryl groups appear to be the principal sites on protein at which covalent binding of these reactive metabolites of 3HAA takes place, a feature shared by the reactive species generated during acetaminophen metabolism.

Quantitative analysis of a synthetic peptide, NR58-3.14.3, in serum by LC-MS with inclusion of a diastereomer as internal standard.

A method for quantifying an intramolecularly linked all-d-amino acid peptide, NR58-3.14.3, in rat serum by LC-MS using selected ion monitoring with inclusion of a diastereomer as internal standard was developed. The reproducible quantitation of multiply charged compounds by LC-MS using single ion or selective reaction monitoring is often a challenge as the intensity ratio of the ions in a series of different charge states can vary. Good precision was obtained in the selected ion monitoring mode by integrating the summed ion currents of the singly, doubly, and triply charged molecular ions. Since stable isotope analogs are costly and integration of residual unlabeled material can be of concern, a diastereomer of NR58-3.14.3, NR58-3.14.5, was used as internal standard. The diastereomers were indistinguishable by electrospray MS, but fully separated by reversed-phase LC. Consequently, interference due to isotopic impurities or coelution was not encountered. The calibration plot was linear throughout a concentration range of 0.2 to 200.0 microg/ml (r(2) = 0.9996). Intraday precision of the standards analyzed was less than 12% RSD over the calibration range and the accuracy within +/-11% RE. Serum pharmacokinetics were in good agreement with the pharmacokinetic profiles of small, ionic, and polar molecules.

Trisomy 8. Report of a mosaic human male with near-normal phenotype and normal IQ, ascertained through infertility.

Trisomy 8, in mosaic or non-mosaic form is an extremely rare chromosomal condition in man. Liveborn subjects usually present with mental retardation, bone and joint anomalies and a variety of other physical anomalies. The mental retardation associated with the condition is, however, usually moderate compared to that found in other viable human autosomal trisomic conditions. The present report describes a trisomy 8 mosaic male subject with normal IQ and near-normal phenotype, ascertained through infertility. Chromosome studies on peripheral blood lymphocytes reveal a pure trisomy 8 constitution; cultured skin fibroblasts show 46,XY/47,XY+8 mosaicism. At meiosis, the extra No. 8 chromosome is missing from the germ line. The testicular histology indicates a germ cell maturation arrest in many spermatocytes and the patient is severely oligospermic. Biochemical studies to assay levels of glutathione reductase, a red cell enzyme, the gene for which resides in chromosome 8, show increased levels in the trisomy 8 patient compared with controls.