Model-informed drug development approach supporting approval of the 4-week (Q4W) dosing schedule for nivolumab (Opdivo) across multiple indications: a regulatory perspective.

BACKGROUND: A nivolumab dosage regimen of 480 mg intravenously (i.v.) every 4 weeks (Q4W) was approved by FDA for the majority of the approved indications for nivolumab. METHODS: The proposed new dosage regimen was supported by pharmacokinetic modeling and simulation, dose/exposure-response relationships for efficacy and safety in the indicated patient populations, and the clinical safety data with the 480 mg Q4W dosage regimen. Pharmacokinetic exposures achieved with 480 mg Q4W were predicted for 4166 patients in 21 clinical studies with various types of solid and hematological tumors. Exposure-response analyses were conducted to predict 480 mg Q4W safety and efficacy across all FDA-approved indications for nivolumab. RESULTS: For the overall population, the geometric mean exposure achieved with 480 mg i.v. Q4W was 5.2% higher for steady state Cavg and 15.6% lower for Ctrough than those with 3 mg/kg i.v. Q2W, the approved dosage regimen. The simulated concentration-time course achieved with 480 mg Q4W regimen was below the median concentration achieved with 10 mg/kg i.v. Q2W that was also studied in clinical trials. The predicted probability of adverse events was similar between 480 mg Q4W and that observed with the 3 mg/kg Q2W regimen. Efficacy results were found to be similar between Q2W and Q3W dosage regimens in patients with renal cell carcinoma. The predicted efficacy for each indication suggested that the efficacy with 480 mg Q4W is unlikely to be compromised compared with that observed with 3 mg/kg Q2W. CONCLUSIONS: The model-informed analyses of predicted exposure, efficacy and safety based on data from extensive clinical experience with nivolumab suggest that the benefit-risk profile of 480 mg Q4W regimen is comparable to the approved 3 mg/kg Q2W regimen, thus providing the regulatory basis for the approval of 480 mg Q4W regimen in the absence of clinical efficacy data with this new dosage regimen.

An unusual rash for Royal: a case series.

Eight patients, whilst on exercise in Albania, presented with a blistering, erythematous and itchy rash, consistent with caustic burns, after living in dense vegetation for a few days. All patients were found to have been living and operating under fig trees and had come into contact with the sap of Ficus carica, which on exposure to ultraviolet A (UVA) radiation, can cause a process of phytophotodermatitis leading to a blistering rash.

Can adenocarcinoma in situ of the uterine cervix be treated safely by conisation in combination with endocervical curettage?

OBJECTIVE: To evaluate the prognostic value of endocervical curettage (ECC) after conisation in patients treated for adenocarcinoma in situ (AIS) of the uterine cervix. MATERIALS AND METHODS: Patients with AIS diagnosed between 1990 and 2010 and with a minimum of 1.5 years of follow-up were retrospectively identified using computerised clinical files. RESULTS: The authors identified 195 patients (median age 32 years) with a median follow-up of 6.4 years. ECC was performed in 165 patients. In 144 (87%) the initial ECC was normal. In 129 no recurrence was observed during follow-up (90%). A positive ECC was observed in 21. Thirteen patients had hysterectomies; six hysterectomies were normal. Eight patients treated conservatively developed no recurrent disease. Two patients with a positive ECC did not have a hysterectomy and developed recurrent disease. In patients with affected margins, 17% developed recurrent disease. CONCLUSION: ECC performed during initial conisation is a prognostic tool for the treatment ofAIS. Close follow-up is recommended in patients treated conservatively.

Mixed state discrimination using optimal control.

We present theory and experiment for the task of discriminating two nonorthogonal states, given multiple copies. We implement several local measurement schemes, on both pure states and states mixed by depolarizing noise. We find that schemes which are optimal (or have optimal scaling) without noise perform worse with noise than simply repeating the optimal single-copy measurement. Applying optimal control theory, we derive the globally optimal local measurement strategy, which outperforms all other local schemes, and experimentally implement it for various levels of noise.

Current Perspectives on Ligand-Binding Assay Practices in the Quantification of Circulating Therapeutic Proteins for Biosimilar Biological Product Development.

Bioanalysis in biosimilar biological product development (BPD) plays a critical role in demonstrating pharmacokinetic (PK) similarity across products. The 2018 FDA Bioanalytical Method Validation guidance for industry provides general principles in the development, validation, and conduct of bioanalytical assays. Given that the PK similarity assessment in BPD programs involves two or more non-identical products, there are additional considerations for bioanalytical methods. Here in, we provide our perspectives on the definition of (1) a single bioanalytical method in the context of BPD in supporting a PK similarity study, (2) bioanalytical method comparability during accuracy and precision experiments to determine the potential bias difference prior to assessing other validation parameters, and (3) bioanalytical method validations that support PK similarity assessments.

Vitamin D status regulates 25-hydroxyvitamin D3-1 alpha-hydroxylase and its responsiveness to parathyroid hormone in the chick.

We asked this question: Under normal or near-normal metabolic conditions, does the prevailing normal or near-normal vitamin D status dampen the activity of 25-hydroxyvitamin-D3-1 alpha-hydroxylase (1 alpha-hydroxylase) such that it determines not only its "basal" activity but also its responsiveness to stimulation by increased circulating concentrations of parathyroid hormone (PTH)? To answer this question, we measured the activity of 1 alpha-hydroxylase in chicks, with and without administration of PTH, immediately before and during deprivation of vitamin D. Before deprivation of vitamin D, 1 alpha-hydroxylase activity increased only slightly with administration of PTH. With deprivation of vitamin D for 5 and 10 d, while the plasma concentrations of calcium and phosphorus persisted normal and unchanged, 1 alpha-hydroxylase activity not only increased progressively but also became sharply and increasingly responsive to stimulation by administration of PTH. But after 15 d of vitamin D deprivation, and the supervention of hypocalcemia, 1 alpha-hydroxylase activity was not further increased by the administration of PTH. With deprivation of vitamin D, the progressive increase in 1 alpha-hydroxylase correlated inversely with circulating levels of 1,25-dihydroxyvitamin D (1,25-[OH]2D), and the decreasing calcemic response to PTH correlated inversely with the responsiveness of 1 alpha-hydroxylase to PTH (in chicks deprived of vitamin D for 1-10 d). These results demonstrate that: under normal metabolic conditions, the normal vitamin D status regulates the activity of 1 alpha-hydroxylase so as to dampen both its "basal" activity and its responsiveness to stimulation by PTH; and vitamin D deprivation insufficient to cause hypocalcemia enhances both the "basal" activity of 1 alpha-hydroxylase and its responsiveness to stimulation by PTH. The results suggest that the normal dampening of 1 alpha-hydroxylase and both of the demonstrated enhancements of its activity are mediated by normal and reduced levels of circulating 1,25-(OH)2D, respectively. The finding that PTH fails to further stimulate 1 alpha-hydroxylase when vitamin D deprivation is sufficient in duration to cause hypocalcemia confirms the findings of other investigators and again demonstrates that observations made during abnormal metabolic circumstances may bear little on the physiologic regulation of 1 alpha-hydroxylase under normal or near-normal metabolic circumstances.

Parathyroidectomy reduces 25-hydroxyvitamin D3-1 alpha-hydroxylase activity in the hypocalcemic vitamin D-deficient chick.

To test the hypothesis that in the vitamin D-deficient state the activity of 25-hydroxyvitamin D3-1 alpha-hydroxylase (25-OHD3-1 alpha-hydroxylase) is modulated by parathyroid hormone and the plasma concentration of phosphate only in the presence of small amounts of 1,25-dihydroxyvitamin D3 (or some other metabolite of vitamin D), we measured the activity of this enzyme 24 h after parathyroidectomy (PTX) in frankly hypocalcemic, vitamin D-deficient chicks that were not supplemented with vitamin D or one of its metabolites. The otherwise predictable complications of PTX in this metabolic setting (hypocalcemia of increasing severity, tetany, moribundity, and death) were prevented by continuous intravenous administration of calcium (as a solution of calcium chloride/calcium gluconate 1:1) through a catheter in the external jugular vein placed at the time of PTX. The findings were as follows: (a) The activity of 25-OHD3-1 alpha-hydroxylase was significantly less in the parathyroidectomized group than in the sham-operated control chicks (P less than 0.001). (b) The reductive effect of PTX on the activity of this enzyme was significantly attenuated when hypophosphatemia was increased in severity by administration of glucose. (c) In the post-PTX state the activity of 25-OHD3-1 alpha-hydroxylase and plasma concentration of phosphate were significantly, inversely related (P less than 0.001). (d) In the sham-operated control group the activity of this enzyme and the plasma concentration of phosphate were not significantly correlated. These findings indicate that in the vitamin D-deficient state, both circulating parathyroid hormone and the plasma concentration of phosphate can significantly modulate the activity of 25-OHD3-1 alpha-hydroxylase in the absence of vitamin D or its metabolites. The findings also suggest that in the vitamin D-deficient state the plasma concentration of phosphate modulates the activity of this enzyme only when the concentration of circulating parathyroid hormone is not increased.

Effect of dietary phosphorus on circulating concentrations of 1,25-dihydroxyvitamin D and immunoreactive parathyroid hormone in children with moderate renal insufficiency.

The hyperparathyroidism characteristic of patients with moderate renal insufficiency could be caused by decreases in the plasma concentration of ionized calcium (Ca++) evoked by: (a) recurring increases in the plasma concentration of inorganic phosphorus that may be detectable only in the post-prandial period; (b) a reversible, phosphorus-mediated suppression of renal 25-hydroxyvitamin D-1 alpha-hydroxylase that decreases the plasma concentration of 1,25-dihydroxyvitamin D (1,25-(OH)2D) enough to decrease both gut absorption and bone resorption of Ca++; (c) both of these. In a group of eight children with moderate renal insufficiency, mean glomerular filtration rate (GFR) 45 +/- 4 (SE) ml/min per 1.73 M2, ages 6-17 yr, we tested these hypotheses by determining the effect of short term (5 d) restriction and supplementation of dietary intake of phosphorus on the plasma concentration of 1,25-(OH)2D, the serum concentrations of immunoreactive parathyroid hormone (iPTH) and phosphorus, and the fractional renal excretion of phosphorus ( FEPi ). When dietary phosphorus was normal, 1.2 g/d, the serum concentrations of phosphorus throughout the day were not greater than those of normal control children, and the serum concentrations of carboxyl-terminal iPTH (C-iPTH) were greater, 59 +/- 9 vs. 17 +/- 3 mu leq/ml, and unchanging; the serum concentration of intact-iPTH was also greater, 198 +/- 14 vs. 119 +/- 8 pg/ml. The plasma concentration of 1,25-(OH)2D was lower than that of age-matched controls, 27 +/- 3 vs. 36 +/- 2 pg/ml (P less than 0.01). When dietary phosphorus was restricted to 0.35 g/d, the plasma concentration of 1,25-(OH)2D increased by 60% to a mean value not different from that of normal controls, while serum concentrations of C-iPTH and intact-iPTH decreased by 25%, the latter concentration to a mean value not different from that of controls. FEPi decreased from 31 to 9%. When dietary phosphorus was supplemented to 2.4 g/d, the plasma concentration of 1,25-(OH)2D decreased 32%, while those of C-iPTH and intact-iPTH increased by 131 and 45%, respectively; FEPi increased from 27 to 53%. Plasma concentrations of 25-hydroxyvitamin D remained normal and unchanged, and GFR did not change when dietary phosphorus was manipulated. The data demonstrate that in children with moderate renal insufficiency: (a) A normal dietary intake of phosphorus in attended by a decreased circulating concentration of 1,25-(OH)2D and an increased concentration of iPTH, but not by recurring increases in the serum concentration of phosphorus at any time of the day; (b) Dietary phosphorus is, however, a major determinant of the circulating concentrations of both 1,25-(OH)2D and iPTH, which vary inversely and directly, respectively, with dietary intake of phosphorus, and increase and decrease, respectively, to normal values when phosphorus is restricted for 5 d; (c) Restriction and supplementation of dietary phosphorus induces changes in the serum concentration of iPTH that correlate strongly but inversely with those induced in the plasma concentration of 1,25-(OH)2D (r = -0.88, P < 0.001); and (d) The physiologic responsiveness of the renal tubule to changes in dietary phosphorus is to a substantial extent intact. The data provide support for the second hypothesis stated.

Toward greater insights on pharmacokinetics and exposure-response relationships for therapeutic biologics in oncology drug development.

There has been increased interest in optimizing dosing regimens for oncology products over the past decade. Investigations to refine dosing regimens often occur after new drug approval. There is growing focus on the use of exposure-response (ER) approaches to identify optimal dosing regimens for therapeutic biologics. Herein, we describe several recent observations that have informed our thinking on the use of ER analyses in the dose regimen optimization of therapeutic biologics developed to treat cancer.

Association of time-varying clearance of nivolumab with disease dynamics and its implications on exposure response analysis.

Nivolumab is a human monoclonal antibody that blocks the interaction between PD-1 programmed death-1 (PD-1) and its ligands, PD-L1 and PD-L2. Nivolumab demonstrated efficacy in clinical trials for various types of cancer. A time-varying clearance was identified for nivolumab. We show that the change of clearance over time is associated with the post-treatment effects: clearance decreases when disease status improves. This interaction between posttreatment effects and drug exposure may lead to a biased steep estimate of the exposure-response (E-R) relationship for efficacy. Under this scenario, simulations were performed to develop a proposed methodology to assess the causal effect of drug exposure upon clinical response. Data from nivolumab trials were subsequently used to verify the proposed methodology for E-R analysis. The results showed that E-R analysis results based on pharmacokinetic (PK) metrics derived from the first dose are more consistent with the true E-R or dose-response relationship than the steady-state PK metrics.

Genetically engineered tetravalent single-chain Fv of the pancarcinoma monoclonal antibody CC49: improved biodistribution and potential for therapeutic application.

Failure of radiolabeled monoclonal antibodies (MAbs) in the treatment of solid tumors, for the most part, is a result of undesirable pharmacokinetics that lead to significant radiation exposure of normal tissues and an inadequate delivery of radiation doses to tumors. Using genetic engineering, antitumor MAbs can be optimized for desirable clinical applications. In the present study, we report the generation of a tetravalent single-chain Fv [[sc(Fv)2]2] of the murine MAb CC49 that recognizes the tumor-associated glycoprotein, TAG-72. [Sc(Fv)2]2 was expressed as a secreted soluble protein in Pichia pastoris under the regulation of alcohol oxidase 1 promoter. The in vitro binding properties of the tetravalent construct were analyzed by solid-phase RIA and surface plasmon resonance studies using BIAcore. The binding affinity constant (K(A)) for the [sc(Fv)2]2 and CC49 IgG were similar, i.e., 1.02 x 10(8) M(-1) and 1.14 x 10(8) M(-1), respectively, and were 4-fold higher than its divalent scFv [sc(Fv)2; 2.75 x 10(7) M(-1)]. At 6 h postadministration, the percentage of injected dose accumulated/g of LS-174T colon carcinoma xenografts was 21.3+/-1.3, 9.8+/-1.3, and 17.3+/-1.1 for radioiodinated [sc(Fv)2]2, sc(Fv)2, and IgG, respectively. Pharmacokinetic analysis of blood clearance studies showed the elimination half-life for [sc(Fv)2]2, sc(Fv)2, and IgG as 170, 80, and 330 min, respectively. The gain in avidity resulting from multivalency along with an improved biological half-life makes the tetravalent construct an important reagent for cancer therapy and diagnosis in MAb-based radiopharmaceuticals.

Collagen biosynthesis by human skin fibroblasts. III. The effects of ascorbic acid on procollagen production and prolyl hydroxylase activity.

Human skin fibroblasts were cultured under conditions optimized for collagen synthesis, and the effects of ascorbic acid on procollagen production, proline hydroxylation and the activity of prolyl hydroxylase were examined in cultures. the results indicated that addition of ascorbic acid to confluent monolayer cultures of adult human skin fibroblasts markedly increased the amount of [3H]hydroxyproline synthesized. Ascorbic acid, however, did not increase the synthesis of 3H-labeled collagenous polypeptides assayed independently of hydroxylation of proline residues, nor did it affect the amount of prolyl hydroxylase detectable by an in vitro enzyme assay. Also long-term cultures of the cells or initiation of fibroblast cultures in the presence of ascorbic acid did not lead to an apparent selection of a cell population which might be abnormally responsive to ascorbic acid. Thus, ascorbic acid appears to have one primary action on the synthesis of procollagen by cultured human skin fibroblasts: it is necessary for synthesis of hydroxyproline, and consequently for proper triple helix formation and secretion of procollagen.

Collagen biosynthesis by human skin fibroblasts. II. Isolation and further characterization of type I and type III procollagens synthesized in culture.

Human skin fibroblasts in culture have previously been shown to synthesize genetically distinct procollagens type I and type III. In the present study, cultured human skin fibroblasts were incubated under conditions optimized for synthesis of these procollagens in medium containing [3H]proline. The newly synthesized type I and type III 3H-labeled procollagens in the culture medium were then isolated as native proteins by DEAE-cellulose chromatography, or by gel filtration and SDS-polyacrylamide slab gel electrophoresis under denaturing conditons after limited pepsin proteolysis. The chromatographic procedures were optimized to yield reliable and reporducible results with good recoveries. The isolated procollagens were identified by cyanogen bromide peptide mapping and characterized by cleavage with highly purified collagenase synthesized by human skin fibroblasts. Assay of the relative synthesis of type I/III procollagens by normal human skin fibroblasts using DEAE-cellulose chromatography indicated that 80% of the procollagen in the medium was type I while the remaining 20% consisted of type III. When the ratio of newly-synthesized type I/III collagens was estimated by gel filtration or using SDS-polyacrylamide slab gel electrophoresis after limited pepsin proteolysis, relatively fewer type III collagen alpha-chains were recovered. This observation suggests that some of the type of the type III collagen molecules are in a conformation which is less resistant to digestion by pepsin than the triple-helix of type I procollagen. The coefficient of variation for the relative synthesis of type I and type III procollagens by control cultures was relatively small (16%), indicating that the phenotypic expression of type I and type III procollagen genes, under optimized culture conditions, is under a relatively tight control. The results further suggest that the optimized methodology developed for assay of the relative synthesis of type I and type III procollagens and collagens by cultured human skin fibroblasts can be utilized in studies on collagen aberrations in acquired and inherited diseases of connective tissue.

Collagen biosynthesis by human skin fibroblasts. I. Optimization of the culture conditions for synthesis of type I and type III procollagens.

Skin fibroblasts in culture can provide a convenient means to study aberrations of collagen metabolism in a variety of clinical conditions. In the present study, the culture conditions for the synthesis of procollagen by cultured human skin fibroblasts were optimized by independently varying parameters in the cell culture environment. To study the synthesis of procollagen the cell cultures were labeled with [3H]proline and the collagenous polypeptides were determined either by measuring the synthesis of hydroxy[3H]proline or by assaying the 3H-labeled proteins digested into dialyzable 3H-labeled peptides by bacterial collagenase. On the basis of the experimental results, the following culture conditions are suggested for optimal synthesis of procollagen: (a) cell culture medium should be supplemented with ascorbic acid (25--50 micrograms/ml) and fetal calf serum (20%); (b) the pH of the culture medium should be kept above 7.2 and preferably in the pH range 7.5--7.8; (c) the cell cultures should be used one to two days after reaching visual confluency. Under these conditions the synthesis and secretion of [3H]procollagen was found to be linear through a 24 h incubation period, and procollagen was demonstrated to be a major gene product of the fibroblasts. The relative synthesis of type I and type III procollagens was also monitored by isolating these genetically distinct procollagens by DEAE-cellulose chromatography or by measuring type I and III collagens by sodium dodecyl sulfate polyacrylamide gel electrophoresis after limited pepsin proteolysis. No marked changes were observed in type I/III procollagen ratios in situations where the total formation of hydroxy[3H]proline was significantly affected. The average coefficient of variance for procollagen synthesis between replicate cultures was found to be relatively small (14%), and the optimization of the culture conditions for the control cells has, therefore, created a reliable and reproducible basis for employing human skin fibroblasts to study collagen metabolism in acquired and inherited diseases.

Relative position of the hexahistidine tag effects binding properties of a tumor-associated single-chain Fv construct.

Hexahistidine tag (His-tag) is the most widely used tag for affinity purification of recombinant proteins for their structural and functional analysis. In the present study, single chain Fv (scFv) constructs were engineered form the monoclonal antibody (MAb) CC49 which is among the most extensively studied MAb for cancer therapy. For achieving efficient purification of scFvs by immobilized metal-ion affinity chromatography (IMAC), a His-tag was placed either at the C-terminal (scFv-His6) or N-terminal (His6-scFv) of the coding sequence. Solid-phase radioimmunoassay for scFv-His6 showed only 20-25% binding whereas both His6-scFv and scFv (no His-tag) showed 60-65% binding. Surface plasmon resonance studies by BIAcore revealed the binding affinity constant (KA) for His6-scFv and scFv as 1.19 x 10(6) M(-1) and 3.27 x 10(6) M(-1), respectively. No K(A) value could be calculated for scFv-His6 due to poor binding kinetics (kon and koff). Comparative homology modeling for scFv and scFv-His6 showed that the C-terminal position of the His-tag partially covered the antigen-binding site of the protein. The study demonstrates that the C-terminal position of His-tag on the CC49 scFv adversely affects the binding properties of the construct. The results emphasize the importance of functional characterization of recombinant proteins expressed with purification tags.

Radioimmunotherapy of human colon cancer xenografts using a dimeric single-chain Fv antibody construct.

Progress in the use of monoclonal antibodies (MAbs) for the treatment of solid tumors is limited by a number of factors, including poor penetration of the labeled IgG molecule into the tumors, their inability to reach the tumor in sufficient quantities without significant normal tissue toxicity, and the development of a human antimouse antibody response to the injected MAb. One possible way to alter the pharmacology of antibodies is via the use of smaller molecular weight antibody fragments called single-chain Fvs (scFvs). A divalent construct of MAb CC49, CC49 (scFv)2, composed of two noncovalently associated scFvs, was generated and shown to bind a tumor-associated antigen (TAG-72) epitope with a similar binding affinity to that of the murine IgG. The therapeutic potential of this construct after labeling with 131I was examined in athymic mice bearing established s.c. human colon carcinoma (LS-174T) xenografts. Treatment groups (n = 10) received a single dose of 131I-labeled CC49 (scFv)2 (500-2000 microCi) or 131I-labeled CC49 IgG (250 and 500 microCi). The group of mice treated with the lowest dose of 131I-(scFv)2 (500 microCi) showed statistically significant prolonged survival, compared with controls (P = 0.036). Complete tumor regression was observed in 20% of mice given 1500 microCi of labeled (scFv)2 and 30 and 60% of mice treated with 250 and 500 microCi of labeled IgG, respectively. In conclusion, the CC49 (scFv)2 construct provides a promising delivery vehicle for therapeutic applications.

Single-Dose versus fractionated radioimmunotherapy of human colon carcinoma xenografts using 131I-labeled multivalent CC49 single-chain fvs.

The prospects of radiolabeled antibodies in cancer detection and therapy remain promising. However, efforts to achieve cures, especially of solid tumors, with the systemic administration of radiolabeled monoclonal antibodies (MAbs) have met with limited success. Using genetic engineering techniques, MAbs have been tailored to improve the therapeutic index (tumor:normal tissue ratio) in clinical radioimmunotherapy. In the present study, we investigated the potential of tetravalent ([sc(Fv)2]2) and divalent [sc(Fv)2] single chain Fvs of MAb CC49 for therapy in athymic mice bearing s.c. LS-174T human colon carcinoma xenografts. Mice received 1,000 microCi of 131I-labeled [sc(Fv)2]2 or 131I-labeled sc(Fv)2, either as a single injection on day 6 or as four injections (250 microCi each) on days 6, 7, 8, and 9; the day of tumor implantation was taken as day 0. The median survival for the control group was 26 days. Comparisons of single and fractionated therapeutic regimens showed median survival as 32 (P < 0.001) and 53 days (P < 0.0001), respectively for [sc(Fv)2]2 and 26 (P > 0.5) and 38 days (P < 0.0001), respectively for sc(Fv)2 when compared with the control groups. The time for the quadrupling of tumor volume for single and fractionated therapeutic treatments were: 9.0 +/- 0.8 and 21.1 +/- 2.9 days respectively for sc(Fv)2; 16.6 +/- 1.9 and 32.9 +/- 2.7 days respectively for [sc(Fv)2]2; and 8.3 +/- 0.7 and 8.4 +/- 0.6 days respectively for the control group. No 131I-labeled systemic toxicity was observed in any treatment groups. The results show that radioimmunotherapy delivery for sc(Fv)2 and [sc(Fv)2]2 in a fractionated schedule clearly presented a therapeutic advantage over single administration. The treatment group receiving tetravalent scFv showed a statistically significant prolonged survival with both single and fractionated administrations suggesting a promising prospect of this reagent for cancer therapy and diagnosis in MAb-based radiopharmaceuticals.

Endothelial cells express insulin-like growth factor-binding proteins 2 to 6.

Cultured endothelial cells have been shown to produce insulin-like growth factor-binding proteins (IGFBPs); however, the identity of these BPs has not been defined. We now demonstrate that cultured bovine endothelial cells produce IGFBP2, IGFBP3, and IGFBP4 and have mRNA specific for IGFBP2, -3, -4, -5 and -6. DNA probes for bovine IGFBP2-6 were obtained by polymerase chain reaction (PCR) amplification of cDNA from bovine large vessel pulmonary artery and aortic endothelial cells as well as omental and periaortic fat microvessel cells, using oligonucleotide primers whose sequences were based on the reported cDNA sequences of IGFBP2-6. The PCR-derived probes were labeled with 32P and used for Northern blot analysis of RNAs obtained from the four bovine endothelial cell types. Transcripts corresponding to IGFBP2-6 were found in RNA from large vessel endothelial cells (bovine pulmonary artery and bovine aorta) and microvessel cells (periaortic and omental fat). The PCR-derived probe for IGFBP4 was used to screen a bovine pulmonary artery cDNA library for a full-length bovine IGFBP4 cDNA clone. One positive clone, containing a single EcoRI insert of approximately 2.0 kilobases, was selected for further characterization by DNA sequence analysis. This clone contained an open reading frame encoding a 258-amino acid protein that was 97% identical to human IGFBP4, 268 basepairs of 5'-untranslated region, and a longer 1044 basepairs of 3'-untranslated region. IGFBP4 protein was purified from bovine pulmonary artery-conditioned medium, shown to have N-terminal amino acid sequence DEAIHCPPCSEEKLARCR (identical to human IGFBP4) and to be secreted in glycosylated and nonglycosylated forms. Immunoblots further demonstrated that microvessel cells, at early passage, secrete predominantly IGFBP2 and IGFBP3, while large vessel cells, at early and late passages, secrete IGFBP3 and IGFBP4. Thus, cultured bovine endothelial cells synthesize and secrete IGFBP2, IGFBP3, and IGFBP4 and have mRNA encoding IGFBP2-6. The production of specific IGFBPs by endothelial cells raises the interesting possibility that the vascular endothelium contributes to circulating and tissue levels of specific IGFBPs in vivo.