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Biomedical subjects

R C Ladner

Publications and source records attributed to R C Ladner.

At least 19 recordsLinked to original sources
PubMed2000

Inflammation and infection imaging with a 99mTc-neutrophil elastase inhibitor in monkeys.

UNLABELLED: A radiolabeled human neutrophil elastase inhibitor (EPI-HNE-2) may represent an improved nuclear medicine imaging agent for inflammation and infection. This peptide displays rapid pharmacokinetics due to its low molecular weight and localizes specifically on neutrophil elastase released in inflammatory sites by activated neutrophils. METHODS: In this investigation, the peptide was radiolabeled with 99mTc using N-hydroxysuccinimidyl S-acetylmercaptoacetyltriglycline (NHS-MAG3) as a bifunctional chelator and was administered on 18 occasions to 5 rhesus monkeys with inflammation/infection. RESULTS: Plasma clearance was rapid, with liver and kidneys representing the major organs of accumulation. No evidence of toxicity, dosage effects, or circulating antiMAG3-EPI-HNE-2 antibodies was observed. Specificity of localization was established using radiolabeled bovine pancreatic trypsin inhibitor (a non-hNE-binding peptide of similar size) as a nonspecific negative control peptide and by predosing with unlabeled EPI-HNE-2 to block receptor sites before the administration of radiolabeled EPI-HNE-2. The ability of radiolabeled EPI-HNE-2 to image inflammation/infection was evaluated in 12 studies in monkeys receiving only radiolabeled EPI-HNE-2 and with lesions in the arm, shoulder, or lower back. Positive images were obtained in all studies, uptake was apparent almost immediately, and images were still positive 24 h later. As a positive control, animals also received nonspecific IgG antibody radiolabeled with 99mTc either directly or by NHS-MAG3. Compared with labeled antibody, plasma clearance of 99mTc was faster with labeled EPI-HNE-2 and accumulation in liver and heart was lower. Uptake of radioactivity in the inflammation was higher during the first hour with EPI-HNE-2 versus antibody but lower thereafter. CONCLUSION: When radiolabeled with 99mTc, EPI-HNE-2 localized specifically in inflammations in a monkey model and provided early images of diagnostic quality.

Animals↗
PubMed1999

Gene transfer to mammalian cells using genetically targeted filamentous bacteriophage.

We have genetically modified filamentous bacteriophage to deliver genes to mammalian cells. In previous studies we showed that noncovalently attached fibroblast growth factor (FGF2) can target bacteriophage to COS-1 cells, resulting in receptor-mediated transduction with a reporter gene. Thus, bacteriophage, which normally lack tropism for mammalian cells, can be adapted for mammalian cell gene transfer. To determine the potential of using phage-mediated gene transfer as a novel display phage screening strategy, we transfected COS-1 cells with phage that were engineered to display FGF2 on their surface coat as a fusion to the minor coat protein, pIII. Immunoblot and ELISA analysis confirmed the presence of FGF2 on the phage coat. Significant transduction was obtained in COS-1 cells with the targeted FGF2-phage compared with the nontargeted parent phage. Specificity was demonstrated by successful inhibition of transduction in the presence of excess free FGF2. Having demonstrated mammalian cell transduction by phage displaying a known gene targeting ligand, it is now feasible to apply phage-mediated transduction as a screen for discovering novel ligands.

Animals↗
PubMed1999

Polypeptides from phage display. A superior source of in vivo imaging agents.

To decide whether experience teaches that small proteins and constrained peptides having high affinity for molecular targets can be engineered to have suitable pharmacokinetics for imaging. Phage display, a molecular diversity technology, allows selection of polypeptides having high affinity and specificity for almost any target. These polypeptides can be modified in ways that improve pharmacokinetics with acceptable impact on binding. Often, relatively few changes confers pharmacokinetics suitable for imaging on polypeptides selected for affinity and specificity to a target and for stability. It is likely that few variants of phage-selected proteins and constrained peptides will need to be tested to obtain a useful imaging agent.

Affinity Labels↗
PubMed1998

Labeling peptides with technetium-99m using a bifunctional chelator of a N-hydroxysuccinimide ester of mercaptoacetyltriglycine.

UNLABELLED: A modified mercaptoacetyltriglycine (MAG3) chelator, which has acetyl S-protection and which is derivitized with N-hydroxysuccinimide (NHS) ester for conjugation, has been used to radiolabel four small (approximately 6- to 7-kDa) peptides, bovine pancreatic trypsin inhibitor, epidermal growth factor, human neutrophil elastase inhibitor and plasmin inhibitor, with 99mTc. METHODS: Each peptide was specifically labeled at the MAG3 chelation sites at ambient temperature and neutral pH. Specific activities of 100-150 mCi/mg were achieved at labeling efficiencies of about 50%, but specific activities of 3500 mCi/micromol could be attained. RESULTS: By a variety of assays, protein activity was unimpaired by the conjugation and labeling for two of the four peptides. The activities for plasmin of the plasmin inhibitor and bovine pancreatic trypsin inhibitor were reduced by conjugation, presumably because of a sensitive lysine residue in the structure of each of these two peptides. Multiple peaks were present in the high-performance liquid chromatography radiochromatograms, especially of human neutrophil elastase inhibitor; however, most peaks could be shown to be labeled active peptide. Stability during cysteine challenge at modest cysteine-to-peptide molar ratios and during incubation in serum was observed in each case. Large differences among the labeled peptides were apparent in the 3-hr biodistributions of 99mTc in normal mice. CONCLUSION: The use of NHS-S-acetyl-MAG3 may be a convenient method of radiolabeling peptides with 99mTc.

Animals↗
PubMed1996

Iterative optimization of high-affinity proteases inhibitors using phage display. 1. Plasmin.

We generated a series of libraries having variants of the first Kunitz domain of human lipoprotein-associated coagulation inhibitor (LACI-D1, also known as tissue-factor pathway inhibitor-I) displayed on bacteriophage M13 as pIII-fusions. We varied LACI-DI iteratively in two regions: the P1 region (positions 10-21) and the "second loop", (positions 31-39), which together form one end of the domain. Display-phage library Lib#1 allows 31 200 amino-acid sequences in P1 region (residues 13, 16-19). Preliminary, we screened Lib#1 against human plasmin (PLA, EC 3.4.21.7) immobolized on agarose to enrich for phage displaying variants with PLA affinity. We introduced a 1600-fold increase in second-loop diversity (residues 31, 32, 34, 39) into the population of selectants from Lib#1, yielding Lib#2. Lib#2 (allowing approximately 50 million amino-acid sequences) was screened against PLA-agarose to isolate highest affinity binders. Protein EPI-P211, derived from the best isolate of Lib#2, inhibits PLA with Ki = 2 nM (at least 500-fold better than LACI-D1) and with high specificity. We used amino-acid sequences of PLA-binding selectants to design a PLA-biased library (Lib#3) which we screened against PLA. The protein EPI-P302 (derived from the best binder obtained from Lib#3) has Ki for PLA inhibition of 87 pM, which is 25-fold better than the first-round best binder and > or = 12 500-fold better than LACI-D1. EPI-P302 also shows very high specificity for PLA vs other human proteases and is resistant to inactivation by oxidants and extremes of temperature or pH. Thus, one can use selectants from one library to design target-tailored combinatorial libraries and obtain quite stable, highly specific, very high-affinity binding molecules while maintaining an essentially human framework.

Amino Acid Sequence↗
PubMed1996

Iterative optimization of high-affinity protease inhibitors using phage display. 2. Plasma kallikrein and thrombin.

As discussed in the accompanying paper [Markland, W., Ley, A. C., & Ladner, R. C. (1996) Biochemistry 35, 8045-8057], we generated libraries from the first Kunitz domain of human lipoprotein-associated coagulation inhibitor (LACI-D1) using multivalent M13 III display and derived potent inhibitors of human plasmin (PLA) by iterative variegation and selection. Here, we show that high-affinity, high-specificity binders to human plasma kallikrein (pKAL) and human thrombin (THBN) can be obtained starting from the identical library and employing the same iterative variegation procedures used to obtain PLA inhibitors. Lib#1 (allowing 31 200 variants involving five positions near the P1 residue of LACI-D1) and its pKAL-biased derivative, Lib#4 (allowing an additional 1600 variants at residues 31, 32, 34, and 39), were screened against pKAL, yielding potent inhibitors. One of these, EPI-K401, has Ki = 284 pM, very high specificity, and excellent stability. We used information from Lib#4 selectants to design Lib#5 (allowing 1.5 x 10(6) amino-acid sequences involving nine varied positions) from which we obtained an inhibitor (EPI-K503) having high affinity for pKAL (Ki = 40 pM) and retaining the high specificity of EPI-K401. When we screened Lib#1 and its THBN-tailored derivative, Lib#6, against THBN, we obtained a different and very homogeneous population of selected molecules. The purified proteins derived from Lib#6 selectants bound to THBN-agarose beads but did not inhibit proteolytic activity of THBN, suggesting that these selectants bind to a site on THBN other than the catalytic site. Thus, a single large combinatorial library can serve as a source to obtain highly specific, high-affinity binding molecules for each of several targets. Furthermore, the results with THBN show that the binding of Kunitz domains to other proteins is not limited to the catalytic sites of trypsin-homologous proteases.

Amino Acid Sequence↗
PubMed1996

Obtaining a family of high-affinity, high-specificity protein inhibitors of plasmin and plasma kallikrein.

Human lipoprotein-associated coagulation inhibitor (LACI) is a serum protein containing three Kunitz domains. We displayed the first domain (LACI-D1) on the III protein of phage M13 and made libraries of this domain. We iteratively varied 13 residues in the region corresponding to the BPTI-trypsin interface and selected for binding to human plasmin (PLA) and human plasma kallikrein (pKAL). For PLA, our first-round best binder, EPI-P211, had KD = 2 nM. Using information from the first selection, we made a PLA-biased library containing approximately 500,000 proteins and selected from these a protein, EPI-P302, having a KD for PLA of 87 pM. EPI-P302 inhibits pKAL with KD approximately 250 nM (approximately 2800-fold higher than for PLA) and KD values for other proteases are higher yet. From the same initial LACI-D1 library, we selected an inhibitor of pKAL, EPI-K401, with a KD for pKAL of 287 pM. We used information from this selection to construct a pKAL-biased library from which we selected EPI-K502, which has a KD for pKAL of 40 pM. EPI-K502 inhibits PLA with KD approximately 20 nM (500-fold higher than for pKAL); KD values for other proteases are much higher. For both targets and for both selections, there are families of proteins having a few differences and a range of affinities for their targets. These proteins are candidate drugs and imaging agents for indications involving excess PLA or pKAL. Structure-activity relationships of PLA and pKAL binders will allow design of small molecules that are specific for these targets.

Amino Acid Sequence↗
PubMed1996

Affinity maturation of proteins displayed on surface of M13 bacteriophage as major coat protein fusions.

This chapter described the preparation and fractionation of libraries of M13 phage displaying proteins as fusions to the major coat protein. High titer (10(13) pfu/ml) phage libraries can readily be generated using a single vector and the level of display surpasses that of gene III fusion phage. Since the synthetic VIII fusion gene can be customized, this system should provide the flexibility required to construct phage libraries displaying a variety of different peptides and proteins and to select variants possessing the highest affinity for target molecules of a diverse chemical nature.

Bacteriophage M13↗
PubMed1995

Constrained peptides as binding entities.

Displaying proteins and peptides on genetic packages and selecting packages that display high-affinity binders allows large numbers of peptidyl compounds to be tested for binding to targets. Phage-displayed libraries of unstructured peptides (UPs) have yielded binders for some targets, but not for many others. Therefore, more attention is being paid to peptidyl compounds that have varied regions that are subject to conformational constraint.

Amino Acid Sequence↗
PubMed1993

M13 bacteriophage displaying disulfide-constrained microproteins.

A display-phage library (TN2), displaying an 18-residue peptide fused to coat protein III, represents a collection of up to 8.55 x 10(6) peptides encoded by only 1.68 x 10(7) DNA sequences. Each displayed peptide has two fixed cysteine residues (allowing disulfide formation) and six variegated residues, four between the cysteines and one either side of the cysteines. Screening this library against streptavidin (Sv) and the anti-beta-endorphin monoclonal antibody, 3-E7, yielded phage displaying disulfide-constrained microproteins with sequences similar to those published for the linear-peptide display phage. Analysis of selected clones indicated that a disulfide bond is required for high-affinity binding to each of the target proteins. The microproteins selected for binding to Sv and 3-E7 show more stringent sequence specificity than do linear peptides selected for binding to the same targets.

Amino Acid Sequence↗
PubMed1992

Protease inhibitor display M13 phage: selection of high-affinity neutrophil elastase inhibitors.

We report display of the complete protease inhibitor (Kunitz) domain, BPTI, on the surface of bacteriophage M13 as a fusion to the gene III product. Phage that display BPTI bind specifically to anti-BPTI antibodies, trypsin and anhydrotrypsin. A point mutation of BPTI [Lys15-->Leu(K15L)] alters the binding specificity of fusion phage such that a human neutrophil elastase-binding phenotype is conferred while a trypsin-binding phenotype is eliminated. Phage were eluted from an immobilized protease with step gradients of decreasing pH. Phage that display Kunitz domains having higher affinity for the immobilized protease exhibit characteristic pH elution phenotypes, indicating that bound display phage can be selectively recovered from an affinity matrix. Utilization of this technology should enable the selection of remodeled protease inhibitors exhibiting novel binding specificities.

Amino Acid Sequence↗
PubMed1992

Directed evolution of a protein: selection of potent neutrophil elastase inhibitors displayed on M13 fusion phage.

Inhibitors of human neutrophil elastase were engineered by designing and producing a library of phage-displayed protease inhibitory domains derived from wild-type bovine pancreatic trypsin inhibitor and fractionating the library for binding to the target protease. The affinity of one of the engineered variants for human neutrophil elastase (Kd = 1.0 pM) is 3.6 x 10(6)-fold higher than that of the parental protein and exceeds the highest affinity reported for any reversible human neutrophil elastase inhibitor by 50-fold. Thus the display phage method has allowed us to obtain protein derivatives that exhibit greatly increased affinity for a predetermined target. The technology can be applied to design high-affinity proteins for a wide variety of target molecules.

Amino Acid Sequence↗
PubMed1991

Design, construction and function of a multicopy display vector using fusions to the major coat protein of bacteriophage M13.

Incorporation of numerous copies of a heterologous protein (bovine pancreatic trypsin inhibitor; BPTI) fused to the mature major coat protein (gene VIII product; VIII) of bacteriophage M13 has been demonstrated. Optimization of the promoter, signal peptide and host bacterial strain allowed for the construction of a working vector consisting of the M13 genome, into which was cloned a synthetic gene composed of a lac (or tac) promoter, and sequences encoding the bacterial alkaline phosphatase signal peptide, mature BPTI and the mature coat protein. Processing of the BPTI-VIII fusion protein and its incorporation into the bacteriophage were found to be maximal in a host bacterial strain containing a prlA/secY mutation. Functional protein is displayed on the surface of M13 phage, as judged by specific interactions with antiserum, anhydrotrypsin, and trypsin. Such display vectors can be used for epitope mapping, production of artificial vaccines and the screening of diverse libraries of proteins or peptides having affinity for a chosen ligand. The VIII display phage system has practical advantages over the III display phage system in that many more copies of the fusion protein can be displayed per phage particle and the presence of the VII fusion protein has little or no effect on the infectivity of the resulting bacteriophage.

Amino Acid Sequence↗
PubMed1987

Protein engineering of subtilisin BPN': enhanced stabilization through the introduction of two cysteines to form a disulfide bond.

Introduction of a disulfide bond by site-directed mutagenesis was found to enhance the stability of subtilisin BPN' (EC 3.4.21.14) under a variety of conditions. The location of the new disulfide bond was selected with the aid of a computer program, which scored various sites according to the amount of distortion that an introduced disulfide linkage would create in a 1.3-A X-ray model of native subtilisin BPN'. Of the several amino acid pairs identified by this program as suitable candidates, Thr-22 and Ser-87 were selected by using the additional requirement that the individual cysteine substitutions occur at positions that exhibit some degree of variability in related subtilisin amino acid sequences. A subtilisin variant containing cysteine residues at positions 22 and 87 was created by site-directed mutagenesis and was shown to have an activity essentially equivalent to that of the wild-type enzyme. Differential scanning calorimetry experiments demonstrated the variant protein to have a melting temperature 3.1 degrees C higher than that of the wild-type protein and 5.8 degrees C higher than that of the reduced form (-SH HS-) of the variant protein. Kinetic experiments performed under a variety of conditions, including 8 M urea, showed that the Cys-22/Cys-87 disulfide variant undergoes thermal inactivation at half the rate of that of the wild-type enzyme. The increased thermal stability of this disulfide variant is consistent with a decrease in entropy for the unfolded state relative to the unfolded state that contains no cross-link, as would be predicted from the statistical thermodynamics of polymers.

Anti-Bacterial Agents↗