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P Carter

Publications and source records attributed to P Carter.

At least 91 records · Page 5Linked to original sources
PubMed1994

Generation of soluble interleukin-1 receptor from an immunoadhesin by specific cleavage.

The extracellular portion of the interleukin-1 receptor (IL-1R) is sufficient for high-affinity binding to IL-1; however, the structural basis for binding of the receptor to IL-1 is not known. To produce individual domains of IL-1 receptor for structural studies, we constructed a molecular fusion of IL-1 receptor with immunoglobulin G heavy chain that contains a protease specific sequence joining the two portions of the molecule (IL-1R-G-IgG). We introduced the hexapeptide sequence AAHY:TL (where ":" denotes the scissile bond) at the junction of the IL-1R and IgG regions, for specific cleavage by an H64A variant of subtilisin BPN' (Genenase I), an endoprotease that cleaves selectively at this sequence (Carter et al., (1989) Proteins 6, 240-248). Plasmid DNA encoding the fusion protein was used to transfect human embryonic kidney 293 cells transiently, and secreted IL-1R-G-IgG was purified from cell supernatants by protein A chromatography. The IL-1 receptor's extracellular region was then generated by enzymatic cleavage with Genenase I which was immobilized on controlled-pore glass. Incubation of IL-1R-G-IgG with immobilized Genenase I resulted in specific cleavage at the target site, as confirmed by SDS-PAGE, immunoblotting and direct sequencing of the newly generated termini. The resulting soluble IL-1R was separated from the immunoglobulin Fc cleavage product by re-chromatography on protein A. The purified, soluble IL-1R retained quantitatively the ability to bind to its ligand, IL-1 beta. This approach offers a generic means by which the extracellular region of a given type I transmembrane receptor can be expressed as an immunoadhesin, released enzymatically and then easily purified for crystallographic or ligand binding studies.

Amino Acid Sequence↗
PubMed1994

A three month double-blind study of doxazosin as treatment for benign prostatic bladder outlet obstruction.

OBJECTIVE: To evaluate the efficacy and tolerability of doxazosin in the treatment of bladder outflow obstruction resulting from benign prostatic hyperplasia (BPH). PATIENTS AND METHODS: One-hundred and thirty-five patients with symptomatic urodynamically confirmed obstructive BPH were treated for 12 weeks with either doxazosin (67 patients) or placebo (68 patients) after an initial 2 week baseline evaluation. The main outcome measures were urodynamic and symptomatic evaluation for efficacy. Blood pressure and adverse events were monitored. RESULTS: Data were obtained in 122 patients (60 doxazosin, 62 placebo). Doxazosin produced increases in both mean and maximum urinary flow rates of 1.01 ml/s and 3.2 ml/s respectively, compared with 0.21 ml/s and 2.2 ml/s on placebo. The increase in mean flow rate was statistically significant (P = 0.04), while that for maximum flow rate approached significance (P = 0.09). The maximum subtracted voiding pressure was substantially reduced (P = 0.007) and 19 of 53 (36%) patients had an increase in maximum flow rate of 50% or more compared with 9 of 54 (17%) on placebo (P = 0.024). Twelve weeks' therapy with doxazosin resulted in significant improvements (compared with placebo) in: hesitancy (doxazosin 26 of 46, placebo 11 of 43; P = 0.003), impaired urinary stream (doxazosin 31 of 55, placebo 16 of 48; P = 0.019) nocturia (doxazosin 22 of 56, placebo 10 of 54; P = 0.017) and urgency (doxazosin 27 of 45, placebo 16 of 42; P = 0.041). Frequency improved with doxazosin therapy (doxazosin 26 of 59, placebo 15 of 55; P = 0.062). Adverse events, most frequently dizziness and headache, were usually mild and transient and led to a discontinuation of doxazosin therapy in one patient. No clinically significant changes in sexual function or blood pressure were seen. CONCLUSION: Doxazosin was well-tolerated and produced both urodynamic and symptomatic improvement in men with BPH, thereby providing a satisfactory alternative to existing drugs with the additional benefit of once daily dosage.

Aged↗
PubMed1994

A comparison between cytology and histology to detect anal intraepithelial neoplasia.

INTRODUCTION: Anal intraepithelial neoplasia (AIN), which may be a precursor of anal carcinoma, has been identified on histology following minor anal surgical procedures, in particular the removal of perianal condylomata, in increasing numbers of homosexual and bisexual men. Anal cytology has recently been proposed as a useful method of identifying AIN lesions. OBJECTIVE: To compare anal cytology with histology as a method of detecting AIN. METHODS: 215 homosexual and bisexual men attending a central London sexually transmitted diseases clinic had an anal cytological smear performed under standard conditions. The perianal area and anal canal were then examined using a colposcope, and areas macroscopically suggestive of intraepithelial neoplasia were biopsied. RESULTS: 176 of the 215 patients were biopsied of whom 76 had AIN on histology. 154 of the 215 patients had an adequate anal smear of whom 46 and 85 had cytological features of both HPV and AIN, or HPV alone respectively. Including features of HPV alone as an abnormal smear, anal cytology, when compared with anoscopy and histology as the gold standard for diagnosing AIN, resulted in a sensitivity of 87.5%, a specificity of 16.3%, a positive predictive value of 37.4% and a negative predictive value of 69.6%. Restricting abnormal smears to those with features of both HPV and AIN resulted in a sensitivity of 33.9%, a specificity of 72.5%, a positive predictive value of 41.3% and a negative predictive value of 65.7%. CONCLUSION: Anal cytology is a sensitive but nonspecific method of identifying patients with biopsy proven AIN if cytological features of HPV alone are included as abnormal smears. Specificity is improved by restricting abnormal smears to those with features of both HPV and AIN but this markedly lowers the sensitivity of the test. At present, anoscopy and histology are required in addition to anal cytology to differentiate between patients who simply have anal condylomata and those who also have AIN.

Anal Canal↗
PubMed1993

Engineering Fab' fragments for efficient F(ab)2 formation in Escherichia coli and for improved in vivo stability.

We previously developed an efficient route to humanized F(ab')2 fragments by high level secretion of the Fab' arms from Escherichia coli followed by directed chemical coupling in vitro. Here the number and type of interchain linkages in F(ab')2 molecules has been modified to simplify their production and improve their serum stability. All F(ab')2 variants had comparable binding affinity for the p185HER2 Ag and antiproliferative activity against p185HER2-overexpressing tumor cells. This was anticipated since the modifications are distant from the Ag-binding loops. Replacement of a single disulfide bridge between Fab' arms with a more stable thioether bridge increased the serum permanence time in normal mice by threefold to 2.1 h. Removal of the disulfide bond between L and H chains in the thioether-bridged F(ab')2 did not affect the pharmacokinetics, suggesting that the L chain remains associated with the H chain. An additional Fab' variant containing three repeats of the motif, CysProPro, was constructed with the aim of promoting efficient formation of F(ab')2 in E. coli. This Fab' (CPP)3 variant was recovered predominantly (up to 70%) as F(ab')2 directly from fermentation cell pastes, thus circumventing the need for in vitro coupling. The F(ab')2 (CPP)3 variant has a similar serum pharmacokinetics to the thioether-bridged molecules. The improvements described here for deriving F(ab')2 fragments from E. coli should enhance the clinical potential of these molecules.

Amino Acid Sequence↗
PubMed1993

X-ray structures of the antigen-binding domains from three variants of humanized anti-p185HER2 antibody 4D5 and comparison with molecular modeling.

The X-ray structures of 1 Fv and 2 Fab humanized anti-p185HER2 antibody fragments (IgG1-kappa) have been determined at a resolution between 2.7 A and 2.2 A. The antibodies are three different versions of a human antibody framework onto which the antigen recognition loops from a murine antibody (4D5) have been grafted. The sequences of the three versions differ in the framework region at positions L55, H78 and H102. The version 8 Fv fragment crystallizes in space group P2(1) with cell parameters a = 37.6 A, b = 63.4 A, c = 90.2 A, beta = 98.2 degrees, with two molecules per asymmetric unit, and has been refined against data 10.0 A-2.2 A to an R-factor of 18.3%. Versions 4 and 7 Fabs crystallize in space group P1 with cell parameters a = 39.2 A, b = 80.2 A, c = 86.1 A, alpha = 113.1 degrees, beta = 92.7 A, gamma = 102.6 A and two molecules per asymmetric unit. Version 4 has been refined against data 10.0 A-2.5 A resolution to an R-factor of 17.9%. Version 7 has been refined against data 10 A-2.7 A to an R-factor of 17.1%. The X-ray structures have been used to assess the accuracy of structural predictions made via molecular modeling, and they confirm the structural role of certain framework residues and the conformations of five of six complementarity determining regions (CDRS). The average deviation of the model from the X-ray structures is within the range observed among the X-ray structures for 81% of the C alpha atoms. Of the hydrogen bonds common to the X-ray structures, 94% of the main-chain-main-chain and 79% of the main-chain-side-chain ones were predicted by the model. The side-chain conformation was predicted correctly for 79% of the buried residues. The third CDR in the heavy chain is variable, differing by up to 8 A between molecules within an asymmetric unit. The structural relationship between variable domains of light and heavy chains is not significantly altered by the absence of constant domains in the Fv molecule. The antigen-binding potential of an unusual light chain sequence has been confirmed. The arginine at position 66 interacts with the first light chain CDR, but in a fashion somewhat different than predicted. A substitution of a leucine for an alanine side-chain directed between the beta-sheets has only relatively small and local effects.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1993

Cysteine pairing in the glycoprotein IIbIIIa antagonist kistrin using NMR, chemical analysis, and structure calculations.

The pairing of the cysteines in disulfide bonds was investigated for the 68-residue RGD-containing protein kistrin, a potent antagonist of the integrin GP IIbIIIa and an inhibitor of platelet aggregation. Kistrin belongs to a family of homologous proteins found in snake venoms termed disintegrins, all of which have a cysteine content. The disulfide pairing of the 12 cysteines was investigated by chemical analysis, NMR spectroscopy, and distance geometry calculations. The data show that the disulfide pairs are 4-19, 6-14, 13-36, 27-33, 32-57, and 45-64. The various means for assigning the disulfide bonds are described, and the results are compared with the cysteine pairings reported for other disintegrin proteins.

Amino Acid Sequence↗
PubMed1993

Binding interactions of kistrin with platelet glycoprotein IIb-IIIa: analysis by site-directed mutagenesis.

The binding interactions between platelet fibrinogen receptor, glycoprotein (GP) IIb-IIIa, and kistrin, a snake venom disintegrin protein that contains the adhesion site recognition sequence Arg-Gly-Asp (RGD) and potently inhibits platelet aggregation, have been investigated by site-directed mutagenesis of a synthetic kistrin gene. Kistrin was expressed as a fusion protein in Escherichia coli under control of the alkaline phosphatase promoter. This construction included the stII signal sequence to direct secretion to the periplasmic space and one synthetic (Z) domain of Staphylococcal protein A to allow affinity purification using IgG Sepharose. Kistrin was cleaved from the Z-domain by site-specific proteolysis using a mutant subtilisin BPN' and purified by reverse-phase HPLC. This approach facilitated the rapid purification of a set of 43 alanine replacement mutants whose relative affinity for GP IIb-IIIa was measured by competition with immobilized kistrin and by inhibition of platelet aggregation in human platelet-rich plasma. Alanine replacements at R49, G50, and D51 led to weaker inhibitors of platelet aggregation by 90-fold, 2-fold, and > 200-fold, respectively. The conservative D51E mutant was still > 100-fold less potent whereas R49K had a minor effect (1.8-fold), implying the critical nature of the aspartate for high affinity binding. However, mutations outside of the RGD region led to proteins indistinguishable from kistrin, suggesting no substantial secondary binding interactions. Furthermore, reduced kistrin is not active. We therefore propose that a favorable conformation of the RGD region alone is responsible for the high affinity binding of kistrin to GP IIb-IIIa.

Amino Acid Sequence↗
PubMed1993

Differential responses of human tumor cell lines to anti-p185HER2 monoclonal antibodies.

The HER2 protooncogene encodes a receptor tyrosine kinase, p185HER2. The overexpression of p185HER2 has been associated with a worsened prognosis in certain human cancers. In the present work we have screened a variety of different tumor cell lines for p185HER2 expression using both enzyme-linked immunosorbent and fluorescence-activated cell sorting assays employing murine monoclonal antibodies directed against the extracellular domain of the receptor. Increased levels of p185HER2 were found in breast (5/9), ovarian (1/6), stomach (2/3) and colorectal (5/16) carcinomas, whereas all kidney and submaxillary adenocarcinoma cell lines tested were negative. Some monoclonal antibodies directed against the extracellular domain of p185HER2 inhibited growth in monolayer culture of breast and ovarian tumor cell lines overexpressing p185HER2, but had no effect on the growth of colon or gastric adenocarcinomas expressing increased levels of this receptor. The most potent growth-inhibitory anti-p185HER2 monoclonal antibody in monolayer culture, designated mumAb 4D5 (a murine IgG1 kappa antibody), was also tested in soft-agar growth assays for activity against p185HER2-overexpressing tumor cell lines of each type, with similar results. In order to increase the spectrum of tumor types potentially susceptible to monoclonal antibody-mediated anti-p185HER2 therapies, to decrease potential immunogenicity issues with the use of murine monoclonal antibodies for human therapy, and to provide the potential for antibody-mediated cytotoxic activity, a mouse/human chimeric 4D5 (chmAb 4D5) and a "humanized" 4D5 (rhu)mAb 4D5 HER2 antibody were constructed. Both engineered antibodies, in combination with human peripheral blood mononuclear cells, elicited antibody-dependent cytotoxic responses in accordance with the level of p185HER2 expression. Since this cytotoxic activity is independent of sensitivity to mumAb 4D5, the engineered monoclonal antibodies expand the potential target population for antibody-mediated therapy of human cancers characterized by the overexpression of p185HER2.

Antibodies, Monoclonal↗
PubMed1993

Fine needle aspiration in the clinical management of mammary masses.

The role of fine needle aspiration (FNA) for clinical decision-making in patients with nodules of the breast remains in evolution. We retrospectively studied FNA outcomes in 183 patients, stratified by decades of age, to determine the reliability of FNA cytologic examination. Using Bayesian analysis, the predictive value of a positive FNA for women more than 60 years of age is 98 to 99 percent. In women 41 to 60 years of age, those with a first-degree relative with carcinoma of the breast and those with a prior biopsy with increased risk have an 88 to 95 percent likelihood of carcinoma. In women younger than 41 years of age, this likelihood decreases to 65 percent. Additionally, the probability of a lump being carcinoma, given the FNA was negative, ranges from 3 to 80 percent. Therefore, biopsy of a dominant nodule should not be deferred based only on a negative FNA. We can conclude that women 61 years of age or older could be counseled for definitive care at the first operation. Women 41 to 60 years of age, those with a positive family history or those with a past biopsy with increased risk, could be counseled for definitive therapy, but have the diagnosis confirmed with frozen section. Women less than 40 years of age without other risk factors receive no diagnostic benefit from FNA and should only be evaluated with open biopsy.

Adult↗
PubMed1992

Antigen binding thermodynamics and antiproliferative effects of chimeric and humanized anti-p185HER2 antibody Fab fragments.

The murine monoclonal antibody 4D5 (anti-p185HER2) inhibits the proliferation of human tumor cells overexpressing p185HER2 in vitro and has been "humanized" [Carter, P., Presta, L., Gorman, C. M., Ridgway, J. B. B., Henner, D., Wong, W.-L. T., Rowland, A. M., Kotts, C., Carver, M. E., & Shepard, H. M. (1992) Proc. Natl. Acad. Sci. U.S.A. (in press)] for use in human cancer therapy. We have determined the antigen binding thermodynamics and the antiproliferative activities of chimeric 4D5 Fab (ch4D5 Fab) fragment and a series of eight humanized Fab (hu4D5 Fab) fragments differing by amino acid substitutions in the framework regions of the variable domains. Fab fragments were expressed by secretion from Escherichia coli and purified from fermentation supernatants by using affinity chromatography on immobilized streptococcal protein G or staphylococcal protein A for ch4D5 and hu4D5, respectively. Circular dichroism spectroscopy indicates correct folding of the E. coli produced Fab, and scanning calorimetry shows a greater stability for hu4D5 (Tm = 82 degrees C) as compared with ch4D5 Fab (Tm = 72 degrees C). KD values for binding to the extracellular domain (ECD) of p185HER2 were determined by using a radioimmunoassay; the delta H and delta Cp for binding were determined by using isothermal titration calorimetry. ch4D5 Fab and one of the humanized variants (hu4D5-8 Fab) bind p185HER2-ECD with comparable affinity (delta G degrees = -13.6 kcal mol-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Humanization of an anti-p185HER2 antibody for human cancer therapy.

The murine monoclonal antibody mumAb4D5, directed against human epidermal growth factor receptor 2 (p185HER2), specifically inhibits proliferation of human tumor cells overexpressing p185HER2. However, the efficacy of mumAb4D5 in human cancer therapy is likely to be limited by a human anti-mouse antibody response and lack of effector functions. A "humanized" antibody, humAb4D5-1, containing only the antigen binding loops from mumAb4D5 and human variable region framework residues plus IgG1 constant domains was constructed. Light- and heavy-chain variable regions were simultaneously humanized in one step by "gene conversion mutagenesis" using 311-mer and 361-mer preassembled oligonucleotides, respectively. The humAb4D5-1 variant does not block the proliferation of human breast carcinoma SK-BR-3 cells, which overexpress p185HER2, despite tight antigen binding (Kd = 25 nM). One of seven additional humanized variants designed by molecular modeling (humAb4D5-8) binds the p185HER2 antigen 250-fold and 3-fold more tightly than humAb4D5-1 and mumAb4D5, respectively. In addition, humAb4D5-8 has potency comparable to the murine antibody in blocking SK-BR-3 cell proliferation. Furthermore, humAb4D5-8 is much more efficient in supporting antibody-dependent cellular cytotoxicity against SK-BR-3 cells than mumAb4D5, but it does not efficiently kill WI-38 cells, which express p185HER2 at lower levels.

Adenocarcinoma↗
PubMed1992

CD20 and CD5 expression in B-chronic lymphocytic leukemia.

In order to quantitate a previously noted decrease in CD20 fluorescence intensity (FI) on B-CLL lymphocytes, binding capacities [BC x 10(3) +/- 1SD = number of antibodies bound per cell] were calculated. The mean (N = 5) BC x 10(3) +/- 1SD of CD20 reagents for normal B-PBL and B-CLL lymphocytes confirmed this observation. B-PBL and B-CLL were 56 +/- 11 and 61 +/- 14, and 19 +/- 15 and 18 +/- 16, respectively, for Leu 16 and B1. Although adequate compensation standards for the determination of CD5 and CD20 coexpression are not available, qualitatively, the density of CD5 on both normal B-PBL and B-CLL is less compared to the expression of CD5 by normal T cells. CD5 expression on B-CLL seems to be linked to the lower levels of CD20, whereas CD5 expression may appear to be absent on CLL lymphocytes expressing normal levels of CD20. Levels of CD20 in B-CLL suggest involvement of one or two genes (alleles) whose decreased expression may be linked to CD5 expression.

Antigens, CD↗
PubMed1992

Development of humanized bispecific antibodies reactive with cytotoxic lymphocytes and tumor cells overexpressing the HER2 protooncogene.

The HER2 protooncogene encodes a 185-kD transmembrane phosphoglycoproteins, human epidermal growth factor receptor 2 (p185HER2), whose amplified expression on the cell surface can lead to malignant transformation. Overexpression of HER2/p185HER2 is strongly correlated with progression of human ovarian and breast carcinomas. Recent studies have shown that human T cells can be targeted with bispecific antibody to react against human tumor cells in vitro. We have developed a bispecific F(ab')2 antibody molecule consisting of a humanized arm with a specificity to p185HER2 linked to another arm derived from a murine anti-CD3 monoclonal antibody that we have cloned from UCHT1 hybridoma. The antigen-binding loops for the anti-CD3 were installed in the context of human variable region framework residues, thus forming a fully humanized BsF(ab')2 fragment. Additional variants were produced by replacement of amino acid residues located in light chain complementarity determining region 2 and heavy chain framework region 3 of the humanized anti-CD3 arm. Flow cytometry analysis showed that the bispecific F(ab')2 molecules can bind specifically to cells overexpressing p185HER2 and to normal human peripheral blood mononuclear cells bearing the CD3 surface marker. In additional experiments, the presence of bispecific F(ab')2 caused up to fourfold enhancement in the cytotoxic activities of human T cells against tumor cells overexpressing p185HER2 as determined by a 51Cr release assay. These bispecific molecules have a potential use as therapeutic agents for the treatment of cancer.

Amino Acid Sequence↗
PubMed1992

High level Escherichia coli expression and production of a bivalent humanized antibody fragment.

Many clinical uses of antibodies will require large quantities of fragments which are bivalent and humanized. We therefore attempted to generate humanized F(ab')2 fragments by secretion from E. coli. Titers of 1-2 g l-1 of soluble and functional Fab' fragments have been routinely achieved as judged by antigen-binding ELISA. Surprisingly, this high expression level of Fab' in the periplasmic space of E. coli does not drive dimerization. However, we have developed a protocol to directly and efficiently recover Fab' with the single hinge cysteine in the free thiol state, allowing F(ab')2 formation by chemically-directed coupling in vitro. The E. coli derived humanized F(ab')2 fragment is indistinguishable from F(ab')2 derived from limited proteolysis of intact antibody in its binding affinity for the antigen, p185HER2, and anti-proliferative activity against the human breast tumor cell line, SK-BR-3, which over-expresses p185HER2. This system makes E. coli expression of bivalent antibody fragments for human therapy (or other uses) practical.

Amino Acid Sequence↗