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J D Marks

Publications and source records attributed to J D Marks.

At least 73 records · Page 4Linked to original sources
PubMed1995

In vitro and in vivo characterization of a human anti-c-erbB-2 single-chain Fv isolated from a filamentous phage antibody library.

BACKGROUND: Antibody-based reagents have failed to live up to their anticipated role as highly specific targeting agents for cancer therapy. Targeting with human single-chain Fv (sFv) molecules may overcome some of the limitations of murine IgG, but are difficult to produce with conventional hybridoma technology. Alternatively, phage display of antibody gene repertoires can be used to produce human sFv. OBJECTIVES: To isolate and characterize human single chain Fvs which bind to c-erbB-2, an oncogene product overexpressed by 30-50% of breast carcinomas and other adenocarcinomas. STUDY DESIGN: A non-immune human single-chain Fv phage antibody library was selected on human c-erbB extracellular domain and sFv characterized with respect to affinity, binding kinetics, and in vivo pharmacokinetics in tumor-bearing scid mice. RESULTS: A human single-chain Fv (C6.5) was isolated which binds specifically to c-erbB-2. C6.5 is entirely human in sequence, expresses at high level as native protein in E. coli, and is easily purified in high yield in two steps. C6.5 binds to immobilized c-erbB-2 extracellular domain with a Kd of 1.6 x 10(-8) M and to c-erbB-2 on SK-OV-3 cells with a Kd of 2.0 x 10(-8) M, an affinity that is similar to sFv produced against the same antigen from hybridomas. Biodistribution studies demonstrate 1.47% injected dose/g tumor 24 h after injection of 125I-C6.5 into scid mice bearing SK-OV-3 tumors. Tumor:normal organ ratios range from 8.9:1 for kidney to 283:1 for muscle. CONCLUSIONS: These results are the first in vivo biodistribution studies using an sFv isolated from a non-immune human repertoire and confirm the specificity of sFv produced in this manner. The use of phage display to produce C6.5 mutants with higher affinity and slower k(off) would permit rigorous evaluation of the role of antibody affinity and binding kinetics in tumor targeting, and could result in the production of a therapeutically useful targeting protein for radioimmunotherapy and other applications.

Amino Acid Sequence↗
PubMed1995

Molecular characteristics of anti-self antibody fragments against neutrophil cytoplasmic antigens from human V gene phage display libraries.

Recently it has been demonstrated that human antibody fragments with binding activities against self antigens can be isolated from repertoires of rearranged V genes from non-immunized humans. We have applied phage display technology to study the B cell repertoire for antibody activity against neutrophil cytoplasmic antigens. These antibodies may play an important role in Wegener's granulomatosis (WG) and related forms of vasculitides. Autoantibodies in patients with WG are directed against proteinase 3. The immunodominant antigen in other forms of vasculitis is myeloperoxidase, but the B cell response can also be directed against other neutrophil enzymes, e.g. lysozyme, human neutrophil elastase, lactoferrin and cathepsin G. We show here that anti-self reactivity against neutrophil cytoplasmic antigens can be detected in the rearranged V gene repertoire of healthy individuals and that the reactivity can be directed against structural related epitopes which are present on different neutrophil cytoplasmic antigens. The scFv with binding activities were sequenced and the V gene usage, the level of somatic mutations and the immunoserological characteristics of the antibody fragments are discussed. Further evidence is presented that antibody fragments consisting only of a heavy chain variable domain can recognize neutrophil cytoplasmic antigens in a specific manner. These single-domain antibody fragments were used in experiments designed to establish the relative role of the light chain variable domains in antigen binding.

Amino Acid Sequence↗
PubMed1995

Cannula selection and cannulation techniques for nonpulsatile mechanical ventricular assistance.

The successful use of mechanical ventricular assistance is, in large part, dependent upon easy insertion of well designed inflow and outflow cannulas. This manuscript describes a family of cannulas specifically designed for use in a nonpulsatile ventricular assist device (VAD) circuit. Although a variety of commercially available cannulas can be employed in a nonpulsatile VAD circuit, the cannulas described in this manuscript possess a number of unique design features. The reinforced thin-walled design and end hole construction optimize fluid flow characteristics in both the inflow and outflow cannula. The extended length allows the cannulas to transverse the skin at a point far distant from the mediastinum, reducing the potential for ascending cannula site infection. The purpose of this manuscript is not to review all cannulas available for nonpulsatile VAD circuits, but rather to describe in detail a family of cannulas that are ideal VAD cannulas based on their design characteristics. In addition individual cardiac surgeons implant very few of these devices annually. Thus the advantages and disadvantages of common cannulation sites and a proven technique for cannula insertion will also be presented.

Aorta↗
PubMed1994

In vitro assembly of repertoires of antibody chains on the surface of phage by renaturation.

Antibodies can be made from repertoires of associated heavy and light chains displayed on the surface of bacteriophage, and are readily diversified by random point mutation or by chain shuffling. To make extensive variations around the "core" antigen binding contacts of a crystallographically solved mouse antibody NQ10/12.5 (gamma l, kappa), the NQ10 light chain was assembled in vitro with a repertoire of about 10(7) human heavy chains displayed on the surface of phage, and selected by binding to hapten. An antibody with a much improved affinity was isolated from the repertoire (K(a) = 10(9) M-1 compared with 10(8) M-1 for NQ10). The sequence of the human heavy chain (VH-IL) was highly related to NQ10. It conserved the same folds for the H1, H2 and H3 loops, six of the seven contact residues for hapten, and also a phOx binding motif (Asp-X-Gly-X-X) in the H3 loop. It appears that the new heavy chain partners for the NQ10 light chain often retain many critical antigen binding features found in the NQ10 heavy chain.

Amino Acid Sequence↗
PubMed1994

Characterization of human blood group scFv antibodies derived from a V gene phage-display library.

We previously reported the initial characterization of five human single-chain Fv (scFv) antibody fragments specific for the blood group antigens B, D(Rh), E(Rh), Kpb and HI. The scFvs were isolated from a phage-antibody library constructed from the variable region genes of two non-immunized donors. In this paper we report the specificity, affinity and kinetics of antigen binding of these scFv fragments. All five scFvs agglutinated the appropriate red cell phenotype following the addition of a monoclonal antibody which recognizes a peptide tag incorporated into the scFv. The anti-B and anti-HI scFv molecules, which recognize high density carbohydrate antigens, spontaneously polymerized and agglutinated red cells directly. None of the antibody fragments showed cross-reactivity with other red cell antigens, with the exception of the anti-E which reacted weakly with E-negative cells. Specific scFv binding was confirmed by ELISA, flow cytometry and radioactive labelling. The anti-D scFv recognized 17,600 sites on cDE/cDE red cells with an association constant (Ka), of 5.2 x 10(7) M-1 and a rate constant for dissociation (koff) of 1.9 x 10(-2) s-1. The anti-E scFv recognized 29,800 and 39,800 sites on cDE/cDE red cells in two experiments with Kas of 8.4 x 10(6) and 4.4 x 10(7) M-1. The koff for this antibody was 2.7 x 10(-2) s-1. The results demonstrate that scFv antibody fragments specific for cell surface antigens and possessing affinities typical of the primary immune response can be obtained from a phage-display library.

ABO Blood-Group System↗
PubMed1993

Human anti-self antibodies with high specificity from phage display libraries.

Recently we demonstrated that human antibody fragments with binding activities against foreign antigens can be isolated from repertoires of rearranged V-genes derived from the mRNA of peripheral blood lymphocytes (PBLs) from unimmunized humans. The heavy and light chain V-genes were shuffled at random and cloned for display as single-chain Fv (scFv) fragments on the surface of filamentous phage, and the fragments selected by binding of the phage to antigen. Here we show that from the same phage library we can make scFv fragments encoded by both unmutated and mutated V-genes, with high specificities of binding to human self-antigens. Several of the affinity purified scFv fragments were shown to be a mixture of monomers and dimers in solution by FPLC gel filtration and the binding kinetics of the dimers were determined using surface plasmon resonance (k(on) = 10(5)-10(6) M-1s-1, k(off) = 10(-2)s-1 and Ka = 10(7) M-1). The kinetics of association are typical of known Ab-protein interactions, but the kinetics of dissociation are relatively fast. For therapeutic application, the binding affinities of such antibodies could be improved in vitro by mutation and selection for slower dissociation kinetics.

Amino Acid Sequence↗
PubMed1993

Human antibody fragments specific for human blood group antigens from a phage display library.

We have isolated human antibody fragments with binding specificities against the blood group antigens of the ABO and I blood group systems (B and HI), of the Rh system (D and E) and of the Kell system (Kpb), by selecting a phage-antibody library on human red cells. The fragments, expressed in bacteria, were antigen-specific and effective in assays including agglutination and immunohistochemistry. Selection of phage-antibody libraries using intact cells seems to offer a promising alternative to hybridoma technology for the production of antibodies against cell surface molecules.

ABO Blood-Group System↗
PubMed1993

Adenosine-induced inhibition of vagal motoneuron excitability: receptor subtype and mechanisms.

With the use of intracellular techniques and a medullary slice preparation, we examined the changes in cellular and membrane properties of single adult rat vagal motoneurons during exposure to potent and specific agonists and antagonists of adenosine A1 and A2 receptors. A1 receptor stimulation increased input resistance, but markedly reduced spontaneous neuronal firing and increased rheobase. A1 agonists also increased the amplitude of the action potential afterhyperpolarization (AHP) in a dose-dependent manner. Prior treatment with A1 antagonists blocked these electrophysiological effects. Blockade of Ca2+ entry with Co2+ abolished the AHP increase. Synaptic blockade with both tetrodotoxin and high Mg2+, low Ca2+ solutions prevented the increase in input resistance. A2 receptor stimulation was without effect. Perfusion with adenosine in the presence of dipyridamole caused effects similar to A1 agonist stimulation, but no effect in the absence of dipyridamole. These results show that adenosine decreases vagal motoneuron excitability by stimulating A1 receptors. Our data also support the idea that the adenosine-induced decrease in excitability is presynaptic in nature. In addition, the AHP increase may be mediated by enhanced Ca2+ entry into the postsynaptic neuron.

Adenosine↗
PubMed1992

The repertoire of human germline VH sequences reveals about fifty groups of VH segments with different hypervariable loops.

We have used the polymerase chain reaction and VH family-based primers to clone and sequence 74 human germline VH segments from a single individual and built a directory to include all known germline sequences. The directory contains 122 VH segments with different nucleotide sequences, 83 of which have open reading frames. The directory indicates that the structural diversity of the germline repertoire for antigen binding is fixed by about 50 groups of VH segments: each group encodes identical hypervariable loops. The directory should help in mapping the VH locus, in estimating somatic mutation and VH segment usage and in designing and constructing synthetic antibody libraries.

Amino Acid Sequence↗
PubMed1992

Structural repertoire of the human VH segments.

The VH gene segments produce the part of the VH domains of antibodies that contains the first two hypervariable regions. The sequences of 83 human VH segments with open reading frames, from several individuals, are currently known. It has been shown that these sequences are likely to form a high proportion of the total human repertoire and that an individual's gene repertoire produces about 50 VH segments with different protein sequences. In this paper we present a structural analysis of the amino acid sequences produced by the 83 segments. Particular residue patterns in the sequences of V domains imply particular main-chain conformations, canonical structures, for the hypervariable regions. We show that, in almost all cases, the residue patterns in the VH segments imply that the first hypervariable regions have one of three different canonical structures and that the second hypervariable regions have one of five different canonical structures. The different observed combinations of the canonical structures in the first and second regions means that almost all sequences have one of seven main-chain folds. We describe, in outline, structures of the antigen binding site loops produced by nearly all the VH segments. The exact specificity of the loops is produced by (1) sequence differences in their surface residues, particularly at sites near the centre of the combining site, and (2) sequence differences in the hypervariable and framework regions that modulate the relative positions of the loops.

Amino Acid Sequence↗
PubMed1992

Mediastinitis in heart transplantation.

Between March 1985 and December 1991, mediastinitis developed in 12 of 420 cardiac transplantation patients (2.8%). The mortality rate in this group of patients was 8.3% (1/12). Actuarial survival (1 year, 75%; and 5 years, 65%) was not significantly different from that of the group without mediastinitis (1 year, 88%; and 5 years, 75%). A higher percentage of the patients in the group with mediastinitis were listed as UNOS status 1 (50% versus 35%) and had a history of previous sternotomies (58% versus 44%). The presentation of mediastinitis was typical. Computed tomographic scanning with or without aspiration was a valuable adjunct in the diagnosis of mediastinitis. Induction immunotherapy with minimal steroids in the perioperative period was used in all patients. This may contribute to the patients' ability to mount an appropriate and effective response to infection, permitting earlier diagnosis. The debridement irrigation technique used in 8 of 12 patients had a low success rate of 33%, whereas the debridement muscle flap technique used in 4 of 12 was 100% successful in eliminating infection.

Actuarial Analysis↗
PubMed1992

Cardiac retransplantation: a viable option? The Registry of the International Society for Heart and Lung Transplantation.

To evaluate cardiac retransplantation as an appropriate utilization of scarce donor organs we analyzed data from the registry of the International Society for Heart and Lung Transplantation (ISHLT) (n = 449) and the Utah Cardiac Transplant Program (n = 20). Actuarial survival among retransplants was lower than in patients who received only one transplant in both the ISHLT registry patients (1 year survival, 48% versus 78%; p = 0.001) and the Utah series (1 year survival, 74% versus 88%; p = 0.06). Uncontrolled rejection, short interval (< 6 months) between transplantations, and the need for mechanical circulatory support were identified as risk factors for retransplantation. The incidence of rejection and infection was similar in first and second transplant recipients. Second transplant recipients had a higher level of sensitization, a greater incidence of donor-specific positive crossmatches, and an increased early mortality. Repetition in the second donor of mismatched HLA antigens present in the first donor did not adversely affect survival. If patients who underwent retransplantation within 6 months of their initial transplantation, those receiving transplants for uncontrolled rejection, and those requiring mechanical assistance were eliminated from the study, the short-term and long-term survival after cardiac retransplantation does not differ from that in patients having a single transplant.

Actuarial Analysis↗