Biomedical subjects

J Sharon

Publications and source records attributed to J Sharon.

At least 19 recordsLinked to original sources

PubMed2000

Serpins identified as cell growth inhibitors in human plasma.

We have identified a new factor, CFX, in human serum and plasma that inhibits the growth of cultured human and mouse cell lines. CFX was determined to be a negatively charged, hydrophobic glycoprotein, with a native molecular weight of 110-120 kDa and a minimal active subunit of 55 kDa. It is precipitated by 60% ammonium sulfate and is resistant to heat treatment at 100 degrees C for 30 min. CFX was purified from human plasma to a single band on a gel which retained the cell growth inhibitory activity. Amino acid sequence analysis of the CFX band revealed sequences from four human glycoproteins, alpha1-antichymotrypsin, C1-esterase inhibitor, alpha1-antitrypsin, and alpha2-antiplasmin, all members of the superfamily of serpins. Of the four, C1-esterase inhibitor was shown to be the most potent cell growth inhibitor. These results suggest that serpins may play a cell growth inhibitory role in vivo, in addition to their role as protease inhibitors.

Cell Division↗

PubMed2000

Generation of a polyclonal fab phage display library to the human breast carcinoma cell line BT-20.

We have previously described a vector system for generating recombinant polyclonal antibody libraries. This system uses bidirectional phagemid and mammalian expression vectors to facilitate mass transfer of selected variable light and variable heavy (VL-VH) region gene pairs from the phagemid to the mammalian vector, to express polyclonal libraries of whole IgG antibodies. We report here the first stage of generating a polyclonal antibody library to the human breast carcinoma cell line BT-20, using this vector system. VL and VH region gene pairs were obtained from a mouse immunized with BT-20 cells, and cloned, in opposite transcriptional orientations, in the bidirectional phagemid vector, to produce an Fab phage display library. This library was selected by panning on BT-20 cells and shown to bind specifically to BT-20 cells. Such libraries, after suitable negative selection to eliminate major reactivities against normal tissue, could be transferred in mass to our bidirectional mammalian expression vector for production of libraries of chimeric antibodies with mouse V regions and human constant (C) regions. These polyclonal antibody libraries will mediate effector functions and are expected to be useful for breast cancer therapy, as well as diagnosis.

Animals↗

PubMed2000

Recombinant polyclonal antibody libraries.

We describe a technology for generating recombinant polyclonal antibody libraries (PCALs) that enables the creation and perpetuation of standardized mixtures of polyclonal whole antibodies specific for a multiantigen (or polyantigen). Therefore, this technology combines the advantages of targeting multiple antigenic determinants -- high avidity, low likelihood of antigen 'escape variants', and efficient mediation of effector functions, with the advantages of using monoclonal antibodies -- unlimited supply of standardized reagents and the availability of the genetic material for desired manipulations. The technology for generating recombinant polyclonal antibody libraries begins with the creation of phage display Fab (antibody) libraries. This is followed by selection of sublibraries with desired antigen specificities, and mass transfer of the variable region gene pairs of the selected sublibraries to a mammalian expression vector for generation of libraries of polyclonal whole antibodies. We review here our experiments for selection of phage display antibody libraries against microbes and tumor cells, as well as the recent literature on the selection of phage display antibody libraries to multiantigen targets.

Animals↗

PubMed1999

A bidirectional phage display vector for the selection and mass transfer of polyclonal antibody libraries.

An approach to the creation of antigen-specific polyclonal libraries of intact antibodies is presented. A polyclonal library of Fab antibody fragments would be expressed using a phage display vector, and selected for reactivity with an antigen or group of antigens. For conversion into a sublibrary of intact polyclonal antibodies, the selected heavy (H) and light (L) chain variable (V) region gene combinations would be transferred in mass, as linked pairs, to a eukaryotic expression vector which provides immunoglobulin (Ig) constant (C) region genes. To enable this selection and transfer, a bidirectional phage display vector was generated, in which the V region gene pairs are linked head to head in opposite transcriptional orientations. The functionality of this vector was demonstrated by the selection, transfer and expression of linked V region gene pairs derived from an A/J mouse that had been immunized with p-azophenylarsonate (Ars)-coupled keyhole limpet hemocyanin (KLH). As expected, the expressed IgG2b anti-Ars antibodies with selected V region gene pairs were shown to have V region sequences and Ars-binding characteristics similar to those of anti-Ars hybridoma antibodies. The technology presented here has potential for many diagnostic and therapeutic applications. These include the generation of polyclonal antibody libraries against multiple epitopes on infectious agents or cancer cells, and of polyclonal libraries encoding chimeric molecules composed of antibody V regions and T cell receptor C regions.

Amino Acid Sequence↗

PubMed1999

Generation of a polyclonal Fab phage display library to the protozoan parasite Cryptosporidium parvum.

We had developed a technology for creation of recombinant polyclonal antibody libraries, standardized perpetual mixtures of polyclonal whole antibodies for which the genes are available and can be altered as desired. We report here the first phase of generating a polyclonal antibody library to Cryptosporidium parvum, a protozoan parasite that causes severe disease in AIDS patients, for which there is no effective treatment. BALB/c mice, immunized by neonatal oral infection with oocysts followed by intraperitoneal immunization with a sporozoite/oocyst preparation of C. parvum, were used for construction of a Fab phage display library in a specially-designed bidirectional vector. This library was selected for reactivity to an oocyst/sporozoite preparation, and was shown to be antigen-specific and diverse. Following mass transfer of the selected variable region gene pairs to appropriate mammalian expression vectors, such anti-C. parvum Fab phage display libraries could be used to develop chimeric polyclonal antibody libraries, with mouse variable regions and human constant regions, for passive immunotherapy of C. parvum infection.

Animals↗

PubMed1996

Analysis of antigen binding and idiotypic expression by antibodies with polyglycine-replaced complementarity-determining regions.

We investigated the feasibility and usefulness, for structure-function studies, of removing the side chains of entire complementarity-determining regions (CDRs) of Abs by replacement with polyglycine. The CDRs of a murine Ab specific for p-azophenylarsonate (Ars) were replaced with polyglycine, one CDR at a time and in combinations, by oligonucleotide-directed mutagenesis of the V region genes. Mutant Abs were expressed in transfected hybridoma cells and analyzed for Ars binding and for idiotypic expression. The results suggest that, except for the longest CDRs, polyglycine replacement does not alter the general structure of the Ab molecule. However, for analysis of functional contributions of a CDR, the polyglycine replacement method appears to be most useful for CDRs with extended structures whose replacement by polyglycine does not affect the structure of other parts of the variable regions. In the current studies, such CDRs were CDR1 of the heavy chain (H1) and CDR2 of the light chain (L2). The polyglycine replacement of L2, which does not contain an Ag-contacting residue, allowed the formation of an Ars binding Ab. Furthermore, this mutant Ab revealed previously uncharacterized contributions of L2 to idiotypic expression. Polyglycine replacement of H1 abolished Ars binding as expected, because H1 contains an Ag-contacting residue. However, introduction of the contacting residue (Asn) on the polyglycine-replaced H1 background restored the ability of the Ab to bind Ars. The results suggest that polyglycine replacement of CDRs can provide structural information that complements and extends the information obtained by other methods.

Amino Acid Sequence↗

PubMed1996

Experimental immunization with a monoclonal anti-idiotope antibody that mimics the Neisseria gonorrhoeae lipooligosaccharide epitope 2C7.

An anti-idiotope monoclonal antibody (MAb), called CA1 (Ab2), was produced in mice against MAb 2C7, which recognizes a widely in vivo-expressed gonococcal lipooligosaccharide (LOS) epitope. Mice immunized with MAb CA1 initially had a 2.5-fold increase in IgG (12-fold after a booster) but no increase in IgM anti-LOS (Ab1') antibody. Control mice immunized with LOS had a 4.5-fold rise in IgG and 4-fold rise in IgM anti-LOS antibody. In rabbits, MAb CA1 elicited a 9-fold rise in IgG and a 3.3-fold rise in IgM anti-LOS (Ab1') antibody. Ab1' antibody bactericidal activity was 1-2 logs greater than that produced by immunization with LOS. Ab1' mediated complete human polymorphonuclear leukocyte phagocytosis of 2C7 epitope-positive (but not 2C7 epitope-negative) gonococci. MAb CA1 acts as a molecular surrogate (Ab2beta) for the nominal LOS antigen and may form the basis for vaccine candidates for human immunization against Neisseria gonorrhoeae.

Animals↗

PubMed1994

A method for linking VL and VH region genes that allows bulk transfer between vectors for use in generating polyclonal IgG libraries.

Libraries of Ab fragments have been produced by others from light and heavy chain cDNAs derived from populations of B lymphocytes and expressed in bacteria. However, such libraries have not been transferred to eukaryotic expression vectors to generate polyclonal libraries of intact glycosylated Abs that can mediate effector functions. We present a method for transferring pairs of linked VL-VH region genes between circular prokaryotic and eukaryotic vectors. The key feature of the transfer is that the VL and VH region genes are linked head to head (<-->) in opposite transcriptional orientations. To illustrate this method, a pair of VL and VH region cDNAs derived from an existing hybridoma cell line were linked head to head by PCR, transferred as a unit between vectors, and expressed as an IgG Ab with Ag binding activity. Although we tested the transfer of a single VL-VH region gene pair, this system is expected to allow the bulk transfer of physically linked VL-VH region gene combinations between different circular vectors and the expression of the same library as either Ab fragments or intact Abs.

Animals↗

PubMed1994

An isotype switched and somatically mutated rheumatoid factor clone isolated from a MRL-lpr/lpr mouse exhibits limited intraclonal affinity maturation.

Employing site-directed mutagenesis we have reconstructed and expressed the germ-line precursor of an expanded rheumatoid factor (RF) clone. This RF clone, designated clone F, was isolated from an autoimmune MRL/MpJ-lpr/lpr mouse. Most of the clone members were extensively mutated and isotyped-switched. The predominant isotype of clone F was gamma 3. The RF bound specifically to the MRL gamma 2 a allotype (Igh-1j) but not to the B6 gamma 2a allotype (Igh-1b). The germ-line antibody was also found to bind gamma 2a in an RF assay. The affinities of the germ-line RF and representative members of the clone were measured in an ELISA-based equilibrium binding assay. The dissociation constant (Kd) of the germ-line RF was 2.5 x 10(-6) M. All of the expressed clone members had affinities within a two- to sixfold range of the germ line, indicating that the mechanisms of somatic hypermutation and selection resulted in only limited affinity maturation of this autoantibody clone.

Amino Acid Sequence↗

PubMed1993

Chimeric antibodies with anti-dextran-derived complementarity-determining regions and anti-p-azophenylarsonate-derived framework regions.

The framework regions of antibodies fold into a conserved beta-sheet structure that acts as scaffolding for the antigen-contacting complementarity-determining regions (CDR). To test the structural equivalence of the frameworks between two antibodies with widely different combining sites, we created chimeric H and L chains by grafting the CDR of an alpha(1-->6)dextran specific antibody onto the framework of a p-azophenylarsonate (Ars) specific antibody through oligonucleotide-directed mutagenesis of the anti-Ars variable region genes. Antibodies consisting of various chain combinations of the chimeric, anti-dextran, and anti-Ars derived H and L chains were generated in transfectomas and tested for binding to dextran and Ars. Of the newly created chimeric and/or hybrid antibodies, an antibody with the chimeric H chain and the anti-dextran L chain bound to dextran with the same association constant as the parental anti-dextran antibody, and like the anti-dextran antibody was shown by immunochemical mapping to have a site complementary to six glucose residues. None of the other new variable region combinations, including the all-chimeric combination, showed binding to either dextran or Ars. These results indicate that the H chain but not the L chain anti-dextran and anti-Ars frameworks are functionally equivalent. Attempts to confer dextran binding on the H and L chain chimeric antibody, by mutagenizing selected framework residues, were unsuccessful. This study demonstrates the important role of the frameworks in the precise alignment of the CDR for Ag binding.

Amino Acid Sequence↗

PubMed1993

Verification of a model of a F(ab) complex with phenylarsonate by oligonucleotide-directed mutagenesis.

A model of the combining site of a mouse antibody specific for p-azophenylarsonate was tested by oligonucleotide-directed mutagenesis of the proposed hapten-contacting residues, Arg96 in the L chain, and Asn35, Trp47, Tyr50, Ser95, and Tyr100b in the H chain. The affinity and relative affinity for p-azophenylarsonate-N-acetyl-L-tyrosine of mutant antibodies expressed in transfectomas were determined by fluorescence quenching and by inhibition ELISA, respectively. The results show that alteration of the proposed contacting residues has drastic effects on hapten binding, and that the hydroxyl groups of Tyr50 and Tyr100b appear to orient the phenyl rings for optimal aromatic-aromatic interactions with the phenyl ring of the hapten. They further indicate a tight packing of the contacting residues around the hapten, which cannot accommodate changes in the positions of the functional groups of Asn35 and Ser95.

Antibody Affinity↗

PubMed1993

An analysis of idiotype expression in a high-affinity, somatically mutated variant of a germline-encoded anti-p-azobenzenearsonate antibody.

Using two polyclonal (rabbit) and two monoclonal anti-idiotype (anti-Id) reagents, we investigated structural correlates of the Id of mAb 36-71, a somatically mutated member of the CRIA Id family that has an exceptionally high affinity for the p-azobenzenearsonate (Ars) hapten. The two monoclonal anti-Ids reacted principally with the L chain of 36-71. The polyclonal anti-Ids interacted with both the H and L chain. The amino acid sequences of the VH and VL regions of 36-71 differ in eight and 11 positions respectively from those of the anti-Ars mAb 36-65, an unmutated prototype of the CRIA family. In the presence of 36-71L only three substitutions in 36-65 VH, introduced by mutagenesis, sufficed to restore full expression of the 36-71 Id. The same three substitutions had previously been shown to increase the affinity of 36-65 by a factor of 200, to a level equivalent to that of 36-71. X-ray crystallography had indicated that two of these substitutions introduce conformational changes consistent with the increase in affinity. We propose that these conformational changes may also account for the critical role of the three amino acids in Id expression. We also found that 36-65 is a very poor inhibitor of the interaction of 36-71 with its polyclonal anti-Ids, despite identity of the hapten-contacting residues in the two mAbs and evidence (from hapten inhibition) that the hapten-binding region is part of an important Id. Again, a difference in conformation at the binding site of the two mAbs could account for these observations.

Amino Acid Sequence↗

PubMed1993

Oligonucleotide-directed mutagenesis of antibody combining sites.

We review here our attempts to achieve a better understanding of the structure--function relationship of antibody combining sites, and to gain insights into the engineering of antibodies with desired specificity and affinity. We have focused on a model system--antibodies to the hapten p-azophenylarsonate (Ars) derived from A/J mice. Oligonucleotide-directed mutagenesis was used to alter the sequence of the variable region genes of such anti-Ars antibodies. Mutant antibodies were generated in hybridoma cells following transfection of the altered genes, and the effects of the primary structure changes on antibody specificity, affinity, and idiotypic expression were assessed. These studies suggest that an antibody combining site with basic specificity for an antigen could be created by introducing a set of a few amino acid residues in the complementarity determining regions, and that the affinity of such a site could be improved one substitution at a time in a sequential manner.

Amino Acid Sequence↗

PubMed1992

Comparison of GK1.5 and chimeric rat/mouse GK1.5 anti-CD4 antibodies for prolongation of skin allograft survival and suppression of alloantibody production in mice.

GK1.5 is a rat mAb that recognizes the mouse CD4 Ag. It has been shown to deplete CD4+ cells in vivo and to be immunosuppressive. To evaluate the effect of the C region of this antibody in achieving cell depletion, chimeric antibodies, each having the rat GK1.5 V regions and one of the four mouse IgG C region isotypes, were compared with the native rat antibody. The chimeric antibodies and the native antibody were tested for their ability to mediate in vitro C-dependent cytotoxicity, in vivo cell depletion, and prolongation of allogeneic skin graft survival and suppression of alloantibody production. In vitro C-dependent cytotoxicity assays revealed that rat IgG2b and the chimeric antibodies containing mouse IgG2a, mouse IgG2b, and mouse IgG3 were effective in lysing CD4+ lymphocytes whereas mouse IgG1 was ineffective. In vivo studies of CD4+ cell depletion showed that mouse IgG2a, rat IgG2b, and mouse IgG2b were effective isotypes, mouse IgG1 was less effective, and mouse IgG3 did not deplete CD4+ cells. A correlation was found between the ability of an isotype to deplete CD4+ cells in vivo and its ability to prolong the survival of skin allografts and to suppress alloantibody production. The nondepleting mouse IgG3 was ineffective in these assays. Overall the most effective mouse isotype was IgG2a which was as effective as rat IgG2b. These results indicate 1) that syngeneic isotypes of mAb can cause cell depletion and consequently the prolongation of allograft rejection and suppression of alloantibody production; 2) that not all isotypes are equally effective; and 3) that the ability of a given isotype to deplete cells in vivo does not correlate with its ability to mediate C-dependent lysis in vitro. Our results are consistent with the hypothesis that in vivo depletion of cells is mediated by opsonization and binding through the FcR.

Animals↗

PubMed1991

Three-dimensional structure of murine anti-p-azophenylarsonate Fab 36-71. 1. X-ray crystallography, site-directed mutagenesis, and modeling of the complex with hapten.

The structure of the antigen-binding fragment (Fab) of an anti-p-azophenylarsonate monoclonal antibody, 36-71, bearing a major cross-reactive idiotype of A/J mice has been refined to an R factor of 24.8% at a resolution of 1.85 A. The previously solved partial structure of this Fab at a resolution of 2.9 A (Rose et al., 1990) was used as an initial model for refinement against the high-resolution data. The complex with hapten has been modeled by docking the small-molecule crystal structure of phenylarsonic acid into the structure of the native Fab on the basis of a low-resolution electron density map of the complex. In this model, residue Arg-96 in the light chain and residues Asn-35, Trp-47, and Ser-99 in the heavy chain contact the arsonate moiety of the hapten; an additional bond is found between the arsonate group and a tightly bound water molecule. The phenyl moiety of the hapten packs against two tyrosine side chains at positions 50 and 106 in the heavy chain. Residue Arg-96 in the light chain had been implicated as involved in hapten binding on the basis of previous experiments, and indeed, this residue appears to play a crucial role in this model. Experiments employing site-directed mutagenesis directly support this conclusion. The heavy-chain complementarity-determining regions have novel conformations not previously observed in immunoglobulins except for the recently solved anti-p-azophenylarsonate Fab R 19.9 (Lascombe et al., 1989).

Amino Acid Sequence↗

PubMed1991

Three-dimensional structure of murine anti-p-azophenylarsonate Fab 36-71. 2. Structural basis of hapten binding and idiotypy.

Comparison between the structures and solvent-accessible surfaces of the antigen-binding fragments of two murine anti-p-azophenylarsonate monoclonal antibodies, one bearing a major cross-reactive idiotype of A/J strain mice (36-71) and one lacking the idiotype (R19.9; Lascombe et al., 1989), highlight the structural basis for the determination of hapten affinity and idiotypy. Since the sequence of R 19.9 is identical with the germline-encoded sequence at 16 positions in both heavy-chain and light-chain variable regions where somatic mutations and junctional differences have occurred to produce the 36-71 sequence, the structure of R 19.9 can be used to model the structure of the germline-encoded antibody (36-65) in the regions around these sites. These 16 sequence differences exclude the third heavy-chain complementarity-determining region because R 19.9 utilizes a D gene segment not associated with the predominant idiotype, which is 4 residues longer than the canonical D gene segment utilized in the sequences of 36-71 and 36-65. This difference between the structures of R 19.9 and 36-71 does not affect the validity of using the structure of R 19.9 to model the structure of 36-65 since the third heavy-chain complementarity-determining region is highly solvent-exposed in both 36-71 and R 19.9, and does not interact with any of these 16 sites. Comparing the structures of 36-71 and R 19.9 suggests that only three of the differences in the heavy-chain sequences, and three of the differences in the light-chain sequences of 36-71 and 36-65, increase the affinity for hapten.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1991

Different epitope structures select distinct mutant forms of an antibody variable region for expression during the immune response.

Antibody variable (V) regions that initially differ from one another by only single amino acid residues at VH-D and D-JH segment junctions (termed canonical V regions) can be elicited in strain A/J mice by three different haptens. Among such V regions an amino acid substitution due to somatic mutation is recurrently observed at VH CDR2 position 58, regardless of which of these haptens is used for immunization. This substitution confers upon a canonical V region a generic increase in affinity for all the haptens. Conversely, the type of amino acid substitution at VH position 59 resulting from somatic mutation that is recurrently observed among such V regions changes with the eliciting hapten, in a manner that correlates directly with the cognate affinity increases (or decreases) for hapten conferred by the observed substitutions. This small subregion of VH CDR2 therefore plays a major role in determining both affinity and specificity for antigen. The data confirm that affinity for antigen is of pivotal importance in determining the degree of selection of different mutant forms of a V region. Moreover, during an immune response a sufficiently diverse mutant repertoire can be generated from a single canonical V region to allow adaptation to increase affinity for three different epitopes.

Amino Acid Sequence↗

PubMed1990

Clustered H chain somatic mutations shared by anti-p-azophenylarsonate antibodies confer enhanced affinity and ablate the cross-reactive idiotype.

A cluster of four consecutive CDR2 somatic mutations are shared by the VH regions of two independently isolated hybridoma antibodies with specificity for p-azophenylarsonate (Ars). The mutations appear to be derived by a series of independent events. To assess the influence of these shared somatic mutations on antibody affinity for Ars and on idiotypy, we introduced them, via site-directed mutagenesis, into the V region of an anti-Ars antibody that was otherwise unmutated and we eliminated them from the mutated context of one of the two antibodies in which they were originally found. Results of affinity measurements by fluorescence quenching and of idiotypic binding assays performed on these engineered mutants demonstrated that the shared mutations increased affinity for Ars and eliminated the predominant Id associated with strain A anti-Ars antibodies and four of five idiotypes defined by anti-idiotypic mAb. These results support the interpretation that a strong affinity-based selection pressure has favored the clonal expansion of B cells with receptors containing these mutations despite the loss of a predominant Id. Thus, in producing antibodies containing V regions conferring high affinity for Ag, the combined processes of somatic mutation and clonal selection have generated a common structural solution through parallel repeats.

Amino Acid Sequence↗