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C F Barbas

Publications and source records attributed to C F Barbas.

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

Efficient aldolase catalytic antibodies that use the enamine mechanism of natural enzymes.

Antibodies that catalyze the aldol reaction, a basic carbon-carbon bond-forming reaction, have been generated. The mechanism for antibody catalysis of this reaction mimics that used by natural class I aldolase enzymes. Immunization with a reactive compound covalently trapped a Lys residue in the binding pocket of the antibody by formation of a stable vinylogous amide. The reaction mechanism for the formation of the covalent antibody-hapten complex was recruited to catalyze the aldol reaction. The antibodies use the epsilon-amino group of Lys to form an enamine with ketone substrates and use this enamine as a nascent carbon nucleophile to attack the second substrate, an aldehyde, to form a new carbon-carbon bond. The antibodies control the diastereofacial selectivity of the reaction in both Cram-Felkin and anti-Cram-Felkin directions.

Acetone↗
PubMed1995

CDR walking mutagenesis for the affinity maturation of a potent human anti-HIV-1 antibody into the picomolar range.

We describe the investigation of methodologies for the creation of very high affinity human antibodies. The high affinity human antibody b4/12 was optimized for its affinity to the human envelope glycoprotein gp120 of human immunodeficiency virus type 1 (HIV-1). Five libraries of b4/12 were constructed by saturation mutagenesis of complementarity-determining regions (CDRs). Libraries of antibody Fab fragments were displayed on the surface of filamentous phage and selected in vitro for binding to immobilized gp120. Sequential and parallel optimization strategies of CDRs were examined. The sequential CDR walking strategy consistently yielded b4/12 variants of improved affinity in each of the four different optimization sequences examined. This resulted in a 96-fold improvement in affinity. Additivity effects in the antibody combining site were explored by combining independently optimized CDRs in the parallel optimization strategy. Six variants containing optimized CDRs were constructed. Improvement of affinity based on additivity effects proved to be unpredictable but did lead to a modest improvement in affinity. Indeed, only one of the six combinations demonstrated additivity. The highest affinity Fab prepared using this strategy was improved 420-fold in affinity. The affinity of this Fab was 15 pM as compared to 6.3 nM for b4/12. Examination of the kinetics of Fab binding to gp120 revealed that improvements in affinity were dominated by a slowing of the off-rate of the Fab. The methodology presented here provides a route for the improvement of the affinities of antibodies typical of tertiary immune responses into the picomolar range. Such improvements may have profound effects on the utility of antibodies as therapeutic and prophylactic agents.

Amino Acid Sequence↗
PubMed1995

Surface plasmon resonance based kinetic studies of zinc finger-DNA interactions.

Libraries of the zinc finger DNA binding protein, Zif268, have been constructed and selected for affinity and specificity toward DNA targets using the phage display technique (Wu et al., 1995). Mutant proteins were purified to homogeneity and were characterized for their ability to interact with their DNA targets using a real-time biomolecular interaction assay (BIA). One mutant protein, C7, bound the Zif268 consensus binding sequence with a 13-fold increase in affinity as compared to the wild-type Zif268 protein. Mutant proteins with moderate affinity for new DNA targets within a consensus sequence of HIV-1 have also been obtained. Surface plasmon resonance based BIA has provided invaluable kinetic information which offers insights into the mechanism of protein-DNA interactions.

Base Sequence↗
PubMed1995

Building zinc fingers by selection: toward a therapeutic application.

A phage display approach was utilized to modify the specificity of each of the three fingers of the murine transcription factor Zif268. Selections were performed by using the consensus binding sequence of the natural protein and a conserved sequence in the genome of the type 1 human immunodeficiency virus. By using an extensive randomization strategy, the entire 3-bp specificity of a finger has been changed. Rapid analysis of selected zinc fingers was facilitated by the development of an immunoscreening assay for DNA binding and specificity. To investigate the mechanism of binding and specificity, the binding kinetics of Zif268 and 10 selected variants were determined in real time with an assay based on surface plasmon resonance. Differential mechanisms for sequence-specific recognition were observed. No evidence in support of a single general coding relationship between zinc finger and target DNA sequence was observed. The prospects for the development of this class of proteins in human therapy are considered.

Animals↗
PubMed1995

Neutralizing recombinant human antibodies to a conformational V2- and CD4-binding site-sensitive epitope of HIV-1 gp120 isolated by using an epitope-masking procedure.

As part of the goal of assembling a mixture of neutralizing human mAbs for possible prophylaxis and therapy of HIV-1 disease, we describe a strategy by which neutralizing human Abs to a weakly immunogenic epitope can be accessed. From a phage display library derived from an asymptomatic HIV-1 seropositive donor, a panel of recombinant Fabs against the CD4 binding site (CD4bs) of gp120 was retrieved by affinity selection using recombinant gp120 (strain LAI). Two Fabs corresponding to the dominant clones were used to mask the CD4bs epitope(s) before repeating the selection procedure. Four Fabs were then retrieved that had novel heavy chain sequences. Three recognized a novel epitope distinct from that recognized by conventional CD4bs Abs and were defined by the following criteria: 1) second V region (V2 region) dependence indicated by sensitivity to amino acid changes in the V2 loop and by competition with murine anti-V2 mAbs; 2) CD4bs dependence indicated by sensitivity to amino acid changes usually associated with CD4 binding and by inhibition of Fab binding to gp120 by soluble CD4; this dependence seemed to arise via conformational changes rather than by direct binding, as CD4bs Abs enhanced binding of two of the novel Fabs and, in a reversal of the competition format, the novel Fabs did not inhibit soluble CD4 binding to gp120; and 3) equivalent binding to glycosylated and deglycosylated gp120 and significant, although much reduced, binding to denatured gp120 in contrast with CD4bs Abs, which do not bind to deglycosylated or denatured gp120. One of the novel Fabs efficiently neutralized the MN and LAI strains of HIV-1. These results indicate the presence of a novel neutralizing conformational epitope on gp120 sensitive to the V2 loop and the CD4bs and further highlight the conformational flexibility of gp120. The strategy of masking highly immunogenic epitopes with Abs to rescue a broader range of specific Abs from combinatorial libraries should be widely applicable.

Amino Acid Sequence↗
PubMed1995

Human monoclonal Fab fragments specific for viral antigens from combinatorial IgA libraries.

BACKGROUND: IgA constitutes the first line of immune defense, interacting with a variety of environmental antigens. Following infection with respiratory syncytial virus (RSV) individuals frequently exhibit elevated serum IgA titers specific for the virus. Previously combinatorial IgG libraries have successfully been used to clone such human antibody responses. OBJECTIVES: Here we evaluate the possibility of constructing combinatorial IgA libraries on the surface of filamentous phage to retrieve human viral-specific IgA Fab fragments. STUDY DESIGN: Bone marrow from an HIV-1 seropositive donor was used as RNA source to construct combinatorial IgA kappa and lambda libraries of approximately 10(7) clones. RESULTS: By affinity selection using an immobilized recombinant RSV FG protein, two unique IgA Fab fragments producing clones (AD5 and AD23) reactive with the selecting antigen were isolated. One of the Fab fragments was found to be specific for RSV F glycoprotein and bind with high apparent affinity (2 x 10(8) M-1). The other binds with lower affinity and exhibits cross-reactivity with other antigens. CONCLUSION: The strategy described, involving construction of combinatorial IgA libraries on the surface of filamentous phage, should be generally applicable to the investigation of both mucosal and systemic human IgA immune responses, and may become an important tool for evaluation of mucosal vaccine regimes.

Amino Acid Sequence↗
PubMed1995

Protection against HIV-1 infection in hu-PBL-SCID mice by passive immunization with a neutralizing human monoclonal antibody against the gp120 CD4-binding site.

OBJECTIVE: Mice with severe combined immunodeficiency (SCID) transplanted with human peripheral blood lymphocytes (hu-PBL) have been shown to be useful as an animal model for HIV-1 infection. This model was used to assess the ability of a human anti-gp120 antibody to protect against HIV-1 infection. DESIGN AND METHODS: hu-PBL-SCID mice were injected with an HIV-1 broadly neutralizing human monoclonal antibody against the gp120 CD4-binding site prior to challenge with HIV-1SF2. The antibody b12, employed for these studies, was isolated from an antibody phage-display library prepared from bone-marrow of a long-term asymptomatic HIV-1-seropositive donor. Both Fab fragments and whole immunoglobulin (Ig) G1 b12 antibody were assessed for protection. RESULTS: Fab b12, tested at a dose approximately 1.9 mg/kg, was able to protect 25% of hu-PBL-SCID mice from HIV-1 infection. IgG1 b12, which displayed favorable pharmacokinetic properties, showed a dose-dependent protection that was complete with a regimen of two injections of 100 micrograms per mouse. The in vivo protective dose of antibody at the time of virus challenge was estimated to be 4.5-7 mg/kg from antibody clearance data. CONCLUSIONS: This study demonstrates for the first time that complete protection against HIV-1 infection can be achieved in the hu-PBL-SCID model by passive immunization with physiologically relevant doses of a human gp120 CD4-binding site antibody derived from natural infection.

Acquired Immunodeficiency Syndrome↗
PubMed1995

Primary isolates of human immunodeficiency virus type 1 are relatively resistant to neutralization by monoclonal antibodies to gp120, and their neutralization is not predicted by studies with monomeric gp120.

A panel of anti-gp120 human monoclonal antibodies (HuMAbs), CD4-IgG, and sera from people infected with human immunodeficiency virus type 1 (HIV-1) was tested for neutralization of nine primary HIV-1 isolates, one molecularly cloned primary strain (JR-CSF), and two strains (IIIB and MN) adapted for growth in transformed T-cell lines. All the viruses were grown in mitogen-stimulated peripheral blood mononuclear cells and were tested for their ability to infect these cells in the presence and absence of the reagents mentioned above. In general, the primary isolates were relatively resistant to neutralization by the MAbs tested, compared with the T-cell line-adapted strains. However, one HuMAb, IgG1b12, was able to neutralize most of the primary isolates at concentrations of < or = 1 microgram/ml. Usually, the inability of a HuMAb to neutralize a primary isolate was not due merely to the absence of the antibody epitope from the virus; the majority of the HuMAbs bound with high affinity to monomeric gp120 molecules derived from various strains but neutralized the viruses inefficiently. We infer therefore that the mechanism of resistance of primary isolates to most neutralizing antibodies is complex, and we suggest that it involves an inaccessibility of antibody binding sites in the context of the native glycoprotein complex on the virion. Such a mechanism would parallel that which was previously postulated for soluble CD4 resistance. We conclude that studies of HIV-1 neutralization that rely on strains adapted to growth in transformed T-cell lines yield the misleading impression that HIV-1 is readily neutralized. The more relevant primary HIV-1 isolates are relatively resistant to neutralization, although these isolates can be potently neutralized by a subset of human polyclonal or monoclonal antibodies.

Amino Acid Sequence↗
PubMed1995

Cross-clade neutralization of primary isolates of human immunodeficiency virus type 1 by human monoclonal antibodies and tetrameric CD4-IgG.

We have tested three human monoclonal antibodies (MAbs) IgG1b12, 2G12, and 2F5) to the envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1), and a tetrameric CD4-IgG molecule (CD4-IgG2), for the ability to neutralize primary HIV-1 isolates from the genetic clades A through F and from group O. Each of the reagents broadly and potently neutralized B-clade isolates. The 2F5 MAb and the CD4-IgG2 molecule also neutralized strains from outside the B clade, with the same breadth and potency that they showed against B-clade strains. The other two MAbs were able to neutralize a significant proportion of strains from outside the B clade, although there was a reduction in their efficacy compared with their activity against B-clade isolates. Neutralization of isolates by 2F5 correlated with their possession of the LDKW motif in a segment of gp41 near the membrane-spanning domain. The other two MAbs and CD4-IgG2 recognize discontinuous binding sites on gp120, and so no comparison between genetic sequence and virus neutralization was possible. Our data show that a vaccine based on the induction of humoral immunity that is broadly active across the genetic clades is not impossible if immunogens that express the epitopes for MAbs such as 2F5, 2G12, and IgG1b12 in immunogenic configurations can be created. Furthermore, if the three MAbs and CD4-IgG2 produce clinical benefit in immunotherapeutic trials in the United States or Europe, they may also do so elsewhere in the world.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Building synthetic antibodies as adhesive ligands for integrins.

An antibody engineering strategy was employed to build high affinity ligands and antagonists of integrins alpha v beta 3 and alpha IIb beta 3. Previously, we inserted the integrin recognition motif, RGD, into the antigen binding site of a human antibody and selected the optimal flanking sequences from a phage-display library (Barbas, C. F., Languino, L. R., and Smith, J. W. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 10003-10007). The resulting antibody, Fab-9, blocked the function of integrin alpha v beta 3 but also bound to the ligand binding site of platelet integrin alpha IIb beta 3. In this report, the antibody engineering effort has been extended by 1) redesigning Fab-9 to achieve specificity for platelet integrin alpha IIb beta 3, 2) building non-RGD-containing antibodies that bind the ligand binding site of both beta 3-integrins, and 3) testing the hypothesis that peptides derived from complementarity determining regions (CDR) can be used to emulate the activity of the parent synthetic antibody. These goals were accomplished by subjecting the original antibody, Fab-9, to a "motif optimization" (MTF). A phage library was constructed in which the residues flanking the RGD motif in Fab-9 were maintained, but the RGDX sequence was randomized. This library was panned on purified alpha IIb beta 3 to identify high affinity binders. Four function-blocking antibodies lacking RGD, but with specificity for alpha IIb beta 3, were characterized. The antibody with the highest preference for alpha IIb beta 3, MTF-10, had an adhesion sequence of KGDN. This sequence is similar in primary structure to the active sequence within the disintegrin barbourin, which also antagonizes alpha IIb beta 3 (Scarborough, R. M., Rose, J. W., Hsu, M. A., Phillips, D. R., Fried, V. A., Campbell, A. M., Nannizzi, L., and Charo, I. F. (1991) J. Biol. Chem. 266, 9359-9362). MTF-10 had a 70-fold higher affinity for alpha IIb beta 3 than alpha v beta 3. Through our selection strategy, we also identified several antibodies that lack RGD but still blocked ligand binding to both integrins with high affinity. Therefore, the RGD sequence is not necessary for a high affinity interaction with the ligand binding site of beta 3-integrins. Further investigation showed that the activity of inhibitory antibodies could be emulated by synthetic peptides derived from the protein sequences of the antibody's HCDR3. CDR-derived peptides blocked ligand binding to integrins and maintained essentially the same specificity as the parent antibody.

Amino Acid Sequence↗
PubMed1994

Human monoclonal antibodies: recent achievements.

The latest developments include an anti-HIV antibody that has 1,000 times the affinity of natural antibody and that neutralizes primary clinical isolates, and an anti-RSV antibody that reduces viral titers in mice by 12,000-fold within 24 hours.

Animals↗
PubMed1994

The nature of the autoimmune antibody repertoire in human immunodeficiency virus type 1 infection.

Human immunodeficiency virus type 1 (HIV-1) seropositive donors typically have high serum antibody titers to a range of autoantigens, and the corresponding autoantibodies have been suggested to be of importance in the pathogenesis of HIV-1 infection. We have prepared 38 IgG human monoclonal autoantibodies from asymptomatic HIV-1 seropositive donors with elevated serum titers to autoantigens by construction of Fab combinatorial libraries on the surface of phage and affinity selection using a range of autoantigens, including double-stranded DNA, major histocompatibility complex class II, CD14, epidermal growth factor receptor, and ganglioside GD2. The autoantibodies are shown to be of moderate affinity and exhibit marked cross-reactivity with a range of antigens. This contrasts with the specific high-affinity antibodies selected (i) against infectious agents using the same libraries and (ii) against one of the autoantigens using a library from a donor with established autoimmune disease. The results lend no support to the presence of specific autoantibodies in HIV-1 infection and instead suggest attention should be focused on the pathological significance of high serum levels of antibodies capable of interacting with multiple molecular species.

Amino Acid Sequence↗
PubMed1994

In vitro evolution of a neutralizing human antibody to human immunodeficiency virus type 1 to enhance affinity and broaden strain cross-reactivity.

A method is described that allows for the improvement of antibody affinity. This method, termed complementary-determining region (CDR) walking, does not require structural information on either antibody or antigen. Complementary-determining regions are targeted for random mutagenesis followed by selection for fitness, in this case increased binding affinity, by the phage-display approach. The current study targets a human CD4-binding-site anti-gp120 antibody that is potently and broadly neutralizing. Evolution of affinity of this antibody demonstrates in this case that affinity can be increased while reactivity to variants of human immunodeficiency virus type 1 is broadened. The neutralizing ability of this antibody is improved, as assayed with laboratory and primary clinical isolates of human immunodeficiency virus type 1. The ability to produce human antibodies of exceptional affinity and broad neutralizing ability has implications for the therapeutic and prophylactic application of antibodies for human immunodeficiency virus type 1 infection.

Amino Acid Sequence↗
PubMed1994

Direct selection for a catalytic mechanism from combinatorial antibody libraries.

Semisynthetic combinatorial antibody library methodology in the phage-display format was used to select for a cysteine residue in complementarity-determining regions. Libraries were panned with an alpha-phenethyl pyridyl disulfide that undergoes disulfide interchange. Out of 10 randomly picked clones, two contained an unpaired cysteine, one of which was studied. The antibody catalyzed the hydrolysis of the corresponding thioester where the electrophilic carbonyl occupies the three-dimensional space that was defined by the reactive sulfur atom during selection. The reaction operates by covalent catalysis. Although the steady-state rate enhancement relative to the activated thiol ester substrate is modest, hydrolysis of the acylated cysteine intermediate is remarkably efficient with a catalytic advantage of about four orders of magnitude. The results suggest that iterative mechanism-based selection procedures can recapitulate the enzymatic mechanisms refined through evolution.

Amino Acid Sequence↗
PubMed1994

Recombinant human respiratory syncytial virus (RSV) monoclonal antibody Fab is effective therapeutically when introduced directly into the lungs of RSV-infected mice.

Previously, recombinant human respiratory syncytial virus (RSV) monoclonal antibody Fabs were generated by antigen selection from random combinatorial libraries displayed at the tip of filamentous phage. Two such Fabs, which exhibited high binding affinity for RSV F glycoprotein (a major protective antigen), were evaluated for therapeutic efficacy in infected mice just before or at the time of peak virus replication in the lungs. Fab 19, which neutralized RSV infectivity with high efficiency in tissue culture, was effective therapeutically when delivered directly into the lungs by intranasal instillation under anesthesia. In contrast, RSV Fab 126, which failed to neutralize virus in cell culture, did not exhibit a therapeutic effect under these conditions. The amount of Fab 19 required to effect a 5000- to 12,000-fold reduction in titer of RSV in the lungs within 24 hr was rather small. In four separate experiments, a single instillation of 12.9-50 micrograms of RSV Fab 19 was sufficient to achieve such a reduction in pulmonary virus in a 25g mouse. The use of Fabs instead of the whole immunoglobulin molecules from which they are derived reduced the protein content of a therapeutic dose. This is important because the protein load that can be delivered effectively into the lungs is limited. The therapeutic effect of a single treatment with Fab 19 was not sustained, so that a rebound in pulmonary virus titer occurred on the 2nd day after treatment. This rebound in pulmonary RSV titer could be prevented by treating infected mice with a single dose of Fab 19 daily for 3 days. These observations suggest that human monoclonal Fabs grown in Escherichia coli may prove useful in the treatment of serious RSV disease as well as diseases caused by other viruses where replication in vivo is limited primarily to the lumenal lining of the respiratory tract.

Administration, Intranasal↗
PubMed1994

Recognition properties of a panel of human recombinant Fab fragments to the CD4 binding site of gp120 that show differing abilities to neutralize human immunodeficiency virus type 1.

Six recombinant human Fab fragments that were derived from the same human immunodeficiency virus type 1 (HIV-1)-infected individual and are directed against the CD4 binding site (CD4bs) of the gp120 envelope glycoprotein were studied. A range of neutralizing activity against the HIV-1 (HXBc2) isolate was observed, with Fab b12 exhibiting the greatest potency among the Fabs tested. The neutralizing potency of Fab b12 was better than that of monoclonal whole antibodies directed against the third variable (V3) region of gp120. To explore the basis for the efficient neutralizing activity of b12, the recognition of a panel of HIV-1 gp120 mutants by the six Fabs was studied. The patterns of sensitivity to particular gp120 amino acid changes were similar for all six Fabs to those seen for anti-CD4bs monoclonal antibodies derived from HIV-1-infected individuals by conventional means. In addition, recognition by Fab b12 demonstrated an atypical sensitivity to changes in the V1 and V2 variable regions. Next, the binding of the Fabs to monomeric gp120 and to the envelope glycoprotein complex was examined. Neither the binding properties of the b12 Fab to monomeric gp120 nor the ability of the Fab to compete with soluble CD4 for monomeric gp120 binding appeared to account for the greater neutralizing potency. However, both quantitative and qualitative differences between the binding of b12 and that of less potent Fabs to the cell surface envelope glycoprotein complex were observed. Relative to less potently neutralizing Fabs, Fab b12 exhibited a higher affinity for a subpopulation of cell surface envelope glycoproteins, the conformation of which was best approximated by the mature gp120 glycoprotein. Apparently, subtle differences in the gp120 epitope recognized allow some members of the group of anti-CD4bs antibodies to bind to the functionally relevant envelope glycoprotein complex and to neutralize virus more efficiently.

Animals↗