Production of stable anti-digoxin Fv in escherichia coli

Anthony, James; Near, Richard I.; Sui-lam, Wong; Iida, Eric; Ernst, Eileen; Wittekind, Michael; Haber, Edgar; Ng, Shi-Chung

doi:10.1016/0161-5890(92)90060-b

Cited by 31 publications

(9 citation statements)

References 23 publications

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“…A previous report described a different specificity profile between the scFv expressed in bacteria and the F ab obtained from the same mAb [10], but no specificity difference has been described until now between scFvs showing the same nucleotidic sequence and expressed in eucaryotic and procaryotic vectors [27, 28]. Nevertheless, the majority of scFvs described in the literature do not show any change in the specificity profile compared with the parental mAb [60, 61]. The possibility cannot excluded however that fine variations do exist but have not yet been identified.…”

Section: Discussionmentioning

confidence: 95%

Anti‐digoxin scFv fragments expressed in bacteria and in insect cells have different antigen binding properties

Lemeulle¹,

Chardès²,

Montavon³

et al. 1998

FEBS Letters

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A gene encoding a single-chain antibody fragment directed against digoxin (named 1C10 scFv) was cloned in two expression systems. For this purpose, a new baculovirus transfer cassette fully compatible with the procaryotic pHEN vector was constructed. Baculovirus production led to higher yield than did Escherichia coli expression. The procaryotic fragment showed variations in the fine specificity profile but an affinity constant nearly identical to that of the 1C10 F ab , whereas the eucaryotic scFv fragment had a lower affinity with a specificity profile identical to original mAb. The half-lives of the digoxin:scFv complexes and the global specificity are compatible with therapeutic use of this antibody fragment.z 1998 Federation of European Biochemical Societies.

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Section: Discussionmentioning

confidence: 95%

Anti‐digoxin scFv fragments expressed in bacteria and in insect cells have different antigen binding properties

Lemeulle¹,

Chardès²,

Montavon³

et al. 1998

FEBS Letters

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“…1, and the modified phagemids were transformed into XL1-Blue. The transformants were grown in 2x YT (1.6% tryptone, 1% yeast extract, 0.5% sodium chloride, 0.2% glycerol, 50 mM potassium phosphate, pH 7.2) at 30~ to an ODsgs of 0.1, induced with 1 mM isopropyl ~-D-thiogalactoside, and further incubated for 18-36 h. Periplasmic contents were extracted as described (20). Fabs were affinity purified by chromatography on Arsbovine gamma globulin Sepharose 4B (Pharmacia Biotech, Inc., Piscataway, NJ) as previously described (21).…”

Section: Expression Characterization and Purification Of Fabs Phagmentioning

confidence: 99%

Random mutagenesis of two complementarity determining region amino acids yields an unexpectedly high frequency of antibodies with increased affinity for both cognate antigen and autoantigen.

Casson

Manser

1995

The Journal of Experimental Medicine

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SummaryTo gain insight into the mechanism and limitations of antibody affinity maturation leading to memory B cell formation, we generated a phage display library of random mutants at heavy chain variable (V) complementarity determining region 2 positions 58 and 59 of an anti-pazophenylarsonate (Ars) Fab. Single amino acid substitutions at these positions resulting from somatic hypermutation are recurrent products of affinity maturation in vivo. Most of the ex vivo mutants retained specificity for Ars. Among the many mutants displaying high Ars-binding activity, only one contained a position 58 and 59 amino acid combination that has been previously observed among monoclonal antibodies (mAbs) derived from Ars-immunized mice. Affinity measurements on 14 of the ex vivo mutants with high Ars-binding activity showed that 11 had higher intrinsic affinities for Ars than the wild-type V region. However, nine of these Fabs also bound strongly to denatured DNA, a property neither displayed by the wild-type V region nor observed among the mutants characteristic of in vivo affinity maturation. These data suggest that ex vivo enhancement of mAb affinity via site-directed and random mutagenesis approaches may often lead to a reduction in antibody specificity that could complicate the use of the resulting mAbs for diagnostic and therapeutic applications. Moreover, the data are compatible with a hypothesis proposing that increased specificity for antigen, rather than affinity per se, is the driving force for formation of the memory B cell compartment.D uring the course of a T cell-dependent antibody response, the V regions of antibodies expressed by B cells differentiating to memory are structurally altered because of hypermutation of the genes encoding them. Working in concert with hypermutation, a stringent process of antigen selection results in the affinity maturation of the antibody response. Frequent end products of the mutation-selection process are V regions containing recurrent amino acid changes at particular positions (1-3). Site-directed mutagenesis studies have shown that many of these recurrent mutations individually result in increased affinity for the eliciting antigen (2-4).Affinity for antigen is only one of the factors that could influence the composition of the somatically mutated antibody repertoire expressed by the memory B cell compartment, however. Somatic mutation takes place throughout the length of V region genes, but is not a random process; hotspots and hot areas for mutation clearly exist (1, 5-7). In some responses, mutants with altered idiotypy (8) or antigen binding on-rate (9) may be differentially selected. It has also been suggested that somatic mutation will create autoantibodies de novo (10-13), and that peripheral tolerance mechanisms must eliminate B cells expressing these autoreactive antibodies (14).Secondary anti-p-azophenylarsonate (Ars) 1 antibodies from A/J mice are largely encoded by a single combination of V gene segments (termed "canonical"). Recurrent amino acid substitutions du...

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“…The requirement for antigen binding capacity supports this mechanism. Both hapten and protein antigens have been shown to facilitate the pairing and folding in vitro of several V H and V L (22,23,39). Similarly, peptide has been shown to directly facilitate class I heavy chain association with ␤ 2 -microglobulin and folding in cell lysates in vitro (40,41).…”

Section: Nppc Rescues Ig Secretion From Ilementioning

confidence: 99%

Recovering Antibody Secretion Using a Hapten Ligand as a Chemical Chaperone

Wiens¹,

O’Hare²,

Rittenberg³

2001

Journal of Biological Chemistry

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Engineered antibodies have come to the forefront as research reagents and clinical therapeutics. However, reduced stability or expression levels pose a major problem with many engineered antibodies. As a model for understanding functional consequences of variable region mutation, we have studied the assembly and trafficking of anti-phenylphosphocholine antibodies. Previously, we identified severe secretion defects because of mutations in the heavy chain second complementarity determining region, which is involved in antigen binding. Here we demonstrate that immunoglobulin secretion is increased up to 27-fold by incubating stably transfected PCG1-1 cells with cognate hapten p-nitrophenylphosphocholine. Secretion was unaffected by nonbinding analogs. Radiotracer and metabolic labeling experiments demonstrated specific cellular uptake of p-nitrophenylphosphocholine and increased intracellular heavy and light chain assembly. Brefeldin A inhibited hapten-mediated immunoglobulin secretion but not assembly, indicating that assembly occurs early within the biosynthetic pathway. Recovery of secretion correlated with antigen binding capacity, suggesting that the rescue mechanism involves stabilization of heavy and light chain variable domains. This model system provides the first demonstration that cognate ligands can increase intracellular assembly of functional anti-hapten antibody within mammalian cells and suggests that small molecules of appropriate specificity and affinity acting as chemical chaperones may find application for increasing or regulating immunoglobulin expression.

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Production of stable anti-digoxin Fv in escherichia coli

Cited by 31 publications

References 23 publications

Anti‐digoxin scFv fragments expressed in bacteria and in insect cells have different antigen binding properties

Anti‐digoxin scFv fragments expressed in bacteria and in insect cells have different antigen binding properties

Random mutagenesis of two complementarity determining region amino acids yields an unexpectedly high frequency of antibodies with increased affinity for both cognate antigen and autoantigen.

Recovering Antibody Secretion Using a Hapten Ligand as a Chemical Chaperone

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