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Query: UNIPROT:P06889 (Mol)
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Humanization of monoclonal antibodies by complementary determinant region (CDR)-grafting has become a standard procedure to improve the clinical usage of animal antibodies. However, antibody humanization may result in loss of activity that has been attributed to structural constraints in the framework structure. In this paper, we report the complete humanization of the 6.7 anti-human CD18 monoclonal antibody in a scFv form. We used a germline-based approach to design a humanized VL gene fragment and expressed it together with a previously described humanized VH. The designed humanized VL has only 14 mutations compared to the closest human germline sequence. The resulting humanized scFv maintained the binding capacity and specificity to human CD18 expressed on the cell surface of peripheral blood mononuclear cells (PBMC), and showed the same pattern of staining T-lymphocytes sub-populations, in comparison to the original monoclonal antibody. We observed an unexpected effect of a conserved mouse-human framework position (L37) that hinders the binding of the humanized scFv to antigen. This paper reveals a new framework residue that interferes with paratope and antigen binding and also reinforces the germline approach as a successful strategy to humanize antibodies.
Mol Immunol 2003 May
PMID:Humanization of the anti-CD18 antibody 6.7: an unexpected effect of a framework residue in binding to antigen. 1269 20

Protein and other antigens typically have a number of different epitopes. This presents an opportunity for designing high-affinity antibodies by connecting via a flexible peptide linker two antibody fragments recognizing non-overlapping epitopes on the same antigen. The same strategy was employed in natural and designed DNA-binding proteins. According to a previous theory, the linking enhances the antigen-binding affinity over those of the individual antibody fragments (with association constants K(A) and K(B)) by p(d(0))K(B) or p(d(0))K(A), where p(d(0))=(3/4pil(p)bL)(3/2)exp(-3d(0)(2)/4l(p)bL)(1-5l(p)/4bL+ cdots, three dots, centered ) is the probability density for the end-to-end vector of the flexible linker with L residues to have a distance d(0). The predicted affinity enhancement is found to be actually approached by a bi-specific antibody against hen egg lysozyme consisting of scFv fragments of D1.3 and HyHEL-10. The wide applicability of the theory is demonstrated by diverse examples of protein-protein interactions constrained by flexible linkers.
J Mol Biol 2003 May 23
PMID:Quantitative account of the enhanced affinity of two linked scFvs specific for different epitopes on the same antigen. 1274 13

Many attempts have been made to develop antibody fragments that can be expressed in the cytoplasm ("intrabodies") in a stable and functional form. The recombinant antibody fragment scFv(F8) is characterised by peculiarly high in vitro stability and functional folding in both prokaryotic and eukaryotic cytoplasm. To dissect the relative contribution of different scFv(F8) regions to cytoplasmic stability and specificity we designed and constructed five chimeric molecules (scFv-P1 to P5) in which several groups of residues important for antigen binding in the poorly stable anti-hen egg lysozyme (HEL) scFv(D1.3) were progressively grafted onto the scFv(F8) scaffold. All five chimeric scFvs were expressed in a soluble form in the periplasm and cytoplasm of Escherichia coli. All the periplasmic oxidised forms and the scFv(P3) extracted from the cytoplasm in reducing conditions had HEL binding affinities essentially identical (K(d)=15nM) to that of the cognate scFv(D1.3) fragment (K(d)=16nM). The successful grafting of the antigen binding properties of D1.3 onto the scFv(F8) opens the road to the exploitation of this molecule as a scaffold for the reshaping of intrabodies with desired specificities to be targeted to the cytoplasm.
J Mol Biol 2003 Jul 04
PMID:Engineering stable cytoplasmic intrabodies with designed specificity. 1282 71

The scFv antibody towards the Burkholderia pseudomallei exotoxin was previously constructed by phage display and exhibited good specificity towards the exotoxin. We report here the optimization of the scFv expression in an E. coli expression system. Four different E. coli strains (ER2537, TG1, HB2151, and XL1-Blue) were examined for optimal expression of the scFv protein. Two types of carbon source (i.e. 0.2% glucose and 0.2% glycerol) were also tested for their ability to induce the scFv expression. Cells that carried the scFv construct were grown at 30 degrees C and induced with 0.05 mM IPTG. The expression was then monitored by SDS-PAGE, Western blotting, and indirect ELISA. The Western blot profile showed different levels of the scFv expression among the host strains; XL1-Blue exhibited the highest level of the scFv protein expression. Glycerol at a concentration of 0.2% (v/v) significantly increased the scFv protein expression level when compared to 0.2% (w/v) glucose. Further optimization demonstrated that the scFv protein expression in XL1-Blue was the most optimal with a glycerol concentration as low as 0.05%. However, by indirect ELISA, only the scFv protein that was expressed in 0.2% (v/v) glycerol exhibited high specificity towards the Burkholderia pseudomallei exotoxin.
J Biochem Mol Biol 2003 Sep 30
PMID:Bacterial expression of the scFv fragment of a recombinant antibody specific for Burkholderia pseudomallei exotoxin. 1453 33

A recombinant scFv antibody against human spleen ferritin was expressed as a barstar-fused protein in Escherichia coli and in Nicotiana tabacum plants and suspension cell cultures. As demonstrated by immunoblotting with antibarstar antibodies, direction of the recombinant protein to the endomembrane system of plant cells ensured its stability and solubility. Production of the recombinant protein did not differ between parental transgenic plants and their first-generation progeny. Fusion with barstar allowed not only immunochemical detection of the recombinant scFv antibody, but also their purification from the plant material by affinity chromatography with barnase-His6 immobilized on a metal-affinity carrier.
Mol Biol (Mosk)
PMID:[Biosynthesis of mini-antibodies to human ferritin in plant and bacterial producers]. 1459 30

We raised a mouse monoclonal antibody (5S) against the 'a' epitope of the Hepatitis B surface antigen (HBsAg) by selecting for binding of the hybridoma supernatant in conditions that usually destabilize protein-protein interactions. This antibody, which was protective in an in vitro assay, had a high affinity with a relative dissociation constant in the nanomolar range. It also displayed stable binding to antigen in conditions that usually destabilize antigen-antibody interactions, like 30% DMSO, 8 M urea, 4 M NaCl, 1 M guanidium HCl and extremes of pH. The variable regions of the antibody were cloned and expressed as an single chain variable fragment (scFv) (A5). A5 had a relative affinity comparable to the mouse monoclonal and showed antigen binding in presence of 20% DMSO, 8 M urea and 3 M NaCl. It bound the antigen in the pH range of 6-8, though its tolerance for guanidium HCl was reduced. Sequence analysis demonstrated a significant increase in the frequency of somatic replacement mutations in CDRs over framework regions in the light but not in the heavy chain. A comparison of the molecular models of the variable regions of the 5S antibody and its germ-line precursor revealed that critical mutations in the heavy and light chains interface resulted in better inter-chain packing and in the movement of CDR H3 and CDR L1 from their germline positions, which may be important for better antigen binding. In addition to providing a reagent for neutralizing for the virus, such an antibody provides a model for the evolution of stable high affinity interaction during antibody maturation.
Mol Immunol 2003 Dec
PMID:Characterization and molecular modeling of a highly stable anti-Hepatitis B surface antigen scFv. 1459 65

We describe a novel approach for high-throughput screening of recombinant antibodies, based on their immobilization on solid cellulose-based supports. We constructed a large human synthetic single-chain Fv antibody library where in vivo formed complementarity determining regions were shuffled combinatorially onto germline-derived human variable-region frameworks. The arraying of library-derived scFvs was facilitated by our unique display/expression system, where scFvs are expressed as fusion proteins with a cellulose-binding domain (CBD). Escherichia coli cells expressing library-derived scFv-CBDs are grown on a porous master filter on top of a second cellulose-based filter that captures the antibodies secreted by the bacteria. The cellulose filter is probed with labeled antigen allowing the identification of specific binders and the recovery of the original bacterial clones from the master filter. These filters may be simultaneously probed with a number of antigens allowing the isolation of a number of binding specificities and the validation of specificity of binders. We screened the library against a number of cancer-related peptides, proteins, and peptide-protein complexes and yielded antibody fragments exhibiting dissociation constants in the low nanomolar range. We expect our new antibody phage library to become a valuable source of antibodies to many different targets, and to play a vital role in facilitating high-throughput target discovery and validation in the area of functional cancer genomics.
J Mol Biol 2004 Jan 02
PMID:A human synthetic combinatorial library of arrayable single-chain antibodies based on shuffling in vivo formed CDRs into general framework regions. 1465 49

Several human monoclonal antibodies can neutralize a range of human immunodeficiency virus type 1 (HIV-1) primary isolates but their potency and related ability to suppress generation of HIV-1 escape mutants is significantly lower than the activity of antiretroviral drugs currently in clinical use. Recently, a human Fab, X5, was identified and found to neutralize primary isolates from different clades. Further improvement of the potency and breadth of HIV-1 neutralization by this antibody could be critical for its potential use in the treatment of HIV-1-infected patients. However, increasing potency of an antibody by selection from libraries may lead to a decrease in the breadth of neutralization. In an attempt to solve this problem, we subjected a random mutagenesis library of the scFv X5 to sequential rounds of selection on non-homologous HIV-1 envelope glycoproteins (Envs) dubbed sequential antigen panning (SAP). By using SAP, we identified two scFv antibodies, m6 and m9, that were tested with a panel of 33 diverse primary HIV-1 infectious isolates in an assay based on a reporter cell-line expressing high levels of CD4, CCR5 and CXCR4. The IC(50) was less than 50 microg/ml for 21 (m6) and 19 (m9) out of 29 isolates from group M (subtypes A-C, F, G and CRF-01AE) and one isolate from group N; three isolates from group O were not significantly inhibited at 50 microg/ml. The average IC(50) values for the two antibodies were significantly (p<0.001, n=29) lower compared to scFv X5. Their inhibitory activity does not appear to be related to the HIV-1 subtype, coreceptor usage or the disease stage. m9 inhibited infection of peripheral blood mononuclear cells by the primary isolates JRCSF, 89.6 and BR020 with IC(90) of 4, 6 and 25 microg/ml, respectively; for a single-round infection by pseudovirus, the IC(90) for JRSCF, 89.6, YU2 and HXBc2 was 15, 5, 15 and 5 microg/ml, respectively. In these two assays the IC(90) for m9 was, on average, two- to threefold lower than for scFv X5. These results demonstrate that both the potency and the breadth of HIV-1 neutralization of one of the few known potent broadly cross-reactive human monoclonal antibodies, scFv X5, could be improved significantly. However, only experiments in animal models and clinical trials in humans will show whether these new scFvs and the approach for their identification have potential in the development of prophylactics and therapeutics for HIV-1 infections.
J Mol Biol 2004 Jan 02
PMID:Improved breadth and potency of an HIV-1-neutralizing human single-chain antibody by random mutagenesis and sequential antigen panning. 1465 51

Antibodies or antibody fragments represent a powerful class of targeting moieties to specifically attach proteins to the surface of a given cell or tissue. Since the presence of constant antibody domains in these targeted fusion proteins may have certain disadvantages, we report here the reduction of the targeting epitope to the variable regions of an Ab by the generation of a single chain antibody fragment (scFv). As an effector molecule, we attached the human complement regulatory protein (CRP) decay-accelerating factor (DAF) at its amino-terminus with a scFv specific for TER-119, a red blood cell (RBC) restricted surface antigen of the mouse. This heterologous system enabled us to study (a) the applicability of a scFv as a targeting domain, (b) the functionality of the effector molecule with respect to regulation of the complement cascade in vitro, and (c) the in vivo biodistribution characteristics of a scFv-DAF fusion protein attached to a clinically relevant target cell type. RBCs from C57BL/6 mice loaded in vitro or in vivo with this fusion protein were significantly protected against lysis by human complement. After intravenous injection, a homogeneous population of in vivo tagged RBCs was maintained throughout a 6-day follow-up. This result and in vitro mixing experiments indicated that there was an equilibration of the fusion protein between tagged and non-tagged RBCs. Thus, scFv-mediated targeting of proteins to a selected cell or tissue surface has promise as a means to supplement absent or defective plasma membrane constituents. This approach should therefore be applicable for diseases caused by a membrane protein deficiency such as paroxysmal nocturnal hemoglobinuria (PNH).
Mol Immunol 2004 Feb
PMID:ScFv-mediated in vivo targeting of DAF to erythrocytes inhibits lysis by complement. 1472 87

Site-directed mutagenesis (SDM) is a powerful tool for exploring protein structure and function, and several procedures adjusted to specific purposes are still being developed. Herein we describe a straightforward and efficient method with versatile applications for introducing site-specific alterations in any deoxyribonucleic acid (DNA) sequence cloned in a plasmidic expression vector. In this polymerase chain reaction (PCR)-based SDM method, forward and reverse primers are used to amplify the plasmid containing the sequence of interest. The primers are designed so that the desired modifications are introduced at the 5' end of one of the primers, whereas the other primer starts with the nucleotide at position (-1) of the one to be modified. The PCR is carried out using Pfu DNA polymerase. The blunt-ended PCR-generated DNA fragment is self-ligated and used to transform Escherichia coli. Mutant clones are screened by colony hybridization using the mutagenic primer as probe and the presence of the mutation is confirmed by direct DNA sequencing. This procedure was used efficiently to introduce substitutions, deletions, and insertions in the DNA sequences coding for a recombinant form (scFv) of antibody 107 specific of the human CR3 molecule, the rat alpha integrin CD11b A-domain and the human CD8beta cloned in pPICZalphaB, pGEX-2T, and CDM8 expression vectors, respectively.
Mol Biotechnol 2004 Jan
PMID:A novel simple and rapid PCR-based site-directed mutagenesis method. 1473 21


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