Directed in vitro evolution and crystallographic analysis of a peptide-binding single chain antibody fragment (scFv) with low picomolar affinity

We generated a single chain Fv fragment of an antibody (scFv) with a binding affinity of about 5 pm to a short peptide by applying rigorous directed evolution. Starting from a high affinity peptide binder, originally obtained by ribosome display from a murine library, we generated libraries of mutants with error-prone PCR and DNA shuffling and applied off-rate selection by using ribosome display. Crystallographic analysis of the scFv in its antigen-bound and free state showed that only few mutations, which do not make direct contact to the antigen, lead to a 500-fold affinity improvement over its potential germ line precursor. These results suggest that the affinity optimization of very high affinity binders is achieved by modulating existing interactions via subtle changes in the framework rather than by introducing new contacts. Off-rate selection in combination with ribosome display can evolve binders to the low picomolar affinity range even for peptide targets.     

Immunolabeling of CD3-positive lymphocytes with a recombinant single-chain antibody/alkaline phosphatase conjugate

G3(3) is a novel murine monoclonal antibody directed against the CD3 antigen of human T lymphocytes which could be used to analyze lymphoid malignancies. We have produced and characterized a recombinant colorimetric immunoconjugate with the antigen-binding specificity of antibody G3(3). A gene encoding a single-chain antibody variable fragment (scFv) was assembled using the original hybridoma cells as a source of antibody variable heavy (VH) and variable light (VL) chain genes. The chimeric gene was introduced into a prokaryotic expression vector in order to produce a soluble scFv fused to bacterial alkaline phosphatase. DNA sequencing and Western blotting analyses demonstrated the integrity of the soluble immunoconjugate recovered from induced recombinant bacteria. The scFv/AP protein was bifunctional and similar in immunoreactivity to the parent G3(3) antibody. Flow cytometry and immunostaining experiments confirmed that the activity of the scFv/AP protein compares favourably with that of the parent antibody. The scFv/AP conjugate was bound to CD3 antigen at the surface of T cells and was directly detected by its enzymatic activity. Thus this novel fusion protein has potential applications as an immunodiagnostic reagent.     

A kinetic trap is an intrinsic feature in the folding pathway of single-chain Fv fragments

We have studied the equilibrium unfolding and the kinetics of folding and unfolding of an antibody scFv fragment devoid of cis-prolines. An anti-GCN4 scFv fragment carrying a VL lambda domain, obtained by ribosome display, served as the model system together with an engineered destabilized mutant in VH carrying the R66K exchange. Kinetic and equilibrium unfolding experiments indicate that the VH mutation also affects VL unfolding, possibly by partially destabilizing the interface provided by VH, even though the mutation is distant from the interface. Upon folding of the scFv fragment, a kinetic trap is populated whose escape rate is much faster with the more stable VH domain. The formation of the trap can be avoided if refolding is carried out stepwise, with VH folding first. These results show that antibody scFv fragments do not fold by the much faster independent domain folding, but instead form a kinetically trapped off-pathway intermediate, which slows down folding under native conditions. This intermediate is characterized by premature interaction of the unfolded domains, and particularly involving unfolded VH, independent of proline cis-trans isomerization in VL. This work also implies that VH should be a prime target in engineering well behaving antibody fragments.     

单链抗体展示系统研究进展

单链抗体(single chain antibody fragment,scFv)是由抗体重链可变区(variable region of heavy chain,VH)和轻链可变区(variable region of light chain,VL)通过柔性短肽连接组成的小分子,是具有完整抗原结合活性的最小功能片段,包含抗体识别及抗原结合部位。相比于其他抗体,scFv具有分子量小、穿透性强、免疫原性弱、易构建表达等优点。目前,scFv最常用的展示系统主要有噬菌体展示系统、核糖体展示系统、mRNA展示系统、酵母细胞表面展示系统和哺乳动物细胞展示系统等。近年来,随着scFv在医学、生物学、食品安全学等领域的发展,使得其在生物合成和应用研究方面备受关注。本文对近年来scFv展示系统的研究进展作一综述,以期为scFv的筛选及应用提供理论基础。     

A new approach for rapidly reshaping single-chain antibody in vitro by combining DNA shuffling with ribosome display

Antibody reshaping is an effective way to reduce the immunogenicity while maintaining or improving the affinity of murine antibodies. This paper describe a new in vitro approach for rapidly reshaping murine antibodies by combining DNA shuffling with ribosome display. With the new method, a reshaping anti-4-1BB single-chain antibody (scFv), Re-4B4-1 scFv, which bound to its antigen (4-1BB) specifically and strongly, was selected from a reshaping library. These results proved definitely the feasibility of the new designed approach for antibody reshaping.     

Synthesis, cloning and expression of the single-chain Fv gene of the HPr-specific monoclonal antibody, Jel42. Determination of binding constants with wild-type and mutant HPrs.

The monoclonal antibody Jel42 is specific for the Escherichia coli histidine-containing protein, HPr, which is an 85 amino acid phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system. The binding domain (Fv) has been produced as a single chain Fv (scFv). The scFv gene was synthesized in vitro and coded for pelB leader peptide-heavy chain-linker-light chain-(His)(5) tail. The linker is three repeats from the C-terminal repetitive sequence of eukaryotic RNA polymerase II. This linker acts as a tag; it is the antigen for the monoclonal antibody Jel352. The codon usage was maximized for E.coli expression, and many unique restriction endonuclease sites were incorporated. The scFv gene incorporated into pT7-7 was highly expressed, yielding 10-30% of the cell protein as the scFv, which was found in inclusion bodies with the leader peptide cleaved. Jel42 scFv was purified by denaturation/renaturation yielding preparations with K(d) values from 20 to 175 nM. However, based upon an assessment of the amount of active refolded scFv, the binding dissociation constant was estimated to be 2.7 +/- 2.0 nM compared with 2.8 +/- 1.6 and 3.7 +/- 0.3 nM previously determined for the Jel42 antibody and Fab fragment respectively. The effect of mutation of the antigen HPr on the binding constant of the scFv was very similar to the properties determined for the antibody and the Fab fragment. It was concluded that the small percentage ( approximately 6%) of refolded scFv is a true mimic of the Jel42 binding domain and that the incorrectly folded scFv cannot be detected in the binding assay.     

利用核糖体展示技术筛选口蹄疫病毒 单链抗体基因

目的:利用核糖体展示技术筛选口蹄疫病毒特异性单链抗体基因。方法:在已构建好的核糖体展示文库的基础上,利用核糖体展示技术,经过5轮的体外转录、体外转译、亲和筛选和RT-PCR,将得到的序列进行测序分析。结果:筛选到FMDV scFv基因,且基因得到富集。结论:实验运用核糖体展示技术,以FMDV抗原和纯化的146S病毒粒子为靶标筛选到了FMDV scFv基因,将为scFv用于FMD的基础研究、免疫学研究以及为预防、治疗和诊断提供帮助,也为研制FMD的快速诊断技术奠定先前基础。     

Ribosome display and selection of human anti-cD22 scFvs derived from an acute lymphocytic leukemia patient

Novel in vitro methods for the display of antibody libraries against disease-related antigens have led to the development of powerful protein-based biotherapeutics. Eukaryotic ternary ribosome complexes can be used to display human single chain antibodies (scFvs) to isolate specific binding reagents to these antigens. Here, we present the isolation of human scFv against the immunotherapeutic target antigen CD22 from a patient-derived human scFv library using ribosome display technology. The ribosome complexes were enriched against the extra-cellular domain of human CD22 conjugated to magnetic beads. Isolated constructs were further affinity-matured and specific binding activity was demonstrated by surface plasmon resonance and validated using in vitro cell assays. The isolated human anti-CD22 scFvs can provide a basis for the development of new immunotherapeutic strategies in CD22-expressing malignant diseases.     

Very high expression of an anti-carcinoembryonic antigen single chain Fv antibody fragment in the yeast Pichia pastoris

In this paper we report the development of a recombinant strain of the yeast Pichia pastoris, which secretes an anti-carcinoembryonic antigen single chain Fv (scFv) antibody fragment to the culture supernatant as a biologically active protein, at levels of 1.2 g l(-1). The yeast scFv was purified by IMAC, with a final yield of approximately 0.440 g of 93% pure scFv per liter of culture supernatant. The specific activity in ELISA of the yeast scFv was almost three times higher than that of a bacterial periplasmic counterpart. These results reaffirm that the yeast P. pastoris is a suitable host for high level production of scFv antibody fragments with potential in vivo diagnostic and therapeutic applications.     

Cloning,expression, and identification of anti‐carbofuran single chain Fv gene

Phage display method was used to clone anti‐carbofuran (CBF) single chain Fv (scFv) gene. The heavy chain and light chain variable region genes were amplified by the polymerase chain reaction from the CBF‐specific hybridoma cell lines 5D3 and assembled as a scFv DNA fragment with linker peptide (Gly₄Ser)₃. The scFv DNA fragment was cloned into M13 phagemid vector pCANTAB5E and the anti‐CBF antibody libraries were then constructed. After one round of panning with CBF‐ovalbumin (CBF‐OVA) as a conjugate, antigen‐binding positive recombinant phage clones were successfully selected by enzyme‐linked immunosorbent assay (ELISA). The positive phages were used to infect Escherichia coli HB2151 cells and the expression of the soluble scFv antibodies was then induced by IPTG. The scFv antibody was about 31 kDa by SDS‐PAGE and showed HRP‐anti‐E‐tag antibody‐recognized activity by Western blotting. The indirect competitive ELISA (icELISA) showed that the recombinant scFv antibody could competitively combine with CBF, with the IC₅₀ value of 1.07 ng/mL. The cross reactivity studies showed that the anti‐CBF scFv antibody, similar to the parent monoclonal antibody, poses high specificity to CBF and has little reactivity to the analogs. Taken together, these findings suggest that the recombinant scFv antibody can be used for further developing immunoassay method for CBF. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Domain interactions in antibody Fv and scFv fragments: effects on unfolding kinetics and equilibria

The equilibrium denaturation and unfolding kinetics of the domains V(L) and V(H) have been compared with those of the Fv and single-chain Fv (scFv) fragment of an engineered variant of the antibody McPC603 in the presence and absence of the antigen phosphorylcholine. The scFv fragment is significantly more stable than the isolated constituting domains. Antigen binding stabilizes the heterodimeric assembly even further. Domain dissociation and domain unfolding are coupled processes, giving rise to a highly cooperative unfolding transition. For the Fv fragment, cooperative unfolding is only observed in the presence of antigen. At low protein concentrations and in the absence of antigen, the Fv fragment is significantly destabilized, leading to quantitative domain dissociation before significant domain unfolding takes place. The kinetic unfolding of V(H), V(L) and the scFv fragment is monophasic. Unfolding of the scFv fragment is much slower, when extrapolated to zero denaturant, than either of the isolated domains, suggesting that the higher thermodynamic stability of the scFv fragment is at least partially due to a high-energy transition state for unfolding. These studies emphasize the enormous importance of mutual domain stabilization in engineering stable antibodies.     

Generation of a murine single chain Fv (scFv) antibody specific for cucumber mosaic virus (CMV) using a phage display library

With the long-term goal of generating CMV-resistant transgenic plants using antibody genes, a single-chain variable fragment (scFv) antibody that binds to the cucumber mosaic virus was isolated from a scFv phage display library by four rounds of affinity selection with CMV-Mf as an antigen. The scFv has the identical binding specificity to CMV as a monoclonal antibody that is generated by the hybridoma fusion technique, and recognized purified preparations of CMV isolates belonging to either subgroup I or II in immunoblotting. The nucleotide sequences of the recombinant antibody showed that a heavy chain variable region (V(H)) gene belonged to the VH3 subgroup and the kappa light chain variable region (V kappa) came from the Vkappa4 subgroup. Our results demonstrate that the scFv phage display library, an alternative approach to the traditional hybridoma fusion technique, has a potential applicability in the study of plant virus and plant pathology.     

Construction and characterization of a pseudo-immune human antibody library using yeast surface display

Lymphocytes from eight individuals out of 60 healthy donors, whose plasmas showed relatively higher antibody titer for a target antigen of death receptor 5 (DR5), were selected for the source of antibody genes to construct so called an anti-DR5 pseudo-immune human single-chain fragment variable (scFv) library on the yeast cell surface (approximately 2x10(6) diversity). Compared with a large nonimmune human scFv library (approximately 1x10(9) diversity), the repertoire of the pseudo-immune scFv library was significantly biased toward the target antigen, which facilitated rapid enrichments of the target-specific high affinity scFvs during selections by fluorescence activated cell sortings. Isolated scFvs, HW5 and HW6, from the pseudo-immune library showed much higher specificity and affinity for the targeted antigen than those from the nonimmune library. Our results suggest that a pseudo-immune antibody library is very efficient to isolate target-specific high affinity antibody from a relatively small sized library.     

Functional construction of the anti-mucin core protein (MUC1) antibody MUSE11 variable regions in a bacterial expression system

A bacterial expression system for the variable region fragments (Fvs) of the anti-MUC1 tumor antigen antibody MUSE11 has been constructed. The Fv fragment showed binding specificity toward TFK-1 cells, with slightly reduced affinity compared to its parent IgG. The single-chain Fv fragment was arranged in two orders, VH-linker-VL and VL-linker-VH. However, linking the regions with a flexible peptide linker (GGGGS)(3) or with a shorter linker (GGGGS) led to a dramatic decrease in the biological activity toward the target antigen in both arrangements, suggesting that the MUSE11 antibody loses its activity when the domains are linked with polypeptide linkers. These results indicate that the variable region domains of the anti-MUC1 antibody MUSE11 have specificity only in the Fv form, and that linking the domains strongly reduces the association with its target antigen. Gel filtration analysis indicates that the scFv has a dimeric structure, suggesting that the inactivation of MUSE11 scFv is due to unfavorable intermolecular associations of the scFv chains. To our knowledge, this is the first report of a significant reduction in affinity caused by linking the variable domains in both arrangements, i.e., VH-VL and VL-VH.     

A novel single-chain Fv antibody for connective tissue growth factor against the differentiation of fibroblast into myofibroblast

This study was aimed to investigate the effect of a single-chain fragment variable antibody of connective tissue growth factor (anti-CTGF scFv) against the differentiation of fibroblast into myofibroblast. The scFv antibody was firstly expressed in Escherichia coli cells and was then purified by affinity chromatography. The yield scFv protein reached a purity over 95% after purification. Immunoreactivity assay demonstrated that scFv possessed a special affinity toward CTGF. RT-PCR, western blot, and immunofluorescence experiments showed that increased expression of α-smooth muscle actin induced by TGF-β1 could be suppressed by this scFv antibody through inhibiting the phosphorylation of Akt.     

Inhibition of Candida albicans adhesion by recombinant human antibody single-chain variable fragment specific for Als3p

The Candida albicans adhesin, Als3p, was identified as a potential cognate antigen for previously described human antibody fragments [single-chain variable fragment (scFv)] based on similarity of the binding pattern of the scFv to the distribution of this protein on the hyphal surface. Although all scFv bound avidly to wild type, scFv3 showed no detectable binding via immunofluorescence assay to strain 1843, containing a homozygous deletion of ALS3. Binding to the ALS3 reintegrant strain, 2322, was preserved, and scFv3 also bound to Saccharomyces cerevisiae expressing ALS3. Other scFv retained binding to 1843, but with a markedly altered pattern. To determine if scFv3 could interfere with Als3p function, adhesion assays were conducted using human epithelial or endothelial cells as target. Treatment of wild-type C. albicans with scFv3 reduced adhesion of the fungus to both cell types to levels comparable to the als3Delta/als3Delta mutant. These experiments confirm that phage display is a viable method to isolate human scFv specific to an antigen implicated in C. albicans virulence, and that the scFv interfere with adhesion to human cells. The altered pattern of immunostaining with other scFv that retain binding to the als3Delta/als3Delta mutant suggest that Als3p may also have a role in structural organization of the C. albicans cell surface.     

Engineering stable cytoplasmic intrabodies with designed specificity

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.