Expression and functional analysis of recombinant scFv and diabody fragments with specificity for human RhD

In an attempt to generate recombinant anti-D reagents for possible diagnostic and therapeutic use we cloned the genes encoding the variable (V) domains of a human anti-D antibody secreted by the lymphoblastoid cell line BTSN4. A single-chain Fv (scFv) fragment was constructed using a 21 amino acid linker to join the genes encoding the variable domains of the BTSN4 heavy (VH) and light chains (VL). A diabody construct was also generated by reducing the length of the scFv linker from 21 to 10 residues. The scFv and diabody constructs were cloned into the pFLAG-CTS vector, expressed in E. coli host cells and the recombinant proteins were affinity-isolated from bacterial culture medium. Analysis of the recombinant proteins indicated that they retained the D antigen binding specificity of the parental BTSN4 IgG. Furthermore, both fragments mediated agglutination of papain-treated D positive erythrocytes in the absence of a cross-linking second antibody. While the agglutinating property of BTSN4 diabody was readily explained by the non-covalent association of this protein as a bivalent dimer, oligomeric forms of BTSN4 scFv were not detected when the protein was analysed by size exclusion chromatography. Thus, the agglutinating property of the scFv is not the result of the formation of non-covalently associated multimeric forms of the antibody fragment.     

ScFv multimers of the anti-neuraminidase antibody NC10: shortening of the linker in single-chain Fv fragment assembled in V(L) to V(H) orientation drives the formation of dimers, trimers, tetramers and higher molecular mass multimers

Synthetic genes encoding single-chain variable fragments (scFvs) of NC10 anti-neuraminidase antibody were constructed by joining the V(L) and V(H) domains with linkers of fifteen, five, four, three, two, one and zero residues. These V(L)-V(H) constructs were expressed in Escherichia coli and the resulting proteins were characterized and compared with the previously characterized NC10 scFv proteins assembled in V(H)-V(L) orientation. Size-exclusion chromatography and electron microscope images of complexes formed between various NC10 scFvs and anti-idiotype Fab' were used to analyse the oligomeric status of these scFvs. The result showed that as the linker length between V(L) and V(H) was reduced, different patterns of oligomerization were observed compared with those with V(H)-V(L) isomers. As was the case for V(H)-V(L) orientation, the scFv-15 V(L)-V(H) protein existed mainly as a monomer whereas dimer (diabody) was a predominant conformation for the scFv-5, scFv-4 and scFv-3 V(L)-V(H) proteins. In contrast to the V(H)-V(L) isomer, direct ligation of V(L) to V(H) led to the formation of predominantly a tetramer (tetrabody) rather than to an expected trimer (triabody). Furthermore, the transition between dimers and higher order oligomers was not as distinct as for V(H)-V(L). Thus reducing the linker length in V(L)-V(H) from three to two residues did not precisely dictate a transition between dimers and tetramers. Instead, two-residue as well as one-residue linked scFvs formed a mixture of dimers, trimers and tetramers.     

The crystal structure of an anti-CEA scFv diabody assembled from T84.66 scFvs in V(L)-to-V(H) orientation: implications for diabody flexibility

Diabodies (scFv dimers) are small, bivalent antibody mimetics of approximately 55kDa in size that possess rapid in vivo targeting pharmacokinetics compared to the intact parent antibody, and may prove highly suitable for imaging and therapeutic applications. Here, we describe T84.66Di, the first diabody crystal structure in which the scFvs comprise V domains linked in the V(L)-to-V(H) orientation. The structure was determined by X-ray diffraction analysis to 2.6 A resolution. The T84.66Di scFv was constructed from the anti-carcinoembryonic antigen (anti-CEA) antibody T84.66 variable domains connected by an eight residue peptide linker to provide flexibility between Fv modules and promote dimer formation with bivalent affinity to the cell-surface target, CEA. Therefore, it was surprising to observe a close association of some Fv module complementarity-determining regions in the T84.66 diabody crystal, especially compared to other diabody structures all of which are linked in the opposite V(H)-to-V(L) orientation. The differences between the arrangement of Fv modules in the T84.66Di V(L)-to-V(H) linked diabody structure compared to the crystal structure of L5MK16 and other proposed V(H)-to-V(L) linked diabodies has been investigated and their potential for flexibility discussed. The comparison between V(H)-to-V(L) and V(L)-to-V(H) linked diabodies revealed in this study represents a limited repertoire of possible diabody Fv orientations, but one that reveals the potential flexibility of these molecules. This analysis therefore provides some signposts that may impact on future molecular designs for these therapeutic molecules with respect to diabody flexibility and avidity.     

Dimeric and trimeric antibodies: high avidity scFvs for cancer targeting

Recombinant antibody fragments can be engineered to assemble into stable multimeric oligomers of high binding avidity and specificity to a wide range of target antigens and haptens. This review describes the design and expression of diabodies (dimers), triabodies (trimers) and tetrabodies (tetramers). In particular we discuss the role of linker length between V-domains and the orientation of the V-domains to direct the formation of either diabodies (60 kDa), triabodies (90 kDa) or tetrabodies (120 kDa), and how the size, flexibility and valency of each molecules is suited to different applications for in vivo imaging and therapy. Single chain Fv antibody fragments joined by polypeptide linkers of at least 12 residues irrespective of V-domains orientation predominantly form monomers with varying amounts of dimer and higher molecular mass oligomers in equilibrium. A scFv molecule with a linker of 3-12 residues cannot fold into a functional Fv domain and instead associates with a second scFv molecule to form a bivalent dimer (diabody, approximately 60 kDa). Reducing the linker length below three residues can force scFv association into trimers (triabodies, approximately 90 kDa) or tetramers ( approximately 120 kDa) depending on linker length, composition and V-domain orientation. A particular advantage for tumour targeting is that molecules of 60-100 kDa have increased tumour penetration and fast clearance rates compared with the parent Ig (150 kDa). We highlight a number of cancer-targeting scFv diabodies that have undergone successful pre-clinical trials for in vivo stability and efficacy. We also briefly review the design of multi-specific Fv modules suited to cross-link two or more different target antigens. Bi-specific diabodies formed by association of different scFv molecules have been designed as cross-linking reagents for T-cell recruitment into tumours (immunotherapy), viral retargeting (gene therapy) and as red blood cell agglutination reagents (immunodiagnostics). The more challenging trispecific multimers (triabodies) remain to be described.     

Effect of domain order on the activity of bacterially produced bispecific single-chain Fv antibodies

Bispecific single-chain Fv antibodies comprise four covalently linked immunoglobulin variable (VH and VL) domains of two different specificities. Depending on the order of the VH and VL domains and on the length of peptides separating them, the single-chain molecule either forms two single-chain Fv (scFv) modules from the adjacent domains of the same specificity, a so-called scFv-scFv tandem [(scFv)(2)], or folds head-to-tail with the formation of a diabody-like structure, a so-called bispecific single-chain diabody (scBsDb). We generated a number of four-domain constructs composed of the same VH and VL domains specific either for human CD19 or CD3, but arranged in different orders. When expressed in bacteria, all (scFv)(2) variants appeared to be only half-functional, binding to CD19 and demonstrating no CD3-binding activity. Only the diabody-like scBsDb could bind both antigens. Comparison of the scBsDb with a structurally similar non-covalent dimer (diabody) demonstrated a stabilizing effect of the linker in the middle of the scBsDb molecule. We demonstrated that the mechanism of inactivation of CD19xCD3 diabody under physiological conditions is initiated by a dissociation of the weaker (anti-CD3) VH/VL interface followed by domain swapping with the formation of non-active homodimers. The instability of one homodimer makes the process of diabody dissociation/reassociation irreversible, thus gradually decreasing the fraction of active molecules. The structural parameters influencing the formation of functional bispecific single-chain antibodies are indicated and ways of making relatively stable bispecific molecules are proposed.     

Construction of a diabody (small recombinant bispecific antibody) using a refolding system

Diabodies are the recombinant bispecific antibodies (BsAbs), constructed from heterogeneous single-chain antibodies. Usually, diabodies have been prepared from bacterial periplasmic fraction using a co-expression vector (i.e. genes encoding two chains were tandemly located under the same promoter). Some diabodies, however, cannot be expressed as a soluble material owing to inclusion body formation, which limits the utilization of diabodies in various fields. Here we report an improved method for the construction of diabodies using a refolding system. As a model, a bispecific diabody binding to adenocarcinoma-associated antigen MUC1 and to CD3 on T cells was studied. One chain consisted of a VH specific for MUC1 linked to a VL specific for CD3 with a short polypeptide linker (GGGGS). The second was composed of a VL specific for MUC1 linked to a VH specific for CD3. The two hetero scFvs were independently obtained from intracellular insoluble fractions of Escherichia coli, purified, mixed stoichiometrically (at an equivalent molar ratio of 1:1) and refolded. The refolded two hetero scFv has a hetero-dimeric structure, with complete specificity for both target cells [i.e. MUC1 positive cells and CD3 positive lymphokine-activated killer cells with a T cell phenotype (T-LAK)]. Evaluation of the in vitro efficacy of T-LAK with the diabody by growth inhibition assay of cancer cells demonstrated maximum growth inhibition of cancer cells to reach approximately 98% at an effector:target ratio (E:T ratio) of 10, almost identical with that with anti-MUC1xanti-CD3 chemically synthesized BsAbs (c-BsAbs). This is the first report of the construction of a diabody using a refolding system.     

Noncovalent scFv multimers of tumor-targeting anti-Lewis(y) hu3S193 humanized antibody

Single-chain variable fragments (scFvs) of anti-Lewis(y) hu3S193 humanized antibody were constructed by joining the V(H) and V(L) domains with either +2 residues, +1 residue, or by directly linking the domains. In addition two constructs were synthesized in which one or two C-terminal residues of the V(H) domain were removed (-1 residue, -2 residue) and then joined directly to the V(L) domain. An scFv construct in the reverse orientation with the V(L) joined directly to the V(H) domain was also synthesized. Upon transformation into Escherichia coli all scFv constructs expressed active protein. Binding activity, multimeric status, and multivalent properties were assessed by flow cytometry, size exclusion chromatography, and biosensor analysis. The results for hu3S193 scFvs are consistent with the paradigm that scFvs with a linker of +3 residues or more associate to form a non-covalent dimer, and those with a shorter linker or directly linked associate predominantly to form a non-covalent trimer and tetramer that are in equilibrium. While the association of V domains to form either a dimer or trimer/tetramer is governed by the length of the linker, the stability of the trimer/tetramer in the equilibrium mixture is dependent on the affinity of the interaction of the individual V domains to associate to form the larger Fv module.     

一种核糖体展示单链抗体文库的构建与鉴定

用柑桔溃疡病致病菌Xanthomonas axonopodis pv. citri（Xac）全菌免疫小鼠,提取小鼠脾细胞mRNA,RT-PCR扩增小鼠抗体重链可变区（VH）和轻链可变区（VL）,采用linker (Gly4Ser)3连接VH和VL,构建用于核糖体展示方法筛选阳性单链抗体（scFvs）的文库。通过将scFv文库DNA转化大肠杆菌JM109,随机挑取克隆子测序以分析单链抗体文库的多样性。结果显示,用柑桔溃疡病菌免疫后的小鼠抗血清效价为2500倍左右,扩增的VH大小为350bp左右,VL的大小为650bp左右,linker连接后的单链抗体文库DNA大小为1200bp左右。测序结果显示,单链抗体文库多样性好。以Xac为靶,筛选到了抗Xac的特异性抗体,说明该抗体库可用于柑桔溃疡病菌单链抗体的筛选。     

Di-, tri- and tetrameric single chain Fv antibody fragments against human CD19: effect of valency on cell binding.

Single chain variable fragments (scFv) of the murine monoclonal antibody HD37 specific to human B-cell antigen CD19 were constructed by joining the VH and VL domains with linkers of 18, 10, 1 and 0 residues. ScFv-18 formed monomers, dimers and small amounts of tetramers; scFv-10 formed dimers and small amounts of tetramers; scFv-1 formed exclusively tetramers; scFv-0 formed exclusively trimers. The affinities of the scFv-10 (diabody) and scFv-1 (tetrabody) were approximately 1.5- and 2.5-fold higher, respectively, than that of the scFv-0 (triabody). The tetrabody displayed a significantly prolonged association with cell-bound antigen (t1/2 cell surface retention at 37 degrees C of 26.6 min) compared to both the diabody (13.3 min) and triabody (6.7 min). This increase in avidity of the tetrabody combined with its larger size could prove to be particularly advantageous for imaging and the immunotherapy of B-cell malignancies.     

A diabody that dissociates to monomer forms at low concentration: effects on binding activity and tumor targeting

A human scFv, 15-9, was selected from a phage display library for binding to murine laminin-1. A diabody was made from the scFv by shortening the linker from 15 to 5 amino acids between the VH and VL sequence. Radioiodinated scFv and diabody were analyzed for size, binding to laminin, and biodistribution in tumor bearing mice. Diabody preparations at concentrations greater than 10 nM were largely dimer forms (approximately 60 kDa) as judged by gel filtration, but diluted diabody was eluted as a monomer (approximately 30 kDa). At low concentrations the radiolabeled diabody did not bind well to laminin. The (125)I diabody had significantly lower accumulation in tumors than did the scFv when injected at lower concentrations. These data indicate that the diabody dimer dissociates at concentrations of about 10nM resulting in monomers with no binding activity for laminin and poor tumor homing properties.     

Remodeling domain interfaces to enhance heterodimer formation.

An anti-p185HER2/anti-CD3 humanized bispecific diabody was previously constructed from two cross-over single-chain Fv in which YH and VL domains of the parent antibodies are present on different polypeptides. Here this diabody is used to evaluate domain interface engineering strategies for enhancing the formation of functional heterodimers over inactive homodimers. A disulfide-stabilized diabody was obtained by introducing two cysteine mutations, VL L46C and VH D101C, at the anti-p185HER2.VL/VH interface. The fraction of recovered diabody that was functional following expression in Escherichia coli was improved for the disulfide-stabilized compared to the parent diabody (> 96% versus 72%), whereas the overall yield was > 60-fold lower. Eleven "knob-into-hole" diabodies were designed by molecular modeling of sterically complementary mutations at the two VL/VH interfaces. Replacements at either interface are sufficient to improve the fraction of functional heterodimer, while maintaining overall recoverable yields and affinity for both antigens close to that of the parent diabody. For example, diabody variant v5 containing the mutations VL Y87A:F98M and VH V37F:L45W at the anti-p185HER2 VL/VH interface was recovered as 92% functional heterodimer while maintaining overall recovered yield within twofold of the parent diabody. The binding affinity of v5 for p185HER2 extracellular domain and T cells is eightfold weaker and twofold stronger than for the parent diabody, respectively. Domain interface remodeling based upon either sterically complementary mutations or interchain disulfide bonds can facilitate the production of a functional diabody heterodimer. This study expands the scope of domain interface engineering by demonstrating the enhanced assembly of proteins interacting via two domain interfaces.     

Antigen binding and stability properties of non-covalently linked anti-CD22 single-chain Fv dimers

By varying linker length and domain orientation three multivalent derivatives of a monovalent anti-CD22 single-chain fragment variable (scFv) antibody were generated. Shortening the linker of the V(H)-V(L) oriented scFv to 5 or 0 residues resulted in the formation of diabodies or a mixture of tetramers and trimers, respectively. Unexpectedly, a V(L)-0-V(H) scFv assembled to homogenous dimers, remained substantially more stable than the V(H)-5-V(L) diabody when incubated in human serum at 37 degrees C, and retained its dimeric state when concentrated up to 4 mg/ml. These properties suggest the V(L)-0-V(H) scFv could become an attractive vehicle for the selective delivery of multiple effector molecules to CD22(+) tumor cells.     

肝癌细胞特异性结合单链抗体三维结构的同源模建

用MSI公司开发的计算机辅助分子设计系统模建肝癌细胞表面抗原特异性单链抗体三维结构。先分别模建VH(variable region of the heave chain)和VL(variable region of the light chain)两个结构域,然后搭建出scFv(single chain variable fragment)片段的整体三维结构,并对模建的结构进行分子力学和动力学优化;对结构的合理性验证显示模建结构是合理的。文章可为预测该特异性单链抗体的生物活性以及研制高亲和力、高特异性的双价抗体提供结构信息。     

Improving the productivity of single-chain Fv antibody against c-Met by rearranging the order of its variable domains

Single-chain Fv (scFv) antibody against c-Met is expected to be employed in clinical treatment or imaging of cancer cells owing to the important biological roles of c-Met in the proliferation of malignancies. Here, we show that the productivity of scFv against c-Met in Escherichia coli is significantly influenced by the orientation of its variable domains. We generated anti-c-Met scFv antibodies with two different domain orders (i.e., VL-linker-VH and VHlinker- VL), expressed them in the cytoplasm of E. coli trx/ gor deleted mutant, and compared their specific activities as well as their productivities. Productivity of total and functional anti-c-Met scFv with VH/VL orientation was more than five times higher than that with VL/VH format. Coexpression of DsbC enhanced the yield of soluble amounts of anti-c-Met scFv protein for both constructs. The purified scFv antibodies of the two different formats exhibited almost the same antigen-binding activities. We also compared the productivities and specific activities of anti-c-Met diabodies with VH/VL or VL/VH formats and obtained similar results to the case of scFv antibodies.     

Efficient construction of a diabody using a refolding system: anti-carcinoembryonic antigen recombinant antibody fragment

Recombinant fragments of the variable region of antibodies are useful in many experimental and clinical applications. However, it can be difficult to obtain these materials in soluble form after their expression in bacteria. Here, we report an efficient procedure for preparing several variable-domain fragments (Fv), single-chain Fv (scFv), and a diabody (the smallest functional bispecific antibody) of anti-carcinoembryonic antigen (CEA) antibody by overexpression in Escherichia coli in inclusion bodies, using a refolding system to obtain renatured proteins. Two types of refolded Fv were prepared: (i) Heavy and light chains of the immunoglobulin variable regions (VH and VL, respectively) were coexpressed with a dicistronic expression vector (designated Fv(co)); (ii) VH and VL were expressed separately, mixed stoichiometrically, and refolded (designated Fv(mix)). All samples refolded with high efficiency; Fv(co), Fv(mix), scFv, and the bispecific diabody bound to several CEA-positive cell lines, exactly as did soluble Fv fragments secreted by E. coli (Fv(sol)) and the parent IgG. The refolded fragments inhibited binding of the parent IgG to CEA-positive cell lines, indicating that their epitope is identical to that of IgG. The bispecific diabody, which combined variable-region fragments of anti-CEA antibody with variable-region fragments of anti-CD3 antibody, was also prepared using the refolding system. This refolded diabody could bind to lymphokine-activated killer cells. In addition, its cytotoxicity toward human bile duct carcinoma TFK-1 and other several other CEA-positive cell lines was concentration-dependent. Taken together, our results suggest that a refolding procedure can be used to prepare various functional antibody fragments (Fv, scFv, and diabody).     

Construction of multiform scFv antibodies using linker peptide

Multiform single chain variable fragments (acFvs) including different length linker scFvs and bispecific seFv were constructed. The linker lengths of 0, 3, 5, 8, 12, and 15 amino acids between VH and VL of antideoxynivalenol (anti-DON) scFv were used to analyze the affinities of scFvs. The affinity constants of these scFvs increased when the linker was lower than 12 amino acids. The affinity constant would not change when the linker was longer than 12 amino acids. Fusion gene of anti-DON seFv and antizearalenone (anti-ZEN) scFv was also constructed through eormection by a short peptide tinker DNA to express a bispecific scFv. The affinity constants assay showed that the two scFvs of fusion bispecific scFv remained their own affinity compared to their parental scFvs. Competitive direct enzyme linked immunosorbent assay was used to detect DON and ZEN in contaminated wheat (Triticum aestivum L.) samples, and the results indicated that this bispecifie acFv was applicable in DON and ZEN detection. This work confirmed that bispecific scFv could be successfully obtained, and might also have an application in diagnosing fungal infection, and breeding transgertic plants.     

Recognition of DNA by VH and Fv domains of an IgG anti-poly(dC) antibody with a singly mutated VH domain

Secondary antigen stimulation usually produces IgG antibodies with hypermutated V segments. Studying a strong secondary response to the polynucleotide antigen poly(dC), however, we found a highly selective IgG antibody (mAb dC7) with only one mutation (a conservative Leu to Ileu substitution) throughout the whole VH domain. To investigate the roles of VH and VL domains in selective binding by this mAb, we prepared its VH, VL and single-chain Fv (scFv) fragments. A bacterial expression system produced soluble monomeric V region proteins. CD spectra confirmed that they had the beta-secondary structure expected for Ig domains. Both the scFv and VH fragments bound to single-stranded non-protonated poly(dC) and to ssDNA but not to protonated, more structured poly(dC) or dsDNA. The VL domain alone did not bind to nucleic acids, but VL association modified the VH binding, giving the scFv a 10-fold higher affinity than the VH for poly(dC) and greatly increasing the cytosine-dependent selectivity. Non-ionic interactions were prominent in the Fv reaction with a (dC)( n) sequence. Ionic interactions were revealed in Fv cross-reactions with ssDNA, and were more prominent in binding of either poly(dC) or ssDNA by VH alone, consistent with the lesser base selectivity of the VH. Thus, the Fv and VH alone bind to a single antigen, poly(dC), but mechanistic differences result from additional subsites in the Fv. Generation of a selective IgG with very few CDR mutations in either VH or VL, which was accompanied by IgM antibodies with unmutated V regions, also suggests that nucleic acid binding activity is a property of the B cell repertoire even before immunization.     

Recombinant antineuraminidase single chain antibody: Expression,characterization, and crystallization in complex with antigen

The variable heavy (VH) and variable light (VL) genes of NC10, a monoclonal antibody with specificity toward N9 neuraminidase (NA), were cloned and sequenced. A single chain Fv (scFv) fragment of NC10, consisting of VH and VL domains joined by a peptide linker, was designed, constructed and expressed in the E. coli expression vector pPOW. The N-terminal secretion signal PelB directed the synthesized protein into the periplasm where it was associated with the insoluble membrane fraction. An octapeptide (FLAG) tail was fused to the C-terminus of the single chain Fv to aid in its detection and remained intact throughout the protein purification process. NC10 scFv was purified by solubilization of the E. coli membrane fraction with guanidinium hydrochloride followed by column chromatography. The purified NC10 scFv showed binding affinity for its antigen, NA, 2-fold lower than that of the parent Fab. The complex between NA and the scFv has been crystallized by the vapor diffusion method. The crystals are tetragonal, space group P42₁2, with unit cell dimensions a = b = 141 Å, c = 218 Å. © 1993 Wiley-Liss, Inc.     

Homology modelling and bivalent single-chain Fv construction of anti-HepG2 single-chain immunoglobulin Fv fragments from a phage display library

We prepared single-chain immunoglobulin Fv fragments (scFv) SLH10 specific for the HepG2 cell line after biopanning from a large human-naïe phage display library (Griffin. 1 Library). The three-dimensional (3D) structure of SLH10 was modelled by the Insight II molecule simulation software. The structure was refined using the molecular dynamics method. The structures with the least steric clashes and lowest energy were determined finally. The optimized structures of heavy (VH) and light (VL) variable chains of SLH10 scFv were obtained. Then SLH10 bivalent single-chain Fv (BsFv) was constructed that would be suitable for high-affinity targeting. SLH10 BsFv was generated by linking scFvs together and identified by sequencing. Its expression products were confirmed by western blot analysis. The relative molecular masses of scFv and BsFv were approximately 30 kDa and 60 kDa, respectively. Flow cytometry revealed that SLH10 BsFv bound the selected cell lines with greater signal intensity than the parental scFv. The improved antigen binding of SLH10 BsFv may be useful for immunodiagnostics or targeted gene therapy for liver cancer.     

Recombinant single-chain Fv antibodies that recognize the p25 protein of the Maedi-Visna virus

Single chain Fv (scFv) antibodies (generated by phage display technology, molecules representing new and efficient tools in the research and diagnostics of infectious diseases) against the capsid protein (p25) of Maedi-Visna virus were selected. Several clones of p25 specific scFv antibodies were identified; one of them was expressed as a soluble scFv molecule, purified by immobilized metal-affinity chromatography and further characterized by sequencing and determination of the kinetic equilibrium association constant. Sequence analysis showed that the rearranged VL and VH domains of the analyzed scFv clone used sequences from the VL3 family (germline DPL16/VL3.1) and VH1 family (germline VH20), respectively. The kinetic equilibrium association constant was determined as KA = 1.12 +/- 0.52 L/mumol.