ZHANG, Guo-min; CHEN, Yü-ping; GUAN, Yuan-zhi; WANG, Yan; AN, Yun-qing

Mouse-sourced mAb as a heterogenous protein is constrained in clinical application as therapeutic antibody. Human-sourced gene engineering antibody is expected to be used in human body directly because of lower immunogenicity and higher affinity. The phage antibody library is an important technology to obtain human antibodies without immunization. It has been used to prepare human antibodies against a variety of antigens.^1-5 Since the affinity of the antibodies is positively correlated to the capacity of the antibody library,⁶ constructing a large antibody library is essential to obtain various high-affinity antibodies. In order to increase the antibody library capacity, researchers have used in vivo recombination to construct the antibody library. The loxp/cre site-specific recombination system has shown a bright prospect of application. In 1994, Griffiths et al⁷ used double vectors and the loxp/cre recombination system to construct a large antibody library. However, the use of double vectors may deliver non-functional clones while lowering the efficiency. The method was not accepted extensively. In 2000, Sblattero et al⁸ used a single-vector intracellular recombination to clone light and heavy chain genes into the same vector, and inserted loxp511 and loxpwt sequences on both sides of heavy chain V-region, which recombine cre + cells to construct a large antibody library, thus suggesting the potential application.

Vector pDF was constructed from the vector pDAN5 by Prof. WANG Yan.⁹ It can be used as an expression vector to make a large phage antibody library through the loxp/cre site-specific recombination and express Fab and scFv (single chain variable fragment).⁹ In the present study we tried to reconstruct the vector. Appropriate restriction sites were designed in the linker sites of scFv, without influencing its homologous recombination and scFv function to be used for creation of a large phage antibody library. Meanwhile, the linkers of the scFv antibodies selected from the library can be shortened by simple endonuclease digestion to construct diabodies.

METHODS

Materials

E. coli XL1-blue, Cre recombinase expressesing E. coli BS1365, vector pDF, and proteins used for screening the library (digoxin, pepsin, ovalbumin and keratin) were kindly provided by Prof. WANG Yan (Navy General Hospital, Beijing). The phage display vector of anti-keratin scFv with the standard glycine-serine linker, pKscFv, was constructed at our laboratory.¹⁰ TA and IK (N-terminal dominant epitopes of bactericidal/ permeability-increasing protein) were commercially available as a product from Shanghai Sango Biotech Co., Ltd. China.

Primer design and vector construction

In order to splice VL and VH into the scFv gene, two linker overlap primers, VH 5′-terminal extension primer PVHL2 and VL 3′-terminal extension primer PVLEX, were designed according to the universal primer sequences for light and heavy chain V-regions for the construction of antibody libraries.⁹ PVHL2: 5′-GGAGGGTCGACCATAACTTCGTATAATGTATAC TATACGAAGTTATCCGGAGGCGGTACC-3′; PVLEX: 5′-GGATAACTTCGTATAGTATACATTATACGAAGTT AT-GGTCGACCCTCCGGAAC-3′; Primer PVHL2 contained loxp511 sequence and an Acc III restriction site. Primer PVLEX also contained an Acc III restriction site. Its 5′-terminal and PVHL2 3′-terminal sequences were partially complementary. With VL and VH genes (as templates) and extension primers, nested polymerase chain reaction (PCR) was carried out to obtain VL' and VH' containing the loxp511 sequence and restriction sites. The nested PCR was followed by over-lap PCR to get scFv. After purification by electrophoresis, the scFv gene and vector pDF were digested with BssH II+Nhe I and combined to create the vector pDscFv.

Identification of the expression function of a vector

In order to determine whether the vector pDscFv can express functional scFv, the linker of anti-keratin scFv, pKscFv, was replaced by cloning VL and VH of pKscFv into the vector pDscFv respectively. Primers were designed according to the V-region sequence of pKscFv and appropriate restriction sites were introduced to amplify VL and VH respectively. The products were digested and then cloned into pDscFv to prepare pDKscFv. Phage antibodies were expressed and their keratin-binding activity was detected by enzyme linked immunosorbent assay (ELISA). The expression function of pDscFv was evaluated by comparing the keratin-binding activity of the products of pDKscFv with that of pKscFv.

To determine the feasibility of constructing diabodies of the vector, one pDKscFv clone was selected to prepare diabody. Its plasmids were extracted and digested with Acc III and then spontaneously ligated to shorten the linker. The products were transformed into E. coli XL1-blue to express phage antibody. The antigen-binding specificity was analyzed by ELISA. In addition, the vector was compared with the original clone.

Construction of a primary phage antibody library

Total RNA from peripheral lymphocytes was conventionally extracted using Trizol reagent. Single-stranded cDNA was synthesized by reverse transcriptase (RT)-PCR. Light and heavy chain V-region genes were amplified by PCR with cDNA as a template. The obtained VL and VH fragments of various subgroups were subjected to nested PCR using extension primers to obtain VL' and VH' fragments containing the loxp511 sequence and Acc III restriction sites. Then VL' and VH' fragments were subjected to overlap PCR to splice the scFv gene. After isolation and purification by electrophoresis, the scFv gene was digested with BssH II+ Nhe I and cloned into the vector pDscFv. The recombinants were used to transform E. coli XL1-blue by electroporation. The bacterial suspension was diluted and spread on ampicillin-containing plates to calculate the library capacity. Most of the remaining bacteria were subcultured and rescued by helper viruses VCSM13 (PFU =1.7×10¹²). The bacteria were incubated at 30°C overnight. The culture supernatants were collected and then precipitated with PEG to obtain the primary phage antibody library.

Construction of a large antibody library

E. coli BS1365 was grown in a 2YT medium containing 50 μg/ml kanamycin and 10 g/L glucose till the log phase (A₆₀₀ = 0.5). The primary phage antibody library was used to super-infect BS1365 (multiplicity of infection (MOI) = 100) by resting for 1 hour without shaking at 37°C to let multiple copies enter the same bacteria. Light and heavy chain exchanges were carried out by the loxp/cre site-specific recombination system to increase the diversity of combination of VL and VH. The bacteria were cultured on a swing bed till the log phase, and then helper viruses VCSM13 was added to rescue. Being cultured overnight at 30°C, the recombination phage antibody library was collected to infect the bacteria not expressing cre (XL1-blue) at MOI ≤1 to couple phenotype and genotype. The infection was allowed by lefting without shaking for 30 minutes at 37°C. Some bacteria suspension was spread on ampicillin- containing culture plates to calculate the library capacity. The remaining bacteria were rescued by VCSM13 and then incubated at 30°C overnight. The supernatants were collected and precipitated with PEG to obtain the large phage antibody library. The antibody titer was measured and the clones were selected to extract plasmids for digestion with BssH II+Nhe I for determining the recombination rate. The method for the construction of a large antibody library by loxp/cre mediated intracellular recombination is shown in Fig. 1.

Fig. 1.

Screening of a large phage antibody library

The library was tested by screening upon several antigens such as digoxin, pepsin, ovalbumin, keratin, TA and IK. Immunotubes (NUNC Company, Denmark) were coated with 1 ml of 50-100 μg/ml antigen solution diluted with 0.05 mol/ L of carbonate buffer at 4°C overnight, then blocked with 2.5% of skimmed milk at 37°C for 2 hours and incubated with 1 ml of the phage antibody library solution at 37°C for 2 hours. The tube was rinsed twice with 0.05% tween-20 phosphate buffered saline (PBS) (10 times in the 2nd round and more than 20 times afterwards) and once with distilled water. Then phagemid binding on the wall of immunotubes was eluted with the methods previously described.⁹ Appropriate aliquots of the eluted bacterial suspension were inoculated over ampicillin-containing plates to determine colony forming unit (CFU). The remaining bacteria were cultured at 37°C for 2 hours and amplified to 50 ml. VCSM13 was added to rescue in the log phase and the bacteria were cultured at 30°C overnight. The phage was collected for further cycles of screening.

Test of phage antibody binding activity and analysis of gene diversity

Colonies were randomly selected from culture plates of the 3rd and 4th screening rounds and cultured in ampicillin-containing 2YT medium at 37°C till the log phase. Helper viruses VCSM13 were added and bacteria were cultured at 30°C overnight. The supernatants were collected for detection of the protein binding specificity by ELISA of double wells. Positive clones gave A₄₉₀ signals at least three times the background A₄₉₀ signal. Plasmids were extracted from the screened positive clones and identified by enzymatic digestion. DNA fingerprint analysis was made to confirm the gene diversity of the obtained antibodies. The scFv gene was PCR amplified using positive clones as a template, and purified by centrifugation using the CL-6B gel microcolumn. The products were digested with restriction endonuclease Mva I and then subjected to polyacrylamide gel electrophoresis. Analysis of gene diversity was based on the electrophoresis results.

Antibody V-region sequence analysis

The V-regions of screened different positive clones were sequenced by Shanghai Sango Biotech Co., Ltd. Sequence analysis and the homological comparison of different V genes were carried out with the Pcgene software (IntelliGenetics Co., USA).

Construction of anti-digoxin diabody

Anti-digoxin antibody may provide a potential reagent for the diagnosis and therapy of digoxin toxication. Here, we prepared anti-digoxin diabody from screened anti-digoxin scFv. Plasmids were extracted from one of the clones expressing anti-digoxin scFv and digested with Acc III and then spontaneously ligated. The recombinants were used to transform E. coli XL1-blue. Enzymatic digestion and sequencing analysis were carried out to confirm the length of the scFv linker that had been shortened to 15 bp after incision of the loxP511 recombination site. The phage antibody was expressed and the specific binding activity was tested by ELISA of double wells, with other antigens such as keratin, pepsin and ovalbumin as controls. Each test was repeated for three times.

RESULTS

Construction of the phage antibody expression vector

VL' and VH' were obtained and then spliced into the scFv gene by over-lap PCR (Fig. 2). The scFv gene was cloned into vector pDF via BssH II+Nhe I sites to construct the expression vector pDscFv (Fig. 3). The amino acid sequence of the scFv linker expressed by the vector was SGGSTITSYNVYYTKLSGGGT (underlined, product encoded by the loxp511 sequence). After Acc III endonuclease excision, the linker was shortened to 5 amino acid residues (SGGGT). After verification by enzymatic digestion and DNA sequencing, the vector pDscFv was used as the phage antibody library expression vector.

Fig. 2.

Fig. 3.

Determination of expression function of the vector

VL and VH of pKscFv were cloned into the vector pDscFv to prepare pDKscFv. Enzymatic digestion and sequencing confirmed that pDKscFv was obtained successfully. Keratin binding activity of the expressed phage antibodies was determined by ELISA. The results demonstrated that after the replacement of the linker, scFv remained its specific binding activity to keratin without changing in binding affinity.

One pDKscFv clone was selected to extract plasmids. Following digestion with Acc III, the products ligated spontaneously. Enzymatic digestion and sequencing of the extracted plasmids confirmed that the short linkers were successfully obtained. The expression phage antibody supernatants were subjected to analysis of antigen-binding specificity by ELISA. The results demonstrated that the linkers with 5 amino acid residues kept their binding activities high (Fig. 4).

Fig. 4.

Construction of a primary phage antibody library

To guarantee the antibody library diversity, we collected 2 ×10⁹ mononuclear cells from peripheral blood of 6 healthy people and 2×10⁹ mononuclear cells from umbilical blood of 10 neonates. Approximately 20 mg of total RNA was extracted. Totally 200 μg of total RNA was reverse-transcribed into cDNA. V-region primers for different subsets were used to amplify VH and VL genes. The results of electrophoresis showed bands at approximately 350 bp (Fig. 5A). The products were subjected to nested PCR with extension primers to obtain VL' and VH' and then spliced into scFv by overlap PCR (Fig. 5B). The obtained scFv gene was cloned into the vector pDscFv. The recombinants were transformed into E. coli XL1-blue by electroporation. A small number of bacteria were spread on plates and the library capacity was shown to be 7×10⁵ by colony counting. The remaining bacteria were subcultured and rescued by VCSM13 to obtain the primary phage antibody library. The titer of the concentrated precipitates was 2×10¹³/ml.

Fig. 5.

Construction of a large phage antibody library

A total of 200 μl of the primary phage antibody library (containing 4×10¹² phage particles) was used to infect 20 ml of BS1365 bacteria (containing 4×10¹⁰ bacteria). After proliferation of bacteria in a liquid medium and rescued by VCSM13, the recombination library was derived with a titer of 1.63×10¹³/ml. The phagemids were then used to infect E. coli XL1-blue at MOI ≤ 1 and a 1.2×10¹⁰ large phage antibody library was obtained with a titer of 1.16×10¹³/ml. One hundred clones were randomly selected to extract plasmids, which were subsequently identified by enzymatic digestion and electrophoresis. Ninety-nine clones yielded a band at approximately 1000 bp and the effective library recombination rate was 99%.

Identification of a large phage antibody library

The antibody library was screened for enrichment by using digoxin, keratin, pepsin, ovalbumin, synthesized polypeptides TA and IK as antigens respectively. Specific binding activity to these antigens of the antibodies was performed using clones selected from culture plates of the 3rd and 4th screening rounds. The results are shown in Table. Antibodies against all 6 antigens were successfully screened, with a range of 4 to 10 antibodies per antigen.

Table

The results of DNA fingerprint analysis of anti-IK antibody genes indicated that the antibodies against six antigens had 1 to 4 different V-region genes respectively (Fig. 6).

Fig. 6.

The different V-region genes of obtained antibodies against six antigens were sequenced. Sequence analysis and homology comparison were carried out with the Pcgene software. The results demonstrated a high diversity, with the light-chain genes belonging to subgroups Vλ I, Vλ II and Vλ III and the heavy chain genes belonging to subgroups VH1, VH2, VH3 and VH4, respectively.

Construction of anti-digoxin diabody

One clone was selected from the anti-digoxin scFv to construct diabody. Plasmids were digested with Acc III and then ligated spontaneously. Enzymatic digestion and sequencing analysis confirmed the presence of a 15 bp short linker. Then the recombinant was induced to express, and the supernatant was subjected to antigen-binding specificity analysis by ELISA. The results demonstrated that its activity remained unchanged, suggesting the generation of functional diabodies, which is potentially important for clinical diagnosis and treatment of digoxin toxication.

DISCUSSION

Phage antibody library technology simulates the process of antibody generation in vivo and makes it possible to prepare antibodies without antigen immunization. Hence, it is one of the important tools to prepare human antibodies. As demonstrated, it is a prerequisite for constructing a large, high-diversity phage antibody library to obtain high-affinity antibodies. Nevertheless, it is difficult to obtain a large antibody library because of such limitations as low transformation efficiencies with E. coli. Multiple transformations may increase the library capacity, but it is likely to produce repeated and futile clones and thus is a non-economic approach. The Loxp/cre recombination system is a cre protein-mediated, site-specific recombination system.^11,12 Sblattero et al⁸ constructed light and heavy chain genes in a single phasmid vector and introduced heterogenetic loxp sites on each side of the heavy chain gene. In this way, extensive sequence exchanges may occur between VL and VH in the presence of the cre protein, thus effectively increasing the diversity of combination of VL and VH.

Prof. WANG Yan and coworkers⁹ constructed a phage antibody library vector pDF based on pDAN5, which can be used for the construction of a large phage antibody library. pDF can express small univalent antibodies such as Fab and scFv. In this study, we reconstructed the vector pDF. In brief, two Acc III restriction sites were introduced by PCR-mediated, site-directed mutagenesis. Hence scFv obtained from the antibody library can be conveniently constructed into their diabodies simply by enzymatic digestion with negative impact on their functions. Diabody, a kind of small molecule antibody with two antigen binding sites,¹³ has a molecular weight equivalent to 1/3 of that of IgG. It can penetrate into tissues and be metabolized from serum easily.¹⁴ Its molecular weight is twice that of scFv, and thus its retention time in tissues and serum half-life are relatively long.¹⁵ In addition, as compared with long scFv linkers, short diabody linkers show a strong resistance to protease digestion. In some clinical trials, diabodies demonstrated an excellent biological profile in terms of tumor targeting, tumor tissue permeation, and blood clearance. So diabody technology highlights clinical application of antibody therapy against malignant diseases.^16-19

In order to investigate the effect of reconstruction on vector functions, we evaluated the recombination capability of the reconstructed vector, the binding activity of scFv, and the feasibility of diabody construction. The results showed that the two designed restriction sites did not change the Loxp511 sequence and its site-specific recombination capability. A large antibody library can ever be constructed with the intracellular recombination method but no influence on scFv's binding activity. The design of scFv linker may be significant to maintain the binding activity of parent antibody. Most linkers are reported to comprise 14-15 amino acid residues. Currently the most widely used linker comprises four glycine residues and one serine residue (GGGGS)₃.²⁰ Glycine has the smallest molecular weight and the shortest side-chain, and serine is a hydrophilic amino acid, which can increase the linker's hydrophilicity. The sequence of the amino acid encoded by loxp511 was ITSYNVYYTKL, which can be used as a scFv linker with no influence on the scFv's antigen-binding activity. In the present study, restriction sites were introduced on both sides of the loxp511 sequence to conveniently construct diabodies. As a result, the length of scFv linker was increased to 21 amino acids. The introduced amino acid sequences were SGGST and SGGGT, both comprising hydrophilic amino acids. The original pKscFv linker was SR (GGGGS)₃.¹⁰ After sequence modifications, scFv's antigen-binding activity remained unchanged.

Shortening of the linker between VL and VH can pair VL and VH of different molecules to form diabody. The linker length is a key to the formation of diabodies. Previous studies^21,22 showed that diabodies dominate when the linker is composed of 3-12 amino acid residues, structures similar to scFv dominate when the linker is composed of more than 12 amino acid residues, and trimers or multimers are likely to form when the linker is composed of 1 or 2 amino acid residue(s). Presently the majority of diabodies have linkers comprising 5-6 amino acid residues.^23,24 The frequently reported linker peptide has a length of 5 amino acid residues, namely, GGGGS.^20,25 In this study, the linker of diabody comprised 5 amino acid residues, that is, SGGGT. Analysis with anti-keratin and anti-digoxin scFv confirmed that the linker can be used to construct ordinary diabodies.

Using a new vector, we constructed a large antibody library with a single-vector, intracellular recombination method. The primary antibody library had a capacity of 7×10⁵. After random recombination and pairing of light and heavy chains, the secondary library had a capacity of 1.2×10¹⁰. For each of the six antigens used in the study, specific antibodies we obtained showed that the constructed large phage antibody library using vector pDscFv is of good diversity and that human antibodies can be isolated against virtually any antigens. Meanwhile, as required, their diabodies can be constructed for further clinical study and application.

REFERENCES