Pseudomonas aeruginosa, a Gram-negative pathogen, causes life-threatening infections. Lung injury and the development of sepsis depend largely on expression of the virulence genes associated with the type III secretion system of this bacterium. The type III secretion system functions as a molecular syringe to deliver type III secretory toxins directly into the cytosol of eukaryotic cells and also acts to inhibit innate immune mechanisms, thereby preventing bacterial clearance. Antibodies against PcrV, the cap structure in the translocational needle of type III secretory apparatus of P. aeruginosa, block toxin translocation of the type III secretion system. We have been investigating the therapeutic use of a recombinant anti-PcrV single-chain antibody. In this chapter, as a preliminary step toward an antibody-based immunotherapy against bacterial infections, we summarize our experience of constructing a recombinant single-chain antibody (called scFv166), in which the heavy (VH) and light chain (VL) variable regions of the anti-PcrV monoclonal IgG are joined by a flexible peptide linker. The practical methodologies used to make recombinant scFv166 against a bacterial protein component are described in detail.
- single-chain antibody
- Pseudomonas aeruginosa
- type III secretion system
Bacterial infections still frequently cause life-threatening diseases in humans. New pathogens have emerged, old pathogens have reemerged, and the prevalence of multidrug resistant microorganisms has increased despite the introduction of various new antibiotics since antibacterial agents were first developed in the early twentieth century. The difficulties associated with treating infections in immunocompromised patients have increased the need for new adjunctive immunotherapies. During the last 20 years, major advances in the techniques used to generate human antibodies and humanize murine monoclonal antibodies have seen antibody-based therapies to arrive as potential candidates for adjuvant therapies for infectious diseases. However, today, antibody therapy for bacterial infections is still indicated in relatively few situations, although more attention should be focused on it because of the increased levels of bacterial drug resistance and higher numbers of immunocompromised patients.
We have been investigating the therapeutic use of recombinant antibodies against the Gram-negative pathogen,
Here, as a preliminary step toward antibody-based immunotherapy against bacterial infections, we summarize our trial to block the TTSS-associated virulence of
2. Antibody-based blockade of
P. aeruginosatype III secretion
In the TTSS, the translocated toxins are not exposed extracellularly and evade direct recognition by the host immune system. Therefore, targeting the protein factors involved in the “
In our previous study, the Mab166 murine monoclonal antibody, which has neutralizing effects on virulence of the
In the next chapter, we describe the methods used to clone the variable antibody domains VH and VL from hybridoma cells and assembly of a single-chain antibody as an
3. Methods for construction of a single-chain antibody
3.1. Cloning the variable VH and VL domains from hybridoma cells
3.1.1. Poly A+ RNA extraction
The anti-PcrV IgG Mab166 hybridoma cell line  was cultured in a standard culture medium. After the cells had reached confluence in a 75 cm2 flask, they were harvested by centrifugation at 600 rpm for 5 min. The cell pellet was homogenized in 2 mL of TRIzol™ reagent (Thermo Fisher Scientific, Waltham, MA, USA), and total RNA extracted after chloroform fractionation, isopropanol precipitation, and washing with 70% ethanol. Poly A+ RNA was extracted with an oligotex mRNA spin-column (Qiagen, Valencia, CA).
3.1.2. RNA oligo-capping
To clone the variable VH and VL domains from the total RNA, the oligo-capping method reported by Maruyama and Sugano  using a GeneRacer™ kit (Thermo Fisher Scientific) was used. mRNA (250 ng) was incubated with calf intestinal phosphatase at 50 °C for 1 h to dephosphorylate non-mRNA or truncated mRNA species. After the reaction, phenol-chloroform extraction and ethanol precipitation were performed, and the dephosphorylated RNA was incubated with tobacco acid pyrophosphatase at 37°C for 1 h to remove the 5’-cap structure from the full-length mRNA. After phenol-chloroform extraction and ethanol precipitation, the synthetic RNA oligo (GeneRacer™ RNA Oligo, Thermo Fisher Scientific) was ligated to the decapped RNA with T4 RNA ligase at 37°C for 1 h. After phenol-chloroform extraction and ethanol precipitation, the RNA was suspended in diethylpyrocarbonate-treated water.
3.1.3. Reverse transcription of mRNA
The RNA-oligo ligated, full-length mRNA was reverse transcribed using a 54 base-pair primer containing an 18 nucleotide dT tail (GeneRacer™ Oligo-dT, Thermo Fisher Scientific) and avian myeloblastosis virus reverse transcriptase at 42°C for 1 h. After the reaction, the sample was diluted four times with sterile water.
3.1.4. Construction of a single-chain antibody gene
The cDNAs encoding V regions of the heavy and light (kappa) chains were PCR-amplified using a set of primers (VH forward: 5'-TGA GGA GAC GGT GAC TGA GGT TCC-3', VH reverse : 5'-CAG GTG CAG CTG AAG CAG TCA GG-3', Vk2 forward: 5'-CCG TTT TAT TTC CAG CTT GGT CCC-3', Vk reverse : 5'-GAC ATC CAG ATG ACT CAG TCT CCA-3'). PCRs were run over 30 cycles (94°C for 30 sec, 60°C for 40 sec, and 72°C for 40 sec). VH and VL fragment-amplified PCR products were purified separately by agarose gel electrophoresis. The PCR products derived from the murine immunoglobulin VH and VL domain of Mab166 were subcloned into a pCR2.1 vector (TOPO cloningTM, Thermo Fisher Scientific) and submitted to a DNA sequencing service for DNA sequence acquisition and analysis. Sequencing of the immunoglobulin variable genes for Mab166 was analyzed by The International imMunoGeneTics Database IMGT (http://www.imgt.org).
The purified VH and VL cDNAs were each assembled into a single gene using a DNA linker fragment-encoding a glycine-serine (Gly4Ser)3 linker peptide, thereby connecting the two cDNAs in the correct reading frame. Assembly PCR was run with a set of primers to multiply VH-linker-VL. The assembled fragment was amplified using two oligonucleotide primers with either an
3.2. Expression and purification of recombinant single-chain antibody fragments
3.2.1. Expression and purification of scFv166
scFv166 protein expression was induced in the
The column was washed twice with washing buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, 0.05% Tween 20, pH 8.0), and the bound scFv166 antibodies were eluted with elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, 0.05% Tween 20, pH 8.0). The eluate was dialyzed against PBS overnight and applied to an endotoxin removal column (Detoxi-Gel, Thermo Fisher Scientific) to get rid of the contaminating endotoxin. The purified antibodies were stored at −80°C until use.
In this study, we assembled the variable regions of the heavy and light chains of the anti-PcrV monoclonal IgG together with a glycine-serine linker in a single-chain antibody format. First, we assembled scFv166 in two different formats: one with VH-linker-Vk positioned between the two variable segments (Figure 2), the other with Vk-linker-VH positioned between two variable segments. The assembled scFv166 gene was subcloned into the
3.3. Protein gels and immunoblot analyses
The purity of scFv166 was evaluated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie Blue staining (Figure 3). Briefly, samples of
3.4. Affinity determination of scFv166
The affinity of scFv166 for its cognate antigen was determined by competition ELISA, and the result was compared with that of the hybridoma-derived parental Mab166, as described previously , Figure 5. Briefly, in the first step, the total antibody concentration range in which the absorbance correlates proportionately with the free antibody concentration was measured by indirect ELISA with the PcrV antigen coated at 1 μg/mL. In the second step,
4.1. Aminoacid sequence of VH, VL of scFv166
The sequence of the Mab166 heavy chain region is shown in Figure 2. The DNA sequence of the 5’-untranslational region and a V-region segment in the heavy chain-containing complementarity determining regions (CDRs) 1 and 2 is identical (except two amino acids in the frame 3 region) to germline Musmus IGHV2S2 (IGHV subgroup 2, VH#101, Accession #J00502). The V-region sequence also shows the same level of homology as that reported for pseudogene, IGHV2S5 (Accession #M21165). Transcription starts 24 nucleotides downstream of the TATA box of germline IGHV2S2. Nucleotides differ from the germline sequence at 10 positions, and these cause the following amino acid changes: position #61 in CDR2 S->D, #87 in FR3 V->L, #95 Q->R, and #96 S->A, #97 N->T. The first 15 nucleotides in the D-region encode the first 5 unique amino acids in CDR3, and the region consists of 16 amino acids in total. The J-region DNA sequence is identical to the IGHJ4 germline sequence (Accession #V00770). The unique CDR3 sequence includes the Arg-Gly-Asp (RGD) sequence, which functions as a recognition sequence for adhesion receptors in many adhesive proteins including fibrinogen, fibronectin, von Willebrand factor, and vitronectin.
The nucleotide sequence of the variable region of the kappa light chain, along with its predicted amino acid sequence, is shown in Figure 2. The CDRs are underlined, and the amino acids are numbered according to a convention. This kappa variable chain is a class II mouse kappa variable region. Although its sequence is not identical to any germline variable regions present in the data bank (The International ImMunoGeneTics Database IMGT), the DNA sequence of the 5’-untranslational region, and V-region of the kappa light chain shows the highest homology to germline Musumus IGKV12-41*01F (IGKV subgroup 12, Accession #AJ235953). Transcription starts from nine nucleotides downstream of the TATA box. Nucleotides differ from the germline sequence at four positions (+#192, C->A), (+#218, A->C), (+#250, A->T, +#251, A->C), and they cause amino acid changes at the following positions: #30 in CDR1 H->Q, #45 in FR2 K->T, and #56 in CDR2 N->S. The DNA sequence in the J-region is identical to germline IGKJ2 (Accession #V00777).
4.2. Evaluation of the expressed scFv166
Immunoblot to the anti-cMyc tag visualized the secreted scFv166 (298 amino acids) as a predicted 29.8 kD-band in the eluted solution from Ni-NTA agarose as shown in Figure 3. The bindings of scFv166 to both native PA103 PcrV (294 amino acids, 32.4 kD) and recombinant PcrV(rPcrV, 306 amino acids, 33.8 kD) were confirmed as shown in Figure 4. The binding affinity of Mab166 was 1 × 10−8 M, while that of scFv166 was 5 × 10−6 M (Figure 5).
4.3. Humanization and affinity maturation
The next step, for human use, after testing the binding affinity of scFv166 to a target molecule, together with the affinity maturation steps, is the elimination of the human-specific antigenic mouse amino acid sequence. In fact, Mab166 has already been humanized by antibody affinity engineering by serial epitope-guided complementarity replacement (SECR) which is a licensed humanization/affinity maturation technique of KaloBios Pharmaceutical Inc (Brisbane, California, USA) [33, 37] (Figure 6). In brief, SECR provides for a method for obtaining human idiologs for any nonhuman antibody to any target by epitope-guided replacement of variable regions using competitive cell-based methods in which the competitor can be either the reference antibody or a ligand that binds to the same epitope on the target as the reference antibody . Fab 1A8 of humanized Mab166 by SECR bound to PcrV with approximately a twofold-higher affinity than the original murine Mab166 Fab . Therefore, a further modification of scFv166 can be done by referring to the existing information available for the modified amino acid sequences in Fab 1A8 .
We have shown in an
This work was supported by the Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (KAKENHI No. 24390403, 26670791, and 15H05008) and by The Ministry of Education, Culture, Sports, Science and Technology, Japan to Teiji Sawa. The research studies associated with this chapter were carried out in the University of California San Francisco (UCSF) when Teiji Sawa was an Anesthesia/UCSF faculty member, under the generous support of Dara W. Frank, Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, and Jeanine P. Wiener-Kronish, Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital.
|CDR||complementarity determining region|
|P. aeruginosa||Pseudomonas aeruginosa|
|SECR||serial epitope-guided complementarity replacement|
|TTS||type III secretory|
|TTSS||type III secretion system|
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