**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).

**5. Conclusion**

**Acknowledgements**

**Abbreviations**

**Author details**

Teiji Sawa1

We have shown in an *in vivo* study that instillation of a single dose of Fab into the lungs of mice protected them against a lethal pulmonary challenge with *P. aeruginosa* [36]. The ability to use a recombinant Fab fragment for the treatment of *P. aeruginosa* infection in patients with ventilator-associated pneumonia or chronically infected cystic fibrosis patients has potential to minimize acute lung injury and mortality associated with TTS virulence of *P. aeruginosa*. Further optimization, such as the affinity maturation and PEGylation, will be the next step to achieve clinical application in humans. An engineered single-chain antibody that binds to the *P. aeruginosa* PcrV protein with high affinity has strong potential to be an effective new

Construction and Characteristics of a Recombinant Single-Chain Antibody Fragment...

http://dx.doi.org/10.5772/intechopen.70316

129

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

therapeutic reagent for infections caused by *P. aeruginosa*.

and Pain Medicine, Massachusetts General Hospital.

CDR complementarity determining region

\*Address all correspondence to: anesth@koto.kpu-m.ac.jp

SECR serial epitope-guided complementarity replacement

, Kiyoshi Moriyama2

1 Department of Anesthesiology, Kyoto Prefectural University of Medicine, Kyoto, Japan

2 Department of Anesthesiology, School of Medicine, Korin University, Mitaka, Japan

and Yoshifumi Naito1

*P. aeruginosa Pseudomonas aeruginosa*

TTSS type III secretion system

\*, Atsushi Kainuma1

TTS type III secretory

#### **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 [37]. Fab 1A8 of humanized Mab166 by SECR bound to PcrV with approximately a twofold-higher affinity than the original murine Mab166 Fab [37]. 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 [33].


**Figure 6.** Amino acid sequence differences between murine Mab166 and humanized Fab 1A8. CDR3 in the heavy chain, CDR3 and FR4 in the light chain (*k*) of humanized Fab 1A8 have sequence modifications following humanization and affinity maturation compared to corresponding sequences of Mab166.
