**5. Conclusion**

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

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

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

(X D S/G)

QHFWSTPYT QQFWXTPYT

FGGGTKLEIKR

(X D M/F, Z D I/S/Q)

NRGDIYYDFTYAMDY NRGDIYYDFTYAXDZ

FR4 FGGGTKLTVLR

murine (Mab166) humanized (1A8)

murine (Mab166) humanized (1A8)

murine (Mab166) humanized (1A8)

**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 of Mab166 was 1 × 10−8 M, while that of scFv166 was 5 × 10−6 M (**Figure 5**).

IGKJ2 (Accession #V00777).

128 Antibody Engineering

Fab 1A8 [33].

**4.2. Evaluation of the expressed scFv166**

**4.3. Humanization and affinity maturation**

light chain κ

heavy chain IgG2b CDR3

affinity maturation compared to corresponding sequences of Mab166.

CDR3

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