**2. Antibody-based blockade of** *P. aeruginosa* **type III secretion**

*P. aeruginosa* translocates its virulent TTS toxins (ExoS, ExoT, ExoU, and ExoY) directly into eukaryotic cells to disrupt their normal cellular processes [12, 13]. The translocation of ExoS and ExoT proteins, which both have ADP-ribosyltransferase and GTPase activities, inhibits the phagocytic activities of macrophages [14, 15]. The translocation of ExoU, which has patatin-like phospholipase A2 activity, is correlated with acute cytotoxicity *in vitro* and lung damage, sepsis, and mortality in animal models [7, 16–20]. The translocation of ExoY, which possesses adenylate cyclase activity, causes an increase of cytosolic cyclic AMP in eukaryotic cells and affects cell morphology [21]. In our past clinical study, we discovered an association between patients infected with TTSS-expressing *P. aeruginosa* strains and mortality [22], and other reports from various countries have supported the association of TTSS with severe clinical outcomes in patients infected with this bacterium [19].

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 immuno-

We have been investigating the therapeutic use of recombinant antibodies against the Gramnegative pathogen, *P. aeruginosa*. *P. aeruginosa* is an opportunistic pathogen responsible for a variety of acute infections in immunocompromised patients, and chronic infections in those with cystic fibrosis [1, 2]. *P. aeruginosa* is also highly resistant to various antibiotics and causes nosocomial pneumonia with an associated high mortality rate despite aggressive treatment with antimicrobial drugs [3, 4]. We have been studying the pathogenesis of acute infections caused by *P. aeruginosa* to identify a therapeutic target in this pathogen, and have reported that its ability to cause epithelial injury, to disseminate into the circulation, and to avoid host innate immune responses is highly associated with its type III secretion system (TTSS) [5–9]. The TTSS of Gram-negative bacteria mediates the translocation of toxins from the bacterial cytoplasm directly into the cytosol of host eukaryotic cells [10, 11]. Once inside the eukaryotic cell, these translocated bacterial toxins interfere with signal transduction. TTSSs with homology to *P. aeruginosa* have been described in most Gram-negative bacterial pathogens (e.g., *Yersinia*, *Shigella*, *Salmonella*, and *Escherichia coli*), and all of them are associated with

Here, as a preliminary step toward antibody-based immunotherapy against bacterial infections, we summarize our trial to block the TTSS-associated virulence of *P. aeruginosa* using recombinant antibody technologies. Especially, cloning of the variable domains of the light and heavy chain from a hybridoma cell and assembling the cloned VH and VL domains to recombinant single-chain antibody (scFv) to confirm the binding to the target antigen are required steps to humanize murine antibody. In addition to a brief explanation of the *P. aeruginosa* TTSS and the concept of a virulence blockade, the advantage of a recombinant single-chain antibody (scFv), the detailed methods to clone the variable domains from hybridoma cells and construction of scFv166, in which the heavy (VH) and light chain (VL) variable regions of the anti-PcrV monoclonal IgG molecule are joined by a flexible peptide

**2. Antibody-based blockade of** *P. aeruginosa* **type III secretion**

*P. aeruginosa* translocates its virulent TTS toxins (ExoS, ExoT, ExoU, and ExoY) directly into eukaryotic cells to disrupt their normal cellular processes [12, 13]. The translocation of ExoS and ExoT proteins, which both have ADP-ribosyltransferase and GTPase activities, inhibits

compromised patients.

120 Antibody Engineering

pathogenicity [12].

linker, will be described.

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 "*secretion*" or the "*translocation*" process of the TTSS seems a rational approach for blocking TTS virulence. To target the TTSS of *P. aeruginosa*, we have been developing neutralizing antibodies capable of blocking the translocation process of the TTSS [23]. An obvious candidate for a protective antigen was PcrV as it shares relatively high homology with the protective antigen from *Yersinia* sp., LcrV [6, 24-33]. Using genetic analyses, we demonstrated that PopD and PcrV were required for the delivery of *P. aeruginosa*-encoded TTS toxins [23]. In addition, recombinant TTS proteins, such as ExoU, PcrV, and PopD, were produced, purified, and tested for their protective capacities in a model of acute lung infection in mice. Only PcrV was protective in these experiments. Antibodies to PcrV protected against type III intoxication as shown by the inhibition of translocation of ExoY and by the inhibition of macrophage cytotoxicity mediated by ExoU. Passive protection with anti-PcrV reduced the inflammatory response, minimized bacteremia, and prevented septic shock [23]. Moreover, the protective capacity of the antibody was Fc-independent because F(ab')2 fragments of polyclonal anti-PcrV were also effective [34–36].

In our previous study, the Mab166 murine monoclonal antibody, which has neutralizing effects on virulence of the *P. aeruginosa* TTSS, was developed [35]. Also, the Fab fragments of the Mab166 had comparable therapeutic effects to the whole IgG of Mab166 in preventing *P. aeruginosa*-induced acute lung injury, and the Fc-dependent opsonization of the bacteria does not seem critical for the efficacy of the Mab166 [36]. These results implicate that the blockade of type III secretion-associated virulence can be attained by the effective Fab fragment of IgG molecules. Because the Fc-portion of IgG may induce unfavorable inflammatory responses such as complement fixation, activation of macrophages, the administration of the whole IgG may cause some inflammatory side effects. If the Fab fragment had the same therapeutic potency as the whole IgG, the therapeutic administration of either Fab fragments or scFv might overcome the disadvantages of the intratracheal administration of whole IgG. Therefore, the *E. coli*-derived recombinant scFv against PcrV is attractive to be an effective therapeutic agent against *P. aeruginosa* pneumonia.

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 *E. coli*-derived recombinant protein. Previously, the engineered recombinant Fab fragment against PcrV was humanized to allow it to be considered for adjunctive therapy in patients [37–39].
