NZ624072B2 - Combination therapies using anti- pseudomonas psl and pcrv binding molecules - Google Patents

Abstract

Disclosed are bispecific antibodies comprising a binding domain that binds to Pseudomonas Psl and a binding domain that binds to Pseudomonas PcrV.

Description

COMBINATION THERAPIES USING ANTI— PSEUDOMONAS PSL AND PCRV BINDING MOLECULES REFERENCE TO CE LISTING SUBMITTED ONICALLY The t of the electronically submitted sequence listing in ASCII text file entitled sequencelisting_PCTascii.txt created on November 6, 2012 and having a size of 382 kilobytes filed with the application is incorporated herein by reference in its entirety.

BACKGROUND Field of the Disclosure This disclosure relates to combination therapies using seudomonas Psl and Pch binding domains for use in the prevention and treatment of Pseudomonas infection.

Furthermore, the disclosure provides compositions useful in such ies.

Background of the Disclosure Pseudomonas aeruginosa (P. aeruginosa) is a gram—negative opportunistic pathogen that causes both acute and chronic infections in compromised individuals (Ma et al., Journal of iology 189(22):8353—8356 (2007)). This is partly due to the high innate resistance of the bacterium to clinically used antibiotics, and partly due to the formation of highly antibiotic-resistant biofilms (Drenkard E., Microbes Infect 531213— 1219 (2003); Hancokc & Speert, Drug Resist Update 3247—255 ).

P. aeruginosa is a common cause of hospital—acquired infections in the Western world. It is a frequent causative agent of bacteremia in burn victims and immune compromised individuals (Lyczak et al., Microbes Infect 231051—1060 (2000)). It is also the most common cause of nosocomial gram—negative pneumonia (Craven et al., Semin Respir Infect 11:32—53 ), especially in mechanically ated patients, and is the most prevalent pathogen in the lungs of individuals with cystic fibrosis (Pier et al., ASM News 6:339—347 (1998)).

Pseudomonas Ps1 exopolysaccharide is reported to be anchored to the e of P. nosa and is thought to be important in facilitating colonization of host tissues and in establishing/maintaining biofilm formation (Jackson, K.D., et al., J Bacteriol 186, 4466—4475 (2004)). Its structure comprises mannose-rich repeating pentasaccharide (Byrd, M.S., et al., Mol Microbiol 73, 622—638 (2009)).

Pch is a relatively conserved component of the type III secretion . Pch appears to be an integral component of the translocation apparatus of the type III secretion system mediating the ry of the type III secretory toxins into target eukaryotic cells (Sawa T., et al. Nat. Med. 5, 392—398 ). Active and passive immunization against Pch improved acute lung injury and mortality of mice ed with cytotoxic P. aeruginosa (Sawa et a]. 2009). The major effect of zation t Pch was due to the blockade of translocation of the type III secretory toxins into eukaryotic cells.

Due to increasing rug resistance, there remains a need in the art for the development of novel strategies for the identification of new Pseudomonas-specific prophylactic and therapeutic agents.

BRIEF SUMMARY The disclosure provides a binding molecule or antigen binding fragment f that specifically binds Pseudomonas Pch, which comprises: (a) a heavy chain CDRl comprising SYAMN (SEQ ID NO:218), or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; a heavy chain CDR2 comprising AITISGITAYYTDSVKG (SEQ ID NO: 219), or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; and a heavy chain CDR3 comprising EEFLPGTHYYYGMDV (SEQ ID NO: 220), or a t thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; (b) a light chain CDRl comprising RASQGIRNDLG (SEQ ID NO: 221), or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; a light chain CDR2 comprising SASTLQS (SEQ ID NO: 222), or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; and a light chain CDR3 comprising LQDYNYPWT (SEQ ID NO: 223), or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; or combinations of (a) and (b). In one embodiment, the binding molecule or antigen binding fragment thereof specifically binds Pseudomonas Pch, and comprises: (a) a heavy chain CDRl comprising SYAMN (SEQ ID NO: 218), a heavy chain CDR2 comprising AITISGITAYYTDSVKG (SEQ ID NO: 219), and a heavy chain CDR3 comprising EEFLPGTHYYYGMDV (SEQ ID NO: 220); and (b) a light chain CDRl comprising RNDLG (SEQ ID NO: 221), a light chain CDR2 comprising S (SEQ ID NO: 222), and a light chain CDR3 comprising LQDYNYPWT (SEQ ID NO: 223). In one embodiment, the isolated binding le or antigen binding fragment thereof specifically binds Pseudomonas Pch and comprises (a) a heavy chain variable region having at least 90% sequence identity to SEQ ID NO: 216; (b) a light chain le region having at least 90% sequence identity to SEQ ID NO: 217; or combinations of (a) and (b). In another embodiment, the binding molecule or fragment f comprises: (a) a heavy chain variable region having at least 95% sequence identity to SEQ ID NO: 216; (b) a light chain variable region having at least 95% sequence identity to SEQ ID NO: 217; or combinations of (a) and (b). In another embodiment, the binding molecule or fragment thereof is V2L2 and comprises: (a) a heavy chain variable region comprising SEQ ID NO: 216; and (b) a light chain variable region comprising SEQ ID NO: 217.

In one ment, the disclosure provides an isolated binding molecule or antigen binding fragment thereof that specifically binds to the same Pseudomonas Pch epitope as an antibody or antigen-binding fragment f comprising the VH and VL region of V2L2. In another embodiment, the disclosure provides an ed binding molecule or antigen binding fragment thereof that specifically binds to Pseudomonas Pch, and competitively inhibits Pseudomonas Pch g by an antibody or antigen- binding fragment thereof comprising the VH and VL of V2L2. In one embodiment, the binding le or fragment thereof is a recombinant antibody. In one ment, the binding molecule or fragment thereof is a monoclonal antibody. In one embodiment, the binding molecule or fragment thereof is a chimeric antibody. In one embodiment, the binding molecule or nt thereof is a humanized antibody. In one embodiment, the g molecule or fragment thereof is a human antibody. In one embodiment, the binding molecule or fragment thereof is a bispeciflc antibody.

In one embodiment, the g molecule or fragment thereof inhibits delivery of type III secretory toxins into target cells.

In one embodiment, the sure provides a bispecif1c antibody comprising a binding domain that binds to Pseudomonas Psl and a binding domain that binds to Pseudomonas Pch. In one embodiment, the Ps1 binding domain comprises a scFv nt and the Pch binding domain comprises an intact immunoglobulin. In one embodiment, the Psl binding domain comprises an intact immunoglobulin and said Pch g domain comprises a scFv nt. In one embodiment, the scFv is fused to the amino-terminus of the VH region of the intact immunoglobulin. In one embodiment, the scFv is fused to the carboxy—terminus of the CH3 region of the intact immunoglobulin. In one embodiment, the scFV is inserted in the hinge region of the intact immunoglobulin.

In one embodiment, the anti-Psl binding domain cally binds to the same Pseudomonas Psl epitope as an antibody or antigen-binding fragment f comprising the heavy chain variable region (VH) and light chain le region (VL) region at least 90% identical to the ponding region of WapR—004. In one embodiment, the anti—Psl binding domain specifically binds to Pseudomonas Psl, and competitively inhibits Pseudomonas Psl binding by an antibody or antigen-binding nt thereof comprising a VH and VL region at least 90% identical to the corresponding region of WapR—004. In one embodiment, the VH and VL of WapR—004 se SEQ ID NO:11 and SEQ ID NO:12, respectively. In one embodiment, the WapR—004 sequence is selected from the group consisting of: SEQ ID NO:228, SEQ ID NO:229, and SEQ ID NO:235. In one embodiment, the anti-Pch binding domain specifically binds to the same Pseudomonas Pch epitope as an antibody or antigen-binding fragment thereof comprising the VH and VL region of V2L2. In one embodiment, the anti-Pch binding domain specifically binds to Pseudomonas Pch, and competitively inhibits Pseudomonas Pch binding by an antibody or antigen-binding fragment thereof comprising the VH and VL of V2L2. In another embodiment, the anti-Pch binding domain specifically binds to the same Pseudomonas Pch epitope as an antibody or antigen-binding fragment thereof comprising a VH and VL region at least 90% identical to the corresponding region of V2L2. In one embodiment, the VH and VL of V2L2 comprise SEQ ID NO:216 and SEQ ID NO:217, respectively. In one embodiment, the VH and VL of WapR—004 (SEQ ID NOs:11 and 12, respectively) and the VH and VL of V2L2 (SEQ ID NOs: 216 and 217, respectively). In one embodiment, the bispecific antibody comprises an amino acid sequence selected from the group consisting of: SEQ ID , SEQ ID NO:229, and SEQ ID NO:235.

In one embodiment, the sure provides a polypeptide comprising an amino acid sequence of SEQ ID NO:216 or SEQ ID . In one embodiment, the polypeptide is an antibody.

In one embodiment, the disclosure es a cell comprising or producing the binding le or polypeptide sed herein.

In one embodiment, the disclosure provides an isolated polynucleotide molecule sing a cleotide that encodes a binding molecule or polypeptide described herein. In one embodiment, the polynucleotide le comprises a polynucleotide sequence selected from the group consisting of: SEQ ID NO:238 and SEQ ID NO:239.

In another embodiment, the disclosure provides a vector comprising a polynucleotide described herein. In another embodiment, the disclosure provides a cell sing a polynucleotide or vector.

In one embodiment, the disclosure provides a ition comprising a binding le, bispecif1c antibody, or polypeptide described herein and a pharmaceutically acceptable carrier.

In one embodiment, the disclosure provides a composition comprising a binding domain that binds to Pseudomonas Psl and a binding domain that binds to Pseudomonas Pch. In one embodiment, the anti-Psl binding domain specifically binds to the same Pseudomonas Psl epitope as an dy or antigen-binding fragment thereof comprising the heavy chain variable region (VH) and light chain variable region (VL) region at least 90% identical to the corresponding region of WapR—004, Cam—003, Cam—004, Cam—005, WapR—OOl, WapR—OOZ, WapR—003, or WapR—Ol6. In one embodiment, the anti-Psl binding domain specifically binds to Pseudomonas Psl, and competitively inhibits Pseudomonas Psl binding by an antibody or antigen-binding fragment thereof comprising a VH and VL region at least 90% identical to the corresponding region of WapR—004, Cam—003, Cam—004, Cam—005, WapR—OOl, WapR—002, WapR—003, or WapR—Ol6. In one embodiment, the VH and VL of WapR-004 comprise SEQ ID NO: 11 and SEQ ID I\O: 12, tively, the VH and VL of Cam—003 comprise SEQ ID NO:1 and SEQ ID \IO:2, respectively, the VH and VL of Cam—004 comprise SEQ ID NO:3 and SEQ ID \IO:2, respectively, the VH and VL of Cam—005 comprise SEQ ID NO:4 and SEQ ID \IO:2, respectively, the VH and VL of WapR—OOl se SEQ ID NO:5 and SEQ ID \IO:6, respectively, the VH and VL of WapR—002 se SEQ ID NO:7 and SEQ ID \IO:8, respectively, the VH and VL of WapR—003 comprise SEQ ID N09 and SEQ ID l\O:lO, respectively, and the VH and VL of WapR—Ol6 se SEQ ID NO: 15 and SEQ ID NO: 16, respectively. In one embodiment, the anti-Pch binding domain specifically binds to the same Pseudomonas Pch epitope as an antibody or antigen-binding nt thereof comprising the VH and VL region of V2L2. In one ment, the anti-Pch binding domain specifically binds to Pseudomonas Pch, and competitively inhibits Pseudomonas Pch binding by an antibody or antigen-binding fragment thereof comprising the VH and VL of V2L2. In one ment, the anti-Pch binding domain specif1cally binds to the same Pseudomonas Pch e as an antibody or antigenbinding fragment thereof comprising a VH and VL region at least 90% identical to the corresponding region of V2L2. In one embodiment, the VH and VL of V2L2 comprise SEQ ID NO:2l6 and SEQ ID NO:217, respectively. In one embodiment, the anti-Psl binding domain comprises the VH and VL region of WapR-004, and said anti-Pch binding domain comprises the VH and VL region of V2L2, or antigen-binding fragments thereof In one embodiment, the composition comprises a first binding molecule comprising said anti Psl-binding domain, and a second binding molecule comprising a Pch-binding domain. In one embodiment, the first g molecule is an antibody or antigen binding fragment f, and said second binding molecule is an antibody or antigen g fragment thereof. In one embodiment, the antibodies or antigen binding fragments are independently ed from the group consisting of: monoclonal, humanized, ic, human, Fab fragment, Fab' fragment, F(ab)2 fragment, and scFv fragment. In one embodiment, the binding s, binding molecules or fragments thereof, bind to two or more, three or more, four or more, or five or more different P. aeruginosa serotypes. In one embodiment, the binding domains, binding molecules or fragments thereof, bind to at least 80%, at least 85%, at least 90% or at least 95% of P. aeruginosa strains isolated from infected patients. In one embodiment, the P. aeruginosa strains are isolated from one or more of lung, sputum, eye, pus, feces, urine, sinus, a wound, skin, blood, bone, or knee fluid. In one embodiment, the antibody or antigen g fragment thereof is conjugated to an agent selected from the group consisting of antimicrobial agent, a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a lipid, a biological response modifier, ceutical agent, a lymphokine, a heterologous antibody or nt thereof, a detectable label, hylene glycol (PEG), and a combination of two or more of any said . In one embodiment, the detectable label is selected from the group consisting of an enzyme, a fluorescent label, a chemiluminescent label, a bioluminescent label, a radioactive label, or a combination of two or more of any said detectable labels.

(Followed by page 7A) ] In one embodiment the disclosure provides for the use of the binding molecule or fragment thereof, a bispecific antibody, a polypeptide, or a composition thereof as described herein in the manufacture of a ment for preventing or treating a Pseudomonas infection in a subject in need thereof. [0020b] In a preferred embodiment, the sure provides for the use of a composition comprising a bispecific antibody comprising a binding domain that binds to monas Psl and a binding domain that binds to Pseudomonas PcrV in the manufacture of a medicament for preventing or treating a Pseudomonas infection in a subject in need thereof. -7A- wed by page 8) aeruginosa 3064 A WapR (1) or P. aeruginosa PAOl MexAB OprM A WapR (2) in suspension. Bars te the output titers (in CPU) at each round of selection, and circles indicate the proportion of duplicated VH CDR3 sequences, an indication of clonal ment. (E) ELISA screen of scFv from phage display to test binding to multiple strains of P. aeruginosa. ELISA data (absorbance at 450 nm) are shown for eight individual phage-scFvs from selections and one irrelevant phage-scFv. (F) FACS binding of P. nosa c antibodies with representative s from unique P. aeruginosa serotypes. For each antibody tested a human IgG negative control antibody is shown as a shaded peak.

Figure 2 (A-B): Evaluation of mAbs promoting OPK of P. aeruginosa (A) Opsonophagocytosis assay with luminescent P. aeruginosa serogroup 05 strain (PAOl.lux), with dilutions of purified monoclonal antibodies derived from phage panning. (B) phagocytosis assay with luminescent P. nosa serogroup Oll strain (9882-80.lux), with dilutions of purified WapR-004 and Cam-003 monoclonal antibodies derived from phage panning. In both A and B, R347, an isotype matched human monoclonal antibody that does not bind P. nosa antigens, was used as a negative control.

Figure 3 (A—I): Identification of the P. aeruginosa Psl exopolysaccharide target of antibodies derived from phenotypic screening. Reactivity of dies was determined by indirect ELISA on plates coated with indicated P. aeruginosa strains: (A) wild type PAOl, PAOlAwpr, PAOlArmZC and PAOlAgaZU. (B) PAOlApslA. The Genway antibody is specific to a P. aeruginosa outer ne protein and was used as a positive control. (C) FACS binding analysis of Cam-003 to PAOl and PAOlApSZA. Cam—003 is indicated by a solid black line and clear peak; an isotype matched non—P. aeruginosa— specific human IgG1 dy was used as a negative control and is indicated by a gray line and shaded peak. (D) LPS purified from PAOl and PAOlApsZA was resolved by SDS-PAGE and bloted with antisera derived from mice vaccinated with PAOlAwapRAaZgD, a mutant strain deficient in O-antigen transport to the outer membrane and alginate production. (E) Cam-003 ELISA binding data with isogenic mutants of PAOl. Cam-003 is only capable of binding to strains expressing Psl. pPWl45 is a pUCP expression vector containing psZA. (F and G) phagocytosis assays indicating that Cam-003 only es killing of strains capable of producing Psl (wild type PAOl and PAOlApslA complemented in trans with the pslA gene). (H and I) ELISA data indicating reactivity of anti-Psl antibodies WapR—OOl, WapR—004, and WapR—Ol6 with PAOl AwprAaZgD and PAOl AwprAalgDApslA. R347 was used as a negative control in all experiments.

Figure 4: Anti-Psl mAbs inhibit cell ment of luminescent P. aeruginosa strain PAOl.lux to A549 cells. ase PAOl.lux were added to a confluent monolayer of A549 cells at an MOI of 10 followed by analysis of RLU after repeated washing to remove unbound P. aeruginosa. Results are representative of three independent experiments performed in duplicate for each antibody concentration.

Figure 5 (A—C): In vivo passaged P. aeruginosa strains maintain/increase expression of Psl. The Cam—003 antibody is shown by a solid black line and a clear peak; the human IgG negative control antibody is shown as a gray line and a shaded peak. (A) For the positive control, Cam-003 was assayed for binding to strains grown to log phase from an ght culture (~5 x lOS/ml). (B) The a for each strain were prepared to 5 x 108 CFU/ml from an overnight TSA plate grown to lawn and tested for reactivity to Cam—003 by flow cytometry. (C) Four hours post intraperitoneal challenge, bacteria was harvested from mice by peritoneal lavage and d for the presence of Psl with Cam- 003 by flow cytometry.

Figure 6 (A-F): Survival rates for animals treated with anti-Psl monoclonal antibodies Cam—003 or WapR—004 in a P. nosa acute pneumonia model. (A—D) Animals were d with Cam-003 at 45, 15, and 5mg/kg and R347 at 45mg/kg or PBS 24 hours prior to intranasal ion with (A) PAOl (1.6 x 107 CPU), (B) 33356 (3 x 107 CFU), (C) 6294 (7 x 106 CFU), (D) 6077 (l x 106 CFU). (E-F) Animals were treated with 04 at 5 and lmg/kg as indicated followed by infection with 6077 at (E) (8 x 105 CFU), or (F) (6 x 105 CFU). Animals were carefully monitored for survival up to 72 hours (A-D) or for 120 hours (E-F). In all experiments, PBS and R347 served as negative controls. Results are represented as Kaplan—Meier survival curves; differences in survival were calculated by the nk test for Cam—003 vs. R347. (A) Cam—003 kg — P<0.0001; 15mg/kg — 03; 5mg/kg — P=0.0033). (B) 3 (45mg/kg — P=0.0012; 15mg/kg — P=0.0012; 5mg/kg — P=0.0373). (C) Cam—003 (45mg/kg — P=0.0007; 15mg/kg — P=0.0019; 5mg/kg — P=0.0212). (D) Cam—003 (45mg/kg — P<0.0001; 15mg/kg — P<0.0001; 5mg/kg — P=0.0001). Results are representative of at least two independent experiments. (E) [Cam—003 (5mg/kg) vs. R347 (5mg/kg): P=0.02; Cam—003 (lmg/kg) vs. R347 (5mg/kg): 48; WapR—004 (5mg/kg) vs. R347 (5mg/kg): P<0.0001; WapR—004 (lmg/kg) vs. R347 (5mg/kg): P=0.0886; 04 (5mg/kg) vs. Cam—003 (5mg/kg): P=0.0017; WapR—004 (lmg/kg) vs. Cam—003 (lmg/kg): P=0.2468; R347 g) vs. PBS: P=0.6676] (F) [Cam—003 (5mg/kg) vs. R347 (5mg/kg): P=0.0004; Cam—003 g) vs. R347 (5mg/kg): P<0.0001; WapR—004 g) vs. R347 (5mg/kg): P<0.0001; WapR-004 (lmg/kg) vs. R347 (5mg/kg): 01; WapR—004 (5mg/kg) vs. Cam—003 (5mg/kg): P=0.0002; WapR—004 (lmg/kg) vs. Cam—003 (lmg/kg): P=0.2628; R347 (5mg/kg) vs. PBS: 76]. Results are representative of five independent experiments.

Figure 7 (A—F): Anti—Ps1 monoclonal antibodies, Cam—003 and WapR—004, reduce organ burden after induction of acute pneumonia. Mice were treated with Cam—003 antibody 24 hours prior to infection with (A) PAOl (1.1 x 107 CFU), (B) 33356 (1 X 107 CFU), (C) 6294 (6.25 x 106 CFU) (D) 6077 (l x 106 CFU), and WapR-004 antibody 24 hours prior to infection with (E) 6294 (~1 x 107 CFU), and (F) 6206 (~1 x 106 CFU). 24 hours post-infection, animals were euthanized ed by harvesting or organs for identification of viable CFU. Differences in viable CFU were determined by the Mann- Whitney U—test for Cam—003 or WapR—004 vs. R347. (A) Lung: Cam—003 (45mg/kg — ; lSmg/kg — P=0.0021; 5mg/kg — 15); Spleen: Cam—003 (45mg/kg — P=0.0120; lSmg/kg — P=0.0367); s: Cam—003 (45mg/kg — P=0.0092; lSmg/kg — P=0.0056); (B) Lung: Cam—003 (45mg/kg — P=0.0010; lSmg/kg — P<0.0001; 5mg/kg — P=0.0045); (C) Lung: 3 kg — P=0.0003; lSmg/kg — P=0.0039; 5mg/kg — P=0.0068); Spleen: Cam—003 (45mg/kg — P=0.0057; lSmg/kg — 30; 5mg/kg — P=0.0012); (D) Lung: Cam—003 (45mg/kg — P=0.0005; lSmg/kg — P=0.0003; 5mg/kg — P=0.0007); Spleen: Cam—003 (45mg/kg — P=0.0015; lSmg/kg — P=0.0089; 5mg/kg — 89); Kidneys: Cam—003 (45mg/kg — P=0.019l; lSmg/kg — P=0.0355; 5mg/kg — P=0.002l). (E) Lung: WapR—004 (lSmg/kg — P=0.0011; 5mg/kg — 04; lmg/kg — P=0.0002); Spleen: WapR—004 (lSmg/kg — P<0.0001; 5mg/kg — P=0.0014; lmg/kg — P<0.0001); F) Lung: WapR—004 (lSmg/kg — P<0.0001; 5mg/kg — P=0.0006; lmg/kg — P=0.0079); Spleen: WapR—004 (lSmg/kg — P=0.0059; 5mg/kg — P=0.026l; lmg/kg — P=0.0047); Kidney: WapR—004 (lSmg/kg — P=0.0208; 5mg/kg — P=0.0268.

Figure 8 (A-G): Anti-Psl monoclonal antibodies Cam-003 and WapR—004 are active in a P. aeruginosa keratitis model and l injury model. Mice were treated with a control IgGl antibody or Cam-003 at 45mg/kg (A, B) or lSmg/kg (C, D) or PBS or a control IgGl antibody or Cam-003 at 45mg/kg or WapR—004 at 45mg/kg or lSmg/kg or 5mg/kg (F, G) 24 hours prior to infection with 6077 (Oll-cytotoxic — 2XlO6 CFU).

Immediately before infection, three 1 mm scratches were made on the left cornea of each animal followed by topical application of P. aeruginosa in a 5 ul inoculum. 24 hours after infection, the corneal pathology scores were calculated followed by l of the eye for determination of viable CFU. Differences in pathology scores and viable CFU were determined by the Mann—Whitney U—test. (A) P=0.0001, (B) P<0.0001, (C) P=0.0003, (D) P=0.0015. (F) and (G) Cam—003 (45mg/kg) vs. WapR—004 (45mg/kg): P=0.018; 3 (45mg/kg) vs. WapR—004 kg): P=0.0025; WapR—004 (45mg/kg) vs. WapR—004 (lSmg/kg): 3l; WapR—004 (5mg/kg) vs. Ctrl: P<0.0001.

Results are representative of five ndent experiments. (E) Survival analysis from Cam—003 and R347 treated CF—l mice in a P. aeruginosa thermal injury model after 6077 infection (2 x 105 CFU) ank: R347 vs. Cam—003 lSmg/kg, P=0.0094; R347 vs.

Cam—003 5mg/kg, P=0.0017). Results are representative of at least three independent experiments. (n) refers to number of animals in each group. Figure 8 (H): Anti-Psl and anti-Pch monoclonal antibodies are active in a P. aeruginosa mouse ocular keratitis model. Mice were injected intraperitoneally (IP) with PBS or a control IgGl antibody (R347) at 45mg/kg or 04 (ct-Ps1) at 5mg/kg or V2L2 (oc-Pch) at 5mg/kg, 16 hours prior to infection with 6077 (Oll-cytotoxic — 1x106 CFU). Immediately before infection, mice were anesthetized followed by initiation of three 1 mm scratches on the cornea and superficial stroma of one eye of each mouse using a ge needle under a dissection microscope, followed by topical ation of P. aeruginosa 6077 strain in a 5 ul inoculum.

Figure 9 (A—C): A Cam—003 Fc mutant dy, CamTM, has diminished OPK and in viva efficacy but maintains anti-cell attachment activity. (A) PAOl.lux OPK assay with Cam-003 and CamTM, which harbors ons in the Fc domain that prevents Fc interactions with Fcy ors (Oganesyan, V., et al., Acta Crystallogr D Biol Crystallogr 64, 4 (2008)). R347 was used as a negative control. (B) PAOl cell attachment assay with 3 and Cam-003—TM. (C) Acute pneumonia model comparing efficacy of Cam—003 vs. Cam—003—TM.

Figure 10 (A-C): A: Epitope mapping and fication of the relative binding affinity for anti-Psl monoclonal antibodies. Epitope mapping was performed by competition ELISA and confirmed using an OCTET® flow system with Psl derived from the supernatant of an overnight culture of P. aeruginosa strain PAOl. Relative binding affinities were measured on a FORTEBIO® OCTET® 384 instrument. Also shown are antibody concentrations where cell attachment was maximally inhibited and OPK EC50 values for each dy. B, C. Relative binding affinities of various WapR—004 mutants %nwfimwonaFmUEmO®OCUH®3Mdmmmwm.AbommmamCWKECw values for the various mutants.

Figure 11 (A-M): Evaluation of WapR-004 (W4) mutants clones in the P. aeruginosa opsonophagocytic killing (OPK) assay (A—M) OPK assay with luminescent P. nosa serogroup 05 strain (PAOl.luX), with dilutions of different W4 mutant clones in scFv-Fc format. In some instances, W4 IgGl was included in the assay and is indicated as W4-IgGl. W4-RAD-Cam and W4-RAD-GB represent the same WapR- 004RAD sequence described . "W4-RAD" is a shorthand name for WapR- 004RAD, and W4-RAD-Cam and W4-RAD-GB designations in panels D through M ent two ent preparations of WapR-004RAD. (N—Q): Evaluation of the optimized anti-Psl mAbs derived from lead (WapR-004) zation in the P. aeruginosa OPK assay. (N—O) OPK assay with luminescent PAOl.lux using dilutions of purified lead zed monoclonal antibodies. (P-Q) Repeat OPK assay with PAOl.lux with dilutions of ed mAbs to confirm results. (N—Q): W4—RAD was used as a ative positive control. In all experiments, R347, a human IgG1 monoclonal antibody that does not bind P. aeruginosa ns, was used as a negative control.

Figure 12 (A-H): (A) The Pch epitope diversity. . (B) Percent inhibition of cytotoxicity analysis for the parental V2L2 mAb, mAbl66 (positive control) and R347 (negative control). (C) Evaluation of the V2L2 mAb, mAbl66 (positive control) and R347 (negative control) ability to prevent lysis of RBCs. (D) Evaluation of the V2L2- germlined mAb (V2L2-GL) and optimized V2L2-GL mAbs (V2L2—P4M, V2L2-MFS, V2L2-MD and V2L2-MR) to prevent lysis of RBCs. (E) tion of mAbs 1E6, lF3, llA6, 29D2, PCRV02 and V2L7 to prevent lysis of RBCs (F) Evaluation of mAbs V2L2 2012/063722 and 29D2 to prevent lysis of RBCs. (G-H) Relative binding ties of L and V2L2—MD antibodies.

Figure 13 (A—I): In vivo survival study of anti-Pch antibody treated mice. (A) Mice were treated 24 hours prior to infection with: 1.03 X 106 CFU 6077 (exoU+) with 45 mg/kg R347 (negative control), 45 mg/kg, 15.0 mg/kg, 5.0 mg/kg, or 1.0 mg/kg mAb166 (positive control), or 15 mg/kg, 5.0 mg/kg, 1.0 mg/kg, or 0.2 mg/kg V2L2. Survival was monitored for 96 hours. (B) Mice were treated 24 hours prior to infection with: 2.1 x 107 CPU 6294 (exoSl) with 15 mg/kg R347 (negative control), 15.0 mg/kg, 5.0 mg/kg, or 1.0 mg/kg mAb166 (positive control), or 15 mg/kg, 5.0 mg/kg, or 1.0 mg/kg V2L2. Survival was monitored for 168 hours. Mice were d 24 hours prior to infection with: (C) 6294 (06) or (D) PA103A with R347 (negative control), 5mg/kg of the Pch antibody Pch—02, or 5mg/kg, 1.0mg/kg, 0.2mg/kg, or 0.04mg/kg V2L2. Mice were treated 24 hours prior to infection with strain 6077 with R347 (negative control), 5mg/kg of the Pch antibody Pch-02, V2L7 (5mg/kg or 1mg/kg), 3G5 (5mg/kg or 1mg/kg), or 11A6 (5mg/kg or ) (E), or 25mg/kg of the V2L7, 1E6, 1F3, 29D2, R347 or 1mg/kg of the Pch antibody Pch-01 (F), or 25mg/kg of the 21F1, V2L2, 2H3, 4A8, SH3, LE10, R347 or 1mg/kg of the Pch-02 (G), or the 29D2 (1 mg/kg, 3mg/kg or 10 mg/kg), the V2L2 (1 mg/kg, 3mg/kg or 10 mg/kg) R347 or 1mg/kg of the Pch—02 (H). Mice were treated 24 hours prior to ion with: 6294 (06) or PA103A with the V2L2 (0.04mg/kg, 0.2mg/kg, 1 mg/kg or 5 mg/kg), R347 or 5mg/kg of the Pch-02. Percent survival was assayed in an acute pneumonia model.

Figure 14: Organ burden analysis of V2L2 treated mice. Mice were treated 24 hours prior to infection with 6206 with (A) R347 (negative control), 1 mg/kg, 0.2 mg/kg, or 0.07 mg/kg V2L2 and (B) 15 mg/kg R347 (negative l); 15.0 mg/kg, 5.0 mg/kg, or 1.0 mg/kg mAb166 (positive control); or 5.0 mg/kg, 1.0 mg/kg, or 0.2 mg/kg V2L2.

Colony forming units were identified per gram of tissue in lung, spleen, and kidney.

Figure 15: Organ burden analysis of V2L2 and WapR—004 (W4) treated mice.

Mice were treated 24 hours prior to infection with 6206 (Oll-ExoU+) with R347 (negative control), V2L2 alone, or V2L2 (0.1mg/kg) in ation with increasing concentrations of W4 (0.1, 0.5, 1.0, or 2.0 mg/kg). Colony g units were identified per gram of tissue in lung, spleen, and kidney. .

Figure 16 (A—G): Survival rates for animals treated with ch monoclonal antibody V2L2 in a P. aeruginosa acute pneumonia model. V2L2-GL, V2L2-MD, V2L2-PM4, V2L2-A and V2L2-MFS designations in panels A through G represent different ations of V2L2. (A-C) Animals were treated with V2L2 at lmg/kg, 0.5mg/kg or R347 at 0.5mg/kg prior to asal infection with (A) 6077 (9.75 x 105 CFU), (B, C) 6077 (9.5 X 105 CFU). (D—F) Animals were treated with V2L2 at 0.5mg/kg, 0.1mg/kg or R347 at 0.5mg/kg followed by infection with 6077 (D) (l x 106 CFU), (E) (9.5 x 105 CPU) or F (1.026 x 106 CPU). (G) Animals were treated with V2L2—MD at (0.04mg/kg, 0.2mg/kg, lmg/kg or 5mg/kg), mAbl66 (positive control) at (0.2mg/kg, lmg/kg, 5mg/kg or lSmg/kg), or R347 at 0.5mg/kg followed by infection with 6206 (2 x 107+ CFU).

Figure 17 (A—B): Schematic representation of (A) oc/W4, Bs2—TNFOt/W4, BS3-TNFOt/W4 and (B) Bs2-V2L2/W4-RAD, Bs3-V2L2/W4-RAD, and Bs4-V2L2-W4- RAD Psl/Pch bispeciflc antibodies. (A) For oc/W4, the W4 scFv is fused to the amino-terminus of TNFoc VL through a (G4S)2 linker. For BS2—TNFOL/W4, the W4 scFv is fused to the amino-terminus of TNFoc VH through a (G4S)2 . For Bs3- TNFOL/W4, the W4 scFv is fused to the carboxy—terminus of CH3 through a (G4S)2 linker. (B) For Bs2—V2L2-2C, the W4-RAD scFv is fused to the amino-terminus of V2L2 VH through a (G4S)2 linker. For Bs2-W4-RAD-2C, the V2L2 scFv is fused to the amino-terminus of W4-RAD VH through a (G4S)2 linker. For Bs3-V2L2-2C, the W4- RAD scFv is fused to the carboxy-terminus of CH3 through a (G4S)2 . For Bs4- V2L2-2C, the W4-RAD scFv is inserted in the hinge region, linked by (G4S)2 linker on the N—terminal and C-terminal of the scFv.

Figure 18: Evaluation of WapR—004 (W4) scFv activity in a bispeciflc constructs depicted in Figure 17A. The W4 scFv was ligated onto two different ific constructs (in alternating N— or C—terminal orientations) having a TNFoc binding arm. Each W4- TNFOL bispecific construct (le—TNFoc/W4, FOL/W4 and Bs3—TNF0t/W4) retained the ability to inhibit cell ment similarly as W4 using the ux (05) assay indicating that the W4 scFv retains its activity in a bispecif1c format. R347 was used as a negative control.

Figure 19 (A-C): Anti-Psl and anti-Pch binding domains were combined in the bispecif1c format by replacing the TNFoc antibody of Figure 17B with V2L2. These constructs are identical to those depicted in Figure 17B with the exception of using the non-stabilized W4-scFv in place of the ized W4-RAD scFv. Both W4 and W4-RAD target identical epitopes and have identical onal activities. Percent inhibition of cytotoxicity was analysed for both BS2-V2L2 and BS3-V2L2 using both (A) 6206 and (B) SZA treated A549 cells. (C) L2, BS3-V2L2, and Bs4-V2L2 were evaluated for their ability to t lysis of RBCs compared to the parental control. All bi—specif1c constructs retained anti—cytotoxicity activity similar to the parental V2L2 antibody using 6206 and 6206ApslA infected cells and prevented lysis of RBCs similar to the parental control (V2L2). R347 was used as a negative control in all ments.

Figure 20 (A-C): Evaluation of sl/anti-Pch bispecif1c ucts for promoting OPK of P. aeruginosa. Opsonophagocytosis assay is shown with luminescent P. aeruginosa serogroup 05 strain (PAOl.lux), with dilutions of purified Psl/TNFoc bispeciflc dies (BS2-TNFOL and Bs3-TNFoc); the W4-RAD or V2L2-IgGl parental antibodies; the Psl/Pch bispecif1c antibodies Bs2- V2L2 or Bs3-V2L2, or the Bs2-V2L2- 2C, Bs3-V2L2-2C, Bs4-V2L2-2C or the Bs4-V2L2-2C antibody harboring a YTE mutation (Bs4-V2L2-2C-YTE). (A) While the Bs2-V2L2 antibody showed similar killing compared to the parental W4-RAD antibody, the killing for the L2 antibody was sed. (B) While the Bs2—V2L2—2C and Bs4—V2L2—2C antibodies showed similar killing compared to the parental W4-RAD antibody, the killing for the Bs3-V2L2-2C antibody was decreased. (C) W4—RAD and W4-RAD-YTE designations represent different ations of W4-RAD. Bs4-V2L2-2C (old lot) and Bs4-V2L2-2C (new lot), designations represent different preparations of Bs4-V2L2-2C. The YTE modification in Bs4-V2L2-2C-YTE is a modification made to dies that increases the half-life of antibodies. Different preparations of Bs4 antibodies (old lot vs. new lot) showed similar killing compared to the parental W4-RAD antibody, r the Bs4-V2L2-2C—YTE antibodies had a 3-fold drop in OPK activity when compared to Bs4—V2L2—2C (See EC50 table). R347 was used as a negative control in all experiments.

Figure 21 (A-I): In vivo survival study of anti-Psl/anti-Pch bispecif1c antibodies Bs2-V2L2, Bs3-V2L2, Bs4-V2L2-2C and Bs4-V2L2-2C-YTE-treated mice in a 6206 acute pneumonia model system. Mice (n=10) were treated with (A): R347 (negative control, 0.2 mg/kg), Bs2-V2L2 (0.28 mg/kg), Bs3-V2L2 (0.28 mg/kg), V2L2 (0.2 mg/kg) or W4-RAD (0.2 mg/kg); (B-C): R347 (negative control, 1 mg/kg), Bs2-V2L2 (0.5 mg/kg or 1 mg/kg), or Bs4-V2L2-2C (0.5 mg/kg or 1 mg/kg); (D): R347 (negative control, 1 mg/kg), L2 (0.5 mg/kg or 1 mg/kg), or Bs4-V2L2-2C (0.5 mg/kg or 1 mg/kg); (E): R347 (negative control, 2 mg/kg), a combination of the individual W4 and V2L2 antibodies (0.5 mg/kg or 1 mg/kg each) or Bs4-V2L2-2C (1 mg/kg or 2 mg/kg); (F): R347 (negative l, 1 , a mixture of the individual W4 and V2L2 antibodies (0.5 mg/kg or 1 mg/kg each) or Bs4-V2L2-2C (1 mg/kg or 0.5 mg/kg). Twenty-four hours post-treatment, all mice were infected with ~ (6.25x105-1x106 CFU/animal) 6206 (Oll-ExoU+). All mice were monitored for 120 hours. (A): All of the control mice succumbed to ion by approximately 30 hours post—infection. All of the Bs3—V2L2 animals survived, along with those which received the V2L2 control. Approximately 90% of the W4-RAD immunized animals survived. In contrast, approximately 50% of the Bs2-V2L2 animals succumbed to infection by 120 hours. (B-F): All of the control mice succumbed to infection by approximately 48 hours post—infection. (B): L2— 2C had greater activity in comparison to Bs2-V2L2 at both 1.0 and 0.5 mg/kg. (C): Bs4— V2L2-2C appeared to have r activity in comparison to Bs2-V2L2 at 1.0 mg/kg (results are not statistically significant). (D): Bs4-V2L2-2C had greater activity in comparison to L2 at 0.5 mg/kg. (E): Bs4-V2L2-2C at both 2 mg/kg and 1 mg/kg had greater activity in comparison to the antibody mixture at both 1.0 and 0.5 mg/kg. (F): Bs4-V2L2 (1 mg/kg) has similar activity at both 1.0 and 0.5 mg/kg. (G-H): Both Bs4— V2L2-2C and Bs4-V2L2-2C—YTE had similar activity at both 1.0 and 0.5 mg/kg. Results are represented as Kaplan—Meier survival curves; ences in al were calculated by the Log-rank test for (B) Bs4-V2L2-2C vs. Bs2-V2L2 (1 mg/kg — P=0.034; 0.5mg/kg — P=0.0002); (D) L2—2C vs. Bs3—V2L2 (0.5 mg/kg — P<0.0001); (E): L2— 2C (2 mg/kg) vs. antibody e (1 mg/kg each)-P=0.0012; Bs4-V2L2-2C (1 mg/kg) vs. antibody mixture (0.5 mg/kg each)—P=0.0002. (G—H): Mice (n=8) were treated with: R347 (negative control, 1 mg/kg), Bs4-V2L2-2C (1 and 0.5 mg/kg), and Bs4-V2L2-2C— YTE (1 and 0.5 mg/kg) and 6206 (9e5 CFU). No difference in al between Bs4— V2L2—2C and Bs4—V2L2—2C—YTE at either dose were observed by Log—Rank. (I): To analyze the cy of each antibody construct, mice were treated with 0.1 mg/kg, 0.2 mg/kg, 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 5 mg/kg, 10 mg/kg or 15 mg/kg and analyzed for survival in a 6206 lethal pneumonia model. The percent survival is indicated in the table with the number of animals for each comparison indicated in parentheses.

Figure 22: Organ burden analysis of anti-Psl/Pch bispecific antibody-treated s using the 6206 acute pneumonia model. Mice were treated 24 hours prior to infection with 6206 (01 l-ExoU+) with R347 (negative control), V2L2 or W4-RAD alone (0.2 mg/kg), Bs2-V2L2 (0.28 , or BS3-V2L2 (0.28 . Colony forming units were identified per gram of tissue in lung, , and kidney. At the concentration tested, both Bs2-V2L2 and Bs3-V2L2 significantly decreased organ burden in lung.

However, neither of the bispeciflc constructs was able to significantly affect organ burden in spleen or kidney compared to the parental antibodies.

Figure 23 (A-B): Organ burden analysis of anti-Psl/Pch bispecif1c antibody- treated animals using a 6294 model system. Mice were d 24 hours prior to infection with 6294 with R347 (negative control), V2L2 or W4-RAD alone (0.5 mg/kg), Bs2-V2L2 (0.7 mg/kg), or Bs3—V2L2 (0.7 mg/kg) (A), or V2L2 or W4-RAD alone (0.2 , Bs2- V2L2 (0.2 mg/kg), L2 (0.2 mg/kg) or a combination of the individual W4-RAD and V2L2 antibodies (0.1 mg/kg each) (B). Twenty-four hours post-administration of antibody, all mice were infected with an inoculum containing 2.5 x 107 CFU 6294 (A) or 1.72 x 107 CFU 6294 (B). Colony forming units were identified per gram of tissue in lung, spleen, and kidney. Using the 6294 model system, (A) both the BS2-V2L2 and BS3-V2L2 significantly decreased organ burden in all of the tissues to a level comparable to that of the V2L2 parental dy. The W4-RAD parental antibody had no effect on decreasing organ burden. (B) Bs2—V2L2, Bs3—V2L2, and +V2L2 combination significantly decreased organ burden in all of the tissues to a level comparable to that of the V2L2 parental antibody.

Figure 24: In vivo survival study of Bs2—W4/V2L2 and Bs3-W4/V2L2-treated mice in a 6294 model system. Mice were treated with R347 (negative control, 0.2 mg/kg), Bs2-V2L2 (0.28 mg/kg), Bs3-V2L2 (0.28 mg/kg), V2L2 (0.2 mg/kg) or W4- RAD (0.2 . Twenty-four hours post-treatment, all mice were infected with 6294.

All mice were monitored for 120 hours. All of the control mice succumbed to ion by approximately 75 hours post-infection. Sixty percent of the Bs3-V2L2 and 50% of the Bs2-V2L2 animals survived after 120 hours post-inoculation. As was seen in the organ burden studies, W4-RAD immunization did not affect al with all mice succumbing to infection at approximately the same time as the controls.

Figure 25 (A-D): Organ burden analysis of anti-Psl/Pch bispecific antibody or W4 + V2L2 combination therapy in the 6206 model system. Suboptimal concentrations of antibody were used (A—C) to enable the ability to decipher dy activity. (D) High concentrations of Bs4 were used. Mice were treated 24 hours prior to infection with 6206 with R347 (negative control), V2L2 or W4-RAD alone (0.2 mg/kg), L2 (0.2 mg/kg), Bs3—V2L2 (0.2 , Bs4 (15.0, 5.0 and 1.0 mg/kg) or a combination of the individual W4 and V2L2 dies (0.1 mg/kg each). Twenty—four hours post— administration of antibody, all mice were infected with an um containing (A), (B) 4.75 x 105 CPU 6206 (Oll—ExoU+), or (C) 7.75 x 105 CPU 6206 (Oll—ExoU+) or (D) 9.5 X 105 CPU 6206 (Oll—ExoU+). Colony g units were identified per gram of tissue in lung, spleen, and kidney. Using the 6206 model system, both the BS2-V2L2 and BS3—V2L2 decreased organ burden in the lung, spleen and kidneys to a level comparable to that of the W4 + V2L2 combination. In the lung, the combination significantly reduced bacterial CFUs Bs2- and Bs3—V2L2 and V2L2 using the Kruskal-Wallis with Dunn’s post test. Significant differences in bacterial burden in the spleen and kidney were not observed, although a trend towards reduction was noted. (D) When optimal concentrations of Bs4—V2L2—2C were used (15.0, 5.0, and 1.0), rapid and efficient bacterial nce was observed from the lung. In on, bacterial dissemination to the spleen and s were also ablated. Asterisks indicate statistical significance when compared to the R347 control using the Kruskal-Wallis with Dunn’s post test.

Figure 26 (A—J): Therapeutic adjunctive therapy: Bs4-V2L2—2C + antibiotic. (A)— (B) Mice were treated 24 hours prior to infection with 1 x 106 CPU 6206 with 0.5 mg/kg R347 (negative control) or Bs4-V2L2-2C (0.2 mg/kg or 0.5 mg/kg) or Ciprofloxacin (CIP) (20 mg/kg or 6.7 mg/kg) 1 hour post infection, or a combination of the Bs4-V2L2- 2C 24 hours prior to infection and CIP 1 hour post infection (0.5 mg/kg + 20 mg/kg or 0.5 mg/kg + 6.7 mg/kg or 0.2 mg/kg + 20 mg/kg or 0.2 mg/kg + 6.7 mg/kg, respectively). (C) Mice were treated 1 hour post infection with 9.5 x 105 CPU 6206 with 5 mg/kg R347 or CIP (20 mg/kg or 6.7 mg/kg) or Bs4-V2L2-2C (1 mg/kg or 5 mg/kg), or a combination of the Bs4-V2L2-2C and CIP (5 mg/kg + 20 mg/kg or 5 mg/kg + 6.7 mg/kg or 1 mg/kg + 20 mg/kg or 1 mg/kg + 6.7 mg/kg, respectively). (D) Mice were treated 2 hours post infection with 9.5 X 105 CPU 6206 with 5 mg/kg R347 or CIP (20 mg/kg or 6.7 mg/kg) or Bs4-V2L2-2C (1 mg/kg or 5 mg/kg), or a combination of the Bs4-V2L2-2C and Cipro (5 2012/063722 mg/kg + 20 mg/kg or 5 mg/kg + 6.7 mg/kg or 1 mg/kg + 20 mg/kg or 1 mg/kg + 6.7 mg/kg, respectively). (E) Mice were treated 2 hours post ion with 9.75 x 105 CPU 6206 with 5 mg/kg R347or Bs4-V2L2-2C (1 mg/kg or 5 mg/kg) or CIP (20 mg/kg or 6.7 mg/kg) 1 hour post infection, or a combination of the Bs4-V2L2-2C 2 hours post ion and CIP 1 hour post infection (5 mg/kg + 20 mg/kg or 5 mg/kg + 6.7 mg/kg or 1 mg/kg + 20 mg/kg or 1 mg/kg + 6.7 mg/kg, respectively). (F) Mice were treated 1 hour post infection with 9.5 x 105 CPU 6206 with 5 mg/kg R347 or Meropenem (MEM) (0.75 mg/kg or 2.3 mg/kg) or Bs4-V2L2-2C (1 mg/kg or 5 mg/kg), or a combination of the Bs4—V2L2—2C and MEM (5 mg/kg + 2.3 mg/kg or 5 mg/kg + 0.75 mg/kg or 1 mg/kg + 2.3 mg/kg or 1 mg/kg + 0.75 mg/kg, respectively). (G) Mice were treated 2 hours post infection with 9.75 x 105 CPU 6206 with 5 mg/kg R347 or Bs4-V2L2-2C (1 mg/kg or 5 mg/kg) or MEM (0.75 mg/kg or 2.3 mg/kg) 1 hour post infection, or a combination of the Bs4-V2L2-2C 2 hours post infection and MEM 1 hour post ion (5 mg/kg + 2.3 mg/kg or 5 mg/kg + 0.75 mg/kg or 1 mg/kg + 2.3 mg/kg or 1 mg/kg + 0.75 mg/kg, respectively). (H) Mice were treated 2 hours post infection with l x 106 CPU 6206 with 5 mg/kg R347 or Bs4-V2L2-2C (1 mg/kg or 5 mg/kg) or MEM (0.75 mg/kg or 2.3 mg/kg), or a combination of the Bs4-V2L2-2C 2 and MEM (5 mg/kg + 2.3 mg/kg or 5 mg/kg + 0.75 mg/kg or 1 mg/kg + 2.3 mg/kg or 1 mg/kg + 0.75 mg/kg, respectively). (I) Mice were treated 4 hour post infection with 9.25 x 105 CPU 6206 with 5 mg/kg R347 or CIP (6.7 mg/kg) or Bs4-V2L2-2C (1 mg/kg or 5 mg/kg) or a combination of the Bs4-V2L2- 2C and CIP (5 mg/kg + 6.7 mg/kg or 1 mg/kg + 6.7 mg/kg, respectively) Mice were , (J) treated 4 hour post infection with 1.2 x 106 CPU 6206 with 5 mg/kg R347 + CIP (6.7 , CIP (6.7 mg/kg), or Bs4-V2L2-2C (1 mg/kg or 5 mg/kg) or a ation of the Bs4—V2L2—2C and CIP (5 mg/kg + 6.7 mg/kg or 1 mg/kg + 6.7 mg/kg, respectively). (A— J) Bs4 antibody combined with either CIP or MEM increases efficacy of antibiotic therapy, indicating synergistic protection when the molecules are combined. In addition, gh antibiotic delivered by itself or in combination with a P. aeruginosa non— specific antibody can reduce or control bacterial CFU in the lung, antibiotic alone does not protect mice from lethality in this setting. Optimal protection in this setting requires including L2-2C in combination with antibiotic.

Figure 27 (A-C): Difference in onal activity of bi-specific antibodies BS4- WT, BS4—GL and BS4-GLO: opsonophagocytic killing assay (A), anti-cell attachment assay (B), and a RBC lysis anti-cytotoxicity assay (C).

Figure 28 (A-B): Percent protection against lethal pneumonia in mice challenged in lactic (A) or therapeutic (B) settings with P. aeruginosa strains. The t survival is indicated in the table with the number of animals for each comparison indicated in parentheses. The dashes indicate not tested.

Figure 29 (A—B): Survival rates for animals treated with bispecif1c antibody Bs4- GLO in a P. aeruginosa lethal emia model. (A) Animals were treated with Bs4- GLO at 15mg/kg, 5mg/kg, lmg/kg or R347 at g 24 hours prior to intraperitoneal ion with 6294 (06) (5.58 x 107 CFU). (B) Animals were treated with Bs4-GLO at 5 mg/kg, 1 mg/kg, 0.2 mg/kg or R347 at 5mg/kg 24 hours prior to intraperitoneal infection with 6206 (Oll—ExoU+) (6.48 X 106 CFU). Results are represented as Kaplan-Meier survival curves; differences in survival were calculated by the Log—rank test for BS4— GLO at each concentration vs. R347. (A) Bs4-GLO at all concentrations vs. R347 P<0.0001. (B) Bs4—GLO at all concentrations vs. R347 P=0.0003. Results are representative of three independent experiments.

Figure 30 (A—C): Survival rates for s prophylactically treated (prevention) with Bs4-GLO in a P. aeruginosa thermal injury model. (A) Animals were treated with Bs4-GLO at 15mg/kg, 5mg/kg or R347 at 15mg/kg 24 hours prior to induction of thermal injury and subcutaneous infection with P. aeruginosa strain 6077 (01 l-ExoU+) with 1.4 x 105 CFU directly under the wound. (B) Animals were treated with Bs4-GLO at 15mg/kg or R347 at 15mg/kg 24 hours prior to induction of thermal injury and subcutaneous infection with P. aeruginosa strain 6206 xoU+) with 4.15 x 104 CFU ly under the wound. (C) Animals were treated with O at 15mg/kg, 5mg/kg or R347 at 15mg/kg 24 hours prior to induction of thermal injury and subcutaneous infection with P. aeruginosa strain 6294 (06) with 7.5 x 101 CFU directly under the wound. Results are represented as Kaplan—Meier survival curves; differences in survival were calculated by the Log-rank test for Bs4-GLO at each tration vs. R347. (A-C) Bs4-GLO at all trations vs. R347 — P<0.0001. Results are representative of two independent experiments for each P. aeruginosa strain.

Figure 31 (A-B): Survival rates for animals therapeutically treated (treatment)) with Bs4-GLO in a P. aeruginosa thermal injury model. (A) Animals were treated with Bs4-GLO at 42.6mg/kg, 15 mg/kg or R347 at 45mg/kg 4h hours after ion of thermal injury and subcutaneous infection with P. aeruginosa strain 6077 (Oll—EXOU+) with 1.6 x 105 CFU directly under the wound. (B) Animals were d with Bs4-GLO at 15mg/kg, 5 mg/kg or R347 at 15mg/kg 12h hours after induction of thermal injury and subcutaneous infection with P. nosa strain 6077 (01 l—ExoUT) with 1.0 x 105 CFU directly under the wound. s are represented as Kaplan—Meier survival curves; differences in survival were ated by the Log—rank test for BS4-GLO at each concentration vs. R347. (A) Bs4—GLO at both concentrations vs. R347 — P=0.0004. (B) Bs4—GLO at 5mg/kg vs. R347 — P=0.048. Results are entative of two independent experiments.

Figure 32 (A—B): Therapeutic tive therapy: Bs4GLO + ciprofloxacin (CIP): (A) Mice were treated 4 hour post infection with 9.5 x 105 CFU 6206 with 5 mg/kg R347 + CIP (6.7 mg/kg) or Bs4-WT (1 mg/kg or 5 mg/kg) or a combination of the Bs4-WT and CIP (5 mg/kg + 6.7 mg/kg or 1 mg/kg + 6.7 mg/kg, respectively). (B) Mice were treated 4 hour post infection with 9.5 x 105 CFU 6206 with 5 mg/kg R347 + CIP (6.7 mg/kg) or Bs4-GLO (1 mg/kg or 5 mg/kg) or a combination of the Bs4-GLO and CIP (5 mg/kg + 6.7 mg/kg or 1 mg/kg + 6.7 mg/kg, respectively Figure 33 (A—B): Therapeutic adjunctive therapy: Bs4-GLO + meropenem (MEM): (A) Mice were treated 4 hour post infection with 9.5 X 105 CFU 6206 with 5 mg/kg R347 + MEM (0.75 mg/kg) or Bs4-WT (1 mg/kg or 5 mg/kg) or a combination of the Bs4-WT and MEM (5 mg/kg + 0.75 mg/kg or 1 mg/kg + 0.75 mg/kg, respectively).

(B) Mice were treated 4 hour post ion with 9.5 x 105 CFU 6206 with 5 mg/kg R347 + MEM (0.75 mg/kg) or Bs4-GLO (1 mg/kg or 5 mg/kg) or a combination of the Bs4- GLO and MEM (5 mg/kg + 0.75 mg/kg or 1 mg/kg + 0.75 mg/kg, tively).

Figure 34 (A—C): Therapeutic adjunctive therapy: Bs4—GLO + antibiotic in a lethal bacteremia model. Mice were treated 24 hours prior to eritoneal infection with P. aeruginosa strain 6294 (06) 9.3 x 107 with Bs4—GLO at (0.25mg/kg or 0.5mg/kg) or R347 (negative control). One hour post infection, mice were treated subcutaneously with (A) 1mg/kg CIP, (B) 2.5mg/kg MEM or (C) 2.5mg/kg TOB. Results are represented as Kaplan-Meier survival curves; differences in survival were calculated by the Log-rank test for Bs4-GLO at each concentration vs. R347.

Figure 35 (A-B) Schematic representation of alternative s for Bs4 constructs (A) anti-Pch variable regions are present separately on the heavy and light chains while the anti-Psl variable regions are present as an scFv within the hinge region of the heavy chain and (B) anti-Psl variable regions are present separately on the heavy and light chains while the anti-Pch variable regions are present as an scFv within the hinge region of the heavy chain.

DETAILED DESCRIPTION I. DEFINITIONS It is to be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "a binding molecule which specifically binds to Pseudomonas Ps1 and/or Pcr ," is understood to represent one or more binding molecules which specifically bind to Pseudomonas Psl and/or Pch. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

As used herein, the term “polypeptide” is intended to encompass a singular “polypeptide” 3 as well as plural “polypeptides,’ and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds).

The term "polypeptide" refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the t. Thus, peptides, dipeptides, tides, oligopeptides, “protein,3’ “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids are included within the definition of "polypeptide,” and the term “polypeptide” can be used d of, or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to the products of post—expression modifications of the polypeptide, including t limitation glycosylation, ation, phosphorylation, ion, derivatization by known ting/blocking , proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide can be derived from a natural biological source or produced by inant technology, but is not necessarily translated from a designated nucleic acid sequence. It can be generated in any manner, including by chemical synthesis.

A ptide as disclosed herein can be of a size of about 3 or more, 5 or more, or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides can have a defined three—dimensional ure, although they do not necessarily have such structure.

Polypeptides with a defined three—dimensional structure are referred to as folded, and polypeptides which do not possess a defined three—dimensional structure, but rather can adopt a large number of different conformations, and are referred to as ed. As used herein, the term glycoprotein refers to a protein coupled to at least one ydrate moiety that is attached to the protein via an oxygen-containing or a nitrogen-containing side chain of an amino acid residue, e. g., a serine residue or an asparagine residue.

By an "isolated" polypeptide or a fragment, variant, or derivative f is intended a polypeptide that is not in its natural milieu. No particular level of purification is required. For example, an ed polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated as sed herein, as are native or recombinant polypeptides which have been ted, fractionated, or partially or ntially purified by any suitable technique.

Other polypeptides disclosed herein are fragments, derivatives, analogs, or variants of the foregoing polypeptides, and any combination thereof. The terms "fragment," nt," "derivative" and "analog" when referring to a binding molecule such as an dy which specifically binds to Pseudomonas Psl and/or Pch as disclosed herein e any polypeptides which retain at least some of the antigenbinding properties of the corresponding native antibody or polypeptide. Fragments of polypeptides include, for example, proteolytic fragments, as well as deletion fragments, in addition to specific antibody fragments discussed elsewhere herein. Variants of a binding molecule, e.g., an antibody which specifically binds to Pseudomonas Psl and/or Pch as disclosed herein include fragments as bed above, and also polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. Variants can occur naturally or be non-naturally occurring. Non-naturally occurring variants can be produced using art—known mutagenesis techniques. Variant polypeptides can comprise conservative or non—conservative amino acid substitutions, deletions or additions.

Derivatives of a binding le, e.g., an antibody which specifically binds to Pseudomonas Psl and/or Pch as disclosed herein are polypeptides which have been altered so as to exhibit additional es not found on the native polypeptide. Examples include fusion proteins. t polypeptides can also be referred to herein as "polypeptide analogs. H As used herein a "derivative" of a binding molecule, e.g., an antibody which specifically binds to Pseudomonas Psl and/or Pch refers to a subject polypeptide having one or more residues chemically tized by reaction of a functional side group. Also included as "derivatives" are those peptides which contain one or more naturally occurring amino acid derivatives of the twenty rd amino acids.

For example, 4-hydroxyproline can be substituted for proline; 5-hydroxylysine can be substituted for lysine; 3-methylhistidine can be substituted for histidine; homoserine can be substituted for serine; and ornithine can be substituted for lysine.

The term ucleotide" is intended to encompass a singular nucleic acid as well as plural nucleic acids, and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA (pDNA). A polynucleotide can comprise a conventional phosphodiester bond or a non—conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (PNA)). The term "nucleic acid" refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide.

By "isolated" nucleic acid or polynucleotide is ed a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. For example, a recombinant polynucleotide encoding a binding molecule, e.g., an dy which ically binds to Pseudomonas Psl and/or Pch contained in a vector is considered ed as disclosed herein. Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or ed (partially or substantially) polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides. Isolated polynucleotides or nucleic acids further e such molecules produced synthetically. In on, polynucleotide or a nucleic acid can be or can include a regulatory element such as a promoter, ribosome binding site, or a ription terminator.

As used herein, a "coding " is a portion of nucleic acid which consists of codons translated into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is not translated into an amino acid, it can be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional 2012/063722 terminators, introns, and the like, are not part of a coding region. Two or more coding regions can be present in a single cleotide construct, e.g., on a single vector, or in te polynucleotide constructs, e.g., on separate (different) vectors. rmore, any vector can contain a single coding region, or can comprise two or more coding regions, e.g., a single vector can separately encode an globulin heavy chain variable region and an immunoglobulin light chain variable region. In addition, a vector, polynucleotide, or c acid can encode heterologous coding regions, either fused or unfused to a nucleic acid encoding an a binding molecule which specifically binds to Pseudomonas Psl and/or Pch, e.g., an antibody, or antigen-binding fragment, variant, or derivative thereof. Heterologous coding regions include without limitation specialized elements or motifs, such as a secretory signal peptide or a heterologous onal domain.

In n embodiments, the polynucleotide or nucleic acid is DNA. In the case of DNA, a polynucleotide comprising a nucleic acid which encodes a polypeptide normally can include a promoter and/or other ription or translation control elements operably associated with one or more coding regions. An operable association is when a coding region for a gene product, e.g., a polypeptide, is associated with one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the tory sequence(s). Two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are "operably associated" if induction of promoter function results in the transcription of mRNA encoding the desired gene product and if the nature of the e between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product or interfere with the y of the DNA template to be ribed. Thus, a promoter region would be operably associated with a nucleic acid encoding a polypeptide if the promoter was capable of effecting transcription of that nucleic acid. The promoter can be a cell—specific promoter that directs substantial transcription of the DNA only in predetermined cells. Other transcription l elements, besides a promoter, for example enhancers, ors, repressors, and transcription termination signals, can be operably ated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcription control regions are disclosed herein.

A variety of transcription control regions are known to those skilled in the art.

These include, without limitation, transcription control regions which on in vertebrate cells, such as, but not limited to, promoter and er segments from galoviruses (the immediate early promoter, in conjunction with intron-A), simian virus 40 (the early promoter), and retroviruses (such as Rous sarcoma virus). Other transcription l regions e those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit B-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue—specific promoters and enhancers as well as lymphokine—inducible promoters (e.g., promoters inducible by interferons or interleukins).

Similarly, a variety of ation control ts are known to those of ry skill in the art. These include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence).

In other embodiments, a polynucleotide can be RNA, for example, in the form of messenger RNA (mRNA). cleotide and c acid coding regions can be associated with additional coding regions which encode secretory or signal peptides, which direct the ion of a polypeptide encoded by a polynucleotide as disclosed herein, e.g., a polynucleotide encoding a binding molecule which specifically binds to Pseudomonas Ps1 and/or Pch, e. g., an antibody, or antigen-binding fragment, variant, or derivative thereof. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Those of ordinary skill in the art are aware that polypeptides secreted by vertebrate cells generally have a signal peptide fused to the inus of the polypeptide, which is cleaved from the complete or "full length" polypeptide to produce a secreted or "mature" form of the polypeptide. In certain embodiments, the native signal peptide, e.g., an immunoglobulin heavy chain or light chain signal peptide is used, or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is ly associated with it. Alternatively, a heterologous mammalian signal peptide, or a functional derivative thereof, can be used. For example, the wild—type leader sequence can be substituted with the leader sequence of human tissue plasminogen activator (TPA) or mouse B—glucuronidase.

Disclosed herein are certain binding molecules, or antigen—binding fragments, variants, or derivatives thereof. Unless ically referring to full—sized antibodies such as naturally—occurring antibodies, the term "binding molecule" encompasses full—sized antibodies as well as antigen-binding fragments, variants, analogs, or derivatives of such antibodies, e.g., naturally occurring antibody or immunoglobulin molecules or engineered antibody les or fragments that bind antigen in a manner similar to dy molecules.

As used herein, the term “binding molecule” refers in its broadest sense to a molecule that specifically binds an antigenic determinant. As described further herein, a binding molecule can comprise one of more of the binding domains bed herein. As used herein, a ng " includes a site that specifically binds the antigenic determinant. A non-limiting example of an antigen g molecule is an antibody or nt thereof that retains antigen-specific binding.

The terms "antibody" and "immunoglobulin" can be used interchangeably herein.

An antibody (or a fragment, variant, or derivative thereof as disclosed herein comprises at least the variable domain of a heavy chain and at least the variable s of a heavy chain and a light chain. Basic immunoglobulin structures in vertebrate s are relatively well understood. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988).

As will be discussed in more detail below, the term oglobulin” comprises various broad classes of polypeptides that can be guished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon, (y, u, 0L, 8, a) with some subclasses among them (e. g., yl—y4). It is the nature of this chain that ines the "class" of the dy as IgG, IgM, IgA IgG, or IgE, respectively. The immunoglobulin subclasses (isotypes) e.g., IgGl, Ing, IgG3, IgG4, IgA1, etc. are well characterized and are known to confer functional specialization.

Modified versions of each of these classes and isotypes are readily discernible to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of this disclosure.

WO 70615 Light chains are classified as either kappa or lambda (K, 9»). Each heavy chain class can be bound with either a kappa or lambda light chain. In general, the light and heavy chains are covalently bonded to each other, and the "tail" portions of the two heavy chains are bonded to each other by covalent disulflde linkages or non—covalent linkages when the immunoglobulins are generated either by hybridomas, B cells or genetically engineered host cells. In the heavy chain, the amino acid sequences run from an N- terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain.

Both the light and heavy chains are diVided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it will be appreciated that the variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the nt domains of the light chain (CL) and the heavy chain (CH1, CH2 or CH3) confer important biological ties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like. By convention the numbering of the constant region s increases as they become more distal from the antigen g site or amino-terminus of the antibody. The N—terminal portion is a variable region and at the C- terminal portion is a constant region; the CH3 and CL domains actually se the carboxy-terminus of the heavy and light chain, respectively.

As indicated above, the variable region allows the binding molecule to selectively recognize and specifically bind epitopes on antigens. That is, the VL domain and VH , or subset of the complementarity determining regions (CDRs), of a binding molecule, e.g., an antibody combine to form the variable region that s a three dimensional antigen binding site. This quaternary binding le structure forms the antigen binding site present at the end of each arm of the Y. More ically, the antigen binding site is defined by three CDRs on each of the VH and VL chains.

In naturally occurring antibodies, the six “complementarity ining regions” or “CDRs” present in each antigen binding domain are short, non—contiguous sequences of amino acids that are specifically positioned to form the antigen binding domain as the antibody assumes its three dimensional configuration in an aqueous environment. The remainder of the amino acids in the n binding s, referred to as "framework" regions, show less inter-molecular variability. The framework regions largely adopt a [3- sheet conformation and the CDRs form loops which connect, and in some cases form part of, the B—sheet ure. Thus, framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions. The antigen binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary e promotes the valent binding of the antibody to its cognate epitope. The amino acids comprising the CDRs and the framework regions, respectively, can be readily identified for any given heavy or light chain variable region by one of ordinary skill in the art, since they have been precisely defined (see, "Sequences of Proteins of Immunological Interest," Kabat, E., et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. M01. Biol., [96:901—917 (1987), which are incorporated herein by reference in their entireties).

In the case where there are two or more definitions of a term which is used and/or accepted within the art, the definition of the term as used herein is intended to include all such meanings unless explicitly stated to the contrary. A specific example is the use of the term "complementarity determining region" ("CDR") to describe the non—contiguous n ing sites found within the variable region of both heavy and light chain polypeptides. This particular region has been bed by Kabat et al., U.S. Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and by Chothia et al., J. M01. Biol. 196:901—917 (1987), which are incorporated herein by nce, where the definitions include overlapping or subsets of amino acid residues when ed against each other. Nevertheless, application of either definition to refer to a CDR of an dy or ts thereof is intended to be within the scope of the term as defined and used herein. The riate amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth below in Table I as a comparison. The exact residue numbers which encompass a ular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of the antibody.

:CDR[mfimfimm1 Kabat Chothia VH CDRl VH CDR2 VH CDR3 VL CDRl VL CDR2 50—56 50—52 VL CDR3 89—97 91—96 1Numbering of all CDR definitions in Table l is ing to the numbering conventions set forth by Kabat et all. (see below).

Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody. One of ordinary skill in the art can unambiguously assign this system of "Kabat numbering" to any variable domain sequence, without ce on any experimental data beyond the sequence . As used herein, "Kabat numbering" refers to the numbering system set forth by Kabat et al., US. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). Unless otherwise specified, references to the numbering of specific amino acid residue positions in a binding le which specifically binds to Pseudomonas Ps1 and/or Pch, e.g, an dy, or antigen-binding fragment, variant, or derivative thereof as disclosed herein are according to the Kabat numbering system.

Binding molecules, e.g., antibodies or antigen-binding nts, ts, or derivatives thereof include, but are not limited to, polyclonal, monoclonal, human, humanized, or chimeric antibodies, single chain antibodies, e-binding fragments, e.g., Fab, Fab' and F(ab')2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulflde—linked Fvs (dev), fragments comprising either a VL or VH domain, fragments produced by a Fab expression y. ScFv molecules are known in the art and are described, e.g., in US patent 5,892,019. Immunoglobulin or antibody molecules encompassed by this disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.

By "specifically binds," it is lly meant that a binding molecule, e.g., an antibody or fragment, variant, or derivative thereof binds to an epitope via its antigen binding , and that the binding entails some complementarity between the antigen binding domain and the epitope. According to this definition, a binding molecule is said to "specifically bind" to an e when it binds to that e, via its antigen binding domain more y than it would bind to a random, unrelated epitope. The term "specificity" is used herein to qualify the relative affinity by which a certain binding molecule binds to a certain epitope. For example, binding molecule "A" may be deemed to have a higher specificity for a given epitope than binding molecule "B," or binding molecule "A" may be said to bind to epitope "C" with a higher specificity than it has for related epitope "D." By "preferentially binds," it is meant that the antibody specifically binds to an epitope more y than it would bind to a d, similar, homologous, or analogous epitope. Thus, an antibody which "preferentially binds" to a given epitope would more likely bind to that epitope than to a related epitope, even though such an antibody can cross—react with the related epitope.

By way of non-limiting example, a binding molecule, e.g., an dy can be considered to bind a first epitope preferentially if it binds said first epitope with a dissociation constant (KD) that is less than the antibody’s KD for the second epitope. In another non-limiting example, a binding le such as an antibody can be considered to bind a first antigen preferentially if it binds the first epitope with an affinity that is at least one order of magnitudeless than the dy’s KD for the second epitope. In another non-limiting example, a binding molecule can be considered to bind a first e preferentially if it binds the first epitope with an affinity that is at least two orders of magnitude less than the antibody’s KD for the second epitope.

In r non-limiting example, a binding molecule, e. g., an antibody or fragment, variant, or derivative thereof can be considered to bind a first epitope preferentially if it binds the first epitope with an off rate (k(off)) that is less than the antibody’s k(off) for the second epitope. In another non-limiting example, a g molecule can be considered to bind a first epitope preferentially if it binds the first epitope with an ty that is at least one order of magnitude less than the antibody’s k(off) for the second epitope. In another non-limiting example, a binding molecule can be considered to bind a first epitope preferentially if it binds the first epitope with an affinity that is at least two orders of magnitude less than the antibody’s k(off) for the second epitope.

A binding molecule, e. g., an antibody or nt, variant, or derivative thereof disclosed herein can be said to bind a target antigen, e.g., a polysaccharide disclosed herein or a fragment or variant thereof with an off rate (k(off)) of less than or equal to 5 X '2 sec'l, 10'2 sec'l, 5 X 10'3 sec'1 or 10'3 sec'l. A binding molecule as disclosed herein can be said to bind a target antigen, e. g., a polysaccharide with an off rate (k(off)) less than or equal to 5 X 10'4 sec'l, 10'4 sec'l, 5 X 10'5 sec'l, or 10'5 sec'1 5 X 10'6 sec'l, 10'6 sec'l, 5 X 10'7 sec"1 or 10'7 sec'l.

A binding molecule, e.g., an antibody or antigen-binding nt, variant, or derivative disclosed herein can be said to bind a target antigen, e. g., a polysaccharide with an on rate (k(on)) of greater than or equal to 103 M"1 sec'l, 5 X 103 M'1 sec'l, 104 M'1 sec'1 or 5 X 104 M'1 sec'l. A binding molecule as disclosed herein can be said to bind a target antigen, e. g., a polysaccharide with an on rate (k(on)) greater than or equal to 105 M"1 sec' 1, 5 X 105 M'1 sec'l, 106 M'1 sec'l, or 5 X 106 M'1 sec'1 or 107 M'1 sec'l.

A binding molecule, e.g., an antibody or fragment, variant, or derivative thereof is said to competitively inhibit binding of a reference antibody or antigen binding fragment to a given epitope if it preferentially binds to that epitope to the extent that it blocks, to some degree, binding of the reference antibody or antigen binding fragment to the epitope. Competitive inhibition can be determined by any method known in the art, for example, competition ELISA assays. A g molecule can be said to competitively t binding of the reference antibody or n binding fragment to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

As used , the term "affinity" refers to a e of the strength of the g of an individual epitope with the CDR of an immunoglobulin molecule. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) at pages 27—28. As used herein, the term "avidity" refers to the overall stability of the complex n a population of globulins and an antigen, that is, the functional combining strength of an immunoglobulin mixture with the antigen. See, e. g. Harlow at pages 29-34.

, Avidity is related to both the affinity of individual immunoglobulin molecules in the population with specific epitopes, and also the valencies of the immunoglobulins and the antigen. For example, the interaction n a bivalent monoclonal antibody and an antigen with a highly repeating epitope structure, such as a polymer, would be one of high avidity.

Binding molecules or antigen-binding fragments, variants or derivatives thereof as disclosed herein can also be described or specified in terms of their cross—reactivity. As used herein, the term "cross-reactivity" refers to the ability of a g molecule, e. g., an antibody or fragment, variant, or derivative f, specific for one antigen, to react with a second antigen; a measure of relatedness between two ent antigenic substances.

Thus, a binding molecule is cross ve if it binds to an epitope other than the one that induced its formation. The cross reactive epitope generally contains many of the same complementary structural features as the inducing epitope, and in some cases, can actually fit better than the original.

A g molecule, e. g., an antibody or fragment, variant, or derivative thereof can also be described or specified in terms of their binding ty to an n. For example, a binding molecule can bind to an n with a dissociation constant or KD no greater than 5 x 10'2 M, 10'2 M, 5 x 10'3 M, 10'3 M, 5 x 10'4 M, 10'4 M, 5 x 10'5 M, 10'5 M, x 10‘6 M, 10‘6 M, 5 x 10‘7 M, 10‘7 M, 5 x 10‘8 M, 10‘8 M, 5 x 10‘9 M, 10‘9 M, 5 x 10'10M, '10M, 5 x 10'11M, , 5 x 10'12M, , 5 x 10‘13 M, 10‘13 M, 5 x 10'14M, 10‘14 M, 5 x 10'15 M, or 10'15 M.

Antibody fragments including single-chain dies can se the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CH1, CH2, and CH3 domains. Also included are antigen-binding fragments also comprising any combination of variable (s) with a hinge region, CH1, CH2, and CH3 domains. Binding molecules, e. g., antibodies, or antigen-binding fragments thereof disclosed herein can be from any animal origin including birds and mammals. The antibodies can be human, , donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In another embodiment, the variable region can be condricthoid in origin (e. g., from sharks). As used herein, "human" antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulins and that do not express endogenous immunoglobulins, as described infra and, for example in, US. Pat. No. 5,939,598 by Kucherlapati et a1.

As used herein, the term “heavy chain portion” includes amino acid sequences derived from an immunoglobulin heavy chain. a binding molecule, e.g., an antibody comprising a heavy chain portion comprises at least one of: a CHl domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment f. For example, a binding molecule, e.g., an dy or fragment, variant, or derivative thereof can comprise a ptide chain comprising a CHl domain; a polypeptide chain comprising a CHl domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CHl domain and a CH3 domain; a ptide chain comprising a CHl domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain comprising a CHl domain, at least a portion of a hinge , a CH2 domain, and a CH3 domain. In another ment, a binding molecule, e.g., an antibody or nt, variant, or derivative thereof comprises a polypeptide chain comprising a CH3 domain. Further, a binding molecule for use in the disclosure can lack at least a portion of a CH2 domain (e.g., all or part of a CH2 domain).

As set forth above, it Will be understood by one of ordinary skill in the art that these domains (e.g., the heavy chain portions) can be modified such that they vary in amino acid sequence from the lly occurring immunoglobulin molecule.

The heavy chain portions of a binding molecule, e.g., an antibody as disclosed herein can be derived from different immunoglobulin molecules. For example, a heavy chain portion of a polypeptide can comprise a CHl domain derived from an IgGl molecule and a hinge region derived from an IgG3 molecule. In another example, a heavy chain portion can comprise a hinge region derived, in part, from an IgGl molecule and, in part, from an IgG3 le. In another example, a heavy chain portion can comprise a chimeric hinge derived, in part, from an IgGl molecule and, in part, from an IgG4 molecule.

As used herein, the term “light chain portion” includes amino acid sequences d from an immunoglobulin light chain. The light chain portion comprises at least one of a VL or CL domain.

Binding molecules, e.g., antibodies or antigen-binding fragments, variants, or derivatives thereof disclosed herein can be described or specified in terms of the epitope(s) or portion(s) of an antigen, e. g., a target ccharide that they recognize or specifically bind. The portion of a target polysaccharide which specifically cts with the n binding domain of an antibody is an "epitope," or an "antigenic determinant." A target antigen, e.g., a polysaccharide can comprise a single e, but typically ses at least two epitopes, and can include any number of epitopes, ing on the size, conformation, and type of antigen.

As previously indicated, the subunit ures and three dimensional configuration of the constant regions of the various immunoglobulin s are well known. As used herein, the term “VH domain” includes the amino terminal variable domain of an immunoglobulin heavy chain and the term “CHl domain” includes the first (most amino terminal) constant region domain of an immunoglobulin heavy chain. The CHl domain is adjacent to the VH domain and is amino terminal to the hinge region of an immunoglobulin heavy chain molecule.

As used herein the term “CH2 domain” includes the portion of a heavy chain molecule that s, e.g., from about residue 244 to residue 360 of an antibody using conventional numbering schemes ues 244 to 360, Kabat numbering system; and residues 231—340, EU numbering system; see Kabat EA et a1. op. cit. The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N—linked branched carbohydrate chains are interposed n the two CH2 domains of an intact native IgG molecule. It is also well documented that the CH3 domain s from the CH2 domain to the C-terminal of the IgG molecule and comprises approximately 108 residues.

As used herein, the term “hinge region” includes the portion of a heavy chain molecule that joins the CH1 domain to the CH2 domain. This hinge region comprises approximately 25 residues and is flexible, thus allowing the two N—terminal antigen binding regions to move independently. Hinge regions can be subdivided into three distinct s: upper, middle, and lower hinge domains (Roux et al., J. Immunol. [61:4083 (1998)).

As used herein the term “disulfide bond” includes the covalent bond formed n two sulfur atoms. The amino acid cysteine comprises a thiol group that can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG les, the CH1 and CL regions are linked by a disulfide bond and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system ion 226 or 229, EU numbering system).

As used herein, the term “chimeric antibody” will be held to mean any antibody wherein the immunoreactive region or site is obtained or derived from a first species and WO 70615 the constant region (which can be intact, partial or modified) is ed from a second species. In some embodiments the target binding region or site will be from a non—human source (6. g. mouse or primate) and the constant region is human.

The term "bispecific antibody" as used herein refers to an antibody that has binding sites for two different antigens within a single antibody molecule. It will be appreciated that other molecules in addition to the canonical antibody structure can be constructed with two g speciflcities. It will further be appreciated that antigen binding by bispeciflc antibodies can be simultaneous or sequential. Triomas and hybrid hybridomas are two examples of cell lines that can secrete bispeciflc antibodies.

Bispeciflc antibodies can also be constructed by recombinant means. (Strohlein and Heiss, Future Oncol. 6:1387—94 ; Mabry and Snavely, [Drugs. 13 :543—9 (2010)).

As used herein, the term "engineered antibody" refers to an antibody in which the variable domain in either the heavy and light chain or both is altered by at least partial replacement of one or more CDRs from an antibody of known specificity and, if necessary, by l ork region replacement and sequence ng. gh the CDRs can be derived from an antibody of the same class or even subclass as the antibody from which the framework regions are derived, it is envisaged that the CDRs will be derived from an antibody of different class and preferably from an antibody from a different species. An engineered antibody in which one or more "donor" CDRs from a non-human dy of known specificity is d into a human heavy or light chain framework region is referred to herein as a "humanized dy." It may not be necessary to replace all of the CDRs with the complete CDRs from the donor variable region to transfer the antigen binding capacity of one variable domain to another. Rather, it may only be necessary to transfer those residues that are necessary to maintain the activity of the target g site. Given the explanations set forth in, e. g., U. S. Pat. Nos. ,585,089, 5,693,761, 5,693,762, and 6,180,370, it will be well within the competence of those d in the art, either by carrying out routine experimentation or by trial and error testing to obtain a functional engineered or humanized antibody.

As used herein the term “properly folded polypeptide” includes polypeptides (e.g., anti—Pseudomonas Ps1 and Pch antibodies) in which all of the functional domains comprising the polypeptide are distinctly active. As used herein, the term “improperly folded polypeptide” includes ptides in which at least one of the functional domains of the ptide is not active. In one embodiment, a properly folded polypeptide comprises polypeptide chains linked by at least one disulfide bond and, conversely, an improperly folded polypeptide comprises polypeptide chains not linked by at least one disulfide bond.

As used herein the term “engineered” includes manipulation of nucleic acid or polypeptide molecules by synthetic means (e.g. by recombinant techniques, in vitro peptide synthesis, by enzymatic or chemical coupling of es or some combination of these techniques).

As used herein, the terms "linked," "fused" or "fusion" are used interchangeably.

These terms refer to the joining er of two more elements or ents, by whatever means including chemical conjugation or recombinant means. An "in-frame fusion" refers to the joining of two or more polynucleotide open reading frames (ORFs) to form a continuous longer ORF, in a manner that maintains the correct translational reading frame of the original ORFs. Thus, a inant fusion protein is a single protein ning two or more segments that correspond to polypeptides encoded by the original ORFs (which ts are not normally so joined in .) Although the reading frame is thus made continuous throughout the fused segments, the segments can be physically or spatially separated by, for example, in-frame linker ce. For example, polynucleotides encoding the CDRs of an immunoglobulin variable region can be fused, in-frame, but be separated by a polynucleotide encoding at least one immunoglobulin framework region or additional CDR regions, as long as the "fused" CDRs are co— translated as part of a continuous polypeptide.

In the context of polypeptides, a "linear ce" or a nce" is an order of amino acids in a polypeptide in an amino to carboxyl terminal direction in which residues that neighbor each other in the sequence are contiguous in the primary structure of the polypeptide.

The term “expression” as used herein refers to a process by which a gene produces a biochemical, for example, a polypeptide. The process includes any manifestation of the functional presence of the gene within the cell including, without tion, gene knockdown as well as both transient expression and stable expression. It includes without limitation transcription of the gene into ger RNA (mRNA), and the translation of such mRNA into polypeptide(s). If the final desired product is a biochemical, expression includes the creation of that biochemical and any precursors. Expression of a gene produces a "gene t." As used herein, a gene product can be either a nucleic acid, e.g., a messenger RNA produced by ription of a gene, or a polypeptide which is translated from a transcript. Gene products described herein further include nucleic acids with post transcriptional modifications, e.g., polyadenylation, or polypeptides with post translational modifications, e. g., ation, glycosylation, the addition of lipids, association with other protein subunits, lytic cleavage, and the like.

As used herein, the terms "treat" or "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change, infection, or disorder. cial or desired al results include, but are not limited to, alleviation of symptoms, shment of extent of disease, stabilized (i.e., not ing) state of disease, clearance or reduction of an infectious agent such as P. aeruginosa in a subject, a delay or slowing of disease ssion, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected al if not receiving treatment. Those in need of ent include those y with the infection, condition, or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented, e.g., in burn patients or immunosuppressed patients susceptible to P. aeruginosa infection.

By "subject" or "individual" or "animal" or "patient" or “mammal,” is meant any t, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, s, rats, mice, horses, cattle, cows, bears, and so on.

As used herein, phrases such as “a subject that would benefit from administration of anti—Pseudomonas Psl and Pch binding domains or binding molecules” and “an animal in need of treatment” includes subjects, such as mammalian subjects, that would benefit from administration of anti-Pseudomonas Psl and Pch binding domains or a binding molecule, such as an antibody, comprising one or more of the binding s.

Such binding s, or binding molecules can be used, e.g., for detection of Pseudomonas Psl or Pch (e.g., for a diagnostic procedure) and/or for treatment, i.e., palliation or prevention of a disease, with seudomonas PSI and Pch binding molecules. As described in more detail herein, the seudomonas Ps1 and Pch binding molecules can be used in unconjugated form or can be conjugated, e.g., to a drug, prodrug, or an e.

The term "synergistic effect", as used herein, refers to a greater-than-additive therapeutic effect produced by a combination of compounds wherein the therapeutic effect obtained with the combination s the additive s that would otherwise result from individual administration the compounds alone. Certain embodiments include methods of producing a synergistic effect in the treatment of Pseudomonas infections, wherein said effect is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500%, or at least 1000% greater than the corresponding additive . ministration" refers to the administration of different nds, such as an anti-Psl and an anti-Pch binding domain, or binding molecule comprising one or both an anti-Ps1 and anti-Pch binding domain, such that the compounds elicit a synergistic effect on anti—Pseudomonas immunity. The compounds can be administered in the same or different compositions which if separate are administered proximate to one another, generally within 24 hours of each other and more typically within about 1-8 hours of one another, and even more typically within 1-4 hours of each other or close to simultaneous administration. The relative amounts are s that achieve the desired synergism.

II. BINDING DOMAINS AND BINDING MOLECULES dies that bind Ps1 and formats for using these antibodies have been described in the art. See, for example, International Application Nos. 2012/041538, filed June 8, 2012, and , filed November 6, 2012 (attorney docket no. AEMS-115WOl, entitled “MULTISPECIFIC AND MULTIVALENT BINDING PROTEINS AND USES THEREOF”), which are herein incorporated in their entireties by reference.

One embodiment is directed to binding domains that specifically bind to Pseudomonas Pch, n binding can t the activity of the type III toxin secretion system. In certain embodiments, the binding domains have the same Pseudomonas binding specificity as the antibody V2L2. 2012/063722 r embodiment is directed to binding domains that specifically bind to Pseudomonas Psl or Pch, wherein administration of both binding domains results in synergistic effects against Pseudomonas infections by (a) inhibiting attachment of Pseudomonas aeruginosa to epithelial cells, (b) promoting, mediating, or enhancing opsonophagocytic killing (OPK) of P. nosa, (c) inhibiting ment of P. aeruginosa to epithelial cells, or (d) disrupting the ty of the type III toxin secretion system. In certain ments, the binding domains have the same Pseudomonas binding specificity as the antibodies 3, WapR-004, V2L2, or 29D2.

Other embodiments are directed to an isolated binding molecule(s) comprising one or both g domains that specifically bind to Pseudomonas Psl and/or Pch, wherein administration of the binding molecule results in synergistic effects t Pseudomonas infections. In certain embodiments, the binding molecule can comprise a binding domain from the antibodies or fragments thereof that include, but are not limited to Cam—003,WapR—004, V2L2, or 29D22.

As used herein, the terms "binding domain" or "antigen binding " includes a site that specifically binds an epitope on an antigen (e.g., an epitope of Pseudomonas Psl or Pch). The antigen binding domain of an antibody typically includes at least a portion of an immunoglobulin heavy chain variable region and at least a portion of an immunoglobulin light chain variable region. The binding site formed by these variable regions ines the specificity of the antibody.

The disclosure is more specifically directed to a composition comprising at least two anti—Pseudomonas binding s, wherein one binding domain specifically binds Psl and the other binding domain ically binds Pch. In one embodiment, the composition comprises one binding domain that specifically binds to the same Pseudomonas Psl e as an antibody or antigen-binding fragment thereof comprising the heavy chain variable region (VH) and light chain variable region (VL) region of WapR—004, Cam—003, Cam—004, Cam—005, WapR—OOl, WapR—002, WapR—003, or WapR—Ol6. In certain embodiments, the second binding domain specifically binds to the same Pseudomonas Pch epitope as an antibody or antigen binding fragment thereof comprising the heavy chain variable region (VH) and light chain variable region (VL) of V2L2 or 29D2.

In one embodiment, the composition ses one binding domain that specifically binds to Pseudomonas Psl and/or competitively inhibits Pseudomonas Psl binding by an antibody or antigen-binding fragment thereof comprising the VH and VL of WapR—004, Cam—003, Cam—004, Cam—005, WapR—OOl, WapR—002, WapR—003, or WapR—Ol6. In certain embodiments, the second binding domain specifically binds to the same Pseudomonas Pch epitope and/or competitively inhibits Pseudomonas Pch g by an antibody or antigen g fragment thereof comprising the heavy chain variable region (VH) and light chain variable region (VL) of V2L2 or 29D2. r embodiment is directed to an isolated g molecule, e.g., an antibody or antigen-binding fragment thereof which specifically binds to the same Pseudomonas Pch epitope as an antibody or antigen-binding fragment thereof comprising the VH and VL region ofV2L2 or 29D2.

Also included is an isolated binding molecule, e. g., an antibody or fragment thereof which specifically binds to Pseudomonas Pch and itively ts Pseudomonas Pch binding by an antibody or antigen-binding fragment thereof comprising the VH and VL ofV2L2 or 29D2.

One embodiment is directed to an isolated binding molecule, e.g., an dy or antigen-binding fragment thereof which specifically binds to the same Pseudomonas Psl e as an antibody or antigen-binding fragment thereof comprising the VH and VL region of WapR—OOl, WapR—002, or WapR—003.

Also ed is an isolated binding molecule, e. g., an antibody or fragment f which specifically binds to Pseudomonas Psl and competitively inhibits monas Psl binding by an dy or antigen-binding fragment thereof comprising the VH and VL of WapR—OOl, WapR—002, or WapR—003.

Further included is an isolated binding molecule, e.g., an antibody or fragment thereof which specifically binds to the same monas Psl epitope as an antibody or antigen-binding fragment thereof comprising the VH and VL of WapR—Ol6.

Also included is an isolated binding molecule, e. g., an antibody or fragment thereof which specifically binds to Pseudomonas Psl and competitively inhibits Pseudomonas Psl binding by an antibody or antigen-binding fragment thereof comprising the VH and VL of WapR—Ol6.

Methods of making dies are well known in the art and described .

Once antibodies to various fragments of, or to the full—length Pseudomonas Psl or Pch without the signal sequence, have been produced, determining which amino acids, or epitope, of Pseudomonas Psl or Pch to which the antibody or antigen binding nt binds can be determined by epitope mapping ols as described herein as well as methods known in the art (e. g. double antibody—sandwich ELISA as described in er 11 - Immunology," Current Protocols in Molecular Biology, Ed. Ausubel et al., v.2, John Wiley & Sons, Inc. (1996)). Additional epitope mapping protocols can be found in Morris, G. Epitope Mapping Protocols, New Jersey: Humana Press (1996), which are both orated herein by reference in their entireties. Epitope mapping can also be performed by commercially available means (i.e. ProtoPROBE, Inc. (Milwaukee, Wisconsin)).

In certain aspects, the disclosure is directed to a binding molecule, e.g., an dy or fragment, variant, or derivative thereof which specifically binds to Pseudomonas Psl and/or Pch with an affinity characterized by a dissociation nt (KD) which is less than the KD for said reference monoclonal antibody.

In certain embodiments an anti—Pseudomonas Psl and/or Pch binding molecule, e. g., an antibody or antigen-binding fragment, variant or derivative thereof as disclosed herein binds specifically to at least one epitope of Psl or Pch, i.e., binds to such an epitope more readily than it would bind to an unrelated, or random epitope; binds preferentially to at least one e of Psl or Pch, i.e., binds to such an epitope more y than it would bind to a related, similar, homologous, or analogous epitope; competitively inhibits binding of a reference antibody which itself binds specifically or preferentially to a certain epitope of Psl or Pch; or binds to at least one epitope of Psl or Pch with an affinity characterized by a iation constant KD of less than about 5 X ‘2 M, about 10‘2 M, about 5 x 10‘3 M, about 10‘3 M, about 5 x 10‘4 M, about 10‘4 M, about 5 x 10'5 M, about 10'5 M, about 5 x 10'6 M, about 10'6 M, about 5 x 10'7 M, about 10' M, about 5 x 10‘8 M, about 10‘8 M, about 5 x 10‘9 M, about 10‘9 M, about 5 x 10‘10 M, about 10'10 M, about 5 x 10'11 M, about 10'11 M, about 5 x 10'12 M, about 10'12 M, about 5 x 10-13 M, about 10-13 M, about 5 x 10-14 M, about 10-14 M, about 5 x 10‘15 M, or about 10‘ WO 70615 As used in the context of binding dissociation constants, the term " allows for the degree of variation inherent in the methods utilized for measuring antibody affinity. For example, depending on the level of precision of the instrumentation used, standard error based on the number of samples measured, and rounding error, the term “about 10‘2 M” might e, for example, from 0.05 M to 0.005 M.

In specific embodiments a binding molecule, e. g., an antibody, or antigen-binding fragment, variant, or derivative thereof binds Pseudomonas Psl and/or Pch with an off rate (k(off)) of less than or equal to 5 X 10'2 sec'l, 10'2 sec'l, 5 X 10'3 sec"1 or 10'3 sec'l.

Alternatively, an antibody, or antigen-binding fragment, variant, or derivative thereof binds Pseudomonas Psl and/or Pch with an off rate (k(off)) of less than or equal to 5 X '4 sec'l, 10'4 sec'l, 5 X 10'5 sec'l, or 10'5 sec'1 5 X 10'6 sec'l, 10'6 sec'l, 5 X 10'7 sec'1 or '7 sec'l.

In other embodiments, a binding molecule, e. g., an antibody, or antigen-binding fragment, variant, or derivative thereof as disclosed herein binds monas Psl and/or Pch with an on rate (k(on)) of greater than or equal to 103 M"1 sec'l, 5 X 103 M"1 sec'l, 104 M'1 sec'1 or 5 X 104 M'1 sec'l. Alternatively, a binding molecule, e.g., an antibody, or antigen—binding fragment, variant, or derivative thereof as disclosed herein binds Pseudomonas Psl and/or Pch with an on rate (k(on)) r than or equal to 105 M"1 sec' 1, 5 X 105 M'1 sec'l, 106 M'1 sec'l, or 5 X 106 M'1 sec'1 or 107 M'1 sec'l.

In various embodiments, an seudomonas Psl and/or Pch binding molecule, e. g., an antibody, or antigen-binding fragment, variant, or derivative thereof as described herein promotes opsonophagocytic killing of Pseudomonas, or ts Pseudomonas binding to epithelial cells. In certain embodiments described herein, the Pseudomonas Psl or Pch target is Pseudomonas aeruginosa Psl or Pch. In other embodiments, certain binding molecules bed herein can bind to structurally related polysaccharide molecules regardless of their source. Such Psl—like les would be expected to be identical to or have sufficient structural relatedness to P. aeruginosa Psl to permit specif1c recognition by one or more of the binding molecules disclosed. In other embodiments, certain binding molecules described herein can bind to structurally related polypeptide molecules regardless of their source. Such ke molecules would be expected to be cal to or have sufficient structural relatedness to P. aeruginosa Pch to permit specific recognition by one or more of the g les disclosed. Therefore, for example, certain binding molecules described herein can bind to Psl-like and/or Pch-like molecules produced by other bacterial species, for example, Psl—like or Pch—like molecules produced by other Pseudomonas species, e.g., monas fluorescens, Pseudomonas putida, 0r Pseudomonas alcaligenes. Alternatively, certain binding molecules as described herein can bind to Psl—like and/or Pch—like molecules ed synthetically or by host cells genetically modified to produce Psl-like or Pch-like molecules.

Unless it is ically noted, as used herein a "fragment thereof' in reference to a binding molecule, e. g., an antibody refers to an antigen-binding fragment, i.e., a portion of the antibody which ically binds to the antigen.

Anti—Pseudomonas Psl and/or Pch g molecules, e.g., antibodies or antigen- binding fragments, variants, or derivatives f can comprise a constant region which mediates one or more effector functions. For example, binding of the Cl component of complement to an antibody constant region can activate the complement .

Activation of complement is important in the zation and lysis of pathogens. The activation of complement also ates the inflammatory se and can also be involved in autoimmune hypersensitivity. Further, antibodies bind to receptors on various cells via the Fc region, with a PC receptor binding site on the antibody Fc region binding to a PC receptor (FcR) on a cell. There are a number of Fc receptors which are ic for different classes of antibody, ing IgG (gamma receptors), IgE (epsilon receptors), IgA (alpha receptors) and IgM (mu receptors). Binding of antibody to Fc ors on cell surfaces rs a number of important and diverse biological responses including ment and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody—coated target cells by killer cells (called antibody—dependent cell—mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer and control of immunoglobulin production.

Accordingly, certain embodiments disclosed herein include an anti—Pseudomonas Psl and/or Pch binding molecule, e.g., an antibody, or antigen-binding fragment, variant, or derivative thereof, in which at least a fraction of one or more of the nt region domains has been deleted or ise altered so as to provide desired biochemical characteristics such as reduced effector functions, the ability to non—covalently dimerize, increased ability to localize at the site of a tumor, reduced serum half-life, or increased serum half-life when compared with a whole, unaltered antibody of imately the same immunogenicity. For example, certain binding molecules bed herein are domain deleted antibodies which comprise a polypeptide chain similar to an immunoglobulin heavy chain, but which lack at least a portion of one or more heavy chain domains. For instance, in certain antibodies, one entire domain of the constant region of the modified antibody will be deleted, for example, all or part of the CH2 domain will be deleted.

Modified forms of anti—Pseudomonas Psl and/or Pch binding molecules, e.g., antibodies or antigen-binding fragments, variants, or derivatives thereof can be made from whole precursor or parent antibodies using techniques known in the art. Exemplary techniques are discussed elsewhere herein.

In certain embodiments both the variable and constant regions of anti- Pseudomonas Psl and/or Pch binding molecules, e.g., dies or n—binding nts are fully human. Fully human antibodies can be made using techniques that are known in the art and as described herein. For example, fully human antibodies against a specific antigen can be prepared by administering the antigen to a transgenic animal which has been modified to produce such antibodies in response to antigenic nge, but whose nous loci have been disabled. Exemplary techniques that can be used to make such antibodies are described in US patents: 6,150,584; 6,458,592; 6,420,140. Other techniques are known in the art. Fully human anti bodies can likewise be produced by various display technologies, e.g., phage display or other viral display systems, as described in more detail elsewhere herein.

Anti—Pseudomonas Psl and/or Pch binding molecules, e.g., antibodies or antigen- binding fragments, variants, or derivatives thereof as disclosed herein can be made or manufactured using techniques that are known in the art. In certain ments, binding molecules or fragments thereof are “recombinantly ed,” i.e., are produced using recombinant DNA technology. Exemplary techniques for making antibody molecules or fragments thereof are discussed in more detail elsewhere herein.

In certain anti—Pseudomonas Ps1 and/or Pch binding les, e.g., antibodies or antigen-binding fragments, variants, or tives thereof bed herein, the Fc portion can be mutated to decrease effector function using ques known in the art.

For e, the deletion or inactivation (through point mutations or other means) of a constant region domain can reduce Fc receptor binding of the circulating modified antibody y increasing tumor localization. In other cases it can be that constant region modifications te complement binding and thus reduce the serum half-life and nonspecific association of a conjugated xin. Yet other modifications of the constant region can be used to modify disulf1de linkages or oligosaccharide moieties that allow for enhanced localization due to increased antigen specificity or antibody lity. The resulting physiological profile, bioavailability and other mical effects of the modifications, such as localization, biodistribution and serum half-life, can easily be measured and quantified using well known immunological techniques without undue experimentation.

In certain embodiments, anti—Pseudomonas Ps1 and/or Pch binding molecules, e.g., antibodies or antigen-binding nts, variants, or derivatives thereof will not elicit a deleterious immune response in the animal to be treated, e. g., in a human. In one ment, anti—Pseudomonas Ps1 and/or Pch binding molecules, e.g., antibodies or antigen-binding fragments, ts, or derivatives thereof are modified to reduce their immunogenicity using art-recognized techniques. For example, antibodies can be humanized, de—immunized, or chimeric antibodies can be made. These types of dies are derived from a non-human antibody, typically a murine or primate antibody, that s or substantially retains the antigen-binding properties of the parent antibody, but which is less immunogenic in humans. This can be achieved by various methods, including (a) grafting the entire non-human variable domains onto human constant s to generate chimeric antibodies; (b) grafting at least a part of one or more of the non-human complementarity determining regions (CDRs) into a human framework and constant s with or without retention of critical framework residues; or (c) transplanting the entire non-human variable domains, but "cloaking" them with a like section by replacement of surface residues. Such s are disclosed in Morrison et al., Proc. Natl. Acad. Sci. 81:6851—6855 (1984); Morrison et al., Adv.

Immunol. 92 (1988); Verhoeyen et al., Science 239:1534—1536 (1988); Padlan, Molec. Immun. 28:489—498 (1991); Padlan, Molec. Immun. 31:169—217 , and US.

Pat. Nos. 5,585,089, 5,693,761, 5,693,762, and 6,190,370, all of which are hereby incorporated by reference in their entirety.

De-immunization can also be used to decrease the immunogenicity of an dy.

As used herein, the term “de-immunization” includes tion of an antibody to modify T cell epitopes (see, e.g., WO9852976A1, WOOO34317A2). For example, VH and VL sequences from the starting antibody are analyzed and a human T cell epitope "map" from each V region g the location of epitopes in relation to mentarity- determining regions (CDRs) and other key residues within the sequence. Individual T cell epitopes from the T cell epitope map are analyzed in order to identify alternative amino acid substitutions with a low risk of altering activity of the final antibody. A range of alternative VH and VL sequences are designed comprising combinations of amino acid substitutions and these sequences are subsequently incorporated into a range of binding polypeptides, e.g., Pseudomonas Psl— and/or eciflc antibodies or antigen—binding fragments thereof disclosed herein, which are then tested for function. Complete heavy and light chain genes comprising modified V and human C s are then cloned into expression vectors and the subsequent plasmids introduced into cell lines for the production of whole antibody. The antibodies are then compared in riate mical and biological , and the optimal variant is identified.

Anti—Pseudomonas Psl and/or Pch binding molecules, e.g., antibodies or antigen— binding fragments, variants, or derivatives thereof can be generated by any suitable method known in the art. Polyclonal antibodies to an antigen of interest can be produced by various procedures well known in the art. For example, an anti—Pseudomonas Ps1 and/or Pch antibody or n-binding nt thereof can be administered to various host animals including, but not limited to, rabbits, mice, rats, chickens, hamsters, goats, donkeys, etc., to induce the production of sera containing polyclonal antibodies specific for the antigen. s adjuvants can be used to se the immunological response, depending on the host species, and include but are not d to, Freund's (complete and incomplete), l gels such as aluminum hydroxide, surface active substances such as cithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette—Guerin) and Corynebacterium parvum. Such adjuvants are also well known in the art.

Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. (1988) DNA encoding dies or antibody fragments (e. g., antigen binding sites) can also be derived from antibody libraries, such as phage display libraries. In a particular, such phage can be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e. g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or fied with n, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M13 g s expressed from phage with scFv, Fab, Fv OE DAB idual Fv region from light or heavy ) or disulf1de stabilized Fv antibody domains inantly fused to either the phage gene III or gene VIII protein. Exemplary s are set forth, for example, in EP 368 684 B1; US. patent. 5,969,108, Hoogenboom, HR. and Chames, Immunol. Today 21:371 (2000); Nagy et al. Nat. Med. 8:801 (2002); Huie et al., Proc. Natl. Acad. Sci. USA 982682 (2001); Lui et al., J. Mol.

Biol. 315:1063 (2002), each of which is incorporated herein by reference. Several publications (e.g., Marks et al., chnology 10:779—783 (1992)) have described the production of high affinity human antibodies by chain shuffling, as well as combinatorial infection and in vivo recombination as a strategy for constructing large phage libraries. In another embodiment, Ribosomal display can be used to replace bacteriophage as the display rm (see, e.g., Hanes et al., Nat. Biotechnol. 18:1287 (2000); Wilson et al., Proc. Natl. Acad. Sci. USA 98:3750 (2001); or Irving et al., J. Immunol. s 248:31 (2001)). In yet another embodiment, cell surface libraries can be screened for antibodies (Boder et al., Proc. Natl. Acad. Sci. USA 97:10701 ; Daugherty et al., J. Immunol.

Methods 243:211 (2000)). Such procedures provide alternatives to traditional hybridoma techniques for the isolation and uent cloning of monoclonal antibodies.

In phage display s, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. For example, DNA sequences encoding VH and VL regions are amplified from animal cDNA libraries (e. g., human or murine cDNA libraries of lymphoid tissues) or synthetic cDNA ies. In certain embodiments, the DNA encoding the VH and VL regions are joined together by an scFv linker by PCR and cloned into a phagemid vector (e.g., p CANTAB 6 or pComb 3 HSS). The vector is oporated in E. coli and the E. coli is ed with helper phage. Phage used in these methods are typically ntous phage including fd and M13 and the VH or VL regions are usually recombinantly fused to either the phage gene III or gene VIII. Phage expressing an antigen binding domain that binds to an antigen of st (i.e., Pseudomonas Ps1 or Pch) can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid e or bead.

Additional examples of phage display methods that can be used to make the antibodies include those sed in Brinkman et al., J. Immunol. Methods [82:41—50 (1995); Ames et al., J. Immunol. Methods 184: 177—186 (1995); Kettleborough et al., Eur.

J. Immunol. 24:952—958 (1994); Persic et al., Gene 187:9—18 (1997); Burton et al., Advances in logy 57:191—280 (1994); PCT Application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and US. Pat. Nos. 5,698,426; 5,223,409; 484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; ,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety.

As described in the above references and in the examples below, after phage selection, the dy coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria. For example, techniques to recombinantly e Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al., BioTechniques 12(6):864—869 (1992); and Sawai et al., AJRI 34:26—34 (1995); and Better et al., Science 240: 1041—1043 (1988) (said references incorporated by reference in their entireties).

Examples of ques which can be used to e single—chain Fvs and antibodies include those described in US. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203:46—88 (1991); Shu et al., PNAS 90:7995—7999 (1993); and Skerra et al., e 240:1038—1040 (1988). In certain embodiments such as eutic administration, chimeric, humanized, or human antibodies can be used. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine onal antibody and a human immunoglobulin nt region. Methods for producing chimeric antibodies are known in the art. See, e. g., Morrison, Science 229: 1202 (1985); Oi et al., BioTechniques 4:214 ; s et al., J. Immunol. Methods 1—202 (1989); US. Pat. Nos. 5,807,715; 4,816,567; and 4,816397, which are incorporated herein by reference in their entireties. Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human s and framework regions from a human immunoglobulin molecule. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably e, antigen binding. These framework tutions are identified by methods well known in the art, e. g., by modeling of the interactions of the CDR and framework residues to identify framework residues ant for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., US. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entireties.) Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR—grafting (EP 239,400; PCT publication WO 91/09967; US. Pat. Nos. ,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 6; EP 6; Padlan, Molecular Immunology 28(4/5):489—498 (1991); Studnicka et al., Protein Engineering 7(6):805—814 (1994); Roguska. et al., PNAS 91:969—973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332).

Fully human dies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art including phage display methods described above using dy libraries derived from human immunoglobulin sequences. See also, US. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 54, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety.

Human antibodies can also be produced using transgenic mice which are incapable of expressing functional nous globulins, but which can express human immunoglobulin genes. For example, the human heavy and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. In on, various ies can be engaged to provide human antibodies produced in transgenic mice ed against a selected antigen using technology similar to that described above.

Fully human antibodies which recognize a selected epitope can be generated using a que referred to as "guided selection." In this approach a selected non-human onal dy, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al., Bio/Technology 12:899-903 (1988). See also, U.S. Patent No. 5,565,332.) In another embodiment, DNA encoding desired monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Isolated and subcloned hybridoma cells or isolated phage, for example, can serve as a source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into prokaryotic or eukaryotic host cells such as E. coli cells, simian COS cells, e Hamster Ovary (CHO) cells or myeloma cells that do not otherwise produce globulins. More particularly, the isolated DNA (which can be synthetic as described herein) can be used to clone constant and variable region sequences for the manufacture antibodies as described in Newman et al., U.S. Pat. No. 5,658,570, filed January 25, 1995, which is incorporated by reference herein. Transformed cells expressing the d antibody can be grown up in relatively large quantities to provide clinical and commercial supplies of the globulin.

In one ment, an isolated binding molecule, e.g., an antibody ses at least one heavy or light chain CDR of an antibody molecule. In another embodiment, an isolated binding molecule comprises at least two CDRs from one or more antibody molecules. In another embodiment, an ed binding molecule comprises at least three CDRs from one or more antibody molecules. In another embodiment, an isolated binding molecule comprises at least four CDRs from one or more antibody molecules. In another 2012/063722 embodiment, an ed binding le comprises at least five CDRs from one or more antibody molecules. In another embodiment, an isolated g molecule of the ption comprises at least six CDRs from one or more antibody molecules.

In a specific embodiment, the amino acid ce of the heavy and/or light chain variable domains can be ted to identify the sequences of the complementarity determining s (CDRs) by methods that are well-known in the art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability. Using routine recombinant DNA techniques, one or more of the CDRs can be inserted within framework regions, e. g., into human framework regions to humanize a non-human antibody. The framework regions can be lly occurring or consensus framework regions, and preferably human ork regions (see, e.g., Chothia et al., J. M01. Biol. 278:457—479 (1998) for a listing of human framework regions). The polynucleotide generated by the ation of the framework regions and CDRs encodes an antibody that specifically binds to at least one epitope of a desired antigen, e.g., Psl or Pch. One or more amino acid substitutions can be made within the framework regions, and, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such s can be used to make amino acid substitutions or ons of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds. Other alterations to the polynucleotide are encompassed by the present disclosure and are within the capabilities of a person of skill of the art.

Also ed are binding molecules that comprise, consist essentially of, or consist of, variants (including derivatives) of antibody molecules (e.g., the VH regions and/or VL regions) described herein, which binding molecules or fragments thereof specifically bind to Pseudomonas Psl or Pch. Standard techniques known to those of skill in the art can be used to introduce mutations in the nucleotide sequence encoding a binding molecule or fragment thereof which specifically binds to Pseudomonas Psl and/or Pch, including, but not limited to, site-directed mutagenesis and PCR-mediated mutagenesis which result in amino acid substitutions. The variants ding derivatives) encode polypeptides comprising less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the reference VH , VHCDRl, VHCDRZ, VHCDR3, VL region, VLCDRl, VLCDRZ, or . A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. es of amino acid residues having side chains with r charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, ic acid), uncharged polar side chains (e. g., glycine, asparagine, ine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e. g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta- branched side chains (e. g., threonine, valine, isoleucine) and aromatic side chains (e. g., tyrosine, phenylalanine, tryptophan, histidine). atively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant s can be screened for ical activity to identify mutants that retain activity (e. g., the ability to bind an Pseudomonas Psl or Pch).

For example, it is possible to introduce mutations only in framework regions or only in CDR s of an antibody molecule. Introduced mutations can be silent or neutral missense mutations, i.e., have no, or little, effect on an dy’s ability to bind antigen. These types of mutations can be useful to optimize codon usage, or e a hybridoma’s dy production. Alternatively, non-neutral missense mutations can alter an antibody’s y to bind n. The location of most silent and neutral missense mutations is likely to be in the framework regions, while the location of most non-neutral missense mutations is likely to be in CDR, though this is not an absolute requirement.

One of skill in the art would be able to design and test mutant molecules with desired properties such as no alteration in antigen binding activity or alteration in binding activity (e. g., improvements in antigen binding activity or change in antibody specificity).

Following mutagenesis, the encoded protein can routinely be expressed and the functional and/or biological activity of the encoded protein, (e.g., ability to bind at least one e of Pseudomonas Psl or Pch) can be determined using techniques described herein or by routinely modifying techniques known in the art.

WO 70615 One embodiment provides a bispecif1c antibody comprising an anti—Pseudomonas Psl and Pch binding domain disclosed herein. In n embodiments, the bispecific antibody contains a first Psl binding domain, and the second Pch binding domain.

Bispeciflc antibodies with more than two ies are plated. For example, trispecific antibodies can also be prepared using the methods described herein. (Tutt et al., J. Immunol., 147:60 (1991)).

One embodiment provides a method of producing a bispecif1c antibody, that utilizes a single light chain that can pair with both heavy chain variable domains t in the bispecif1c molecule. To fy this light chain, various strategies can be employed. In one embodiment, a series of monoclonal antibodies are identified to each antigen that can be targeted with the bispecif1c antibody, followed by a determination of which of the light chains utilized in these antibodies is able to function when paired with the heavy chain of any of the antibodies identified to the second target. In this manner a light chain that can function with two heavy chains to enable binding to both antigens can be fied. In another embodiment, display techniques, such as phage display, can enable the identification of a light chain that can function with two or more heavy chains.

In one embodiment, a phage library is ucted which comprises a diverse repertoire of heavy chain variable s and a single light chain variable . This library can further be utilized to identify antibodies that bind to various antigens of interest. Thus, in certain embodiments, the antibodies identif1ed will share a common light chain.

In certain embodiments, the bispecif1c antibody comprises at least one single chain Fv (scFv). In n embodiments the bispecif1c antibody comprises two scFvs.

For example, a scFv can be fused to one or both of a CH3 domain-containing polypeptide contained within an antibody. Some methods comprise producing a bispecif1c molecule wherein one or both of the heavy chain constant regions comprising at least a CH3 domain is ed in conjunction with a single chain Fv domain to provide antigen binding.

III. DY POLYPEPTIDES The disclosure is further directed to isolated polypeptides which make up binding molecules, e.g., antibodies or antigen-binding fragments thereof, which specifically bind to Pseudomonas Ps1 and/or Pch and polynucleotides encoding such polypeptides.

Binding molecules, e.g., antibodies or fragments thereof as disclosed herein, comprise polypeptides, e.g., amino acid sequences encoding, for example, Psl—specific and/or Pch— specific n binding regions derived from immunoglobulin molecules. A polypeptide or amino acid sequence "derived from" a designated protein refers to the origin of the polypeptide. In certain cases, the polypeptide or amino acid sequence which is derived from a particular starting polypeptide or amino acid sequence has an amino acid sequence that is essentially identical to that of the starting sequence, or a portion thereof, wherein the n consists of at least 10-20 amino acids, at least 20-30 amino acids, at least 30- 50 amino acids, or which is otherwise identifiable to one of ordinary skill in the art as having its origin in the starting ce.

Also disclosed is an ed binding molecule, e.g., an antibody or antigen- binding fragment thereof which specifically binds to Pseudomonas Psl comprising an immunoglobulin heavy chain variable region (VH) amino acid sequence at least 80%, 85%, 90% 95% or 100% identical to one or more of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 74 as shown in Table 2.

Further disclosed is an isolated binding molecule, e.g., an antibody or antigen- binding fragment thereof which specifically binds to Pseudomonas Psl comprising a VH amino acid ce identical to, or identical except for one, two, three, four, five, or more amino acid substitutions to one or more of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 74 as shown in Table 2.

Some embodiments include an isolated g molecule, e.g., an antibody or antigen-binding fragment thereof which ically binds to Pseudomonas Psl comprising a VH, where one or more of the VHCDRl, VHCDR2 or VHCDR3 regions of the VH are at least 80%, 85%, 90%, 95% or 100% identical to one or more reference heavy chain VHCDRl, VHCDR2 or VHCDR3 amino acid sequences of one or more of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 74 as shown in Table 2.

Further disclosed is an ed binding molecule, e.g., an antibody or n- binding fragment thereof which specifically binds to Pseudomonas Psl comprising a VH, 2012/063722 —56— where one or more of the VHCDRl, VHCDR2 or VHCDR3 regions of the VH are identical to, or identical except for four, three, two, or one amino acid substitutions, to one or more reference heavy chain VHCDRl, VHCDR2 and/or VHCDR3 amino acid sequences of one or more of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: , or SEQ ID NO: 74 as shown in Table 2. Thus, according to this embodiment the VH comprises one or more of a VHCDRl, VHCDR2, or VHCDR3 identical to or identical except for four, three, two, or one amino acid substitutions, to one or more of the VHCDRl, , or VHCDR3 amino acid sequences shown in Table 3.

Also disclosed is an isolated binding molecule, e.g., an antibody or antigenbinding fragment thereof which specifically binds to Pseudomonas Psl comprising an globulin light chain variable region (VL) amino acid sequence at least 80%, 85%, 90% 95% or 100% identical to one or more of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16 as shown in Table 2.

Some embodiments se an isolated binding le, e.g., an antibody or antigen-binding fragment thereof which specifically binds to Pseudomonas Psl comprising a VL amino acid sequence identical to, or identical except for one, two, three, four, five, or more amino acid substitutions, to one or more of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16 as shown in Table 2.

Also provided is an isolated binding molecule, e.g., an antibody or antigenbinding fragment thereof which specifically binds to Pseudomonas Psl comprising a VL, where one or more of the VLCDRl, VLCDR2 or VLCDR3 regions of the VL are at least 80%, 85%, 90%, 95% or 100% identical to one or more reference light chain VLCDRl, VLCDR2 or VLCDR3 amino acid sequences of one or more of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16 as shown in Table 2.

Further provided is an isolated binding molecule, e.g., an antibody or antigen- binding fragment thereof which ically binds to Pseudomonas Psl comprising a VL, where one or more of the VLCDRl, VLCDR2 or VLCDR3 s of the VL are identical to, or identical except for four, three, two, or one amino acid substitutions, to one or more reference heavy chain VLCDRl, VLCDR2 and/or VLCDR3 amino acid ces of one or more of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16 as shown in Table 2. Thus, according to this embodiment the VL comprises one or more of a VLCDRl, VLCDR2, or VLCDR3 identical to or identical except for four, three, two, or one amino acid substitutions, to one or more of the VLCDRl, VLCDR2, or VLCDR3 amino acid sequences shown in Table 3.

In other embodiments, an isolated antibody or antigen-binding fragment thereof which specifically binds to Pseudomonas Psl, comprises, consists essentially of, or consists of VH and VL amino acid sequences at least 80%, 85%, 90% 95% or 100% identical to: (a) SEQ ID NO: 1 and SEQ ID NO: 2, respectively,(b) SEQ ID NO: 3 and SEQ ID NO:2, respectively,(c) SEQ ID NO: 4 and SEQ ID NO: 2 , respectively, (d) SEQ ID NO: 5 and SEQ ID NO: 6, respectively,(e) SEQ ID NO: 7 and SEQ ID NO: 8, tively,(f) SEQ ID NO: 9 and SEQ ID NO: 10, respectively,(g) SEQ ID NO: 11 and SEQ ID NO: 12, respectively,(h) SEQ ID NO: 13 and SEQ ID NO: 14, tively; (i) SEQ ID NO: 15 and SEQ ID NO: 16, respectively; or (j) SEQ ID NO: 74 and SEQ ID NO: 12, respectively. In certain embodiments, the above—described antibody or antigen-binding fragment thereof comprises a VH with the amino acid sequence SEQ ID NO: 11 and a VL with the amino acid sequence of SEQ ID NO: 12. In some embodiments, the above—described antibody or antigen-binding nt thereof comprises a VH with the amino acid sequence SEQ ID NO: 1 and a VL with the amino acid ce of SEQ ID NO: 2. In other embodiments, the above—described antibody or antigen—binding fragment thereof comprises a VH with the amino acid sequence SEQ ID NO: 11 and a VL with the amino acid ce of SEQ ID NO: 12.

Certain embodiments provide an isolated binding molecule, e.g, an antibody, or antigen-binding fragment thereof which ically binds to monas Psl, comprising an immunoglobulin VH and an immunoglobulin VL, each comprising a mentarity determining region 1 (CDRl), CDR2, and CDR3, wherein the VH CDRl is PYYWT (SEQ ID NO:47), the VH CDR2 is YIHSSGYTDYNPSLKS (SEQ ID NO: 48), the VH CDR3 is selected from the group consisting of ADWDRLRALDI 2012/063722 —58— (P510096, SEQ ID NO:258), AMDIEPHALDI (P510225, SEQ ID NO:267), ADDPFPGYLDI (P510588, SEQ ID NO:268), ADWNEGRKLDI 67, SEQ ID ), ADWDHKHALDI (P510337, SEQ ID NO:270), ATDEADHALDI (P510170, SEQ ID NO:271), ADWSGTRALDI (P510304, SEQ ID NO:272), GLPEKPHALDI (P510348, SEQ ID NO:273), SLFTDDHALDI (P510573, SEQ ID NO:274), ASPGVVHALDI 74, SEQ ID NO:275), AHIESHHALDI (P510582, SEQ ID NO:276), ATQAPAHALDI (P510584, SEQ ID NO:277), SQHDLEHALDI (P510585, SEQ ID ), and AMPDMPHALDI (P510589, SEQ ID NO:279), the VL CDR1 is RASQSIRSHLN (SEQ ID , the VL CDR2 is GASNLQS (SEQ ID NO:51), and the VL CDR3 is selected from the group consisting of QQSTGAWNW (P510096, SEQ ID NO:280), QQDFFHGPN (P510225, SEQ ID NO:281), QQSDTFPLK (P510588, SEQ ID NO:282), QQSYSFPLT (WapR0004, P510567, P510573, P5100574, P510582, P510584, P510585, SEQ ID NO:52), PLT (P510337, SEQ ID NO:283), SQSDTFPLT (P510170, SEQ ID NO:284), GQSDAFPLT (P510304, SEQ ID NO:285), LQGDLWPLT (P510348, SEQ ID NO:286), and QQSLEFPLT (P510589, SEQ ID NO:287), n the VH and VL CDR5 are ing to the Kabat numbering system.

Certain embodiments provide an ed binding molecule, e.g, an antibody, or antigen-binding fragment thereof which specifically binds to Pseudomonas P51, comprising an globulin VH and an immunoglobulin VL, each comprising a complementarity determining region 1 (CDR1), CDR2, and CDR3, wherein the VH CDR1 is PYYWT (SEQ ID NO:47), the VH CDR2 is YIHSSGYTDYNPSLKS (SEQ ID NO: 48), the VL CDR1 is RASQSIRSHLN (SEQ ID NO:50), the VL CDR2 is GASNLQS (SEQ ID N051), and the VH CDR3 and the VL CDR3 se, tively, ADWDRLRALDI (P510096, SEQ ID NO:258) and WNW (P510096, SEQ ID NO:280); AMDIEPHALDI (P510225, SEQ ID NO:267) and QQDFFHGPN (P510225, SEQ ID NO:281); ADDPFPGYLDI (P510588, SEQ ID NO:268) and QQSDTFPLK (P510588, SEQ ID NO:282); ADWNEGRKLDI (P510567, SEQ ID NO:269) and the VL CDR3 is QQSYSFPLT (WapR0004, P510567, P510573, 74, 2, P510584, P510585, SEQ ID NO:52); ADWDHKHALDI (P510337, SEQ ID NO:270) and QDSSSWPLT (P510337, SEQ ID NO:283); ATDEADHALDI (P510170, SEQ ID NO:271) and SQSDTFPLT (P510170, SEQ ID NO:284); ADWSGTRALDI (P510304, SEQ ID NO:272) and GQSDAFPLT (P510304, SEQ ID NO:285); GLPEKPHALDI (P510348, SEQ ID NO:273) and 48, SEQ ID NO:286); SLFTDDHALDI (Ps10573, SEQ ID NO:274) and SEQ ID NO:52; ASPGVVHALDI (P510574, SEQ ID NO:275) and SEQ ID NO:52; AHIESHHALDI (P510582, SEQ ID NO:276) and SEQ ID NO:52; ATQAPAHALDI (P510584, SEQ ID NO:277) and SEQ ID NO:52; SQHDLEHALDI (P510585, SEQ ID ) and SEQ ID NO:52; or AMPDMPHALDI (P510589, SEQ ID NO:279) and PLT (P510589, SEQ ID NO:287).

Certain ments provide an isolated binding le, e.g., an antibody or antigen-binding fragment thereof which specifically binds to Pseudomonas Psl, comprising an immunoglobulin VH and an immunoglobulin VL, wherein the VH comprises QVQLQESGPGLVKPSETLSLTCTVSGGSISPYYWTWIRQPPGKX_1LELIGYIHSSGY TDYNPSLKSRVTISGDTSKKQFSLKLSSVTAADTAVYYCARADWDRLRALDIWG QGTMVTVSS, wherein X1 is G or C (P510096, SEQ ID NO:288), and the VL ses DIQLTQSPSSLSASVGDRVTITCRASQSIRSHLNWYQQKPGKAPKLLIYGASNLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSTGAWNWFGX_2GTKVEIK, wherein X2 is G or C (P510096, SEQ ID NO:289); wherein the VH comprises SGPGLVKPSETLSLTCTVSGGSISPYYWTWIRQPPGKGLELIGYIHSSGY TDYNPSLKSRVTISGDTSKKQFSLKLSSVTAADTAVYYCARAMDIEPHALDIWGQ GTMVTVSS (P510225, SEQ ID NO:290), and the VL comprises DIQLTQSPSSLSASVGDRVTITCRASQSIRSHLNWYQQKPGKAPKLLIYGASNLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSDDGFPNFGGGTKVEIK (P510225, SEQ ID NO:291); wherein the VH comprises QVQLQESGPGLVKPSETLSLTCTVSGGSISPYYWTWIRQPPGKGLELIGYIHSSGY TDYNPSLKSRVTISGDTSKKQFSLKLSSVTAADTAVYYCARADDPFPGYLDIWGQ GTMVTVSS (P510588, SEQ ID NO:292), and the VL comprises DIQLTQSPSSLSASVGDRVTITCRASQSIRSHLNWYQQKPGKAPKLLIYGASNLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSDTFPLKFGGGTKVEIK (P510588, SEQ ID NO:293); wherein the VH comprises QVQLQESGPGLVKPSETLSLTCTVSGGSISPYYWTWIRQPPGKGLELIGYIHSSGY TDYNPSLKSRVTISGDTSKKQFSLKLSSVTAADTAVYYCARADWNEGRKLDIWG QGTMVTVSS (P510567, SEQ ID NO:294), and the VL comprises SEQ ID NO:11; herein the VH comprises SGPGLVKPSETLSLTCTVSGGSISPYYWTWIRQPPGKGLELIGYIHSSGY TDYNPSLKSRVTISGDTSKKQFSLKLSSVTAADTAVYYCARADWDHKHALDIWG QGTMVTVSS (Ps10337, SEQ ID NO:295), and the VL comprises DIQLTQSPSSLSASVGDRVTITCRASQSIRSHLNWYQQKPGKAPKLLIYGASNLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQDSSSWPLTFGGGTKVEIK (Ps10337, SEQ ID ); wherein the VH comprises EVQLLESGPGLVKPSETLSLTCNVAGGSISPYYWTWIRQPPGKGLELIGYIHSSGY TDYNPSLKSRVTISGDTSKKQFSLHVSSVTAADTAVYFCARATDEADHALDIWG QGTLVTVSS (Ps10170, SEQ ID NO:297), and the VL comprises EIVLTQSPSSLSTSVGDRVTITCRASQSIRSHLNWYQQKPGKAPKLLIYGASNLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCSQSDTFPLTFGGGTKLEIK (Ps10170, SEQ ID NO:298); wherein the VH comprises EVQLLESGPGLVKPSETLSLTCNVAGGSISPYYWTWIRQPPGKGLELIGYIHSSGY TDYNPSLKSRVTISGDTSKKQFSLHVSSVTAADTAVYFCARADWSGTRALDIWG QGTLVTVSS 04, SEQ ID NO:299), and the VL comprises EIVLTQSPSSLSTSVGDRVTITCWASQSIRSHLNWYQQKPGKAPKLLIYGASNLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCGQSDAFPLTFGGGTKLEIK (Ps10304, SEQ ID NO:300); wherein the VH comprises EVQLLESGPGLVKPSETLSLTCNVAGGSISPYYWTWIRQPPGKGLELIGYIHSSGY TDYNPSLKSRVTISGDTSKKQFSLHVSSVTAADTAVYFCARGLPEKPHALDIWGQ GTLVTVSS (Ps10348, SEQ ID NO:301), and the VL ses EIVLTQSPSSLSTSVGDRVTITCRASQSIRSHLNWYQQKPGKAPKLLIYGASNLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQGDLWPLTFGGGTKLEIK (Ps10348, SEQ ID NO:302); wherein the VH comprises EVQLLESGPGLVKPSETLSLTCNVAGGSISPYYWTWIRQPPGKGLELIGYIHSSGY TDYNPSLKSRVTISGDTSKKQFSLHVSSVTAADTAVYFCARSLFTDDHALDIWGQ GTLVTVSS (Ps10573, SEQ ID NO:303), and the VL comprises SEQ ID NO:11; wherein the VH comprises EVQLLESGPGLVKPSETLSLTCNVAGGSISPYYWTWIRQPPGKGLELIGYIHSSGY LKSRVTISGDTSKKQFSLHVSSVTAADTAVYFCARASPGVVHALDIWGQ GTLVTVSS (Ps10574, SEQ ID NO:304), and the VL ses SEQ ID NO:11; wherein the VH comprises EVQLLESGPGLVKPSETLSLTCNVAGGSISPYYWTWIRQPPGKGLELIGYIHSSGY TDYNPSLKSRVTISGDTSKKQFSLHVSSVTAADTAVYFCARAHIESHHALDIWGQ SS (Ps10582, SEQ ID NO:305), and the VL ses SEQ ID NO: 1 1; wherein the VH comprises EVQLLESGPGLVKPSETLSLTCNVAGGSISPYYWTWIRQPPGKGLELIGYIHSSGY TDYNPSLKSRVTISGDTSKKQFSLHVSSVTAADTAVYFCARATQAPAHALDIWG QGTLVTVSS (Ps10584, SEQ ID NO:306), and the VL comprises SEQ ID NO:ll; n the VH comprises EVQLLESGPGLVKPSETLSLTCNVAGGSISPYYWTWIRQPPGKGLELIGYIHSSGY TDYNPSLKSRVTISGDTSKKQFSLHVSSVTAADTAVYFCARSQHDLEHALDIWGQ GTLVTVSS 85, SEQ ID NO:307), and the VL comprises SEQ ID NO:ll; or wherein the VH ses EVQLLESGPGLVKPSETLSLTCNVAGGSISPYYWTWIRQPPGKGLELIGYIHSSGY TDYNPSLKSRVTISGDTSKKQFSLHVSSVTAADTAVYFCARAMPDMPHALDIWG QGTLVTVSS (Ps10589, SEQ ID NO:308), and the VL comprises EIVLTQSPSSLSTSVGDRVTITCRASQSIRSHLNWYQQKPGKAPKLLIYGASNLQS SGSGSGTDFTLTISSLQPEDFATYYCQQSLEFPLTFGGGTKLEIK (Ps10589, SEQ ID NO:325).

Also disclosed is an isolated antibody single chain FV (ScFV) fragment which specifically binds to Pseudomonas Psl (an "anti-Psl ScFV"), comprising the formula VH- L-VL or alternatively VL-L-VH, where L is a linker sequence. In n aspects the linker can comprise (a) [GGGGS]n, wherein n is 0, l, 2, 3, 4, or 5, (b) [GGGG]n, wherein n is 0, l, 2, 3, 4, or 5, or a combination of (a) and (b). For example, an exemplary linker comprises: GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID ). In certain embodiments the linker further comprises the amino acids ala-leu at the C—terminus of the linker. In n embodiments the anti—Psl ScFV comprises the amino acid sequence of SEQ ID NO:240, SEQ ID NO:24l, SEQ ID NO:242, SEQ ID NO:243, SEQ ID NO:244, SEQ ID NO:245, SEQ ID NO:246, SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:252, SEQ ID NO:253, SEQ ID NO:254, or SEQ ID NO:262. 2012/063722 Also disclosed is an ed antibody single chain Fv (ScFv) fragment which specifically binds to Pseudomonas Pch (an "anti-Pch ScFv"), comprising the formula VH-L-VL or alternatively H, where L is a linker sequence. In certain aspects the linker can comprise (a) [GGGGS]n, wherein n is 0, 1, 2, 3, 4, or 5, (b) [GGGG]n, wherein n is 0, 1, 2, 3, 4, or 5, or a combination of (a) and (b). For example, an exemplary linker comprises: GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:326). In certain embodiments the linker further comprises the amino acids ala-leu at the C—terminus of the linker.

Also disclosed is an isolated g molecule, e.g., an antibody or antigenbinding fragment thereof which specifically binds to monas Pch comprising an immunoglobulin heavy chain variable region (VH) and/or light chain variable region (VL) amino acid sequence at least 80%, 85%, 90% 95% or 100% identical to SEQ ID NO: 216 or SEQ ID NO: 217.

Further disclosed is an isolated binding molecule, e.g., an antibody or antigen- binding fragment thereof which specifically binds to Pseudomonas Pch comprising a VH, where one or more of the VHCDRl, VHCDR2 or VHCDR3 regions of the VH are cal to, or identical except for four, three, two, or one amino acid substitutions, to one or more reference heavy chain VHCDRl, VHCDR2 and/or VHCDR3 amino acid ces of one or more of: SEQ ID NOs: 218—220 as shown in Table 3. Thus, according to this embodiment the VH comprises one or more of a VHCDRl, VHCDR2, or VHCDR3 identical to or identical except for four, three, two, or one amino acid substitutions, to one or more of the VHCDRl, VHCDR2, or VHCDR3 amino acid sequences shown in Table 3.

Further provided is an isolated g molecule, e.g., an antibody or antigen- binding fragment thereof which specifically binds to monas Pch sing a VL, where one or more of the VLCDRl, VLCDR2 or VLCDR3 regions of the VL are identical to, or cal except for four, three, two, or one amino acid substitutions, to one or more reference heavy chain VLCDRl, VLCDR2 and/or VLCDR3 amino acid sequences of one or more of: SEQ ID NOs: 221—223 as shown in Table 3. Thus, according to this embodiment the VL comprises one or more of a VLCDRl, VLCDR2, or VLCDR3 identical to or identical except for four, three, two, or one amino acid substitutions, to one or more of the VLCDRl, VLCDR2, or VLCDR3 amino acid sequences shown in Table 3.

Also provided is an isolated binding molecule, e. g., an antibody or n- binding fragment thereof which specifically binds to Pseudomonas Pch comprising a VH and a VL, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO:255 and SEQ ID NO:257, and wherein the VL comprises the amino acid sequence of SEQ ID NO:256.

Further provided is an isolated binding molecule, e.g., an antibody or antigen- binding fragment thereof which specifically binds to Pseudomonas Pch comprising a VH and a VL, each comprising a CDRl, CDR2, and CDR3, wherein the VH CDRl is (a) SYAMS (SEQ ID NO:311), or a variant thereof sing 1, 2, 3, or 4 conservative amino acid substitutions, the VH CDR2 is AISGSGYSTYYADSVKG (SEQ ID NO: 312), or a t f comprising 1, 2, 3, or 4 vative amino acid substitutions, and the VHCDR3 is EYSISSNYYYGMDV (SEQ ID NO: 313), or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; or (b) wherein the VL CDRl is WASQGISSYLA (SEQ ID NO:314), or a variant thereof sing 1, 2, 3, or 4 conservative amino acid substitutions, the VL CDR2 is AASTLQS (SEQ ID NO:315), or a variant f comprising 1, 2, 3, or 4 conservative amino acid substitutions, and the VL CDR3 is QQLNSSPLT (SEQ ID NO:316), or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; or (c) a combination of (a) and (b); wherein the VH and VL CDRs are according to the Kabat numbering . In certain aspects of this embodiment, (a) the VH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% 99%, or 100% identical to SEQ ID NO:317, (b) the VL ses an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% 99%, or 100% identical to SEQ ID ; or (c) a combination of (a) and (b).

Also disclosed is an isolated bispeciflc binding molecule, e.g., a bispeciflc antibody or antigen-binding fragment f which ically binds to both Pseudomonas Psl and Pseudomonas Pch comprising an immunoglobulin heavy chain variable region (VH) and/or light chain variable region (VL) amino acid sequence at least 80%, 85%, 90% 95% or 100% identical to SEQ ID NO: 228, SEQ ID NO:229, or SEQ ID NO: 235.

In certain embodiments, a bispecific antibody as disclosed herein has the structure of BSl, BS2, BS3, or BS4, all as shown in . In certain bispecific dies disclosed herein the binding domain which specifically binds to Pseudomonas Psl ses an anti-Psl ScFv molecule. In other aspects the binding domain which specifically binds to Pseudomonas Psl comprises a tional heavy chain and light chain. Similarly in certain bispecific antibodies disclosed herein the binding domain which specifically binds to Pseudomonas Pch comprises an anti—Pch ScFv molecule.

In other aspects the binding domain which specifically binds to Pseudomonas Pch comprises a conventional heavy chain and light chain.

In certain aspects a bispecific antibody as disclosed herein had the BS4 structure, disclosed in detail in US. Provisional Appl. No. ,651filed on April 16, 2012 and International Application No: PCT/US2012/ 63639, filed er 6, 2012 (attorney docket no. AEMS—l lSWOl, entitled “MULTISPECIFIC AND ALENT BINDING PROTEINS AND USES THEREOF”), which is incorporated herein by reference in its entirety. For example, this disclosure provides a bispecific antibody in which an anti-Psl ScFv molecule is inserted into the hinge region of each heavy chain of an anti-Pch antibody or fragment thereof.

This disclosure provides an isolated binding molecule, e.g., a bispecfic antibody comprising an antibody heavy chain and an antibody light chain, where the antibody heavy chain ses the formula VH-CHl-Hl-Ll-S-L2-H2-CH2-CH3, wherein CHl is a heavy chain constant region domain-l, H1 is a first heavy chain hinge region fragment, L1 is a first linker, S is an anti-Pch ScFv molecule, L2 is a second linker, H2 is a second heavy chain hinge region fragment, CH2 is a heavy chain constant region domain-2, and CH3 is a heavy chain nt region domain-3. In certain aspects the VH comprises the amino acid sequence of SEQ ID NO:255, SEQ ID NO:257, or SEQ ID NO:3l7. In certain s L1 and L2 are the same or different, and independently comprise (a) [GGGGS]n, wherein n is 0, l, 2, 3, 4, or 5, (b) [GGGG]n, wherein n is 0, l, 2, 3, 4, or 5, or a combination of (a) and (b). In certain embodiments Hl comprises EPKSC (SEQ ID ), and H2 comprises DKTHTCPPCP (SEQ ID NO:32l).

In certain aspects, S comprises an anti-Psl ScFv molecule having the amino acid ce of SEQ ID NO:240, SEQ ID , SEQ ID NO:242, SEQ ID NO:243, SEQ ID NO:244, SEQ ID NO:245, SEQ ID NO:246, SEQ ID NO:247, SEQ ID NO:248, SEQ —65— ID NO:249, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:252, SEQ ID NO:253, SEQ ID NO:254, or SEQ ID NO:262, or any ation of two or more of these amino acid sequences.

In further aspects, CH2-CH3 comprises (SEQ ID NO:322), wherein X1 is M or Y, X2 is S or T, and X3 is T or E. In further aspects the antibody light chain comprises VL- CL, wherein CL is an antibody light chain kappa constant region or am an antibody light chain lambda constant region. In further aspects VL comprises the amino acid sequence of SEQ ID NO:256 or SEQ ID NO:318. CL can comprise, e. g., the amino acid ce of SEQ ID NO:323.

Further provided is an ed binding molecule, e. g., a bispeciflc antibody which specifically binds to both Pseudomonas Psl and Pseudomonas Pch comprising a VH comprising the amino acid sequence SEQ ID NO:264, and a VL comprising the amino acid sequence SEQ ID NO:263.

In some embodiments, the bispecif1c antibodies of the invention can be a tandem single chain (sc) Fv fragment, which contain two different scFv fragments (i.e., V2L2 and W4) covalently tethered together by a linker (e.g., a polypeptide linker). (Ren- Heidenreich et al. Cancer [00:1095—1103 (2004); Korn et al. J Gene Med 6:642—651 (2004)). In some embodiments, the linker can contain, or be, all or part of a heavy chain polypeptide constant region such as a CH1 . In some embodiments, the two antibody nts can be ntly tethered together by way of a polyglycine-serine or polyserine—glycine linker as described in, e.g., US. Pat. Nos. 324 and 491, respectively. Methods for generating bispeciflc tandem scFv antibodies are described in, e. g., Maletz et al. Int J Cancer 93 :409—416 (2001); and Honemann et al. Leukemia 18:636—644 (2004). Alternatively, the antibodies can be "linear dies" as described in, e.g., Zapata et al. Protein Eng. 8:1057—1062 . Briefly, these antibodies comprise a pair of tandem Fd segments (VH-CHl-VH-CHl) that form a pair of antigen binding s.

The disclosure also embraces variant forms of bispeciflc antibodies such as the tetravalent dual variable domain immunoglobulin (DVD—Ig) molecules described in Wu et al. (2007) Nat Biotechnol 25(1 1): 1290—1297. The DVD—Ig molecules are designed such that two different light chain le s (VL) from two different parent antibodies are linked in tandem directly or via a short linker by recombinant DNA techniques, followed by the light chain constant domain. For example, the DVD-Ig light chain polypeptide can contain in tandem: (a) the VL from V2L2; and (b) the VL from WapR- 004. rly, the heavy chain comprises the two different heavy chain variable domains (VH) linked in tandem, ed by the constant domain CH1 and Fc region.

For example, the DVD-Ig heavy chain polypeptide can contain in tandem: (a) the VH from V2L2; and (b) the VH from WapR-004. In this case, expression of the two chains in a cell results in a heterotetramer containing four antigen combining sites, two that specifically bind to V2L2 and two that ically bind to Psl. Methods for generating DVD—Ig les from two parent antibodies are further described in, e.g., PCT Publication Nos. and WO 24715.

In certain embodiments, an isolated binding le, e. g., an antibody or n-binding fragment thereof as described herein specifically binds to Pseudomonas Psl and/or Pch with an affinity characterized by a dissociation constant (KD) no r than 5 x 10‘2 M, 10‘2 M, 5 x 10‘3 M, 10‘3 M, 5 x 10‘4 M, 10‘4 M, 5 x 10‘5 M, 10‘5 M, 5 x ‘6 M, 10‘6 M, 5 x10"7 M, 10‘7 M, 5 x10"8 M, 10‘8 M, 5 x10"9 M, 10-9 M, 5 x10"10 M, '10 M, 5 x 10'11 M, 10'11 M, 5 x 10'12 M, 10'12 M, 5 x 10'13 M, 10'13 M, 5 x 10'14 M, 10'14 M, 5 x 10'15 M, or 10'15 M.

In specific embodiments, an isolated binding molecule, e. g., an antibody or antigen-binding fragment thereof as described herein specifically binds to Pseudomonas Psl and/or Pch, with an affinity characterized by a dissociation constant (KD) in a range of about 1 x 10'10 to about 1 x 10'6 M. In one embodiment, an isolated binding molecule, e.g., an dy or antigen-binding fragment f as described herein specifically binds to Pseudomonas Psl and/or Pch, with an affinity characterized by a KD of about 1.18 x 10'7 M, as determined by the OCTET® binding assay described herein. In another embodiment, an isolated binding le, e. g., an antibody or antigen-binding fragment thereof as described herein specifically binds to Pseudomonas Psl and/or Pch, with an affinity characterized by a KD of about 1.44 x 10'7 M, as determined by the OCTET® g assay described herein.

Some embodiments include the isolated binding molecules e.g., an antibody or nt thereof as described above, which (a) can inhibit attachment of Pseudomonas aeruginosa to epithelial cells, (b) can promote OPK of P. aeruginosa, or (c) can inhibit attachment of P. aeruginosa to epithelial cells and can promote OPK of P. aeruginosa. —67— In some embodiments the isolated binding le e.g., an antibody or fragment thereof as described above, where maximum inhibition of P. aeruginosa attachment to epithelial cells is achieved at an antibody concentration of about 50 ug/ml or less, 5.0 [Lg/ml or less, or about 0.5 [1ng or less, or at an antibody concentration ranging from about 30 ug/ml to about 0.3 [Lg/ml, or at an antibody concentration of about 1 ug/ml, or at an antibody tration of about 0.3 [Lg/ml.

Certain embodiments include the ed binding molecule e.g., an antibody or fragment thereof as described above, where the OPK EC50 is less than about 0.5 [Lg/ml, less than about 0.05 [Lg/ml, or less than about 0.005 [Lg/ml, or where the OPK EC50 ranges from about 0.001 ug/ml to about 0.5 ug/ml, or where the OPK EC50 ranges from about 0.02 [1le to about 0.08 [Lg/ml, or where the OPK EC50 ranges from about 0.002 ug/ml to about 0.01 ug/ml or where the OPK EC50 is less than about 0.2 ug/ml, or wherein the OPK EC50 is less than about 0.02 ug/ml. In certain embodiments, an anti- Pseudomonas Psl binding molecule, e.g., antibody or fragment, variant or derivative thereof described herein specifically binds to the same Ps1 e as monoclonal antibody WapR—004, WapR-004RAD, 3, Cam—004, or Cam—005, or will competitively inhibit such a monoclonal dy from binding to Pseudomonas Psl.

WapR-004RAD is identical to WapR-004 except for an amino acid substitution G98A of the VH amino acid sequence of SEQ ID NO:11.

Some ments include 04 (W4) mutants comprising an scFv—Fc molecule amino acid sequence identical to, or identical except for one, two, three, four, five, or more amino acid substitutions to one or more of: SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145; or SEQ ID NO: 146.

Other embodiments include WapR-004 (W4) mutants comprising an scFv-Fc molecule amino acid sequence at least 80%, 85%, 90% 95% or 100% identical to one or more of: SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145; or SEQ ID NO: 146.

In some embodiments, an anti—Pseudomonas Ps1 binding molecule, e.g., dy or fragment, variant or derivative thereof described herein ically binds to the same epitope as monoclonal antibody WapR—OOl, WapR-002, or WapR-003, or will competitively inhibit such a onal dy from binding to Pseudomonas Psl.

In certain embodiments, an anti—Pseudomonas Ps1 binding molecule, e.g., antibody or fragment, variant or derivative thereof bed herein specifically binds to the same epitope as monoclonal antibody WapR-016, or will competitively inhibit such a monoclonal antibody from binding to Pseudomonas Psl.

TABLE 2: Reference VH and VL amino acid sequences* Antibody VH VL Name Cam—003 QVRLQQSGPGLVKPSET SSELTQDPAVSVALGQTVRITCM VSGGSTSM LRSYYASWYQQKPGQAPVLVIYGfl fiWLRQPPGKGLEWIGfl NRPSGIPDRFSGS SSGNTASLTITGAQ HSNGGTNYNPSLKSRL AEDEADYYCNSRDSSGNHVVFGGGT TISGDTSKNQFSLNLSF KLTVL ALYYCARM SEQ ID NO:2 DVYGPAFDIWGQGTM SEQ ID NO:1 QVQLQQSGPGRVKPSE SSELTQDPAVSVALGQTVRITCM TLSLTCTVSGYSVSSG_Y LRSYYASWYQQKPGQAPVLVIYGfl YWGWIRQSPGTGLEWI PDRFSGS SSGNTASLTITGAQ GSISHSGSTYYNPSLKS AEDEADYYCNSRDSSGNHVVFGGGT RVTISGDASKNQFFLRL KLTVL TSVTAADTAVYYCARQ SEQ ID NO:2 EATANFDSWGRGTLVT SEQ ID NO:3 QVQLQQSGPGLVKPSET DPAVSVALGQTVRITCM LSLTCTVSGGSVSM LRSYYASWYQQKPGQAPVLVIYGfl MWIRQPPGKGLEWI NRPSGIPDRFSGSSSGNTASLTITGAQ GSIYSSGSTYYSPSLKS AEDEADYYCNSRDSSGNHVVFGGGT RVTISGDTSKNQFSLKL KLTVL SSVTAADTAVYYCARL SEQ ID NO:2 NWGTVSAFDIWGRGTL SEQ ID NO:4 Antibody VL Name WapR—OOl EVQLLESGGGLVQPGG QAGLTQPASVSGSPGQSITISCTGTSS SLRLSCSASGFTFSM DIATYNYVSWYQQHPGKAPKLMIYE MWVRQAPGKGLEYV GTKRPSGVSNRFSGSKSGNTASLTIS SDIGTNGGSTNYADSV GLQAEDEADYYCSSYARSYTYVFGT ERFTISRDNSKNTVYL GTELTVL AEDTAVYHCV SEQ ID NO:6 AGIAAAYGFDVWGQG TMVTVSS SEQ ID NO:5 SGGGLVQPGG QTVVTQPASVSGSPGQSITISCTGTSS SLRLSCSASGFTFSSY_P DVGGYNYVSWYQQHPGKAPKLMIY MWVRQAPGKGLDYV EVSNRPSGVSNHFSGSKSGNTASLTIS SDISPNGGSTNYADSV GLQAEDEADYYCSSYTTSSTYVFGT K_GRFTISRDNSKNTLFL GTKVTVL QMSSLRAEDTAVYYCV SEQ ID NO:8 MGLVPYGFDIWGQGTL VTVSS SEQ ID NO:7 QMQLVQSGGGLVQPGG QTVVTQPASVSASPGQSITISCAGTSG SLRLSCSASGFTFSSY_P DVGNYNFVSWYQQHPGKAPKLLIYE MWVRQAPGKGLDYV GSQRPSGVSNRFSGSRSGNTASLTIS SDISPNGGATNYADSV EADYYCSSYARSYTYVFGT ERFTISRDNSKNTVYL L QMSSLRAEDTAVYYCV SEQ ID NO:10 MGLVPYGFDNWGQGT MVTVSS SEQ ID NO:9 dy VL Name WapR—004 EVQLLESGPGLVKPSET EIVLTQSPSSLSTSVGDRVTITCM LSLTCNVAGGSISM SIRSHLNWYQQKPGKAPKLLIYw IWIRQPPGKGLELIGE MGVPSRFSGSGSGTDFTLTISSLQ HSSGYTDYNPSLKSRV PEDFATYYCQQSYSFPLTFGGGTKLE TISGDTSKKQFSLHVSS 1K VTAADTAVYFCARG_D SEQ ID N012 LDIWGQGTL VTVSS SEQ ID NO:11 EVQLVQSGADVKKPGA SSELTQDPAVSVALGQTVRITCM KASGYTFTG_H LRSYYTNWFQQKPGQAPLLVVYA_K wWVRQAPGQGLEW NKRPPGIPDRFSGSSSGNTASLTITGA MGWINPDSGATSYAQ QAEDEADYYCHSRDSSGNHVVFGG MRVTMTRDTSITT GTKLTVL AYMDLSRLRSDDTAVY SEQ ID NO:14 YCATDTLLSNHWGQGT LVTVSS SEQ ID NO:13 EVQLVESGGGLVQPGGSL QSVLTQPASVSGSPGQSITISCTGTSSDVG RLSCAASGYTFSMWV GYNYVSWYQQ RQAPGKGLEWVAGISGSG GVSNRFSGSKSGNTASLTISGLQAEDEAD DTTDYVDSVKGRFTVSRD YCSSYSSGTVVFGGGTELTVL NSKNTLYLQMNSLRADDT SEQ ID NO: 16 AVYYCASRGGLGGYYRG GFDFWGQGTMVTVSS SEQ ID NO:15 Antibody VL Name WapR— EVQLLESGPGLVKPSET EIVLTQSPSSLSTSVGDRVTITCM 004RAD LSLTCNVAGGSISM SIRSHLNWYQQKPGKAPKLLIYw IWIRQPPGKGLELIGE FSGSGSGTDFTLTISSLQ HSSGYTDYNPSLKSRV PEDFATYYCQQSYSFPLTFGGGTKLE SKKQFSLHVSS 1K VTAADTAVYFCARA_D SEQ ID NO:12 WDLLHALDIWGQGTL VTVSS SEQ ID NO:74 V2L2 EMQLLESGGGLVQPGG AIQMTQSPSSLSASVGDRVTITCLAS SLRLSCAASGFTFSfl g[GIRNDLGWYQQKPGKAPKLVIYE MWVRQAPGEGLEWV STLQSGVPSRFSGSGSGTDFTLSISSL SAITISGITAYYTDSVK QPDDFATYYCLQDYNYPWTFGQGT QRFTISRDNSKNTLYLQ KVEIK GDTAVYYCA SEQ ID NO:217 KEEFLPGTHYYYGMD XWGQGTTVTVSS SEQ ID NO:216 *VH and VL CDRl, CDR2, and CDR3 amino acid sequences are underlined TABLE 3: Reference VH and VL CDRl, CDR2, and CDR3 amino acid sequences Antibody VHCDRl VHCDR2 VHCDR3 VLCDRl VLCDR2 VLCDR3 Name Cam—003 YI TNYNPSL GPAFDI VV KS SEQ ID SEQ ID NO:22 SEQ ID NO: 19 NO: 18 Antibody VHCDRl VHCDR2 VHCDR3 VLCDRl VLCDR2 VLCDR3 Name Cam-004 SGYYW SISHSGST SEATAN QGDSLRSY NSRDSSGNH G YYNPSLK FDS YAS VV SEQ ID S SEQ ID SEQ ID SEQ ID N022 N023 SEQ ID NO:25 N020 N024 Cam—005 SSGYYW SIYSSGST LNWGTV QGDSLRSY GNH YAS VV T YYSPSLKS SAFDI SEQ ID SEQ ID N022 N020 SEQ ID SEQ ID SEQ ID N026 NO:27 NO:28 WapR-OO 1 RYPMH DIGTNG GIAAAY TGTSSDIAT EGTKRPS SSYARSYT SEQ ID GSTNYA GFDV YNYVS SEQ ID YV N029 DSVKG SEQ ID SEQ ID NO:33 SEQ ID NO:34 SEQ ID NO:3 1 NO:32 NO:30 WapR—OOZ SYPM SEQ ID STNYAD GGYNYVS SEQ ID V NO:35 SVKG SEQID NO:39 SEQ ID NO:40 SEQ ID N038 NO:36 WapR—003 SYPMH DISPNGG AGTSGDV EGSQRPS YT SEQ ID ATNYAD GNYNFVS SEQ ID YV NO:41 SVKG SEQ ID NO:45 SEQ ID NO:46 SEQ ID NO:44 NO:42 PCT/U82012/063722 Antibody VHCDRl VHCDR2 VHCDR3 VLCDRl VLCDR2 VLCDR3 Name WapR-004 PYYWT YIHSSGY GDWDL RASQSIRS GASNLQS QQSYSFPLT SEQID TDYNPSL LHALDI HLN SEQID SEQIDNO:52 NO:47 KS SEQID SEQID NO:51 SEQ ID NO:49 NO:50 NO:48 WapR-007 GHNIH WINPDS DTLLSN QGDSLRS AKNKRPP HSRDSSGN SEQ ID GATSYA H YYTN SEQ ID HVV NO:53 QKFQG SEQID SEQID NO:57 SEQIDNO:58 SEQ ID NO:55 NO:56 NO:54 WapR-016 SYATS GISGSGDT RGGLGG TGTSSDVG EVSNRPS SSYSSGTVV SEQ ID TDYVDSV YYRGGF GYNYVS SEQ ID SEQ ID NO:64 NO:59 KG DF SEQ ID NO:63 SEQ ID SEQ ID NO:62 NO:60 NO:61 $2113- PYYWT YIHSSGY ADWDL RS GASNLQS QQSYSFPLT SEQID TDYNPSL LHALDI HLN SEQID SEQIDNO:52 NO:47 KS SEQID SEQID NO:51 SEQ ID NO:75 NO:50 NO:48 V2L2 SYAMN AITISGIT EEFLPG RASQGIRN SASTLQS LQDYNYP SEQ ID AYYTDS THYYY DLG SEQ ID SEQ ID NO:218 VKG GMDV NO‘223 SEQ ID NO:222 SEQID SEQID NO:221 NO:219 NO:220 In n embodiments, an anti—Pseudomonas Pch g le, e.g., antibody or fragment, variant or derivative thereof described herein specifically binds to the same Pch epitope as monoclonal antibody V2L2, and/or will competitively inhibit such a monoclonal antibody from binding to monas Pch.

For example, in certain aspects the anti—Pseudomonas Pch binding molecule, e.g., antibody or fragment, variant or derivative thereof comprises V2L2-GL and/or V2L2—MD.

In certain embodiments, an anti—Pseudomonas Pch g molecule, e.g., antibody or fragment, variant or tive thereof described herein specifically binds to the same Pch epitope as monoclonal antibody 29D2, and/or will competitively inhibit such a monoclonal antibody from g to Pseudomonas Pch.

Any seudomonas Psl and/or Pch binding molecules, e.g., antibodies or fragments, variants or derivatives thereof described herein can further include additional ptides, e.g., a signal peptide to direct secretion of the d polypeptide, antibody constant regions as described herein, or other heterologous polypeptides as bed herein. Additionally, binding molecules or fragments thereof of the description include polypeptide fragments as described elsewhere. Additionally anti—Pseudomonas Psl and/or Pch binding molecules, e. g., antibodies or fragments, variants or derivatives thereof bed herein can be fusion polypeptides, Fab fragments, scFvs, or other derivatives, as bed herein.

Also, as described in more detail elsewhere herein, the disclosure includes itions comprising anti—Pseudomonas Psl and/or Pch binding molecules, e.g., dies or fragments, variants or derivatives thereof described herein.

It will also be understood by one of ordinary skill in the art that seudomonas Psl and/or Pch binding molecules, e. g., antibodies or fragments, variants or derivatives thereof described herein can be modified such that they vary in amino acid sequence from the naturally occurring binding polypeptide from which they were derived. For example, a polypeptide or amino acid sequence derived from a designated protein can be similar, e. g., have a certain percent identity to the starting sequence, e.g., it can be 60%, 70%, 75%, 80%, 85%, 90%, or 95% identical to the starting sequence.

As known in the art, "sequence identity" between two polypeptides is determined by comparing the amino acid ce of one polypeptide to the sequence of a second polypeptide. When discussed herein, whether any particular polypeptide is at least about 70%, 75%, 80%, 85%, 90% or 95% identical to r polypeptide can be determined —76— using methods and computer ms/software known in the art such as, but not limited to, the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). BESTFIT uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482—489 (1981), to find the best segment of homology between two sequences. When using T or any other ce alignment program to determine whether a particular sequence is, for example, 95% cal to a reference sequence, the parameters are set, of course, such that the tage of identity is ated over the full length of the reference polypeptide sequence and that gaps in homology of up to 5% of the total number of amino acids in the nce sequence are allowed.

Percentage of “sequence identity” can also be determined by comparing two optimally aligned sequences over a comparison window. In order to optimally align sequences for comparison, the portion of a polynucleotide or polypeptide sequence in the comparison window can comprise additions or deletions termed gaps while the reference sequence is kept constant. An optimal alignment is that alignment which, even with gaps, produces the greatest possible number of “identical” ons between the reference and comparator sequences. tage “sequence identity” between two sequences can be determined using the version of the program “BLAST 2 Sequences” which was ble from the National Center for Biotechnology Information as of September 1, 2004, which program incorporates the programs BLASTN (for nucleotide sequence comparison) and BLASTP (for polypeptide sequence ison), which programs are based on the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90(12):5873—5877, 1993).

When utilizing “BLAST 2 Sequences,” parameters that were default ters as of September 1, 2004, can be used for word size (3), open gap penalty (11), extension gap penalty (1), gap drop-off (50), expect value (10) and any other required parameter including but not limited to matrix option. rmore, nucleotide or amino acid substitutions, deletions, or insertions leading to conservative substitutions or changes at "non-essential" amino acid regions can be made. For example, a polypeptide or amino acid sequence derived from a designated protein can be identical to the ng sequence except for one or more individual amino acid substitutions, insertions, or deletions, e.g., one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty or more individual amino acid substitutions, insertions, or deletions. In certain embodiments, a polypeptide or amino acid sequence derived from a designated n has one to five, one to ten, one to fifteen, or one to twenty dual amino acid substitutions, ions, or deletions relative to the ng sequence.

An anti—Pseudomonas Ps1 and/or Pch g molecule, e.g., an antibody or fragment, variant or derivative thereof described herein can comprise, consist essentially of, or consist of a fusion protein. Fusion proteins are chimeric molecules which se, for example, an immunoglobulin antigen-binding domain with at least one target binding site, and at least one heterologous portion, i.e., a n with which it is not naturally linked in nature. The amino acid sequences can normally exist in te proteins that are brought together in the fusion polypeptide or they can normally exist in the same protein but are placed in a new arrangement in the fusion polypeptide. Fusion proteins can be created, for example, by chemical synthesis, or by creating and translating a cleotide in which the peptide s are encoded in the desired relationship.

The term "heterologous" as applied to a polynucleotide, polypeptide, or other moiety means that the polynucleotide, polypeptide, or other moiety is derived from a distinct entity from that of the rest of the entity to which it is being compared. In a non- limiting example, a "heterologous ptide" to be fused to a binding molecule, e.g., an antibody or an antigen-binding fragment, variant, or derivative thereof is derived from a non-immunoglobulin polypeptide of the same species, or an immunoglobulin or nonimmunoglobulin polypeptide of a different species.

IV. FUSION PROTEINS AND ANTIBODY CONIUGATES In some embodiments, the seudomonas Ps1 and/or Pch binding molecules, e. g., antibodies or fragments, variants or derivatives thereof can be administered multiple times in conjugated form. In still another embodiment, the anti—Pseudomonas Psl and/or Pch binding molecules, e. g., antibodies or fragments, variants or derivatives thereof can be administered in unconjugated form, then in conjugated form, or vice versa.

In specific embodiments, the anti—Pseudomonas Psl and/or Pch binding molecules, e.g., antibodies or nts, variants or derivatives thereof can be conjugated to one or more antimicrobial , for e, Polymyxin B (PMB). PMB is a small lipopeptide antibiotic approved for treatment of multidrug-resistant Gram-negative WO 70615 —78— infections. In addition to its icidal activity, PMB binds lipopolysaccharide (LPS) and lizes its proinflammatory effects. (Dixon, R.A. & Chopra, I. J Antimicrob Chemother 18, 557—563 (1986)). LPS is thought to significantly contribute to inflammation and the onset of Gram-negative sepsis. (Guidet, B., et al., Chest 106, 1194— 1201 (1994)). Conjugates of PMB to carrier molecules have been shown to neutralize LPS and mediate protection in animal models of endotoxemia and infection. (Drabick, J.J., et a]. crob Agents Chemother 42, 583—588 (1998)). Also sed is a method for attaching one or more PMB molecules to cysteine residues uced into the Fc region of monoclonal antibodies (mAb) of the disclosure. For example, the Cam—003— PMB conjugates retained specific, mAb—mediated binding to P. aeruginosa and also retained OPK activity. Furthermore, mAb-PMB conjugates bound and neutralized LPS in vitro. In specific embodiments, the anti—Pseudomonas Ps1 and/or Pch binding molecules, e.g., antibodies or fragments, variants or derivatives thereof can be combined with antibiotics (e.g., Ciprofloxacin, Meropenem, Tobramycin, Aztreonam).

In certain embodiments, an anti—Pseudomonas Ps1 and/or Pch binding molecule, e. g., an antibody or fragment, variant or derivative f described herein can comprise a heterologous amino acid sequence or one or more other moieties not ly associated with an antibody (e. g., an antimicrobial agent, a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a lipid, a biological response modifier, ceutical agent, a lymphokine, a heterologous antibody or fragment thereof, a detectable label, polyethylene glycol (PEG), and a ation of two or more of any said agents). In further embodiments, an anti—Pseudomonas Ps1 and/or Pch binding molecule, e.g., an antibody or fragment, variant or derivative thereof can comprise a detectable label selected from the group consisting of an enzyme, a fluorescent label, a chemiluminescent label, a bioluminescent label, a radioactive label, or a ation of two or more of any said detectable labels.

V. POLYNUCLEOTIDES ENCODING BINDING MOLECULES Also ed herein are c acid molecules encoding the anti—Pseudomonas Ps1 and/or Pch binding molecules, e. g., antibodies or fragments, variants or tives thereof described herein. .

One embodiment provides an isolated polynucleotide comprising, consisting essentially of, or ting of a nucleic acid encoding an immunoglobulin heavy chain variable region (VH) amino acid sequence at least 80%, 85%, 90% 95% or 100% identical to one or more of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: , SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ IS NO: 74, or SEQ ID NO:216 as shown in Table 2.

One embodiment provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin heavy chain variable region (VH) amino acid sequence of SEQ ID NO:257 or SEQ ID NO:259. For example the nucleic acid sequences of SEQ ID NO:261, and SEQ ID NO:: 259, respectively.

Another embodiment provides an isolated polynucleotide comprising, ting essentially of, or consisting of a nucleic acid encoding a VH amino acid ce identical to, or identical except for one, two, three, four, five, or more amino acid substitutions to one or more of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: , SEQ ID NO: 74, or SEQ ID NO:216 as shown in Table 2.

Further embodiment provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid ng a VH, where one or more of the , VHCDR2 or VHCDR3 regions of the VH are identical to, or identical except for four, three, two, or one amino acid tutions, to one or more reference heavy chain VHCDRl, VHCDR2 and/or VHCDR3 amino acid sequences of one or more of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 74, or SEQ ID NO:216 as shown in Table 2.

Another embodiment provides an ed polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an isolated binding molecule, e.g., an antibody or antigen-binding fragment thereof which specifically binds to Pseudomonas Psl sing a VH, where one or more of the VHCDRl, VHCDR2 or VHCDR3 s of the VH are identical to, or identical except for four, three, two, or one amino acid substitutions, to one or more reference heavy chain VHCDRl, VHCDR2 and/or VHCDR3 amino acid sequences of one or more of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ 2012/063722 ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 74 as shown in Table 2.

A r embodiment provides an isolated binding molecule e.g., an antibody or antigen—binding fragment comprising the VH encoded by the polynucleotide specifically or entially binds to monas Ps1 and/or Pch.

Another embodiment provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin light chain variable region (VL) amino acid sequence at least 80%, 85%, 90% 95% or 100% identical to one or more of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO:217 as shown in Table 2.

Another ment provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid ng the immunoglobulin light chain variable region (VL) amino acid sequence of SEQ ID NO:256, e.g., the c acid sequence SEQ ID NO:260..

A further embodiment provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid ng a VL amino acid sequence identical to, or identical except for one, two, three, four, five, or more amino acid substitutions to one or more of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO:217 as shown in Table 2.

Another embodiment provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a c acid encoding a VL, where one or more of the VLCDRl, VLCDR2 or VLCDR3 regions of the VL are at least 80%, 85%, 90%, 95% or 100% identical to one or more reference light chain VLCDRl, VLCDR2 or VLCDR3 amino acid sequences of one or more of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16, or SEQ ID NO:217 as shown in Table 2.

A further embodiment provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a c acid encoding an isolated binding molecule, e.g., an antibody or antigen-binding fragment thereof which specifically binds to Pseudomonas Ps1 comprising an VL, where one or more of the VLCDRl, VLCDR2 or VLCDR3 2012/063722 regions of the VL are identical to, or identical except for four, three, two, or one amino acid substitutions, to one or more reference heavy chain VLCDRl, VLCDR2 and/or VLCDR3 amino acid sequences of one or more of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO:217 as shown in Table 2.

In another embodiment, isolated binding molecules e. g., an antibody or antigen- binding fragment comprising the VL encoded by the polynucleotide ically or entially bind to Pseudomonas Psl and/or Pch.

One embodiment provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid which encodes an scFV molecule including a VH and a VL, where the scFV is at least 80%, 85%, 90% 95% or 100% identical to one or more of SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, or SEQ ID NO:70 as shown in Table 4.

WO 70615 TABLE 4: Reference scFV nucleic acid sequences dy scFV nucleotide sequences Name CAGCCGGCCATGGCCCAGGTACAGCTGCAGCAGTCAGGCCCAGG ACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCT GGTGGCTCCACCAGTCCTTACTTCTGGAGCTGGCTCCGGCAGCCC CCAGGGAAGGGACTGGAGTGGATTGGTTATATCCATTCCAATGGG GGCACCAACTACAACCCCTCCCTCAAGAGTCGACTCACCATATCA GGAGACACGTCCAAGAACCAATTCTCCCTGAATCTGAGTTTTGTG ACCGCTGCGGACACGGCCCTCTATTACTGTGCGAGAACGGACTAC GATGTCTACGGCCCCGCTTTTGATATCTGGGGCCAGGGGACAATG GTCACCGTCTCGAGTGGTGGAGGCGGTTCAGGCGGAGGTGGCAG CGGCGGTGGCGGATCGTCTGAGCTGACTCAGGACCCTGCTGTGTC TGTGGCCTTGGGACAGACAGTCAGGATCACATGCCAAGGAGACA GCCTCAGAAGCTATTATGCAAGCTGGTACCAGCAGAAGCCAGGA CAGGCCCCTGTACTTGTCATCTATGGTAAAAACAACCGGCCCTCA GGGATCCCAGACCGATTCTCTGGCTCCAGCTCAGGAAACACAGCT TCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTAT TACTGTAACTCCCGGGACAGCAGTGGTAACCATGTGGTATTCGGC GGAGGGACCAAGCTGACCGTCCTAGGTGCGGCCGCA SEQ ID NO:65 WO 70615 Antibody scFV nucleotide sequences Name Cam—004 CAGCCGGCCATGGCCCAGGTACAGCTGCAGCAGTCAGGCCCAGG ACGGGTGAAGCCTTCGGAGACGCTGTCCCTCACCTGCACTGTCTC TGGTTACTCCGTCAGTAGTGGTTACTACTGGGGCTGGATCCGGCA GTCCCCAGGGACGGGGCTGGAGTGGATTGGGAGTATCTCTCATAG TGGGAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCAT ATCAGGAGACGCATCCAAGAACCAGTTTTTCCTGAGGCTGACTTC CGCCGCGGACACGGCCGTTTATTACTGTGCGAGATCTGA GGCTACCGCCAACTTTGATTCTTGGGGCAGGGGCACCCTGGTCAC CGTCTCTTCAGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCG GTGGCGGATCGTCTGAGCTGACTCAGGACCCTGCCGTGTCTGTGG CCTTGGGACAGACAGTCAGGATCACATGCCAAGGAGACAGCCTC AGAAGCTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAGGC CCCTGTACTTGTCATCTATGGTAAAAACAACCGGCCCTCAGGGAT CCCAGACCGATTCTCTGGCTCCAGCTCAGGAAACACAGCTTCCTT GACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACTG TAACTCCCGGGACAGCAGTGGTAACCATGTGGTATTCGGCGGAGG GACCAAGCTGACCGTCCTAGGTGCGGCCGCA SEQ ID NO:66 Antibody scFV nucleotide ces Nmne CmnflOS CAGCCGGCCATGGCCCAGGTACAGCTGCAGCAGTCAGGCCCAGG ACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCT GGTGGCTCCGTCAGCAGTAGTGGTTATTACTGGACCTGGATCCGC CAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTCT AGTGGGAGCACATATTACAGCCCGTCCCTCAAGAGTCGAGTCACC ATATCCGGAGACACGTCCAAGAACCAGTTCTCCCTCAAGCTGAGC TCTGTGACCGCCGCAGACACAGCCGTGTATTACTGTGCGAGACTT AACTGGGGCACTGTGTCTGCCTTTGATATCTGGGGCAGAGGCACC CTGGTCACCGTCTCGAGTGGTGGAGGCGGTTCAGGCGGAGGTGGC AGCGGCGGTGGCGGATCGTCTGAGCTGACTCAGGACCCTGCTGTG GCCTTGGGACAGACAGTCAGGATCACATGCCAAGGAGAC AGCCTCAGAAGCTATTATGCAAGCTGGTACCAGCAGAAGCCAGG ACAGGCCCCTGTACTTGTCATCTATGGTAAAAACAACCGGCCCTC AGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAGGAAACACAGC TTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTA TTACTGTAACTCCCGGGACAGCAGTGGTAACCATGTGGTATTCGG CGGAGGGACCAAGCTGACCGTCCTAGGTGCGGCCGCA SEQ ID NO:67 —85— Antibody scFV nucleotide sequences Nmne kaLmH TCTATGCGGCCCAGCCGGCCATGGCCGAGGTGCAGCTGTTGGAGT CTGGGGGAGGTTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCT CCTCTGGGTTCACCTTCAGTCGGTATCCTATGCATTGGGT CCGCCAGGCTCCAGGGAAGGGACTGGAATATGTTTCAGATATTGG TACTAATGGGGGTAGTACAAACTACGCAGACTCCGTGAAGGGCA GATTCACCATCTCCAGAGACAATTCCAAGAACACGGTGTATCTTC AAATGAGCAGTCTGAGAGCTGAGGACACGGCTGTGTATCATTGTG TGGCGGGTATAGCAGCCGCCTATGGTTTTGATGTCTGGGGCCAAG TGGTCACCGTCTCGAGTGGAGGCGGCGGTTCAGGCGGA GGTGGCTCTGGCGGTGGCGGAAGTGCACAGGCAGGGCTGACTCA GCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCC TGCACTGGAACCAGCAGTGACATTGCTACTTATAACTATGTCTCCT GGTACCAACAGCACCCAGGCAAAGCCCCCAAACTCATGATTTATG AGGGCACTAAGCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCT CCAAGTCTGGCAACACGGCCTCCCTGACAATCTCTGGGCTCCAGG CTGAGGACGAGGCTGATTATTACTGTTCCTCATATGCACGTAGTT ACACTTATGTCTTCGGAACTGGGACCGAGCTGACCGTCCTAGCGG CCGC SEQ ID NO:68 Antibody scFV nucleotide sequences Name WapR—002 CTATGCGGCCCAGCCGGCCATGGCCCAGGTGCAGCTGGTGCAGTC TGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTG TTCAGCCTCTGGATTCACCTTCAGTAGCTATCCTATGCACTGGGTC CGCCAGGCTCCAGGGAAGGGACTGGATTATGTTTCAGACATCAGT CCAAATGGGGGTTCCACAAACTACGCAGACTCCGTGAAGGGCAG CATCTCCAGAGACAATTCCAAGAACACACTGTTTCTTCA AATGAGCAGTCTGAGAGCTGAGGACACGGCTGTGTATTATTGTGT GATGGGGTTAGTACCCTATGGTTTTGATATCTGGGGCCAAGGCAC CCTGGTCACCGTCTCGAGTGGAGGCGGCGGTTCAGGCGGAGGTGG CTCTGGCGGTGGCGGAAGTGCACAGACTGTGGTGACCCAGCCTGC CTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCACT GGAACCAGCAGTGACGTTGGTGGTTATAACTATGTCTCCTGGTAC CAACAGCACCCAGGCAAAGCCCCCAAACTCATGATTTATGAGGTC AGTAATCGGCCCTCAGGGGTTTCTAATCACTTCTCTGGCTCCAAGT CTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGG CTGATTATTACTGCAGCTCATATACAACCAGCAGCACTT ATGTCTTCGGAACTGGGACCAAGGTCACCGTCCTAGCGGCCG SEQ ID NO:69 —87— Antibody scFV nucleotide sequences Name WapR—003 AGCCGGCCATGGCCCAGATGCAGCTGGTGCAGTCGGGG GGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTTCA GCCTCTGGATTCACCTTCAGTAGCTATCCTATGCACTGGGTCCGCC AGGCTCCAGGGAAGGGACTGGATTATGTTTCAGACATCAGTCCAA ATGGGGGTGCCACAAACTACGCAGACTCCGTGAAGGGCAGATTC ACCATCTCCAGAGACAATTCCAAGAACACGGTGTATCTTCAAATG AGCAGTCTGAGAGCTGAAGACACGGCTGTCTATTATTGTGTGATG GGGTTAGTGCCCTATGGTTTTGATAACTGGGGCCAGGGGACAATG GTCTCGAGTGGAGGCGGCGGTTCAGGCGGAGGTGGCTCT GGCGGTGGCGGAAGTGCACAGACTGTGGTGACCCAGCCTGCCTCC GTGTCTGCATCTCCTGGACAGTCGATCACCATCTCCTGCGCTGGA ACCAGCGGTGATGTTGGGAATTATAATTTTGTCTCCTGGTACCAA CAACACCCAGGCAAAGCCCCCAAACTCCTGATTTATGAGGGCAGT CAGCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAGGTCTG GCAACACGGCCTCCCTGACAATCTCTGGGCTCCAGGCTGAGGACG AGGCTGATTATTACTGTTCCTCATATGCACGTAGTTACACTTATGT CTTCGGAACTGGGACCAAGCTGACCGTCCTAGCGGCCGCA SEQ ID NO:70 Antibody scFV nucleotide sequences Name WapR—004 TATGCGGCCCAGCCGGCCATGGCCGAGGTGCAGCTGTTGGAGTCG GGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGC AATGTCGCTGGTGGCTCCATCAGTCCTTACTACTGGACCTGGATCC GGCAGCCCCCAGGGAAGGGCCTGGAGTTGATTGGTTATATCCACT GGTACACCGACTACAACCCCTCCCTCAAGAGTCGAGTCA CCATATCAGGAGACACGTCCAAGAAGCAGTTCTCCCTGCACGTGA TGACCGCTGCGGACACGGCCGTGTACTTCTGTGCGAGAG GCGATTGGGACCTGCTTCATGCTCTTGATATCTGGGGCCAAGGGA CCCTGGTCACCGTCTCGAGTGGAGGCGGCGGTTCAGGCGGAGGTG GCTCTGGCGGTGGCGGAAGTGCACTCGAAATTGTGTTGACACAGT CTCCATCCTCCCTGTCTACATCTGTAGGAGACAGAGTCACCATCA CTTGCCGGGCAAGTCAGAGCATTAGGAGCCATTTAAATTGGTATC AGCAGAAACCAGGGAAAGCCCCTAAACTCCTGATCTATGGTGCAT CCAATTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGAT CTGGGACAGATTTCACTCTCACCATTAGTAGTCTGCAACCTGAAG ATTTTGCAACTTACTACTGTCAACAGAGTTACAGTTTCCCCCTCAC TTTCGGCGGAGGGACCAAGCTGGAGATCAAAGCGGCCGC SEQ ID NO:71 Antibody scFV nucleotide sequences Nmne WMpR£07 GCGGCCCAGCCGGCCATGGCCGAAGTGCAGCTGGTGCAGTCTGG GGCTGACGTAAAGAAGCCTGGGGCCTCAGTGAGGGTCACCTGCA AGGCTTCTGGATACACCTTCACCGGCCACAACATACACTGGGTGC CCCCTGGACAAGGGCTTGAATGGATGGGATGGATCAAC CCTGACAGTGGTGCCACAAGCTATGCACAGAAGTTTCAGGGCAGG GTCACCATGACCAGGGACACGTCCATCACCACAGCCTACATGGAC CTGAGCAGGCTGAGATCTGACGACACGGCCGTATATTACTGTGCG ACCGATACATTACTGTCTAATCACTGGGGCCAAGGAACCCTGGTC ACCGTCTCGAGTGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGG CGGTGGCGGATCGTCTGAGCTGACTCAGGACCCTGCTGTGTCTGT GGCCTTGGGACAGACAGTCAGGATCACTTGCCAAGGAGACAGTCT CAGAAGCTATTACACAAACTGGTTCCAGCAGAAGCCAGGACAGG CCCCTCTACTTGTCGTCTATGCTAAAAATAAGCGGCCCCCAGGGA ACCGATTCTCTGGCTCCAGCTCAGGAAACACAGCTTCCT TGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACT GTCATTCCCGGGACAGCAGTGGTAACCATGTGGTATTCGGCGGAG GGACCAAGCTGACCGTCCTAGGTGCGGCCGCA SEQ ID NO:72 Antibody scFVINKfleofidesequences Nmne VVapR:016 CAGCCGGCCATGGCCGAGGTGCAGCTGGTGGAGTCTGGGGGAGG CTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTG TGCAGCCTCTGGATACACCTTTAGCAGCTATGCCACGAGCTGGGT CCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCG CAGGTATTAGTGGTAGTGGTGATACCACAGACTACGTAGACTCCG TGAAGGGCCGGTTCACCGTCTCCAGAGACAATTCC AAGAACACCCTATATCTGCAAATGAACAGCCTGAGAGCCGACGA CACGGCCGTGTATTACTGTGCGTCGAGAGGAGGTTT AGGGGGTTATTACCGGGGCGGCTTTGACTTCTGGGGCCAGGGGAC AATGGTCACCGTCTCGAGTGGAGGCGGCGGTTCAG GCGGAGGTGGCTCTGGCGGTGGCGGAAGTGCACAGTCTGTGCTGA CTGCCTCCGTGTCTGGGTCTCCTGGACAG TCGATCACCATCTCCTGCACTGGAACCAGCAGTGACGTTGGTGGT TATGTCTCCTGGTACCAACAGCACCCAGG CAAAGCCCCCAAACTCATGATTTATGAGGTCAGTAATCGGCCCTC AGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTG GCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACG AGGCTGATTATTACTGCAGCTCATATACAAGCAGC GGCACTGTGGTATTCGGCGGAGGGACCGAGCTGACCGTCCTAGCG GCCGCA SEQ ID NO:73 Antibody Name V2L2 — VH GAGATGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGG GGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCA GCTATGCCATGAACTGGGTCCGCCAGGCTCCAGGGGAGGGGCTGG AGTGGGTCTCAGCTATTACTATTAGTGGTATTACCGCATACTACAC CGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAA GAACACGCTATATCTGCAAATGAACAGCCTGAGGGCCGGGGACAC GGCCGTATATTACTGTGCGAAGGAAGAATTTTTACCTGGAACGCA CTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCAC CGTCTCCTCA SEQ ID NO: 238 d Name V2L2 — VL GCCATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAG GAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGAA TAGGCTGGTATCAACAGAAGCCAGGGAAAGCCCCTAAAC TCGTGATCTATTCTGCATCCACTTTACAAAGTGGGGTCCCATCAAG GTTCAGCGGCAGTGGATCTGGCACAGATTTCACTCTCTCCATCAGC AGCCTGCAGCCTGACGATTTTGCAACTTATTACTGTCTACAAGATT ACAATTACCCGTGGACGTTCGGCCAAGGGACCAAGGTTGAAATCA SEQ ID NO: 239 In some embodiments, an isolated antibody or antigen-binding fragment f encoded by one or more of the polynucleotides bed above, which specifically binds to Pseudomonas Ps1 and/or Pch, comprises, consists essentially of, or consists of VH and VL amino acid ces at least 80%, 85%, 90%, 95% or 100% identical to: (a) SEQ ID NO: 1 and SEQ ID NO: 2, respectively, (b) SEQ ID NO: 3 and SEQ ID NO: 2, respectively, (c) SEQ ID NO: 4 and SEQ ID NO: 2 , respectively, (d) SEQ ID NO: 5 and SEQ ID NO: 6 ID NO: 7 and SEQ ID , respectively, (e) SEQ NO: 8, respectively, (f) SEQ ID NO: 9 and SEQ ID NO: 10, respectively, (g) SEQ ID NO: 11 and SEQ ID NO: 12 ID NO: 13 and SEQ ID , respectively, (h) SEQ NO: 14, respectively; (i) SEQ ID NO: 15 and SEQ ID NO: 16, respectively; or (j) SEQ ID NO: 74 and SEQ ID NO: 12 , respectively.

In certain embodiments, an isolated binding molecule, e. g., an antibody or antigen—binding fragment f encoded by one or more of the polynucleotides described above, specifically binds to Pseudomonas Ps1 and/or Pch with an ty characterized by a dissociation constant (KD) no greater than 5 x 10'2 M, 10'2 M, 5 x 10'3 M, 10‘3 M, 5 x104 M, 10‘4 M, 5 x10'5 M, 10‘5 M, 5 x106 M, 10‘6 M, 5 x10'7 M, 10‘7 M, x 10‘8 M, 10‘8 M, 5 x109 M, 10‘9 M, 5 x1010 M, 10‘10 M, 5 x10'11M, 10'11M, 5 x10- M, 10'12 M, 5 x 10'13 M, 10'13 M, 5 x 10'14 M, 10'14 M, 5 x 10'15 M, or 10'15 M.

In specific embodiments, an isolated binding molecule, e. g., an antibody or antigen—binding fragment thereof encoded by one or more of the polynucleotides described above, specifically binds to Pseudomonas Ps1 and/or Pch, with an affinity characterized by a dissociation constant (KD) in a range of about 1 x 10'10 to about 1 x 10' M. In one embodiment, an isolated binding molecule, e.g., an antibody or antigen- binding fragment thereof encoded by one or more of the polynucleotides described above, specifically binds to Pseudomonas Psl and/or Pch, with an affinity characterized by a KD of about 1.18 x 10'7 M, as determined by the OCTET® binding assay described herein. In another embodiment, an isolated binding molecule, e. g., an antibody or antigen-binding fragment thereof encoded by one or more of the polynucleotides described above, specifically binds to Pseudomonas Psl and/or Pch, with an affinity characterized by a KD of about 1.44 x 10'7 M, as determined by the OCTET® binding assay described herein.

In certain ments, an anti—Pseudomonas Psl and/or Pch g molecule, e. g., antibody or fragment, t or derivative thereof encoded by one or more of the polynucleotides described above, specifically binds to the same Ps1 epitope as monoclonal dy WapR—004, WapR—004RAD, Cam—003, Cam—004, or Cam—005, or will competitively inhibit such a monoclonal antibody from binding to Pseudomonas Psl; and/or specifically binds to the same Pch e as monoclonal antibody V2L2, or will competitively inhibit such a monoclonal antibody from binding to Pseudomonas Pch.

WapR—004RAD is identical to 04 except for a nucleic acid substitution G293C of the VH nucleic acid sequence encoding the VH amino acid sequence of SEQ ID NO:11 (a tution of the nucleotide in the VH-encoding portion of SEQ ID NO:71 at position 317). The nucleic acid sequence encoding the WapR—004RAD VH is presented as SEQ ID NO 76.

Some embodiments provide an isolated cleotide sing, consisting essentially of, or consisting of a nucleic acid encoding a W4 mutant scFv—Fc molecule amino acid sequence identical to, or identical except for one, two, three, four, five, or more amino acid substitutions to one or more of: SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145; or SEQ ID NO: 146.

Other embodiments provide an isolated cleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding a W4 mutant scFV—Fc molecule amino acid ce at least 80%, 85%, 90% 95% or 100% identical to one or more of: SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145; or SEQ ID .

One embodiment provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid which encodes a W4 mutant scFV—Fc molecule, Where the nucleic acid is at least 80%, 85%, 90% 95% or 100% identical to one or more of SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, or SEQ ID NO: 152, SEQ IS NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214; or SEQ ID NO: 215.

One embodiment provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid which encodes a V2L2 polypeptide, where the c acid is at least 80%, 85%, 90% 95% or 100% cal to one or more of SEQ ID NO: 238 or SEQ ID NO: 239.

In other embodiments, an anti—Pseudomonas Psl and/or Pch binding molecule, e. g., antibody or fragment, variant or derivative f encoded by one or more of the polynucleotides described above, specifically binds to the same epitope as monoclonal antibody WapR—001, WapR—002, or WapR—003, or will competitively inhibit such a monoclonal antibody from binding to Pseudomonas Psl.

In certain embodiments, an anti—Pseudomonas Psl and/or Pch binding molecule, e. g., antibody or fragment, variant or derivative f encoded by one or more of the polynucleotides described above, specifically binds to the same epitope as onal antibody WapR-016, or will competitively inhibit such a monoclonal antibody from binding to Pseudomonas Psl.

The disclosure also includes fragments of the polynucleotides as described elsewhere herein. Additionally polynucleotides which encode fusion polynucleotides, Fab fragments, and other tives, as described herein, are also provided.

The polynucleotides can be produced or ctured by any method known in the art. For example, if the nucleotide sequence of the antibody is known, a polynucleotide encoding the antibody can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly, es the synthesis of pping ucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR. atively, a polynucleotide encoding an anti—Pseudomonas Psl and/or Pch binding molecule, e. g., antibody or fragment, variant or tive f can be generated from nucleic acid from a suitable source. If a clone containing a c acid encoding a particular antibody is not available, but the sequence of the antibody le is known, a nucleic acid encoding the antibody can be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+RNA, isolated from, any tissue or cells expressing the antibody or such as hybridoma cells selected to express an antibody) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e. g., a cDNA clone from a cDNA library that encodes the antibody.

Amplified nucleic acids generated by PCR can then be cloned into replicable cloning vectors using any method well known in the art.

Once the tide sequence and corresponding amino acid sequence of an anti— monas Psl and/or Pch binding molecule, e.g., antibody or fragment, variant or derivative thereof is determined, its nucleotide ce can be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for e, the techniques described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. (1990) and Ausubel et al., eds., Current Protocols in lar Biology, John Wiley & Sons, NY , which are both incorporated by reference herein in their entireties ), to generate antibodies having a different amino acid ce, for example to create amino acid substitutions, deletions, and/or insertions.

A polynucleotide encoding an anti—Pseudomonas Psl and/or Pch binding molecule, e. g., antibody or fragment, variant or derivative thereof can be composed of any polyribonucleotide or polydeoxribonucleotide, which can be unmodifled RNA or DNA or modified RNA or DNA. For example, a polynucleotide encoding an anti- Pseudomonas Psl and/or Pch binding molecule, e.g., dy or fragment, variant or derivative thereof can be composed of — and double-stranded DNA, DNA that is a mixture of single— and double—stranded regions, single— and double—stranded RNA, and RNA that is mixture of — and double—stranded regions, hybrid molecules comprising DNA and RNA that can be single-stranded or, more typically, double—stranded or a mixture of single- and double-stranded s. In addition, a polynucleotide encoding an anti—Pseudomonas Psl and/or Pch binding molecule, e. g., antibody or fragment, variant or derivative thereof can be ed of triple—stranded regions comprising RNA or DNA or both RNA and DNA. A polynucleotide encoding an anti—Pseudomonas Psl and/or Pch binding molecule, e. g., antibody or fragment, variant or derivative thereof can also n one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. "Modified" bases e, for e, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically, or metabolically modifled forms.

An isolated polynucleotide encoding a non-natural variant of a ptide derived from an immunoglobulin (e. g., an immunoglobulin heavy chain portion or light chain portion) can be created by introducing one or more nucleotide substitutions, additions or deletions into the tide sequence of the immunoglobulin such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. ons can be introduced by standard techniques, such as site—directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid tutions are made at one or more non-essential amino acid es.

VI. EXPRESSION OF ANTIBODY POLYPEPTIDES As is well known, RNA can be isolated from the original hybridoma cells or from other transformed cells by standard techniques, such as guanidinium isothiocyanate extraction and precipitation ed by centrifugation or chromatography. Where desirable, mRNA can be isolated from total RNA by standard techniques such as chromatography on oligo dT cellulose. le techniques are familiar in the art.

In one embodiment, cDNAs that encode the light and the heavy chains of the anti- Pseudomonas Psl and/or Pch binding molecule, e.g., antibody or nt, variant or derivative thereof can be made, either aneously or separately, using reverse transcriptase and DNA polymerase in accordance with well-known methods. PCR can be initiated by consensus constant region primers or by more specific primers based on the published heavy and light chain DNA and amino acid sequences. As discussed above, PCR also can be used to isolate DNA clones ng the antibody light and heavy chains. In this case the libraries can be screened by consensus primers or larger homologous probes, such as mouse constant region probes.

DNA, typically plasmid DNA, can be isolated from the cells using techniques known in the art, restriction mapped and sequenced in accordance with standard, well known techniques set forth in detail, e.g., in the foregoing references relating to recombinant DNA techniques. Of course, the DNA can be synthetic according to the present disclosure at any point during the isolation process or uent analysis.

Following manipulation of the isolated genetic al to provide an anti- Pseudomonas Psl and/or Pch binding molecule, e.g., antibody or fragment, variant or derivative thereof of the disclosure, the cleotides encoding anti—Pseudomonas Psl and/or Pch binding molecules, are typically inserted in an expression vector for introduction into host cells that can be used to produce the desired ty of anti- Pseudomonas Psl and/or Pch binding molecules.

Recombinant expression of an antibody, or fragment, derivative or analog f, e.g., a heavy or light chain of an antibody which binds to a target molecule described herein, e. g., Psl and/or Pch, requires construction of an expression vector containing a polynucleotide that encodes the antibody. Once a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or n thereof (containing the heavy or light chain variable domain), of the disclosure has been obtained, the vector for the tion of the antibody molecule can be produced by recombinant DNA logy using techniques well known in the art. Thus, methods for preparing a protein by sing a polynucleotide containing an antibody encoding nucleotide sequence are bed herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing dy coding sequences and appropriate transcriptional and translational control signals. These s include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in viva genetic recombination. The sure, thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the disclosure, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter. Such vectors can include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT ation WO 86/05807; PCT ation WO 89/01036; and US. Pat. No. 5,122,464) and the variable domain of the antibody can be cloned into such a vector for expression of the entire heavy or light chain.

The term “vector” or ssion ” is used herein to mean vectors used in accordance with the present disclosure as a vehicle for introducing into and expressing a desired gene in a host cell. As known to those skilled in the art, such s can easily be selected from the group ting of plasmids, phages, viruses and retroviruses. In general, vectors compatible with the instant disclosure will comprise a ion marker, appropriate restriction sites to facilitate cloning of the desired gene and the ability to enter and/or replicate in eukaryotic or prokaryotic cells.

For the purposes of this disclosure, numerous expression vector systems can be employed. For example, one class of vector utilizes DNA elements which are derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV or MOMLV) or SV40 virus. Others involve the use of polycistronic systems with internal ribosome binding sites. onally, cells which have ated the DNA into their chromosomes can be selected by introducing one or more markers which allow selection of ected host cells. The marker can provide for prototrophy to an auxotrophic host, biocide resistance (e.g., antibiotics) or resistance to heavy metals such as copper. The selectable marker gene can either be ly linked to the DNA ces to be expressed, or introduced into the same cell by cotransformation. Additional elements can also be needed for optimal synthesis of mRNA. These elements can include signal sequences, splice s, as well as transcriptional promoters, enhancers, and termination signals.

In some embodiments the cloned variable region genes are inserted into an expression vector along with the heavy and light chain constant region genes (e.g., human) synthetic as discussed above. Of course, any expression vector which is capable of eliciting expression in eukaryotic cells can be used in the present disclosure. Examples of le vectors include, but are not limited to plasmids pcDNA3, pHCMV/Zeo, pCR3.l, pEFl/His, pIND/GS, pRc/HCMV2, pSV40/Ze02, pTRACER-HCMV, pUB6/V5-His, pVAXl, and pZeoSV2 (available from Invitrogen, San Diego, CA), and plasmid pCI (available from Promega, Madison, WI). In l, screening large numbers of transformed cells for those which s suitably high levels if immunoglobulin heavy and light chains is routine experimentation which can be carried out, for example, by robotic systems.

More generally, once the vector or DNA sequence encoding a monomeric subunit of the anti—Pseudomonas Psl and/or Pch binding molecule, e.g., antibody or fragment, variant or derivative thereof of the disclosure has been prepared, the sion vector can be uced into an appropriate host cell. Introduction of the d into the host cell can be accomplished by various techniques well known to those of skill in the art.

These include, but are not limited to, transfection ding electrophoresis and electroporation), last fusion, calcium phosphate precipitation, cell fusion with enveloped DNA, microinjection, and ion with intact Virus. See, Ridgway, A. A. G.

"Mammalian Expression Vectors" Vectors, uez and Denhardt, Eds., Butterworths, Boston, Mass., Chapter 24.2, pp. 470-472 (1988). Typically, plasmid introduction into the host is Via electroporation. The host cells ing the expression construct are grown under conditions appropriate to the tion of the light chains and heavy chains, and assayed for heavy and/or light chain protein synthesis. Exemplary assay ques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or fluorescence—activated cell sorter analysis (FACS), immunohistochemistry and the like.

The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by tional techniques to produce an antibody for use in the methods described herein. Thus, the disclosure includes host cells containing a polynucleotide encoding an anti—Pseudomonas Psl and/or Pch binding molecule, e. g., antibody or fragment, variant or derivative f, or a heavy or light chain thereof, operably linked to a heterologous promoter. In some embodiments for the expression of double—chained antibodies, vectors encoding both the heavy and light chains can be co—expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.

Certain embodiments include an ed polynucleotide sing a nucleic acid which encodes the above—described VH and VL, wherein a binding molecule or antigen- g fragment thereof expressed by the polynucleotide specifically binds Pseudomonas Ps1 and/or Pch. In some embodiments the polynucleotide as described encodes an scFV molecule including VH and VL, at least 80%, 85%, 90% 95% or 100% —100— identical to one or more of SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, or SEQ ID NO: 70 as shown in Table 4.

Some embodiments include vectors sing the above—described polynucleotides. In further embodiments, the polynucleotides are operably associated with a er. In additional embodiments, the disclosure provides host cells sing such vectors. In further embodiments, the disclosure provides vectors where the polynucleotide is operably associated with a promoter, wherein vectors can s a binding molecule which specifically binds Pseudomonas Psl and/or Pch in a suitable host cell.

Also provided is a method of producing a binding molecule or fragment thereof which specifically binds Pseudomonas Psl and/or Pch, comprising culturing a host cell containing a vector comprising the above—described polynucleotides, and ring said antibody, or fragment thereof. In further embodiments, the disclosure provides an isolated binding molecule or fragment thereof produced by the above—described method.

As used , “host cells” refers to cells which harbor vectors constructed using recombinant DNA techniques and encoding at least one heterologous gene. In descriptions of processes for isolation of antibodies from recombinant hosts, the terms "cell" and "cell culture" are used interchangeably to denote the source of antibody unless it is y specified otherwise. In other words, recovery of polypeptide from the "cells" can mean either from spun down whole cells, or from the cell culture containing both the medium and the suspended cells.

A y of xpression vector systems can be utilized to express antibody molecules for use in the methods described herein. Such host—expression systems represent vehicles by which the coding ces of interest can be produced and subsequently purified, but also ent cells which can, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the disclosure in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. is) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems ed with recombinant virus sion vectors (e.g., baculovirus) containing antibody coding sequences; plant —101— cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression s (e. g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e. g., COS, CHO, BLK, 293, 3T3 cells) harboring recombinant sion constructs containing ers derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). Bacterial cells such as Escherichia coli, or eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule. For example, mammalian cells such as e hamster ovary cells (CHO), in conjunction with a vector such as the major ediate early gene promoter element from human cytomegalovirus is an effective sion system for antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2 (1990)).

The host cell line used for n expression is often of mammalian ; those skilled in the art are credited with ability to determine particular host cell lines which are best suited for the desired gene product to be expressed therein. Exemplary host cell lines include, but are not limited to, CH0 (Chinese Hamster Ovary), DG44 and DUXBll (Chinese r Ovary lines, DHFR minus), HELA (human cervical carcinoma), CV1 (monkey kidney line), COS (a derivative of CV1 with SV40 T antigen), VERY, BHK (baby hamster kidney), MDCK, 293, W13 8, R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast), HAK (hamster kidney line), SP2/O (mouse myeloma), P3x63—Ag3.653 (mouse myeloma), BFA—lclBPT (bovine endothelial cells), RAJI (human cyte) and 293 (human kidney). Host cell lines are typically available from commercial services, the American Tissue Culture Collection or from published literature.

In on, a host cell strain can be chosen which modulates the sion of the inserted sequences, or modif1es and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e. g., cleavage) of protein products can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the t modification and processing of the foreign n expressed. —102— To this end, otic host cells which possess the ar machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the dy molecule can be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e. g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells can be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid s resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method can advantageously be used to engineer cell lines which stably express the antibody molecule.

A number of selection systems can be used, ing but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell [1:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase lska & ski, Proc. Natl. Acad. Sci. USA 48202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 1980) genes can be employed in tk-, hgprt- or aprt-cells, tively. Also, tabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 782072 (1981)); neo, which confers resistance to the aminoglycoside G—418 Clinical Pharmacy 12:488—505; Wu and Wu, Biotherapy 3:87—95 (1991); Tolstoshev, Ann. Rev. Pharmacol. l. 32:573—596 (1993); Mulligan, Science 260:926—932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191—217 (1993);, T13 TECH II(5):155-215 (May, 1993); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:147 (1984).

Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. , Current ols in Molecular Biology, John Wiley & Sons, NY ; Kriegler, Gene Transfer and Expression, A Laboratory —103— Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which are incorporated by nce herein in their entireties.

The expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Academic Press, New York, Vol. 3. (1987)). When a marker in the vector system expressing antibody is amplif1able, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).

In vitro production allows scale—up to give large amounts of the d polypeptides. Techniques for ian cell cultivation under tissue culture ions are known in the art and include homogeneous suspension culture, e.g. in an airlift reactor or in a continuous stirrer reactor, or immobilized or entrapped cell culture, e. g. in hollow fibers, microcapsules, on e microbeads or ceramic cartridges. If necessary and/or desired, the solutions of polypeptides can be d by the customary chromatography s, for example gel ion, ion-exchange chromatography, chromatography over DEAE-cellulose or (immuno-)aff1nity chromatography, e. g., after ential thesis of a synthetic hinge region polypeptide or prior to or subsequent to the HIC chromatography step described herein.

Constructs encoding anti—Pseudomonas Psl and/or Pch binding molecules, e. g., antibodies or fragments, variants or derivatives f, as disclosed herein can also be expressed non-mammalian cells such as bacteria or yeast or plant cells. ia which y take up nucleic acids include members of the enterobacteriaceae, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus, and Haemophilus influenzae. It will further be appreciated that, when expressed in ia, the heterologous polypeptides lly become part of inclusion bodies. The heterologouspolypeptides must be isolated, purified and then assembled into functional molecules. Where tetravalent forms of antibodies are desired, the subunits will then self-assemble into tetravalent antibodies (W002/096948A2). —104— In ial systems, a number of expression vectors can be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified can be desirable. Such s include, but are not d, to the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791 (1983)), in which the antibody coding sequence can be ligated individually into the vector in frame with the lacZ coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13 :3101— 3109 (1985); Van Heeke & er, J. Biol. Chem. 24:5503—5509 (1989)); and the like. pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione- agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

In addition to prokaryotes, eukaryotic microbes can also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among eukaryotic microorganisms although a number of other strains are commonly available, e.g., Pichia pastoris.

For expression in Saccharomyces, the plasmid YRp7, for e, (Stinchcomb et al., Nature 282:39 (1979); Kingsman et al., Gene 7:141 (1979); Tschemper et al., Gene :157 ) is commonly used. This d already contains the TRPl gene which es a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for e ATCC No. 44076 or PEP4—1 (Jones, Genetics 85:12 (1977)). The presence of the trpl lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.

In an insect system, apha californica r polyhedrosis virus (AcNPV) is lly used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence can be cloned individually into non— —105— essential regions (for e the drin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).

Once the anti—Pseudomonas Ps1 and/or Pch binding molecule, e.g., antibody or fragment, variant or derivative thereof, as disclosed herein has been recombinantly expressed, it can be purified by any method known in the art for cation of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, ularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Another method for increasing the affinity of antibodies of the disclosure is disclosed in US 2002 0123057 Al.

VII. IDENTIFICATION OF SEROTYPE—INDIFFERENT BINDING MOLECULES The disclosure encompasses a target erent whole—cell approach to identify pe independent therapeutic binding molecules e. g., antibodies or fragments thereof with superior or desired therapeutic activities. The method can be utilized to identify binding les which can antagonize, neutralize, clear, or block an undesired activity of an infectious agent, e. g., a bacterial pathogen. As is known in the art, many ious agents exhibit significant variation in their dominant surface antigens, allowing them to evade immune surveillance. The identification method described herein can identify binding molecules which target antigens which are shared among many different Pseudomonas species or other Gram—negative pathogens, thus providing a therapeutic agent which can target le pathogens from multiple s. For example, the method was utilized to identify a series of binding molecules which bind to the surface of P. aeruginosa in a serotype-independent manner, and when bound to bacterial pathogens, mediate, promote, or enhance opsonophagocytic (OPK) activity against bacterial cells such as bacterial pathogens, 6.g. opportunistic Pseudomonas species (e.g., Pseudomonas aeruginosa, Pseudomonas fluorescens, monas putida, and Pseudomonas alcaligenes) and/or inhibit the attachment of such bacterial cells to epithelial cells.

Certain embodiments disclose a method of identifying serotype-indifferent binding molecules comprising: (a) ing na'ive and/or convalescent antibody libraries in phage, (b) ng serotype-specific antibodies from the library by depletion panning, (c) screening the library for dies that specifically bind to whole cells —lO6— independent of serotype, and (d) ing of the resulting antibodies for desired functional properties.

Certain embodiments provide a whole—cell phenotypic screening approach as disclosed herein with antibody phage libraries derived from either naive or P. aeruginosa infected convalescing patients. Using a panning strategy that initially selected against serotype-specific reactivity, different clones that bound P. aeruginosa whole cells were isolated. Selected clones were converted to human IgG1 antibodies and were confirmed to react with P. aeruginosa clinical isolates regardless of serotype classification or site of tissue isolation (See Examples). Functional activity screens bed herein indicated that the antibodies were effective in preventing P. aeruginosa attachment to mammalian cells and mediated opsonophagocytic (OPK) killing in a concentration-dependent and pe—independent manner.

In further embodiments, the above—described binding molecules or fragments f, antibodies or fragments f, or compositions, bind to two or more, three or more, four or more, or five or more different P. nosa serotypes, or to at least 80%, at least 85%, at least 90% or at least 95% of P. aeruginosa strains isolated from infected patients. In further embodiments, the P. aeruginosa strains are isolated from one or more of lung, sputum, eye, pus, feces, urine, sinus, a wound, skin, blood, bone, or knee fluid.

VIII. PHARMACEUTICAL COMPOSITIONS COMPRISING ANTI— PSEUDOMONAS PSL AND/OR PCRV BINDING MOLECULES The pharmaceutical compositions used in this disclosure comprise pharmaceutically acceptable carriers well known to those of ordinary skill in the art.

Preparations for parenteral administration include sterile aqueous or non—aqueous ons, suspensions, and emulsions. Certain ceutical compositions as disclosed herein can be orally administered in an acceptable dosage form including, e.g., capsules, tablets, aqueous sions or solutions. Certain pharmaceutical compositions also can be administered by nasal aerosol or inhalation. Preservatives and other additives can also be present such as for example, crobials, antioxidants, chelating , and inert gases and the like. Suitable formulations for use in the therapeutic s sed herein are described in Remington's Pharmaceutical Sciences, Mack Publishing Co., 16th ed.(1980) —lO7— The amount of an anti—Pseudomonas Psl and/or Pch binding molecule, e.g., dy or fragment, variant or tive thereof, that can be combined with the carrier materials to e a single dosage form will vary depending upon the host treated and the particular mode of administration. Dosage regimens also can be ed to provide the optimum d response (e.g., a therapeutic or prophylactic response). The compositions can also comprise the anti—Pseudomonas Psl and/or Pch binding molecules, e.g., antibodies or fragments, variants or derivatives thereof dispersed in a biocompatible carrier material that functions as a suitable delivery or support system for the compounds.

IX. TREATMENT METHODS USING THERAPEUTIC BINDING MOLECULES Methods of preparing and administering anti-Pseudomonas Psl and/or Pch binding molecules, e. g., an antibody or fragment, variant or derivative thereof, as disclosed herein to a subject in need thereof are well known to or are readily determined by those skilled in the art. The route of administration of the anti-Pseudomonas Psl and/or Pch binding les, e. g., antibody or fragment, t or derivative thereof, can be, for example, oral, parenteral, by inhalation or topical. The term eral as used herein includes, e.g., intravenous, intraarterial, intraperitoneal, intramuscular, or subcutaneous administration. A suitable form for administration would be a solution for injection, in particular for intravenous or intraarterial injection or drip. However, in other methods compatible with the teachings herein, an anti-Pseudomonas Psl and/or Pch binding molecules, e. g., antibody or fragment, t or derivative thereof, as disclosed herein can be delivered directly to the site of the adverse cellular population e.g., infection thereby increasing the exposure of the ed tissue to the therapeutic agent.

For example, an anti—Pseudomonas Psl and/or Pch g molecule can be directly administered to ocular tissue, burn injury, or lung tissue.

Anti—Pseudomonas Psl and/or Pch g molecules, e.g., antibodies or nts, variants or derivatives thereof, as disclosed herein can be administered in a pharmaceutically ive amount for the in viva treatment domonas infection. In this regard, it will be appreciated that the disclosed binding molecules will be formulated so as to facilitate administration and promote stability of the active agent. For the purposes of the instant application, a ceutically effective amount shall be held to —108— mean an amount sufficient to achieve effective binding to a target and to achieve a benefit, e. g., treat, ameliorate, lessen, clear, or prevent Pseudomonas infection.

Some embodiments are directed to a method of preventing or treating a Pseudomonas ion in a subject in need thereof, comprising stering to the subject an effective amount of the binding molecule or fragment thereof, the antibody or fragment thereof, the ition, the polynucleotide, the vector, or the host cell described herein. In further embodiments, the Pseudomonas ion is a P. aeruginosa infection. In some embodiments, the subject is a human. In certain embodiments, the infection is an ocular infection, a lung infection, a burn infection, a wound infection, a skin infection, a blood infection, a bone infection, or a combination of two or more of said infections. In further embodiments, the t suffers from acute nia, burn injury, corneal infection, cystic fibrosis, or a ation thereof Certain embodiments are ed to a method of blocking or preventing ment of P. aeruginosa to epithelial cells sing contacting a mixture of epithelial cells and P. aeruginosa with the binding molecule or fragment thereof, the antibody or fragment thereof, the composition, the polynucleotide, the vector, or the host cell bed herein.

Also disclosed is a method of enhancing OPK of P. aeruginosa comprising contacting a mixture of phagocytic cells and P. aeruginosa with the binding molecule or fragment thereof, the antibody or fragment thereof, the composition, the polynucleotide, the vector, or the host cell bed herein. In further embodiments, the phagocytic cells are differentiated HL—60 cells or human polymorphonuclear leukocytes (PMNs).

In keeping with the scope of the disclosure, anti—Pseudomonas Psl and/or Pch binding molecules, e. g., antibodies or fragments, variants or derivatives thereof, can be administered to a human or other animal in accordance with the aforementioned methods of treatment in an amount ient to produce a therapeutic effect. The anti- monas Psl and/or Pch binding molecules, e.g., dies or fragments, variants or derivatives thereof, disclosed herein can be administered to such human or other animal in a conventional dosage form prepared by combining the antibody of the disclosure with a conventional pharmaceutically able carrier or diluent according to known techniques. 2012/063722 —lO9— Effective doses of the compositions of the present disclosure, for treatment of Pseudomonas infection vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. y, the patient is a human but non-human mammals including transgenic mammals can also be treated. Treatment dosages can be titrated using routine methods known to those of skill in the art to optimize safety and efficacy.

Anti—Pseudomonas Psl and/or Pch binding molecules, e.g., antibodies or fragments, variants or tives thereof can be administered multiple occasions at various frequencies depending on various factors known to those of skill in the art..

Alternatively, anti—Pseudomonas Psl and/or Pch binding molecules, e.g., antibodies or fragments, variants or derivatives thereof can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and ncy vary depending on the half-life of the antibody in the patient.

The compositions of the disclosure can be stered by any suitable method, e. g., parenterally, intraventricularly, orally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term “parenteral” as used herein includes subcutaneous, intravenous, intramuscular, articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.

X. SYNERGY Chou and Talalay (Adv. Enzyme Regal, 22:27—55 ) developed a mathematical method to describe the experimental gs of combined drug effects in a qualitative and quantitative manner. For ly ive drugs, they showed that the generalized isobol on applies for any degree of effect (see page 52 in Chou and y). An isobol or isobologram is the c representation of all dose combinations of two drugs that have the same degree of effect. In isobolograms, a straight line indicates additive effects, a e curve (curve below the straight line) represents synergistic effects, and a convex curve (curve above the straight line) represents antagonistic effects. These curves also show that a combination of two mutually exclusive drugs will show the same type of effect over the whole concentration range, —110— either the combination is additive, synergistic, or antagonistic. Most drug combinations show an additive effect. In some instances r, the combinations show less or more than an additive effect. These combinations are called antagonistic or synergistic, respectively. A combination manifests therapeutic synergy if it is therapeutically superior to one or other of the constituents used at its m dose. See, T. H. Corbett et al., Cancer Treatment Reports, 66, 1187 . Tallarida R] (J Pharmacol Exp Ther. 2001 Sep; 298 (3):865—72) also notes "Two drugs that produce overtly similar effects will sometimes produce exaggerated or shed effects when used concurrently. A quantitative ment is necessary to distinguish these cases from simply additive action." A synergistic effect can be measured using the combination index (CI) method of Chou and Talalay (see Chang et al., Cancer Res. 45: 2434—2439, (1985)) which is based on the median-effect principle. This method calculates the degree of synergy, additivity, or antagonism between two drugs at various levels of cytotoxicity. Where the CI value is less than 1, there is synergy n the two drugs. Where the CI value is 1, there is an additive effect, but no synergistic . CI values greater than 1 indicate antagonism.

The smaller the CI value, the greater the synergistic effect. In another embodiment, a synergistic effect is ined by using the fractional inhibitory concentration (FIC).

This fractional value is determined by expressing the IC50 of a drug acting in combination, as a function of the IC50 of the drug acting alone. For two interacting drugs, the sum of the PIC value for each drug represents the measure of synergistic ction.

Where the PIC is less than 1, there is synergy between the two drugs. An FIC value of 1 indicates an additive effect. The smaller the PIC value, the greater the synergistic interaction.

In some embodiments, a synergistic effect is obtained in Pseudomonas treatment wherein one or more of the g agents are administered in a "low dose" (i.e., using a dose or doses which would be considered non—therapeutic if administered alone), wherein the administration of the low dose binding agent in combination with other g agents (administered at either a low or eutic dose) results in a synergistic effect which exceeds the additive effects that would ise result from individual administration of the binding agent alone. In some embodiments, the synergistic effect is achieved via —lll— administration of one or more of the binding agents administered in a "low dose" wherein the low dose is provided to reduce or avoid toxicity or other undesirable side effects.

XI. IMMUNOASSAYS Anti—Pseudomonas Psl and/or Pch binding molecules, e.g., antibodies or fragments, variants or tives thereof can be assayed for immunospecif1c binding by any method known in the art. The assays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation , precipitin reactions, gel diffusion precipitin reactions, immunodiffusion , agglutination , complement-fixation assays, immunoradiometric assays, cent immunoassays, protein A immunoassays, to name but a few. Such assays are e and well known in the art (see, e.g., Ausubel et al., eds, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, Vol. 1 (1994), which is incorporated by reference herein in its entirety). Exemplary immunoassays are described briefly below (but are not intended by way of limitation).

There are a variety of methods available for measuring the affinity of an antibody- n interaction, but relatively few for determining rate constants. Most of the methods rely on either labeling antibody or antigen, which inevitably complicates routine measurements and introduces uncertainties in the measured ties. Antibody affinity can be measured by a number of methods, including OCTET®, BIACORE®, ELISA, and FACS.

The OCTET® system uses biosensors in a 96-well plate format to report kinetic analysis. Protein binding and iation events can be monitored by ing the binding of one protein in solution to a second protein immobilized on the ForteBio biosensor. In the case of measuring binding of anti-Psl or Pch antibodies to Psl or Pch, the Psl or Pch is immobilized onto OCTET® tips followed by analysis of binding of the antibody, which is in solution. Association and disassociation of antibody to immobilized Psl or Pch is then detected by the instrument sensor. The data is then collected and exported to GraphPad Prism for affinity curve fitting. —112— Surface plasmon nce (SPR) as performed on BIACORE® offers a number of advantages over conventional methods of measuring the affinity of antibody-antigen interactions: (i) no requirement to label either antibody or antigen; (ii) antibodies do not need to be purified in advance, cell culture supernatant can be used directly; (iii) real-time measurements, allowing rapid semi-quantitative comparison of different monoclonal antibody interactions, are enabled and are sufficient for many evaluation purposes; (iv) ciflc e can be regenerated so that a series of different monoclonal antibodies can easily be compared under identical conditions; (v) analytical procedures are fully automated, and extensive series of measurements can be performed without user intervention. BIAapplications Handbook, n AB (reprinted 1998), E® code No. BR—lOOl—86; hnology Handbook, version AB (reprinted 1998), BIACORE® code No. BR—1001—84.

SPR based binding studies require that one member of a binding pair be immobilized on a sensor surface. The g r immobilized is referred to as the ligand. The binding partner in solution is referred to as the analyte. In some cases, the ligand is attached indirectly to the surface h binding to r immobilized molecule, which is referred as the capturing molecule. SPR response reflects a change in mass concentration at the or surface as analytes bind or dissociate.

Based on SPR, real-time BIACORE® measurements monitor interactions directly as they . The technique is well suited to determination of kinetic parameters.

Comparative affinity ranking is extremely simple to perform, and both kinetic and affinity constants can be derived from the sensorgram data.

When analyte is ed in a discrete pulse across a ligand surface, the resulting gram can be divided into three essential phases: (i) Association of analyte with ligand during sample injection; (ii) Equilibrium or steady state during sample injection, where the rate of analyte binding is balanced by dissociation from the complex; (iii) iation of analyte from the surface during buffer flow.

The association and dissociation phases provide information on the kinetics of analyte-ligand interaction (ka and kd, the rates of complex formation and dissociation, kd/ka = KD). The equilibrium phase provides information on the affinity of the analyte- ligand interaction (KD). —ll3— BIAevaluation software provides comprehensive facilities for curve fitting using both numerical integration and global fitting algorithms. With suitable analysis of the data, separate rate and affinity nts for interaction can be obtained from simple BIACORE® investigations. The range of affinities measurable by this technique is very broad ranging from mM to pM.

Epitope specificity is an important characteristic of a monoclonal antibody.

Epitope mapping with E®, in contrast to conventional ques using radioimmunoassay, ELISA or other surface adsorption s, does not require labeling or purified antibodies, and allows site specificity tests using a sequence of several monoclonal antibodies. Additionally, large numbers of es can be processed automatically.

Pair-wise binding experiments test the ability of two MAbs to bind simultaneously to the same n. MAbs directed against separate epitopes will bind independently, whereas MAbs ed against identical or closely related epitopes will interfere with each s binding. These binding ments with BIACORE® are straightforward to carry out.

For example, one can use a capture molecule to bind the first Mab, followed by addition of antigen and second MAb sequentially. The sensorgrams will reveal: 1. how much of the antigen binds to first Mab, 2. to what extent the second MAb binds to the surface—attached n, 3. if the second MAb does not bind, whether reversing the order of the ise test alters the results.

Peptide inhibition is another technique used for epitope mapping. This method can complement pair-wise antibody binding studies, and can relate functional es to structural features when the primary sequence of the antigen is known. Peptides or antigen fragments are tested for inhibition of binding of different MAbs to immobilized antigen. Peptides which interfere with binding of a given MAb are assumed to be structurally related to the epitope defined by that MAb.

XII. ADMINISTRATION A composition comprising either an anti-Psl binding domain or anti-Pch binding domain, or a composition comprising both an anti -Psl and anti-Pch binding domain are administered in such a way that they provide a synergistic effect in the treatment of —114— Pseudomonas in a patient. stration can be by any suitable means provided that the administration provides the desired therapeutic effect, i.e., synergism. In certain ments, the antibodies are administered during the same cycle of therapy, e.g., during one cycle of therapy during a prescribed time period, both of the antibodies are administered to the subject. In some embodiments, administration of the antibodies can be during sequential administration in separate therapy cycles, e. g., the first therapy cycle ing administration of an anti-Psl antibody and the second therapy cycle involving administration of an anti-Pch antibody. The dosage of the binding domains stered to a patient will also depend on frequency of administration and can be readily determined by one of ry skill in the art.

In other embodiments the binding domains are administered more than once during a treatment cycle. For example, in some embodiments, the binding domains are stered weekly for three consecutive weeks in a three or four week treatment cycle.

Administration of the ition comprising one or more of the binding domains can be on the same or different days provided that stration es the desired therapeutic effect.

It will be readily apparent to those skilled in the art that other doses or frequencies of administration that provide the desired therapeutic effect are suitable for use in the present ion.

XII. KITS In yet other embodiments, the present invention provides kits that can be used to perform the methods described herein. In certain embodiments, a kit comprises a g molecule disclosed herein in one or more containers. One d in the art will readily ize that the disclosed binding domains, polypeptides and antibodies of the present invention can be readily incorporated into one of the established kit formats which are well known in the art.

* ** The practice of the disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such ques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., Sambrook et al., ed., Cold Spring Harbor —115— tory Press: (1989); Molecular Cloning: A Laboratory Manual, Sambrook et al., ed., Cold Springs Harbor Laboratory, New York (1992), DNA Cloning, D. N. Glover ed., Volumes I and 11 (1985); Oligonucleotide Synthesis, M. J. Gait ed., ; Mullis et al.

US. Pat. No: 4,683,195; Nucleic Acid Hybridization, B. D. Hames & S. J. Higgins eds. (1984); Transcription And Translation, B. D. Hames & S. J. Higgins eds. ; Culture Of Animal Cells, R. I. Freshney, Alan R. Liss, Inc., (1987); Immobilized Cells And Enzymes, IRL Press, (1986); B. Perbal, A Practical Guide To Molecular Cloning ; the treatise, Methods In Enzymology, Academic Press, Inc., N.Y.; Gene Transfer Vectors For Mammalian Cells, J. H. Miller and M. P. Calos eds., Cold Spring Harbor Laboratory (1987); s In logy, Vols. 154 and 155 (Wu et al. eds.); Immunochemical Methods In Cell And Molecular Biology, Mayer and Walker, eds., Academic Press, London (1987); Handbook 0fExperimental Immunology, Volumes l—IV, D. M. Weir and C. C. Blackwell, eds., (1986); Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1986); and in Ausubel et al., Current Protocols in lar y, John Wiley and Sons, Baltimore, Maryland (1989).

General principles of dy engineering are set forth in Antibody Engineering, 2nd n, C.A.K. Borrebaeck, Ed., Oxford Univ. Press (1995). General principles of protein engineering are set forth in Protein Engineering, A Practical Approach, Rickwood, D., et al., Eds., IRL Press at Oxford Univ. Press, Oxford, Eng. (1995).

General ples of antibodies and antibody-hapten binding are set forth in: Nisonoff, A., Molecular Immunology, 2nd ed., Sinauer Associates, Sunderland, MA (1984); and Steward, M.W., Antibodies, Their Structure and Function, Chapman and Hall, New York, NY (1984). Additionally, standard s in immunology known in the art and not specifically described are generally followed as in Current ols in Immunology, John Wiley & Sons, New York; Stites et al. (eds) Basic and Clinical -Immunology (8th ed.), Appleton & Lange, Norwalk, CT (1994) and Mishell and Shiigi (eds), Selected Methods in Cellular Immunology, W.H. Freeman and Co., New York (1980).

Standard reference works setting forth general principles of immunology include t Protocols in Immunology, John Wiley & Sons, New York; Klein, J Immunology: The Science ofSelf-NonselfDiscrimination, John Wiley & Sons, New York (1982); Kennett, R., et al., eds., Monoclonal Antibodies, Hybridoma.‘ A New ion in Biological Analyses, Plenum Press, New York (1980); Campbell, A., “Monoclonal 2012/063722 —116— Antibody Technology” in Burden, R., et al., eds., Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 13, Elsevere, Amsterdam (1984), Kuby Immunnology 4th ed.

Ed. Richard A. Goldsby, Thomas J. Kindt and Barbara A. Osborne, H. nd & Co. (2000); Roitt, 1., Brostoff, J. and Male D., Immunology 6th ed. London: Mosby (2001); Abbas A., Abul, A. and Lichtman, A., Cellular and Molecular Immunology Ed. 5, Elsevier Health es Division (2005); mann and Dubel, Antibody Engineering, Springer Verlan ; Sambrook and Russell, Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press (2001); Lewin, Genes VIII, Prentice Hall (2003); Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988); Dieffenbach and Dveksler, PCR Primer Cold Spring Harbor Press (2003).

EXAMPLES Example 1: Construction and screening of human antibody phage display libraries This e describes a target indifferent whole cell panning approach with human antibody phage ies derived from both naive and P. aeruginosa infected convalescing patients to identify novel protective antigens against Pseudomonas infection (Figure 1A). Assays included in the in vitro functional screens included phagocytosis (OPK) killing assays and cell attachment assays using the epithelial cell line A549. The lead candidates, based on superior in vitro activity, were tested in P. aeruginosa acute pneumonia, keratitis, and burn infection models.

Figure 1B shows construction of patient antibody phage display library. Whole blood was pooled from 6 recovering patients 7—10 days post diagnosis followed by RNA extraction and phage library construction as previously bed (Vaughan, T.J., et al., Nat Biotechnol 14, 309—314 (1996); Wrammert, J., et al., Nature 453, 1 (2008)).

Figure 1C shows that the final cloned scFv library contained 5.4 X 108 transformants and cing revealed that 79% of scFv genes were full—length and in frame. The VH CDR3 loops, often important for determining epitope icity, were 84% diverse at the amino acid level prior to library selection.

In on to the t library, a naive human scFv phage display library containing up to 1x1011 binding members (Lloyd, C., et al., Protein Eng Des Sel 22, 159— 168 (2009)) was used for antibody isolation (Vaughan, T.J., et al., Nat Biotechnol I 4, 309—314 ). Heat killed P.aeruginosa (1X109) was immobilized in IMMUNOTM 2012/063722 —117— Tubes (Nunc; MAXISORPTM) followed for phage display selections as described (Vaughan, T.J., et al., Nat Biotechnol 14, 309—314 (1996)) with the exception of triethanolamine (100nM) being used as the elution buffer. For selection on P. aeruginosa in suspension, heat killed cells were blocked followed by addition of blocked phage to cells. After g, eluted phage was used to infect E. coli cells as described (Vaughan, 1996). Rescue of phage from E. coli and binding to heat—killed P. aeruginosa by ELISA was performed as described (Vaughan, 1996).

Following development and validation of the cell affinity ion methodology, both the new convalescing patient library and a previously constructed naive y (Vaughan, T.J., et al., Nat Biotechnol 14, 309—314 (1996)) underwent affinity selection on suspensions of P. nosa strain 3064 possessing a complete 0— antigen as well as an isogenic wapR mutant strain which lacked surface expression of 0— antigen. Figure 1D shows that output titers from successive patient library selections were found to increase at a greater rate for the patient library than for the naive library (1x107 vs 3x105 at round 3, respectively). In on, duplication of VH CDR3 loop sequences in the ies (a measure of clonal enrichment during ion), was also found to be higher in the patient y, reaching 88-92%, compared to 15-25% in the naive library at round 3 (Figure 1D). Individual scFv phage from affinity selections were next screened by ELISA for reactivity to P. aeruginosa heterologous serotype strains (Figure 1E). ELISA plates (Nunc; MAXISORPTM) were coated with P. aeruginosa strains from overnight cultures as described (DiGiandomenico, A., et al., Infect Immun 72, 7012—7021 ). d antibodies were added to d plates for 1 hour, , and treated with HRP-conjugated anti-human secondary antibodies for 1 hour followed by development and analysis as described (Ulbrandt, N.D., et al., J Virol 80, 7799—7806 (2006)). The dominant species of phage obtained from whole cell selections with both libraries yielded serotype specific reactivity (data not shown). Clones exhibiting serotype independent binding in the absence of nonspecific binding to E. coli or bovine serum albumin were selected for further evaluation.

For IgG expression, the VH and VL chains of selected antibodies were cloned into human IgG1 expression vectors, co—expressed in HEK293 cells, and purified by protein A affinity chromatography as described (Persic, L., et al., Gene 187, 9—18 ). Human IgG1 antibodies made with the variable regions from these selected serotype independent —118— phage were ed for P. aeruginosa specificity and prioritized for subsequent analysis by whole cell binding to dominant clinically relevant serotypes by FACS analysis (Figure 1F), since this method is more stringent than ELISA. For the flow cytometry based binding assays mid—log phase P. aeruginosa s were concentrated in PBS to an OD650 of 2.0. After incubation of antibody (10 ug/mL) and bacteria (~l x 107 cells) for 1 hr at 4°C with shaking, washed cells were ted with an ALEXA FLUOR 647® goat anti- human IgG antibody (Invitrogen, ad, CA) for 0.5 hr at 4°C.

Washed cells were stained with BACLIGHTTM green bacterial stain as recommended (Invitrogen, Carlsbad, CA). Samples were run on a LSR II flow cytometer (BD Biosciences) and analyzed using BD FacsDiva (V. 6.1.3) and FlowJo (V. 9.2; TreeStar).

Antibodies exhibiting binding by FACS were further prioritized for functional activity testing in an opsonophagocytosis killing (OPK) assay.

Example 2: Evaluation of mAbs ing OPK of P. aeruginosa This example describes the evaluation of prioritized human IgG1 antibodies to promote OPK of P. aeruginosa. Figure 2A shows that with the exception of WapR—007 and the ve control antibody R347, all antibodies mediated concentration dependent killing of luminescent P. aeruginosa serogroup 05 strain (PAOl.lux). WapR—004 and Cam—003 exhibited superior OPK activity. OPK assays were performed as described in (DiGiandomenico, A., et al., Infect Immun 72, 7012—7021 (2004)), with modifications.

Briefly, assays were performed in 96-well plates using 0.025 ml of each OPK component; P. aeruginosa strains; diluted baby rabbit serum; differentiated HL-60 cells; and monoclonal antibody. In some OPK , luminescent P. nosa strains, which were constructed as described (Choi, KH., et al., Nat Methods 2, 443—448 (2005))., were used. Luminescent OPK assays were performed as bed above but with determination of relative luciferase units (RLUs) using a Perkin Elmer ENVISION Multilabel plate reader (Perkin Elmer).

The ability of the WapR-004 and Cam-003 antibodies to mediate OPK activity against r clinically relevant O-antigen pe strain, 9882—80.lux, was evaluated.

Figure 2B shows that enhanced WapR-004 and Cam-003 OPK activity extends to strain 9882—80 (01 l). —ll9— In addition, this example describes the evaluation of WapR-004 (W4) mutants in scFv—Fc format to promote OPK of P. aeruginosa. One mutant, Wap—004RAD (W4— RAD), was specifically created through site-directed mutagenesis to remove an RGD motif in VH. Other W4 mutants were prepared as follows. Nested PCR was performed as described (Roux, K.H., PCR Methods App] 4, 8185—194 (1995)), to amplify W4 variants (derived from somatic hypermutation) from the scFv library d from the escing P. aeruginosa ed patients for analysis. This is the library from which WapR—004 was derived. W4 variant fragments were subcloned and sequenced using standard procedures known in the art. W4 mutant light chains (LC) were recombined with the WapR-004 heavy chain (HC) to produce W4 s in scFv-Fc format. In addition WapR-004 RAD heavy chain (HC) mutants were recombined with parent LCs of M7 and M8 in the c format. Constructs were prepared using standard procedures known in the art. Figures 11 (A-M) show that with the exception of the negative control antibody R347, all WapR-004 (W4) mutants mediated concentration dependent killing of luminescent P. aeruginosa serogroup 05 strain (PAOl.lux).

The WapRRAD variable region was germ-lined to reduce potential immunogenicity, producing WapRgermline ("WapRGL"), and was lead optimized via site-directed mutagenesis. Clones with improved affinity for Psl were selected in competition—based s. Top clones were ranked by affinity improvement and analyzed in an in vitro functional assay. The 14 lead optimized clones are: 6, Plel70, Ps10225, Ple304, , Psl348, Ps10567, Ps10573, 4, Ps10582, Ps10584, 5, Ps10588 and Ps10589.

Example 3: Serotype independent anti—P.aerugin0sa antibodies target the Psl exopolysaccharide This example describes fication of the target of anti—P. nosa antibodies derived from phenotypic screening. Target analysis was performed to test whether the serotype independent antibodies ed protein or carbohydrate antigens.

No loss of g was observed in ELISA toPAOl whole cell extracts exhaustively digested with proteinase K, suggesting that vity targeted surface accessible carbohydrate residues (data not shown). Isogenic mutants were constructed in genes responsible for O-antigen, alginate, and LPS core biosynthesis; wpr igendeficient ); wpr/aZgD (O-antigen and alginate deficient); rmZC (O-antigen-deficient and truncated outer core); and gaZU (O—antigen—deficient and truncated inner core). P.

WO 70615 —120— aeruginosa mutants were constructed based on the allele replacement strategy described by Schweizer (Schweizer, H.P., M01 Microbiol 6, 204 (1992); Schweizer, H.D., Biotechniques 15, 831—834 (1993)). s were mobilized from E. coli strain Sl7.1 into P. aeruginosa strain PAOl; inants were isolated as described (Hoang, T.T., et al., Gene 212, 77—86 (1998)). Gene deletion was confirmed by PCR. P. aeruginosa mutants were complemented with pUCP30T—based constructs harboring wild type genes.

Reactivity of antibodies was determined by indirect ELISA on plates coated with above indicated P. nosa strains: Figure 3A shows that Cam—003 binding to the wpr or the wpr/algD double mutant was unaffected, however binding to the rmlC and gaZU mutants were abolished. While these results were consistent with binding to LPS core, vity to LPS purified from PAOl was not observed. The rmZC and galU genes were recently shown to be required for thesis of the Psl exopolysaccharide, a repeating pentasaccharide polymer consisting of D-mannose, nose, and D—glucose. Cam— 003 binding to an isogenic pslA knockout PAOlApsZA, was tested, as psZA is required for Psl biosynthesis (Byrd, M.S., et al., M01 Microbiol 73, 622—638 ). g of Cam—003 to PAOlApsZA was abolished when tested by ELISA (Figure 3B) and FACS (Figure 3C), while the LPS molecule in this mutant was cted (Figure 3D). Binding of Cam—003 was restored in a PAOlAwpr/aZgD/pslA triple mutant complemented with pslA (Figure 3E) as was the ability of 3 to mediate opsonic killing to complemented SlA in contrast to the mutant (Figure 3F and 3G). Binding of Cam-003 antibody to a Pel ysaccharide mutant was also unaffected further confirming Psl as our antibody target (Figure 3E). Binding assays confirmed that the remaining antibodies also bound Psl (Figure 3H and 31).

Example 4: Anti—Ps1 mAbs block attachment of P. aeruginosa to cultured epithelial cells.

This example shows that anti—Ps1 antibodies blocked P. aeruginosa association with epithelial cells. Anti—Ps1 antibodies were added to a confluent monolayer of A549 cells (an adenocarcinoma human alveolar basal epithelial cell line) grown in opaque 96- well plates (Nunc Nunclon Delta). Log—phase luminescent P. aeruginosa PAOl strain (PAOl.lux) was added at an MOI of 10. After incubation of PAOl.lux with A549 cells at 37°C for 1 hour, the A549 cells were washed, followed by addition of LB+0.5% glucose.

Bacteria were quantified following a brief incubation at 37°C as performed in the OPK assay described in Example 2. ements from wells without A549 cells were used —l2l— to correct for non-specific binding. Figure 4 shows that with the exception of Cam-005 and WapR—007, all antibodies reduced association of PAOl.lux to A549 cells in a dose— dependent manner. The mAbs which performed best in OPK assays, WapR-004 and Cam—003 (see Figures 2A-B, and Example 2), were also most active at inhibiting P. nosa cell attachment to A549 lung lial cells, providing up to ~80% reduction compared to the negative control. WapR-016 was the third most active antibody, showing similar inhibitory activity as WapR-004 and Cam-003 but at 10-fold higher dy concentration. e 5: In vivo passaged P. aeruginosa strains maintain/increase expression of Psl To test if Psl expression in vivo is maintained, mice were injected intraperitoneally with P. aeruginosa isolates followed by harvesting of bacteria by peritoneal lavage four hours post—infection. The presence of Psl was analyzed with a control antibody and Cam- 003 by flow cytometry as conditions for antibody binding are more stringent and allow for fication of cells that are positive or negative for Psl expression. For ex vivo binding, bacterial inocula (0.1ml) was prepared from an ght TSA plate and delivered intraperitoneally to BALB/c mice. At 4 hr. ing challenge, bacteria were ted, RBCs lysed, sonicated and resuspended in PBS supplemented with 0.1% Tween—20 and 1% BSA. Samples were stained and analyzed as previously described in Example 1. Figure 5 shows that ia harvested after peritoneal lavage with three wild type P. aeruginosa strains showed strong 3 staining, which was comparable to log phase cultured bacteria (compare Figures 5A and 5C). In vivo passaged wild type bacteria ted enhanced staining when compared to the inoculum (compare Figures 5B and 5C). Within the inocula, Psl was not detected for strain 6077 and was minimally detected for strains PAOl (05) and 6206 (Oll—cytotoxic). The binding of Cam—003 to ia increased in on to the a indicating that Psl expression is maintained or increased in vivo. Wild type strains 6077, PAOl, and 6206 express Psl after in vivo passage, however strain PAOl harboring a deletion of pslA (PAOlApsZA) is unable to react with Cam-003. These results further emphasize Psl as the target of the monoclonal antibodies. —l22— Example 6: Survival rates for animals treated with anti-Psl monoclonal antibodies Cam-003 and WapR—004 in a P. aeruginosa acute pneumonia model Antibodies or PBS were administered 24 hours before infection in each model. P. aeruginosa acute pneumonia, keratitis, and thermal injury infection models were performed as described (DiGiandomenico, A., et al., Proc Natl Acad Sci U S A 104, 4624-4629 (2007)), with cations. In the acute pneumonia model, BALB/c mice (The Jackson Laboratory) were infected with P. aeruginosa strains suspended in a 0.05 ml inoculum. In the thermal injury model, CF—l mice (Charles River) received a 10% total body surface area burn with a metal brand heated to 92°C for 10 seconds. Animals were infected subcutaneously with P. aeruginosa strain 6077 at the indicated dose. For organ burden experiments, acute pneumonia was induced in mice followed by harvesting of lungs, spleens, and kidneys 24 hours post-infection for determination of CFU.

Monoclonal antibodies Cam—003 and WapR—004 were evaluated in an acute lethal pneumonia model against P. aeruginosa strains representing the most frequent serotypes associated with clinical disease. Figures 6A and 6C show significant concentration- dependent survival in Cam—003—treated mice infected with strains PAOl and 6294 when compared to controls. Figures 6B and 6D show that complete tion from challenge with 33356 and cytotoxic strain 6077 was afforded by Cam—003 at 45 and 15 mg/kg while 80 and 90% survival was ed at 5mg/kg for 33356 and 6077, respectively. Figures 6E and 6F show significant tration-dependent survival in WapRtreated mice in the acute pneumonia model with strain 6077 (01 l) (8 x 105 CFU) e 6E), or 6077 (01U(6x105CFU)Gfigne6F) Cam-003 and 04 were next examined for their ability to reduce P. aeruginosa organ burden in the lung and spread to distal , and later the animals were treated with various concentrations of WapR-004, Cam-003, or control antibodies at l different concentrations. Cam—003 was effective at ng P. aeruginosa lung burden against all four strains tested. Cam-003 was most effective against the highly pathogenic cytotoxic strain, 6077, where the low dose was as effective as the higher dose (Figures 7D). Cam-003 also had a marked effect in reducing dissemination to the spleen and kidneys in mice infected with PAOl (Figure 7A), 6294 e 7C), and 6077 (Figure 7D), while dissemination to these organs was not observed in 33356 infected mice e 7B). Figures 7E and 7F show that similarly, WapR—004 reduced organ burden after induction of acute nia with 6294 (O6) and 6206 (01 1). Specifically, WapR- —123— 004 was effective at reducing P. nosa dissemination to the spleen and s in mice infected.

Example 7: Construction of anti-Pch monoclonal antibody V2L2 VelocImmune® mice (Regeneron Pharmaceuticals) were immunized by Ultra- Short immunization method with r-Pch and serum titers were followed for binding to Pch and neutralizing the hemolytic activity of live Raeruginosa. Mice showing anti— hemolytic activity in the serum were sacrificed and the spleen and lymph nodes (axial, inguinal and popliteal) were harvested. The cell populations from these organs were panned with biotinylated r—Pcrv to select for anti-Pch specif1c B—cells. The selected cells were then fused with mouse myeloma partner P3X63—Ag8 and seeded at 25Kcells/well in hybridoma selection . After 10 days the medium from the hybridoma wells were completely changed with fresh medium and after r 3-4 days the hybridoma supematants were assayed for anti-hemolytic ty. Colonies showing emolytic activity were d dilution cloned at 0.2 cells/well of 96-well plates and the anti- hemolytic activity assay was repeated. Clones showing anti-hemolytic activity were d to Ultra-low IgG containing hybridoma culture medium. The IgG from the ioned media were purified and d for in vitro anti—hemolytic activity and in vivo for protection t infection by P.aerugin0sa. The antibodies were also categorized by competition assay into different groups. The variable (V) domains from the antibodies of interest were subcloned from the cDNA derived from their different respective clones. The subcloned V—segments were fused in frame with the cDNA for the corresponding constant domain in a mammalian expression plasmid. Recombinant IgG were expressed and purified from HEK293 cells. In instances where more than one cDNA V— sequence was obtained from a particular clone, all combinations of variable heavy and light chains were expressed and characterized to identify the onal IgG.

Example 8: Survival rates for animals treated with anti-Psl monoclonal antibodies Cam-003, WapR—004 and anti-Pch monoclonal antibody V2L2 in a P. aeruginosa corneal infection model Cam—003 and WapR—004 efficacy was next ted in a P. nosa corneal infection model which emphasizes the pathogens ability to attach and ze damaged tissue. Figures 8 A—D and 8 F—G show that mice receiving Cam-003 and WapR-004 had significantly less pathology and reduced bacterial counts in total eye homogenates than —124— was observed in ve control—treated animals. Figure 8E shows that Cam—003 was also effective when tested in a thermal injury model, providing significant protection at and 5mg/kg when compared to the dy-treated control. Figure 8 (H): The activity of anti-Psl and anti-Pch monoclonal antibodies V2L2 was tested in a P. aeruginosa mouse ocular keratitis model. C3H/HeN mice were injected intraperitoneally (IP) with PBS or a control IgGl antibody (R347) at 45mg/kg or WapR—004 (ct-Ps1) at 5mg/kg or V2L2 (oc-Pch) at 5mg/kg, 16 hours prior to infection with 6077 (01 1-cytotoxic — 1x106 CFU). Immediately before infection, mice were anesthetized followed by initiation of three 1 mm scratches on the cornea and superficial stroma of one eye of each mouse using a 27—gauge needle under a dissection microscope, followed by topical application of P. aeruginosa 6077 strain in a 5 pl um. Eyes were photographed at 48 hours post infection followed by corneal grading by visualization of eyes under a dissection cope. Grading of corneal infection was performed as previously described by Preston et a]. (Preston, MJ., 1995, Infect. Immun. 7). Briefly, infected eyes were graded 48 h after infection with strain 6077 by an investigator who was unaware of the animal treatments. The following grading scheme was used: grade 0, eye macroscopically identical to an uninfected eye; grade 1, faint opacity partially covering the pupil; grade 2, dense opacity covering the pupil; grade 3, dense y covering the entire pupil; grade 4, perforation of the cornea kage of the eyeball). Mice receiving systemically dosed (1P) 3 or WapR—004RAD showed significantly less pathology and reduced bacterial colony g units (CFU) in total eye homogenates than was observed in the R347 control mAb-treated animals. Similar results were observed in V2L2—treated animals when ed to R3 47—treated controls.

Example 9: A Cam-003 Fc mutant dy, CamTM, has diminished OPK and in viva efficacy but ins anti-cell attachment activity.

Given the potential for dual mechanisms of action, a Cam-003 Fc mutant, Cam- 003 —TM, was created which harbors mutations in the Fc domain that reduces its interaction with Fcy ors (Oganesyan, V., et al., Acta Crystallogr D Biol Crystallogr 64, 700—704 (2008)), to identify if protection was more correlative to anti—cell attachment or OPK activity. P. nosa s were constructed based on the allele replacement strategy described by Schweizer (Schweizer, H.P., M01 Microbiol 6, 1195—1204 (1992); Schweizer, H.D., Biotechniques 15, 831—834 (1993)). Vectors were mobilized from E. 2012/063722 —l25— coli strain S17.1 into P. aeruginosa strain PAOl; recombinants were isolated as described (Hoang, T.T., et al., Gene 212, 77—86 (1998)). Gene deletion was confirmed by PCR. P. aeruginosa mutants were complemented with pUCP30T—based constructs harboring wild type genes. Figures 9A shows that Cam—003—TM ted a 4—fold drop in OPK activity compared to Cam—003 (EC50 of 0.24 and 0.06, respectively) but was as effective in the cell ment assay (Figure 9B). Figure 9C shows that Cam—003—TM was also less effective against nia suggesting that optimal OPK activity is necessary for optimal protection. OPK and cell attachment assays were performed as previously described in Examples 2 and 4, respectively.

Example 10: Epitope mapping and relative affinity for anti-Psl antibodies Epitope mapping was performed by competition ELISA and confirmed using an OCTET® flow system with Psl derived from the supernatant of an overnight e of P. aeruginosa strain PAOl. For competition ELISA, antibodies were biotinylated using the EZ-Link NHS-Biotin and Biotinylation Kit (Thermo Scientific). Antigen coated plates were treated with the EC50 of biotinylated antibodies bated with unlabeled antibodies. After incubation with HRP-conjugated streptavidin (Thermo Scientific), plates were developed as bed above. Competition experiments between anti—Psl mAbs determined that dies targeted at least three unique epitopes, referred to as class 1, 2, and 3 antibodies (Figure 10A). Class 1 and 2 antibodies do not compete for binding, r the class 3 antibody, 16, partially inhibits binding of the Class 1 and 2 antibodies.

Antibody affinity was determined by the OCTET® binding assays using Psl derived from the supernatant of overnight PAOl cultures. Antibody KD was determined by averaging the binding kinetics of seven concentrations for each dy. Affinity measurements were taken with a FORTEBIO® OCTET® 384 instrument using 384 slanted well plates. The supernatant from overnight PAOl es :: the psZA gene were used as the Psl source. Samples were loaded onto OCTET® AminoPropylSilane (hydrated in PBS) sensors and blocked, followed by measurement of anti-Psl mAb binding at several concentrations, and disassociation into PBS + 1% BSA. All procedures were performed as described (Wang, X., et al., J Immunol s 362, 151—160).

Association and disassociation raw AnM data were curve-fitted with GraphPad Prism.

Figure 10A shows the relative binding affinities of anti-Psl antibodies characterized —l26— above. Class 2 antibodies had the highest affinities of all the anti-Psl antibodies. Figure 10A also shows a summary of cell attachment and OPK data experiments. Figure 10B shows the relative binding affinities and OPK EC50 values of the Wap-004RAD ) mutant as well as other W4 mutants lead optimized via site—directed mutagenesis as bed in Example 2. Figure 10C shows the relative binding affinities of the Wap-004RAD (W4RAD), Wap-004RAD-Germline (W4RAD-GL) as well as lead optimized anti-Psl monoclonal antibodies (Ps10096, Plel70, Ple225, Ple304, Ple337, Psl348, Ps10567, Ps10573, 4, Ps10582, Ps10584, Ps10585, Ps10588 and Ps10589).

Highlighted clones Ps10096, Ps10225, Ps10337, Ps10567 and Ps10588 were selected based on their enhanced OPK activity, as shown in Example 10 below.

Example 11: Evaluation of lead zed WapR-004 (W4) mutant clones and lead optimized anti-Psl monoclonal antibodies in the P. aeruginosa opsonophagocytic killing (OPK) assay This example describes the tion of lead optimized WapR-004 (W4) mutant clones and lead optimized anti-Psl monoclonal antibodies to promote OPK of P. aeruginosa using the method described in Example 2. Figures llA—Q show that with the exception of the negative control antibody R347, all antibodies mediated concentration dependent killing of luminescent P. aeruginosa serogroup 05 strain (PAOl.lux).

Example 12: Anti-Pch monoclonal antibody V2L2 reduces lethality from acute pneumonia from multiple strains The Pch epitope diversity was analyzed using three approaches: bead based flow try method, ition ELISA and western blotting of nted rPch.

Competition experiments between anti-Pch mAbs determined that antibodies targeted at least six unique epitopes, referred to as class 1, 2, 3, 4, 5 and 6 dies (Figure 12A).

Class 2 and 3 antibodies partially compete for binding. mAbs representing additional epitope classes: class 1 (V2L7, 3G5, 4C3 and llA6), class 2 (IE6 and 1F3), class 3 (29D2, 4A8 and 2H3), class 4 (V2L2) and class 5 (2lFl, LElO and SH3) were tested for in viva protection as below described.

Novel anti—Pch mAbs were isolated using oma technology and the most potent T3 SS inhibitors were selected using a rabbit red blood cell lysis tion assay.

Percent inhibition of xicity analysis was analysed for the parental V2L2 mAb, mAbl66 (positive control) and R347 (negative l), where the antibodies were administered to cultured broncho-epithelial cell line A549 ed with log-phase P. —l27— nosa strain 6077 (exoU+) at a MOI of imately 10. A549 lysis was d by measuring released lactate dehydrogenase (LDH) activity and lysis in the presence of mAbs was compared to wells without mAb to determine percent inhibition. The V2L2 mAb, mAbl66 (positive control) and R347 (negative control) were evaluated for their ability to prevent lysis of RBCs, where the antibodies were mixed with log—phase P. aeruginosa 6077 (exoU+) and washed rabbit red blood cells (RBCs) and incubated for 2 hours at 37°. Intact RBCs were ed and the extent of lysis determined by measuring the OD405 of the cell-free supernatant. Lysis in the presence of anti-Pch mAbs was compared to wells without mAb to determine percent inhibition. The positive control antibody, mAbl66, is a previously characterized ch antibody (J Infect Dis. 186: 64—73 , Crit Care Med. 40: 2320-2326 (2012)).(B) The parental V2L2 mAb demonstrated inhibition of cytotoxicity with an IC50 of 0. 10 ug/ml and exhibited an IC50 tration 28-fold lower than mAbl66 (IC50 of 2.8 ug/ml). (C) V2L2 also demonstrated prevention of RBC lysis with an IC50 of 0.37 ug/ml and exhibited an IC50 concentration 10-fold lower than mAbl66 (IC50 of 3.7 ug/ml).

The V2L2 variable region was fully germlined to reduce potential genicity. V2L2 was affinity matured using the parsimonious mutagenesis approach to randomize each position with 20 amino acids for all six CDRs, identifying affinity-improved single mutations. A combinatorial library was then used, encoding all possible combinations of affinity-improved single mutations. Clones with improved affinity to Pch were selected using g ELISA in IgG format. Top clones were ranked by affinity ement and analyzed in an in vitro functional assay.V2L2 CDRs were systematically mutagenized and clones with improved affinity to Pch were selected in competition—based screens. Clones were ranked by ses in affinity and analyzed in a functional assay. As shown in Figure 12D, RBC lysis was analyzed for V2L2-germlined MAb (V2L2—GL), V2L2—GL optimized mAbs (V2L2—P4M, V2L2—MFS, D and V2L2-MR), and a negative control antibody R347 using Pseudomonas strain 6077 infected A549 cells. V2L2—GL, V2L2—P4M, V2L2—MFS, V2L2—MD and V2L2—MR demonstrated prevention of RBC lysis.. As shown in Figure 12E, mAbs 1E6, lF3, llA6, 29D2, PCRV02 and V2L7 trated prevention of RBC lysis. As shown in Figure l2F, V2L2 was more potent in prevention of RBC lysis than the 29D2. —l28— Binding kinetics of L and V2L2-MD were measured using a Bio-Rad ProteOnTM XPR36 instrument. dies were ed on a GLC bisensor chip using anti-human IgG reagents. rPch protein was injected at multiple concentrations and the dissociation phase followed for 600 seconds. Data was captured and ed using ProteOn r software. Figure 12 (G-H) shows the relative binding affinities of (G) V2L2—GL and (H) V2L2—MD antibodies. The clone V2L2—MD had increased Kd by 2—3 folds over V2L2—GL.

The in vivo effect of administration of an anti-Pch antibodies was studied in mice using an acute pneumonia model. Groups of mice were treated with either sing concentrations of the V2L2 antibody, a positive control anti-Pch antibody (mAbl66), or a negative control (R347), as shown in Figure 13 (A—B). Groups of mice were also treated with either increasing concentrations of the V2L2 antibody, the Pch antibody Pch-02, or a negative control (R347), as shown in Figure 13 (C-D). Twenty—four hours after treatment, all mice were infected with 5 X 107 CFU (C) Pseudomonas aeruginosa 6294 (06) or (D) PA103A (011). As shown in Figure 13, nearly all control treated animals succumbed to infection by 48 hours post infection. However, V2L2 showed a dose—dependent effect on ed survival even out to 168 hours nfection.

Further, V2L2 provided icantly more potent protection than mAbl66 at similar doses (P=0.025, 5 mg/kg for strain 6077; P < 0.0001, 1 mg/kg for strain 6294).

Groups of mice were d with either increasing concentrations of the 11A6, 3G5 or V2L7, the same concentrations of 29D2, 1F3, 1E6, V2L2, LE10, SH3, 4A8, 2H3, or 21F1, increasing concentrations of the 29D2, increasing concentrations of the V2L2, the Pch antibody Pch-02, or a negative control (R347), as shown in Figure 13 (E-H).

Mice were injected intraperitoneally (IP) with mAbs 24 hours prior to to intranasal infection with Pseudomonas strain 6077 (1 X 106 CFU/animal). As shown in Figure 13E mAbs 11A6, 3G5 and V2L7 did not provide protection in vivo. As shown in Figure 13F, mAb 29D2 provides protection in vivo. As shown in Figure 13G, mAb V2L2 also provides protection in vivo. Figure 13H shows in vivo comparison of 29D2 and V2L2.

Figure 131 shows that mAb V2L2 protects against additional Pseudomonas s (i.e.,6294 and PA103A). —129— Organ burden of Pseudomonas—infected mice was also studied in response to administration of V2L2. Figure 14 (A) Mice were treated with either 1 mg/kg R347 (control), or 1 mg/kg, 0.2 mg/kg, or 0.07 mg/kg of V2L2 and then were infected intranasally with 1.2 x 106 cfu of monas 6206. Figure 14 (B) Mice were also treated with either 15 mg/kg R347 (negative l); 15.0 mg/kg, 5.0 mg/kg, or 1.0 mg/kg mAb166 (positive control); or 5.0 mg/kg, 1.0 mg/kg, or 0.2 mg/kg V2L2 and then were infected intranasally with 5.5 x 106 cfu of Pseudomonas 6206. As shown in Figure 14 (A-B), while V2L2 had little effect on clearance in the kidney, it y reduced dissemination to both the lung and spleen in a dose-dependent manner. In addition, V2L2 provided significantly greater reduction in organ CFU than mAb166 at similar doses (P < 0.0001, 1 mg/kg, lung).

Example 13: In vivo activity of combination y using 04 (anti-Ps1) and V2L2 (anti- Pch) antibodies The in vivo effect of combination administration of anti-Ps1 and anti-Pch binding domains was further studied in mice using the dies V2L2 and WapR-004 (RAD).

Groups of mice were treated with R347 (2.1 mg/kg - negative control), V2L2 (0.1mg/kg), W4—RAD (0.5 mg/kg), or V2L2/W4 combination (either 0.1, 0.5, 1.0 or 2.0 mg/kg each).

Twenty-four hours post-administration of antibody, all mice were infected with an inoculum containing 5.25 x 105 cfu 6206 (01 1—ExoU+). Twenty—four hours post infection, lungs, spleens, and kidneys were harvested, homogenized, and plated for colony forming unit (CFU) identification per gram of . As shown in Figure 15, at the concentrations tested, both V2L2 and W4 were effective in lowering organ burden, the V2L2/W4 ation showed an additive effect in tissue clearance. Histological examination of lung tissue revealed less hemorrhaging, less edema, and less inflammatory infiltrate compared to mice receiving V2L2 or WapR-004 alone (Table 5).

Similarly immunized animals were also assessed for survival from acute pneumonia infections.

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Example 15: Construction of WapR—004/V2L2 bispeciflc antibodies Figure 17A shows TNFOL bispeciflc model constructs. For le—TNFoc/W4, the W4 scFv is fused to the amino-terminus of TNFoc VL through a (G4S)2 linker. For Bs2- TNFOL/W4, the W4 scFv is fused to the amino-terminus of TNFoc VH through a (G4S)2 linker. For BS3—TNFOL/W4, the W4 scFv is fused to the carboxy—terminus of CH3 through a (G4S)2 linker.

Since the combination of WapR—004 + V2L2 provide protection against Pseudomonas challenge, bispeciflc constructs were generated comprising a WapR—004 scFv D) and V2L2 IgG (Figure 17B). To generate Bs2-V2L2—2C, the W4-RAD scFv is fused to N—terminal of V2L2 VH through (G4S)2 linker. To generate L2- 2C, W4—RAD scFv was fused to C—terminal of CH3 through (G4S)2 linker. To generate L2-2C, the W4-RAD scFv was inserted in hinge , linked by (G4S)2 linker on N—terminal and C—terminal of scFv. To generate Bs2-W4-RAD-2C, the V2L2 scFv was fused to the amino-terminus of W4-RAD VH through a (G4S)2 linker.

To generate the W4-RAD scFv for the Bs3 construct, the W4-RAD VH and VL were amplified by PCR. The primers used to amplify the W4-RAD VH were: W4-RAD VH forward primer: includes (G4S)2 linker and 22bp of VH N—terminal sequence (GTAAAGGCGGAGGGGGATCCGGCGGAGGGGGCTCTGAGGTGCAGCTGTTGG 2012/063722 —l32— AGTCGG (SEQ ID NO:224)); and W4-RAD VH reverse primer: includes part of (G4S)4 linker and 22 bp of VH C—terminal sequence (GATCCTCCGCCGCCGCTGCCCCCTCCCCCAGAGCCCCCTCCGCCACTCGAGA CGGTGACCAGGGTC (SEQ ID NO:225). Similarly, the W4-RAD VL was amplified by PCR using the primers: W4-RAD VL forward primer: includes part of (G4S)2 linker and 22 bp of VL N—terminal sequence (AGGGGGCAGCGGCGGCGGAGGATCTGGGGGAGGGGGCAGCGAAATTGTGTT GACACAGTCTC (SEQ ID )); and W4-RAD VL reverse primer: includes part of vector sequence and 22 bp of VL C—terminal sequence (CAATGAATTCGCGGCCGCTCATTTGATCTCCAGCTTGGTCCCAC SEQ ID NO:227)). The overlapping fragments were then fused together to form the W4-RAD scFv.

W4—RAD scFv sequence in BS3 vector: underlined ces are G4S linker GGGGSGGGGSEVQLLESGPGLVKPSETLSLTCNVAGGSISPYYWTWIRQPPGKCLELIGY TDYNPSLKSRVTISGDTSKKQFSLHVSSVTAADTAVYFCARADWDLLHALDIW GQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQSPSSLSTSVGDRVTITCRASQSIRS HLNWYQQKPGKAPKLLIYGASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQS YSFPLTFGCGTKLEIK (SEQ ID NO:228) After the W4-RAD scFv nt was amplified, it was then gel purified and ligated into the Bs3 vector which had been digested with BamHI/NotI. The ligation was done using the In-Fusion system, followed by transformation in Stellar competent cells.

Colonies were sequenced to confirm the correct W4—RAD scFv insert.

To generate the L2—2C, the IgG portion in the Bs3 vector was replaced with V2L2 IgG. Briefly, the Bs3 vector which contains W4—RAD scFv was digested with BssHII / SalI and the resultant vector band was gel purified. Similarly, the vector containing V2L2 vector was digested with BssHII / SalI and the V2L2 insert was gel ed. The V2L2 insert was then ligated with the Bs3-W4-RAD scFv vector and colonies were sequenced to confirm the correct V2L2 IgG insert.

A r approach was used to generate Bs2-V2L2-2C.

W4—RAD scFv-V2L2 VH sequences in Bs2 : underlined sequences are G4S linker EVQLLESGPGLVKPSETLSLTCNVAGGSISPYYWTWIRQPPGKCLELIGYIHSSGYTDYNP SLKSRVTISGDTSKKQFSLHVSSVTAADTAVYFCARADWDLLHALDIWGQGTLVTVSSQ GGGSGGGGSGGGGSGGGGSEIVLTQSPSSLSTSVGDRVTITCRASQSIRSHLNWYQQKPG —l33— KAPKLLIYGASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSFPLTFGCGT KLEIKGGGGSGGGGSEMQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGE GLEWVSAITISGITAYYTDSVKGRFTISRDNSKNTLYLQMNSLRAGDTAVYYCAKEEFLP GTHYYYGMDVWGQGTTVTVSS (SEQ ID NO:229) The following primers were used to amplify W4—RAD scFv. VH rd ) and VL (reverse primer): W4-RAD VH forward primer for Bs2 vector which includes some intron, 3' signal e and 22bp of W4-RAD VH inal sequence (TTCTCTCCACAGGTGTACACTCCGAGGTGCAGCTGTTGGAGTCGG (SEQ ID NO:230)) and W4—RAD VL reverse primer for Bs2 vector: include (G4S)2 linker and 32 bp of VL C—terminal sequence (CCCCCTCCGCCGGATCCCCCTCCGCCTTTGATCTCCAGCTTGGTCCCACAGCC GAAAG (SEQ ID NO:23 l)) To amplify the V2L2 VH region the ing primers were used: V2L2 VH forward primer: includes (G4S)2 linker and 22 bp of V2L2 VH N—terminal sequence (GGCGGAGGGGGATCCGGCGGAGGGGGCTCTGAGATGCAGCTGTTGGAGTCT GG (SEQ ID NO:232)), and V2L2 VH reverse primer: includes some of CH1 N—terminal sequence and 22 bp of V2L2 VH C—terminal sequence (ATGGGCCCTTGGTCGACGCTGAGGAGACGGTGACCGTGGTC (SEQ ID NO: 233)).

These primers were then used to amplify V2L2 VH, which was then joined by overlap with W4-RAD scFv and V2L2 VH to get W4-RAD scFv-V2L2-VH. The W4- RAD scFv-V2L2 VH was then ligated into Bs2 vector by gel purifying W4-RAD scFv — V2L2 VH (from overlap PCR); digesting Bs2 vector with BerI/SalI, and gel purifying vector band. The W4—RAD scFv-V2L2—VH was then ligated with Bs2 vector by In— Fusion system and ormed into r competent cells and the colonies were confirmed for the correct W4-RAD scFv-V2L2 VH insert. To e VL in Bs2 vector with V2L2 VL, the Bs2 vector which contains W4-RAD scFv—V2L2—VH was digested with BssHII / BsiWI and the vector band was gel purified. The pOE—V2L2 vector was then digested with BssHII / BsiWI and the V2L2 VL insert was gel purified. The V2L2 VL insert was then d with Bs2—W4—RAD scFv-V2L2—VH vector and the colonies were sequenced for correct V2L2 IgG insert.

Finally, a similar PCR-based approach was used to generate the Bs4-V2L2-2C construct. The hinge region with linker sequence is shown below: —134— Hinge region with linker sequence: GGSGGGGS — N-terminus ofscFv (SEQ ID NO:329) CHl hinge linker C-terminus ofscFv — GGGGSGGGGSDKTHTCPPC (SEQ ID NO:330) linker hinge CH2 W4-RAD scFv sequences in BS4 vector: W4-RAD scFv is in bolded italics with the G4S linkers underlined in bolded s; hinge regions are dmibleddemed KVDKRV]EPKSCGGGGSGGGGSEVQLLESGPGLVKPSETLSLTCNVAGGSISPYYWTWIR QPPGKCLELIGYIHSSGYTDYNPSLKSRVTISGDTSKKQFSLHVSSVTAADTA VYFCARAD WDLLHALDIWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQSPSSLSTSVGDRV TITCRASQSIRSHLNWYQQKPGKAPKLLIYGASNLQSGVPSRFSGSGSGTDFTLTISSLQP EDFATYYCQQSYSFPLTFGCGTKLEIKGGGGSGGGGS DKTHTCPPCPAPELMSEQ ID W4-RAD scFv is presented in bolded italics with the G4S linkers underlined in bolded italics EVQLLESGPGLVKPSETLSLTCNVAGGSISPYYWTWIRQPPGKCLELIGYIHSSGYTDYNP SLKSRVTISGDTSKKQFSLHVSSVTAADTA VYFCARADWDLLHALDIWGQGTLVTVSSfl GGSGGGGSGGGGSGGGGSEIVLTQSPSSLSTSVGDRVTITCRASQSIRSHLNWYQQKPGK APKLLIYGASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSFPLTFGCGTK LEIK W4—RAD scFv was generated using PCR and the following primers: W4-RAD VH forward primer for BS4 vector: includes some of linker sequences and 24 bp of W4— RAD VH N—terminal sequence (GAGGTGCAGCTGTTGGAGTCGGGC (SEQ ID NO:236)); and W4—RAD VL e primer for BS4 vector: includes some hinge sequence, linker and 21 bp of W4-RAD VL C-terminal sequence (GTGTGAGTTTTGTngatccCCCTCCGCCAGAGCCACCTCCGCCTTTGATCTCCA GCTTGGTCCC (SEQ ID NO: 237)).

W4—RAD scFv was then ligated into BS4 vector to get Bs4—V2L2—2C by gel purifying W4-RAD scFv (from PCR); the Bs4-V2L2 vector was digested with BamHI and the vector band was gel purified. The W4—RAD scFv was ligated with BS4 vector by ion system and the vector transform Stellar competent cells. Colonies were ced for the correct W4—RAD scFv insert. —l35— The sequences for the light chain and heavy chain of the Bs4-V2L2-2C construct are provided in SEQ ID NOS: 327 and 328, respectively.

Example 16: A Psl/Pch bispecif1c dy promotes survival in pneumonia models As an initial matter, the Bs2 and Bs3 bispecific antibodies were tested to e whether they retained their W4 or V2L2 activity in a bispecif1c format. For the parental W4 scFV, a bispeciflc antibody was generated having W4 and a pha binding arm.

A cell attachment assay was performed as described above using the luminescent P. aeruginosa strain PAOl.lux. As shown in Figure 18, all bispecific ucts performed similarly to the parent W4—IgGl construct.

As shown in Figure 19 (A-C), percent inhibition of cytotoxicity was analyzed for both Bs2—V2L2 and Bs3—V2L2 using both (A) 6206 and (B) 6206ApslA infected cells, and (C) percent inhibition of RBC lysis was analyzed for Bs2-V2L2-2C, Bs3-V2L2-2C and Bs4-V2L2-2C using 6206 infected cells. As shown in Figure 19 (A-C), all ific antibodies retained anti-cytotoxicity actiVity and inhibited RBC lysis at levels similar to the parental V2L2 antibody using 6206 and slA infected cells.

The ability of the Bs2 and Bs3 bispecif1c antibodies to mediate OPK of P. aeruginosa was assessed using the method bed in Example 2. While the Bs2—V2L2 antibody showed similar killing compared to the parental W4-RAD antibody, the killing for the Bs3-V2L2 dy was decreased (Figure 20A). While the Bs2-V2L2-2C and L2-2C antibodies showed similar killing compared to the parental W4-RAD antibody, the killing for the L2—2C antibody was decreased (Figure 20B). Figure 20C shows that different preparations of Bs4 antibodies (old lot vs. new lot) showed similar killing compared to the parental W4-RAD dy, however the Bs4-V2L2-2C— YTE dies had a 3-fold drop in OPK actiVity when compared to Bs4-V2L2-2C. A YTE mutant comprises a combination of three "YTE mutations": M252Y, S254T, and T256E, wherein the numbering is according to the EU index as set forth in Kabat, introduced into the heavy chain of an IgG. See U.S. Patent No. 7,658,921, which is incorporated by reference herein. The YTE mutant has been shown to increase the serum half-life of antibodies approximately four-times as compared to wild—type versions of the same antibody. See, e.g., Dall'Acqua et al., J. Biol. Chem. 281:23514—24 (2006) and U.S.

Patent No. 7,083,784, which are hereby incorporated by reference in their entireties. —l36— Following confirmation that both W4 and V2L2 retained ty in a ific format, the Bs2-V2L2, Bs3—V2L2 and Bs4—V2L2 constructs were assessed for survival from acute pneumonia infections. As shown in Figure 2lA, all of the control mice succumbed to infection by approximately 30 hours post—infection. All of the Bs3—V2L2 animals survived, along with those which received the V2L2 control. Approximately 90% of the W4-RAD immunized animals survived. In contrast, Figures B-F show that approximately 50% of the Bs2-V2L2 animals bed to infection by 120 hours. All of the control mice succumbed to infection by approximately 48 hours post—infection.

Figures G—H do not show difference in survival between Bs4-V2L2—2C and Bs4—V2L2— 2C—YTE treated mice at either dose. These s suggest that both antibodies on equivalently in the 6206 acute nia model. Figure 21 I shows that Bs2-V2L2, Bs4- V2L2-2C, and W4-RAD + V2L2 antibody mixture are the most ive in protection against lethal pneumonia in mice challenged with P. aeruginosa strain 6206 (ExoU+).

Organ burden was also assessed for similar immunized mice as described above. ing immunization as above, mice were challenged with 2.75 x 105 CFU 6206. As shown in Figure 22, at the concentration tested, both Bs2-V2L2 and Bs3-V2L2 significantly sed organ burden in lung. However, neither of the bispeciflc constructs was able to icantly affect organ burden in spleen or kidney compared to the parental antibodies due to the use of suboptimal concentrations of the bispeciflc constructs. Suboptimal concentrations were used to enable the y to decipher antibody activity.

Survival and organ burden effects of the bispeciflc antibodies were also addressed using the 6294 strain. Using the 6294 model system, both the BS2-V2L2 and BS3-V2L2 significantly decreased organ burden in all of the tissues to a level comparable to that of the V2L2 al antibody. The W4-RAD parental antibody had no effect on decreasing organ burden (Figure 23A). As shown in Figure 23B, Bs2-V2L2, Bs3-V2L2, and W4- RAD+V2L2 combination significantly decreased organ burden in all of the tissues to a level comparable to that of the V2L2 parental antibody.

The survival data for immunized mice was similar in the 6294 nged mice as before. As shown in Figure 24, BS3-V2L2 showed similar survival activity to V2L2 alone-treated mice, while L2 treated mice showed a slightly lower level of protection from challenge. —l37— Organ burden was also assessed in bispecif1c antibodies treated in comparison with combination—treated animals as described above. As shown in Figures 25 (A—C), both the BS2-V2L2 and BS3-V2L2 decreased organ burden in the lung, spleen and s to a level comparable to that of the W4 + V2L2 combination. In the lung, the combination icantly reduced bacterial CFUs Bs2- and Bs3-V2L2 and V2L2 using the Kruskal-Wallis with Dunn’s post test. Significant differences in bacterial burden in the spleen and kidney were not observed, although a trend towards reduction was noted.

An organ burden study was also performed with Bs4-GLO using 6206 in the pneumonia model. As shown in Figure 25 (D), when higher concentrations of antibody are used in prophylaxis of mice, a significant al-Wallis with Dunn’s post test) level of reduction in bacterial burden from the lung was observed. Significant reductions in bacterial dissemination to the spleen and kidneys were also observed when using higher concentrations of Bs4-GLO in this model.

These results were med by histological examination of lung tissue of immunized BALB/c mice challenged with 1.3 3x107 CFU using P. aeruginosa strain 6294 awnomuijCFUmngaW%mmammnmauTwm6mamy25xmfimU using P. aeruginosa strain 6206 (Table 7).

Example 17: Therapeutic adjunctive therapy: Bs4-V2L2-2C + antibiotic Survival effect of the Bs4 bispecif1c antibody and antibiotic adjunctive therapy was evaluated in an acute lethal pneumonia model against P. aeruginosa 6206 strain as previously described in Example 6 (Figure 26 (A—J)). (A—B) Mice were treated 24 hours prior to infection with 6206 with R347 ive control) or Bs4-V2L2-2C or loxacin (CIP) 1 hour post infection, or a combination of the L2-2C 24 hours prior to infection and Cipro 1 hour post infection. (C) Mice were treated 1 hour post infection with 6206 with R347 or CIP or Bs4-V2L2-2C, or a combination of the Bs4- V2L2-2C and CIP. (D) Mice were treated 2 hours post infection with 6206 with R347 or CIP or L2-2C, or a combination of the Bs4-V2L2-2C and CIP. (E) Mice were treated 2 hours post infection with 6206 with R347or Bs4-V2L2-2C or CIP 1 hour post ion, or a combination of the Bs4-V2L2-2C 2 hours post infection and CIP 1 hour post infection. (F) Mice were d 1 hour post infection with 6206 with R347 or Meropenem (MEM) or Bs4-V2L2-2C, or a combination of the Bs4-V2L2-2C and MEM. 2012/063722 —l38— (G) Mice were d 2 hours post infection with 6206 with R347 or Bs4—V2L2—2C or MEM 1 hour post infection, or a combination of the Bs4-V2L2-2C 2 hours post infection and MEM 1 hour post infection. (H) Mice were treated 2 hours post infection with 6206 with R347 or Bs4-V2L2-2C or MEM, or a combination of the Bs4-V2L2-2C 2 and MEM.

(I) Mice were treated 4 hour post infection with 6206 with R347 or Cipro or Bs4-V2L2- 2C or a combination of the Bs4—V2L2—2C and Cipro. All of the control mice succumbed to infection by approximately 24 hours post-infection. As shown in Figures 26 (A-I) Bs4 antibody combined with either CIP or MEM increases efficacy of antibiotic therapy, indicating synergistic protection when the molecules are combined. Further studies focused on the level of bacterial burden in mice treated with Bs4 or CIP alone or in combination (Bs4+CIP). As shown in Figure 26 (J), the level of bacterial burden in all organs (lung, spleen and kidneys) were similar in R347+CIP and Bs4+CIP, however only mice where Bs4 was included in the combination with CIP survive the infection (Figures 26 (A-E, 1)). Altogether, these data indicate the otics are important for reducing the bacterial burden in this animal model setting, however the specific antibody is required to reduce bacterial pathogenicity, thus protecting normal host immunity.

Survival effect of the Bs4 bispecif1c antibody and Tobramycin antibiotic adjunctive therapy will be evaluated in an acute lethal nia model against P. aeruginosa 6206 strain as previously described in Example 6. Mice will be d 24 hours prior to infection with 6206 with R347 ive control) or Bs4-V2L2-2C or Tobramycin 1 hour post infection, or a combination of the Bs4-V2L2-2C 24 hours prior to infection and Tobramycin 1 hour post infection. Mice will also be treated 1 hour post infection with 6206 with R347 or Tobramycin or L2-2C, or a combination of the L2-2C and Tobramycin. In on, mice will be treated 2 hours post infection with 6206 with R347 or Tobramycin or Bs4-V2L2-2C, or a combination of the Bs4- V2L2-2C and Tobramycin. Furthermore, mice will be treated 2 hours post infection with 6206 with R347 or Bs4-V2L2-2C or Tobramycin 1 hour post infection, or a combination of the Bs4-V2L2-2C 2 hours post infection and Tobramycin 1 hour post infection. Mice will be d 4 hour post infection with 6206 with R347 or ycin or Bs4-V2L2- 2C or a combination of the Bs4-V2L2-2C and Tobramycin.

Survival effect of the Bs4 bispecific antibody and nam antibiotic adjunctive therapy will be evaluated in an acute lethal pneumonia model against P. aeruginosa 6206 2012/063722 —l39— strain as previously described in Example 6. Mice will be treated 24 hours prior to infection with 6206 with R347 (negative l) or Bs4-V2L2-2C or Aztreonam 1 hour post infection, or a combination of the Bs4-V2L2-2C 24 hours prior to ion and Aztreonam 1 hour post ion. Mice will also be treated 1 hour post infection with 6206 with R347 or Aztreonam or L2-2C, or a combination of the Bs4-V2L2-2C and Aztreonam. In addition, mice will be treated 2 hours post infection with 6206 with R347 or Aztreonam or Bs4-V2L2-2C, or a combination of the Bs4-V2L2-2C and Aztreonam. Furthermore, mice will be treated 2 hours post infection with 6206 with R347 or L2-2C or Aztreonam 1 hour post infection, or a combination of the Bs4- V2L2-2C 2 hours post infection and Aztreonam 1 hour post infection. Mice will be treated 4 hour post infection with 6206 with R347 or Aztreonam or Bs4-V2L2-2C or a combination of the Bs4-V2L2-2C and Aztreonam.

Example 18: Construction of the BS4-GLO bispecific antibody The BS4—GLO (Germlined Lead thimized) bispecific construct was generated comprising anti-Psl scFv (Ps10096 scfv) and V2L2-MD (VH+VL) as shown in Figure 35A. The BS4-GLO light chain comprises ined lead optimized anti-Pch antibody light chain variable region (i.e., V2L2-MD). The BS4-GLO heavy chain comprises the formula VH-CHl-Hl-Ll-S-L2-H2-CH2-CH3, n CH1 is a heavy chain nt region domain-l, H1 is a first heavy chain hinge region fragment, L1 is a first linker, S is an anti-Pch ScFv molecule, L2 is a second linker, H2 is a second heavy chain hinge region fragment, CH2 is a heavy chain constant region domain-2, and CH3 is a heavy chain constant region domain-3.

Bs4—GLO light chain: AIS 2MT§ QSPSSLSASVGDRVTITCRASS QGIRNDLGWYS 2g QKPGKAPKLLIYSASTLS QS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDYNYPWTFGQGTKVEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID GLO (germlined lead optimized) V2L2 (i.e., V2L2-MD) light chain variable region is underlined Bs4—GLO heavy chain: —140— EM LLESGGGLV PGGSLRLSCAASGFTFSSYAMNWVR APGEGLEWVSAITIS GITAYYTDSVKGRFTISRDNSKNTLYLS zMNSLRAGDTAVYYCAKEEFLPGTHYY YGMDVWGS [GTTVTVSS[ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKRV]MGGGGSGGGGSEVQLLESGPGLVKPSETLSLTCNVAGGSISPYYW TWIRQPPGKCLELIGYIHSSGYTDYNPSLKSRVTISGDTSKKQFSLHVSSVTAADTA VYFCARADWDLLHALDIWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQ SPSSLSTSVGDRVTITCRASQSIRSHLNWYQQKPGKAPKLLIYGASNLQSGVPSRFS GSGSGTDFTLTISSLQPEDFATYYCQQSYSFPLTFGCGTKLEIKGGGGSGGGGSD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIfiRIPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK GLO ined -lead optimized) V2L2 (i.e., V2L2—MD) heavy chain variable region is underlined; CH1 is bracketed []; GLO (germlined-lead optimized) W4- RAD (i.e., Ps10096) scFv is in bolded italics with the G48 linkers underlined in bolded italics; hinge regions are doubled underlined.

An alternative Bs4-GLO bispecif1c construct comprising an anti-Pch ScFv and an anti-Psl (VH+VL) is shown in Figure 35B, and is generated similarly.

Example 19: Evaluation of the functional activity and efficacy of the Bs4—GLO bispecific Bispeciflc antibodies Bs4—WT (also referred to herein as Bs4—V2L2—2C), Bs4—GL ising germlined anti-Pch and anti-Ps1 variable regions) and O produced as described in Example 18 were tested for differences in functional activity in an opsonophagocytic killing assay (Figure 27A), as usly bed in Example 2, anti- cell attachment assay (Figure 27B), as previously bed in Example 4 and a RBC lysis anti-cytotoxicity assay (Figure 27C), as previously described in Example 12. No in vitro difference in functional activities between the antibodies was ed.

In vivo efficacy of Bs4—GLO was ed as follows. For prophylactic evaluation, mice were prophylactically d with several trations of the Bs4- GLO (i.e., 0.007mg/kg, 0.02mg/kg, 0.07mg/kg, 0.2mg/kg, 0.5mg/kg, 1mg/kg, 3mg/kg, —141— 5mg/kg, 10mg/kg or 15mg/kg) (Figure 28A), 24 hours before ion with the following P. aeruginosa strains (6206 (1.0 x 106), 6077 (1.0 x 106), 6294 (2.0 x 107) or PA103 (1.0 x 106)). For therapeutic evaluation, mice were eutically treated with several concentrations of the Bs4—GLO (i.e., 0.03mg/kg, 0.3mg/kg, 0.5mg/kg, 1mg/kg, 2mg/kg, 5mg/kg, 10mg/kg, g, or 45mg/kg) (Figure 28B), at one hour after infection with the following P. nosa strains (6206 (1.0 x 106), 6077 (1.0 x 106), 6294 (2.0 x 107) or PA103 (1.0 x106».

Survival effect of the Bs4—GLO bispecific antibody was evaluated in an acute lethal pneumonia model against different P. aeruginosa strains as usly described in Example 6. Figure 29 shows survival rates for animals treated with the Bs4-GLO in a P. aeruginosa lethal bacteremia model. Aspects of the emia model are disclosed in detail in US. Provisional Appl. No. 61/723,128, filed November 6, 2012 (attorney docket no. ATOX—500P1, entitled “METHODS OF TREATING S. AUREUS ASSOCIATED DISEASES”), which is incorporated herein by reference in its entirety.

Animals were d with Bs4-GLO or R347, 24 hours prior to intraperitoneal infection with (A) 6294 (O6) or (B) 6206. The BS4-GLO is effective at all tested concentrations in protection against lethal pneumonia in mice challenged with P. aeruginosa strains (A) 6294 and (B) 6206.

Survival effect of the Bs4—GLO bispeciflc antibody was evaluated in a P. aeruginosa thermal injury model against different P. aeruginosa strains. Figure 30 shows al rates for animals lactically treated with the Bs4-GLO in a P. aeruginosa thermal injury model. Animals were treated with Bs4-GLO or R347 hours prior to induction of thermal injury and subcutaneous infection with P. aeruginosa strain (A) 6077 (01 1—ExoU+) or (B) 6206 (01 1—ExoU+) or (C) 6294 (06) directly under the wound.

The BS4-GLO is effective at all tested concentrations in prevention in a P. nosa thermal injury model in mice challenged with P. aeruginosa strains (A) 6077, (B) 6206 and (C) 6294.

Figure 31 shows survival rates for animals therapeutically treated with bispecif1c antibody Bs4-GLO in a P. nosa l injury model. (A) Animals were treated with Bs4-GLO or R347 (A) 4h hours or (B) 12 hours after induction of thermal injury and subcutaneous infection with P. aeruginosa strain 6077 (Oll—ExoU+) directly under the wound. The Bs4—GLO is effective at all tested concentrations in treatment in a P. —142— nosa thermal injury model in mice treated with Bs4-GLO (B) 4h hours or (B) 12 hours after induction of thermal injury and subcutaneous ion with P. nosa strain 6077.

Example 20: Therapeutic adjunctive y: Bs4-GLO + antibiotic Survival effect of the Bs4—GLO bispecif1c antibody and antibiotic adjunctive therapy was evaluated in an acute lethal pneumonia model against P. aeruginosa 6206 strain as previously described in Example 6.

Figure 32 shows therapeutic adjunctive y with ciprofloxacin (CIP). (A) Mice were treated 4 hour post infection with P. aeruginosa strain 6206 with R347 + CIP or Bs4-WT or a combination of the Bs4-WT and CIP. (B) Mice were treated 4 hour post ion with P. aeruginosa strain 6206 with R347 + CIP or Bs4-GLO or a combination of the Bs4-GLO and CIP. (A-B) Bs4-WT or BS4-GLO antibody combined with CIP increased efficacy of antibiotic therapy.

Figure 33 shows therapeutic adjunctive therapy with meropenem (MEM): (A) Mice were treated 4 hour post infection with P. aeruginosa strain 6206 with R347 + MEM or Bs4-WT or a combination of the BS4-WT and MEM. (B) Mice were treated 4 hour post infection with P. aeruginosa strain 6206 with R347 + MEM or BS4 or a combination of the Bs4-GLO and MEM. (A-B) Bs4-WT or Bs4-GLO antibody combined with MEM increases efficacy of antibiotic therapy.

Figure 34 shows therapeutic adjunctive therapy: Bs4—GLO plus antibiotic in a lethal bacteremia model. Mice were treated 24 hours prior to intraperitoneal infection with P. aeruginosa strain 6294 with Bs4-GLO at the indicated concentrations, which were previously determine to be sub—therapeutic protective doses in this model and R347 ive control). One hour post infection, mice were treated subcutaneously with antibiotics at the indicated trations, which were previously determined to be sub— therapeutic protective doses (A) oxacin (CIP), (B) Meropenem (MEM) or (C) ycin (TOB). Animals were carefully red for survival up to 72 hours post— infection. Bs4-GLO antibody combined with either CIP, MEM or TOB, at sub-protective doses, increases efficacy of antibiotic therapy. —143— The sure is not to be limited in scope by the specific embodiments described which are intended as single illustrations of individual aspects of the sure, and any compositions or methods which are functionally equivalent are within the scope of this sure. Indeed, various modifications of the disclosure in addition to those shown and described herein will become nt to those d in the art from the foregoing description and accompanying drawings. Such cations are intended to fall within the scope of the appended .

All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

In addition, US. Provisional Application Nos.: 61/556,645 filed November 7, 2011; 61/624,651 filed April 16, 2012; 61/625,299 filed April 17, 2012; 61/697,585 filed September 6, 2012 and International Application No: , filed November 6, 2012 (attorney docket no. AEMS—115W01, entitled “MULTISPECIFIC AND MULTIVALENT BINDING PROTEINS AND USES THEREOF”) are incorporated by reference in their entirety for all purposes.

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Claims (17)

WHAT IS CLAIMED IS:

1. A bispecific dy comprising a binding domain that binds to Pseudomonas Psl and a binding domain that binds to Pseudomonas PcrV.

2. The bispecific antibody of claim 1, wherein (a) the Psl binding domain ses a scFv fragment and the PcrV g domain comprises an intact immunoglobulin, or (b) the Psl binding domain comprises an intact immunoglobulin and the PcrV binding domain comprises a scFv fragment.

3. The bispecific antibody of claim 2, n (a) the scFv is fused to the amino-terminus of the VH region of the intact immunoglobulin; (b) the scFv is fused to the carboxy-terminus of the CH3 region of the intact immunoglobulin, or (c) the scFv is inserted in the hinge region of the intact globulin.

4. The ific antibody of claim 1, wherein the anti-Psl binding domain (a) binds to the same Pseudomonas Psl epitope as an antibody or antigenbinding fragment thereof comprising a VH comprising the amino acid sequence SEQ ID NO: 11 and a VL region comprising the amino acid sequence SEQ ID NO: 12; (b) can competitively inhibit Pseudomonas Psl binding by an antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence SEQ ID NO: 11 and a VL region comprising the amino acid sequence SEQ ID NO: 12; or (c) a combination of (a) and (b).

5. The bispecific antibody of any one of claims 1 to 4, wherein the anti-PcrV binding domain (a) binds to the same Pseudomonas PcrV epitope as an antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence SEQ ID NO: 216 and a VL comprising the amino acid sequence SEQ ID NO: 217; (b) itively inhibits Pseudomonas PcrV binding by an antibody or antigen-binding fragment thereof comprising a VH comprising the amino acid sequence SEQ ID NO: 216 and a VL sing the amino acid ce SEQ ID NO: 217, or (c) a combination of (a) and (b).

6. The bispecific antibody of claim 5, wherein the crV binding domain comprises a VH comprising the amino acid sequence SEQ ID NO: 216 and a VL comprising the amino acid sequence SEQ ID NO: 217.

7. The bispecific antibody of any one of claims 1 to 6, wherein the anti-Psl binding domain comprises a VH comprising the amino acid sequence SEQ ID NO: 11, and a VL comprising the amino acid sequence SEQ ID NO: 12, and wherein the anti-PcrV binding domain comprises a VH comprising the amino acid sequence SEQ ID NO: 216, and a VL comprising the amino acid sequence SEQ ID NO: 217.

8. An isolated polynucleotide molecule comprising a nucleic acid sequence that encodes the bispecific antibody or nt thereof of any one of claims 1 to

9. A composition comprising the ific antibody or fragment thereof of any one of claims 1 to 7, and a pharmaceutically acceptable r.

10. The composition of claim 9, comprising a binding domain that binds to Pseudomonas Psl and a binding domain that binds to Pseudomonas PcrV.

11. The use of a ific antibody ing to any one of claims 1 to 7, or a composition of claim 9 or 10 in the manufacture of a medicament for preventing or treating a Pseudomonas infection in a subject in need thereof.

12. A kit comprising the composition of claim 9 or 10.

13. A bispecific antibody according to claim 1, substantially as herein bed ore exemplified.

14. An isolated polynucleotide molecule according to claim 8, substantially as herein described or exemplified.

15. A ition according to claim 9, substantially as herein described or exemplified.

16. A use according to claim 11, substantially as herein described or exemplified.

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