NZ618266B2 - Anti-pseudomonas psl binding molecules and uses thereof - Google Patents

Abstract

Disclosed is an isolated antibody or antigen-binding fragment thereof which specifically binds to Pseudomonas Psl polysaccharide, wherein the antibody or antigen-binding fragment thereof promotes opsonophagocytic killing (OPK) of P. aeruginosa, and wherein the isolated antibody or antigen-binding fragment thereof specifically binds to the same Pseudomonas Psl epitope as an antibody or antigen-binding fragment thereof comprising the heavy chain variable region (VH) and the light chain variable region (VL) of WapR-004RAD. Also disclosed is an isolated antibody or antigen-binding fragment thereof which specifically binds to Pseudomonas Psl polysaccharide, comprising a set of complementarity determining regions (CDRs) VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and VLCDR3 having the sequences of PYYWT, YIHSSGYTDYNPSLKS, ADWDLLHALDI, RASQSIRSHLN, GASNLQS and YSFPLT. agment thereof specifically binds to the same Pseudomonas Psl epitope as an antibody or antigen-binding fragment thereof comprising the heavy chain variable region (VH) and the light chain variable region (VL) of WapR-004RAD. Also disclosed is an isolated antibody or antigen-binding fragment thereof which specifically binds to Pseudomonas Psl polysaccharide, comprising a set of complementarity determining regions (CDRs) VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and VLCDR3 having the sequences of PYYWT, YIHSSGYTDYNPSLKS, ADWDLLHALDI, RASQSIRSHLN, GASNLQS and YSFPLT.

Description

ANTI— PSEUDOMONAS PSL BINDING MOLECULES AND USES THEREOF BACKGROUND Field of the sure This disclosure relates to an anti—Pseudomonas Psl binding les and uses thereof, in particular in prevention and treatment of Pseudomonas infection. Furthermore, the disclosure provides compositions and methods for preventing and treating Pseudomonas infection.

Background of the Disclosure Pseudomonas aeruginosa (P. aeraginosa) is a gram—negative opportunistic pathogen that causes both acute and chronic infections in compromised individuals (Ma et al., Journal of Bacteriology 189(22):8353—8356 (2007)). This is partly due to the high innate resistance of the bacterium to clinically used otics, and partly due to the formation of highly antibiotic- resistant biofllms (Drenkard E., Microbes Infect —1219 (2003); Hancokc & , Drug Resist Update 3 :247—255 (2000)).

P. aeraginosa 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 ). It is also the most common cause of nosocomial gram—negative pneumonia (Craven et al., Semin Respir Infect 11:32—53 (1996)), ally in mechanically ventilated patients, and is the most prevalent pathogen in the lungs of individuals With cystic fibrosis (Pier et al., ASM News 6:339—347 (1998)). Serious P. aeraginosa infections can become systemic, resulting in sepsis. Sepsis is characterized by severe systemic inflammation, often resulting in multiple organ failure and death.

Pseudomonas Psl ysaccharide is reported to be anchored to the surface of P. aeruginosa and is thought to be important in facilitating colonization of host tissues and in ishing/maintaining biofilm formation (Jackson, K.D., et al., J iol 186, 4466—4475 (2004)). Its structure comprises mannose—rich repeating pentasaccharide (Byrd, M.S., et al., Mol Microbiol 73, 622—638 (2009)) Due to sing multidrug resistance, there remains a need in the art for the pment of novel strategies for the identification of new Pseudomonas-speciflc prophylactic and therapeutic agents.

BRIEF Y ] The present invention relates to ed antibodies or antigen-binding fragments f which specifically bind to Pseudomonas Psl polysaccharide and which promote opsonophagocytic killing (OPK) of P. aeruginosa. In particular the isolated antibodies or antigen-binding fragments thereof specifically bind to the same Pseudomonas Psl epitope as an antibody or antigen-binding fragment thereof sing the heavy chain variable region (VH) and the light chain variable region (VL) of WapR-004RAD, or competitively inhibit Pseudomonas Psl binding by an dy or antigen-binding fragment comprising the heavy chain variable region (VH) and the light chain variable region (VL) of WapR-004RAD.

Also provided is the use of such antibodies in the manufacture of a medicament for the treatment of a Pseudomonas infection as well as pharmaceutical compositions comprising the antibodies or antigen binding fragments thereof. [0005b] Other aspects and embodiments of the invention are described herein for completeness. (followed by 2A) wed by 3) and SEQ ID NO: 8, tively, and the VH and VL of WapR—003 comprise SEQ ID NO: 9 and SEQ ID NO: 10, tively.

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

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

Some embodiments include the binding molecule e.g., an antibody or fragment thereof as described above, where the VH and VL of WapR—Ol6 comprise SEQ ID NO:SEQ ID NO: 15 and SEQ ID NO: 16, respectively.

Also provided is an isolated binding molecule e.g., an antibody or antigen—binding fragment f which specifically binds to Pseudomonas Psl comprising an antibody VH, where the VH comprises an amino acid sequence at least 90% identical or identical to 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, or SEQ ID NO: 15.

Some embodiments include an isolated binding molecule e.g., an antibody or antigen- binding fragment thereof which specifically binds to Pseudomonas Psl comprising an antibody VL, where the VL comprises an amino acid sequence at least 90% identical or identical to 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, and SEQ ID NO: 16.

Also provided is an isolated dy or antigen-binding fragment f which specifically binds to Pseudomonas psl, comprising VH and VL amino acid sequences at least 90% cal or 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, 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 NO: , respectively, (h) SEQ 14, respectively; or (i) SEQ ID NO: 15 and SEQ ID NO: 16, respectively. In specific embodiments, the described antibody or antigen-binding fragment thereof ses a VH with the amino acid sequence SEQ ID NO: 1 and a VL with the amino acid sequence 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 sequence of SEQ ID NO: 12.

Also disclosed is an isolated binding molecule e.g., an antibody or antigen—binding fragment thereof which specifically binds to Pseudomonas Psl comprising an antibody VH, where the VH comprises a VH complementarity determining region-1 (VHCDRl) amino acid sequence identical to, or identical except for four, three, two, or one amino acid substitutions to: SEQ ID NO: 17, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 35, SEQ ID NO: 41, SEQ ID NO: 47, SEQ ID NO: 53, or SEQ ID NO: 59.

Also provided is an isolated binding molecule e.g., an dy or antigen—binding fragment thereof which specifically binds to Pseudomonas Psl sing an dy VH, where the VH comprises a VH complementarity ining region-2 (VHCDR2) amino acid sequence identical to, or cal except for four, three, two, or one amino acid tutions to: SEQ ID NO: 18, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 36, SEQ ID NO: 42, SEQ ID NO: 48, SEQ ID NO: 54, or SEQ ID NO: 60.

Further provided is an ed binding molecule e.g., an antibody or antigen-binding fragment thereof which specifically binds to Pseudomonas Psl comprising an antibody VH, where the VH comprises a VH complementarity determining region-3 (VHCDR3) amino acid sequence identical to, or identical except for four, three, two, or one amino acid substitutions to: SEQ ID NO: 19, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO: 37, SEQ ID NO: 43, SEQ ID NO: 49, SEQ ID NO: 55, or SEQ ID NO: 61.

Also disclosed is an isolated binding molecule e.g., an antibody or antigen—binding fragment thereof which specifically binds to Pseudomonas psl comprising an antibody VL, where the VL comprises a VL complementarity determining region-1 (VLCDRl) amino acid sequence cal to, or identical except for four, three, two, or one amino acid substitutions to: SEQ ID NO: 20, SEQ ID NO: 32, SEQ ID NO: 38, SEQ ID NO: 44, SEQ ID NO: 50, SEQ ID NO: 56, or SEQ ID NO: 62.

Further provided is an isolated binding molecule e.g., an antibody or antigen—binding fragment thereof which specifically binds to Pseudomonas Psl comprising an antibody VL, where the VL comprises a VL mentarity ining region-2 (VLCDR2) amino acid sequence identical to, or identical except for four, three, two, or one amino acid substitutions to: SEQ ID NO: 21, SEQ ID NO: 33, SEQ ID NO: 39, SEQ ID NO: 45, SEQ ID NO: 51, SEQ ID NO: 57, or SEQ ID NO: 63.

Some embodiments include an isolated binding molecule e.g., an antibody or n- binding fragment thereof which specifically binds to monas Psl sing an antibody VL, where the VL comprises a VL complementarity determining region-3 (VLCDR3) amino acid ce identical to, or identical except for four, three, two, or one amino acid substitutions to: SEQ ID NO: 22, SEQ ID NO: 34, SEQ ID NO: 40, SEQ ID NO: 46, SEQ ID NO: 52, SEQ ID NO: 58, or SEQ ID NO: 64.

Also provided is an ed binding le e. g., an antibody or antigen—binding fragment thereof which specifically binds to Pseudomonas Psl comprising an antibody VH, where the VH comprises VHCDRl, VHCDR2, and VHCDR3 amino acid sequences identical to, or identical except for four, three, two, or one amino acid substitutions in one or more of the VHCDRs to: SEQ ID NOs: 17, 18, and 19, SEQ ID NOs: 23, 24, and 25, SEQ ID NOs: 26, 27, and 28, SEQ ID NOs: 29, 30, and 31, SEQ ID NOs: 35, 36, and 37, SEQ ID NOs: 41, 42, and 43, SEQ ID NOs: 47, 48, and 49, SEQ ID NOs: 53, 54, and 55, or SEQ ID NOs: 59, 60, and 61, respectively.

Some embodiments include an isolated binding molecule e.g., an antibody or antigen- g fragment thereof which specifically binds to Pseudomonas Psl comprising an antibody VL, where the VL comprises VLCDRl, VLCDR2, and VLCDR3 amino acid sequences identical to, or identical except for four, three, two, or one amino acid tutions in one or more of the VHCDRs to: SEQ ID NOs: 20, 21, and 22, SEQ ID NOs: 32, 33, and 34, SEQ ID NOs: 38, 39, and 40, SEQ ID NOs: 44, 45, and 46, SEQ ID NOs: 50, 51, and 52, SEQ ID NOs: 56, 57, and 58, or SEQ ID NOs: 62, 63, and 64, respectively.

Also disclosed is an isolated binding le e.g., an antibody or antigen—binding fragment thereof which specifically binds to Pseudomonas Psl comprising an antibody VL, where the VL comprises VLCDRl, VLCDR2, and VLCDR3 amino acid sequences identical to, or identical except for four, three, two, or one amino acid substitutions in one or more of the VHCDRs to: SEQ ID NOs: 20, 21, and 22, SEQ ID NOs: 32, 33, and 34, SEQ ID NOs: 38, 39, and 40, SEQ ID NOs: 44, 45, and 46, SEQ ID NOs: 50, 51, and 52, SEQ ID NOs: 56, 57, and 58, or SEQ ID NOs: 62, 63, and 64, respectively.

Some embodiments include the ed binding molecule e. g., an antibody or fragment 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.

Other ments e the isolated binding molecule e. g., an antibody or fragment thereof as described above, where maximum inhibition of P. aeruginosa attachment to lial cells is achieved at an antibody concentration of about 50 11ng or less, 5.0ug/ml or less, or about 0.5ug/ml or less, or at an antibody tration ranging from about 30 11ng to about 0.3 ug/ml, or at an dy concentration of about 1 ug/ml, or at an antibody concentration of about 0.3 ug/ml.

Certain ments include the isolated binding molecule e.g., an dy or fragment thereof as described above, where the OPK EC50 is less than about 0.5 ug/ml, less than about 0.05ug/ml, or less than about 0.005ug/ml, or where the OPK EC50 ranges from about 0.001 11ng to about 0.5 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.

Also included is the isolated binding molecule e.g., an antibody or fragment thereof as described above, which ically binds to P. aeruginosa Ps1 with an affinity characterized by a iation constant (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, m4NL5xm5M,m5NL5xm6M,mfiNLSXNJM,MJNLSXM$M,M$NLSXmy NLmgij10mMfl0mij10UMJ0UMJX10uMfl0qux10BMJ0BM, x 10'14 M, 10'14 M, 5 x 10'15 M, or 10'15 M, or wherein KD is in a range of about 1 x 10'10 to about 1 x 10'6 M. In one embodiment, an isolated antibody as described herein specifically binds to Pseudomonas Psl, with an affinity terized by a KD of about 1.18 x 10'7 M, as determined by the OCTET® binding assay. In another embodiment, an isolated antibody as described herein ically binds to Pseudomonas Ps1, with an affinity characterized by a KD of about 1.44 x 10'7 M, as determined by the OCTET® binding assay.

In various embodiments, the above—described binding molecules are humanized.

In various embodiments, the above—described binding molecules are chimeric.

In various embodiments, the above—described binding molecules are fully human.

In certain embodiments, the above—described binding molecules are Fab fragments, Fab' fragments, F(ab)2 fragments, or scFv fragments.

In certain embodiments, the above—described binding molecules comprise light chain constant regions consisting of a human kappa nt region or a human lambda constant region.

In certain embodiments, the above—described binding molecules comprise a heavy chain nt region or fragment thereof. In further embodiments, the heavy chain constant region or fragment thereof is a human IgG1.

In certain embodiments, the above—described g molecules are monoclonal antibodies.

In some embodiments, the above described binding molecules e.g., an antibodies or fragments thereof are 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 modif1er, pharmaceutical agent, a lymphokine, a heterologous antibody or fragment thereof, a detectable label, polyethylene glycol (PEG), and a combination of two or more of any said agents. In further embodiments, detectable label is ed from the group consisting of an , a fluorescent label, a chemiluminescent label, a bioluminescent label, a radioactive label, or a combination of two or more of any said detectable labels.

Additional embodiments include compositions comprising the above—described binding molecules e.g., antibodies or fragments thereof, and a r.

Certain embodiments include an ed polynucleotide sing a nucleic acid which s the above—described VH. In some embodiments, the polynucleotide further comprises a nucleic acid which encodes the above—described VL, where a binding molecule or antigen- binding fragment thereof expressed by the polynucleotide specif1cally binds Pseudomonas Psl.

In some embodiments the polynucleotide as described herein encodes an scFv molecule including VH and VL, comprising the nucleotide sequence 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. In other ments, the disclosure includes an isolated polynucleotide comprising a nucleic acid which encodes the described VL. In further embodiments, the polynucleotide r ses a nucleic acid which encodes the above—described VH, where a binding molecule or antigen—binding fragment thereof sed by the polynucleotide specifically binds Pseudomonas Psl.

Certain embodiments provide vectors comprising the above—described polynucleotides.

In further embodiments, the polynucleotides are operably associated with a promoter. In additional embodiments, the disclosure provides host cells comprising such vectors. In r embodiments, the disclosure provides s where the polynucleotide is ly associated with a promoter, where vectors can express a g molecule e. g., an antibody or fragment thereof as described above which specifically binds Pseudomonas Psl in a suitable host cell.

Some embodiments es a method of producing a g molecule e.g., an antibody or fragment f as described above which specifically binds Pseudomonas Psl, comprising culturing a host cell containing a vector comprising the described polynucleotides, and recovering said antibody, or fragment thereof. Further embodiments provide an isolated binding molecule or fragment thereof produced by the above—described method.

In some embodiments, the Pseudomonas species is Pseudomonas aeruginosa.

In further embodiments, the above—described binding molecules or fragments thereof, antibodies or fragments thereof, or compositions, bind to two or more, three or more, four or more, or five or more different P. aeruginosa 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. P. aeruginosa pes are rized according to an International Antigen Typing System (IATS) originally described in Liu, P.V. et al. Int. J. Syst. Bacteriol. 33:256—264 (1983), as supplemented, e.g., by Liu P.V., Wang S., J. Clin. Microbiol. 28:922—925 (1990).

Some embodiments are directed to a method of preventing or treating a monas infection in a t in need thereof, comprising stering to the subject an effective amount of the binding molecule or fragment thereof, the antibody or fragment f, the composition, the polynucleotide, the vector, or the host cell described herein. In further embodiments, the Pseudomonas infection 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 ion, a wound infection, a skin infection, a blood infection, a bone infection, or a combination of two or more of said ions. In further embodiments, the subject suffers from acute pneumonia, burn injury, corneal infection, cystic is, or a combination thereof.

Some embodiments are directed to a method of blocking or preventing attachment of P. aeruginosa to epithelial cells comprising contacting a mixture of lial cells and P. aeruginosa with the binding molecule or fragment thereof, the antibody or fragment thereof, the composition, the polynucleotide, the , or the host cell described herein.

Also disclosed is a method of promoting OPK of P. aeruginosa comprising contacting a e of phagocytic cells and P. aeruginosa with the binding molecule or fragment thereof, the antibody or fragment thereof, the composition, the cleotide, the vector, or the host cell described . In further ments, the phagocytic cells are entiated HL-60 cells or human polymorphonuclear leukocytes .

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES Figure l (A-F): Phenotypic whole cell screening with human antibody phage libraries identifled P. aeruginosa functionally active specific antibodies. (A) Overview of complete antibody selection strategy. (B) Flow diagram describing the process to isolate antibody variable region genes from patients recently d to P. aeruginosa. (C) Characteristics of the scFv phage display libraries, indicating the size and diversity of the cloned dy repertoire. (D) Comparison of the phage display selection efficiency using either the patient antibody library or a na'1've antibody y, when selected on P. aeruginosa 3064 A WapR (1) or P. aeruginosa PAOl MexAB OprM A WapR (2) in suspension. Bars indicate the output titers (in CFU) at each round of selection, and circles indicate the proportion of duplicated VH CDR3 sequences, an indication of clonal enrichment. (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 scFvs from selections and one irrelevant phage-scFv. (F) FACS g of P. aeruginosa specific antibodies with representative strains from unique P. aeruginosa serotypes.

For each antibody tested a human IgG negative control antibody is shown as a shaded peak.

Figure 2 (A—D): Evaluation of specific 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. aeruginosa serogroup Oll strain (9882—80.lux), with dilutions of purified WapR-004 and Cam-003 onal antibodies derived from phage panning. In both A and B, R347, an isotype matched human monoclonal antibody that does not bind P. aeruginosa ns, was used as a negative control. (A,B) Results are entative data from three independent experiments performed for each antibody. (C-D): Evaluation of Cam—003 promoting opsonophagocytic killing (OPK) of P. aeruginosa (C) Opsonophagocytosis assay with representative non-mucoid strains from clinically relevant O-antigen serotypes (6294 (O6 ExoU'), 6077 (011 ExoUI), 9882—80.lux (Oll ExoU'), 33356 (09 ExoU'), ux (O6) and 6206.1ux (Oll ExoU+)). (D) Opsonophagocytosis assay with representative mucoid strains that were engineered to be luminescent (A004.lux, A010.lux and A015.lux). The lines ent the mean percent killing and error bars represent the standard deviation. Percent killing was calculated ve to results obtained in assays run in the absence of antibody. (C,D) An R347 control was used within individual assays for each strain. For simplicity, the R347 control was not included in the figures. Results are representative data from three independent experiments performed for each strain.

Figure 3 (A—K): Identification of the P. aeruginosa Psl exopolysaccharide target of antibodies d from phenotypic screening. Reactivity of antibodies was determined by ct 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 membrane protein and was used as a positive control. (C) FACS g analysis of Cam—003 to PAOl and PAOlApSlA. Cam—003 is indicated by a solid black line and clear peak; an isotype d non—P. aerugz‘nosa—specific human IgG1 antibody was used as a negative control and is indicated by a gray line and shaded peak. (D) LPS purified from PAOl and PAOlApSlA was resolved by SDS-PAGE and immunobloted with antisera d from mice vaccinated with PAOlAwapRAaZgD, a mutant strain deficient in O-antigen transport to the outer ne and alginate production. (E) Cam-003 ELISA binding data with isogenic mutants of PAOl. Lane 1: PAOlAwprAaZgD; Lane 2: PAOlAwprAaZgDApsZA; Lane 3: PAOlAwprAaZgDApeZA; Lane 4: PAOlAwprAaZgDApSZA + pUCP; Lane 5: PAOlAwprAaZgDApsZA + pPWl45. pPWl45 is a pUCP expression vector containing psZA. * tes P<0.005 using the Mann—Whitney U—test when comparing Cam—003 vs. R347 binding. (F and G) phagocytosis assays indicating that Cam-003 only mediates g 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, 04, and WapR—Ol6 with PAOl AwprAaZgD and PAOl AwprAaZgDApslA. (J) Reactivity of antibodies was determined by indirect ELISA on plates coated with indicated P. aeruginosa strains: wild type PAOl, PAOlAwpr, PAOlAwprAalgD, PAOlArmZC and PAOlAgaZU. R347 was used as a negative control in all experiments. (A, B, C, F, G, H, I, J).

Each panel is a entative data set from three independent experiments.

(K) Anti—Psl antibody capture of enriched Psl isolated from whole P. aeruginosa cells as ed on a FORTEBIO® OCTET® 384 ment. R347 was used as a negative control.

Results are representative data from three independent experiments.

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

Figure 5 (A—U): In vivo ed 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 l, Cam—003 was assayed for binding to strains grown to log phase from an overnight culture (~5 x 108/ml). (B) The inocula 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 eritoneal challenge, bacteria was ted from mice by peritoneal lavage and assayed for the presence of Psl with Cam—003 by flow cytometry. (D) Four hours and (E) twenty four hours post intranasal challenge, bacteria were harvested from mice by bronchoalveolar lavage (BAL) and assayed for the presence of Psl with Cam—003 by flow cytometry. Each flow cytometry panel is a representative data set from five independent experiments (F—U) The binding of P. aeruginosa specific antibodies (Cam—003, Cam—004 and Cam—005) to representative strains from unique P. aeruginosa pes (F) PAOl(05), (G) 2135 (01), (H) 2531 (01), (I) 2410 (06), (J) 2764 (011), (K) 2757 (011), (L) 33356 (09), (M) 33348 (01), (N) 3039 (NT), (0) 3061 (NT), (P) 3064 (NT), (Q) 19660 (NT), (R) 9882—80 (01 1), (S) 6073 (01 1), (T) 6077 (01 1) and (U) 6206 (01 1). Cam—003, Cam—004, and Cam—005 antibodies are shown by as gray line and a clear peak; the human IgG negative control antibody is shown as a solid black line and a shaded peak.

Figure 6 (A-G): Survival rates for animals treated with anti-Psl monoclonal antibodies Cam—003 or WapR—004 in a P. aeruginosa acute pneumonia model. (A-D) Animals were treated with Cam-003 at 45, 15, and 5mg/kg and R347 at g or PBS 24 hours prior to intranasal ion with (A) PAOl (1.6 x 107 CPU), (B) 33356 (3 x 107 CPU), (C) 6294 (7 x 106 CPU), (D) 6077 (1 x 106 CPU). (E-F) Animals were treated with WapR—004 at 5 and 1mg/kg as indicated followed by infection with 6077 at (E) (8 x 105 CPU), or (F) (o x 105 CPU). Animals were carefully monitored for survival up to 72 hours (A—D) or for 120 hours (E—F). (G) Animals were treated with Cam-003 at 15 mg/kg or 5 mg/kg, or R347 at 15 mg/kg 24 hours prior to asal infection with PAOl (4.4 x 107 CPU), and Cam-003 at 15 mg/kg 24 hours prior to intranasal infection with PAOlApslA (3 x 107 CPU). 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 Log—rank test for Cam—003 vs. R347. (A) Cam—003 (45mg/kg — P<0.0001; lSmg/kg — P=0.0003; 5mg/kg — P=0.0033). (B) Cam—003 (45mg/kg — P=0.0012; lSmg/kg — P=0.0012; 5mg/kg — P=0.0373). (C) 3 (45mg/kg — P=0.0007; lSmg/kg — P=0.0019; 5mg/kg — P=0.02l2). (D) Cam—003 (45mg/kg — P<0.0001; lSmg/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): P=0.4848; WapR—004 (5mg/kg) vs. R347 g): P<0.0001; WapR—004 (lmg/kg) vs. R347 (5mg/kg): P=0.0886; WapR—004 (5mg/kg) vs. Cam—003 (5mg/kg): P=0.0017; WapR—004 (lmg/kg) vs. Cam—003 (lmg/kg): P=0.2468; R347 (5mg/kg) vs. PBS: P=0.6676] (F) 03 (5mg/kg) vs. R347 (5mg/kg): P=0.0004; Cam—003 (lmg/kg) vs. R347 g): P<0.0001; WapR—004 (5mg/kg) vs. R347 (5mg/kg): P<0.0001; WapR—004 g) vs. R347 (5mg/kg): P<0.0001; WapR—004 g) 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: P=0.6676. (G) Cam—003 (lSmg/kg — P=0.0028; 5mg/kg — P=0.0028)]. 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 d 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 (1 x 106 CFU), and WapR—004 antibody 24 hours prior to infection with (E) 6294 (~l x 107 CFU), and (F) 6206 (~l 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 3 or WapR—004 vs. R347.

(A) Lung: Cam—003 (45mg/kg — P=0.0015; lSmg/kg — P=0.002l; 5mg/kg — P=0.0015); Spleen: Cam—003 kg — 20; lSmg/kg — P=0.0367); Kidneys: Cam—003 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: Cam—003 (45mg/kg — P=0.0003; g — P=0.003 9; 5mg/kg — P=0.0068); Spleen: Cam—003 (45mg/kg — P=0.0057; lSmg/kg — P=0.0230; 5mg/kg — P=0.0012); (D) Lung: Cam—003 (45mg/kg — 05; lSmg/kg — P=0.0003; 5mg/kg — 07); Spleen: Cam—003 (45mg/kg — P=0.0015; lSmg/kg — P=0.0089; 5mg/kg — P=0.0089); Kidneys: Cam—003 (45mg/kg — P=0.019l; lSmg/kg — P=0.0355; 5mg/kg — P=0.002l). (E) Lung: WapR—004 kg — P=0.0011; 5mg/kg — P=0.0004; lmg/kg — P=0.0002); Spleen: WapR—004 (lSmg/kg — 01; 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); : 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 thermal injury model. Mice were treated with a control IgGl antibody or Cam-003 at g (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 — 2 x 106 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 ion, the corneal pathology scores were ated followed by removal of the eye for ination of viable CFU. ences 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; Cam—003 (45mg/kg) vs. WapR—004 kg): P=0.0025; WapR—004 (45mg/kg) vs. WapR—004 (lSmg/kg): P=0.l33l; 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 , P=0.0017). Results are representative of at least three independent experiments. (n) refers to number of s in each group.

Figure 9 (A—E): A Cam-003 Fc mutant antibody, CamTM, has diminished OPK and in viva eff1cacy but maintains anti-cell attachment ty. (A) PAOl.lux OPK assay with Cam- 003 and CamTM, which harbors mutations in the Fc domain that prevents Fc interactions with Fcy receptors (Oganesyan, V., et al., Acta Crystallogr D Biol Crystallogr 64, 700—704 (2008)). R347 was used as a negative control. Results are representative data from three independent ments. (B) PAOl cell attachment assay with Cam-003 and CamTM.

Results are representative data from two independent experiments. (C-E) Acute pneumonia model comparing efficacy of Cam—003 vs. 3—TM. P. nosa strain 6077 acute pneumonia model using BALB/c mice inoculated with (C) 1.22 x 106 (D) 2.35 x 105 or (E) 1.07 x 106 comparing efficacy of Cam—003 versus Cam-003—TM. Mice were treated with antibody 24 hours before challenge. (C—E) Ten animals were used in each group. Results are representative data from two independent experiments. 2012/041538 Figure 10 (A-B): A: Epitope g and identification 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 ies 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 antibody. B. Relative binding affinities of various WapR-004 mutants as measured on a FORTEBIO® OCTET® 384 instrument. Also shown are OPK EC50 values for the various s.

Figure 11: (A-M): Evaluation of WapR—004 (W4) mutants clones in the P. aeruginosa OPK assay (A—M) Opsonophagocytosis assay with luminescent P. aeruginosa serogroup 05 strain (PAOl.lux), with dilutions of different W4 mutant clones in c format. In some instances, W4 IgGl was included in the assay and is ted as W4-IgGl. W4-RAD-Cam and —GB represent the same WapR—004RAD sequence described herein. "W4—RAD" is a shorthand name for WapR-004RAD, and W4-RAD-Cam and -GB designations in panels D h M represent two ent preparations of WapR—004RAD. In all experiments, R347, a human IgG1 monoclonal antibody that does not bind P. aeruginosa antigens, was used as a negative control.

Figure 12: Method of site-directed conjugation of Polymyxin B (PMB) to mAbs in which a heterobifunctional SM-PEG12 linker (Pierce) was conjugated to a primary amine on PMB under ions determined to favor conjugation of a single . Conjugation efficiency and levels free PMB—linker in the samples were determined by UPLC and mass spectrometry.

Figure 13 (A—B): PMB—mAb site—directed conjugates. Using the developed site—directed conjugation method, PMB was conjugated to CAM-003 and A7 (hIgGl l) mAb variants with either one (SM, NDlO), two (DM, NDlO/l9) or three (TM, ND4/10/19) cysteine engineered into the Fc region. A: Cam—003 and A7 Fc region mutated residues. B: The average number of PMB in PMB-mAb conjugates (single mutant (SM) > double mutant 1 (DMl) > double mutant 2 (DM2)).

Figure 14 (A-B): Evaluation of PMB-mAb conjugates binding to wild-type P. aeruginosa PAOl cells by FACS analysis. A: Cam—003 (Cam—003—SM—PMB, Cam—003-DM1- PMB, Cam—003—DM2—PMB, mock—conjugated wild—type Cam—003 (Cam—003—Mock—PMB)). B: A7 control conjugates -PMB, A7-DMl-PMB, A7-DM2—PMB, mock-conjugated wild- type A7 (A7—Mock—PMB)). R347 was used as a negative control in all experiments.

Figure 15 (A-B): OPK activity of PMB-mAb conjugates against A: P. nosa PAOl wild—type strain and B: against the ApsZA P. aeruginosa strain which does not express the Ps1 target.

Figure 16 (A-B): Neutralization of P. aeruginosa LPS by PMB—mAb ates. A: PMB—Cam—003 conjugates and onjugated wild—type Cam—003 and B: PMB—A7 conjugates and mock—conjugated wild—type A7.

Figure 17: Structure showing polymyxin, a cyclic antibacterial lipopeptide that neutralize the proinflammatory effects of LPS and can be used for the treatment of Gram- negative MDR ions (colistin/polymyxin E). Polymyxins have 5 positively charged diamonbutyric acids (circled) that mediate interactions with vely-charged Lipid A in LPS and lize its proinflammatory activity.

Figure 18 (A-B): OPK activity by human HL-60 neutrophil cell line in the presence of rabbit complement was evaluated using P. aeruginosa strain PAOl expressing bacterial luciferase. A: % killing by CAMPMB Conjugates. B: % killing by A7-PMB Conjugates.

Figure 19 (A—B): A. Percent Survival of C57B1/6 mice dosed with 45 mg/kg CAM—TM— PMB Conjugates. B: Percent Survival of C57B1/6 mice dosed with 45 mg/kg A7—TM-PMB Conjugates.

Figure 20 (A—B): A. Percent Survival of 6 mice dosed with 45 mg/kg, 15 mg/kg and 5 mg/kg CAM-TM—PMB ates. B: Percent Survival of 6 mice dosed with 45 mg/kg, 15 mg/kg and 5 mg/kg A7-TM-PMB Conjugates.

Figure 21 (A—C): Percent survival of C57B1/6 mice dosed with mAb or PMB—mAb conjugates i.p A: 10 mg/kg. B: 1 mg/kg. C: 0.1 mg/kg.

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 g molecule which specifically binds to Pseudomonas Ps1," is understood to represent one or more binding molecules which specifically bind to Pseudomonas Ps1. 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” as well as plural “polypeptides,” and refers to a le composed of monomers (amino acids) -l6- linearly linked by amide bonds (also known as peptide . 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 product. Thus, peptides, dipeptides, tripeptides, 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 ed within the definition of "polypeptide,” and the term “polypeptide” can be used instead 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 without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A ptide can be derived from a l biological source or produced by recombinant logy, 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, 10 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 structure, although they do not necessarily have such structure. Polypeptides with a defined three—dimensional structure are referred to as folded, and ptides 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 d to at least one carbohydrate 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 thereof is intended a polypeptide that is not in its natural milieu. No ular level of purification is required. For example, an isolated polypeptide can be d from its native or natural environment.

Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated as disclosed herein, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.

Other polypeptides disclosed herein are fragments, derivatives, analogs, or variants of the ing polypeptides, and any combination f. The terms "fragment," "varian ," "derivative" and "analog" when referring to a binding le such as an antibody which ically binds to Pseudomonas Psl as disclosed herein include any polypeptides which retain at least some of the antigen-binding ties of the ponding native antibody or polypeptide. Fragments of polypeptides e, for example, proteolytic fragments, as well as deletion fragments, in addition to c antibody fragments discussed ere .

Variants of a binding molecule, e. g., an antibody which specifically binds to Pseudomonas Psl as disclosed herein include fragments as described above, and also polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. Variants can occur lly or be non—naturally occurring. Non—naturally occurring variants can be produced using art—known mutagenesis ques. Variant polypeptides can comprise vative or non— conservative amino acid substitutions, deletions or additions. tives of a binding molecule, e.g., an antibody which specifically binds to Pseudomonas Psl as sed herein are polypeptides which have been altered so as to exhibit additional features not found on the native polypeptide. Examples include fusion ns. Variant polypeptides can also be referred to herein as "polypeptide analogs. H As used herein a "derivative" of a binding le, e.g., an antibody which specifically binds to Pseudomonas Psl refers to a subject polypeptide having one or more residues chemically derivatized by reaction of a functional side group. Also included as "derivatives" are those peptides which contain one or more lly occurring amino acid derivatives of the twenty standard amino acids. For example, 4-hydroxyproline can be substituted for proline; 5—hydroxylysine can be substituted for ; 3—methylhistidine can be substituted for histidine; homoserine can be substituted for serine; and ornithine can be substituted for lysine.

The term "polynucleotide" 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 c acids (PNA)). The term "nucleic acid" refer 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 intended 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 antibody which specifically binds to Pseudomonas Psl contained in a vector is considered isolated as disclosed herein. Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (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 include such molecules produced synthetically. In addition, polynucleotide or a nucleic acid can be or can include a regulatory element such as a promoter, me binding site, or a transcription ator.

As used herein, a "coding region" 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 ators, introns, and the like, are not part of a coding region. Two or more coding s can be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide ucts, e.g., on separate (different) vectors. Furthermore, any vector can contain a single coding , or can comprise two or more coding s, 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 nucleic acid can encode heterologous coding s, either fused or unfused to a nucleic acid encoding an a binding molecule which specifically binds to Pseudomonas Psl, 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 functional domain.

In certain 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 transcription or ation control elements operably associated with one or more coding s. 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 regulatory 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 linkage between the two DNA fragments does not interfere with the ability of the expression regulatory ces to direct the expression of the gene product or ere with the ability of the DNA template to be transcribed. Thus, a promoter region would be operably associated with a nucleic acid encoding a polypeptide if the er was capable of effecting transcription of that nucleic acid. The er can be a cell-specific er that directs substantial transcription of the DNA only in WO 70807 predetermined cells. Other transcription control elements, besides a promoter, for example enhancers, operators, sors, and transcription ation signals, can be operably associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcription control regions are disclosed herein.

A y 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 enhancer segments from cytomegaloviruses (the immediate early promoter, in conjunction with -A), simian virus 40 (the early promoter), and retroviruses (such as Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit B-globin, as well as other sequences e of controlling gene expression in eukaryotic cells. Additional suitable transcription control s include tissue—specific ers and enhancers as well as kine—inducible promoters (e. g., promoters inducible by interferons or interleukins).

Similarly, a variety of translation control elements are known to those of ordinary 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).

Polynucleotide and nucleic acid coding s can be associated with additional coding regions which encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide as disclosed herein, e.g., a polynucleotide encoding a binding le which specifically binds to Pseudomonas Psl, e.g., an antibody, or n—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 g protein chain across the rough endoplasmic reticulum has been initiated. Those of ordinary skill in the art are aware that polypeptides secreted by rate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the complete or "full length" polypeptide to produce a ed 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 operably associated with it.

Alternatively, a heterologous mammalian signal e, 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 n binding molecules, or antigen-binding fragments, variants, or derivatives thereof. Unless specifically ing 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 molecules or nts that bind antigen in a manner similar to antibody molecules.

As used herein, the term “binding molecule” refers in its st sense to a molecule that specifically binds an antigenic determinant. A miting example of an antigen binding molecule is an antibody or fragment thereof that retains antigen-specific binding.

The terms "antibody" and "immunoglobulin" can be used interchangeably herein. An dy (or a fragment, t, or derivative thereof as disclosed herein comprises at least the variable domain of a heavy chain and at least the variable domains of a heavy chain and a light chain. Basic immunoglobulin structures in vertebrate systems are relatively well tood.

See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor tory Press, 2nd ed. 1988).

As will be discussed in more detail below, the term “immunoglobulin” ses various broad classes of polypeptides that can be distinguished 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 determines the "class" of the dy as IgG, IgM, IgA IgG, or IgE, respectively. The immunoglobulin subclasses (isotypes) e.g., IgGl, Ing, IgG3, IgG4, IgAl, etc. are well characterized and are known to confer onal 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.

Light chains are fied as either kappa or lambda (K, 7»). 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 disulf1de linkages or non-covalent es when the immunoglobulins .21. 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 onal 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 properties such as secretion, transplacental mobility, Fc receptor binding, ment binding, and the like. By convention the numbering of the nt region domains increases as they become more distal from the antigen binding 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 comprise 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 defines a three ional antigen binding site. This quaternary binding molecule structure forms the antigen g site present at the end of each arm of the Y. More specifically, the antigen g site is defined by three CDRs on each of the VH and VL chains.

In naturally occurring antibodies, the six “complementarity determining regions” or “CDRs” present in each antigen binding domain are short, non—contiguous sequences of amino acids that are ically positioned to form the n binding domain as the antibody assumes its three dimensional configuration in an aqueous environment. The remainder of the amino acids in the antigen binding domains, referred to as "framework" s, show less inter— molecular variability. The ork regions largely adopt a B-sheet conformation and the CDRs form loops which connect, and in some cases form part of, the B—sheet structure. 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 reactive n.

This complementary surface promotes the non-covalent binding of the antibody to its cognate epitope. The amino acids comprising the CDRs and the framework s, respectively, can be 2012/041538 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., US. 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 e all such gs unless explicitly stated to the contrary. A specific example is the use of the term "complementarity determining region" ("CDR") to describe the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides.

This particular region has been described by Kabat et al., US. Dept. of Health and Human es, "Sequences of Proteins of Immunological Interest" (1983) and by Chothia et al., J.

M01. Biol. 196:901—917 (1987), which are orated herein by reference, where the definitions include overlapping or subsets of amino acid residues when compared against each other. heless, application of either definition to refer to a CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues which ass 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 particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues se a particular CDR given the variable region amino acid sequence of the antibody.

TABLE 1; CDRDefinitions1 Chothia VH CDRl 26—32 VH CDR2 52—58 VH CDR3 VL CDRl VL CDR2 VL CDR3 89—97 91—96 1Numbering of all CDR definitions in Table 1 is according to the numbering conventions set forth by Kabat et all. (see .

Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody. One of ry skill in the art can unambiguously assign this 2012/041538 system of "Kabat numbering" to any variable domain sequence, without reliance on any experimental data beyond the sequence itself. 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 Psl, e. g., an antibody, or n-binding fragment, variant, or derivative thereof as disclosed herein are according to the Kabat numbering system. g molecules, e. g., antibodies or antigen-binding fragments, variants, or derivatives thereof include, but are not limited to, onal, monoclonal, human, humanized, or chimeric antibodies, single chain antibodies, epitope-binding fragments, e.g., Fab, Fab' and F(ab')2, Fd, Fvs, single-chain Fvs , -chain antibodies, disulfide-linked Fvs (dev), fragments comprising either a VL or VH domain, fragments produced by a Fab sion library. 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 generally meant that a binding molecule, e.g., an antibody or fragment, variant, or derivative thereof binds to an epitope via its antigen binding domain, and that the binding entails some complementarity n the antigen binding domain and the epitope. According to this definition, a binding molecule is said to "specifically bind" to an epitope when it binds to that epitope, via its antigen binding domain more readily than it would bind to a random, unrelated e. The term "specificity" is used herein to qualify the ve affinity by which a certain binding molecule binds to a certain epitope. For example, g 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 readily 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 dy can cross-react with the related epitope.

By way of non—limiting example, a binding molecule, e.g., an antibody 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 molecule such as an antibody can be considered to bind a first antigen preferentially if it binds the first epitope with an ty that is at least one order of magnitudeless than the antibody’s KD 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 affinity that is at least two orders of ude less than the antibody’s KD for the second e.

In another non-limiting example, a binding le, 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 A binding molecule, e.g., an antibody or fragment, variant, or derivative thereof disclosed herein can be said to bind a target antigen, e.g., a polysaccharide disclosed herein or a nt or variant thereof 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. 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, '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 fragment, 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 g molecule as sed herein can be said to bind a target antigen, e.g., a polysaccharide with an on rate ) greater than or equal to 105 M'1 sec'l, 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 t 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 nt to the epitope. Competitive inhibition can be determined by any method known in the art, for example, competition ELISA assays. A binding molecule can be said to competitively inhibit binding of the nce antibody or antigen 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 herein, the term "affinity" refers to a measure of the strength of the binding 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 between a population of immunoglobulins 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 between a bivalent monoclonal antibody and an antigen with a highly repeating e structure, such as a r, would be one of high avidity.

Binding molecules or antigen-binding nts, 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 binding molecule, e. g., an antibody or fragment, variant, or derivative thereof, specific for one antigen, to react with a second antigen; a measure of relatedness between two different antigenic substances. Thus, a binding molecule is cross reactive if it binds to an epitope other than the one that d its formation. The cross reactive epitope generally contains many of the same complementary structural es as the inducing epitope, and in some cases, can actually fit better than the al.

A binding molecule, e. g., an antibody or nt, variant, or derivative thereof can also be described or specified in terms of their g affinity to an antigen. For example, a binding molecule can bind to an antigen with a dissociation constant or KD no greater than 5 x 10'2 M, 10' 2M, 5 x 10'3 M, 10'3 M, 5 x 10'4 M, 10'4M, 5 x10'5 M, 10'5 M, 5 x 10'6M, 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, 10'10 M, 5 x 10'11 M, 10'11 M, 5 x 10'12 M, '12M, 5 M, 10'13 M, 5 x 10'14 M, 10'14 M, 5 x10'15 M, or WM.

Antibody fragments ing single-chain antibodies can comprise the variable (s) alone or in ation with the entirety or a portion of the ing: hinge region, CH1, CH2, and CH3 s. Also included are antigen-binding fragments also comprising any combination of variable region(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, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or n antibodies. In another embodiment, the le region can be condricthoid in origin (e.g., from sharks). As used herein, "human" dies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from s 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 al.

As used herein, the term “heavy chain portion” includes amino acid ces 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, , and/or lower hinge region) domain, a CH2 domain, a CH3 , or a variant or fragment f For example, a binding molecule, e.g., an antibody 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 polypeptide 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 domain, a CH2 domain, and a CH3 domain. In another embodiment, a binding molecule, e. g., an antibody or fragment, variant, or derivative f 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 naturally occurring immunoglobulin molecule.

The heavy chain portions of a g molecule, e.g., an dy as disclosed herein can be d 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 molecule. In r example, a heavy chain portion can comprise a chimeric hinge derived, in part, from an IgGl le and, in part, from an IgG4 molecule.

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

Binding molecules, e.g., dies 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 polysaccharide that they recognize or specifically bind. The n of a target polysaccharide which specifically interacts with the antigen binding domain of an antibody is an "epitope," or an "antigenic determinant." A target antigen, e. g., a polysaccharide can comprise a single epitope, but typically comprises at least two epitopes, and can include any number of epitopes, depending on the size, conformation, and type of antigen.

As previously indicated, the subunit structures and three dimensional configuration of the constant regions of the various immunoglobulin classes are well known. As used herein, the term “VH domain” includes the amino terminal variable domain of an immunoglobulin heavy chain and the term “CH1 domain” includes the first (most amino terminal) constant region domain of an immunoglobulin heavy chain. The CH1 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” es the n of a heavy chain le that extends, e.g., from about residue 244 to residue 360 of an antibody using conventional numbering schemes (residues 244 to 360, Kabat numbering system; and es 231—340, EU numbering system; see Kabat EA et a]. 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 extends 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 le that joins the CH1 domain to the CH2 domain. This hinge region comprises approximately 25 residues and is e, thus ng the two N—terminal antigen binding regions to move independently. Hinge regions can be subdivided into three distinct domains: upper, middle, and lower hinge s (Roux et al., J. Immunol. [61:4083 (1998)).

As used herein the term “disulfide bond” includes the covalent bond formed between 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 lly ing IgG molecules, the CH1 and CL regions are linked by a disulfide bond and the two heavy chains are linked by two ide bonds at positions ponding to 239 and 242 using the Kabat numbering system (position 226 or 229, EU ing system).

As used herein, the term ric dy” will be held to mean any antibody wherein the immunoreactive region or site is obtained or derived from a first species and the constant region (which can be intact, l or modified) is obtained 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.

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 partial ork region replacement and sequence changing. Although 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 ably from an antibody from a different species. An ered antibody in which one or more " CDRs from a non-human antibody of known specificity is grafted into a human heavy or light chain framework region is referred to herein as a "humanized antibody." 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 in the activity of the target binding 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 skilled 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— monas Psl antibodies) in which all of the functional domains comprising the polypeptide are ctly active. As used herein, the term “improperly folded polypeptide” includes polypeptides in which at least one of the functional domains of the polypeptide 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 tic or chemical coupling of peptides or some combination of these techniques).

As used herein, the terms "linked," "fused" or "fusion" are used interchangeably. These terms refer to the joining together of two more elements or components, 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 uous longer ORF, in a manner that maintains the correct ational reading frame of the original ORFs. Thus, a recombinant fusion protein is a single protein ning two or more segments that correspond to polypeptides encoded by the original ORFs (which segments are not normally so joined in nature.) Although the reading frame is thus made continuous throughout the fused segments, the segments can be physically or lly separated by, for example, in-frame linker sequence. For example, cleotides ng the CDRs of an immunoglobulin le 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 nslated as part of a continuous ptide.

In the context of polypeptides, a "linear sequence" or a "sequence" 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 ure 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 limitation, gene knockdown as well as both transient sion and stable expression. It es without limitation transcription of the gene into messenger 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 sors. Expression of a gene produces a "gene product." As used herein, a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription 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., methylation, glycosylation, the addition of lipids, association with other protein subunits, proteolytic cleavage, and the like.

As used , 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. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) 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. ment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already 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 ion.

By "subject" or "individual" or "animal" or "patien " or “mammal,” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or y is desired.

Mammalian ts e humans, domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, , cattle, cows, bears, and so on.

As used herein, phrases such as “a subject that would t from administration of an anti—Pseudomonas Psl antibody” and “an animal in need of treatment” includes subjects, such as ian subjects, that would benefit from administration of an anti-Pseudomonas Psl antibody used, e.g., for detection of Pseudomonas Ps1 (e.g., for a diagnostic ure) and/or from treatment, i.e., palliation or prevention of a disease, with an anti—Pseudomonas Psl antibody. As bed in more detail herein, the anti—Pseudomonas Ps1 antibody can be used in ugated form or can be conjugated, e.g., to a drug, prodrug, or an e.

II. BINDING LES One embodiment is directed to an isolated binding molecule e.g., an antibody or n- binding fragment thereof which specifically binds to Pseudomonas Psl, wherein the binding molecule (a) can inhibit attachment of Pseudomonas aeruginosa to epithelial cells, (b) can e, mediate, or enhance opsonophagocytic killing (OPK) of P. aeruginosa, or (c) can inhibit attachment of P. aeruginosa to epithelial cells and can promote, mediate, or enhance OPK of P. aeruginosa. In certain embodiments, the binding molecule or fragment thereof as described above can be antibody or antigen—binding fragment thereof such as Cam—003 or WapR—004.

As used herein, the term “antigen binding domain” includes a site that ically binds an e on an antigen (e. g., an epitope of monas Psl). 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 le regions determines the specificity of the antibody.

The disclosure is more ically directed to an isolated binding molecule, e.g., an antibody or antigen-binding fragment f which specifically binds to the same Pseudomonas Psl epitope as an antibody or antigen-binding fragment thereof sing the heavy chain variable region (VH) and light chain variable region (VL) region of WapR-004, Cam-003, Cam— 004, or Cam—005.

Further 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 04, Cam—003, 4, or 5.

One ment is directed to an isolated binding molecule, e.g., an antibody or n—binding fragment thereof which specifically binds to the same Pseudomonas Psl epitope as an antibody or antigen-binding fragment thereof comprising the VH and VL region of WapR- 001, WapR—002, or 03.

Also included is an isolated g 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—OOl, WapR—002, or 03.

Further included is an isolated binding molecule, e. g., an dy 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.

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 sing the VH and VL of WapRl6.

Methods of making antibodies are well known in the art and described herein. Once antibodies to various nts of, or to the full-length Pseudomonas Psl without the signal sequence, have been ed, determining which amino acids, or epitope, of Pseudomonas Psl to which the antibody or antigen binding fragment binds can be determined by epitope mapping protocols as described herein as well as methods known in the art (6. g. double antibody— sandwich ELISA as bed in "Chapter 11 — Immunology," Current Protocols in Molecular Biology, Ed. l et al., v.2, John Wiley & Sons, Inc. (1996)). onal epitope mapping protocols can be found in Morris, G. Epitope Mapping Protocols, New Jersey: Humana Press (1996), which are both incorporated herein by reference in their entireties. Epitope mapping can also be med by commercially available means (i.e. ProtoPROBE, Inc. (Milwaukee, Wisconsin)).

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

In certain embodiments an anti—Pseudomonas Psl binding molecule, e.g., an antibody or antigen-binding fragment, variant or derivative f as disclosed herein binds specifically to at least one epitope of Psl, i.e., binds to such an epitope more readily than it would bind to an unrelated, or random e; binds preferentially to at least one epitope of Psl, i.e., binds to such an epitope more readily 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 n epitope of Psl; or binds to at least one epitope of Psl with an affinity characterized by a dissociation constant KD of less than about 5 x 10'2 M, about 10'2 M, about 5 X ‘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 ‘6 M, about 10‘6 M, about 5 x 10‘7 M, about 10'7 M, about 5 x 10'8 M, about 10'8 M, about 5 x ‘9 M, about 10‘9 M, about 5 x lO'lOM, 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 10'15 M.

As used in the context of binding dissociation constants, the term “about” allows for the degree of variation inherent in the methods ed for ing 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 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 With an off rate (k(off)) of 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' 1, 10'6 sec'l, 5 X 10'7 sec"1 or 10'7 sec'l.

In other embodiments, a binding molecule, e.g., an antibody, or antigen-binding fragment, variant, or tive thereof as disclosed herein binds Pseudomonas Psl 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 n-binding fragment, t, or derivative thereof as disclosed herein binds Pseudomonas Psl With an on rate (k(on)) greater than or equal to 105 M"1 sec'l, 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 binding molecule, e.g., an antibody, or antigen—binding fragment, variant, or derivative thereof as described herein promotes opsonophagocytic killing ofPseudomonas, or inhibits Pseudomonas binding to lial cells.

In certain embodiments described , the Pseudomonas Psl target is Pseudomonas aeruginosa Psl. In other embodiments, certain binding molecules described herein can bind to structurally related polysaccharide molecules regardless of their source. Such Psl—like molecules would be expected to be identical to or have sufficient structural relatedness to P. aeruginosa Psl to permit ic recognition by one or more of the binding molecules disclosed. For example, certain binding les described herein can bind to Psl—like molecules produced by other bacterial species, for example, Psl—like molecules produced by other Pseudomonas species, e.g., Pseudomonas cens, Pseudomonas putida, 0r Pseudomonas alcaligenes. Alternatively, n g molecules as described herein can bind to Psl—like molecules produced synthetically or by host cells genetically modified to produce Psl—like molecules.

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

An anti—Pseudomonas Psl binding molecules, e.g., antibodies or antigen-binding fragments, variants, or derivatives thereof 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 system. Activation of complement is important in the opsonization and lysis of pathogens. The activation of complement also stimulates the inflammatory response and can also be involved in autoimmune hypersensitivity.

Further, dies bind to ors 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 specific for ent classes of antibody, including IgG (gamma receptors), IgE (epsilon receptors), IgA (alpha receptors) and IgM (mu receptors).

Binding of antibody to Fc receptors on cell surfaces triggers a number of important and diverse biological ses including engulfment and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called dy—dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory ors, placental transfer and control of immunoglobulin production.

Accordingly, certain embodiments disclosed herein include an anti—Pseudomonas Psl binding le, e.g., an antibody, or antigen-binding fragment, variant, or derivative thereof, in which at least a fraction of one or more of the constant region domains has been deleted or otherwise 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 ed with a whole, unaltered antibody of approximately the same immunogenicity. For example, certain binding molecules described 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 n antibodies, one entire domain of the nt region of the modified antibody will be deleted, for example, all or part of the CH2 domain will be deleted. d forms of anti—Pseudomonas Psl g molecules, e.g., antibodies or n— binding fragments, variants, or derivatives f can be made from whole precursor or parent antibodies using techniques known in the art. Exemplary techniques are discussed elsewhere herein.

WO 70807 In certain ments both the variable and constant regions of anti—Pseudomonas Psl binding molecules, e. g., antibodies or antigen-binding fragments 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 dies 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 challenge, but whose endogenous 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 ere herein.

Anti—Pseudomonas Psl binding molecules, e.g., antibodies or antigen-binding fragments, variants, or derivatives thereof as disclosed herein can be made or manufactured using ques that are known in the art. In certain embodiments, binding molecules or fragments thereof are “recombinantly produced,” i.e., are produced using recombinant DNA technology.

Exemplary techniques for making dy les or fragments thereof are discussed in more detail elsewhere herein.

In certain anti-Pseudomonas Psl binding molecules, e.g., antibodies or antigen-binding fragments, variants, or derivatives thereof described herein, the Fc portion can be mutated to decrease or function using techniques known in the art. For example, the deletion or inactivation (through point ons or other means) of a constant region domain can reduce Fc receptor binding of the ating modified antibody thereby sing tumor localization. In other cases it can be that constant region cations moderate ment binding and thus reduce the serum half—life and nonspecific association of a conjugated cytotoxin. Yet other modifications of the constant region can be used to modify disulflde linkages or oligosaccharide moieties that allow for enhanced localization due to increased antigen specificity or antibody flexibility. The resulting physiological profile, bioavailability and other biochemical effects of the modifications, such as zation, tribution and serum half-life, can easily be measured and quantified using well known immunological techniques without undue experimentation.

In certain embodiments, anti—Pseudomonas Psl binding molecules, e.g., antibodies or antigen-binding fragments, variants, or derivatives f will not elicit a deleterious immune response in the animal to be treated, e. g., in a human. In one embodiment, anti—Pseudomonas Psl 2012/041538 binding molecules, e. g., antibodies or antigen-binding fragments, variants, or derivatives thereof are modified to reduce their immunogenicity using art-recognized techniques. For example, dies can be humanized, de—immunized, or chimeric dies can be made. These types of antibodies are derived from a non-human dy, typically a murine or primate antibody, that retains 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 regions 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 regions with or without ion of critical framework residues; or (c) transplanting the entire non- human variable domains, but "cloaking" them with a human-like section by replacement of surface residues. Such methods are disclosed in Morrison et al., Proc. Natl. Acad. Sci. 81:6851— 6855 (1984); on et al., Adv. Immunol. 44:65—92 (1988); Verhoeyen etal., Science 239:1534—1536 (1988); Padlan, Molec. Immun. 28:489—498 (1991); Padlan, Molec. Immun. 31:169—217 (1994), 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 antibody. As used , the term “de-immunization” includes alteration of an antibody to modify T cell epitopes (see, e.g., WO9852976A1, W00034317A2). For example, VH and VL ces from the starting antibody are analyzed and a human T cell epitope "map" from each V region showing the location of epitopes in relation to complementarity-determining regions (CDRs) and other key residues within the sequence. Individual T cell es 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 dy. 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., monas Psl—speciflc antibodies or antigen-binding fragments thereof disclosed herein, which are then tested for on. te heavy and light chain genes comprising modified V and human C regions are then cloned into expression s and the subsequent plasmids introduced into cell lines for the production of whole dy. The antibodies are then compared in riate biochemical and biological assays, and the optimal variant is identified.

Anti—Pseudomonas Psl binding molecules, e.g., dies or antigen-binding fragments, variants, or tives 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 Psl antibody or antigen-binding fragment thereof can be administered to various host animals including, but not limited to, rabbits, mice, rats, chickens, hamsters, goats, s, etc., to induce the production of sera containing polyclonal antibodies specific for the n. s adjuvants can be used to se the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic s, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette—Guerin) and Corynebacteriam parvum. Such adjuvants are also well known in the art.

Monoclonal antibodies can be ed 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 dies can be ed 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 antibodies or antibody fragments (e. g., antigen binding sites) can also be derived from antibody libraries, such as phage display libraries. In a ular, 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 identified with antigen, e. g., using labeled n or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with scFv, Fab, Fv OE DAB (individual Fv region from light or heavy chains) or disulf1de stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Exemplary methods are set forth, for example, in EP 368 684 B1; US. patent. 5,969,108, Hoogenboom, HR. and Chames, Immanol. Today 21:371 (2000); Nagy et al. Nat. Med. 8:801 ; Huie et al., Proc. Natl. Acad. Sci. USA 982682 (2001); Lui et al., J. Mol. Biol. 315:1063 (2002), each of which is orated herein by reference. l publications (e.g., Marks et al., Bio/Technology 10:779—783 ) have described the production of high affinity human antibodies by chain shuffling, as well as combinatorial infection and in vivo recombination as a gy for constructing large phage libraries. In another embodiment, mal display can be used to replace bacteriophage as the display platform (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. Methods 248:31 (2001)). In yet another embodiment, cell surface libraries can be screened for dies (Boder et al., Proc. Natl. Acad. Sci. USA 97: 10701 (2000); Daugherty et al., J. Immunol. Methods 1 (2000)). Such ures provide alternatives to traditional hybridoma techniques for the ion and subsequent cloning of monoclonal dies.

In phage display methods, 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 libraries. 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 electroporated in E. coli and the E. coli is infected 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 sing an antigen binding domain that binds to an antigen of interest (i.e., Pseudomonas Psl) can be selected or fied with antigen, e. g., using d antigen or antigen bound or captured to a solid surface or bead.

Additional examples of phage display methods that can be used to make the antibodies include those disclosed in an et al., J. Immunol. Methods [82:41—50 (1995); Ames et al., J. Immunol. Methods [84:177—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 Immunology 57:191—280 (1994); PCT Application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 37; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and US.

Pat. Nos. 426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; ,571,698; 5,427,908; 5,516,637; 5,780,225; 727; 5,733,743 and 5,969,108; each ofwhich is incorporated herein by reference in its entirety.

As described in the above references and in the es below, after phage selection, the antibody 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 produce 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 ; 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 techniques 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 logy 203:46—88 (1991); Shu et al., PNAS 90:7995—7999 (1993); and Skerra et al., Science 38—1040 (1988). In certain embodiments such as therapeutic administration, ic, 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 monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric dies are known in the art. See, e. g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); s et al., J. Immunol. Methods [25:191—202 (1989); US. Pat. Nos. 5,807,715; 4,816,567; and 4,8163 97, which are orated 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 species and framework regions from a human immunoglobulin le. Often, ork residues in the human framework s will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by s well known in the art, e. g., by modeling of the interactions of the CDR and framework residues to fy framework residues important 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 e, afting (EP 239,400; PCT publication WO 91/09967; US. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489—498 (1991); Studnicka et al., Protein Engineering 7(6):805—814 ; Roguska. et al., PNAS —973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332).

Fully human antibodies 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 antibody 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 98/16654, WO 96/34096, WO 96/3373 5, and W0 91/ 10741; each of which is incorporated herein by reference in its entirety.

Human antibodies can also be ed using transgenic mice which are incapable of expressing onal endogenous immunoglobulins, but which can express human immunoglobulin genes. For e, the human heavy and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. In addition, various companies 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 ize a selected epitope can be generated using a technique referred to as "guided ion." In this approach a selected non—human monoclonal antibody, 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 —903 (1988). See also, US.

Patent No. 5,565,332.) In another embodiment, DNA encoding desired onal antibodies can be readily isolated and sequenced using tional 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 ected into prokaryotic or eukaryotic host cells such as E. coli cells, simian COS cells, Chinese r Ovary (CHO) cells or a cells that do not otherwise produce immunoglobulins. More particularly, the isolated DNA (which can be synthetic as described ) can be used to clone constant and variable region sequences for the manufacture antibodies as described in Newman et al., US. Pat. No. 570, filed January 25, 1995, which is incorporated by reference herein. Transformed cells expressing the desired antibody can be grown up in relatively large quantities to provide clinical and commercial supplies of the immunoglobulin.

In one embodiment, an ed binding molecule, e.g., an antibody ses at least one heavy or light chain CDR of an antibody molecule. In another embodiment, an isolated g 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 embodiment, an isolated binding molecule comprises at least five CDRs from one or more antibody molecules. In another embodiment, an isolated binding molecule of the description comprises at least six CDRs from one or more antibody molecules.

In a specific embodiment, the amino acid sequence of the heavy and/or light chain variable s can be inspected to identify the sequences of the complementarity determining regions (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 inant 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 naturally occurring or sus ork regions, and preferably human framework 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 combination of the framework regions and CDRs encodes an antibody that specifically binds to at least one epitope of a desired antigen, e. g., Psl. One or more amino acid substitutions can be made within the ork regions, and, the amino acid substitutions improve binding of the dy to its antigen. Additionally, such methods can be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an hain disulfide bond to te antibody molecules lacking one or more intrachain ide 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 provided 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 s) described herein, which binding molecules or fragments thereof specifically bind to Pseudomonas Psl. 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 ically binds to Pseudomonas Ps1, including, but not d to, site—directed mutagenesis and PCR-mediated mutagenesis which result in amino acid tutions. The variants ding tives) 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 tutions, 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 region, VHCDRl, VHCDRZ, VHCDR3, VL region, VLCDRl, VLCDRZ, or VLCDR3. 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 s 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, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e. g., e, 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). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by tion mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity (e. g., the y to bind an Pseudomonas Psl).

For example, it is possible to introduce mutations only in ork regions or only in CDR regions of an antibody molecule. Introduced mutations can be silent or neutral se mutations, i.e., have no, or little, effect on an antibody’s ability to bind antigen. These types of mutations can be useful to optimize codon usage, or improve a hybridoma’s antibody production.

Alternatively, non-neutral missense mutations can alter an antibody’s ability to bind antigen. The location of most silent and neutral missense mutations is likely to be in the framework s, 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 les 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 ty of the encoded protein, (e.g., ability to bind at least one epitope of Pseudomonas Psl) can be determined using techniques described herein or by routinely modifying techniques known in the art.

III. ANTIBODY POLYPEPTIDES The disclosure is further directed to ed polypeptides which make up binding molecules, e.g., antibodies or antigen-binding fragments thereof, which specifically bind to Pseudomonas Psl 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 antigen binding regions derived from immunoglobulin molecules. A polypeptide or amino acid ce ed 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 ially identical to that of the ng sequence, or a portion thereof, wherein the portion 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 ng sequence.

Also disclosed is an isolated binding molecule, e.g., an antibody or antigen—binding fragment thereof which ically 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 n—binding fragment thereof which specifically binds to Pseudomonas Psl comprising a VH 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: 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 ed 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 , VHCDR2 or VHCDR3 s 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 le, e.g., an antibody or antigen—binding fragment thereof which cally binds to Pseudomonas Psl comprising a VH, 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 , 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, 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, VHCDR2, or VHCDR3 amino acid sequences shown in Table 3.

Also disclosed is an isolated binding molecule, e.g., an antibody or n—binding fragment thereof which ically binds to Pseudomonas Psl comprising 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, or SEQ ID NO: 16 as shown in Table 2.

Some embodiments disclose an ed binding molecule, 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 ed binding molecule, e.g., an antibody or antigen—binding 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 Further provided is an isolated binding molecule, e.g., an antibody or antigen-binding 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 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, 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 n-binding fragment thereof which specifically binds to Pseudomonas Ps1, 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 ID NO: 5 and SEQ ID , respectively,(d) SEQ NO: 6 ID NO: 7 and SEQ ID NO: , respectively,(e) SEQ 8, respectively,(f) SEQ ID NO: 9 and SEQ ID NO: 10, respectively,(g) SEQ ID NO: 11 and SEQ ID NO: 12 ID , respectively,(h) SEQ NO: 13 and SEQ ID 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 In certain embodiments, the above— , respectively. 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 n—binding nt thereof comprises a VH with the amino acid sequence SEQ ID NO: 1 and a VL with the amino acid sequence of SEQ ID NO: 2. In other embodiments, the above—described antibody or antigen—binding nt 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 n embodiments, an ed binding molecule, e. g., an antibody or antigen- binding fragment thereof as described herein ically binds to Pseudomonas Psl with an affinity characterized by a iation 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 x105 M, 10'5 M, 5 x10'6 M, 10'6 M, 5 x10'7 M, 10'7 M, 5 x10" M, 10'8 M, 5 x109 M, 10'9 M, 5 x1010 M, 10'10 M, 5 x 10'11 M, 10'11 M, 5 x 10'12 M, 10'12 M, 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 n- g fragment thereof as described herein specifically binds to Pseudomonas Psl, with an affinity characterized by a dissociation constant (KD) in a range of about 1 x 10'10 to about 1 x '6 M. In one embodiment, an ed binding molecule, e. g., an antibody or antigen-binding fragment thereof as described herein ically binds to monas Ps1, with an affinity characterized by a KD of about 1.18 x 10'7 M, as determined by the OCTET® binding assay bed herein. In another embodiment, an isolated binding molecule, e.g., an antibody or antigen—binding fragment thereof as described herein specifically binds to Pseudomonas Ps1, with an affinity characterized by a KD of about 1.44 x 10'7 M, as determined by the OCTET® binding assay described herein.

Some ments include the isolated binding molecule e. g., an antibody or fragment 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.

In some embodiments the isolated binding molecule 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 tration of about 50 11ng or less, 5.0ug/ml or less, or about 0.5ug/ml or less, or at an antibody tration ranging from about 30 ug/ml to about 0.3 ug/ml, or at an antibody tration of about 1 ug/ml, or at an antibody concentration of about 0.3 ug/ml.

Certain embodiments include the isolated binding molecule e.g., an antibody or fragment thereof as described above, where the OPK EC50 is less than about 0.5 ug/ml, less than about 0.05ug/ml, or less than about 0.005ug/ml, or where the OPK EC50 ranges from about 0.001 11ng to about 0.5 ug/ml, or where the OPK EC50 ranges from about 0.02 11ng to about 0.08 ug/ml, or where the OPK EC50 ranges from about 0.002 ug/ml to about 0.01 11ng 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 bed herein specifically binds to the same Ps1 e as monoclonal antibody WapR-004, WapR-004RAD, Cam-003, Cam-004, or Cam—005, or will competitively inhibit such a monoclonal antibody from binding to Pseudomonas Psl. WapR— 004RAD is identical to WapR-004 except for an amino acid tution G98A of the VH amino acid sequence of SEQ ID NO:11.

Some embodiments include WapR—004 (W4) mutants sing an scFv—Fc molecule 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: 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 ments e 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 Psl binding molecule, e.g., antibody or fragment, variant or derivative thereof described herein specifically binds to the same epitope as 2012/041538 monoclonal antibody Ol, WapR—OOZ, or WapR—003, or will competitively inhibit such a monoclonal antibody from binding to Pseudomonas Psl.

In certain embodiments, an anti—Pseudomonas Ps1 binding molecule, e.g., dy or fragment, variant or derivative thereof described herein specifically binds to the same epitope as monoclonal antibody WapR-Ol6, or will competitively inhibit such a monoclonal antibody from binding to Pseudomonas Psl.

TABLE 2: Reference VH and VL amino acid se uences* dy VH VL Name Cam—003 QVRLQQSGPGLVKPSET SSELTQDPAVSVALGQTVRITCM LSLTCTVSGGSTSM LRSYYASWYQQKPGQAPVLVIYGfl fiWLRQPPGKGLEWIGfl NRPSGIPDRFSGSSSGNTASLTITGAQ HSNGGTNYNPSLKSRL AEDEADYYCNSRDSSGNHVVFGGGT TISGDTSKNQFSLNLSF KLTVL VTAADTALYYCARM SEQ ID NO:2 DVYGPAFDIWGQGTM SEQ ID NO:1 Cam—004 QVQLQQSGPGRVKPSE SSELTQDPAVSVALGQTVRITCM TLSLTCTVSGYSVSSG_Y LRSYYASWYQQKPGQAPVLVIYGfl YWGWIRQSPGTGLEWI PDRFSGSSSGNTASLTITGAQ GSISHSGSTYYNPSLKS AEDEADYYCNSRDSSGNHVVFGGGT RVTISGDASKNQFFLRL KLTVL TSVTAADTAVYYCARE SEQ ID NO:2 EATANFDSWGRGTLVT SEQ ID NO:3 Antibody VH VL Nmne Cam—005 QVQLQQSGPGLVKPSET SSELTQDPAVSVALGQTVRITCM LSLTCTVSGGSVSSSGY LRSYYASWYQQKPGQAPVLVIYGfl MWIRQPPGKGLEWI NRPSGIPDRFSGSSSGNTASLTITGAQ GSIYSSGSTYYSPSLKS AEDEADYYCNSRDSSGNHVVFGGGT RVTISGDTSKNQFSLKL KLTVL SSVTAADTAVYYCARL SEQ ID NO:2 NWGTVSAFDIWGRGTL SEQ ID NO:4 WapR—OOI EVQLLESGGGLVQPGG QAGLTQPASVSGSPGQSITISCTGTSS SLRLSCSASGFTFSM DIATYNYVSWYQQHPGKAPKLMIYE flWVRQAPGKGLEYV GVSNRFSGSKSGNTASLTIS SDIGTNGGSTNYADSV GLQAEDEADYYCSSYARSYTYVFGT ERFTISRDNSKNTVYL L QMSSLRAEDTAVYHCV SEQ ID NO:6 AGIAAAYGFDVWGQG TMVTVSS SEQ ID NO:5 WapR—OOZ SGGGLVQPGG QTVVTQPASVSGSPGQSITISCTGTSS SLRLSCSASGFTFSSY_P DVGGYNYVSWYQQHPGKAPKLMIY flWVRQAPGKGLDYV EVSNRPSGVSNHFSGSKSGNTASLTIS GGSTNYADSV GLQAEDEADYYCSSYTTSSTYVFGT ERFTISRDNSKNTLFL GTKVTVL QMSSLRAEDTAVYYCV SEQ ID NO:8 MGLVPYGFDIWGQGTL VTVSS SEQ ID NO:7 Antibody VH VL Name WapR—003 QMQLVQSGGGLVQPGG QTVVTQPASVSASPGQSITISCAGTSG SLRLSCSASGFTFSSY_P FVSWYQQHPGKAPKLLIYE MWVRQAPGKGLDYV GS![RPSGVSNRFSGSRSGNTASLTIS SDISPNGGATNYADSV GLQAEDEADYYCSSYARSYTYVFGT KGRFTISRDNSKNTVYL GTKLTVL QMSSLRAEDTAVYYCV SEQ ID NO:10 MGLVPYGFDNWGQGT MVTVSS SEQ ID NO:9 WapR—004 EVQLLESGPGLVKPSET EIVLTQSPSSLSTSVGDRVTITCM LSLTCNVAGGSISM SIRSHLNWYQQKPGKAPKLLIYfl IWIRQPPGKGLELIGE MGVPSRFSGSGSGTDFTLTISSLQ HSSGYTDYNPSLKSRV PEDFATYYCS[SQSYSFPLTFGGGTKLE TISGDTSKKQFSLHVSS IK VTAADTAVYFCARG_D SEQ ID NO:12 WDLLHALDIWGQGTL VTVSS SEQ ID NO:11 WapR—007 EVQLVQSGADVKKPGA DPAVSVALGQTVRITCM SVRVTCKASGYTFTG_H LRSYYTNWFQQKPGQAPLLVVYA_K wWVRQAPGQGLEW NKRPPGIPDRFSGSSSGNTASLTITGA MGWINPDSGATSYAQ QAEDEADYYCHSRDSSGNHVVFGG MRVTMTRDTSITT L RLRSDDTAVY SEQ ID NO:14 YCATDTLLSNHWGQGT LVTVSS SEQ ID NO:13 Antibody VH VL Name WapR—016 EVQLVESGGGLVQPGGSL QSVLTQPASVSGSPGQSITISCTGTSSDVG RLSCAASGYTFSSYATSWV GYNYVSWYQQ RQAPGKGLEWVAGISGSG GVSNRFSGSKSGNTASLTISGLQAEDEAD DTTDYVDSVKGRFTVSRD YCSSYSSGTVVFGGGTELTVL NSKNTLYLQMNSLRADDT SEQ ID NO: 16 AVYYCASRGGLGGYYRG GFDFWGQGTMVTVSS SEQ ID NO: 15 WapR— EVQLLESGPGLVKPSET EIVLTQSPSSLSTSVGDRVTITCM 004RAD LSLTCNVAGGSISM SIRSHLNWYQQKPGKAPKLLIYfl IWIRQPPGKGLELIGE MGVPSRFSGSGSGTDFTLTISSLQ HSSGYTDYNPSLKSRV YYCS[SQSYSFPLTFGGGTKLE TISGDTSKKQFSLHVSS 1K VTAADTAVYFCARA_D SEQ ID NO:12 WDLLHALDIWGQGTL VTVS S SEQ ID NO:74 *VH and VL CDRl, CDR2, and CDR3 amino acid sequences are ined TABLE 3: Reference VH and VL CDRl, CDRZ, and CDR3 amino acid ces Antibody VHCDRl VHCDR2 VHCDR3 VLCDRl VLCDR2 VLCDR3 Name YIHSNGG TDYDVY QGDSLRSY NSRDSSGNH TNYNPSL GPAFDI YAS VV KS SEQ ID SEQ ID SEQ ID N022 SEQ ID NO: 19 N020 NO: 18 SISHSGST SEATAN QGDSLRSY NSRDSSGNH YYNPSLK FDS YAS VV S SEQ ID SEQ ID SEQ ID N022 SEQ ID N025 N020 N024 Antibody VHCDRl VHCDR3 VLCDRl VLCDR3 Name Cam—005 SSGYYW SIYSSGST LNWGTV QGDSLRSY GKNNRPS GNH T YYSPSLKS SAFDI SEQ ID NO:22 SEQ ID SEQ ID NO:27 NO:28 DIGTNG GIAAAY TGTSSDIAT EGTKRPS SSYARSYT GSTNYA GFDV YV DSVKG SEQ ID SEQ ID NO:34 SEQ ID NO:3 1 NO:30 DISPNGG TGTSSDV EVSNRPS SSYTTSSTY STNYAD GGYNYVS SEQ ID V SVKG SEQ ID NO:39 SEQ ID NO:40 SEQ ID NO:38 NO:36 DISPNGG AGTSGDV EGSQRPS SSYARSYT ATNYAD GNYNFVS YV SVKG SEQ ID SEQ ID NO:46 SEQ ID NO:44 NO:42 YIHSSGY GDWDL RASQSIRS S YSFPLT TDYNPSL LHALDI HLN SEQ ID SEQ ID NO:52 KS SEQ ID SEQ ID NO:51 SEQ ID NO:49 NO:50 NO:48 Antibody VHCDRl VHCDR2 VHCDR3 VLCDRl VLCDR2 VLCDR3 Name WapR-007 GHNIH WINPDS DTLLSN QGDSLRS AKNKRPP HSRDSSGN SEQ ID GATSYA H YYTN SEQ ID HVV NO:53 QKFQG SEQ ID SEQ ID NO:57 SEQ ID NO:58 SEQ ID NO:55 NO:56 NO:54 WapR-016 SYATS GISGSGDT RGGLGG TGTSSDVG S 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 WapR- PYYWT YIHSSGY ADWDL RASQSIRS GASNLQS YSFPLT 004RAD SEQID TDYNPSL LHALDI HLN SEQID SEQIDNO:52 NO:47 KS SEQ ID SEQ ID NO:51 SEQ ID NO:75 NO:50 NCX48 Any anti—Pseudomonas Psl binding molecules, e. g., antibodies or fragments, variants or derivatives thereof described herein can further include additional ptides, e.g., a signal e to direct secretion of the encoded polypeptide, antibody constant regions as described , or other heterologous polypeptides as described herein. Additionally, g molecules or fragments thereof of the description include polypeptide fragments as bed elsewhere.

Additionally anti-Pseudomonas Psl binding molecules, e.g., dies or fragments, variants or derivatives thereof described herein can be fusion polypeptides, Fab fragments, scFvs, or other derivatives, as described herein.

Also, as described in more detail elsewhere herein, the sure includes compositions comprising anti—Pseudomonas Psl binding molecules, e. g., antibodies or fragments, ts or derivatives f described herein.

It will also be tood by one of ordinary skill in the art that anti-Pseudomonas Psl binding molecules, e.g., antibodies or fragments, variants or derivatives thereof described herein can be ed 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% cal to the starting ce.

The term nt ce identity" between two polynucleotide or polypeptide sequences refers to the number of identical matched positions shared by the sequences over a comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matched position is any position where an identical nucleotide or amino acid is presented in both the target and reference sequence.

Gaps presented in the target sequence are not counted since gaps are not nucleotides or amino acids. Likewise, gaps presented in the reference sequence are not counted since target sequence nucleotides or amino acids are counted, not nucleotides or amino acids from the reference sequence.

The percentage of sequence identity is calculated by determining the number of positions at which the identical amino—acid residue or nucleic acid base occurs in both sequences to yield the number of d positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the —56— percentage of ce identity. The comparison of ces and ination of t sequence identity between two sequences may be accomplished using readily available software both for online use and for download. Suitable software programs are available from various sources, and for alignment of both n and nucleotide sequences. One le program to determine percent sequence ty is b12seq, part of the BLAST suite of program available from the US. government's National Center for Biotechnology Information BLAST web site (blast.ncbi.nlm.nih. gov). B12seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other le programs are, e.g., Needle, her, Water, or r, part of the EMBOSS suite of bioinformatics programs and also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk/Tools/psa.

Different regions within a single polynucleotide or polypeptide target sequence that aligns with a polynucleotide or polypeptide reference sequence can each have their own percent sequence identity. It is noted that the percent sequence identity value is rounded to the nearest tenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. It also is noted that the length value will always be an integer.

One skilled in the art will appreciate that the generation of a sequence alignment for the calculation of a t sequence identity is not limited to binary sequence—sequence comparisons exclusively driven by primary sequence data. Sequence alignments can be derived from multiple sequence alignments. One suitable program to generate multiple sequence alignments is ClustalW2, available from www.clustal.org. Another suitable program is MUSCLE, available from www.drive5.com/muscle/. ClustalW2 and MUSCLE are alternatively ble, e. g., from the EBI.

It will also be appreciated that sequence alignments can be generated by ating sequence data with data from heterogeneous sources such as ural data (e.g., crystallographic protein structures), functional data (e.g., location of mutations), or phylogenetic data. A suitable program that ates heterogeneous data to generate a multiple sequence alignment is T-Coffee, available at www.tcoffee.org, and alternatively available, e. g., from the EBI. It will also be appreciated that the final alignment used to calculated percent sequence identity may be curated either automatically or manually.

Whether any particular polypeptide is at least about 70%, 75%, 80%, 85%, 90% or 95% identical to another polypeptide can also be determined 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 ch Park, 575 e Drive, Madison, WI 53711). BESTFIT uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 489 , to find the best segment of homology between two sequences. When using BESTFIT or any other sequence alignment m to determine whether a particular sequence is, for example, 95% identical to a reference sequence, the parameters are set, of , such that the tage of identity is calculated 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 reference sequence are allowed.

Furthermore, nucleotide or amino acid substitutions, deletions, or insertions leading to conservative substitutions or changes at "non—essential" amino acid s can be made. For example, a polypeptide or amino acid sequence d from a designated protein can be identical to the starting 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 protein has one to five, one to ten, one to fifteen, or one to twenty individual amino acid substitutions, insertions, or deletions relative to the starting sequence.

An anti—Pseudomonas Psl binding molecule, e.g., an antibody or fragment, variant or derivative thereof described herein can comprise, consist essentially of, or t of a fusion protein. Fusion proteins are chimeric molecules which comprise, for example, an immunoglobulin antigen-binding domain with at least one target binding site, and at least one heterologous n, i.e., a portion with which it is not naturally linked in nature. The amino acid sequences can normally exist in separate 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 d, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship. —58— 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 miting example, a "heterologous polypeptide" 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 s, or an immunoglobulin or munoglobulin polypeptide of a different species.

IV. FUSION PROTEINS AND ANTIBODY CONJUGATES In some embodiments, the anti—Pseudomonas Psl binding molecules, e.g., antibodies or fragments, ts or derivatives f can be administered multiple times in conjugated form.

In still another embodiment, the anti—Pseudomonas Psl 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 ic embodiments, the anti—Pseudomonas Psl binding molecules, e.g., antibodies or fragments, variants or derivatives thereof can be conjugated to one or more antimicrobial agents, for example, Polymyxin B (PMB). PMB is a small lipopeptide antibiotic approved for treatment of multidrug-resistant Gram-negative ions. In addition to its bactericidal activity, PMB binds lipopolysaccharide (LPS) and neutralizes its proinflammatory effects. (Dixon, R.A. & Chopra, I. J Antimicrob Chemother 18, 3 ). LPS is t to significantly contribute to inflammation and the onset of Gram—negative . (Guidet, B., et al., Chest 106, 1194-1201 (1994)). Therapies that neutralize and/or clear LPS from circulation have the potential to prevent or delay the onset of sepsis and improve clinical outcome. Polymyxin B (PMB) is a lipopeptide antibiotic approved for treatment of multidrug-resistant Gram-negative infections. In addition to its bactericidal activity, PMB binds LPS and neutralizes its proinflammatory effects. Conjugates of PMB to carrier molecules have been shown to lize LPS and mediate protection in animal models of endotoxemia and infection. (Drabick, J.J., et a].

Antimicrob Agents Chemother 42, 583—588 (1998)). Also disclosed is a method for attaching one or more PMB molecules to cysteine residues introduced into the Fc region of onal antibodies (mAb) of the disclosure. For example, the Cam—003—PMB conjugates retained specif1c, mAb—mediated binding to P. aeruginosa and also retained OPK ty. Furthermore, mAb—PMB conjugates bound and neutralized LPS in vitro.

In certain embodiments, an anti—Pseudomonas Psl binding molecule, e.g., an antibody or fragment, variant or derivative thereof described herein can comprise a heterologous amino acid sequence or one or more other es not normally associated with an antibody (e.g., an antimicrobial agent, a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a lipid, a ical response modifier, pharmaceutical agent, a lymphokine, a heterologous antibody or fragment f, a detectable label, polyethylene glycol (PEG), and a combination of two or more of any said agents). In further embodiments, an anti—Pseudomonas Psl binding molecule, e.g., an antibody or fragment, variant or tive thereof can comprise a detectable label selected from the group consisting of an enzyme, a cent label, a uminescent label, a bioluminescent label, a radioactive label, or a combination of two or more of any said detectable labels.

V. POLYNUCLEOTIDES ENCODING BINDING MOLECULES Also ed herein are nucleic acid molecules encoding the anti—Pseudomonas Psl binding molecules, e.g., dies or fragments, variants or derivatives thereof described herein.

One embodiment provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an globulin 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 IS NO: 74 as shown in Table 2.

Another embodiment provides an isolated cleotide comprising, consisting 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: 15, or SEQ ID NO: 74 as shown in Table 2.

Further embodiment provides an isolated polynucleotide comprising, consisting essentially of, or ting of a nucleic acid encoding a VH, 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: 15, or SEQ ID NO: 74 as shown in Table 2. r embodiment provides an isolated polynucleotide sing, consisting essentially of, or consisting of a nucleic acid ng an isolated binding molecule, e.g., an antibody or antigen-binding fragment thereof which specifically binds to Pseudomonas Psl comprising a VH, where one or more of the VHCDRl, VHCDR2 or VHCDR3 regions of the VH are identical to, or cal 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: 15, or SEQ ID NO: 74 as shown in Table 2.

A further embodiment provides an isolated binding molecule e.g., an dy or antigen- binding fragment comprising the VH encoded by the polynucleotide specifically or preferentially binds to Pseudomonas Psl.

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 ce 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.

A further embodiment es an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding 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.

Another embodiment provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding a VL, where one or more of the , 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.

A further embodiment es 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 nt thereof which specifically binds to Pseudomonas Psl comprising an 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 tutions, 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, or SEQ ID NO: 16 as shown in Table 2.

In another embodiment, an isolated binding molecule e.g., an antibody or antigen-binding fragment comprising the VL encoded by the polynucleotide specifically or preferentially binds to Pseudomonas Psl.

One embodiment es 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 70807 TABLE 4: Reference scFV nucleic acid sequences Antibody scFV nucleotide sequences Name Cmn£03 CAGCCGGCCATGGCCCAGGTACAGCTGCAGCAGTCAGGCCCAGG ACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCT TCCACCAGTCCTTACTTCTGGAGCTGGCTCCGGCAGCCC CCAGGGAAGGGACTGGAGTGGATTGGTTATATCCATTCCAATGGG GGCACCAACTACAACCCCTCCCTCAAGAGTCGACTCACCATATCA GGAGACACGTCCAAGAACCAATTCTCCCTGAATCTGAGTTTTGTG ACCGCTGCGGACACGGCCCTCTATTACTGTGCGAGAACGGACTAC GATGTCTACGGCCCCGCTTTTGATATCTGGGGCCAGGGGACAATG GTCACCGTCTCGAGTGGTGGAGGCGGTTCAGGCGGAGGTGGCAG CGGCGGTGGCGGATCGTCTGAGCTGACTCAGGACCCTGCTGTGTC TGTGGCCTTGGGACAGACAGTCAGGATCACATGCCAAGGAGACA GCCTCAGAAGCTATTATGCAAGCTGGTACCAGCAGAAGCCAGGA CAGGCCCCTGTACTTGTCATCTATGGTAAAAACAACCGGCCCTCA GGGATCCCAGACCGATTCTCTGGCTCCAGCTCAGGAAACACAGCT TCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTAT TACTGTAACTCCCGGGACAGCAGTGGTAACCATGTGGTATTCGGC GGAGGGACCAAGCTGACCGTCCTAGGTGCGGCCGCA SEQ ID NO:65 2012/041538 ZXnfibody scFVInufleofidesequences Nmne Can}004 CAGCCGGCCATGGCCCAGGTACAGCTGCAGCAGTCAGGCCCAGG ACGGGTGAAGCCTTCGGAGACGCTGTCCCTCACCTGCACTGTCTC TGGTTACTCCGTCAGTAGTGGTTACTACTGGGGCTGGATCCGGCA GTCCCCAGGGACGGGGCTGGAGTGGATTGGGAGTATCTCTCATAG TGGGAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCAT ATCAGGAGACGCATCCAAGAACCAGTTTTTCCTGAGGCTGACTTC TGTGACCGCCGCGGACACGGCCGTTTATTACTGTGCGAGATCTGA GGCTACCGCCAACTTTGATTCTTGGGGCAGGGGCACCCTGGTCAC CGTCTCTTCAGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCG GTGGCGGATCGTCTGAGCTGACTCAGGACCCTGCCGTGTCTGTGG CCTTGGGACAGACAGTCAGGATCACATGCCAAGGAGACAGCCTC AGAAGCTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAGGC ACTTGTCATCTATGGTAAAAACAACCGGCCCTCAGGGAT CCCAGACCGATTCTCTGGCTCCAGCTCAGGAAACACAGCTTCCTT GACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACTG TAACTCCCGGGACAGCAGTGGTAACCATGTGGTATTCGGCGGAGG GACCAAGCTGACCGTCCTAGGTGCGGCCGCA SEQ ID NO:66 WO 70807 Antibody scFV nucleotide sequences Nmne CmnflOS CAGCCGGCCATGGCCCAGGTACAGCTGCAGCAGTCAGGCCCAGG ACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCT GGTGGCTCCGTCAGCAGTAGTGGTTATTACTGGACCTGGATCCGC CAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGAGTATCTATTCT AGTGGGAGCACATATTACAGCCCGTCCCTCAAGAGTCGAGTCACC ATATCCGGAGACACGTCCAAGAACCAGTTCTCCCTCAAGCTGAGC TCTGTGACCGCCGCAGACACAGCCGTGTATTACTGTGCGAGACTT GGCACTGTGTCTGCCTTTGATATCTGGGGCAGAGGCACC CTGGTCACCGTCTCGAGTGGTGGAGGCGGTTCAGGCGGAGGTGGC AGCGGCGGTGGCGGATCGTCTGAGCTGACTCAGGACCCTGCTGTG TCTGTGGCCTTGGGACAGACAGTCAGGATCACATGCCAAGGAGAC AGCCTCAGAAGCTATTATGCAAGCTGGTACCAGCAGAAGCCAGG ACAGGCCCCTGTACTTGTCATCTATGGTAAAAACAACCGGCCCTC AGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAGGAAACACAGC TTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTA TTACTGTAACTCCCGGGACAGCAGTGGTAACCATGTGGTATTCGG CGGAGGGACCAAGCTGACCGTCCTAGGTGCGGCCGCA SEQ ID NO:67 —65— Antibody scFV nucleotide sequences Name WMpRflOl TCTATGCGGCCCAGCCGGCCATGGCCGAGGTGCAGCTGTTGGAGT CTGGGGGAGGTTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCT GTTCAGCCTCTGGGTTCACCTTCAGTCGGTATCCTATGCATTGGGT CCGCCAGGCTCCAGGGAAGGGACTGGAATATGTTTCAGATATTGG TACTAATGGGGGTAGTACAAACTACGCAGACTCCGTGAAGGGCA GATTCACCATCTCCAGAGACAATTCCAAGAACACGGTGTATCTTC GCAGTCTGAGAGCTGAGGACACGGCTGTGTATCATTGTG TGGCGGGTATAGCAGCCGCCTATGGTTTTGATGTCTGGGGCCAAG GGACAATGGTCACCGTCTCGAGTGGAGGCGGCGGTTCAGGCGGA TCTGGCGGTGGCGGAAGTGCACAGGCAGGGCTGACTCA GCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCC TGCACTGGAACCAGCAGTGACATTGCTACTTATAACTATGTCTCCT GGTACCAACAGCACCCAGGCAAAGCCCCCAAACTCATGATTTATG AGGGCACTAAGCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCT CCAAGTCTGGCAACACGGCCTCCCTGACAATCTCTGGGCTCCAGG CTGAGGACGAGGCTGATTATTACTGTTCCTCATATGCACGTAGTT ACACTTATGTCTTCGGAACTGGGACCGAGCTGACCGTCCTAGCGG CCGC SEQ ID NO:68 Antibody scFV nucleotide sequences Nmne “MpR002 CTATGCGGCCCAGCCGGCCATGGCCCAGGTGCAGCTGGTGCAGTC TGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTG TTCAGCCTCTGGATTCACCTTCAGTAGCTATCCTATGCACTGGGTC CGCCAGGCTCCAGGGAAGGGACTGGATTATGTTTCAGACATCAGT CCAAATGGGGGTTCCACAAACTACGCAGACTCCGTGAAGGGCAG ATTCACCATCTCCAGAGACAATTCCAAGAACACACTGTTTCTTCA AATGAGCAGTCTGAGAGCTGAGGACACGGCTGTGTATTATTGTGT GTTAGTACCCTATGGTTTTGATATCTGGGGCCAAGGCAC CCTGGTCACCGTCTCGAGTGGAGGCGGCGGTTCAGGCGGAGGTGG CTCTGGCGGTGGCGGAAGTGCACAGACTGTGGTGACCCAGCCTGC CTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCACT GGAACCAGCAGTGACGTTGGTGGTTATAACTATGTCTCCTGGTAC CAACAGCACCCAGGCAAAGCCCCCAAACTCATGATTTATGAGGTC AGTAATCGGCCCTCAGGGGTTTCTAATCACTTCTCTGGCTCCAAGT CTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGG ACGAGGCTGATTATTACTGCAGCTCATATACAACCAGCAGCACTT ATGTCTTCGGAACTGGGACCAAGGTCACCGTCCTAGCGGCCG SEQ ID NO:69 —67— Antibody scFV nucleotide sequences Name WapR-003 AGCCGGCCATGGCCCAGATGCAGCTGGTGCAGTCGGGG GGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTTCA GCCTCTGGATTCACCTTCAGTAGCTATCCTATGCACTGGGTCCGCC AGGCTCCAGGGAAGGGACTGGATTATGTTTCAGACATCAGTCCAA GTGCCACAAACTACGCAGACTCCGTGAAGGGCAGATTC ACCATCTCCAGAGACAATTCCAAGAACACGGTGTATCTTCAAATG AGCAGTCTGAGAGCTGAAGACACGGCTGTCTATTATTGTGTGATG GGGTTAGTGCCCTATGGTTTTGATAACTGGGGCCAGGGGACAATG GTCACCGTCTCGAGTGGAGGCGGCGGTTCAGGCGGAGGTGGCTCT GGCGGTGGCGGAAGTGCACAGACTGTGGTGACCCAGCCTGCCTCC GTGTCTGCATCTCCTGGACAGTCGATCACCATCTCCTGCGCTGGA ACCAGCGGTGATGTTGGGAATTATAATTTTGTCTCCTGGTACCAA CAACACCCAGGCAAAGCCCCCAAACTCCTGATTTATGAGGGCAGT CAGCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAGGTCTG GCAACACGGCCTCCCTGACAATCTCTGGGCTCCAGGCTGAGGACG AGGCTGATTATTACTGTTCCTCATATGCACGTAGTTACACTTATGT CTTCGGAACTGGGACCAAGCTGACCGTCCTAGCGGCCGCA SEQ ID NO:70 WO 70807 Antibody scFV nucleotide sequences Name WapR—004 TATGCGGCCCAGCCGGCCATGGCCGAGGTGCAGCTGTTGGAGTCG GGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGC AATGTCGCTGGTGGCTCCATCAGTCCTTACTACTGGACCTGGATCC GGCAGCCCCCAGGGAAGGGCCTGGAGTTGATTGGTTATATCCACT CCAGTGGGTACACCGACTACAACCCCTCCCTCAAGAGTCGAGTCA CCATATCAGGAGACACGTCCAAGAAGCAGTTCTCCCTGCACGTGA GCTCTGTGACCGCTGCGGACACGGCCGTGTACTTCTGTGCGAGAG GCGATTGGGACCTGCTTCATGCTCTTGATATCTGGGGCCAAGGGA CCCTGGTCACCGTCTCGAGTGGAGGCGGCGGTTCAGGCGGAGGTG GCTCTGGCGGTGGCGGAAGTGCACTCGAAATTGTGTTGACACAGT CTCCATCCTCCCTGTCTACATCTGTAGGAGACAGAGTCACCATCA CTTGCCGGGCAAGTCAGAGCATTAGGAGCCATTTAAATTGGTATC AGCAGAAACCAGGGAAAGCCCCTAAACTCCTGATCTATGGTGCAT CCAATTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGAT CAGATTTCACTCTCACCATTAGTAGTCTGCAACCTGAAG ATTTTGCAACTTACTACTGTCAACAGAGTTACAGTTTCCCCCTCAC TTTCGGCGGAGGGACCAAGCTGGAGATCAAAGCGGCCGC SEQ ID NO:71 Antibody scFV nucleotide sequences Nmne WMpR007 GCGGCCCAGCCGGCCATGGCCGAAGTGCAGCTGGTGCAGTCTGG GGCTGACGTAAAGAAGCCTGGGGCCTCAGTGAGGGTCACCTGCA AGGCTTCTGGATACACCTTCACCGGCCACAACATACACTGGGTGC GACAGGCCCCTGGACAAGGGCTTGAATGGATGGGATGGATCAAC CCTGACAGTGGTGCCACAAGCTATGCACAGAAGTTTCAGGGCAGG GTCACCATGACCAGGGACACGTCCATCACCACAGCCTACATGGAC CTGAGCAGGCTGAGATCTGACGACACGGCCGTATATTACTGTGCG ACATTACTGTCTAATCACTGGGGCCAAGGAACCCTGGTC ACCGTCTCGAGTGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGG CGGATCGTCTGAGCTGACTCAGGACCCTGCTGTGTCTGT GGCCTTGGGACAGACAGTCAGGATCACTTGCCAAGGAGACAGTCT CAGAAGCTATTACACAAACTGGTTCCAGCAGAAGCCAGGACAGG CCCCTCTACTTGTCGTCTATGCTAAAAATAAGCGGCCCCCAGGGA TCCCAGACCGATTCTCTGGCTCCAGCTCAGGAAACACAGCTTCCT TGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACT GTCATTCCCGGGACAGCAGTGGTAACCATGTGGTATTCGGCGGAG GGACCAAGCTGACCGTCCTAGGTGCGGCCGCA SEQ ID NO:72 Antibody scFv nucleotide sequences Name WMpR£l6 CAGCCGGCCATGGCCGAGGTGCAGCTGGTGGAGTCTGGGGGAGG CTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTG TGCAGCCTCTGGATACACCTTTAGCAGCTATGCCACGAGCTGGGT CCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCG CAGGTATTAGTGGTAGTGGTGATACCACAGACTACGTAGACTCCG TGAAGGGCCGGTTCACCGTCTCCAGAGACAATTCC AAGAACACCCTATATCTGCAAATGAACAGCCTGAGAGCCGACGA CACGGCCGTGTATTACTGTGCGTCGAGAGGAGGTTT TTATTACCGGGGCGGCTTTGACTTCTGGGGCCAGGGGAC AATGGTCACCGTCTCGAGTGGAGGCGGCGGTTCAG GCGGAGGTGGCTCTGGCGGTGGCGGAAGTGCACAGTCTGTGCTGA CGCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAG ACCATCTCCTGCACTGGAACCAGCAGTGACGTTGGTGGT TATAACTATGTCTCCTGGTACCAACAGCACCCAGG CAAAGCCCCCAAACTCATGATTTATGAGGTCAGTAATCGGCCCTC AGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTG GCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACG AGGCTGATTATTACTGCAGCTCATATACAAGCAGC GGCACTGTGGTATTCGGCGGAGGGACCGAGCTGACCGTCCTAGCG GCCGCA SEQ ID NO:73 In some embodiments, an isolated antibody or antigen-binding fragment thereof encoded by one or more of the polynucleotides described above, which specifically binds to Pseudomonas Psl, comprises, consists essentially of, or consists of VH and VL amino acid cesatleast8096,8596,909695960r10096idenficalux (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 ID NO: 5 and SEQ , tively, (d) SEQ ID NO: 6 ID NO: 7 and SEQ ID NO: , respectively, (e) SEQ 8, respectively, (0 SEQ ID NO: 9 and SEQ ID NO: 10, tively, (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 G) SEQ ID NO: 74 and SEQ ID NO: 12 , respectively.

In certain embodiments, an isolated binding molecule, e.g., an dy or n- binding fragment thereof encoded by one or more of the polynucleotides described above, specifically binds to Pseudomonas Psl with an affinity 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 x 10'4 M, 10'4 M, 5 x 10'5 M, 10'5 M, 5 x 10'6 M, 10'6 M, 5 x 10'7 M, 10'7 M, 5 x 10'8 M, 10'8 M, 5 x109 M, 10'9 M, 5 x1010 M, ‘10 M, 5 x 10'11M, 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 x10'15 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 Psl, with an affinity characterized by a iation 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 antibody or antigen-binding fragment thereof encoded by one or more of the polynucleotides described above, specifically binds to Pseudomonas Psl, with an affinity characterized by a KD of about 1.18 x 10'7 M, as determined by the OCTET® g assay described herein. In another embodiment, an ed 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, with an ty characterized by a KD of about 1.44 x 10'7 M, as determined by the OCTET® binding assay described herein.

In certain embodiments, an anti—Pseudomonas Psl binding molecule, e.g., dy or fragment, variant or derivative thereof encoded by one or more of the cleotides described above, 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 antibody from binding to Pseudomonas Psl. WapR—004RAD is identical to WapR—004 except for a nucleic acid substitution G293C of the VH nucleic acid ce encoding the VH amino acid sequence of SEQ ID NO:11 (a substitution of the nucleotide in the VH—encoding portion of SEQ ID NO:71 at position 317). The c acid sequence encoding the WapR—004RAD VH is presented as SEQ ID NO 76.

Some embodiments provide an isolated polynucleotide comprising, 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 polynucleotide comprising, consisting essentially of, or consisting of a c acid encoding a W4 mutant 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.

One embodiment es an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid which encodes a W4 mutant scFv—Fc molecule, where the c 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.

In other embodiments, an anti—Pseudomonas Psl binding molecule, e.g., antibody or fragment, variant or derivative thereof encoded by one or more of the polynucleotides bed 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 g to Pseudomonas Psl.

In certain embodiments, an anti—Pseudomonas Psl binding molecule, e.g., antibody or fragment, variant or derivative thereof encoded by one or more of the polynucleotides bed above, specifically binds to the same epitope as monoclonal antibody WapR—016, or will competitively t such a monoclonal antibody from binding to monas Psl.

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

The polynucleotides can be produced or manufactured 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 sized oligonucleotides (e.g., as described in 2012/041538 Kutmeier et al., BioTechniques I 7:242 (1994)), which, briefly, involves the sis of pping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an anti—Pseudomonas Psl g molecule, e. g., antibody or fragment, variant or derivative thereof can be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid ng a particular antibody is not available, but the sequence of the dy molecule 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 y that s 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 nucleotide ce and corresponding amino acid sequence of an anti— Pseudomonas Psl binding molecule, e.g., antibody or fragment, variant or tive thereof is determined, its nucleotide sequence can be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., inant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, 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 Molecular Biology, John Wiley & Sons, NY (1998), which are both incorporated by reference herein in their ties ), to generate antibodies having a different amino acid sequence, for example to create amino acid tutions, deletions, and/or insertions.

A polynucleotide encoding an anti—Pseudomonas Ps1 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 Ps1 binding molecule, e. g., antibody or fragment, t or derivative thereof can be composed of single— and double—stranded DNA, DNA that is a mixture of single— and double-stranded regions, single— and double—stranded RNA, and RNA that is e 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 regions. In addition, a polynucleotide encoding an anti— Pseudomonas Psl g le, e. g., antibody or fragment, t or tive thereof can be composed of triple—stranded regions comprising RNA or DNA or both RNA and DNA. A polynucleotide encoding an anti—Pseudomonas Psl binding molecule, e. g., antibody or fragment, t or derivative thereof can also contain one or more modifled bases or DNA or RNA backbones modified for stability or for other reasons. "Modified" bases include, for example, 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 modif1ed forms.

An isolated polynucleotide encoding a non-natural variant of a polypeptide 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 nucleotide sequence of the immunoglobulin such that one or more amino acid substitutions, ons or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site—directed mutagenesis and PCR—mediated nesis. vative amino acid substitutions are made at one or more non-essential amino acid residues.

VI. SION OF ANTIBODY PTIDES 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 followed by centrifugation or chromatography. Where desirable, mRNA can be isolated from total RNA by standard techniques such as chromatography on oligo dT cellulose.

Suitable techniques are familiar in the art.

In one embodiment, cDNAs that encode the light and the heavy chains of the anti- Pseudomonas Psl binding molecule, e. g., antibody or fragment, variant or derivative thereof can be made, either simultaneously 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 —76— encoding 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 ing references relating to recombinant DNA techniques. Of course, the DNA can be synthetic according to the t disclosure at any point during the isolation process or subsequent analysis.

Following manipulation of the isolated genetic material to provide an anti—Pseudomonas Psl binding molecule, e. g., antibody or fragment, variant or derivative thereof of the disclosure, the polynucleotides ng anti—Pseudomonas Psl g molecules, are typically inserted in an expression vector for introduction into host cells that can be used to produce the desired quantity of anti—Pseudomonas Psl binding molecules.

Recombinant expression of an antibody, or fragment, derivative or analog thereof, e. g., a heavy or light chain of an antibody which binds to a target molecule bed herein, e.g., Psl, es construction of an expression vector containing a polynucleotide that encodes the antibody. Once a polynucleotide encoding an antibody le or a heavy or light chain of an antibody, or portion thereof (containing the heavy or light chain variable domain), of the disclosure has been obtained, the vector for the production of the antibody molecule can be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide ning an antibody encoding tide ce are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate riptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, tic techniques, and in viva genetic ination.

The disclosure, 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 ce encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and US. Pat. No. 464) and the variable domain of the antibody can be cloned into such a vector for sion of the entire heavy or light chain.

The term “vector” or “expression vector” is used herein to mean vectors used in accordance with the present disclosure as a vehicle for introducing into and sing 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, s and retroviruses. In general, vectors ible with the instant disclosure will comprise a selection 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 ed. For example, one class of vector utilizes DNA elements which are derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, ia virus, baculovirus, retroviruses (RSV, MMTV or MOMLV) or SV40 virus. Others involve the use of polycistronic systems with internal ribosome binding sites. Additionally, cells which have integrated the DNA into their somes can be ed by introducing one or more markers which allow selection of transfected host cells. The marker can provide for prototrophy to an auxotrophic host, biocide ance (e.g., antibiotics) or resistance to heavy metals such as copper. The selectable marker gene can either be directly linked to the DNA sequences to be expressed, or uced into the same cell by cotransformation. Additional elements can also be needed for optimal synthesis of mRNA. These elements can include signal sequences, splice signals, 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 e of eliciting expression in eukaryotic cells can be used in the present disclosure. Examples of suitable vectors include, but are not limited to plasmids , pHCMV/Zeo, pCR3. l, pEFl/His, pIND/GS, pRc/HCMVZ, pSV40/Ze02, R—HCMV, pUB6/V5-His, pVAXl, and pZeoSV2 (available from Invitrogen, San Diego, CA), and plasmid pCI (available from Promega, Madison, WI). In general, screening large numbers of transformed cells for those which express 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 binding molecule, e. g., antibody or fragment, variant or derivative thereof of the disclosure has been prepared, the expression vector can be introduced into an appropriate host cell. Introduction of the plasmid 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, —78— transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped DNA, microinjection, and infection with intact virus.

See, Ridgway, A. A. G. "Mammalian Expression Vectors" Vectors, Rodriguez and Denhardt, Eds., worths, Boston, Mass., Chapter 24.2, pp. 470-472 (1988). Typically, plasmid introduction into the host is via electroporation. The host cells harboring the expression construct are grown under conditions appropriate to the production of the light chains and heavy chains, and assayed for heavy and/or light chain protein sis. Exemplary assay techniques include enzyme-linked immunosorbent assay ), radioimmunoassay (RIA), or fluorescence—activated cell sorter analysis , immunohistochemistry and the like.

The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody for use in the methods bed . Thus, the disclosure es host cells containing a polynucleotide encoding anti—Pseudomonas Ps1 binding molecule, e.g., dy or fragment, variant or tive thereof, or a heavy or light chain thereof, operably linked to a heterologous promoter. In some embodiments for the expression of double—chained antibodies, vectors ng both the heavy and light chains can be co-expressed in the host cell for sion of the entire immunoglobulin molecule, as detailed below. n embodiments include an isolated polynucleotide comprising a nucleic acid which encodes the above—described VH and VL, wherein a binding molecule or antigen—binding fragment thereof expressed by the polynucleotide specifically binds Pseudomonas Psl. In some embodiments the polynucleotide as described encodes an scFv molecule including VH and VL, 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.

Some embodiments include vectors comprising the above—described polynucleotides. In further embodiments, the polynucleotides are operably associated with a er. In additional ments, the disclosure provides host cells comprising such vectors. In further ments, the disclosure provides vectors where the polynucleotide is operably associated with a promoter, wherein vectors can express a binding molecule which ically binds Pseudomonas PS1 in a suitable host cell.

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

As used herein, “host cells” refers to cells which harbor vectors constructed using recombinant DNA ques 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 variety of xpression vector systems can be utilized to express dy molecules for use in the methods described herein. Such host—expression systems represent vehicles by which the coding sequences of interest can be produced and subsequently purified, but also represent 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 ia (e.g., E. coli, B. subtilis) transformed with inant 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 infected with recombinant virus sion vectors (e. g., virus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression s (e. g., cauliflower mosaic virus, CaMV; o mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e. g., COS, CHO, BLK, 293, 3T3 cells) harboring inant expression constructs containing promoters d from the genome of mammalian cells (e. g., metallothionein promoter) or from mammalian viruses (e.g., the irus late promoter; the ia 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 Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., Gene 45: 101 (1986); Cockett et al., Bio/Technology 8:2 (1990)).

The host cell line used for protein expression is often of mammalian origin; 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 Hamster 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 ), MDCK, 293, W138, R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast), HAK er kidney line), SP2/O (mouse myeloma), P3x63—Ag3.653 (mouse myeloma), BFA—lclBPT (bovine endothelial cells), RAJI (human lymphocyte) and 293 (human ). Host cell lines are typically available from cial services, the American Tissue Culture Collection or from published literature.

In addition, a host cell strain can be chosen which modulates the expression of the inserted sequences, or modif1es and processes the gene t in the specific fashion desired.

Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the on 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 correct modification and processing of the n protein sed. To this end, eukaryotic host cells which possess the cellular machinery for proper sing 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 e, cell lines which stably express the antibody 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 able marker in the recombinant plasmid confers resistance to the selection and allows cells to stably ate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and ed into cell lines. This method can ageously be used to engineer cell lines which stably express the antibody molecule.

A number of selection systems can be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell [1:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48202 (1992)), and adenine oribosyltransferase (Lowy et al., Cell 22:817 1980) genes can be employed in tk-, hgprt— or aprt—cells, respectively. Also, antimetabolite 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 77357 (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 ); neo, which confers resistance to the aminoglycoside G—418 Clinical cy —505; Wu and Wu, Biotherapy 3 :87—95 (1991); Tolstoshev, Ann. Rev.

Pharmacol. Toxicol. 32:573—596 ; Mulligan, Science 260:926—932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191—217 (1993);, TIB TECH 155—215 (May, 1993); and hygro, which s resistance to hygromycin (Santerre et al., Gene 30:147 (1984). s commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory 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 reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vector amplif1cation (for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the sion ofcloned genes in mammalian cells in DNA cloning, Academic Press, New York, Vol. 3. (1987)). When a marker in the vector system sing antibody is amplifiable, 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 mammalian cell cultivation under tissue culture conditions are known in the art and include homogeneous sion culture, e. g. in an airlift reactor or in a continuous r reactor, or immobilized or entrapped cell culture, e. g. in hollow fibers, apsules, on agarose microbeads or ceramic cartridges. If necessary and/or desired, the solutions of polypeptides can be purified by the customary chromatography methods, for example gel tion, ion-exchange chromatography, chromatography over DEAE-cellulose or (immuno-)affinity chromatography, e. g., after preferential biosynthesis of a synthetic hinge region polypeptide or prior to or subsequent to the HIC chromatography step described herein.

Constructs encoding anti—Pseudomonas Psl binding molecules, e.g., dies or fragments, variants or derivatives thereof, as disclosed herein can also be expressed non— ian cells such as bacteria or yeast or plant cells. Bacteria 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 bacteria, the heterologous polypeptides typically 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).

In bacterial systems, a number of expression s can be ageously selected ing upon the use intended for the dy 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 vectors include, but are not limited, 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 & Schuster, J. Biol. Chem. 24:5503—5509 (1989)); and the like. pGEX vectors can also be used to express n 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 ed 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 es can also be used. romyces cerevisiae, or common baker's yeast, is the most ly used among otic microorganisms although a number of other strains are commonly available, e.g., Pichia pastoris.

For expression in Saccharomyces, the d YRp7, for example, hcomb et al., Nature 282:39 (1979); Kingsman et al., Gene 7:141 (1979); Tschemper et al., Gene 10:157 (1980)) is commonly used. This plasmid already contains the TRPl gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4—l (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 e of tryptophan.

In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is typically used as a vector to s foreign genes. The virus grows in Spodoptera frugiperda cells. The dy coding sequence can be cloned individually into sential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV er (for example the polyhedrin promoter).

Once the anti—Pseudomonas Psl g molecule, e. g., antibody or fragment, variant or derivative thereof, as sed herein has been recombinantly expressed, it can be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly 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 sed in US 2002 0123057 Al.

VII. IDENTIFICATION OF PE—INDIFFERENT G MOLECULES The disclosure asses a target indifferent whole-cell approach to identify serotype independent therapeutic binding les e. g., antibodies or fragments thereof with superior or desired therapeutic activities. The method can be utilized to identify binding molecules which can antagonize, neutralize, clear, or block an undesired activity of an infectious agent, e.g., a ial pathogen. As is known in the art, many infectious 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 multiple pathogens from multiple s. For example, the method was ed 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 phagocytic (OPK) activity against bacterial cells such as bacterial pathogens, e.g. opportunistic Pseudomonas species (e.g., Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida, and Pseudomonas genes) 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) preparing na'1've and/or convalescent antibody libraries in phage, (b) removing serotype—specific antibodies from the library by depletion panning, (c) screening the library for antibodies that specifically bind to whole cells independent of serotype, and (d) screening 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. ed clones were converted to human IgG1 antibodies and were confirmed to react with P. nosa clinical isolates regardless of serotype fication or site of tissue isolation (See Examples). onal ty screens described 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 thereof, antibodies or fragments thereof, or compositions, bind to two or more, three or more, four or more, or five or more different P. aeruginosa pes, or to at least 80%, at least 85%, at least 90% or at least 95% of P. nosa 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. CEUTICAL COMPOSITIONS COMPRISING ANTI— PSEUDOMONAS PSL BINDING MOLECULES The ceutical compositions used in this disclosure comprise pharmaceutically acceptable carriers well known to those of ordinary skill in the art. Preparations for parenteral administration include e aqueous or ueous solutions, suspensions, and emulsions. n pharmaceutical compositions as disclosed herein can be orally stered in an acceptable dosage form including, e.g., capsules, tablets, aqueous suspensions or solutions.

Certain pharmaceutical compositions also can be administered by nasal aerosol or inhalation.

Preservatives and other ves can also be present such as for example, antimicrobials, —85— idants, chelating agents, and inert gases and the like. Suitable formulations for use in the therapeutic methods disclosed herein are described in Remington's ceutical Sciences, Mack hing Co., 16th ed. (1980).

The amount of an anti—Pseudomonas Psl binding molecule, e. g., antibody or fragment, variant or derivative thereof, that can be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.

Dosage regimens also can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). The compositions can also comprise the anti- Pseudomonas Psl binding les, e.g., antibodies or nts, variants or derivatives thereof dispersed in a biocompatible carrier material that functions as a suitable delivery or t system for the nds.

IX. TREATMENT METHODS USING THERAPEUTIC BINDING MOLECULES Methods of ing and administering an anti-Pseudomonas Psl binding molecule, e.g., an antibody or fragment, variant or derivative thereof, as disclosed herein to a subject in need f are well known to or are readily determined by those d in the art. The route of administration of the anti-Pseudomonas Psl binding molecule, e.g., antibody or fragment, variant or derivative thereof, can be, for example, oral, parenteral, by inhalation or topical. The term parenteral as used herein includes, e. g., intravenous, intraarterial, intraperitoneal, intramuscular, or subcutaneous administration. A suitable form for administration would be a on for injection, in particular for intravenous or intraarterial injection or drip. r, in other methods ible with the teachings herein, an anti—Pseudomonas Psl binding molecule, e. g., antibody or fragment, variant 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 diseased tissue to the therapeutic agent. For e, an anti—Pseudomonas Psl binding molecule can be directly administered to ocular tissue, burn injury, or lung tissue.

Anti—Pseudomonas Psl binding molecules, e.g., antibodies or fragments, ts or derivatives thereof, as disclosed herein can be administered in a pharmaceutically effective amount for the in viva treatment of Pseudomonas infection. In this regard, it will be appreciated that the sed g molecules will be formulated so as to facilitate administration and promote stability of the active agent. For the purposes of the instant application, a pharmaceutically effective amount shall be held to 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 ments are ed to a method of preventing or treating a Pseudomonas infection in a t in need thereof, comprising administering to the t 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 . In further embodiments, the Pseudomonas infection 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 ions. In further embodiments, the subject suffers from acute pneumonia, burn injury, corneal infection, cystic fibrosis, or a combination thereof.

Certain embodiments are directed to a method of blocking or preventing attachment of P. aeruginosa to epithelial cells comprising 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 , or the host cell described herein.

Also disclosed is a method of enhancing OPK of P. aeruginosa comprising contacting a e of phagocytic cells and P. aeruginosa with the binding molecule or nt thereof, the antibody or fragment thereof, the composition, the polynucleotide, the vector, or the host cell described 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 binding les, 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 ent in an amount sufficient to produce a eutic effect. The anti—Pseudomonas Ps1 binding molecules, e.g., antibodies or nts, variants or derivatives thereof, disclosed herein can be stered to such human or other animal in a conventional dosage form prepared by combining the antibody of the disclosure with a conventional pharmaceutically acceptable carrier or diluent according to known ques.

Effective doses of the compositions of the present disclosure, for treatment of Pseudomonas infection vary depending upon many different s, including means of administration, target site, physiological state of the patient, whether the patient is human or an —87— animal, other medications administered, and r treatment is prophylactic or therapeutic.

Usually, the patient is a human but non-human mammals including transgenic mammals can also be treated. ent dosages can be titrated using routine methods known to those of skill in the art to optimize safety and efficacy.

Anti—Pseudomonas Psl binding molecules, e. g., antibodies or fragments, variants or derivatives thereof can be administered le ons at various frequencies depending on s factors known to those of skill in the art.. Alternatively, anti-Pseudomonas Psl binding molecules, e.g., antibodies or fragments, variants or derivatives thereof can be administered as a sustained e formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the ife of the antibody in the patient.

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

X. IMMUNOASSAYS Anti—Pseudomonas Psl binding molecules, e. g., antibodies or fragments, variants or derivatives thereof can be assayed for immunospeciflc binding by any method known in the art.

The immunoassays which can be used include but are not limited to itive and non- competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent assays, protein A immunoassays, to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al., eds, Current ols in Molecular Biology, John Wiley & Sons, Inc., New York, Vol. 1 (1994), which is incorporated by reference herein in its entirety). Exemplary immunoassays are bed briefly below (but are not intended by way of limitation).

There are a variety of methods ble for measuring the ty of an antibody-antigen interaction, but vely 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 quantities. Antibody affinity can be ed 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 g and dissociation events can be monitored by measuring the binding of one protein in solution to a second protein immobilized on the ForteBio sor. In the case of measuring binding of anti-Psl antibodies to Psl, the Psl 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 is then detected by the instrument sensor. The data is then collected and exported to ad Prism for affinity curve fitting.

Surface plasmon resonance (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) biospeciflc surface can be regenerated so that a series of different monoclonal dies can easily be compared under identical conditions; (v) analytical procedures are fully ted, and extensive series of measurements can be performed without user intervention. BIAapplications Handbook, version AB (reprinted 1998), BIACORE® code No. BR—1001—86; BIAtechnology ok, version AB (reprinted 1998), BIACORE® code No. BR—lOOl—84.

SPR based g studies require that one member of a binding pair be immobilized on a sensor e. 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 through binding to another 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, ime BIACORE® measurements monitor interactions ly as they happen. 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 injected in a te pulse across a ligand surface, the resulting sensorgram 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) Dissociation of analyte from the surface during buffer flow.

The association and iation 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).

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 ty constants for interaction can be obtained from simple BIACORE® igations. The range of affinities measurable by this que is very broad g from mM to pM.

Epitope specificity is an important teristic of a monoclonal antibody. Epitope mapping with BIACORE®, in contrast to conventional ques using radioimmunoassay, ELISA or other surface adsorption methods, does not require labeling or purified antibodies, and allows multi—site specificity tests using a sequence of several monoclonal dies.

Additionally, large numbers of analyses can be processed automatically.

Pair-wise binding experiments test the ability of two MAbs to bind simultaneously to the same antigen. MAbs ed against separate epitopes will bind independently, whereas MAbs directed against identical or closely related epitopes will interfere with each other’s binding.

These binding experiments 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 grams will reveal: 1. how much of the antigen binds to first Mab, 2. to what extent the second MAb binds to the surface—attached antigen, 3. if the second MAb does not bind, r 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 dy g studies, and can relate functional epitopes to structural features when the primary sequence of the antigen is known. Peptides or antigen fragments are tested for inhibition of g 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. 2012/041538 The practice of the disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, lar biology, transgenic biology, iology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., Sambrook et al., ed., Cold Spring Harbor Laboratory 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 II ; Oligonucleotide Synthesis, M. J. Gait ed., ; Mullis et a]. 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. (1984); Culture Of Animal Cells, R. I. Freshney, Alan R. Liss, Inc., (1987); lized Cells And Enzymes, IRL Press, (1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, s In logy, Academic Press, Inc., N.Y.; Gene Transfer Vectors For Mammalian Cells, J. H. Miller and M. P. Calos eds., Cold Spring Harbor Laboratory (1987); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.); Immunochemical Methods In Cell And lar Biology, Mayer and Walker, eds., Academic Press, London (1987); Handbook Of Experimental Immunology, Volumes I—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 l et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989).

Standard reference works setting forth general ples of immunology include Current Protocols in Immunology, John Wiley & Sons, New York; Klein, J The Science ., Immunology: of Self-Nonself Discrimination, John Wiley & Sons, New York (1982); Roitt, I., 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 Sciences Division (2005); and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988).

Having now described the disclosure in detail, the same will be more clearly understood by reference to the following examples, which are included th for purposes of ration only and are not intended to be limiting of the disclosure. All patents and ations referred to herein are expressly incorporated by reference in their entireties. _ 91 _ Example 1: Construction and screening of human antibody phage display libraries This e describes a target indifferent whole cell panning ch with human dy phage libraries derived from both naive and P. aeruginosa infected convalescing patients to identify novel tive antigens against Pseudomonas infection (Figure 1A).

Assays included in the in vitro functional screens included opsonophagocytosis (OPK) killing assays and cell attachment assays using the lial cell line A549. The lead candidates, based on superior in vitro ty, 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 ts 7—10 days post diagnosis followed by RNA extraction and phage y construction as previously described (Vaughan, T.J., et al., Nat Biotechnol 14, 309— 314 (1996); Wrammert, J et al., Nature 453, 667—671 (2008)).

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

In addition to the patient library, a naive human scFv phage display library containing up to 1x1011 binding members (Lloyd, G, et al., Protein Eng Des Se] 22, 159—168 (2009)) was used for antibody isolation (Vaughan, T.J., et al., Nat Biotechnol 14, 309—314 (1996)). Heat killed P.aerugin0sa ) was immobilized in IMMUNOTM Tubes (Nunc; MAXISORPTM) ed for phage display ions as described (Vaughan, T.J., et al., Nat Biotechnol 14, 309—314 (1996)) with the exception of anolamine (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 washing, 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 bed (Vaughan, 1996).

Following development and validation of the whole-cell affinity selection methodology, both the new convalescing t library and a previously constructed naive library (Vaughan, T.J., et al., Nat Biotechnol 14, 309—3 14 (1996)) underwent affinity selection on suspensions of P. aeruginosa strain 3064 possessing a complete O-antigen as well as an isogenic wapR mutant strain which lacked surface expression of O-antigen. Figure 1D shows that output titers from WO 70807 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 addition, duplication of VH CDR3 loop sequences in the libraries (a measure of clonal enrichment during ion), was also found to be higher in the patient library, 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 (2004)). d antibodies were added to blocked 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 d serotype specific reactivity (data not shown). Clones exhibiting pe independent g in the absence of nonspecific binding to E. coli or bovine serum albumin were ed for r evaluation.

For IgG expression, the VH and VL chains of selected antibodies were cloned into human IgG1 expression s, co—expressed in HEK293 cells, and ed by protein A affinity chromatography as described (Persic, L., et al., Gene 187, 9—18 (1997)). Human IgG1 antibodies made with the variable regions from these selected serotype independent phage were confirmed for P. aeruginosa specificity and prioritized for subsequent is 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 strains 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 incubated with an ALEXA FLUOR 647® goat anti- human IgG antibody (Invitrogen, Carlsbad, 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 ences) and analyzed using BD FacsDiva (v. 6.1.3) and FlowJo (v. 9.2; TreeStar). Antibodies exhibiting binding by FACS were further tized for functional activity testing in an opsonophagocytosis killing (OPK) assay. _ 93 _ Example 2: Evaluation of mAbs promoting OPK of P. aeruginosa This e describes the evaluation of prioritized human IgG1 antibodies to e OPK of P. aeruginosa. Figure 2A shows that with the exception of WapR-007 and the negative 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 ndomenico, 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 s; diluted baby rabbit serum; differentiated HL-60 cells; and monoclonal antibody. In some OPK assays, luminescent P. aeruginosa strains, which were constructed as described (Choi, K.H., 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 abel plate reader n Elmer).

The ability of the WapR-004 and Cam-003 antibodies to mediate OPK activity against r clinically relevant O-antigen serotype strain, 9882-80.lux, was evaluated. Figure 2B shows that enhanced WapR-004 and Cam-003 OPK ty extends to strain 9882-80 (01 l).

The ability of Cam-003 to mediate OPK activity against representative non-mucoid strains from clinically relevant O-antigen serotypes and against mucoid P. aeruginosa strains which were derived from cystic fibrosis patients was ted. Cam—003 mediated potent OPK of all non-mucoid clinical isolates tested (Figure 2C). In addition, Cam-003 mediated potent killing of all mucoid P. aeruginosa isolates that were tested (Figure 2D).

In addition, this example describes the tion of 04 (W4) mutants in scFv-Fc format to promote OPK of P. aeruginosa. One mutant, Wap-004RAD (W4-RAD), was specifically created through site-directed nesis 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, Sl85—l94 (1995)), to amplify W4 variants ed from somatic hypermutation) from the scFv library derived from the convalescing P. aeruginosa infected patients for analysis. This is the y from which 04 was derived. W4 variant fragments were subcloned and sequenced using rd procedures known in the art. W4 mutant light chains (LC) were recombined with the WapR—004 heavy chain (HC) to produce W4 mutants in scFv-Fc format. In addition WapR-004 RAD heavy chain (HC) mutants were recombined with parent LCs of M7 and M8 in the scFv—Fc 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. nosa oup 05 strain (PAOl.lux). e 3: Serotype independent anti-P. aeruginosa antibodies target the Psl exopolysaccharide This example describes identification of the target of anti-P. aeruginosa antibodies derived from ypic screening. Target is was performed to test whether the serotype independent antibodies targeted protein or carbohydrate antigens. No loss of binding was observed in ELISA toPAOl whole cell extracts exhaustively digested with proteinase K, ting that reactivity targeted e accessible carbohydrate residues (data not shown).

Isogenic mutants were constructed in genes responsible for O—antigen, alginate, and LPS core biosynthesis; wpr (O-antigen-deflcient); wpr/aZgD (O-antigen and te deficient); rmZC (O-antigen-deflcient and truncated outer core); and galU (O-antigen-deflcient and truncated inner core). P. aeruginosa mutants were constructed based on the allele replacement strategy described by zer (Schweizer, H.P., M01 Microbiol 6, 204 (1992); Schweizer, H.D., Biotechniques 15, 831—834 ). Vectors were mobilized from E. coli strain S17.1 into P. nosa 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. Reactivity of antibodies was determined by indirect ELISA on plates coated with above indicated P. aeruginosa strains: Figures 3A and 3J show that Cam—003 binding to the wpr or the wpr/aZgD double mutant was unaffected, however binding to the rmZC and galU mutants were abolished.

While these s were consistent with binding to LPS core, reactivity to LPS purified from PAOl was not observed. The rmZC and gaZU genes were recently shown to be required for thesis of the Psl exopolysaccharide, a repeating pentasaccharide polymer consisting of D- mannose, L—rhamnose, and D—glucose. Cam—003 binding to an isogenic psZA knockout PAOlApsZA, was tested, as psZA is required for Psl biosynthesis (Byrd, M.S., et al., M01 Microbiol 73, 622—638 ). Binding of 3 to PAOlApSlA was abolished when tested by ELISA (Figure 3B) and FACS (Figure 3C), while the LPS molecule in this mutant was unaffected (Figure 3D). Binding of Cam-003 was restored in a PAOlAwpr/aZgD/pslA triple mutant mented with psZA (Figure 3E) as was the ability of Cam—003 to e opsonic killing to mented PAOlApSlA in contrast to the mutant (Figure 3F and 3G). Binding of Cam—003 antibody to a Pel exopolysaccharide 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).

To confirm that all of the antibodies bound to the same antigen, a Psl capture binding assay was performed using a FORTEBIO® OCTET® 384 instrument as described above. The n was nase K-treated ed carbohydrate purified from PAOlAwpr/aZgD/pelA (O—antigen—, alginate— and Pel exopolysaccharide—deficient). Individual dies were bound to aminopropylsilane biosensors followed by blocking and the addition of the enriched carbohydrate antigen. After washing to remove unbound n, binding of unlabelled mAbs to captured antigen was assessed. All bound antibodies (Cam—003, Cam—004, Cam—005, WapR— 001, WapR—002, WapR—003, WapR—007 and l6), with the exception of the control mAb R347, were capable of ing antigen that reacted with each of 3, WapR-OOl, WapR- 002, WapR—003, and WapR—Ol6 (Figure 3K). Minimal reactivity to ed Psl was observed with Cam—004, Cam-005 and WapR—007 even though all three of these antibodies captured suff1cient Psl to potently react with Cam-003, WapR—OOl, OZ, WapR—003, and WapR— 016 e 3K). These results suggest that all of the mAbs derived by phenotypic screening that bound P. aeruginosa independently of serotype, targeted epitopes associated with Psl exopolysaccharide.

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

This example shows that anti-Psl antibodies blocked P. aeruginosa association with epithelial cells. Anti-Psl antibodies were added to a confluent monolayer of A549 cells (an arcinoma human alveolar basal epithelial cell line) grown in opaque 96-well plates (Nunc Nunclon Delta). Log—phase luminescent P. aeruginosa PAOl strain 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. Measurements from wells without A549 cells were used 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 s 2A-B, and Example 2), were also most active at inhibiting P. aeruginosa cell attachment to A549 lung epithelial cells, providing up to ~80% ion compared to the ve control. WapR—016 was the third most active antibody, showing similar inhibitory activity as WapR-004 and Cam-003 but at 10-fold higher antibody concentration.

Example 5: In vivo passaged P. aeruginosa strains maintain/increase expression of Psl To test if Psl sion in vivo is maintained, mice were injected intraperitoneally with P. aeruginosa es followed by harvesting of bacteria by peritoneal lavage four hours post— ion. 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 quantification of cells that are positive or negative for Psl expression. For ex vivo binding, bacterial inocula (0.1ml) was prepared from an overnight TSA plate and delivered intraperitoneally to BALB/c mice. At 4 hr. following challenge, bacteria were harvested, 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 bacteria harvested after peritoneal lavage with three wild type P. aeruginosa strains showed strong Cam—003 staining, which was comparable to log phase cultured bacteria (compare Figures 5A and 5C). In vivo passaged wild type bacteria exhibited 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 s PAOl (05) and 6206 (01 toxic). The binding of Cam—003 to bacteria increased in relation 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 r ize Psl as the target of the monoclonal antibodies.

The level of Psl sion/accessibility on the surface of P. aeruginosa s PAOl and 6206 in the acute pneumonia model was also assessed. Bacteria prepared from ovemight- incubated, confluent plates, as described above, were intranasally administered to BALB/c mice.

At 4 and 24 hours post—infection, bacteria were red from the lungs by bronchoalveolar lavage. Samples were stained and analyzed as usly described in Example 1. Strong Cam- 003 staining was observed for PAOl at 4 hours post—infection, but was minimal for 6206 at this time point (Figure 5D). However, for both strain PAOl and 6206, strong Cam-003 staining was observed at 24 hours post—infection (Figure SE).

The binding of P. aeruginosa specific antibodies (Cam—003, Cam—004 and Cam—005) to representative strains from unique P. aeruginosa serotypes (PAOl(05) (Figure SF), 2135 (01) (Figure 5G), 2531 (01) (Figure SH), 2410 (06) (Figure 51), 2764 (011) (Figure 51), 2757 (01 1) (Figure 5K), 33356 (09) (Figure 5L), 33348 (01) (Figure 5M), 3039 (NT) (Figure SN), 3061 (NT) (Figure 50), 3064 (NT) (Figure 5P), 19660 (NT) e 5Q), 9882—80 (01 1) (Figure SR), 6073 (01 1) (Figure 5S), 6077 (01 1) (Figure ST) and 6206 (01 1) (Figure 5U), was evaluated by flow cytometry as lly described above.

Example 6: Survival rates for animals treated with s1 monoclonal antibodies Cam-003 and WapR—004 in a P. aeruginosa acute nia model Antibodies or PBS were administered 24 hours before infection in each model. P. aeruginosa acute nia, 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 modifications. 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 es 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. onal antibodies Cam—003 and WapR—004 were evaluated in an acute lethal pneumonia model t P. aeruginosa strains representing the most frequent serotypes associated with clinical disease. Figures 6A and 6C show significant tration-dependent al in Camtreated mice infected with strains PAOl and 6294 when compared to controls. Figures 6B and 6D show that complete protection 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 observed at 5mg/kg for 33356 and 6077, respectively. Figures 6E and 6F show significant concentration-dependent survival in WapRtreated mice in the acute pneumonia model with strain 6077 (011) (8 x 105 CFU) e 6B), or 6077 (011) (6 x 105 CFU) (Figure 6F). Figure 6G shows that at 120 hours Cam—003 provided 100 % survival following infection with strain PAOl. Increased survival was not observed against the Psl mutant strain, SlA, used as a negative l in the PAOl acute pneumonia study (Figure 6G), confirming the lack of Cam- 003 activity against strains deficient in Psl expression.

Cam—003 and WapR—004 were next examined for their ability to reduce P. aeruginosa organ burden in the lung and spread to distal organs, and later the animals were treated with various concentrations of WapR—004, Cam—003, or control antibodies at several ent concentrations. Cam—003 was ive at reducing P. aeruginosa lung burden against all four strains tested. Cam-003 was most effective against the highly pathogenic xic , 6077, where the low dose was as effective as the higher dose es 7D). Cam—003 also had a marked effect in reducing dissemination to the spleen and kidneys in mice infected with PAOl e 7A), 6294 (Figure 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 pneumonia with 6294 (O6) and 6206 (01 1).

Specifically, WapR—004 was effective at reducing P. aeruginosa dissemination to the spleen and kidneys in mice infected.

Example 7: Survival rates for animals treated with anti-Psl monoclonal antibodies Cam-003 and WapR—004 in a P. aeruginosa corneal infection model Cam—003 and WapR—004 efficacy was next evaluated in a P. aeruginosa corneal infection model which izes the pathogens ability to attach and colonize 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 was observed in negative control-treated animals. Figure 8E shows that Cam-003 was also effective when tested in a l injury model, ing significant tion at 15 and 5mg/kg when compared to the antibody—treated control.

Example 8: A Cam-003 Fc mutant antibody, Cam-003 -TM, has diminished OPK and in viva efficacy but maintains anti-cell attachment activity.

Given the potential for dual mechanisms of action, a Cam-003 Fc mutant, CamTM, was created which harbors mutations in the Fc domain that reduces its interaction with Fcy receptors (Oganesyan, V., et al., Acta llogr D Biol Crystallogr 64, 700—704 (2008))., to identify if protection was more correlative to anti-cell attachment or OPK activity. P. aeruginosa mutants were constructed based on the allele replacement gy 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. coli strain $17.1 into P. aeruginosa strain PAOl; recombinants were isolated as described , T.T., et al., Gene 212, 77—86 (1998)). Gene deletion was confirmed by PCR. P. aeruginosa mutants were mented with pUCP30T—based constructs harboring wild type genes. Figures 9A shows that 3-TM exhibited a 4-fold drop in OPK activity compared to Cam—003 (ECso of 0.24 and 0.06, respectively) but was as effective in the cell attachment assay e 9B). Figure 9C shows that Cam—003—TM was also less effective against pneumonia 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. When tested in the mouse acute pneumonia model, CamTM was similar in y to 3 at a low infectious inoculum of 6077 (2.4x105 CFU) (Figure 9D). r, further titration of the antibody dose followed by challenge with a larger infectious inoculum (1.07x106) revealed Cam—003 activity was superior to Cam—003—TM, suggesting OPK activity significantly contributes to optimal protection in viva (Figure 9E).

Example 9: Epitope mapping and relative affinity for anti-Ps1 antibodies Epitope mapping was med 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. For competition ELISA, antibodies were biotinylated using the EZ- Link Sulfo-NHS-Biotin and Biotinylation Kit (Thermo Scientific). Antigen coated plates were treated with the EC50 of biotinylated antibodies coincubated with led antibodies. After incubation with HRP—conjugated streptavidin (Thermo Scientific), plates were developed as bed above. Competition experiments between anti—Ps1 mAbs determined that antibodies 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, however the class 3 antibody, WapR—016, partially inhibits binding of the Class 1 and 2 antibodies.

Antibody affinity was ined by the OCTET® binding assays using Psl derived from the supernatant of ght PAOl es. Antibody KD was determined by averaging the binding kinetics of seven concentrations for each dy. Affinity measurements were taken —100— with a IO® OCTET® 384 instrument using 384 slanted well plates. The supernatant from ght PAOl cultures :: 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 med as described (Wang, X., et al., J Immunol Methods 362, 151—160). Association and ociation raw AnM data were curve-fitted with ad Prism. Figure 10A shows the ve binding affinities of anti-Psl antibodies characterized 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 (W4RAD) mutant as well as other W4 s prepared as described in Example 1.

Example 10: Binding of Polymyxin B (PMB)-mAb conjugates to P. nosa PAOl cells was evaluated by FACS In this Example, PMB conjugated to an c monoclonal antibody (mAb) that was capable of ing bacterial clearance was evaluated to determine whether the conjugate would improve and/or expand mAb functionality, while also reducing the toxicity of PMB.

CAM—003, a mAb targeting the P. aeruginosa Psl surface exopolysaccharide, which mediates potent phagocytic killing (OPK) activity and protection in viva, was selected for conjugate evaluation.

This example evaluates binding of various Polymyxin B (PMB)—mAbs conjugates to P. nosa PAOl cells. Using a two—step site—directed conjugation method (Figure 12), xin B (PMB) was conjugated to the Cam-003 and A7 (hIgGl control) mAb variants with either a single or double cysteine engineered into the Fc region. Cam—003 and A7 mAbs Fc variants were prepared using standard protocols as described in (Dimasi, N. et al., J M01 Biol. 393(3):672—92 (2009)). The heterobifunctional SM(PEG)12 linker (Pierce) was initially conjugated to one of the primary amines in PMB via the NHS group in the linker under conditions ined to favor conjugation of a single linker. Polymyxin B sulfate (Sigma) was dissolved in PBS pH 7.2 at 2 mg/ml and reacted with SM(PEG)12 linker at a 4:1 PMB:linker ratio. The reaction was carried out at room temperature for 30 min and stopped with 50 mM glycine. The efficiency of SM(PEG)12 linker conjugation to PMB was approximately 25%. —lOl— Crude preparations of PMB-PEGlz were then reacted with deprotected Fc cysteine mAb variants and conjugated via maleamide in the PEG12 linker ( see, e.g., and WO 2009/092011). The PMB—mAb conjugates were purified by extensive dialysis. The conjugates were initially dialyzed in 3.3X PBS pH 7.2 with 0.7% CHAPS with four buffer exchanges, ed by dialysis in 1X PBS pH 7.2 with additional four buffer exchanges. Conjugation efficiency and levels free PMB-linker in the samples were determined by UPLC and mass spectrometry.

CAM—003 is specific for the P. aeruginosa Psl surface exopolysaccharide and mediates potent OPK ty and protection in multiple in vivo models. Figure 13A shows Cam—003 and A7 Fc region mutated es. SM (A339C), DMl (T289C/A339C), DMZ (A339C/S442C). ation efficiency of PMB-mAbs variants was ined by mass spectrometry analysis of heavy chains in purified conjugates. (see, e.g., and ). The overall conjugation efficiency was 75—85%. Purity of constructs was >95% relative to conjugated vs. free nker. Figure 13B shows the e number of PMB in PMB-Cam-003 and PMB-A7 conjugates (double mutant 2 (DMZ) > double mutant 1 (DMl) > single mutant (SM)).

A7 conjugates exhibited greater conjugation eff1ciency compared to Cam—003 conjugates.

Contamination with free PMB in the purified preparations was determined to be negligible.

Binding of PMB—Cam—003 and PMB—A7 conjugates to P. aeruginosa PAOl cells was evaluated by FACS. R347 was used as a negative control in all experiments. Samples were stained and analyzed as previously described in Example 1. No significant difference in binding of Cam-003 conjugates compared to unconjugated or mock—conjugated Cam—003 was observed (Figure 14A).

Binding of A7 control conjugates was proportional to the number of PMB molecules per conjugate (Figure 14B). This analysis indicates that conjugation of PMB to Cam—003 does not significantly impact whole-cell binding and that conjugated PMB can mediate direct binding to cells, presumably by binding LPS. e 11: Evaluation of PMB-mAb conjugates promoting OPK of P. aeruginosa This example describes two series of ments evaluating the y of PMB—mAb conjugates to promote OPK of P. aeruginosa. In the first experiments es 15A-B), conjugate-mediated OPK activity by human HL-60 neutrophil cell line in the presence of rabbit ment was evaluated using P. aeruginosa strains expressing bacterial luciferase as —102— described in Example 2. R347 was used as a negative l in these experiments. The CAM- 003 conjugates retained potent OPK activity, although it diminished with sing number of PMB per conjugate (SM > DMl > DM2) (Figure 15A). The CAM—003 conjugates did not exhibit OPK activity against the ApsZA P. aeruginosa strain which does not express the Psl , indicating that mAb-mediated binding was required for killing (Figure 15B).

In the second series of experiments, reduction in luminescence following 2 h incubation relative to control lacking mAb was used to determine % g. Figure 18A shows that the CAM—003 conjugates retained OPK activity, although it diminished with increasing number of PMB per conjugate, particularly in DM and TM constructs >DM>TM). The CAM—003 conjugates did not exhibit OPK activity against the PAC! ApslA strain which does not express the Psl target (not . Figure 18B shows that A7—PMB ates did not e OPK indicating that mAb-mediated binding was ed for killing.

Example 12: Neutralization of P. aeruginosa LPS by PMB—mAb conjugates Neutralization of P. aeruginosa OlO LPS activity was ted by preincubating the PMB-mAb conjugates or PMB alone with LPS for 1h, followed by stimulation of murine RAW 264.7 macrophages and quantification of TNF secretion. Final concentration of LPS was 2ng/ml.

TNF was quantified by the FACS-based BDTM Cytometric Bead Array (CBA) method (BD Biosciences) after 6h stimulation. LPS neutralization was measured by a decrease in TNF production relative to the LPS maximal response. PMB—Cam—003 ates, but not mock— conjugated wild—type Cam-003 exhibited LPS neutralization. Efficiency of neutralization was directly proportional to the average number of PMB in the conjugate (DM2 > DMl > SM) (Figure 16A). PMB-A7 conjugates, but not mock-conjugated wild-type A7 exhibited LPS neutralization (Figure 16B). A7 conjugates exhibited better neutralization than CAM-003 conjugates. A7 conjugates exhibited better neutralization than CAM-003 conjugates likely due to greater conjugation efficiency ed with these molecules. Approximately 2 conjugated PMB molecules/mAb are required to neutralize the amount of LPS neutralized by a free PMB molecule. — 103 - Example 13: Evaluation of Cam—003—PMB site—directed conjugates in murine models The efficacy of 3—PMB conjugates were evaluated in two types of murine models: 1) endotoxemia (LPS) nge model, to determine the ability of the conjugates to neutralize and/or detoxify LPS in vivo; and 2) in P. aeruginosa sepsis model, to evaluate if Cam— 003—PMB conjugates effect improved protection against bacterial challenge relative to the antibody alone through PMB-mediated LPS neutralization and/or clearance, in addition to the antibody—mediated bacterial clearance. Other P. aeruginosa challenge models can also be used to test the cy of Cam—003—PMB conjugates (see below).

A. Endotoxemia model It is well established that PMB can bind and neutralize LPS in vivo, and mediate protection against LPS challenge son, DC. et a]. J. Immunochemistry 13(10):813—818 (1976), Drabick, JJ. et al., Antimicrob Agents Chemother. 42(3):583—588 (1998)). In the endotoxemia model, Cam—003—PMB conjugates will be ted for their ability to protect animals from LPS challenge. Purified LPS from Gram-negative bacteria, including P. aeruginosa and E. coli, will be used to challenge mice at the established minimal lethal doses (LD100). As mice are relatively resistant to LPS, D-galactosamine may also be coadministered, as it y increases the sensitivity of mice to LPS to roughly that of humans (Galanos, C. et al., Proc Natl Acad Sci U S A. 76(11):5939—5943 (1979)). Such models have been widely used for preclinical efficacy evaluation of LPS neutralizing molecules, including antibodies and polymyxin-protein conjugates (Bailat, S. et al., Infect Immun. 65(2):811—814 (1997), meier, G. et al., J Pharmacol Exp Ther. 318(2):762—771 (2006), Drabick, JJ. et al., Antimicrob Agents Chemother. 42(3):583—588 (1998)). Cam—003—PMB conjugates, l conjugates and unconjugated Cam—003 can be administered either therapeutically or lactically, and their ability to t animals from LPS challenge can be ted. The extent of protection ed by PMB conjugates can be correlated with levels of proinflammatory cytokines and chemokines measured in sera or plasma, including TNF, KC and IL—6. — 104 — B. P. nosa challenge models Several murine models of P. aeruginosa infection can be used to evaluate the ability of Cam—003—PMB conjugates to mediate protection. P. aeruginosa can be stered to mice intraperitoneally (sepsis model), intravenously (bacteremia model) or asally (pneumonia model) at the determined LD100 doses. These models have previously been used for nical eff1cacy studies of passive or active vaccines (Frank, DW. et al., J Infect Dis. 186(1):64—73. (2002), Secher, T. et al., JAntimicrob Chemother. 66(5): 1 100—1 109 (2011), Miyazaki, S. et al., J Med Microbiol. 43(3).‘169—175 (1995), Dunn, DL. et al., y 440—446 (1984)).

As in the endotoxemia model, it may also be necessary to sensitize mice with D- galactosamine prior to bacterial challenge to overcome their innate resistance to LPS toxicity and to be able to evaluate the contribution of LPS lization and/or clearance to in viva efficacy of the PMB conjugates. D-galactosamine has been demonstrated to reduce the LDlOO of Gram- negative bacteria, likely by increasing sensitivity to LPS shed during infection (Bucklin, SE. et al., J Infect Dis. 172(6): 1519—27 (1995)). 3—PMB conjugates, l conjugates and unconjugated Cam—003 can be administered either therapeutically or prophylactically. The ability of CAM-003 conjugates to effect increased protection over Cam-003 alone by neutralizing and/or clearing the bacterial LPS via the conjugated PMB moiety can be determined in al studies. The efficacy of Cam—003— PMB conjugates in mediated bacterial clearance can also be evaluated by fying P. nosa bacteria in serum and organs, including spleen, kidneys and lungs, following infection. Serum or plasma LPS levels can also be quantified to evaluate the extent of bacterial clearance and LPS clearance and/or neutralization by the CamPMB conjugates and compare it to those of unconjugated Cam—003 and control antibody—PMB conjugates.

C. Endotoxemia Model Data In particular, C57Bl/6 mice (10 per group) were dosed i.p. with mAb or PMB—mAb conjugate 6h prior to challenge with P. aeruginosa PAOlO LPS (Sigma) and D-galactosamine.

PMB control was dosed i.p. 2 h prior to challenge at 0.2 mg/kg and typically provides 80—100% protection. l mice dosed with unconjugated CAM—003 all died within 18h. Figures 19A and B show that, at 45 mg/kg, DM and TM conjugates of CAM—003 and A7 provided 90—100% protection, while the SM ates were not protective. —105— TM conjugates were dosed at 45, 15 and 5 mg/kg. As shown in Figures 20A and B, loss of protective activity was more rapid with CAMTM-PMB than with A7-TM-PMB, which retained 80% protection at 5 mg/kg. These differences suggest that unique structural features of a mAb can impact LPS neutralization activity of conjugated PMB, as previously seen in vitro.

D. Sepsis Model Data C57Bl/6 mice (10 per group) were dosed with mAb or PMB—mAb conjugates i.p (10, l and 0.1 mg/kg) 6 h prior to i.p. challenge with LD80_100 dose of P. aeruginosa strain 6294 (4E7 CFU). Data from two studies was combined in this is. Survival was monitored over 72h.

Combined results of two studies are shown in Figures 2lA—C: . Most control mice dosed with A7 or buffer died by 24 h. Unconjugated CAM—003 showed 50—90% protection. Protective activity appeared to be inversely correlated with dose. CAM—003—PMB ates conferred better tion than unconjugated mAb at the high dose of 10 mg/kg, suggesting that neutralization of LPS shed during infection contributed to survival. The PMB control conjugate exhibited 50% protective activity at 10 mg/kg, suggesting that LPS neutralization can provide a survival benef1t. Conversely, the conjugates were less protective than CAM—003 at the low dose of 0.1mg/kg, and protective activity correlated with in vitro OPK activity of the conjugates (WT>SM>DM>TM). Together the results indicate that ated PMB can confer added protective activity to an opsonic dy by mediating neutralization of LPS and complement its bacterial clearance function.

High conjugation efficiency of PMB to engineered Fc ne residues was achieved using the SM-PEG12 heterobifunctional linker. A series of site-directed PMB conjugates of CAM—003, a potent opsonic and protective mAb targeting P. nosa Ps1 exopolysaccharide, was evaluated in vitro and in viva. CAM—003—PMB conjugates retained in vitro OPK activity.

However the OPK ty was impacted by the increase in the average number of PMB per mAb. DM and TM PMB—mAb conjugates red protection in mouse P. aeruginosa endotoxemia model, demonstrating that LPS neutralization function of PMB was conferred onto the mAb. CAM—003—PMB conjugates showed greater tive activity than ugated CAM—003 mAb in the P. aeruginosa sepsis model at high doses (10 mg/kg), and reduced activity at low dose (0.1 . These data suggest that conjugated PMB can complement bacterial clearance mediated by the opsonic CAM—003 mAb and improve protection by LPS neutralization. The improvement in protective activity by 3-PMB conjugates in the —lO6— sepsis model is lost at lower doses, where levels of conjugated PMB are too low to neutralize LPS, and the primary mode of protection is likely mAb-mediated bacterial clearance. The loss of protective activity of the CAM-003—PMB conjugates at lower doses is consistent with the reduction in in vitro OPK ty as a result of PMB conjugation. These studies show that conjugated PMB on an c mAb can confer LPS neutralization ty and result in increased protective activity in a systemic P. aeruginosa ion model. Optimization of conjugation sites to reduce the negative impact on OPK activity may further improve the protective activity of PMB conjugates relative to unconjugated opsonic mAb.

The disclosure is not to be limited in scope by the specific embodiments described which are intended as single illustrations of individual aspects of the disclosure, and any compositions or s which are onally equivalent are within the scope of this disclosure. Indeed, various cations of the disclosure in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

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 dually indicated to be incorporated by reference.

Claims (12)

What we claim is:

1. An isolated antibody or antigen-binding fragment thereof which specifically binds to Pseudomonas Psl polysaccharide, wherein the antibody or antigen-binding nt thereof promotes opsonophagocytic killing (OPK) of P. aeruginosa, and n the isolated antibody or antigen-binding fragment thereof specifically binds to the same Pseudomonas Psl epitope as an antibody or n-binding fragment thereof comprising the heavy chain variable region (VH) and the light chain variable region (VL) of WapR-004RAD.

2. An isolated antibody or antigen-binding fragment thereof which specifically binds to Pseudomonas Psl ccharide, wherein the antibody or n-binding fragment thereof promotes opsonophagocytic killing (OPK) of P. aeruginosa, and n the isolated antibody or antigen-binding fragment thereof competitively inhibits Pseudomonas Psl binding by an antibody or antigen-binding nt comprising the heavy chain variable region (VH) and the light chain variable region (VL) of WapR-004RAD.

3. An ed antibody or antigen-binding fragment thereof which ically binds to Pseudomonas Psl polysaccharide, comprising a set of complementarity determining regions (CDRs) VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and VLCDR3 having SEQ ID NOs: 47, 48, 75, 50, 51, and 52.

4. The isolated antibody or antigen-binding fragment thereof of claim 3, comprising a heavy chain variable region (VH) comprising SEQ ID NO: 74 and a light chain variable region (VL) comprising SEQ ID NO: 12.

5. The antibody or antigen-binding fragment thereof of any one of claims 1 to 4, n the antibody or n-binding fragment thereof is humanized, chimeric or fully human.

6. The antibody or antigen-binding fragment thereof of any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof is monoclonal.

7. The antibody or antigen-binding fragment f of any one of claims 1 to 6, wherein the antibody or antigen-binding fragment thereof is a Fab fragment, a Fab' fragment, a F(ab)2 nt or a single chain Fv (scFv).

8. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-7, further comprising a pharmaceutically acceptable carrier.

9. The use of an antibody or antigen binding fragment thereof of any one of claims 1-7 in the manufacture of a medicament for the treatment of Pseudomonas infection.

10. An antibody or n binding fragment thereof according to claim 1, substantially as herein described or exemplified.

11. A pharmaceutical composition according to claim 8, ntially as herein described or exemplified.

12. A use according to claim 9, substantially as herein described or exemplified.