bispecific antibody, composition, and, use of composition

专利摘要:
ISOLATED BINDING MOLECULE OR BINDING ANTIGEN BINDING FRAGMENT, BIESPECIFIC ANTIBODY, ISOLATED DA POLYNUCLEOTIDE MOLECULE, COMPOSITION, USE OF COMPOSITION, AND, KIT These are combination therapies that comprise anti-Ps1 and P1 binding molecules. Pseudomonas and related compositions for use in the prevention and treatment of Pseudomonas infection.
公开号:BR112014011028B1
申请号:R112014011028-0
申请日:2012-11-06
公开日:2021-03-02
发明作者:Antonio Digiandomenico；Paul Warrener；Charles Stover；Bret Sellman；Ralph Minter；SAndrine Guillard；Steven Rust；Mladen Tomich；Vignesh Venkatraman；Reena Varkey；Li Peng；Melissa Damschroder；Partha Chowdhury；Nazzareno DIMASI；Ryan Fleming；Binyam Bezabeh；Changshou Gao；Cuihua GAO；Godfrey RAINEY
申请人:Medimmune, Llc；Medimmune Limited；
IPC主号:

专利说明:

REFERENCE TO THE ELECTRONICALLY SUBMITTED SEQUENCE LISTING
[0001] The content of the sequence listing submitted electronically in an ASCII text file entitled sequencelisting_PCTascii.txt created on November 6, 2012 and which has a size of 382 kilobytes deposited with the application is incorporated into this document in its entirety, as a reference. BACKGROUND FIELD OF REVELATION
[0002] This description refers to combination therapies using Pseudomonas anti-Psl and PcrVs binding domain for use in the present and treatment of Pseudomonas infection. Furthermore, the disclosure provides compositions useful in such therapies. BACKGROUND OF THE REVELATION
[0003] Pseudomonas aeruginosa (P. aeruginosa) is a gram-negative opportunistic pathogen that causes both acute and chronic infections in individuals (Ma et al., Journal de Bacteriology 189 (22): 8353 to 8356 (2007)). This is partly due to the bacterium's high innate resistance to clinically used antibiotics and partly due to the formation of highly resistant antibiotic biofilms (Drenkard E., Microbes Infect 5: 1213 to 1219 (2003); Hancokc & Speert, Drug Resist Update 3: 247 to 255 (2000)).
[0004] P. aeruginosa is a common cause of hospital-acquired infections in the Western World. It is a frequent causative agent of bacteremia in burn victims and immunodeficient individuals (Lyczak et al., Microbes Infect 2: 1051 to 1060 (2000)). It is also the most common cause of gram-negative nosocomial pneumonia (Craven et al., Semin Respir Infect 77:32 to 53 (1996)), specifically in mechanically ventilated patients and is the most relevant pathogen in the lungs of individuals with cystic fibrosis (Pier et al., ASM News <5: 339 to 347 (1998)).
[0005] Psi exopolysaccharide from Pseudomonas 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 establishing / maintaining biofilm formation (Jackson, KD, et al ., J Bacteriol 186, 4466 to 4475 (2004)). Its structure comprises repeating pentasaccharide rich in mannose (Byrd, M.S., et al., Mol Microbiol 73, 622 to 638 (2009)).
[0006] PcrV is a relatively conserved component of the type III secretion system. PcrV appears to be an integral component of the type III secretion system translocation apparatus that mediates the application of type III secretory toxins within the target eukaryotic cells (Sawa T., et al. Nat. Med. 5, 392 to 398 ( 1999)). Active and passive immunization against acute lung injury enhanced with PcrV and mortality in mice infected with cytotoxic P. aeruginosa (Sawa et al. 2009). The main effect of immunization against PcrV was due to the blocking of translocation of type III secretory toxins within eukaryotic cells.
[0007] Due to the growing resistance to multiple drugs, there remains a need in the art for the development of innovative strategies for the identification of new prophylactics specific to Pseudomonas and therapeutic agents. BRIEF SUMMARY
[0008] The disclosure provides a binding molecule or antigen binding fragment thereof that specifically binds to Pseudomonas PcrV, comprising: (A) a heavy chain CDR1 comprising SYAMN (SEQ ID NO: 218) or a a variant thereof comprising 1, 2, 3 or 4 conservative amino acid substitutions; a heavy chain CDR2 comprising AITISGITAYYTDSVKG (SEQ ID NO: 219) or a variant thereof comprising 1, 2, 3 or 4 conservative amino acid substitutions; and a heavy chain CDR3 comprising EEFLPGTHYYYGMDV (SEQ ID NO: 220) or a variant thereof comprising 1, 2, 3 or 4 conservative amino acid substitutions; (b) a light chain CDR1 comprising RASQGIRNDLG (SEQ ID NO: 221) or a variant thereof comprising 1, 2, 3 or 4 conservative amino acid substitutions; a light chain CDR2 comprising SASTLQS (SEQ ID NO: 222) or a variant thereof comprising 1, 2, 3 or 4 conservative amino acid substitutions; and a light chain CDR3 comprising LQDYNYPWT (SEQ ID NO: 223) or a variant thereof comprising 1, 2, 3 or 4 conservative amino acid substitutions; or combinations of (A) and (b). In one embodiment, the binding molecule or antigen binding fragment thereof specifically binds to PcrV of Pseudomonas and comprises: (A) a heavy chain CDR1 comprising SYAMN (SEQ ID NO: 218), a heavy chain CDR2 comprising AITISGITAYYTDSVKG (SEQ ID NO: 219) and a heavy chain CDR3 comprising EEFLPGTHYYYGMDV (SEQ ID NO: 220); and (b) a light chain CDR1 comprising RASQGIRNDLG (SEQ ID NO: 221), a light chain CDR2 comprising SASTLQS (SEQ ID NO: 222) and a light chain CDR3 comprising LQDYNYPWT (SEQ ID NO: 223 ). In one embodiment, the isolated binding molecule or antigen binding fragment thereof specifically binds to PcrV of Pseudomonas and comprises (A) a heavy chain variable region that has at least 90% sequence identity with SEQ ID NO: 216; (b) a light chain variable region that has at least 90% sequence identity with SEQ ID NO: 217; or combinations of (A) and (b). In another embodiment, the binding molecule or fragment thereof comprises: (A) a heavy chain variable region that has at least 95% sequence identity with SEQ ID NO: 216; (b) a light chain variable region that has at least 95% sequence identity with SEQ ID NO: 217; or combinations of (A) and (b). In another embodiment, the binding molecule or fragment thereof is V2L2 and comprises: (A) a heavy chain variable region comprising SEQ ID NO: 216; and (b) a light chain variable region comprising SEQ ID NO: 217.
[0009] In one embodiment, the disclosure provides an isolated binding molecule or an antigen binding fragment thereof that specifically binds to the same PcrV epitope as Pseudomonas as an antibody or an antigen binding fragment thereof comprising the VH region and VL of V2L2. In another embodiment, the disclosure provides an isolated binding molecule or antigen binding fragment thereof that specifically binds to Pseudomonas PcrV and competitively inhibits Pseudomonas PcrV that binds via an antibody or antigen binding fragment further comprising VH and VL of V2L2. In one embodiment, the binding molecule or fragment thereof is a recombinant antibody. In one embodiment, the binding molecule or fragment thereof is a monoclonal antibody. In one embodiment, the binding molecule or fragment thereof is a chimeric antibody. In one embodiment, the binding molecule or fragment thereof is a humanized antibody. In one embodiment, the binding molecule or fragment thereof is a human antibody. In one embodiment, the binding molecule or fragment thereof is a bispecific antibody.
[0010] In one embodiment, the binding molecule or fragment thereof inhibits the delivery of type III secretory toxins within the target cells.
[0011] In one embodiment, the disclosure provides a bispecific antibody comprising a binding domain that binds to Pseudomonas Psl and a binding domain that binds to PcrV of Pseudomonas. In one embodiment, the Psl binding domain comprises a scFv fragment and the PcrV binding domain comprises an intact immunoglobulin. In one embodiment, the Psl binding domain comprises an intact immunoglobulin and said PcrV binding domain comprises a scFv fragment. In one embodiment, scFv is fused to the amino terminal of the VH region of the intact immunoglobulin. In one embodiment, scFv is fused to the carboxy-terminal of the CH3 region of the intact immunoglobulin. In one embodiment, scFv is inserted into the hinge region of the intact immunoglobulin.
[0012] In one embodiment, the anti-Psl binding domain specifically binds to the same Psl epitope as Pseudomonas as an antibody or antigen binding fragment thereof comprising the heavy chain variable region (VH) and the region light chain variable (VL) at least 90% identical to the corresponding region of WapR-004. In one embodiment, the anti-Psl binding domain specifically binds to Pseudomonas Psl and competitively inhibits Pseudomonas Psl binding through an antibody or an antigen binding fragment thereof comprising at least a VH and VL region 90% identical to the corresponding region of WapR-004. In one embodiment, VH and VL of WapR-004 comprise SEQ ID NO; 11 and SEQ ID NO: 12, respectively. In one embodiment, the WapR-004 sequence is selected from the group consisting of: SEQ ID NO: 228, SEQ ID NO: 229 and SEQ ID NO: 235. In one embodiment, the anti-PcrV binding domain specifically binds to the same PseV epitope of Pseudomonas as an antibody or antigen binding fragment thereof comprising the V2L2 VH and VL region. In one embodiment, the anti-PcrV binding domain specifically binds to Pseudomonas PcrV and competitively inhibits Pseudomonas PcrV that binds via an antibody or an antigen binding fragment thereof comprising VH and V2L2. In another embodiment, the anti-PcrV binding domain specifically binds to the same PseV epitope of Pseudomonas as an antibody or antigen binding fragment thereof that comprises a VH and VL region at least 90% identical to the corresponding region of V2L2. In one embodiment, VH and VL of V2L2 comprise SEQ ID NO: 216 and SEQ ID NO: 217, respectively. In one embodiment, VH and VL of WapR-004 (SEQ ID NOs: 11 and 12, respectively) and VH and VL of V2L2 (SEQ ID NOs: 216 and 217, respectively). In one embodiment, the bispecific antibody comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 228, SEQ ID NO: 229 and SEQ ID NO: 235.
[0013] In one embodiment, the disclosure provides a polypeptide comprising an amino acid sequence of SEQ ID NO: 216 or SEQ ID NO: 217. In one embodiment, the polypeptide is an antibody.
[0014] In one embodiment, the disclosure provides a cell that comprises or produces the binding molecule or polypeptide disclosed in this document.
[0015] In one embodiment, the disclosure provides an isolated polynucleotide molecule that comprises a polynucleotide that encodes a binding molecule or polypeptide described herein. In one embodiment, the polynucleotide molecule comprises a polynucleotide sequence selected from the group consisting of: SEQ ID NO: 238 and SEQ ID NO: 239. In another embodiment, the disclosure provides a vector that comprises a polynucleotide described in the present document. In another embodiment, the disclosure provides a cell that comprises a polynucleotide or a vector.
[0016] In one embodiment, the disclosure provides a composition that comprises a binding molecule, a bispecific antibody or a polypeptide described herein and a pharmaceutically acceptable carrier.
[0017] In one embodiment, the disclosure provides a composition comprising a binding domain that binds to Psi of Pseudomonas and a binding domain that binds to PcrV of Pseudomonas. In one embodiment, the anti-Psl binding domain specifically binds to the same Psi epitope as Pseudomonas as an antibody or antigen binding fragment thereof comprising the heavy chain variable region (VH) and the variable chain region lightweight (VL) at least 90% identical to the region corresponding to WapR-004, Cam-003, Cam-004, Cam-005, WapR-001, WapR-002, WapR-003 or WapR-016. In one embodiment, the anti-Psl binding domain specifically binds to Psi of Pseudomonas and competitively inhibits Psi binding of Pseudomonas through an antibody or an antigen binding fragment thereof comprising at least a VH and VL region 90% identical to the region corresponding to WapR-004, Cam-003, Cam-004, Cam-005, WapR-001, WapR-002, WapR-003 or WapR-016. In one mode, VH and VL of WapR-004 comprise SEQ ID NO: 11 and SEQ ID NO: 12, respectively, VH and VL of Cam-003 comprise SEQ ID NO: 1 and SEQ ID NO: 2, respectively, VH and VL of Cam-004 comprise SEQ ID NO: 3 and SEQ ID NO: 2, respectively, VH and VL of Cam-005 comprise SEQ ID NO: 4 and SEQ ID NO: 2, respectively, VH and VL of WapR-001 comprise SEQ ID NO: 5 and SEQ ID NO: 6 , respectively, VH and VL of WapR-002 comprise SEQ ID NO: 7 and SEQ ID NO: 8, respectively, VH and VL of WapR-003 comprise SEQ ID NO: 9 and SEQ ID NO: 10, respectively and VH and VL of WapR-016 comprise SEQ ID NO: 15 and SEQ ID NO: 16, respectively. In one embodiment, the anti-PcrV binding domain specifically binds to the same PseV epitope of Pseudomonas as an antibody or antigen binding fragment thereof comprising the V2L2 VH and VL region. In one embodiment, the anti-PcrV binding domain specifically binds to Pseudomonas PcrV and competitively inhibits Pseudomonas PcrV that binds via an antibody or an antigen binding fragment thereof comprising VH and V2L2. In one embodiment, the anti-PcrV binding domain specifically binds to the same PseV epitope of Pseudomonas as an antibody or antigen binding fragment thereof that comprises a VH and VL region at least 90% identical to the corresponding V2L2 region . In one embodiment, VH and VL of V2L2 comprise SEQ ID NO: 216 and SEQ ID NO: 217, respectively. In one embodiment, the anti-Ps 1 binding domain comprises the VH and VL region of WapR-004 and said anti-PcrV binding domain comprises the VH and VL region of V2L2 or the antigen binding fragments thereof.
[0018] In one embodiment, the composition comprises a first binding molecule which comprises said Psl anti-binding domain and a second binding molecule which comprises a PcrV binding domain. In one embodiment, the first binding molecule is an antibody or antigen binding fragment thereof and said second binding molecule is an antibody or antigen binding fragment thereof. In one embodiment, the antibodies or antigen binding fragments are independently selected from the group consisting of: monoclonal, humanized, chimeric, human, Fab fragment, Fab 'fragment, F (ab) 2 fragment and scFv fragment . In one embodiment, the binding domains, binding molecules or fragments thereof, bind to two or more, three or more, four or more or five or more different P. aeruginosa serotypes. In one embodiment, the binding domains, binding molecules or fragments thereof, bind to at least 80%, at least 85%, at least 90% or at least 95% strains of P. aeruginosa isolated from infected patients. In one embodiment, strains of P. aeruginosa are isolated from one or more lungs, sputum, eye, pus, feces, urine, bone cavities, an injury, skin, blood, bone or knee fluid. In one embodiment, the antibody or antigen binding fragment thereof is conjugated to an agent selected from the group consisting of an antimicrobial agent, a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a lipid, a biological response modifier, a pharmaceutical agent, a lymphokine, a heterologous antibody or a fragment thereof, a detectable tag, polyethylene glycol (PEG) and a combination of two or more of any of said agents. In one embodiment, the detectable tag is selected from the group consisting of an enzyme, a fluorescent tag, a chemiluminescent tag, a bioluminescent tag, a radioactive tag or a combination of two or more of any of said detectable tags.
[0019] In one embodiment, the disclosure provides a method of preventing or treating a Pseudomonas infection in an individual in need thereof, which comprises administering to the individual an effective amount of a composition described herein, wherein said administration provides a synergistic therapeutic effect in preventing or treating Pseudomonas infection in said individual and wherein said synergistic effect is greater than a sum of the individual effects of administering molar amounts equal to the individual binding domains. In one embodiment, the synergistic therapeutic effect results in percentage survival greater than the additive percentage survival of individuals to whom only one of the binding domains has been administered. In one embodiment, the composition is administered for two or more prevention / treatment cycles. In one embodiment, the binding domains or the binding molecules are administered simultaneously. In one embodiment, the binding domains or binding molecules are administered sequentially. In one embodiment, the Pseudomonas infection is a P. aeruginosa infection. In one embodiment, the individual is a human. In one embodiment, the infection is an eye infection, a lung infection, a burn infection, a wound infection, a skin infection, a blood infection, a bone infection or a combination of two or more of said infections. In one embodiment, the individual has acute pneumonia, burn injury, corneal infection, cystic fibrosis or a combination thereof.
[0020] In one embodiment, the disclosure provides a method of preventing or treating a Pseudomonas infection in an individual in need of it, which comprises administering to the individual an effective amount of the binding molecule or fragment thereof, a bispecific antibody, a polypeptide or composition described herein.
[0021] In one embodiment, the disclosure provides a kit that comprises a composition described in this document. BRIEF DESCRIPTION OF THE DRAWINGS / FIGURES
[0022] Figure 1 (A-F) is a complete phenotypic cell screening with the human antibody phage libraries identified for specific functionally active antibodies of P. aeruginosa. (A) is an overview of the complete antibody selection strategy. (B) is a flow diagram that describes the process for isolating the variable genes from the antibody region from patients recently exposed to P. aeruginosa. (C) are the characteristics of the scFv phage display libraries, which indicate the size and diversity of the cloned antibody repertoire. (D) is the comparison of the efficiency of phage display selection with the use of either the patient's antibody library or a virgin antibody library, when selected in P. aeruginosa 3064 Δ WapR (') or in P. aeruginosa PAO1 MexAB OprM Δ WapR () in suspension. The bars indicate the output titrations (in CFU) in each selection cycle and the circles indicate the proportion of duplicated VH CDR3 sequences, an indication of clonal enrichment. (E) is the scFv ELISA screening of the phage display to test the binding of multiple strains of P. aeruginosa. ELISA data (absorbance at 450 nm) are shown for eight individual phage-scFvs from selections and one irrelevant phage-scFv. (F) is the binding of FACS of specific P. aeruginosa antibodies to strains representative of unique P. aeruginosa serotypes. For each antibody tested, a human IgG negative control antibody is shown as a shaded peak.
[0023] Figure 2 (AB) is the evaluation of mAbs that promote OPK from the P. aeruginosa opsonophagocytosis assay (A) with luminescent P. aeruginosa 05 serogroup strain (PAOl.lux), with dilutions of purified monoclonal antibodies derived phage filtration. (B) is an opsonophagocytosis assay with serogroup strain of P. aeruginosa luminescent OI 1 (9882-80.lux), with dilutions of purified monoclonal antibodies from WapR-004 and Cam-003 derived from phage filtration. In both A and B, R347, an isotype-matched human monoclonal antibody that does not bind to P. aeruginosa antigens, was used as a negative control.
[0024] Figure 3 (A-I) is the identification of the Psi target exopolysaccharide of P. aeruginosa of the antibodies derived from the phenotypic screening. Antibody reactivity was determined by indirect ELISA on plates coated with P. aeruginosa strains indicated: (A) these are wild PAO1, PAOlAwópL, PAOlArm / C and PAOlΔgαZt /. (B) it is PAOXΔpslA. The Genway antibody is specific for a P. aeruginosa outer membrane protein and was used as a positive control. (C) is a FACS linkage analysis from Cam-003 to PAO1 and PAOlΔ / asZzi. Cam-003 is indicated by a solid black line and transparent peak; a specific non-P. aeruginosa human IgGl antibody corresponding to the isotope was used as a negative control and is indicated by a gray line and shaded peak. (D) it is purified LPS from PAO1 and PAOlΔ / zsZ ^ was separated by SDS-PAGE and immunotransferred with antisera derived from mice vaccinated with PAO1 ΔwapRAalgD, a mutant strain deficient in O-antigen transport to the membrane production and alginate production. (E) These are ELISA binding data in Cam-003 with PAO1 isogenic mutants. Cam-003 is only able to bind to strains that express Psl. pPW145 is a pUCP expression vector that contains pslA opsonophagocytosis assays. (F and G) indicate that Cam-003 only mediates the elimination of strains capable of producing Psl (wild PAO1 and PAOlAps / J complemented in trans with the pslA gene). (H and I) are ELISA data that indicate the reactivity of anti-Psl antibodies from WapR-001, WapR-004, and WapR-016 with PAO1 ΔwbpLΔalgD and PAO1 ΔwbpLΔalgDΔpslA. R347 was used as a negative control in all experiments.
[0025] Figure 4 are anti-Psl mAbs that inhibit cell attachment of the P. aeruginosa luminescent PAOl.lux strain to A549 cells. Logarithmic phase PAOl.lux was added to a single confluent layer of A549 cells in an MOI of 10 followed by RLU analysis after repeated washing to remove unbound P. aeruginosa. The results are representative of three independent experiments performed in duplicate for each antibody concentration.
[0026] Figure 5 (A-C) are the strains of P. aeruginosa subcultured in vivo that maintain / increase Psl expression. The antibody of Cam-003 antibody is shown by a solid black line and a transparent peak; the human IgG negative control antibody is shown as a gray line and a shaded peak. (A) is the positive control, an assay was made with Cam-003 to bind the strains grown until the log phase of a culture overnight (~ 5 x 10 / ml). (B) it is an inoculum for each strain that was prepared for 5x10 CFU / ml of a cultivated TSA, overnight, in a plate up to grass and tested for reactivity to Cam-003 through flow cytometry . (C) refers to four hours after the intraperitoneal stimulus, where the bacteria was harvested from mice through peritoneal lavage and tested for the presence of Psl with Cam-003 by flow cytometry.
[0027] Figure 6 (A-F) are survival rates for animals treated with anti-Psl monoclonal antibodies from Cam-003 or WapR-004 in a model of acute P. aeruginosa pneumonia. (AD) are animals that were treated with Cam-003 at 45, 15 and 5 mg / kg and R347 at 45 mg / kg or PBS 24 hours before intranasal infection with (A) PAO1 (1.6 x 107 (B) 33356 (3 x 107 CFU), (C) 6294 (7 x 106 CFU), (D) 6077 (1 x 106 CFU). (EF) are animals that were treated with WapR-004 at 5 and 1 mg / kg as indicated followed by infection with 6077 in (E) (8 x 10 "CFU) or (F) (6 x 105 CFU). The animals were carefully monitored for survival up to 72 hours (AD) or 120 hours (EF). In all experiments, PBS and R347 served as negative controls. The results were represented as Kaplan-Meier survival curves; differences in survival were calculated by the logarithmic classification test for Cam-003 vs. R347. (A) Cam-003 (45 mg / kg - P <0.0001; 15 mg / kg - P = 0.0003; 5 mg / kg - (B) Cam-003 (45 mg / kg - P = 0.0012; 15 mg / kg - P = 0.0012; 5 mg / kg - P = 0.0373). (C ) Cam-003 (45 mg / kg - P = 0.0007; 15 mg / kg - P = 0.0019; 5 mg / kg - P = 0.0212). (D) Cam-003 (45 mg / kg - P <0.0001; 15 mg / kg - P <0.0001; 5 mg / kg - P = 0.0001). The results are representative of at least two independent experiments. (E) [Cam-003 (5 mg / kg) vs. R347 (5 mg / kg): P = 0.02; Cam-003 (1 mg / kg) vs. R347 (5 mg / kg) kg): P = 0.4848; WapR-004 (5 mg / kg) vs. R347 (5 mg / kg): P <0.0001; WapR-004 (1 mg / kg) vs. R347 (5 mg / kg): P = 0.0886; WapR-004 (5 mg / kg) vs. Cam-003 (5 mg / kg): P = 0.0017; WapR-004 (1 mg / kg) vs. Cam-003 (1 mg / kg): P = 0.2468; R347 (5 mg / kg) vs. PBS: P = 0.6667] (F) [Cam-003 (5 mg / kg) vs. R347 (5 mg / kg): P = 0.0004; Cam-003 (1 mg / kg) vs. R347 (5 mg / kg): P <0.0001; WapR-004 (5 mg / kg) vs. R347 (5 mg / kg): P <0.0001; WapR-004 (1 mg / kg) vs. R347 (5 mg / kg): P <0.0001; WapR-004 (5 mg / kg) vs. Cam-003 (5 mg / kg): P = 0.0002; WapR-004 (1 mg / kg) vs. Cam-003 (1 mg / kg): P = 0.2628; R347 (5 mg / kg) vs. PBS: P = 0.6676]. The results are representative of five independent experiments.
[0028] Figure 7 (A-F) are monoclonal antibodies of anti-Psl, Cam-003 and WapR-004, which reduce the load on the organ after the induction of acute pneumonia. The mice were treated with Cam-003 antibody 24 hours prior to infection with (A) PAO1 (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 (~ 1 x 107 CFU) and (F) 6206 (~ 1 x 106 CFU). 24 hours post-infection, the animals were euthanized followed by collection or organs to identify viable CFU. Differences in viable CFU were determined using Mann-Whitney U Test for Cam-003 or WapR-004 vs. R347. (A) Lung: Cam-003 (45 mg / kg - P = 0.0015; 15 mg / kg - P = 0.0021; 5 mg / kg - P = 0.0015); Spleen: Cam-003 (45 mg / kg - P = 0.0120; 15 mg / kg - P = 0.0367); Kidneys: Cam-003 (45 mg / kg - P = 0.0092; 15 mg / kg - P = 0.0056); (B) Lung: Cam-003 (45 mg / kg - P = 0.0010; 15 mg / kg - P <0.0001; 5 mg / kg - P = 0.0045); (C) Lung: Cam-003 (45 mg / kg - P = 0.0003; 15 mg / kg - P = 0.0039; 5 mg / kg - P = 0.0068); Spleen: Cam-003 (45 mg / kg - P = 0.0057; 15 mg / kg - P = 0.0230; 5 mg / kg - P = 0.0012); (D) Lung: Cam-003 (45 mg / kg - P = 0.0005; 15 mg / kg - P = 0.0003; 5 mg / kg - P = 0.0007); Spleen: Cam-003 (45 mg / kg - P = 0.0015; 15 mg / kg - P = 0.0089; 5 mg / kg - P = 0.0089); Kidneys: Cam-003 (45 mg / kg-P = 0.0191; 15 mg / kg - P = 0.0355; 5 mg / kg - P = 0.0021). (E) Lung: WapR-004 (15 mg / kg - P = 0.0011; 5 mg / kg - P = 0.0004; 1 mg / kg - P = 0.0002); Spleen: WapR-004 (15 mg / kg - P <Q, 0001; 5 mg / kg - P = 0.0014; 1 mg / kg - P <Q, 0001); F) Lung: WapR-004 (15 mg / kg - P <0.0001; 5 mg / kg - P = 0.0006; 1 mg / kg - P = 0.0079); Spleen: WapR-004 (15 mg / kg - P = 0.0059; 5 mg / kg - P = 0.0261; 1 mg / kg - P = 0.0047); Kidney: WapR-004 (15 mg / kg - P = 0.0208; 5 mg / kg-P = 0.0268.
[0029] Figure 8 (A-G) are anti-Psl monoclonal antibodies from Cam-003 and WapR-004 that are active in a P. aeruginosa keratitis model and thermal injury model. The mice were treated with a control IgGl antibody or Cam-003 at 45 mg / kg (A, B) or 15 mg / kg (C, D) or PBS or a control IgGl antibody or Cam-003 at 45 mg / kg or WapR-004 in 45 mg / kg or 15 mg / kg or 5 mg / kg (F, G) 24 hours before infection with 6077 (011-cytotoxic - 2x106 CFU). Immediately prior to infection, scratches of 1 mm were made on the left cornea of each animal followed by topical use of P. aeruginosa in a 5 μl inoculum. 24 hours after the infection, the scores of the pathology of the cornea were calculated followed by the removal of the eye to determine viable CFU. Differences in pathology scores and viable CFU were determined using 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 (45 mg / kg) vs. WapR-004 (45 mg / kg): P = 0.018; Cam-003 (45 mg / kg) vs. WapR-004 (15 mg / kg): P = 0.0025; WapR-004 (45 mg / kg) vs. WapR-004 (15 mg / kg): P = 0.1331; WapR-004 (5 mg / kg) vs. Ctrl: P <0.0001. The results are representative of five independent experiments. (E) is survival analysis of Cam-003 and CF-1 mice treated with R347 in a thermal injury model of P. aeruginosa after 6077 infection (2 x 10 CFU) (logarithmic classification: R347 vs. Cam-003 15 mg / kg, P = 0.0094; R347 vs. Cam-003 5 mg / kg, P = 0.0017). The results are representative of at least two three independent experiments, (n) refers to the number of animals in each group. Figure 8 (H) are anti-Psl and anti-PcrV monoclonal antibodies that are active in a model of P. aeruginosa mouse eye keratitis. PBS or a control IgGl antibody (R347) was injected intraperitoneally (IP) at 45 mg / kg or WapR-004 (α-Psl) at 5 mg / kg or V2L2 (a-PcrV) at 5 mg / kg, 16 hours before infection with 6077 (011-cytotoxic - Ix106 CFU). Immediately before infection, the mice were anesthetized followed by the initiation of three 1 mm scratches on the ridge and on the superficial stroma of one eye of each mouse with the use of a 27 gauge needle under a dissecting microscope, followed by topical use of strain of P. aeruginosa 6077 in a 5 μl inoculum.
[0030] Figure 9 (A-C) is a mutant Fc antibody from Cam-003, Cam-003-TM, has decreased OPK and in vivo efficacy, but maintains anti-cell attachment activity. (A) is the OPO assay of PAOl.lux with Cam-003 and Cam-003-TM, which harbor mutations in the Fc domain that prevents Fc interactions with Fc receptors (Oganesyan, V., et al., Acta Crystallogr D Biol Crystallogr 64, 700 to 704 (2008)). R347 was used as a negative control. (B) is the PAO1 cell attachment assay with Cam-003 and Cam-003-TM. (C) is the acute pneumonia model that compares the effectiveness of Cam-003 vs. Cam-003-TM.
[0031] Figure 10 (A-C) is: A is the mapping and identification of the affinity and relative binding epitope for anti-Psl monoclonal antibodies. Epitope mapping was performed using a competition ELISA and confirmed with the use of an OCTET * flow system with Psl derivative from an overnight culture supernatant of P. aeruginosa strain PAO1. The relative binding affinities were measured on a FORTEBIO * OCTET® 384 instrument. Also shown are the antibody concentrations at which cell attachment was maximally inhibited and the EC50 values of OPK for each antibody. B, C are the relative binding affinities of various WapR-004 mutants as measured on a FORTEBIO * OCTET® 384 instrument. The ECK values of OPK for the various mutants are also shown.
[0032] Figure 11 (AM) is about the evaluation of the WapR-004 (W4) mutant clones in the P. aeruginosa (AM) opsonophagocytic elimination (AM) OPK assay with P. serogroup strain P. luminescent aeruginosa (PAOl.lux), with dilution of different W4 mutant clones in scFv-Fc format. In some cases, W4 IgGl has been included in the assay and is indicated as W4-IgGl. W4-RAD-Cam and W4-RAD-GB represent the same sequence as WapR-004RAD described in this document. "W4-RAD" is an abbreviation for the designations WapR-004RAD and W4-RAD-Cam and W4-RAD-GB, in panels D to M it represents two different preparations of WapR-004RAD. (N-Q) is the evaluation of optimized anti-Psl mAbs derived from orientation optimization (WapR-004) in the P. aeruginosa OPK assay. (N-O) is an OPK assay with PAOl.lux luminescent using dilutions of optimized monoclonal antibodies purified for orientation. (P-Q) repeats the OPK assay with PAOl.lux with purified mAbs dilutions to confirm the results. In (N-Q) W4-RAD was used as a positive comparative control. In all experiments, R347, a human IgGl monoclonal antibody that does not bind to P. aeruginosa antigens, was used as a negative control.
[0033] Figure 12 (A-H) is about (A) is the diversity of PcrV epitope. (B) is the percent inhibition of cytotoxicity analysis for parental V2L2 mAb, mAb 166 (positive control) and R347 (negative control). (C) is an assessment of the mAb ability of V2L2, mAb 166 (positive control) and R347 (negative control) to prevent lysis of RBCs. (D) is the evaluation of mAb with V2L2 germline (V2L2-GL) and mAbs of V2L2-GL (V2L2-P4M, V2L2-MFS, V2L2-MD and V2L2-MR) to prevent lysis of RBCs. (E) is the evaluation of mAbs 1E6, 1F3, 11A6, 29D2, PCRV02 and V2L7 to prevent lysis of RBCs (F) is the evaluation of V2L2 of mAbs and 29D2 to prevent lysis of RBCs. (G-H) are the relative binding affinities of V2L2-GL and V2L2-MD antibodies.
[0034] Figure 13 (A-I) is an in vivo survival study of mice treated with anti-PcrV antibody. (A) are the mice that were treated 24 hours before infection with: 1.03 x 106 CFU 6077 (exoU +) with 45 mg / kg R347 (negative control), 45 mg / kg, 15.0 mg / kg , 5.0 mg / kg or 1.0 mg / kg of mAbl66 (positive control) or 15 mg / kg, 5.0 mg / kg, 1.0 mg / kg or 0.2 mg / kg of V2L2. Survival was monitored for 96 hours. (B) are the mice that were treated 24 hours prior to infection with: 2.1 x 107 CFU 6294 (exoS +) with 15 mg / kg R347 (negative control), 15.0 mg / kg, 5.0 mg / kg or 1.0 mg / kg of mAbl66 (positive control) or 15 mg / kg, 5.0 mg / kg or 1.0 mg / kg of V2L2. Survival was monitored for 168 hours. Mice were treated 24 hours prior to infection with: (C) 6294 (06) or (D) PA 103A with R347 (negative control), 5 mg / kg of PcrV-02 PcrV antibody or 5 mg / kg, 1 , 0 mg / kg, 0.2 mg / kg or 0.04 mg / kg of V2L2. The mice were treated 24 hours before infection with strain 6077 with R347 (negative control), 5 mg / kg of PcrV-02 PcrV antibody, V2L7 (5 mg / kg or 1 mg / kg), 3G5 (5 mg / kg) kg or 1 mg / kg) or 11A6 (5 mg / kg or 1 mg / kg) (E) or 25 mg / kg of V2L7, 1E6, 1F3, 29D2, R347 or 1 mg / kg of PcrV-01 PcrV antibody (F) or 25 mg / kg of 21F1, V2L2, 2H3, 4A8, SH3, LEIO, R347 or 1 mg / kg of PcrV-02 (G) or 29D2 (1 mg / kg, 3 mg / kg or 10 mg / kg), V2L2 (1 mg / kg, 3 mg / kg or 10 mg / kg) R347 or 1 mg / kg of PcrV-02 (H). Mice were treated 24 hours before infection with: 6294 (06) or PAI03A with V2L2 (0.04 mg / kg, 0.2 mg / kg, 1 mg / kg or 5 mg / kg), R347 or 5 mg / kg of PcrV-02. Percent survival was tested on a model of acute pneumonia.
[0035] Figure 14 deals with the organ load analysis of mice treated with V2L2. The mice were treated 24 hours before infection with 6206 with (A) R347 (negative control), 1 mg / kg, 0.2 mg / kg or 0.07 mg / kg of V2L2 and (B) 15 mg / kg of R347 (negative control); 15.0 mg / kg, 5.0 mg / kg or 1.0 mg / kg of rnAblóó (positive control); or 5.0 mg / kg, 1.0 mg / kg or 0.2 mg / kg of V2L2. Colony-forming units were identified by gram of tissue in the lung, spleen and kidney.
[0036] Figure 15: Organ load analysis of mice treated with V2L2 and WapR-004 (W4). The mice were treated 24 hours before infection with 6206 (Oll-ExoU +) with R347 (negative control), V2L2 alone or V2L2 (0.1 mg / kg) in combination with increasing concentrations of W4 (0.1, 0.5 , 1.0 or 2.0 mg / kg). Colony-forming units were identified by gram of tissue in the lung, spleen and kidney. .
[0037] Figure 16 (A-G): Survival rates for animals treated with anti-PcrV V2L2 monoclonal antibody in a model of acute P. aeruginosa pneumonia. The designations V2L2-GL, V2L2-MD, V2L2-PM4, V2L2-A and V2L2-MFS on panels A through G represent different preparations from V2L2. (AC) The animals were treated with V2L2 at 1 mg / kg, 0.5 mg / kg or R347 at 0.5 mg / kg before intranasal infection with (A) 6077 (9.75 x 105 CFU), (B , C) 6077 (9.5 x 105 CFU). (DF) The animals were treated with V2L2 at 0.5 mg / kg, 0.1 mg / kg or R347 at 0.5 mg / kg followed by infection with 6077 (D) (1 x 106 CFU), (E) (9.5 x 105 CFU) or F (1.026 x 106 CFU). (G) The animals were treated with V2L2-MD a (0.04 mg / kg, 0.2 mg / kg, 1 mg / kg or 5 mg / kg), mAbl66 (positive control) a (0.2 mg / kg) kg, 1 mg / kg, 5 mg / kg or 15 mg / kg) or R347 at 0.5 mg / kg followed by infection with 6206 (2 x 107 + CFU).
[0038] Figure 17 (AB): Schematic representation of bispecific antibodies (A) Bsl-TNFα / W4, Bs2-TNFα / W4, Bs3-TNFα / W4 and (B) Bs2- V2L2 / W4-RAD, Bs3-V2L2 / W4-RAD, and Bs4-V2L2-W4-RAD Psl / PcrV. (A) For Bsl-TNFa / W4, the W4 scFv is fused to the amino terminus of TNFα VL via a (G4S) 2 linker. For Bsl-TNFa / W4, the W4 scFv is fused to the amino terminus of TNFα VH via a (G4S) 2 linker. For Bsl-TNFa / W4, the W4 scFv is fused to the CH3 carboxy terminus via a (G4S) 2 linker. (B) For Bs2-V2L2-2C, the W4-RAD scFv is fused to the amino terminus of V2L2 VH via a (G4S) 2 linker. For Bs2-W4-RAD-2C, the V2L2 scFv is fused to the amino terminus of W4-RAD VH via a (G4S) 2 linker. For Bs3-V2L2-2C, the W4-RAD scFv is fused to the CH3 carboxy terminus via a (G4S) 2 linker. For Bs4-V2L2-2C, the W4-RAD scFv is inserted in the hinge region, connected by the ligand (G4S) 2 at the N-terminal and C-terminal of the scFv.
[0039] Figure 18: Activity evaluation of WapR-004 (W4) scFv in a bispecific construct represented in Figure 17A. W4 scFv has been linked to two different bispecific constructs (in alternating N or C-terminal directions) that have a TNFα binding arm. Each bispecific construct of W4-TNFα (Bsl-TNFa / W4, Bs2-TNFa / W4 and Bs3-TNFa / W4) maintained the ability to inhibit cell fixation similarly to W4 using the PAOl.lux assay (05) indicating that W4 scFv maintains its activity in a bispecific format. R347 was used as a negative control.
[0040] Figure 19 (A-C): Anti-Psl and anti-PcrV binding domains were combined in bispecific format by replacing the TNFa antibody in Figure 17B with V2L2. These constructs are identical to those represented in Figure 17B with the exception of using unstabilized W4-scFv in place of stabilized W4-RAD scFv. Both W4 and W4-RAD target identical epitopes and have identical functional activities. Percentage inhibition of cytotoxicity was analyzed for both BS2-V2L2 and BS3-V2L2 with the use of A549 cells treated with both (A) 6206 and (B) 6206ΔpslA. (C) BS2-V2L2, BS3-V2L2 and BS4-V2L2 were evaluated for their ability to prevent lysis of RBCs compared to parental control. All bispecific constructs maintained anti-cytotoxicity activity similarly to the parental V2L2 antibody with the use of cells infected with 6206 and 6206ΔpslA and prevented the lysis of RBCs similarly to parental control (V2L2). R347 was used as a negative control in all experiments.
[0041] Figure 20 (A-C): Evaluation of bispecific anti-Psl / anti-PcrV constructs to promote OPK of P. aeruginosa. The opsonophagocytosis assay is shown with a serogroup 05 strain of P. aeruginosa luminescent (PAOl.lux), with dilutions of purified Psl / TNFa bispecific antibodies (Bs2-TNFα and Bs3-TNFa); the parental antibodies W4-RAD or V2L2-IgGl; the Psl / PcrV antibodies Bs2-V2L2 or Bs3-V2L2 or the Bs2-V2L2-2C, Bs3-V2L2-2C, Bs4-V2L2-2C or Bs4-V2L2-2C antibody that houses a YTE mutation (Bs4-V2L2-2C- YTE). (A) Although the Bs2-V2L2 antibody showed similar extermination compared to the parental W4-RAD antibody, the extermination for the Bs3-V2L2 antibody was decreased. (B) Although the Bs2-V2L2-2C and Bs4-V2L2-2C antibodies showed similar extermination compared to the parental W4-RAD antibody, the extermination for the Bs3-V2L2-2C antibody was decreased. (C) The designations W4-RAD and W4-RAD-YTE represent different preparations of W4-RAD. The designations Bs4-V2L2-2C (old batch) and Bs4-V2L2-2C (new batch) represent different preparations from Bs4-V2L2-2C. The YTE modification in Bs4-V2L2-2C-YTE is a modification made to the antibodies that increases the half-life of the antibodies. The different preparations of Bs4 antibodies (old batch vs. new batch) showed similar extermination compared to the parental W4-RAD antibody, however, Bs4-V2L2-2C-YTE antibodies had a 3-fold drop in OPK activity compared a Bs4-V2L2-2C (See table of EC50). R347 was used as a negative control in all experiments.
[0042] Figure 21 (AI): In vivo survival study of mice treated with bispecific anti-Psl / anti-PcrV antibodies Bs2-V2L2, Bs3-V2L2, Bs4-V2L2-2C and Bs4-V2L2-2C-YTE in one 6206 acute pneumonia model system. The mice (n = 10) were translated with (A): R347 (negative control, 0.2 mg / kg), Bs2-V2L2 (0.28 mg / kg), Bs3- V2L2 (0.28 mg / kg), V2L2 (0.2 mg / kg) or W4-RAD (0.2 mg / kg); (BC): R347 (negative control, 1 mg / kg), Bs2-V2L2 (0.5 mg / kg or 1 mg / kg) or Bs4-V2L2-2C (0.5 mg / kg or 1 mg / kg) ; (D): R347 (negative control, 1 mg / kg), Bs3-V2L2 (0.5 mg / kg or 1 mg / kg), or Bs4-V2L2-2C (0.5 mg / kg or 1 mg / kg ); (E): R347 (negative control, 2 mg / kg), a combination of the individual W4 and V2L2 antibodies (0.5 mg / kg or 1 mg / kg each) or Bs4-V2L2-2C (1 mg / kg or 2 mg / kg); (F): R347 (negative control, 1 mg / kg), a mixture of individual W4 and V2L2 antibodies (0.5 mg / kg or 1 mg / kg each) or Bs4-V2L2-2C (1 mg / kg or 2 mg / kg). Twelve hours after treatment, all mice were infected with approximately (6.25x105-1x106 CFU / animal) 6206 (Oll-ExoU +). All mice were monitored for 120 hours. (A): All control mice succumbed to the infection approximately 30 hours after infection. All Bs3-V2L2 animals survived, along with those that received V2L2 control. Approximately 90% of the animals immunized with W4-RAD survived. In contrast, approximately 50% of Bs2-V2L2 animals succumbed to infection within 120 hours. (B-F): All control mice succumbed to the infection approximately 48 hours after infection. (B): Bs4-V2L2-2C had greater activity compared to Bs2-V2L2 at both 1.0 and 0.5 mg / kg. (C): Bs4-V2L2-2C appeared to have greater activity compared to Bs2-V2L2 at 1.0 mg / kg (the results are not statistically significant). (D): Bs4-V2L2-2C had greater activity compared to Bs2-V2L2 at 0.5 mg / kg. (E): Bs4-V2L2-2C at both 2 and 1 mg / kg had greater activity compared to the mixture of antibodies at both 1.0 and 0.5 mg / kg. (F): Bs4-V2L2 (1 mg / kg) had activity similar to both 1.0 and 0.5 mg / kg. (G-H): Both Bs4-V2L2-2C and Bs4-V2L2-2C-YTE had similar activity at both 1.0 and 0.5 mg / kg. The results are represented as Kaplan-Meier survival curves; differences in survival were calculated by the Log-rank test for (B) Bs4-V2L2-2C vs. Bs2-V2L2 (1 mg / kg - P = 0.034; 0.5 mg / kg - P = 0.0002); (D) Bs4-V2L2-2C vs. Bs3-V2L2 (0.5 mg / kg - P <0.0001); (E): Bs4-V2L2-2C (2 mg / kg) vs. mixture of antibodies (1 mg / kg each) -P = 0.0012; Bs4-V2L2-2C (1 mg / kg) vs. mixture of antibodies (0.5 mg / kg each) - P = 0.0002. (GH): The mice (n = 8) were translated with: R347 (negative control, 1 mg / kg), Bs4-V2L2-2C (1 and 0.5 mg / kg) and Bs4-V2L2-2C-YTE ( 1 and 0.5 mg / kg) and 6206 (9e5 CFU). No difference in survival between V2L2-2C and Bs4-V2L2-2C-YTE at any dose was observed by Log-Rank. (I): To analyze the effectiveness of each antibody construct, the mice were tested with 0.1 mg / kg, 0.2 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 5 mg / kg, 10 mg / kg or 15 mg / kg and analyzed for survival in a model of lethal pneumonia by 6206. Percent survival is indicated in the table with the number of animals for each comparison indicated in parentheses.
[0043] Figure 22: Organ load analysis of animals treated with bispecific anti-Psl / PcrV antibody using the 6206 acute pneumonia model. The mice were treated 24 hours before infection with 6206 (Oll-ExoU +) with R347 (negative control), V2L2 or W4-RAD alone (0.2 mg / kg), Bs2-V2L2 (0.28 mg / kg), or BS3-V2L2 (0.28 mg / kg). Colony-forming units were identified by gram of tissue in the lung, spleen and kidney. At the tested concentration, both Bs2-V2L2 and Bs3-V2L2 significantly decreased the organ load in the lung. However, none of the bispecific constructs was able to significantly affect the organ load in the spleen or kidney compared to parental antibodies.
[0044] Figure 23 (AB): Organ load analysis of animals treated with bispecific anti-Psl / PcrV antibody using the 6294 model system. Mice were treated 24 hours before infection with 6294 with R347 (control negative), V2L2 or W4-RAD alone (0.5 mg / kg), Bs2-V2L2 (0.7 mg / kg), or Bs3-V2L2 (0.7 mg / kg) (A), or V2L2 or W4 -RAD alone (0.2 mg / kg), Bs2-V2L2 (0.2 mg / kg), Bs3-V2L2 (0.2 mg / kg) or a combination of the individual W4-RAD and V2L2 antibodies (0.1 mg / kg each) (B). Twelve hours after antibody administration, all mice were infected with an inoculum containing 2.5 x 10 7 CFU of 6294 (A) or 1.72 x 10 7 CFU of 6294 (B). Colony-forming units were identified by gram of tissue in the lung, spleen and kidney. Using the 6294 model system, (A) the organ load of both BS2-V2L2 and BS3-V2L2 increased significantly in all tissues to a level comparable to that of the parental antibody V2L2. The parental antibody W4-RAD had no effect on decreasing organ load. (B) the organ load of the combination of Bs2-V2L2, Bs3-V2L2 and W4-RAD + V2L2 decreased significantly in all tissues to a level comparable to that of the parental antibody V2L2.
[0045] Figure 24: In vivo survival study of mice treated with Bs2-W4 / V2L2 and Bs3-W4 / V2L2 in a 6294 model system. The mice were treated with R347 (negative control, 0.2 mg / kg ), Bs2-V2L2 (0.28 mg / kg), Bs3-V2L2 (0.28 mg / kg), V2L2 (0.2 mg / kg) or W4-RAD (0.2 mg / kg). Twelve hours after treatment, all mice were infected with 6294. All mice were monitored for 120 hours. All control mice succumbed to the infection approximately 75 hours after infection. Sixty percent Bs3-V2L2 and 50% of Bs2-V2L2 animals survived after 120 hours after inoculation. As seen in organ load studies, immunization with W4-RAD did not affect survival in all mice that succumbed to infection at approximately the same time as controls.
[0046] Figure 25 (AD): Bispecific anti-Psl / PcrV antibody load analysis or W4 + V2L2 combination therapy in the 6206 model system. Non-ideal antibody concentrations (AC) were used to enable ability to decipher antibody activity. (D) High concentrations of Bs4 were used. Mice were treated 24 hours before infection with 6206 with R347 (negative control), V2L2 or W4-RAD alone (0.2 mg / kg), Bs2-V2L2 (0.2 mg / kg), Bs3-V2L2 (0 , 2 mg / kg), Bs4 (15.0, 5.0 and 1.0 mg / kg) or a combination of the individual W4-RAD and V2L2 antibodies (0.1 mg / kg each). Twelve hours after antibody administration, all mice were infected with an inoculum containing (A), (B) 4.75 x 105 CFU 6206 (OI l-ExoU +) or (C) 7.75 x 105 CFU 6206 (Oll-ExoU +) or (D) 9.5 x 105 CFU 6206 (OI 1- ExoU +). Colony-forming units were identified by gram of tissue in the lung, spleen and kidney. Using the 6206 model system, both BS2-V2L2 and BS3-V2L2 decreased the organ load in the lung, spleen and kidneys to a level comparable to that of the combination of W4 + V2L2. In the lung, the combination significantly reduced bacterial CFUs Bs2- and Bs3-V2L2 and V2L2 with the use of Dunn's post-test with Kruskal-Wallis. Significant differences in bacterial load in the spleen and kidney were not observed, although a tendency towards reduction was noted. (D) When ideal concentrations of Bs4-V2L2-2C were used (15.0, 5.0, and 1.0), a fast and effective bacterial cleaning was observed in the lung. In addition, the bacterial spread to the spleen and kidneys has also been excised. Asterisks indicate the statistical significance compared to the R347 control with the use of Fo after Kruskal-Wallis test with Dunn.
[0047] Figure 26 (A-J): Therapeutic adjunct therapy: Bs4-V2L2-2C + antibiotic. (A) - (B) the mice were treated 24 hours before infection with 1 x 106 CFU 6206 with 0.5 mg / kg of R347 (negative control) or Bs4-V2L2-2C (0.2 mg / kg or 0.5 mg / kg) or Ciprofloxacin (CIP) (20 mg / kg or 6.7 mg / kg) 1 hour after infection or a combination of Bs4-V2L2-2C 24 hours before infection and CIP 1 hour after infection infection (0.5 mg / kg 4- 20 mg / kg or 0.5 mg / kg + 6.7 mg / kg or 0.2 mg / kg + 20 mg / kg or 0.2 mg / kg + 6, 7 mg / kg, respectively). (C) Mice were treated 1 hour after infection with 9.5 x 105 CFU 6206 with 5 mg / kg of R.347 or CIP (20 mg / kg or 6.7 mg / kg) or Bs4-V2L2-2C (1 mg / kg or 5 mg / kg), or a combination of Bs4-V2L2-2C and CIP (5 mg / kg + 20 mg / kg or 5 mg / kg + 6.7 mg / kg or 1 mg / kg + 20 mg / kg or 1 mg / kg + 6.7 mg / kg, respectively). (D) Mice were treated 2 hours after infection with 9.5 x 105 CFU 6206 with 5 mg / kg R347 or CIP (20 mg / kg or 6.7 mg / kg) or Bs4-V2L2- 2C (1 mg / kg or 5 mg / kg), or a combination of Bs4-V2L2-2C and Cipro (5 mg / kg + 20 mg / kg or 5 mg / kg + 6.7 mg / kg or 1 mg / kg + 20 mg / kg or 1 mg / kg + 6.7 mg / kg, respectively). (E) Mice were treated 2 hours after infection with 9.75 x 105 CFU 6206 with 5 mg / kg of R347or Bs4-V2L2-2C (1 mg / kg or 5 mg / kg) or CIP (20 mg / kg or 6.7 mg / kg) 1 hour after infection, or a combination of Bs4-V2L2-2C 2 hours after infection and CIP 1 hour after infection (5 mg / kg + 20 mg / kg or 5 mg / kg + 6.7 mg / kg or 1 mg / kg + 20 mg / kg or 1 mg / kg + 6.7 mg / kg, respectively). (F) Mice were treated 1 hour after infection with 9.5 x 105 CFU 6206 with 5 mg / kg R347 or Meropenem (MEM) (0.75 mg / kg or 2.3 mg / kg) or Bs4-V2L2 -2C (1 mg / kg or 5 mg / kg), or a combination of Bs4-V2L2- 2C and MEM (5 mg / kg + 2.3 mg / kg or 5 mg / kg + 0.75 mg / kg or 1 mg / kg + 2.3 mg / kg or 1 mg / kg + 0.75 mg / kg, respectively). (G) Mice were treated 2 hours after infection with 9.75 x 105 CFU 6206 with 5 mg / kg of R347 or Bs4-V2L2-2C (1 mg / kg or 5 mg / kg) or MEM (0.75 mg / kg or 2.3 mg / kg) 1 hour after infection, or a combination of Bs4-V2L2-2C 2 hours after infection and MEM 1 hour after infection (5 mg / kg + 2.3 mg / kg or 5 mg / kg + 0.75 mg / kg or 1 mg / kg + 2.3 mg / kg or 1 mg / kg + 0.75 mg / kg, respectively). (H) Mice were treated 2 hours after infection with 1 x 106 CFU 6206 with 5 mg / kg R347 or Bs4-V2L2-2C (1 mg / kg or 5 mg / kg) or MEM (0.75 mg / kg or 2.3 mg / kg), or a combination of Bs4-V2L2-2C 2 and MEM (5 mg / kg + 2.3 mg / kg or 5 mg / kg + 0.75 mg / kg or 1 mg / kg + 2.3 mg / kg or 1 mg / kg + 0.75 mg / kg, respectively). (I) Mice were treated 4 hours after infection with 9.25 x 105 CFU 6206 with 5 mg / kg of R347 or CIP (6.7 mg / kg) or Bs4-V2L2-2C (1 mg / kg or 5 mg / kg) or a combination of Bs4-V2L2-2C and CIP (5 mg / kg + 6.7 mg / kg or 1 mg / kg + 6.7 mg / kg, respectively), (J) Mice were treated 4 hours after infection with 1.2 x 106 CFU 6206 with 5 mg / kg R347 + CIP (6.7 mg / kg), CIP (6.7 mg / kg), or Bs4-V2L2-2C (1 mg / kg or 5 mg / kg) or a combination of Bs4-V2L2-2C and CIP (5 mg / kg + 6.7 mg / kg or 1 mg / kg + 6.7 mg / kg, respectively). (A-J) Bs4 antibody combined with either CIP or MEM increases the effectiveness of antibiotic therapy, indicating synergistic protection when the molecules are combined. In addition, although the antibiotic delivered alone or in combination with a non-specific P. aeruginosa antibody can reduce or control bacterial CFU in the lung, the antibiotic alone does not protect mice from the lethality of this configuration. The ideal protection of this configuration shows the inclusion of Bs4-V2L2-2C in combination with the antibiotic.
[0048] Figure 27 (AC): Difference in the functional activity of bispecific antibodies BS4-WT, BS4-GL and BS4-GLO: opsonophagocytic extermination assay (A), anti-cellular fixation assay (B) and an anti- cytotoxicity of RBC lysis (C).
[0049] Figure 28 (A-B): Percentage protection against lethal pneumonia in mice stimulated in prophylactic (A) or therapeutic configurations (B) with P. aeruginosa strains. Percentage survival is indicated in the table with the number of animals for each comparison indicated in parentheses. The dashes indicate “not tested”.
[0050] Figure 29 (A-B): Survival rates for animals treated with bispecific Bs4-GLO antibody in a model of lethal bacteremia by P. aeruginosa. (A) Animals were treated with Bs4-GLO at 15 mg / kg, 5 mg / kg, 1 mg / kg or R347 at 15 mg / kg 24 hours before intraperitoneal infection with 6294 (06) (5.58 x 107 CFU ). (B) Animals were treated with Bs4-GLO at 5 mg / kg, 1 mg / kg, 0.2 mg / kg or R347 at 5 mg / kg 24 hours before intraperitoneal infection with 6206 (Oll-ExoU +) (6 , 48 x 106 CFU). The results are represented as Kaplan-Meier survival curves; differences in survival were calculated by the Log-rank test for BS4-GLO at each concentration vs. R347. (A) Bs4-GLO in all concentrations vs. R347 P <0.0001. (B) Bs4-GLO in all concentrations vs. R347 P = 0.0003. The results are representative of three independent experiments.
[0051] Figure 30 (A-C): Survival rates for animals prophylactically treated (prevention) with Bs4-GLO in a model of P. aeruginosa thermal injury. (A) The animals were treated with Bs4-GLO at 15 mg / kg, 5 mg / kg or R347 at 15 mg / kg 24 hours before the induction of thermal injury and subcutaneous infection with P. aeruginosa strain 6077 (OI l- ExoU +) with 1.4 x 105 CFU directly under the wound. (B) the animals were treated with Bs4-GLO at 15 mg / kg or R347 at 15 mg / kg 24 hours before the induction of thermal injury and subcutaneous infection with strain 6206 of P. aeruginosa (OI 1- ExoU +) with 4, 15 x 104 CFU directly under the wound. (B) The animals were treated with Bs4-GLO at 15 mg / kg, 5 mg / kg or R347 at 15 mg / kg 24 hours before the induction of thermal injury and subcutaneous infection with strain 6294 of P. aeruginosa (06) with 7 , 5x 101 CFU directly under the wound. The results are represented as Kaplan-Meier survival curves; differences in survival were calculated by the Log-rank test for BS4-GLO at each concentration vs. R347. (A) Bs4-GLO in all concentrations vs. R347 - P <0.0001. The results are representative of two independent experiments for each strain of P. aeruginosa.
[0052] Figure 31 (A-B): Survival rates for animals therapeutically treated (treatment) with Bs4-GLO in a model of P. aeruginosa thermal injury. (A) Animals were treated with Bs4-GLO at 42.6 mg / kg, 15 mg / kg or R347 at 45 mg / kg for 4 hours after induction of thermal injury and subcutaneous infection with 6077 strain of P. aeruginosa (O11 -ExolT) with 1.6 x 105 CFU directly under the wound. (B) Animals were treated with Bs4-GLO at 15 mg / kg, 5 mg / kg or R347 at 15 mg / kg for 12 hours after induction of thermal injury and subcutaneous infection with P. aeruginosa strain 6077 (011-EXOLT ) with 1.0 x 10 'CFU directly under the wound. Results are represented as Kaplan-Meier survival curves; differences in survival were calculated by the Log-rank test for BS4-GLO at each concentration vs. R347. (A) Bs4-GLO in both concentrations vs. R347 - P = 0.0004. (B) Bs4-GLO at 5 mg / kg vs. R347 - P = 0.048. The results are representative of two independent experiments.
[0053] Figure 32 (AB): Therapeutic adjunctive therapy: Bs4GLO + ciprofloxacin (CIP): (A) Mice were treated 4 hours after infection with 9.5 x 105 CFU 6206 with 5 mg / kg R347 + CIP ( 6.7 mg / kg) or Bs4-WT (1 mg / kg or 5 mg / kg) or a combination of Bs4-WT and CIP (5 mg / kg + 6.7 mg / kg or 1 mg / kg + 6 , 7 mg / kg, respectively). (B) Mice were treated 4 hours after infection with 9.5 x 10 'CFU 6206 with 5 mg / kg of R347 + CIP (6.7 mg / kg) or Bs4-GLO (1 mg / kg or 5 mg / kg) kg) or a combination of Bs4-GLO and CIP (5 mg / kg + 6.7 mg / kg or 1 mg / kg + 6.7 mg / kg, respectively
[0054] Figure 33 (AB): Therapeutic adjunctive therapy: Bs4-GLO + meropenem (MEM): (A) Mice were treated 4 hours after infection with 9.5 x 10 'CFU 6206 with 5 mg / kg R347 + MEM (0.75 mg / kg) or Bs4-WT (1 mg / kg or 5 mg / kg) or a combination of Bs4-WT and MEM (5 mg / kg + 0.75 mg / kg or 1 mg / kg) kg + 0.75 mg / kg, respectively). (B) Mice were treated 4 hours after infection with 9.5 x 10 'CFU 6206 with 5 mg / kg of R347 + MEM (0.75 mg / kg) or Bs4-GLO (1 mg / kg or 5 mg / kg) or a combination of Bs4-GLO and MEM (5 mg / kg + 0.75 mg / kg or 1 mg / kg + 0.75 mg / kg, respectively).
[0055] Figure 34 (A-C): Therapeutic adjunctive therapy: Bs4-GLO + antibiotic in a model of lethal bacteremia. Mice were treated 24 hours before for intraperitoneal infection with strain 6294 of P. aeruginosa (06) 9.3 x 107 with Bs4-GLO a (0.25 mg / kg or 0.5 mg / kg) or R347 (negative control) . One hour after infection, mice were treated subcutaneously with (A) 1 mg / kg CIP, (B) 2.5 mg / kg MEM or (C) 2.5 mg / kg TOB. The results are represented as Kaplan-Meier survival curves; differences in survival were calculated by the Log-rank test for Bs4-GLO at each concentration vs. R347.
[0056] Figure 35 (AB) Schematic representation of alternative formats for Bs4 constructs (A) variable regions of anti-PcrV are present separately in the heavy and light chains, while the variable regions of anti-Psl are present as an scFv within of the hinge region of the heavy chain and (B) variable regions of anti-Psl are present separately in the heavy and light chains, while the variable regions of anti-PcrV are present as an scFv within the hinge region of the heavy chain. DETAILED DESCRIPTION I. DEFINITIONS
[0057] It should be noted that the term "one" or "an" entity refers to one or more of that entity; for example, "a binding molecule that specifically binds to Psl of Pseudomonas and / or PcrV”, is understood to represent one or more binding molecules that specifically bind to Psl of Pseudomonas and / or PcrV. As such, the terms "one" (or "one"), "one or more", and "at least one" may be used interchangeably in this document.
[0058] As used in this document, the term "polypeptide" is intended to encompass a single "polypeptide", as well as "polypeptides" in the plural, and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "protein", "chain of amino acids", or any other term used to refer to a chain or chains of two or more amino acids are included within the definition of "polypeptide", and the "polypeptide" may be used instead of, or interchangeably with, any of those terms. The term "polypeptide" is also intended to refer to products of post-expression modifications of the polypeptide, including, without limitation, glycosylation, acetylation, phosphorylation, amidation, derivation by known protection / blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids A polypeptide can be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence, which can be generated in any way, including by chemical synthesis.
[0059] A polypeptide, 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 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 a structure. Polypeptides with a defined three-dimensional structure are referred to as folded, and polypeptides that do not have a defined three-dimensional structure, but, instead, can adopt a large number of different conformations, and are referred to as unfolded. As used herein, the term glycoprotein refers to a protein attached to at least a chemical portion of carbohydrate that is attached to the protein via an oxygen-containing side chain or one containing nitrogen from an amino acid residue, for example , a serine residue or an asparagine residue.
[0060] By an "isolated" polypeptide or a fragment, variation or derivative thereof, a polypeptide that is not in its natural environment is intended. No particular level of purification is required. For example, an isolated polypeptide can be removed 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 that have been separated, fractionated, or partially or substantially purified by any suitable set of procedures.
[0061] Other polypeptides disclosed herein are fragments, derivatives, analogs or variations of the preceding polypeptides and a combination thereof. The terms "fragment", "variation", "derivative" and "analog", when referring to a binding molecule such as an antibody that specifically binds to Psl from Pseudomonas and / or PcrV, as disclosed herein, include any polypeptides that retain at least some of the antigen binding properties of the corresponding native antibody or polypeptide. Polypeptide fragments include, for example, proteolytic fragments, as well as deletion fragments, in addition to specific antibody fragments discussed elsewhere in this document. Variations of a binding molecule, for example, an antibody that specifically binds to Psl of Pseudomonas and / or PcrV, as disclosed herein, include fragments as described above, as well as polypeptides with altered amino acid sequences due to substitutions, deletions or amino acid inserts. Variations can occur naturally or be unnatural. Variations of unnatural occurrence can be produced using sets of mutagenesis procedures known in the art. Varied polypeptides may comprise conservative or non-conservative amino acid substitutions, deletions or additions. Derived from a binding molecule, for example, an antibody that specifically binds to Psi of Pseudomonas and / or PcrV, as disclosed in this document, are polypeptides that have been altered to exhibit additional features not found in the native polypeptide. Examples include fusion proteins. Varied polypeptides may also be referred to herein as "analogous polypeptides." As used herein, a "derivative" of a binding molecule, for example, an antibody that specifically binds to Psi of Pseudomonas and / or PcrV, if refers to an individual polypeptide that has one or more chemically derived residues by reaction of a functional side group. Also included as "derivatives" are those peptides that contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. 4-hydroxyproline can be substituted for proline; 5-hydroxylysine can be substituted for lysine; 3-methylhistidine can be substituted for histidine; homoserine can be substituted for serine; and omitine can be substituted for lysine.
[0062] The term "polynucleotide" is intended to encompass a single nucleic acid, as well as plural nucleic acids, and refers to an isolated nucleic acid molecule or construct, for example, messenger RNA (mRNA) or plasmid DNA (pDNA ). A polynucleotide can comprise a conventional phosphodiester bond or an unconventional bond (for example, an amide bond, as found in peptide nucleic acids (PNA)). The term "nucleic acid" refers to any one or more segments of nucleic acid, for example, fragments of DNA or RNA, present in a polynucleotide. By "isolated" nucleic acid or polynucleotide is meant a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. For example, a recombinant polynucleotide that encodes a binding molecule, for example, an antibody that specifically binds to Psi of Pseudomonas and / or PcrV contained in a vector is considered to be isolated, as disclosed herein. Additional examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified polynucleotides (partially or substantially) in solution. Isolated RNA molecules include RNA transcripts in vivo or in vitro from polynucleotides. Polynucleotides or isolated nucleic acids further include such synthetically produced molecules. In addition, polynucleotide or nucleic acid can be or can include a regulatory element, such as a promoter, ribosome binding site, or a transcription terminator.
[0063] As used herein, a "coding region" is a portion of nucleic acid that 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 as part of a coding region, but any flanking sequences, for example, promoters, binding sites, ribosome, transcriptional terminators, introns and the like, are not part of a coding region. Two or more coding regions can be present in a single polynucleotide construct, for example, in a single vector, or in separate polynucleotide constructs, for example, in separate (different) vectors. In addition, any vector can contain a single coding region, or can comprise two or more coding regions, for example, a single vector can separately encode an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region. In addition, a vector, polynucleotide or nucleic acid can encode heterologous coding regions, fused or not fused to a nucleic acid that encodes a binding molecule that specifically binds to Psi of Pseudomonas dou PcrV, for example, an antibody, or fragment antigen binding, variation, or derivative thereof. Heterologous coding regions include, without limitation, specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain.
[0064] In certain embodiments, the polynucleotide or nucleic acid is DNA. In the case of DNA, a polynucleotide comprising a nucleic acid that encodes a polypeptide can normally include a promoter and / or other transcription or translation control elements operatively associated with one or more coding regions. An operational association is when a coding region for a gene product, for example, a polypeptide, is associated with one or more regulatory sequences in such a way as to position the expression of the gene product under the influence or control of the sequence (s) regulatory (s). Two DNA fragments (such as a polypeptide coding region and a promoter associated with it) are "operationally associated" if induction of promoter function results in the transcription of mRNA encoding the desired gene product, and if the The nature of the link between the two DNA fragments does not interfere with the ability of regulatory expression sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed. Thus, a promoter region would be operatively associated with a nucleic acid encoding a polypeptide if the promoter has the ability to transcribe that nucleic acid. The promoter can be a cell-specific promoter that directs substantial transcription of DNA only in predetermined cells. Other elements of transcription control, in addition to a promoter, for example, enhancers, operators, repressors and transcription termination signals can be operatively associated with the polynucleotide to target cell-specific transcription. Suitable promoters and other transcription control regions are disclosed in this document.
[0065] A variety of transcription control regions are known to those skilled in the art. These include, without limitation, transcriptional control regions that function in vertebrate cells, such as, but not limited to, cytomegalovirus promoter and enhancer segments (the immediate early promoter, in conjunction with A-intron), simian virus 40 (the early promoter), and retrovirus (such as Rous sarcoma virus). Other transcriptional control regions include those derived from vertebrate genes, such as actin, heat shock protein, bovine growth hormone and rabbit β-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcriptional control regions include tissue-specific promoters and enhancers, as well as inducible lymphokine promoters (for example, interferon- or interleukin-inducible promoters).
[0066] 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 start and end codons, and elements derived from picomavirus (particularly an internal ribosome entry site, or IRES, also referred to as an ISCED sequence).
[0067] In other embodiments, a polynucleotide can be RNA, for example, in the form of an RNA messenger (mRNA).
[0068] Polynucleotide and nucleic acid coding regions can be associated with additional coding regions that encode signal or secretory peptides, which direct the secretion of a polypeptide encoded by a polynucleotide, as disclosed in this document, for example, a polynucleotide that encodes a binding molecule that specifically binds to Psi of Pseudomonas and / or PcrV, for example, an antibody, or antigen binding fragment, variation, or derivative thereof. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence that is cleaved from the mature protein once export of the growing protein chain throughout the rough endoplasmic reticulum has been initiated. Those of ordinary skill in the art are aware that polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the full or "full length" polypeptide to produce a secreted or "mature" form of the polypeptide. In certain embodiments, the native signal peptide, for example, an immunoglobulin heavy chain or light chain signal peptide is used, or a functional derivative of that sequence that is capable of directing the secretion of the associated polypeptide. operational to it. Alternatively, a mammalian Werologist signal peptide, or a functional derivative thereof, may be used. For example, the wild type leader sequence can be replaced by the human tissue plasminogen activator (TPA) leader sequence or mouse β-glucuronidase.
[0069] Certain binding molecules, or antigen binding fragments, variations, or derivatives thereof are disclosed in this document. Unless specifically referring to full size antibodies, such as naturally occurring antibodies, the term "binding molecule" encompasses full size antibodies, as well as antigen binding fragments, variations, analogs, or derivatives of such antibodies, for example, naturally occurring immunoglobulin or antibody molecules or engineered antibody molecules or fragments that bind antigen in a similar way to antibody molecules.
[0070] As used in this document, the term "binding molecule" refers in its broadest sense to a molecule that specifically binds to an antigenic determinant. As described later in this document, a binding molecule can comprise one or more of the binding domains described herein. As used herein, a "binding domain" includes a site that specifically binds to the antigenic determinant. A non-limiting example of an antigen binding molecule is an antibody or fragment thereof that retains specific antigen binding.
[0071] The terms "antibody" and "immunoglobulin" can be used interchangeably in this document. An antibody (or a fragment, variation 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 systems vertebrates are relatively well understood, see, for example, Harlow et al., Antibodies: A ∑ aboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd edition, 1988).
[0072] As will be discussed in more detail below, the term "immunoglobulin" comprises several broad classes of polypeptides that can be biochemically distinguished. Those skilled in the art will note that heavy chains are classified as gamma, mu, alpha, delta, or epsilon, (y, μ, α, δ, ε) with some subclasses between them (for example, yl-y4). It is the nature of this chain that determines the "class" of the antibody such as IgG, IgM, IgA IgG, or IgE, respectively. Immunoglobulin subclasses (isotypes), for example, IgG], IgGz, IgGs, IgG4, IgAb, etc. they are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discernible to those skilled in the art in view of the present disclosure and, consequently, are within the scope of this disclosure.
[0073] Light chains are classified as kappa or lambda (K, À). Each heavy chain class can be linked to a kappa or lambda light chain. In general, the light and heavy chains are covalently linked together, and the "tail" portions of the two heavy chains are linked together by covalent disulfide bonds or non-covalent bonds when immunoglobulins are generated by hybridomas, B cells or genetically engineered host cells. In the heavy chain, the amino acid sequences run from an N termination at the forked ends of the Y configuration to the C termination at the bottom of each chain.
[0074] Both light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it will be observed that the variable domains of both light (VL) and heavy (VH) portions determine antigen recognition and specificity. Conversely, the light chain (CL) and heavy chain (CHI, CH2 or CH3) constant domains confer important biological properties, such as secretion, transplacental mobility, Fc receptor binding, complement binding and the like. By convention, the numbering of constant region domains increases as they become more distal from the antigen or amino-terminal binding site of the antibody. The N-terminal portion is a variable region and the C-terminal portion is a constant region; the CH3 and CL domains actually comprise the carboxy-terminal of the heavy and light chain, respectively.
[0075] As indicated above, the variable region allows the binding molecule to selectively recognize and specifically bind epitopes to antigens. That is, the VL domain and VH domain, or subset of the complementarity determining regions (CDRs), of a binding molecule, for example, an antibody combines to form the variable region that defines a three-dimensional antigen binding site. This quaternary binding molecule structure forms the antigen binding site present at the end of each arm of the Y. More specifically, the antigen binding site is defined by three CDRs on each of the VH and VL chains.
[0076] 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 specifically positioned to form the antigen binding domain as the antibody assumes its three-dimensional configuration in an aqueous environment. The remainder of the amino acids in the antigen binding domains, referred to as "frame" regions, show less intermolecular variability. The framework regions widely adopt a conformation of β-ldmina and the CDRs form bonds that connect and, in some cases are part of, the structure of β-ldmina. Thus, framework regions act to form a framework that provides to position the CDRs in the correct orientation by non-covalent inter-chain interactions. The antigen binding domain formed by the positioned CDRs defines a complementary surface to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent attachment of the antibody to its cognate epitope. The amino acids that comprise the CDRs and the framework regions, respectively, can be readily identified for any given variable region of heavy or light chain by a person of ordinary skill in the art, since they have been precisely defined {see, "Sequences of Proteins of Immunological Interest ”, Kabat, E., et al., United States Department of Health and Human Services (US Department of Health and Human Services), (1983); and Chothia and Lesk, J. Mol. Biol., 796: 901 to 917 (1987), which are hereby incorporated by reference in their entirety for reference).
[0077] In the event that there are two or more definitions of a term that is used and / or accepted within the technique, the definition of the term as used in this document is intended to include all such meanings, unless explicitly stated otherwise. A specific example is the use of the term "complementarity determining region"("CDR") to describe the non-contiguous antigen combination sites found within the variable region of both heavy and light chain polypeptides. This particular region has been described by Kabat et al., United States Department of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and by Chothia et al., J Mal. Biol. 196: 901 to 917 (1987), which are incorporated by reference in this document, where the definitions include overlap or subsets of amino acid residues when compared to each other. Regardless, the application of any definition to refer to a CDR of an antibody or variations in it is intended to be within the scope of the term, as defined and used herein. Suitable amino acid residues that comprise CDRs, as defined by each of the references cited above, are shown below in Table I as a comparison. The exact residue numbers that span a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR, given the variable region amino acid sequence of the antibody. TABLE 1: CDR Definitions 1
I The numbering of all CDR definitions in Table 1 is in accordance with the naming conventions presented by Kabat et al. (see below).
[0078] Kabat et al. they also defined a numbering system for variable domain sequences that is applicable to any antibody. A person of ordinary skill in the art can unequivocally assign this system of "Kabat numbering" to any variable domain sequence, without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system presented by Kabat et al., United States Department 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 molecule that specifically binds to Psl from Pseudomonas and / or PcrV, for example, an antibody, or antigen binding fragment, variation, or derived therefrom, as revealed in this document, are in accordance with the Kabat numbering system.
[0079] Binding molecules, for example, antibodies or antigen binding fragments, variations, or derivatives thereof include, but are not limited to, polyclonal, monoclonal, human, humanized or chimeric antibodies, single chain antibodies, fragments of epitope binding, for example, Fab, Fab 'and F (ab') 2, Fd, Fvs, single chain Fvs (scFv), single chain antibodies, disulfide-linked Fvs (sdFv), fragments that comprise a domain of VL or VH, fragments produced by a Fab expression library. ScFv molecules are known in the art and are described, for example, in U.S. Patent No. 5,892,019. Immunoglobulin or antibody molecules covered by this disclosure can be of any type (for example, IgG, IgE, IgM, IgD, IgA, and IgY), class (for example, IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.
[0080] By "specifically binds to", it is generally meant that a binding molecule, for example, an antibody or fragment, variation, or derivative thereof, binds to an epitope through its antigen binding domain, and that binding implies some complementarity between 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, through its domain of antigen binding more readily than it would bind to an unrelated random epitope. The term "specificity" is used throughout this document to qualify the relative affinity through which a certain binding molecule binds to a certain epitope. For example, "A binding molecule "can be considered to have a greater specificity for a given epitope than the" B "binding molecule, or" A "binding molecule can be said to bind the" C "epitope with a greater specificity than for an epitope related option "D".
[0081] By "preferentially binds", it is meant that the antibody specifically binds to an epitope more readily than it would bind to a related, similar, homologous or analogous epitope. Thus, an antibody that "preferably binds" to a given epitope would more likely bind to that epitope than to a related epitope, even though such an antibody may cross-react with the related epitope.
[0082] For the purpose of non-limiting example, a binding molecule, for example, an antibody can be considered to bind to a first epitope, preferably it binds said first epitope with a dissociation constant (KD) that is less than the KD of the antibody for the second epitope. In another non-limiting example, a binding molecule, such as an antibody, can be considered to bind a first antigen, preferably if it binds the first epitope with an affinity that is at least an order of magnitude less than the KD of the antibody. for the second epitope. In another non-limiting example, a binding molecule can be considered to bind a first epitope, preferably it binds the first epitope with an affinity that is at least two orders of magnitude less than the KD of the antibody to the second epitope.
[0083] In another non-limiting example, a binding molecule, for example, an antibody or fragment, variation, or derivative thereof, can be considered to bind a first epitope, preferably the first epitope binds with a constant rate for dissociation (k (off)) which is less than the k (off) of the antibody for the second epitope. In another non-limiting example, a binding molecule can be considered to bind a first epitope, preferably it binds the first epitope with an affinity that is at least an order of magnitude less than the k (off) of the antibody to the second epitope. In another non-limiting example, a binding molecule can be considered to bind a first epitope, preferably it binds the first epitope with an affinity that is at least two orders of magnitude less than the k (off) of the antibody to the second epitope.
[0084] A binding molecule, for example, an antibody or fragment, variation, or derivatives thereof disclosed herein can be said to bind a target antigen, for example, a polysaccharide disclosed herein or a fragment or variation of the even with a dissociation constant rate (k (off)) of less than or equal to 5 X 10'2 s'1, 10'2 s'1, 5 X 10'3 s'1 or 10 3 s' 1. A binding molecule as disclosed in this document can be said to bind a target antigen, for example, a polysaccharide with a dissociation constant rate (k (off) less than or equal to 5 X 10'4 s "1 , 10'4 s "1, 5X10 '5 s'1, or IO'5 s'1 5 X IO'6 s'1, 10'6 s'1, 5 X 10'7 s'1 or 10'7 s'1.
[0085] A binding molecule, for example, an antibody or antigen binding fragment, variation, or derivatives disclosed herein can be said to bind a target antigen, for example, a polysaccharide with a constant rate for association ( k (on)) greater than or equal to 103 M'1 s'1, 5 X 103 M'1 s'1, 104 M'1 s'1 or 5 X 104 M'1 s "1. A molecule of binding as disclosed in this document can be said to bind a target antigen, for example, a polysaccharide with a constant rate of association (k (on)) greater than or equal to 103 M'1 s'1, 5 X 105 M'1 s'1, 106 M'1 s'1, or 5 X 106 M'1 s'1 or 107 M'1 s'1.
[0086] A binding molecule, for example, an antibody or fragment, variation, or derivative thereof, is said to competitively inhibit the binding of an antibody or reference antigen binding fragment to a given epitope if it is preferentially it binds to that epitope to the extent that it blocks, to some degree, the binding of the antibody or reference antigen binding fragment to the epitope. Competitive inhibition can be determined by any method known in the art, for example, competitive ELISA assays. A binding molecule can be said to competitively inhibit binding of the reference 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%.
[0087] As used herein, the term "affinity" refers to a measure of the strength of the binding of an individual epitope to the CDR of an immunoglobulin molecule. See, for example, Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd edition, 1988) on pages 27 to 28. As used herein, the term "greed" refers to general stability of the complex between an immunoglobulin population and an antigen, that is, the strength of the functional combination of an immunoglobulin mixture with the antigen. See, for example, Harlow, on pages 29 to 34. Avidity is related to the affinity of individual immunoglobulin molecules in the population with specific epitopes, as well as the valences of immunoglobulins and antigen. For example, the interaction between a bivalent monoclonal antibody and an antigen with a highly repetitive epitope structure, such as a polymer, would be highly avid.
[0088] Binding molecules or antigen binding fragments, variations or derivatives thereof, as disclosed in this document, can also be described or specified in terms of their cross-reaction. As used herein, the term "cross-reaction" refers to the ability of a binding molecule, for example, an antibody or fragment, variation, or derivatives thereof, specific for an antigen, to react with a second antigen; a measure of kinship between two different antigenic substances. Thus, a cross-reactive binding molecule binds to an epitope in addition to the one that induced its formation. The cross-reactive epitope usually contains many of the same complementary structural features as the induction epitope and, in some cases, may actually fit better than the original.
[0089] A binding molecule, for example, an antibody or fragment, variation, or derivatives thereof, can also be described or specified in terms of its binding affinity to an antigen. For example, a binding molecule can bind to an antigen with a dissociation or KD constant not greater than 5 x 10 '"M, 10'-M, 5 x 10' M, 10'3M, 5 x 10'4M , 1O'4M, 5 x 10'5M, 10'5M, 5 x 10'6M, 10'6M, 5 x 10'7 M, 10'7M, 5 x 10'8M, 1O'8M, 5 x 10 ' 9M, 10'9M, 5 x 10'10M, 10'10M, 5 x 10'11 M, 10'HM, 5 x 10'12M, 1O'12M, 5 x 10'13M, 10'l3M, 5 x 10 '14M, 10'14M, 5 x 10'15M, OR 10O'I5M.
[0090] Antibody fragments, including single chain antibodies, may comprise the variable region (s) alone or in combination with all or a portion of the following: hinge region, CHI, CH2 domains, and CH3. Also included are antigen binding fragments that also comprise any combination of variable region (s) with a hinge region, CHI, CH2, and CH3 domains. Binding molecules, for example, antibodies, or antigen binding fragments thereof disclosed herein can be of any animal origin, including birds and mammals. The antibodies can be human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse or chicken antibodies. In another modality, the variable region can be related to chondrites in their origin (for example, sharks). As used herein, "human" antibodies include antibodies that have the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic to one or more human immunoglobulins and that do not express endogenous immunoglobulins, as described infra and, for example, in US Patent No. 5,939,598 to Kucherlapati et al.
[0091] As used herein, the term "heavy chain moiety" includes amino acid sequences derived from an immunoglobulin heavy chain. A binding molecule, for example, an antibody comprising a heavy chain portion, comprises at least one of: a CHI domain, a hinge domain (for example, upper, middle and / or lower hinge region), a CH2 domain, CH3 domain, or a variation or fragment thereof. For example, a binding molecule, for example, an antibody or fragment, variation, or derivatives thereof, may comprise a polypeptide chain comprising a CHI domain; a polypeptide chain comprising a CHI domain, at least a portion of a hinge domain and a CH2 domain; a polypeptide chain comprising a CHI domain and a CH3 domain; a polypeptide chain comprising a CHI domain, at least a portion of a hinge domain and a CH3 domain, or a polypeptide chain comprising a CHI domain, at least a portion of a hinge domain, a domain CH2 and a CH3 domain. In another embodiment, a binding molecule, for example, an antibody or fragment, variation, or derivatives thereof, comprises a polypeptide chain comprising a CH3 domain. In addition, a binding molecule for use in the development may lack at least a portion of a CH2 domain (for example, all or part of a CH2 domain). As presented above, it will be understood by a person of ordinary skill in the art that these domains (e.g., the heavy chain moieties) can be modified so that they vary in amino acid sequence of the naturally occurring immunoglobulin molecule.
[0092] The heavy chain portions of a binding molecule, for example, an antibody as disclosed herein, can be derived from different immunoglobulin molecules. For example, a heavy chain portion of a polypeptide may comprise a CHI domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, a heavy chain portion may comprise a hinge region derived, in part, from an IgGl molecule and, in part, from an IgG3 molecule. In another example, a heavy chain portion may comprise a chimeric hinge derived, in part, from an IgGl molecule and, in part, from an IgG4 molecule.
[0093] 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.
[0094] Binding molecules, for example, antibodies or antigen binding fragments, variations, or derivatives thereof disclosed herein can be described or specified in terms of the epitope (s) or portion (s) of a antigen, for example, a target polysaccharide that they specifically recognize or bind to. The portion of a target polysaccharide that specifically interacts with the antigen binding domain of an antibody is an "epitope", or an "antigenic determinant". A target antigen, for example, 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.
[0095] As previously indicated, the subunit structures and three-dimensional configuration of the regions contained in the various classes of immunoglobulin are well known. As used herein, the term "VH domain" includes the amino terminal variable domain of an immunoglobulin heavy chain and the term "CHI domain" includes the first constant region (amino plus terminal) domain of a chain heavy dose of immunoglobulin. The CHI domain is adjacent to the VH domain and is amino terminal to the hinge region of an immunoglobulin heavy chain molecule.
[0096] As used herein, the term "CH2 domain" includes the portion of a heavy chain molecule that extends, for example, from about residue 244 to residue 360 of an antibody using numbering schemes conventional (residues 244 to 360, Kabat numbering system; and residues 231 to 340, EU numbering system; see Kabat EA et al. op. cit. The CH2 domain is unique in that it is not closely paired with another domain. instead, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule, and it is also well documented that the CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and comprises approximately 108 residues.
[0097] As used herein, the term "hinge region" includes the portion of a heavy chain molecule that joins the CHI domain to the CH2 domain. This hinge region comprises approximately 25 residues and is flexible, thus allowing the two N-terminal antigen binding regions to move independently. Hinge regions can be subdivided into three distinct domains: upper, median, and lower hinge domains (Roux et al., J. Immunol. 161AW3 (1998)).
[0098] 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 naturally occurring IgG molecules, the CH 1 and CL regions are linked by a disulfide bond and the two heavy chains are linked by two disulfide bonds at positions that correspond to 239 and 242 using the numbering system Rabat (heading 226 or 229, EU numbering system).
[0099] As used herein, the term "chimeric antibody" will be used to mean any antibody in which the immunoreactive region or site is obtained or derived from a first species and the constant region (which can be intact, partial or modified) it is obtained from a second species. In some embodiments, the target binding region or site will be from a non-human source (for example, mouse or primate) and the constant region is human.
[0100] The term "bispecific antibody", as used herein, refers to an antibody that has binding sites for two different antigens within a single antibody molecule. It will be noted that other molecules, in addition to the canonical antibody structure, can be constructed with two binding specificities. It will be further noted that antigen binding by bispecific antibodies can be simultaneous or sequential. Triomas and hybrid hybridomas are two examples of cell lines that can secrete bispecific antibodies. Bispecific antibodies can also be constructed by recombinant means. (Ströhlein and Heiss, Future Oncol. 6: 1,387 to 1,394 (2010); Mabry and Snavely, IDrugs. 73: 543 to 549 (2010)).
[0101] As used herein, the term "engineered antibody" refers to an antibody in which the variable domain in one of the heavy and light chains or both is altered by at least partial replacement of one or more CDRs of an antibody from known specificity and, if necessary, by replacing the partial frame region and changing the sequence. Although 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 conceived that CDRs will be derived from an antibody of a different class and preferably from an antibody of a species different. An engineered antibody in which one or more "donor" CDRs of a non-human antibody of known specificity is grafted onto a human heavy or light chain frame region is referred to herein as a "humanized antibody." It may not be necessary to replace all CDRs with full CDRs from the donor variable region to transfer the antigen binding capacity from one variable domain to another, instead it may only be necessary to transfer those residues that are necessary to maintain the activity of the target binding site. Given the explanations presented in, for example, US Patent Nos. 5,585,089, 5,693,761, 5,693,762 and 6,180,370, it will be well within the competence of those skilled in the art, whether performing routine experimentation or by trial and error to obtain a functional designed or humanized antibody.
[0102] As used herein the term "suitably folded polypeptide" includes polypeptides (for example, PcrV and Psl antibodies to anti-Pseudomonas) in which all functional domains comprising the polypeptide are distinctly active. As used herein, the term "not properly folded polypeptide" includes polypeptides in which at least one of the polypeptide's functional domains is not active. In one embodiment, a suitably 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.
[0103] As used herein, the term "designed" includes manipulation of polypeptide or nucleic acid molecules by synthetic means (for example, by recombinant procedure sets, in vitro peptide synthesis, by enzymatic or chemical coupling of peptides or some combination of these sets of procedures).
[0104] As used herein, the terms "bonded", "fused" or "fusion" are used interchangeably. These terms refer to the joining of two more elements or components, by any means, including chemical conjugation or means A "frame fusion" refers to the joining of two or more open polynucleotide reading frames (ORFs) to form a longer continuous ORF, so that it maintains the correct translational reading frame of the original ORFs. recombinant fusion protein is a single protein that contains two or more segments that correspond to polypeptides encoded by the original ORFs (whose segments are not normally joined in nature). segments can be physically or spatially separated by, for example, ligand sequence in frame, for example, polynucleotides that encode CDRs of a variable region immunoglobulin cells can be fused, in frame, but separated by a polynucleotide that encodes at least one immunoglobulin frame region or additional CDR regions, provided that the "fused" CDRs are co-translated as part of a continuous polypeptide.
[0105] In the context of polypeptides, a "linear sequence" or a "sequence" is an order of amino acids in a polypeptide in an amino-terminal direction for carboxyl in which residues neighboring each other in the sequence are contiguous in the primary structure of the polypeptide.
[0106] The term "expression" as used herein refers to a process by which a gene produces a biochemist, for example, a polypeptide. The process includes any manifestation of the functional presence of the gene within the cell, including, without limitation, reduction of gene expression, as well as both transient and stable expression. It includes, without limitation, transcription of the gene into an RNA messenger (mRNA), and the translation of such mRNA into polypeptide (s). If the desired end product is a biochemist, the term includes the creation of that biochemist and any precursors. The expression of a gene produces a “gene product.” As used herein, a gene product can be a nucleic acid, for example, an RNA messenger produced by transcription of a gene, or a polypeptide that is translated from A transcript The gene products described in this document further include nucleic acids with post-transcriptional modifications, for example, polyadenylation, or polypeptides with post-translational modifications, for example, methylation, glycosylation, the addition of lipids, association with other protein subunits, proteolytic cleavage and the like.
[0107] As used in this document, the terms "treat" or "treatment" refer to both therapeutic treatment and preventive or prophylactic measures, in which the objective is to prevent or slow down (mitigate) a disorder, infection or unwanted physiological change. Beneficial or desired clinical results include, but are not limited to, relieving symptoms, decreasing the extent of the disease, stabilizing (that is, not worsening) the disease, clearing or reducing an infectious agent such as P. aeruginosa in a subject , a delay or delay in the progression of the disease, improvement or palliation of the disease state and remission (either partial or total), or detectable or undetectable. "Treatment" can also mean prolonging survival compared to expected survival if treatment is not received. Those in need of treatment include those already with the infection, condition or disorder as well as those likely to have the condition or disorder or those in which the condition or disorder should be prevented, for example, in burn patients or immunosuppressed patients susceptible to infection by P.aeruginosa.
[0108] By "subject" or "individual" or "animal" or "patient" or "mammal" is meant any subject, particularly a mammal subject, for whom the diagnosis, prognosis or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals and zoo animals, sports or pets such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, bears and so on.
[0109] As used herein, phrases such as "a subject who would benefit from the administration of PcrV and Psi binding domains of anti-Pseudomonas" and "an animal in need of treatment" include subjects, such as mammalian subjects, who would benefit from the administration of PcrV and Psl binding domains of an Î ± -Pseudomonas or a binding molecule, such as an antibody, which comprises one or more of the binding domains. Such binding domains or binding molecules can be used, for example, for the detection of Pseudomonas Psl or PcrV (for example, for a diagnostic procedure) and / or for the treatment, i.e., palliation or prevention of a disease , with anti-Pseudomonas Psl and PcrV binding molecules. As described in more detail in this document, the anti-Pseudomonas Psi and PcrV binding molecules can be used in an unconjugated form or can be conjugated, for example, to a drug, prodrug or an isotope.
[0110] The term "synergistic effect", as used in this document, refers to a therapeutic effect greater than the additive produced by a combination of compounds in which the therapeutic effect obtained with the combination exceeds the additive effects that would result from another manner of individual administration of the compounds alone. Certain modalities include methods to produce a synergistic effect in the treatment of Pseudomonas infections, where said effect is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50 %, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500% or at least 1,000% greater than the corresponding additive effect.
[0111] "Co-administration" refers to the administration of different compounds, such as an anti-Psl and an anti-PcrV binding domain or binding molecule comprising one or both of an anti-Psl and the binding domain of anti-PcrV, so that the compounds achieve a synergistic effect on anti-Pseudomonas immunity. The compounds can be administered in the same or different compositions which, if separated, are administered next to each other, usually within 24 hours of each other and more typically within about 1 to 8 hours between themselves and even more typically within 1 hour. within 4 hours of each other or close to simultaneous administration. The relative quantities are dosages that achieve the desired synergism. II.CONNECTION DOMAINS AND CONNECTION MOLECULES
[0112] The antibodies that bind Psl and formats to use these antibodies have been described in the art. See, for example, International Application No. PCT / US2012 / 041538, filed on June 8, 2012 and PCT / US2012 / 63639, filed on November 6, 2012 (attorney registration number AEMS-115WO1, entitled “MULTISPECIFIC AND MULTIVALENT BINDING PROTEINS AND USES THEREOF ”), which are incorporated in this document in their entirety by way of reference.
[0113] One modality is targeted at binding domains that specifically bind to Pseudomonas PcrV, where binding can disrupt the activity of the type III toxin secretion system. In certain embodiments, the binding domains have the same binding specificity as Pseudomonas as the V2L2 antibody.
[0114] Another modality is targeted at binding domains that specifically bind to PcrV or Pseudomonas Psi, where administration of both binding domains results in synergistic effects against Pseudomonas infections by (a) inhibiting Pseudomonas binding aeruginosa to epithelial cells, (b) promotion, mediation or enhancement of P. aeruginosa opsonophagocytic murder (OPK), (c) inhibition of P. aeruginosa binding to epithelial cells or (d) interruption of the toxin secretion system activity type III. In certain embodiments, the binding domains have the same Pseudomonas binding specificity as the Cam-003, WapR-004, V2L2 or 29D antibodies.
[0115] Other modalities are directed to an isolated binding molecule (s) comprising one or both binding domains that specifically bind to Pseudomonas Psi and / or PcrV, where administration of the binding molecule results in synergistic effects against Pseudomonas infections. In certain embodiments, the binding molecule may comprise an antibody-binding domain or fragments thereof that include, but are not limited to, Cam-003, WapR-004, V2L2 or 29D22.
[0116] As used herein, the terms "binding domain" or "antigen binding domain" include a site that specifically binds an epitope on an antigen (for example, a Pseudomonas Psl or PcrV epitope ' ). The antigen binding domain of an antibody typically includes at least a portion of an immunoglobulin heavy chain variable region and at least a portion of an immunoglobulin light chain variable region. The binding site formed by these variable regions determines the specificity of the antibody.
[0117] The disclosure is more specifically directed to a composition comprising at least two anti-Pseudomonas binding domains, in which one binding domain specifically binds to Psl and the other binding domain specifically binds to PcrV. In one embodiment, the composition comprises a binding domain that specifically binds to the same Psl epitope as Pseudomonas as an antibody or antigen binding fragment thereof comprising the heavy chain variable region (VH) and light chain variable region (VL) of WapR-004, Cam-003, Cam-004, Cam-005, WapR-001, WapR-002, WapR-003 or WapR-016. In certain embodiments, the second binding domain specifically binds to the same PcrV epitope as Pseudomonas as an antibody or antigen binding fragment thereof comprising the heavy chain variable region (VH) and light chain variable region (VL) of V2L2 or 29D2.
[0118] In one embodiment, the composition comprises a binding domain that specifically binds Psl to Pseudomonas and / or competitively inhibits Psl binding to Pseudomonas by an antibody or antigen binding fragment thereof that comprises VH and VL from WapR-004, Cam-003, Cam-004, Cam-005, WapR-001, WapR-002, WapR-003 or WapR-016. In certain embodiments, the second binding domain specifically binds to the same PseV epitope from Pseudomonas and / or competitively inhibits the binding of PseVom PcrV by an antibody or antigen binding fragment thereof that comprises the heavy chain variable region ( VH) and the light chain variable region (VL) of V2L2 or 29D2.
[0119] Another embodiment is directed to an isolated binding molecule, for example, an antibody or antigen binding fragment thereof that specifically binds to the same PcrV epitope as Pseudomonas as an antibody or antigen binding fragment thereof. comprises the VH and VL region of V2L2 or 29D2.
[0120] An isolated binding molecule, for example, an antibody or fragment thereof, which specifically binds to Pseudomonas PcrV and competitively inhibits the binding of Pseudomonas PcrV by an antibody or antigen binding fragment thereof, is also included. comprises the VH and VL of V2L2 or 29D2.
[0121] One embodiment targets an isolated binding molecule, for example, an antibody or antigen binding fragment thereof that specifically binds to the same Psl epitope as Pseudomonas as an antibody or antigen binding fragment thereof comprises the VH and VL region of WapR-001, WapR-002 or WapR-003.
[0122] Also included is an isolated binding molecule, for example, an antibody or fragment thereof that specifically binds to Psl of Pseudomonas and competitively inhibits Psl binding to Pseudomonas by an antibody or antigen binding fragment thereof. comprises the VH and VL of WapR-001, WapR-002 or WapR-003.
[0123] In addition, an isolated binding molecule, for example, an antibody or fragment thereof, which specifically binds to the same Psl epitope of Pseudomonas as an antibody or antigen binding fragment thereof, comprising VHe VL of WapR -016.
[0124] An isolated binding molecule, for example, an antibody or fragment thereof, which specifically binds to Psi of Pseudomonas and competitively inhibits Psl binding of Pseudomonas by an antibody or antigen binding fragment thereof, is also included. comprises the VH and VL of WapR-016.
[0125] The methods for making the antibodies are well known in the art and described herein. Once antibodies to multiple fragments or to the full-length Pseudomonas Psl or PcrV without the signal sequence have been produced, determining which amino acids or epitope of the Pseudomonas Psl or PcrV to which the antibody or antigen binding fragment binding can be determined by epitope mapping protocols as described in this document as well as methods known in the art (for example, double antibody sandwich ELISA as described in "Chapter 11 - Immunology," Current Protocols in Molecular Biology, Ed Ausubel et al., Volume 2, John Wiley & Sons, Inc. (1996)). Additional epitope mapping protocols can be found in Morris, G. Epitope Mapping Protocols, New Jersey: Humana Press (1996), which are both incorporated into this document for reference in their entirety. Epitope mapping can also be performed by commercially available means (i.e., ProtoPROBE, Inc. (Milwaukee, Wisconsin)).
[0126] In certain aspects, the disclosure is directed to a binding molecule, for example, an antibody or fragment, variant or derivative thereof that specifically binds to Psl and / or PcrV of Pseudomonas with an affinity characterized by a constant dissociation (KD) which is less than the KD for said reference monoclonal antibody.
[0127] In certain embodiments, a Psl and / or PcrV binding molecule of anti-Pseudo monas, for example, an antibody or antigen binding fragment, variant or derivative thereof as disclosed herein, specifically binds at least a Psl or PcrV epitope, that is, it binds to such an epitope more readily than it would bind to an unrelated or random epitope; it preferably binds to at least one epitope of Psl or PcrV, that is, it binds to such an epitope more readily than it would bind to a related, similar, homologous or analogous epitope; competitively inhibits the binding of a reference antibody which in turn specifically or preferentially binds to a specific Psl or PcrV epitope; or binds to at least one Psl or PcrV epitope 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 10 ”M, about 10 ”M, about 5 x 10'4M, about 10'4 M, about 5 x 10'5 M, about 10'5 M, about 5 x 10'6 M, about 10'6 M, about 5x 10'7 M, about 10'7 M, about 5 x 10'8 M, about 10'8 M, about 5x10'9 M, about 10'9 M, about 5 x 10 "10 M, about 10'I () M, about 5x10 '" M, about 10' "M, about 5 x 10'12 M, about 10'12 M, about 5x10'13 M, about 10'13 M, about 5 x 10'14 M, about 10'I4M, about 5 x 10'I5M or about 10 '| 3M.
[0128] As used in the context of binding dissociation constants, the term "about" allows for the degree of variation inherent in the methods used to measure antibody affinity. For example, depending on the level of precision of the instrumentation used, the standard error based on the number of samples measured and the rounding error, the term “about 10'2 M” can include, for example, from 0.05 M to 0.005 M.
[0129] In specific embodiments, a binding molecule, for example, an antibody or antigen binding fragment, variant or derivative of the same Pseudomonas Psl and / or PcrV alloy with a constant rate for dissociation (k (off)) less than or equal to 5 X 10 's', 10 's', 5 X 10 's' or 10 's'. Alternatively, an antibody or antigen-binding fragment, variant or derivative thereof, binds Pseudomonas Psl and / or PcrV with a dissociation constant rate (k (off)) less than or equal to 5 X 10'4 s'1 , 10'4 s1, 5 X IO'5 s'1 or 10'5 s'1 5 X 10'6 s'1, IO'6 s'1, 5 X 10'7 s'1 or 10'7 s '1.
[0130] In other embodiments, a binding molecule, for example, an antibody, or antigen binding fragment, variant or derivative thereof as disclosed in this document binds Pseudomonas Psl and / or PcrV with a constant rate to association (k (on)) greater than or equal to 103 M'1 s'1, 5 X 10J M'1 s'1, 104 M'1 s'1 or 5 X 104 M'1 s * 1. Alternatively, a binding molecule, for example, an antibody or antigen binding fragment, variant or derivative thereof as disclosed herein binds Pseudomonas Psl and / or PcrV with a constant rate for association (k (on) ) greater than or equal to 10 M'1 s'1, 5 X 105 M'1 s * 1, 106 M’1 s'1 or 5 X 106 M’1 s’1 or 107 M’1 s’1.
[0131] In various embodiments, a PsX and / or PcrV binding molecule of anXAN-Pseudomonas, for example, an antibody or antigen binding fragment, variant or derivative thereof as described in this document, promotes the opsonophagocytic extermination of Pseudomonas or inhibits the binding of Pseudomonas to epithelial cells. In certain embodiments described in this document, the target of Pseudomonas Psl or PcrV is Pseudomonas aeruginosa Psl or PcrV. In other embodiments, certain binding molecules described in this document can bind to structurally related polysaccharide molecules regardless of their source. Such Psl-type molecules would be expected to be identical or have sufficient structural relativity to the P. aeruginosa Psl to allow specific recognition by one or more of the disclosed binding molecules. In other embodiments, certain binding molecules described in this document can bind to related polypeptide molecules that are structurally independent of their source. Such PcrV-type molecules would be expected to be identical or have sufficient structural relativity to the P. aeruginosa PcrV to allow specific recognition by one or more of the disclosed binding molecules. Therefore, for example, certain binding molecules described in this document can bind to Psl and / or PcrV type molecules produced by other bacterial species, for example, Psl type or PcrV type molecules produced by other species of bacteria. Pseudomonas, for example, Pseudomonas fluorescens, Pseudomonas putida, or Pseudomonas alcaligenes. Alternatively, certain binding molecules as described in this document can bind to Psl-type and / or PcrV-type molecules produced synthetically or by host cells genetically modified to produce Psl-type or PcrV-type molecules.
[0132] Unless specifically noted, as used herein, a "fragment thereof" in reference to a binding molecule, for example, an antibody refers to an antigen binding fragment, that is, a portion of the antibody that specifically binds to the antigen.
[0133] The hx t -Pseudomonas Psl and / or PcrV binding molecules, for example, antibodies or antigen binding fragments, variants or derivatives thereof, may comprise a constant region that mediates one or more effector functions. For example, binding of the complement component C1 to an antibody constant region can activate the complement system. Complement activation is important in the pathogen opsonization and lysis. Complement activation also stimulates the inflammatory response and may also be involved in autoimmune hypersensitivity. In addition, antibodies bind to receptors in various cells via the Fc region, with an Fc receptor binding site in the Fc antibody region that binds to an Fc receptor (FcR) in a cell. There are several Fc receptors that are specific to different classes of antibody, including IgG (gamma receptors), IgE (epsilon receptors), IgA (alpha receptors) and IgM (mu receptors). The binding of the antibody to Fc receptors on cell surfaces triggers several important and diverse biological responses including the engulfment and destruction of antibody-coated particles, elimination of immune complexes, lysis of antibody-coated target cells by killer cells (called cytotoxicity mediated by antibody-dependent cell or ADCC), release of inflammatory mediators, placental transfer and control of immunoglobulin production.
[0134] Consequently, certain embodiments disclosed herein include an anti-Pseudomonas Psl and / or PcrV binding molecule, for example, an antibody or antigen binding fragment, variant or derivative thereof, in which at least one fraction of one or more domains of constant region has been deleted or altered in another way in order to provide the desired biochemical characteristics such as reduced effector functions, the ability to dimerize non-covalently, an increased ability to locate the site of a tumor, half reduced serum life or increased serum half-life compared to a whole, unchanged antibody with approximately equal immunogenicity. For example, certain binding molecules described herein are antibodies deleted from the domain comprising a polypeptide chain similar to an immunoglobulin heavy chain, but which do not have at least a portion of one or more heavy chain domains. For example, in certain antibodies, an entire domain of the modified antibody constant region will be deleted, for example, all or part of the CH2 domain will be deleted.
[0135] The modified forms of Psl and / or PcrV binding molecules of anti-Pseudomonas, for example, antibodies or antigen binding fragments, variants or derivatives thereof, can be made from parent antibodies or whole precursors with the use of sets of procedures known in the art. Exemplary sets of procedures are discussed elsewhere in this document.
[0136] In certain embodiments, both the variable and constant regions of Psi and / or PcrV binding molecules of anü-Pseudomonas, for example, antibodies or antigen binding fragments are completely human. Completely human antibodies can be made using sets of procedures that are known in the art and as described in this document. For example, fully human antibodies to a specific antigen can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to an antigenic stimulus, but whose endogenous sites have been disabled. The sets of exemplary procedures that can be used to make such antibodies are described in US Patent No. 6,150,584; 6,458,592; 6,420,140. Other sets of procedures are known in the art. Fully human antibodies can also be produced by various display technologies, for example, phage display or other viral display systems, as described in more detail elsewhere in this document.
[0137] Psl and / or PcrV binding molecules of antiPseudomonas, for example, antibodies or antigen binding fragments, variants or derivatives thereof as disclosed herein can be made or manufactured using sets of procedures that are known in the art. In certain embodiments, the binding molecules or fragments thereof are "produced recombinantly", that is, they are produced using recombinant DNA technology. The sets of exemplary procedures for making the antibody molecules or fragments thereof are discussed in more detail elsewhere in this document.
[0138] In certain Psi and / or PcrV binding molecules of anü-Pseudomonas, for example, antibodies or antigen binding fragments, variants or derivatives thereof described in this document, the Fc portion can be mutated to decrease function effect using sets of procedures known in the art. For example, the deletion or inactivation (via point mutations or other means) of a constant region domain can reduce the Fc receptor binding of the circulating modified antibody thereby increasing the location of the tumor. In other cases, it is possible that the constant region changes moderate the complement binding and thus reduce the serum half-life and the non-specific association of a conjugated cytotoxin. Still other modifications of the constant region can be used to modify disulfide bonds or chemical portions of oligosaccharides that allow for improved localization due to increased antibody flexibility or antigen specificity. The resulting physiological profile, bioavailability and other biochemical effects of the modifications, such as the location, biodistribution and serum half-life, can be easily measured and quantified using well-known sets of immunological procedures without undue experimentation.
[0139] In certain embodiments, the Psi and / or PcrV binding molecules of anü-Pseudomonas, for example, antibodies or antigen binding fragments, variants or derivatives thereof, will not obtain a deleterious immune response in the animal to be treated, for example, in a human. In one embodiment, the Psl and / or PcrV binding molecules of anü-Pseudomonas, for example, antibodies or antigen binding fragments, variants or derivatives thereof, are modified to reduce their immunogenicity using sets of procedures recognized in the literature. technical. For example, antibodies can be humanized, de-immunized, or chimeric antibodies can be made. These types of antibodies are derived from a non-human antibody, typically a murine or primate antibody, which retains or substantially retains the antigen-binding properties of the parent antibody, but which is less immunogenic in humans. This can be achieved by several methods, including (a) grafting the entire non-human variable domains into human constant regions to generate chimeric antibodies; (b) grafting at least part of one or more non-human complementarity determining regions (CDRs) into a human framework and constant regions with or without retention of critical framework residues; or (c) transplant the entire non-human variable domains, but "hide" them with human-type secretion by replacing surface residues. Such methods are disclosed in Morrison et al., Proc. Natl. Acad. Sci.81: 6,851 to 6,855 (1984); Morrison et al., Adv. Immunol.44: 65 to 92 (1988); Verhoeyen etal., Science239: 534 to 1,536 (1988); Padlan, Molec. Immun. 28: 489 to 498 (1991); Padlan, Molec. Immun.31: 69 to 217 (1994) and in U.S. Patent Nos. 5,585,089, 5,693,761, 5,693,762 and 6,190,370, all of which are incorporated herein by reference in their entirety.
[0140] Deimmunization can also be used to decrease the immunogenicity of an antibody. As used herein, the term "deimmunization" includes the alteration of an antibody to modify T cell epitopes (see, for example, WO9852976A1, W00034317A2). For example, the VH and VL sequences of the initial antibody are analyzed and a human T cell epitope “map” for each V region showing the location of the epitopes in relation to the complementarity determining regions (CDRs) and other key residues within the sequence. The individual T cell epitopes on the T cell epitope map are analyzed to identify alternative amino acid substitutions with a low risk of altering the activity of the final antibody. A range of alternative VH and VL sequences is designed comprising combinations of amino acid substitutions and these sequences are subsequently incorporated into a range of binding polypeptides, for example, specific antibodies to Pseudomonas Psl and / or PcrV or antigen binding fragments of the same disclosed in this document, which are then tested for function. The complete light and heavy chain genes that comprise human C and modified V regions are then cloned into expression vectors and the subsequent plasmids introduced into the cell lines for the production of the entire antibody. The antibodies are then compared in appropriate biochemical and biological assays and the optimal variant is identified.
[0141] PsP and / or PcrV binding molecules of antiPseudomonas, for example, antibodies or antigen binding fragments, variants or derivatives thereof, can be generated by any 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 and / or PcrV antibody or antigen binding fragment thereof can be administered to various host animals including, but not limited to, rabbits, mice, rats, chickens, hamsters, goats, donkeys, etc. to induce the production of sera that contain antigen-specific polyclonal antibodies. Various adjuvants can be used to increase the immune response, depending on the host species and include, but are not limited to, Freund (complete and incomplete), mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols, polyions, peptides, oil emulsions, Califomian keyhole limpet hemocyanins, dinitrophenol and potentially useful human adjuvants such as BCG (bacillus Calmette-Guerin) and Corynebacterium parvum. Such adjuvants are also well known in the art.
[0142] Monoclonal antibodies can be prepared using a wide variety of sets of procedures known in the art including the use of sets of hybridoma, recombinant and phage display procedures or a combination thereof. For example, monoclonal antibodies can be produced using sets of hybridoma procedures including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd edition ( 1988).
[0143] DNA encoding antibodies or antibody fragments (e.g., antigen binding sites) can also be derived from antibody libraries, such as phage display libraries. In particular, such a phage can be used to display antigen binding domains expressed from a repertoire or combinatorial (e.g., human or murine) antibody library. The phage expressing an antigen binding domain that binds to the antigen of interest can be selected or identified with the antigen, for example, using the identified antigen or antigen bound to or captured on a solid surface or microsphere. The phage used in these methods are typically filamentous phages that include fd and M13 binding domains expressed from the phage with scFv, Fab, Fv OE DAB (individual heavy or light chain Fv region) or disulfide-stabilized Fv antibody domains fused. recombinantly or to the phage III gene or to the VIII gene protein. Exemplary methods are set out, for example, in document No. EP 368 684 Bl; U.S. Patent No. 5,969,108, Hoogenboom, H.R. and Chames, Immunol. Today 21: 311 (2000); Nagy et al. Nat. Med. 5: 801 (2002); Huie et al., Proc. Natl. Acad. Know. USA 95: 2682 (2001); Lui et al., J. Mol. Biol.375: 1063 (2002), each of which is incorporated by reference in this document. Various publications (eg, Marks et al., Bio / Technology 10U19 to 783 (1992)) have described the production of high-affinity human antibodies by chain shuffling, as well as combinatorial infection and in vivo recombination as a strategy for build large phage libraries. In another embodiment, ribosomal display can be used to replace the bacteriophage as the display platform (see, for example, Hanes et al., Nat. Biotechnol. 75: 1287 (2000); Wilson et al., Proc. Natl. Acad. Sci. USA 95: 3,750 (2001); or Irving et al., J. Immunol. Methods 248 3 (2001)). In yet another embodiment, cell surface libraries can be screened for antibodies (Boder et al., Proc. Natl. Acad. Sci. USA 97: 10.701 (2000); Daugherty et al., J. Immunol. Methods 243: 2 \ (2000)). Such procedures provide alternatives to sets of traditional hybridoma procedures for the isolation and subsequent cloning of monoclonal antibodies.
[0144] In phage display methods, functional antibody domains are displayed on the surface of phage particles that carry the polynucleotide sequences that encode them. For example, DNA sequences encoding the VH and VL regions are amplified from animal cDNA libraries (for example, human or murine lymphoid cDNA libraries) or synthetic cDNA libraries. In certain embodiments, the DNAs encoding the VH and VL regions are joined together by a scFv linker by PCR and cloned into a phagemid vector (for example, p CANTAB 6 or pComb 3 HSS). The vector is electroporated in E. coli and £. coli is infected with the helper phage. The phage used in these methods is a typically filamentous phage including fd and Ml3 and the VH or VL regions are usually fused recombinantly to either the phage III or the VIII gene. The phage that expresses an antigen binding domain that binds to an antigen of interest (that is, Pseudomonas Psl or PcrV) can be selected or identified with the antigen, for example, using the identified antigen or bound antigen or captured to a solid surface or microsphere.
[0145] Additional examples of phage display methods that can be used to make antibodies include those disclosed in Brinkman et al., J. Immunol. Methods 182A a 50 (1995); Ames et al., J. Immunol. Methods 184: 177 to 186 (1995); Kettleborough et al., Eur. J. Immunol. 24: 952 to 958 (1994); Persic et al., Gene 187: 9 to 18 (1997); Burton et al., Advances in Immunology 57: 191 to 280 (1994); PCT application No. PCT / GB91 / 01134; PCT Publications No. WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Patent No. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is incorporated in this document as a reference in its entirety.
[0146] As described in the references above and in the examples below, after phage selection, the antibody coding regions of 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, sets of procedures for recombinantly producing the Fab, Fab 'and F (ab') 2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication No. WO 92/22324 ; Mullinax et al., BioTechniques 12 (6): 864 to 869 (1992); and Sawai et al., AJRI 34:26 to 34 (1995); and Better et al., Science 240: . 64 to 1,043 (1988) (said references incorporated by reference without their totalities).
[0147] Examples of sets of procedures that can be used to produce single chain Fvs and antibodies include those described in U.S. Patent Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203: 46 to 88 (1991); Shu et al., PNAS 90: 7,995 to 7,999 (1993); and Skerra et al., Science 249: 1,038 to 1,040 (1988). In certain embodiments, such as therapeutic administration, chimeric, humanized or human antibodies can be used. A chimeric antibody is a molecule in which different portions of the antibody are derived from different species of animals, such as antibodies that have a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See, for example, Morrison, Science 229: 1,202 (1985); Oi et al., BioTechniques 4: 214 (1986); Gillies et al., J. Immunol. Methods 725: 191 to 202 (1989); U.S. Patent No. 5,807,715; 4,816,567; and 4,816,397, which are incorporated in this document as a reference in their entirety. Humanized antibodies are antibody molecules from an antibody of a non-human species that binds to the desired antigen that has one or more complementarity determining regions (CDRs) of non-human species and framework regions of a human immunoglobulin molecule. Often, framework residues in human framework regions will be replaced by the corresponding CDR donor antibody residue to alter, preferably improve, antigen binding. These framework substitutions are identified by methods known in the art, for example, by modeling the interactions of framework residues and CDR to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues in particular positions. (See, for example, Queen et al., U.S. Patent No. 55,585,089; Riechmann et al., Nature 332: 323 (1988), which are incorporated herein by reference in their entirety). Antibodies can be humanized using a variety of sets of procedures known in the art including, for example, CDR grafting (EP 239,400; PCT publication No. WO 91/09967; US Patent No. 5,225,539; 5,530,101 ; and 5,585,089), coating and resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28 (4/5): 489 to 498 (1991); Studnicka et al., Protein Engineering 7 (6): 8Q5 to 814 (1994); Roguska. Et al., PNAS 91: 969 to 973 (1994)) and chain shuffling (US Patent No. 5,565,332).
[0148] Fully human antibodies are particularly desirable for the therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art including the phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also U.S. Patent Nos. 4,444,887 and 4,716,111; and PCT publications No. WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735 and WO 91/10741; each of which is incorporated in this document as a reference in its entirety.
[0149] Human antibodies can also be produced using transgenic mice that do not have the ability to express functional endogenous immunoglobulins, but that can express human immunoglobulin genes. For example, human light and heavy chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. In addition, several companies may be committed to supplying human antibodies produced in transgenic mice directed against a selected antigen using the set of procedures similar to the one described above.
[0150] Completely human antibodies that recognize a selected epitope can be generated using a set of procedures referred to as "guided selection". In this approach, a selected non-human monoclonal antibody, for example, a mouse antibody, is used to guide the selection of a completely human antibody that recognizes the same epitope. (Jespers et al., Bio / Technology 12: 899 to 903 (1988). See also U.S. Patent No. 5,565,332.)
[0151] In another embodiment, the DNA encoding the desired monoclonal antibodies can be readily isolated and sequenced using conventional procedures (for example, using oligonucleotide probes that can specifically bind to the genes encoding the light chains and heavy murine antibodies). Isolated and subcloned hybridoma cells or isolated phage, for example, can serve as a source of such DNA. Once isolated, DNA can be placed into expression vectors, which are then transfected into prokaryotic or eukaryotic host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells or myeloma cells that do not otherwise produce immunoglobulins. More particularly, isolated DNA (which can be synthetic as described herein) can be used to clone the variable and constant region sequences for the manufacture of antibodies as described in Newman et al., U.S. Patent No. 5,658,570, filed on January 25, 1995, which is incorporated by reference in this document. Transformed cells that express the desired antibody can be grown in relatively large quantities to provide clinical and commercial supplies of immunoglobulin.
[0152] In one embodiment, an isolated binding molecule, for example, an antibody comprises at least one light or heavy chain CDR of an antibody molecule. In another embodiment, an isolated binding molecule comprises at least two CDRs of one or more antibody molecules. In another embodiment, an isolated binding molecule comprises at least three CDRs of one or more antibody molecules. In another embodiment, an isolated binding molecule comprises at least four CDRs of one or more antibody molecules. In another embodiment, an isolated binding molecule comprises at least five CDRs of one or more antibody molecules. In another embodiment, an isolated binding molecule of the description comprises at least six CDRs of one or more antibody molecules.
[0153] In a specific embodiment, the amino acid sequence of the heavy and / or light chain variable domains can be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well known in the art, for example, by comparing known amino acid sequences from other variable regions of heavy and light chain to determine regions of sequence hypervariability. With the use of routine recombinant DNA techniques, one or more of the CDRs can be inserted within the framework regions, for example, in human framework regions to humanize a non-human antibody. The framework regions can be naturally occurring or consensus framework regions and preferably human framework regions (see, for example, Chothia et al., J. Mol. Biol. 278A51 to 479 (1998) for a listing of regions of human framework). The polynucleotide generated by combining the framework regions and CDRs encodes an antibody that specifically binds to at least one epitope of a desired antigen, for example, Psl or PcrV. One or more amino acid substitutions can be made within the framework regions and the amino acid substitutions improve the binding of the antibody to its antigen. In addition, such methods can be used to make amino acid substitutions or deletions of one or more variable region cysteine residues that participate in a disulfide bond between chains to generate antibody molecules that do not have one or more disulfide bonds between chains . Other changes to the polynucleotide are covered by the present disclosure and are within the capabilities of one skilled in the art.
[0154] Binding molecules are also provided which comprise, essentially consist of or consist of variants (including derivatives) of antibody molecules (for example, the VH regions and / or VL regions) described herein, such binding molecules or fragments of them specifically bind to Pseudomonas Psl or PcrV. Standard sets of procedures known to those skilled in the art can be used to introduce mutations into the nucleotide sequence that encodes a binding molecule or fragment thereof that specifically binds to Pseudomonas Psl and / or PcrV, including, but not limited to, site-directed mutagenesis and PCR-mediated mutagenesis that result in amino acid substitutions. Variants (including derivatives) encode polypeptides that comprise less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions or less than 2 amino acid substitutions in relation to reference region VH, VHCDR1, VHCDR2, VHCDR3, region VL, VLCDR1, VLCDR2 or VLCDR3. A "conservative amino acid substitution" is one in which the amino acid residue is replaced by an amino acid residue that has a side chain with a similar charge. Families of amino acid residues that have side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (for example, lysine, arginine, histidine), acidic side chains (for example, aspartic acid, glutamic acid), uncharged polar side chains (for example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and side chains aromatic (eg tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced at random throughout all or part of the coding sequence, such as by saturation mutagenesis and the resulting mutants can be screened for biological activity to identify mutants that retain activity (for example, the ability to connect a Pseudomonas Psl or PcrV}.
[0155] For example, it is possible to introduce mutations only in framework regions or only in CDR regions of an antibody molecule. The introduced mutations can be silent or neutral missense mutations, that is, they have little or no effect on an antibody's ability to bind the antigen. These types of mutations can be useful for optimizing codon usage or improving hybridoma antibody production. Alternatively, non-neutral missense mutations can alter an antibody's ability to bind the antigen. The location of the most silent and neutral missense mutations is likely to be in the framework regions, while the location of the most non-neutral missense mutations is likely to be in the CDR, although this is not an absolute requirement. One skilled in the art could design and test the mutant molecules with the desired properties such as no change in antigen binding activity or change in binding activity (e.g., improvements in antigen binding activity or change in antibody specificity). Following mutagenesis, the encoded protein can be routinely expressed and the functional and / or biological activity of the encoded protein (for example, ability to bind at least one epitope of Pseudomonas Psl or PcrV} can be determined using sets of procedures described in this document or by routinely modifying the sets of procedures known in the art.
[0156] One embodiment provides a bispecific antibody comprising an anü-Pseudomonas PcrV and Psl binding domain disclosed in the present document. In certain embodiments, the bispecific antibody contains a first Psl binding domain and a second PcrV binding domain. Bispecific antibodies with more than two valences are contemplated. For example, triespecific antibodies can also be prepared using the methods described in this document. (Tutt et al., J. Immunol., 147: 60 (1991)).
[0157] One modality provides a method to produce a bispecific antibody that uses a single light chain that can pair with both heavy chain variable domains present in the bispecific molecule. To identify this light chain, several strategies can be employed. In one embodiment, a series of monoclonal antibodies are identified for each antigen that can be targeted with the bispecific antibody, followed by determining which of the light chains used in these antibodies can work when paired with the heavy chain of any of the antibodies identified at the time. second target. In this way, a light chain that can work with two heavy chains to allow binding to both antigens can be identified. In another embodiment, display techniques, such as phage display, may allow the identification of a light chain that can work with two or more heavy chains. In one embodiment, a phage library is constructed that comprises a diverse repertoire of heavy chain variable domains and a single light chain variable domain. This library can additionally be used to identify antibodies that bind to various antigens of interest. Thus, in certain embodiments, the identified antibodies will share a common light chain.
[0158] In certain embodiments, the bispecific antibody comprises at least one single chain Fv (scFv). In certain embodiments, the bispecific antibody comprises two scFvs. For example, a scFv can be fused to one or both of a polypeptide that contains the CH3 domain contained within an antibody. Some methods comprise producing a bispecific molecule in which one or both heavy chain constant regions comprising at least one CH3 domain are used in conjunction with a single single chain Fv domain to provide antigen binding. III. ANTIBODY POLYPEPTIDS
[0159] The disclosure is additionally directed to isolated polypeptides that form the binding molecules, for example, antibodies or antigen binding fragments thereof, which specifically bind to Pseudomonas Psl and / or PcrV and polynucleotides encoding such polypeptides. The binding molecules, for example, antibodies or fragments thereof as disclosed herein, comprise polypeptides, for example, amino acid sequences that encode, for example, Psl-specific and / or PcrV-specific antigen-binding regions derived from immunoglobulin molecules. A polypeptide or amino acid sequence "derived from" a designated protein refers to the origin of the polypeptide. In certain cases, the polypeptide or amino acid sequence that is derived from a particular initial polypeptide or amino acid sequence has an amino acid sequence that is essentially identical to that of the initial sequence or a portion thereof, where the portion consists of at least 10 to 20 amino acids, at least 20 to 30 amino acids, at least 30 to 50 amino acids, or that is otherwise identifiable to one skilled in the art as having its origin in the initial sequence.
[0160] Also disclosed is an isolated binding molecule, for example, an antibody or antigen binding fragment thereof that specifically binds to Pseudomonas Psl which comprises an amino acid sequence of the immunoglobulin heavy chain (VH) variable region 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.
[0161] An additional binding molecule, for example, an antibody or antigen binding fragment thereof, which specifically binds to Pseudomonas Psl, which comprises an identical or identical VH amino acid sequence except for one, two, is further disclosed. 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.
[0162] Some embodiments include an isolated binding molecule, for example, an antibody or antigen binding fragment thereof that specifically binds to Pseudomonas Psl that comprises a VH, where one or more of the VHCDR1, VHCDR2 or VHCDR3 regions of the VH are at least 80%, 85%, 90%, 95% or 100% identical to one or more amino acid sequences of VHCDR1, VHCDR2 or VHCDR3 heavy chain reference 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.
[0163] Additionally disclosed is an isolated binding molecule, for example, an antibody or antigen binding fragment thereof that specifically binds to Psl of Pseudomonas comprising a VH, wherein one more of the VHCDR1, VHCDR2 or VHCDR3 regions of the VH are identical to, or identical with the exception of four, three, two or an amino acid substitution, to one or more amino acid sequences of reference VHCDR1, VHCDR2 and / or VHCDR3 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 modality the VH comprises one or more of a VHCDR1, VHCDR2, or VHCDR3 identical to or identical with the exception of four, three, two or an amino acid substitution to one or more of the VHCDR1, VHCDR2 or VHCDR3 amino acid sequences shown in Table 3.
[0164] Revealed is also an isolated binding molecule, for example, an antibody or antigen binding fragment thereof that specifically binds to Pseudomonas Psl which comprises an immunoglobulin light chain variable region (VL) amino acid sequence by minus 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.
[0165] Some modalities disclose an isolated binding molecule, for example, an antibody or antigen binding fragment thereof that specifically binds to Psl of Pseudomonas which comprises a VL amino acid sequence identical to or identical with the exception of 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.
[0166] Provided is also an isolated binding molecule, for example, an antibody or antigen binding fragment thereof that specifically binds to Psl of Pseudomonas comprising a VL, wherein one or more of the VLCDR1, VLCDR2 or VLCDR3 regions of VL are at least 80%, 85%, 90%, 95% or 100% identical to one or more VLCDR1, VLCDR2 or VLCDR3 light chain reference sequences from 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.
[0167] Further provided is an isolated binding molecule, for example, an antibody or antigen binding fragment thereof that specifically binds to Psl of Pseudomonas comprising a VL, wherein one or more of the VLCDR1, VLCDR2 or VLCDR3 regions of the VL are identical to or identical with the exception of four, three, two or an amino acid substitution for one or more amino acid sequences of VLCDR1, VLCDR2 and / or VLCDR3 heavy chain reference 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 modality the VL comprises one or more of a VLCDR1, VLCDR2, or VLCDR3 identical to or identical with the exception of four, three, two or an amino acid substitution for one or more of the amino acid sequences of VLCDR1, VLCDR2 , or VLCDR3 shown in Table 3.
[0168] In other embodiments, an isolated antibody or antigen binding fragment thereof that specifically binds to Psl of Pseudomonas, comprises, consists essentially of, or consists of amino acid sequences of VH and VL at least 80%, 85%, 90 % 95% or 100% identical to: (a) SEQ ID NO: 1 and SEQ ID NO: 2, respectively, (b) SEQ ID NO: 3 and SEQ ID NO: 2, respectively, (c) SEQ ID NO: 4 and SEQ ID NO: 2, respectively, (d) SEQ ID NO: 5 and SEQ ID NO: 6, respectively, (e) SEQ ID NO: 7 and SEQ ID NO: 8, respectively, (f) SEQ ID NO. : 9 and SEQ ID NO: 10, respectively, (g) SEQ ID NO: 11 and SEQ ID NO: 12, respectively, (h) SEQ ID NO: 13 and SEQ ID NO: 14, respectively; (i) SEQ ID NO: 15 and SEQ ID NO: 16, respectively; or (j) SEQ ID NO: 74 and SEQ ID NO: 12, respectively. In certain embodiments, the antibody or antigen-binding fragment described above 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 antibody or the antigen binding fragment described above 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 antibody or antigen binding fragment described therein. above comprises a VH with the amino acid sequence SEQ ID NO: He and a VL with the amino acid sequence of SEQ ID NO: 12.
[0169] Certain embodiments provide an isolated binding molecule, for example, an antibody, or antigen binding fragment thereof that specifically binds to Pseudomonas Psl, which comprises an immunoglobulin VH and an immunoglobulin VL, each of which comprises a complementarity determining region 1 (CDR1), CDR2 and CDR3, where VH CDR1 is PYYWT (SEQ ID NO: 47), VH CDR2 is YIHSSGYTDYNPSLKS (SEQ ID NO: 48), CDR3 DE VH is selected from the group consisting of ADWDRLRALDI (Psl0096, SEQIDNO: 258), AMDIEPHALDI (Psl0225, SEQIDNO: 267), ADDPFPGYLDI (Psl0588, SEQ ID NO: 268), ADWNEGRKLDI (Psl0567, SEQIDNO: 269H, AD9 , SEQIDNO: 270), ATDEADHALDI (Psl0170, SEQ ID NO: 271), ADWSGTRALDI (PsI0304, SEQID NO: 272), GLPEKPHALDI (Psl0348, SEQIDNO: 273), SLFTDDHALDI (Psl0573, SEQ ID NO: 274), , SEQID NO: 275), AHIESHHALDI (Psl0582, SEQIDNO: 276), ATQAPAHALDI (Psl0584, SEQ ID NO: 277), SQHDLEHALDI (Psl0585, SEQ ID NO: 278) and AMPDMPHALDI (Psl0589 , SEQ ID NO: 279), VL CDR1 is RASQSIRSHLN (SEQ ID NO: 50), VL CDR2 is GASNLQS (SEQ ID NO: 51) and VL CDR3 is selected from the group consisting of QQSTGAWNW (Psl0096, SEQ ID NO: 280), QQDFFHGPN (Psl0225, SEQ ID NO: 281), QQSDTFPLK (Psl0588, SEQ ID NO: 282), QQSYSFPLT (WapR0004, Psl0567, Psl0573, Psl00574, Psl0584, Psl0582, Psl0582, Psl0582, Psl0582, Psl0582, Psl0582, Psl0584, NO: 52), QDSSSWPLT (Psl0337, SEQ ID NO: 283), SQSDTFPLT (Psl0170, SEQ ID NO: 284), GQSDAFPLT (Psl0304, SEQ ID NO: 285), LQGDLWPLT (Psl0348, SEQ ID NO: 286), and QQSLEFPLT (Psl0589, SEQ ID NO: 287), where the VH and VL CDRs are in accordance with the Kabat numbering system.
[0170] Certain embodiments provide an isolated binding molecule, for example, an antibody, or antigen binding fragment thereof that specifically binds to Pseudomonas Psl, which comprises an immunoglobulin VH and an immunoglobulin VL, each of which comprises a complementarity determining region 1 (CDR1), CDR2 and CDR3, where VH CDR1 is PYYWT (SEQ ID NO: 47), VH CDR2 is YIHSSGYTDYNPSLKS (SEQ ID NO: 48), CDR1 DE VL is RASQSIRSHLN (SEQ ID NO: 50), VL CDR2 is GASNLQS (SEQ ID NO: 51) and VH CDR3 and VL CDR3 comprise, respectively, ADWDRLRALDI (Psl0096, SEQ ID NO: 258) and QQSTGAWNW ( Psl0096, SEQ ID NO: 280); AMDIEPHALDI (Psl0225, SEQ ID NO: 267) and QQDFFHGPN (Psl0225, SEQ ID NO: 281); ADDPFPGYLDI (Psl0588, SEQ ID NO: 268) and QQSDTFPLK (Psl0588, SEQ ID NO: 282); ADWNEGRKLDI (Psl0567, SEQ ID NO: 269) and the VL CDR3 is QQSYSFPLT (WapR0004, Psl0567, Psl0573, Psl00574, Psl0582, Psl0584, Psl0585, SEQ ID NO: 52); ADWDHKHALDI (Psl0337, SEQ ID NO: 270) and QDSSSWPLT (Psl0337, SEQ ID NO: 283); ATDEADHALDI (Psl0170, SEQ ID NO: 271) and SQSDTFPLT (Psl0170, SEQ ID NO: 284); ADWSGTRALDI (Psl0304, SEQ ID NO: 272) and GQSDAFPLT (Psl0304, SEQ ID NO: 285); GLPEKPHALDI (Psl0348, SEQ ID NO: 273) and (Psl0348, SEQ ID NO: 286); SLFTDDHALDI (PslO573, SEQ ID NO: 274) and SEQ ID NO: 52; ASPGVVHALDI (Psl0574, SEQ ID NO: 275) and SEQ ID NO: 52; AHIESHHALD1 (Psl0582, SEQ ID NO: 276) and SEQ ID NO: 52; ATQAPAHALDI (Psl0584, SEQ ID NO: 277) and SEQ ID NO: 52; SQHDLEHALDI (PslO585, SEQ ID NO: 278) and SEQ ID NO: 52; or AMPDMPHALDI (Psl0589, SEQ ID NO: 279) and QQSLEFPLT (Psl0589, SEQ ID NO: 287).
[0171] Certain embodiments provide an isolated binding molecule, for example, an antibody or antigen binding fragment thereof that specifically binds to Pseudomonas Psl, which comprises an immunoglobulin VH and an immunoglobulin VL, where VH comprises QVQLQESGPGLVKPSETLSLTCTVSGGSISPYYWTWIRQPPGKX1LELI GYIHSSGYTDYNPSLKSRVTISGDTSKKQFSLKLSSVTAADTAVYYCA RADWDRLRALDIWGQGTMVTVSS, wherein XI is G or C (Psl0096, SEQ ID NO: 288), and the VL comprises DIQLTQSPSSLSASVGDRVTITC RASQSIRSHLNWYQQKPGKAPKLLIYGASNLQSGVPSRFSGSGSGTDF TLTISSLQPEDFATYYCQQSTGAWNWFGX2GTKVEIK, wherein X2 is G or C (Psl0096, SEQ ID NO: 289); wherein the VH comprises QVQLQESGPGLVKPSETLSLTCTVSGGSISPYYWTWIRQPPGKGLELIG YIHSSGYTDYNPSLKSRVTISGDTSKKQFSLKLSSVTAADTAVYYCAR AMDIEPHALDIWGQGTMVTVSS (Psl0225, SEQ ID NO: 290), and the VL comprises DIQLTQSPSSLSASVGDRVTITCRASQSIRSHLNWYQQKP GKAPKLLIYGASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QSDDGFPNFGGGTKVEIK (Psl0225, SEQ ID NO: 291); wherein the VH comprises QVQLQESGPGLVKPSETLSLTCTVSGGSISPYYWTWIRQ PPGKGLELIGYIHSSGYTDYNPSLKSRVTISGDTSKKQFSLKLSSVTAA DTAVYYCARADDPFPGYLDIWGQGTMVTVSS (Psl0588, SEQ ID NO: 292), and the VL comprises DIQLTQSPSSLSASVGDRVTITCRASQSI RSHLNWYQQKPGKAPKLLIYGASNLQSGVPSRFSGSGSGTDFTLTISS LQPEDFATYYCQQSDTFPLKFGGGTKVEIK (PslO588, SEQ ID NO: 293); where VH comprises QVQLQESGPGLVKPSETLSLTCTVSG GSISPYYWTWIRQPPGKGLELIGYIHSSGYTDYNPSLKSRVTISGDTSK KQFSLKLSSVTAADTAVYYCARADWNEGRKLDIWGQGTMVTVSS (SEL05); herein VH comprises QVQLQESGPGLVKPSETLSLTCT VSGGSISPYYWTWIRQPPGKGLELIGYIHSSGYTDYNPSLKSRVTISGD TSKKQFSLKLSSVTAADTAVYYCARADWDHKHALDIWGQGTMVTV SS (Psl0337, SEQ ID NO: 295), and the VL comprises DIQLTQSPSSLSASVGDRVTITCRASQSIRSHLNWYQQKPGKAPKLLIY GASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQDSSSWPLTF GGGTKVEIK (Psl0337, SEQ ID NO: 296); wherein the VH comprises EVQLLESGPGLVKPSETLSLTCNVAGGSISPYYWTWIRQPPGKGLELIG YIHSSGYTDYNPSLKSRVTISGDTSKKQFSLHVSSVTAADTAVYFCAR ATDEADHALDIWGQGTLVTVSS (Psl0170, SEQ ID NO: 297), and the VL comprises EIVLTQSPSSLSTSVGDRVTITCRASQSIRSHLNWYQQK PGKAPKLLIYGASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC SQSDTFPLTFGGGTKLEIK (Psl0170, SEQ ID NO: 298); wherein the VH comprises EVQLLESGPGLVKPSETLSLTCNVAGGSISPYYWTWIRQ PPGKGLELIGYIHSSGYTDYNPSLKSRVTISGDTSKKQFSLHVSSVTAA DTAVYFCARADWSGTRALDIWGQGTLVTVSS (Psl0304, SEQ ID NO: 299), and the VL comprises EIVLTQSPSSLSTSVGDRVTITCWASQS IRSHLNWYQQKPGKAPKLLIYGASNLQSGVPSRFSGSGSGTDFTLTISS LQPEDFATYYCGQSDAFPLTFGGGTKLEIK (Psl0304, SEQ ID NO: 300); wherein the VH comprises EVQLLESGPGLVKPSETLSLTCNVA GGSISPYYWTWIRQPPGKGLELIGYIHSSGYTDYNPSLKSRVTISGDTS KKQFSLHVSSVTAADTAVYFCARGLPEKPHALDIWGQGTLVTVSS (Psl0348, SEQ ID NO: 301), and the VL comprises EIVLTQSPSSLSTSVGDRVTITCRASQSIRSHLNWYQQKPGKAPKLLIY GASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQGDLWPLTF GGGTKLEIK (Psl0348, SEQ ID NO: 302); where VH comprises EVQLLESGPGLVKPSETLSLTCNVAGGSISPYYWTWIRQPPGKGLELIG YIHSSGYTDYNPSLKSRVTISGDTSKKQFSLHVSSVTAADTAVYFCAR SLFTDDHALDIWGQGTLVTV3: SEQ: NOQ: 11; where VH comprises EVQLLESGPGLVKPSETLSLTCNVAGGSISPYYWTWIRQPPGKGLELIG YIHSSGYTDYNPSLKSRVTISGDTSKKQFSLHVSSVTAADTAVYFCAR ASPGVVHALDIWGQGTLVTVSS: SEQ: NOQ: 11Q; where the VH comprises EVQLLESGPGLVKPSETLSLTCNVAGGSISPYYWTWIRQPPGKGLELIG YIHSSGYTDYNPSLKSRVTISGDTSKKQFSLHVSSVTAADTAVYFCAR AHIESHHALDIWGQGTLVTVSS (SEQ: NOQ); where VH comprises EVQLLESGPGLVKPSETLSLTCNVAGGSISPYYWTWIRQPPGKGLELIG YIHSSGYTDYNPSLKSRVTISGDTSKKQFSLHVSSVTAADTAVYFCAR ATQAPAHALDIWGQGTLVTV6 (SEQ: NOQQ: 58) where VH comprises EVQLLESGPGLVKPSETLSLTCNVAGGSISPYYWTWIRQPPGKGLELIG YIHSSGYTDYNPSLKSRVTISGDTSKKQFSLHVSSVTAADTAVYFCAR SQHDLEHALDIWGQGTLVTV7 (SEL): SEQQQQQQ: PSL05: or wherein the VH comprises EVQLLESGPGLVKPSETLSLTCNVAGGSISPYYWTWIRQPPGKGLELIG YIHSSGYTDYNPSLKSRVTISGDTSKKQFSLHVSSVTAADTAVYFCAR AMPDMPHALDIWGQGTLVTVSS (Psl0589, SEQ ID NO: 308), and the VL comprises EIVLTQSPSSLSTSVGDRVTITCRASQSIRSHLNWYQQKP GKAPKLLIYGASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ QSLEFPLTFGGGTKLEIK (Psl0589, SEQ ID NO: 325).
[0172] Revealed is also a single antibody Fv fragment of isolated antibody (ScFv) that specifically binds to Psse of Pseudomonas (an "anti-Psl ScFv"), which comprises the formula VH-L-VL or alternatively VL -L-VH, where L is a sequence of ligands. In certain respects the linker may comprise (a) [GGGGS] n, where n is 0, 1, 2, 3, 4, or 5, (b) [GGGG] n, where n is 0, 1, 2, 3, 4, or 5, or a combination of (a) and (b). For example, an exemplary linker comprises: GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 326). In certain embodiments, the linker further comprises the amino acids ala-leu at the C-terminus of the linker. In certain embodiments, the anti-Psl ScFv comprises the amino acid sequence of SEQ ID NO: 240, SEQ ID NO: 241, SEQID NO: 242, SEQIDNO: 243, SEQIDNO: 244, SEQIDNO: 245, SEQIDNO: 246, SEQIDNO: 247 , SEQIDNO: 248, SEQIDNO: 249, SEQIDNO: 250, SEQIDNO: 251, SEQIDNO: 252, SEQIDNO: 253, SEQIDNO: 254 or SEQ ID NO: 262.
[0173] Revealed is also a single antibody Fv fragment of isolated antibody (ScFv) that specifically binds to Pseudomonas PcrV (an "anti-PcrV ScFv"), which comprises the formula VH-L-VL or alternatively VL -L-VH, where L is a sequence of ligands. In certain respects the linker may comprise (a) [GGGGS] n, where n is 0, 1, 2, 3, 4, or 5, (b) [GGGG] n, where n is 0, 1, 2, 3, 4, or 5, or a combination of (a) and (b). For example, an exemplary linker comprises: GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 326). In certain embodiments, the linker further comprises the ala-leu amino acids at the C-terminus of the linker.
[0174] Revealed is also an isolated binding molecule, for example, an antibody or antigen binding fragment thereof which specifically binds to Pseudomonas PcrV which comprises a heavy chain variable region (VH) amino acid sequence and / or immunoglobulin light chain (VL) variable region at least 80%, 85%, 90% 95% or 100% identical to SEQ ID NO: 216 or SEQ ID NO: 217.
[0175] Additionally disclosed is an isolated binding molecule, for example, an antibody or antigen binding fragment thereof that specifically binds to PcrV of Pseudomonas comprising a VH, wherein one more of the VHCDR1, VHCDR2 or VHCDR3 regions of the VH are identical to or identical with the exception of four, three, two or an amino acid substitution, to one or more amino acid sequences of VHCDR1, VHCDR2 and / or VHCDR3 heavy chain reference of one or more of: SEQ ID NOs: 218 -220 as shown in Table 3. Thus, according to this modality, the VH comprises one or more of a VHCDR1, VHCDR2, or VHCDR3 identical to or identical with the exception of four, three, two or an amino acid substitution to one or more of the VHCDR1, VHCDR2 or VHCDR3 amino acid sequences shown in Table 3.
[0176] Further provided is an isolated binding molecule, for example, an antibody or antigen binding fragment thereof that specifically binds to PcrV of Pseudomonas comprising a VL, wherein one or more of the VLCDR1, VLCDR2 or VLCDR3 regions of the VL are identical to or identical except for four, three, two or an amino acid substitution for one or more amino acid sequences of VLCDR1, VLCDR2 and / or VLCDR3 heavy chain reference of one or more of: SEQ ID NOs: 221-223 as shown in Table 3. Thus, according to this modality the VL comprises one or more of a VLCDR1, VLCDR2 or VLCDR3 identical to or identical with the exception of four, three, two or an amino acid substitution to one or more of the amino acid sequences of VLCDR1, VLCDR2 or VLCDR3 shown in Table 3.
[0177] Also provided is an isolated binding molecule, for example, an antibody or antigen binding fragment thereof that specifically binds to Pseudomonas PcrV comprising a VH and a VL, where the VH comprises a selected amino acid sequence from the group consisting of SEQ ID NO: 255 and SEQ ID NO: 257, and where the VL comprises the amino acid sequence of SEQ ID NO: 256.
[0178] Further provided is an isolated binding molecule, for example, an antibody or antigen binding fragment thereof that specifically binds to PcrV of Pseudomonas comprising a VH and a VL, each of which comprises a CDR.1, CDR2, and CDR3, where CDR1 DE VH is (a) SYAMS (SEQ ID NO: 311) or a variant thereof comprising 1, 2, 3 or 4 conservative amino acid substitutions, CDR2 DE VH is AISGSGYSTYYADSVKG (SEQ ID NO: 312) or a variant thereof comprising 1, 2, 3 or 4 conservative amino acid substitutions, and VHCDR3 is EYSISSNYYYGMDV (SEQ ID NO: 313) or a variant thereof comprising 1, 2, 3 or 4 conservative substitutions for amino acid; or (b) where CDR1 DE VL is WASQGISSYLA (SEQ ID NO: 314) or a variant thereof comprising 1, 2, 3 or 4 conservative amino acid substitutions, CDR2 DE VL is AASTLQS (SEQ ID NO: 315) or a variant thereof comprising 1, 2, 3 or 4 conservative amino acid substitutions, and CDR3 DE VL is QQLNSSPLT (SEQ ID NO: 316) or a variant thereof comprising 1, 2, 3 or 4 conservative amino acid substitutions; or (c) a combination of (a) and (b); in which the VH and VL CDRs are in accordance with the Kabat numbering system. In certain aspects of this modality, (a) VH comprises an amino acid sequence of at least 80%, 85%, 90%, 95%, 96%, 97%, 98% 99%, or 100% identical to SEQ ID NO: 317, (b) the VL comprises an amino acid sequence of at least 80%, 85%, 90%, 95%, 96%, 97%, 98% 99%, or 100% identical to SEQ ID NO: 318; or (c) a combination of (a) and (b).
[0179] Revealed is also an isolated bispecific binding molecule, for example, a bispecific antibody or antigen binding fragment thereof that specifically binds both Pseudomonas Psl and Pseudomonas PcrV that comprises a variable region amino acid sequence immunoglobulin heavy chain (VH) and / or light chain variable (VL) region at least 80%, 85%, 90% 95% or 100% identical to SEQ ID NO: 228, SEQ ID NO: 229, or SEQ ID NO: 235.
[0180] In certain embodiments, a bispecific antibody as disclosed in this document has the structure of BS1, BS2, BS3, or BS4, all as shown in Figure 17. In certain bispecific antibodies disclosed in this document, the binding domain that is specifically binds the Psl of Pseudomonas comprises an anti-Psl ScFv molecule. In other respects the binding domain that specifically binds to Psl of Pseudomonas comprises a conventional heavy and light chain. Similarly in certain bispecific antibodies disclosed herein, the binding domain that specifically binds to Pseudomonas PcrV comprises an anti-PcrV ScFv molecule. In other respects the binding domain that specifically binds to Pseudomonas PcrV comprises a conventional heavy and light chain.
[0181] In certain respects a bispecific antibody as disclosed in this document had the structure of BS4, revealed in detail in Provisional Application No. US 61 / 624,651, filed on April 16, 2012 and in International Application No. PCT / US2012 / 63639, filed November 6, 2012 (legal registration number AEMS-115WO1, entitled “MULTISPECIFIC AND MULTIVALENT BINDING PROTEINS AND USES THEREOF”), which are incorporated in this document as a reference in their entirety. For example, this disclosure provides a bispecific antibody in which an anti-Psl ScFv molecule is inserted into the hinge region of each heavy chain of an anti-PcrV antibody or fragment thereof.
[0182] This disclosure provides an isolated binding molecule, for example, a bispecific antibody comprising an antibody heavy chain and an antibody light chain, wherein the antibody heavy chain comprises the formula VH-CH1-H1-L1- S-L2-H2-CH2-CH3, where CHI is a heavy chain constant region domain 1, H1 is a first heavy chain hinge region fragment, LI is a first linker, S is a ScFv molecule of anti-PcrV, L2 is a second ligand, H2 is a second heavy chain hinge region fragment, CH2 is a heavy chain constant region 2 domain and CH3 is a heavy chain constant region domain 3. In certain respects , VH comprises the amino acid sequence of SEQ ID NO: 255, SEQ ID NO: 257 or SEQ ID NO: 317. In certain aspects LI and L2 are the same or different and independently comprise (a) [GGGGS] n, where n is 0, 1, 2, 3, 4, or 5, (b) [GGGG] n, where n is 0, 1, 2, 3, 4, or 5 or a combination of (a) and (b). In certain embodiments, H1 comprises EPKSC (SEQ ID NO: 320), and H2 comprises DKTHTCPPCP (SEQ ID NO: 321).
[0183] In certain respects, S comprises an anti-Psl ScFv molecule that has the amino acid sequence of SEQ ID NO: 240, SEQID NO: 241, SEQIDNO: 242, SEQIDNO: 243, SEQIDNO: 244, SEQID NO: 245 , SEQIDNO: 246, SEQIDNO: 247, SEQIDNO: 248, SEQIDNO: 250, SEQIDNO: 250, SEQIDNO: 251, SEQIDNO: 252, SEQIDNO: 253, SEQ ID NO: 254 or SEQ ID NO: 262, or any combination of two or more of these amino acid sequences.
[0184] In additional aspects, CH2-CH3 comprises (SEQ ID NO: 322), where XI is M or Y, X2 is S or T, and X3 is T or E. In additional aspects the antibody light chain comprises VL -CL, where CL is an antibody light chain kappa constant region or an antibody light chain lambda constant region. In additional aspects, VL comprises the amino acid sequence of SEQ ID NO: 256 or SEQ ID NO: 318. CL can comprise, for example, the amino acid sequence of SEQ IDNO: 323.
[0185] Additionally provided is an isolated binding molecule, for example, a bispecific antibody that specifically binds to both Pseudomonas Psl and Pseudomonas PcrV comprising a VH comprising the amino acid sequence SEQ ID NO: 264, and a VL comprising the amino acid sequence SEQ ID NO: 263.
[0186] In some embodiments, the bispecific antibodies of the invention may be a single chain Fv fragment in tandem, which contains two different scFv fragments (i.e., V2L2 and W4) covalently linked together by a linker (for example, a binding polypeptide). (Ren-Heidenreich et al. Cancer 700: 1095 to 1,103 (2004); Kom et al. J Gene Med 6: 642 to 651 (2004)). In some embodiments, the linker may contain, or be, all or part of a heavy chain polypeptide constant region such as a CHI domain. In some embodiments, the two antibody fragments can be covalently linked together by means of a polyglycine-serine or polyserine-glycine linker as described, for example, in Patents Nos. 7,112,324 and 5,525,491, respectively, methods for generating bispecific tandem scFv antibodies are described in, for example, Maletz et al. Int J Cancer 93: 409 to 416 (2001); and Honemann et al. Leukemia 18: 636 to 644 (2004). Alternatively, the antibodies can be "linear antibodies" as described in, for example, Zapata et al. Protein Eng. 8: 1,057 to 1,062 (1995). Briefly, these antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) that form a pair of antigen binding regions.
[0187] The disclosure also encompasses variant forms of bispecific antibodies such as the tetravalent double variable immunoglobulin molecules (DVD-Ig) described in Wu et al. (2007) Nat Biotechnol 25 (11): 1,290 to 1,297. DVD-Ig molecules are designed so that two variable light chain (VL) domains of two different relative antibodies are linked in tandem directly or by means of a short ligand by sets of recombinant DNA procedures, followed by the constant light chain domain . For example, the DVD-Ig light chain polypeptide can contain in tandem: (a) the VL of V2L2; and (b) the VL of WapR-004. Similarly, the heavy chain comprises the two different tandem-linked heavy chain (VH) domains, followed by the constant domain CHI and the Fc region. For example, the DVD-Ig heavy chain polypeptide may contain in tandem: (a) V2L2 VH; and (b) the VH of WapR-004. In that case, the expression of the two chains in a cell results in a heterotetramer that contains four antigen combining sites, two that specifically bind to V2L2 and two that specifically bind to Psl . Methods for generating DVD-Ig molecules from two related antibodies are further described in, for example, PCT Publications No. WO 2008/024188 and WO 2007/024715.
[0188] In certain embodiments, an isolated binding molecule, for example, an antibody or antigen binding fragment thereof as described in this document specifically binds to Pseudomonas Psl and / or PcrV with an affinity characterized by a dissociation constant (KD) 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, IO'8 M, 5 x 10'9 M, 10 ' 9 M, 5 x 10'10 M, 10'10 M, 5 x 10'11 M, 10'11 M, 5 x 10'12 M, 10'12 M, 5 x 10'13 M, 10'13 M , 5 x 10'14 M, 10'14 M, 5 x 10'15 M or 10 ', 5M.
[0189] In specific embodiments, an isolated binding molecule, for example, an antibody or antigen binding fragment thereof as described in this document specifically binds to Psl of Pseudomonas and / or PcrV, with an affinity characterized by a constant dissociation (KD) in a range of about 1 x 10'10 to about 1 x 10'6 M. In one embodiment, an isolated binding molecule, for example, an antibody or antigen binding fragment thereof as described in this document specifically binds to Psl of Pseudomonas and / or PcrV, with an affinity characterized by a KD of about 1.18 x 10 'M, as determined by the OCTET1 binding assay described in this document. In another embodiment, a isolated binding molecule, for example, an antibody or antigen binding fragment thereof as described herein binds specifically to Psl of Pseudomonas and / or PcrV, with an affinity characterized by a KD of about 1.44 x 10'7 M, as determined by the OCTET® binding assay described in this document.
[0190] Some embodiments include the isolated binding molecule for example, an antibody or fragment thereof as described above, which (a) may inhibit attachment of Pseudomonas aeruginosa to epithelial cells, (b) may promote P. aeruginosa OPK or (c ) can inhibit attachment of P. aeruginosa to epithelial cells and can promote OPK of P. aeruginosa.
[0191] In some embodiments, the isolated binding molecule, for example, an antibody or fragment thereof as described above, in which the maximum inhibition of P. aeruginosa attachment to epithelial cells is achieved at an antibody concentration of about 50 μg / ml or less, 5.0 μg / ml or less, or about 0.5 μg / ml or less, or at an antibody concentration in the range of about 30 μg / ml to about 0.3 μg / ml or at an antibody concentration of about 1 μg / ml, or at an antibody concentration of about 0.3 μg / ml.
[0192] Certain embodiments include the isolated binding molecule for example, an antibody or fragment thereof as described above, where the EC50 of OPK is less than about 0.5 μg / ml, less than about 0.05 μg / ml ml or less than about 0.005 μg / ml or where the ECK of OPK is in the range of about 0.001 μg / ml to about 0.5 μg / ml or where the EC50 of OPK is in the range of about from 0.02 μg / ml to about 0.08 μg / ml or where the ECK of OPK is in the range of about 0.002 μg / ml to about 0.01 μg / ml or where the EC50 of OPK is less than about 0.2 μg / ml or where the ECK of OPK is less than about 0.02 μg / ml. In certain embodiments, a Pseudomonas anti-Psl binding molecule, for example, antibody or fragment, variant or derivative described herein, specifically binds to the same Psl epitope as the 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 with the exception of a G98A amino acid substitution of the VH amino acid sequence of SEQ ID NO: 11.
[0193] Some embodiments include mutants of WapR-004 (W4) that comprise an amino acid sequence of the identical or identical scFv-Fc molecule with the exception of one, two, three, four, five or more amino acid substitutions to one or more than: 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, SEQID NO: 103, SEQID NO: 104, SEQID NO: 105, SEQID NO: 106, SEQID NO: 107, SEQID NO: 108, SEQID NO: 109, SEQID NO: 110, SEQID NO: 111, SEQID NO: 112, SEQID NO: 113, SEQID NO: 114, SEQID NO: 115, SEQID NO: 116, SEQID NO: 117, SEQID NO: 118, SEQID NO: 119, SEQID NO: 120, SEQID NO: 121, SEQ ID NO: 122, SEQID NO: 123, SEQID NO: 124, SEQID NO: 125, SEQID NO: 126, SEQID NO: 127, SEQID NO: 128, SEQID NO : 129, SEQ ID NO: 130, SEQID NO: 131, SEQID NO: 132, SEQID 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.
[0194] Other embodiments include mutants of WapR-004 (W4) that comprise an amino acid sequence of the scFv-Fc molecule 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, SEQID NO: 102, SEQID NO: 103 , SEQIDNO: 104, SEQIDNO: 105, SEQID NO: 106, SEQID NO: 107, SEQIDNO: 108, SEQIDNO: 109, SEQID NO: 110, SEQID NO: 111, SEQIDNO: 112, SEQIDNO: 113, SEQID NO : 114, SEQID NO: 115, SEQIDNO: 116, SEQID NO: 117, SEQID NO: 118, SEQID NO: 119, SEQIDNO: 120, SEQID NO: 121, SEQID NO: 122, SEQID NO: 123, SEQIDNO: 124, SEQ IDNO: 125, SEQID NO: 126, SEQID NO: 127, SEQIDNO: 128, SEQID NO: 129, SEQID NO: 130, SEQID NO: 131, SEQIDNO: 132, SEQ IDNO: 133, SEQID NO: 134, SEQID NO: 134 :1 35, SEQIDNO: 136, SEQIDNO: 137, SEQID NO: 138, SEQID NO: 139, SEQIDNO: 140, SEQIDNO: 141, SEQID NO: 142, SEQID NO: 143, SEQIDNO: 144, SEQID NO: 145; or SEQ ID NO: 146.
[0195] In some embodiments, an anti-Psl binding molecule of Pseudomonas, for example, antibody or fragment, variant or derivative described herein, specifically binds to the same epitope as the monoclonal antibody WapR-001, WapR-002 , or WapR-003 or will competitively inhibit such a monoclonal antibody from binding to Psl dePseudomonas.
[0196] In certain embodiments, an anti-Psl binding molecule of Pseudomonas, for example, antibody or fragment, variant or derivative described herein, specifically binds to the same epitope as the monoclonal antibody WapR-016 or will competitively inhibit such a monoclonal antibody binds to Psl from Pseudomonas. TABLE 2: Reference VH and VL amino acid sequences *

* The VH and CDR1 DE VL, CDR2 and CDR3 amino acid sequences are underlinedTable 3: Reference VH and CDR1 DE VL, CDR2 and CDR3 amino acid sequences

[0197] In certain embodiments, an anti-PcrV of Pseudomonas binding molecule, for example, antibody or fragment, variant or derivative described herein, specifically binds to the same PcrV epitope as the V2L2 monoclonal antibody and / or will inhibit such a monoclonal antibody to bind Pseudomonas PcrV competitively.
[0198] For example, in certain aspects, the Pseudomonas anti-PcrV binding molecule, for example, antibody or fragment, variant or derivative thereof, comprises V2L2-GL and / or V2L2-MD.
[0199] In certain embodiments, a Pseudomonas anti-PcrV binding molecule, for example, antibody or fragment, variant or derivative thereof described herein, specifically binds to the same PcrV epitope as 29D2 monoclonal antibody and / or will inhibit such monoclonal antibody to bind to Pseudomonas PcrV competitively.
[0200] Any PcrV and / or Psl binding molecule of Pseudomonas and / or, for example, antibodies or fragments, variants or derivatives thereof described herein can additionally include additional polypeptides, for example, a signal peptide to direct secretion of the encoded polypeptide, antibody constant regions as described herein, or other heterologous polypeptides as described herein. In addition, the binding molecules or fragments thereof of the description include polypeptide fragments as described elsewhere. In addition, anti-Pseudomonas Psl and / or PcrV binding molecules, for example, antibodies or fragments, variants or derivatives thereof described herein may be fusion polypeptides, Fab fragments, scFvs or other derivatives, as described herein .
[0201] Furthermore, as described in more detail elsewhere in this document, the disclosure includes compositions comprising Psl and / or PcrV binding molecules of anti-Pseudomonas, for example, antibodies or fragments, variants or derivatives thereof. in this document.
[0202] It should also be understood by one skilled in the art that Psl and / or PcrV binding molecules of anti-Pseudomonas, for example, antibodies or fragments, variants or derivatives thereof described in this document can be modified so that they vary in amino acid sequence from the naturally occurring binding polypeptide from which they were derived. For example, an amino acid or polypeptide sequence derived from a designated protein can be similar, for example, to have a certain percentage identity to the initial sequence, for example, it can be 60%, 70%, 75%, 80%, 85%, 90% or 95% identical to the initial sequence.
[0203] As known in the art, the "sequence identity" between two polypeptides is determined by comparing the amino acid sequence of one polypeptide with the sequence of a second polypeptide. When discussed in this document, whether any particular polypeptide is at least about 70%, 75%, 80%, 85%, 90% or 95% identical to another polypeptide can be determined using computer / software methods and programs known in the art such as, but without limitation, the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 5371 1). BESTFIT uses Smith and Waterman's local homology algorithm, Advances in Applied Mathematics 2: 482 to 489 (1981), to find the best homology segment between two sequences. When using BESTFIT or any other sequential alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence, the parameters are, of course, established so that the percentage of identity is calculated over the entire 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.
[0204] The percentage of “sequence identity” can also be determined by comparing two sequences favorably aligned over a comparison window. In order to favorably align the sequences for comparison, the portion of a polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions called gaps as long as the reference sequence is kept constant. A favorable alignment is an alignment that, even with gaps, produces the largest possible number of “identical” positions between the reference and comparison sequences. The percentage of “sequence identity” between two sequences can be determined using the version of the “BLAST 2 Sequences” program that was available from the National Center for Biotechnology Information on September 1, 2004, whose program incorporates the BLASTN programs ( for nucleotide sequence comparison) and BLASTP (for polypeptide sequence comparison), whose programs are based on the Karlin and Altschul algorithm (Proc. Natl. Acad. Sci. USA 90 (12): 5873 to 5877, 1993). When using “BLAST 2 Sequences,” parameters that were standard parameters on September 1, 2004, can be used for word size (3), open gap penalty (11), extension gap penalty (1), disappearance of gap (50), expected value (10) and any other required parameters including but not limited to, matrix option.
[0205] In addition, nucleotide or amino acid substitutions, deletions or insertions that result in conservative substitutions or changes in "non-essential" amino acid regions can be made. For example, a polypeptide or amino acid sequence derived from a designated protein may be identical to the initial sequence except for one or more individual amino acid substitutions, insertions or deletions, for example, 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 initial sequence.
[0206] An anti-Pseudomonas Psl and / or PcrV binding molecule, for example, an antibody or fragment, variant or derivative thereof described herein may comprise, consist essentially of or consist of a fusion protein. Fusion proteins are chimeric molecules that comprise, for example, an immunoglobulin antigen binding domain with at least one target binding site and at least one heterologous moiety, that is, a moiety with which it is not naturally bound by 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 created, for example, through chemical synthesis or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship.
[0207] The term "heterologous", as applied to a polynucleotide, polypeptide or other chemical moiety means that the polynucleotide, polypeptide or other chemical moiety is derived from an entity other than that of the rest of the entity to which it is being compared . In a non-limiting example, a "heterologous polypeptide" to be fused to a binding molecule, for example, an antigen or antibody binding derivative, variant or fragment thereof is derived from a non-immunoglobulin polypeptide of the same species or an immunoglobulin or non-immunoglobulin polypeptide of a different species. IV. ANTIBODY CONJUGATES AND FUSION PROTEINS
[0208] In some embodiments, the Psl and / or PcrV binding molecules of anü-Pseudomonas, for example, antibodies or fragments, variants or derivatives thereof, can be administered multiple times in conjugated form. In yet another embodiment, the Psl and / or PcrV binding molecules of anti-Pseudomonas, for example, antibodies or fragments, variants or derivatives thereof, can be administered in the unconjugated form, then in the conjugated form or vice versa.
[0209] In specific embodiments, the Psl and / or PcrV binding molecules of anü-Pseudomonas, for example, 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 the treatment of gram-negative infections resistant to multiple drugs. In addition to its bactericidal activity, PMB binds lipopolysaccharide (LPS) and neutralizes its pro-inflammatory effects. (Dixon, R.UM. & Chopra, I. J Antimicrob Chemother 18, 557 to 563 (1986)). LPS is believed to contribute significantly to inflammation and the onset of gram-negative sepsis. (Guidet, B., et al., Chest 106, 1194-1201 (1994)). The PMB conjugates for carrier molecules have been shown to neutralize LPS and mediate protection in animal models of endotoxemia and infection. (Drabick, J.J., et al. Antimicrob Agents Chemother 42, 583 to 588 (1998)). Also disclosed is a method for attaching one or more PMB molecules to cysteine residues introduced into the Fc region of monoclonal antibodies (mAb) of the disclosure. For example, Cam-003-PMB conjugates retained specific mAb-mediated binding to P. aeruginosa and also retained OPK activity. In addition, the mAb-PMB conjugates bound and neutralized LPS in vitro. In specific embodiments, the Psl and / or PcrV binding molecules of anti-Pseudomonas, for example, antibodies or fragments, variants or derivatives thereof, can be combined with antibiotics (for example, Ciprofloxacin, Meropenem, Tobramycin, Aztreonam).
[0210] In certain embodiments, an anti-Pseudomonas Psl and / or PcrV binding molecule, for example, an antibody or fragment, variant or derivative thereof described herein may comprise a heterologous amino acid sequence or one or more others chemical portions not commonly associated with an antibody (for example, an antimicrobial agent, a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a lipid, a biological response modifier, a pharmaceutical agent, a lymphokine, a antibody or heterologous fragment thereof, a detectable tag, polyethylene glycol (PEG) and a combination of two or more of any of said agents). In additional embodiments, an antiPseudomonas Psl and / or PcrV binding molecule, for example, an antibody or fragment, variant or derivative thereof, may comprise a detectable tag selected from the group consisting of an enzyme, a fluorescent tag, a chemiluminescent tag, a bioluminescent tag, a radioactive tag or a combination of two or more of any of said detectable tags. V. POLYNUCLEOTIDE CODING BINDING MOLECULES
[0211] Nucleic acid molecules encoding the anti-Pseudomonas Psl and / or PcrV binding molecules, for example, antibodies or fragments, variants or derivatives thereof, are also provided herein. .
[0212] One embodiment provides an isolated polynucleotide comprising, consisting essentially of or consisting of a nucleic acid encoding an immunoglobulin heavy chain (VH) variable region amino acid sequence of 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, SEQ ID NO: 74 or SEQ ID NO: 216 as shown in Table 2.
[0213] One embodiment provides an isolated polynucleotide comprising, consisting essentially of or consisting of a nucleic acid encoding an immunoglobulin heavy chain (VH) variable region amino acid sequence of SEQ ID NO: 257 or SEQ ID NO : 259. For example the nucleic acid sequences of SEQ ID NO: 261 and SEQ ID NO :: 259, respectively.
[0214] Another embodiment provides an isolated polynucleotide comprising, consisting essentially of or consisting of a nucleic acid encoding a VH amino acid sequence identical to, or identical except for one, two, three, four, five or more substitutions of amino acid for one or more of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 74 or SEQ ID NO: 216 as shown in Table 2.
[0215] The additional modality provides an isolated polynucleotide comprising, consisting essentially of or consisting of a nucleic acid encoding a VH, in which one or more of the VHCDR1, VHCDR2 or VHCDR3 regions of the VH are identical to, or identical except by four, three, two or an amino acid substitution, for one or more VHCDR1, VHCDR2 and / or VHCDR3 heavy chain reference amino acid sequences of one or more of: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 74, or SEQ ID NO: 216 as shown in Table 2.
[0216] Another embodiment provides an isolated polynucleotide comprising, consisting essentially of or consisting of a nucleic acid encoding an isolated binding molecule, for example, an antibody or antigen binding fragment thereof that specifically binds to Psl of Pseudomonas comprising a VH, where one or more of the VHCDR1, VHCDR2 or VHCDR3 regions of the VH are identical to, or identical except for four, three, two or an amino acid substitution, for one or more amino acid heavy chain sequences of reference VHCDR1, VHCDR2 and / or VHCDR3 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.
[0217] An additional embodiment provides an isolated binding molecule, for example, an antibody or antigen binding fragment comprising the VH encoded by the polynucleotide that specifically or preferentially binds to the Psl and / or PcrV of Pseudomonas.
[0218] Another embodiment provides an isolated polynucleotide that comprises, which essentially consists of or consists of a nucleic acid encoding an immunoglobulin variable region (VL) amino acid sequence of at least 80%, 85%, 90% 95 % or 100% identical to one or more of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO : 14, SEQ ID NO: 16 or SEQ ID NO: 217 as shown in Table 2.
[0219] Another embodiment provides an isolated polynucleotide comprising, consisting essentially of or consisting of a nucleic acid encoding the immunoglobulin light chain variable region (VL) amino acid sequence of SEQ ID NO: 256, for example, the nucleic acid sequence SEQ ID NO: 260.
[0220] An additional embodiment provides 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 substitutions amino acid for one or more of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 217 as shown in Table 2.
[0221] 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 VLCDR1, VLCDR2 or VLCDR3 regions of the VL are at least 80%, 85% , 90%, 95% or 100% identical to one or more VLCDR1, VLCDR2 or VLCDR3 light chain reference sequences of one or more of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 , SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 16, or SEQ ID NO: 217 as shown in Table 2.
[0222] An additional embodiment provides an isolated polynucleotide comprising, consisting essentially of or consisting of a nucleic acid encoding an isolated binding molecule, for example, an antibody or antigen binding fragment thereof that specifically binds to Psl of Pseudomonas comprising a VL, where one or more of the VLCDR1, VLCDR2 or VLCDR3 regions of the VL are identical to or identical except for four, three, two, or an amino acid substitution, for one or more amino acid sequences VLCDR1, VLCDR2 and / or VLCDR3 reference heavy chain of one or more of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 , SEQ ID NO: 14, SEQ ID NO: 16 or SEQ ID NO: 217 as shown in Table 2.
[0223] In another embodiment, isolated binding molecules, for example, an antibody or antigen binding fragment comprising the VL encoded by the polynucleotide specifically or preferentially bind to the Psl and / or PcrV of Pseudomonas.
[0224] One embodiment provides an isolated polynucleotide comprising, consisting essentially of or consisting of a nucleic acid encoding 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.TABLE 4: Reference scFv nucleic acid sequences




[0225] 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 and / or PcrV, comprises, essentially consists of or consists of sequences of amino acids VH and VL at least 80%, 85%, 90%, 95% or 100% identical to: (a) SEQ ID NO: 1 and SEQ ID NO: 2, respectively, (b) SEQ ID NO: 3 and SEQ ID NO: 2, respectively, (c) SEQ ID NO: 4 and SEQ ID NO: 2, respectively, (d) SEQ ID NO: 5 and SEQ ID NO: 6, respectively, (e) SEQ ID NO: 7 and SEQ ID NO: 8, respectively, (f) SEQ ID NO: 9 and SEQ ID NO: 10, respectively, (g) SEQ ID NO: 11 and SEQ ID NO: 12, respectively, (h) SEQ ID NO: 13 and SEQ ID NO: 14, respectively; (i) SEQ ID NO: 15 and SEQ ID NO: 16, respectively; or (j) SEQ ID NO: 74 and SEQ ID NO: 12, respectively.
[0226] In certain embodiments, an isolated binding molecule, for example, an antibody or antigen binding fragment encoded by one or more of the polynucleotides described above, specifically binds to the Psl and / or PcrV of Pseudomonas with a characterized affinity by a dissociation constant (KD) not greater than 5 x 10'2 M, 10'2 M, 5 x 10 '' 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 x IO ' 9 M, 10'9 M, 5 x 10'10 M, 10'10 M, 5 x 10'11 M, 10'11 M, 5 x 10'12 M, 10'12 M, 5 x 10'13 M , 10'13 M, 5 x 10'14 M, 10'14 M, 5x 1 (T15M, OR 10'15M.
[0227] In specific embodiments, an isolated binding molecule, for example, an antibody or antigen binding fragment encoded by one or more of the polynucleotides described above, specifically binds to the Psl and / or PcrV of Pseudomonas, with an affinity characterized by a dissociation constant (KD) in a range of about 1 x 10'l (1 to about 1 x 10'6 M. In one embodiment, an isolated binding molecule, for example, an antibody or binding fragment antigen encoded by one or more of the polynucleotides described above, specifically binds to the Psl and / or PcrV of Pseudomonas, with an affinity characterized by a KD of about 1.18 x 10 'M, as determined by the OCTET binding assay ® described in this document In another embodiment, an isolated binding molecule, for example, an antibody or antigen binding fragment encoded by one or more of the polynucleotides described above, specifically binds to Psl and / or PcrV of Pseudomonas, with an affinity characterized by a KD of about 1.44 x 10 'M, as determined by the OCTET binding assay described in this document.
[0228] In certain embodiments, a Psl and / or PcrV binding molecule of anti-Pseudomonas, for example, antibody or fragment, variant or derivative thereof encoded by one or more of the polynucleotides described above, specifically binds to the same Psl epitope as a monoclonal antibody WapR-004, WapR-004RAD, Cam-003, Cam-004 or Cam-005 or will competitively inhibit such monoclonal antibody from binding to Pseudomonas' Psl, and / or specifically binds to the same PcrV epitope as antibody monoclonal V2L2, or will competitively inhibit such monoclonal antibody from binding to the PcrV of Pseudomonas. WapR-004RAD is identical to WapR-004 except for a G293C nucleic acid substitution of the VH nucleic acid sequence that encodes the VH amino acid sequence of SEQ ID NO: 11 (a nucleotide substitution in the VH coding portion of SEQ ID NO: 71 in position 317). The nucleic acid sequence encoding WapR-004RAD VH is shown as SEQ ID NO 76.
[0229] Some embodiments provide an isolated polynucleotide comprising, consisting essentially of or consisting of a nucleic acid encoding an amino acid sequence of a mutant scFv-Fc molecule identical to, or identical except for one, two, three, four , five or more amino acid substitutions for 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 IDNO: 102, SEQIDNO: 103, SEQID NO: 104, SEQID NO: 105, SEQIDNO: 106, SEQIDNO: 107, SEQID NO: 108, SEQID NO: 109, SEQID NO: 110, SEQIDNO: 111, SEQID NO: 112, SEQID NO: 113, SEQIDNO: 114, SEQIDNO: 115, SEQID NO: 116, SEQID NO: 117, SEQIDNO: 118, SEQIDNO: 119, SEQID NO: 120, SEQID NO: 121, SEQIDNO: 122, SEQ ID NO: 123, SEQI D NO: 124, SEQ ID NO: 125, SEQIDNO: 126, SEQ ID NO: 127, SEQID NO: 128, SEQ ID NO: 129, SEQIDNO: 130, SEQ ID NO: 131, SEQID NO: 132, SEQ ID NO : 133, SEQIDNO: 134, SEQ ID NO: 135, SEQID NO: 136, SEQ ID NO: 137, SEQIDNO: 138, SEQ ID NO: 139, SEQID NO: 140, SEQ ID NO: 141, SEQIDNO: 142, SEQ ID NO: 143, SEQID NO: 144, SEQ ID NO: 145; or SEQ ID NO: 146.
[0230] Other embodiments provide an isolated polynucleotide comprising, consisting essentially of or consisting of a nucleic acid encoding an amino acid sequence of a mutant scFv-Fc molecule W4 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 IDNO: 100, SEQ ID NO: 101 , SEQID NO: 102, SEQID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQID NO: 106, SEQID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQID NO: 110, SEQID NO : 111, SEQ IDNO: 112, SEQ ID NO: 113, SEQID NO: 114, SEQID NO: 115, SEQ IDNO: 116, SEQ ID NO: 117, SEQID NO: 118, SEQID NO: 119, SEQ IDNO: 120, SEQ ID NO: 121, SEQID NO: 122, SEQID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQID NO: 126, SEQID NO: 127, SE Q IDNO: 128, SEQ ID NO: 129, SEQID NO: 130, SEQID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQID NO: 134, SEQID NO: 135, SEQ IDNO: 136, SEQ ID NO : 137, SEQID NO: 138, SEQID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQID NO: 142, SEQID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145; or SEQ ID NO: 146.
[0231] One embodiment provides an isolated polynucleotide comprising, consisting essentially of or consisting of a nucleic acid encoding a mutant scFv-Fc molecule W4, wherein the nucleic acid is at least 80%, 85%, 90% 95 % or 100% identical to one or more of SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, or SEQ ID NO: 152, SEQ IS NO : 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQIDNO: 156, SEQ IDNO: 157, SEQ IDNO: 158, SEQ ID NO: 159, SEQIDNO: 160, SEQ IDNO: 161, SEQ IDNO: 162, SEQ ID NO: 163, SEQIDNO: 164, SEQ IDNO: 165, SEQ IDNO: 166, SEQ ID NO: 167, SEQIDNO: 168, SEQ IDNO: 169, SEQ IDNO: 170, SEQ ID NO: 171, SEQIDNO: 172, SEQ IDNO: 173, SEQ IDNO: 174, SEQ ID NO: 175, SEQIDNO: 176, SEQ IDNO: 177, SEQ IDNO: 178, SEQ ID NO: 179, SEQIDNO: 180, SEQ IDNO: 181, SEQ IDNO: 182, SEQ ID NO: 183, SEQIDNO: 184, SEQ IDNO: 185, SEQ IDNO: 186, SEQ ID NO: 187, SEQIDNO: 188, SEQ IDNO: 189, SEQ IDNO: 190, SEQ ID NO: 191, SEQIDNO: 192, SEQ IDNO: 193, SEQ IDNO: 194, SEQ ID NO: 195, SEQIDNO: 196, SEQ IDNO: 197, SEQ IDNO: 198, SEQ ID NO: 1 99, SEQIDNO: 200, SEQ IDNO: 201, SEQ IDNO: 202, SEQ ID NO: 203, SEQIDNO: 204, SEQ IDNO: 205, SEQ IDNO: 206, SEQ ID NO: 207, SEQIDNO: 208, SEQ IDNO: 209 , SEQ IDNO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214; or SEQ ID NO: 215.
[0232] One embodiment provides an isolated polynucleotide comprising, consisting essentially of or consisting of a nucleic acid encoding a V2L2 polypeptide, wherein the nucleic acid is at least 80%, 85%, 90% 95% or 100% identical to one or more of SEQ ID NO: 238 or SEQ ID NO: 239.
[0233] In other embodiments, an Psi and / or PcrV binding molecule of anü-Pseudomonas, for example, antibody or fragment, variant or derivative thereof encoded by one or more of the polynucleotides described above, specifically binds to the same epitope as monoclonal antibody WapR-001, WapR-002 or WapR-003, or will competitively inhibit such monoclonal antibody from binding to Pseudomonas Psl.
[0234] In certain embodiments, a Psl and / or PcrV binding molecule of anu-Pseudomonas, for example, antibody or fragment, variant or derivative thereof encoded by one or more of the polynucleotides described above, specifically binds to the same epitope as monoclonal antibody WapR-016, or will competitively inhibit such monoclonal antibody from binding to Pseudomonas Psl.
[0235] The disclosure also includes fragments of the polynucleotides as described elsewhere in this document. In addition, polynucleotides encoding fusion polynucleotides, Fab fragments and other derivatives, as described herein, are also provided.
[0236] Polynucleotides can be produced or manufactured using any method known in the art. For example, if the nucleotide sequence of the antibody is known, a polynucleotide that encodes the antibody can be assembled from chemically synthesized oligonucleotides (for example, as described in Kutmeier et al., BioTechniques 77: 242 (1994)), which, concisely, it involves the synthesis of overlapping oligonucleotides that contain portions of the antibody-encoding sequence, slowing down and agglutinating those oligonucleotides and then amplifying the PCR-linked oligonucleotides.
[0237] Alternatively, a polynucleotide encoding an Psi and / or PcrV binding molecule of anü-Pseudomonas, for example, antibody or fragment, variant or derivative thereof, can be generated from nucleic acid from a suitable source. If a clone that contains a nucleic acid that encodes a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid that encodes the antibody can be synthesized chemically or obtained from a suitable source (for example, an antibody cDNA library or a cDNA library generated from, or nucleic acid, preferably poly A + RNA, isolated from any tissue or cells that express the antibody or such as hybridoma cells selected to express an antibody) by amplification PCR using synthetic primers hybridizable to the 3 'and 5' ends of the sequence or cloning using an oligonucleotide probe specific to the particular gene sequence to identify, for example, a cDNA clone from a cDNA library encoding the antibody. The amplified nucleic acids generated by PCR can then be cloned into replicable cloning vectors using any method well known in the art.
[0238] Once the nucleotide sequence and corresponding amino acid sequence of an Psi and / or PcrV binding molecule of anü-Pseudomonas, for example, antibody or fragment, variant or derivative thereof, is determined, the nucleotide sequence thereof be manipulated using methods well known in the art for manipulating nucleotide sequences, for example, recombinant DNA techniques, site-directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd edition, 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 in this document in their entirety), to generate antibodies that have a different amino acid sequence, for example, to create substitutions, amino acid deletions and / or insertions.
[0239] A polynucleotide encoding a Ps1 and / or PcrV binding molecule of artà-Pseudomonas, for example, antibody or fragment, variant or derivative thereof, may be composed of any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, a polynucleotide that encodes an anti-Pseudomonas Psl and / or PcrV binding molecule, for example, antibody or fragment, variant or derivative thereof, may be composed of single and double stranded DNA, DNA that is a mixture of single strand and double strand regions, single and double strand RNA, and RNA which is a mixture of single and double strand regions, hybrid molecules comprising DNA and RNA that may be single stranded or, more typically, double stranded or a mixture of single and double filament regions. In addition, a polynucleotide encoding an anti-Pseudomonas Psl and / or PcrV binding molecule, for example, antibody or fragment, variant or derivative thereof, may be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. A polynucleotide that encodes an anti-Pseudomonas Psl and / or PcrV binding molecule, for example, antibody or fragment, variant or derivative thereof, may also contain one or more modified bases or modified DNA or RNA backbones for stability or for other reasons. "Modified" bases include, for example, tritilates and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically or metabolically modified forms.
[0240] An isolated polynucleotide encoding an unnatural variant of an immunoglobulin-derived polypeptide (for example, an immunoglobulin heavy chain portion or light chain portion) can be created by introducing one or more nucleotide substitutions, additions or deletions to the nucleotide sequence of the immunoglobulin so that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced using standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are made on one or more non-essential amino acid residues. VI.EXPRESSION OF ANTIBODY POLYPEPTIDS
[0241] As is well known, RNA can be isolated from the original hybridoma cells or from other transformed cells using standard techniques, such as guanidinium isothiocyanate extraction and precipitation followed by centrifugation or chromatography. Where desirable, mRNA can be isolated from total RNA using standard techniques such as chromatography on oligo dT cellulose. Suitable techniques are familiar in the art.
[0242] In one embodiment, cDNAs that encode the light and heavy chains of the Psi and / or PcrV binding molecule of anü-Pseudomonas, for example, antibody or fragment, variant or derivative thereof, can be made, both simultaneously and separately, with the use of reverse transcriptase and DNA polymerase according to well-known methods. PCR can be initiated via consensus constant region primers or by more specific primers based on published light and heavy chain DNA and amino acid sequences. As discussed above, PCR can also be used to isolate DNA clones that encode antibody light and heavy chains. In that case, the libraries can be protected by consensus primers or larger homologous probes, such as mouse constant region probes.
[0243] DNA, typically plasmid DNA, can be isolated from cells using techniques known in the art, with restriction mapped and sequenced according to well-known standard techniques set out in detail, for example, in the previous references related to DNA techniques recombinant. Of course, DNA can be synthetic according to the present disclosure at any point during the subsequent isolation or analysis process.
[0244] Following the manipulation of the isolated genetic material to provide an anti-Pseudomonas Psl and / or PcrV binding molecule, for example, antibody or fragment, variant or derivative thereof, the polynucleotides encoding Psl binding molecules and / or PcrV of anü-Pseudo models, are typically inserted into an expression vector for introduction into host cells that can be used to produce the desired amount of Psi and / or PcrV binding molecules of anü-Pseudomonas.
[0245] Recombinant expression of an antibody, or fragment, derivative or analog thereof, for example, an antibody heavy or light chain that binds to a target molecule described herein, for example, Psl and / or PcrV, requires the construction of an expression vector that contains a polynucleotide that encodes the antibody. Once a polynucleotide encoding an antibody molecule or 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 producing the antibody molecule can be produced using recombinant DNA technology using sets of procedures well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide that contains an antibody that encodes a nucleotide sequence are described herein. Methods that are well known to those skilled in the art can be used to construct expression vectors that contain appropriate antibody coding sequences and translational and transcriptional control signals. These methods include, for example, in vitro set of recombinant DNA procedures, sets of synthetic procedures and in vivo genetic recombination. The disclosure thus provides replicable vectors that comprise a nucleotide sequence that encodes 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 may include the nucleotide sequence encoding the constant region of the antibody molecule (see, for example, PCT Application No. WO 86/05807; PCT Application No. WO 89/01036; and U.S. Patent No. 5,122,464) and the variable domain of the antibody can be cloned into such a vector for expression of the entire heavy or light chain.
[0246] The term "vector" or "expression vector" is used herein to mean vectors used in accordance with the present disclosure as a vehicle for introducing and expressing a desired gene into a host cell. As known to those skilled in the art, such vectors can be easily selected from the group consisting of plasmids, phages, viruses and retroviruses. In general, vectors compatible with the present 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.
[0247] For the purposes of this disclosure, several expression vector systems can be employed. For example, a class of vector uses DNA elements that are derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retrovirus (RSV, MMTV or MOMLV) or SV40 virus. Others involve the use of polycistronic systems with internal ribosome binding sites. In addition, cells that integrated the DNA into their chromosomes can be selected by introducing one or more markers that allow the selection of transferred host cells. The marker can provide prototrophy for an auxotrophic host, resistance to biocide (e.g., antibiotics) or resistance to heavy metals such as copper. The selectable marker gene can be directly linked to the DNA sequences to be expressed or introduced into the same cell through cotransformation. Additional elements may also be needed for optimal mRNA synthesis. These elements can include signal sequences, union signals, as well as transcriptional promoters, enhancers and termination signals.
[0248] In some embodiments the cloned variable region genes are inserted into an expression vector along with the synthetic heavy and light chain constant genes (for example, humans) as discussed above. Of course, any expression vector that has the ability to elicit expression in eukaryotic cells can be used in the present disclosure. Examples of suitable vectors include, but are not limited to, plasmids pcDNA3, pHCMV / Zeo, pCR3.1, pEFl / His, pIND / GS, pRc / HCMV2, pSV40 / Zeo2, pTRACER-HCMV, pUB6 / V5-His, pVAXl and pZeoSV2 (available from Invitrogen, San Diego, CA), and the plasmid pCI (available from Promega, Madison, WI). In general, screening large numbers of transformed cells for those that express suitably high levels of immunoglobulin heavy and light chains is a routine experiment that can be carried out, for example, by robotic systems.
[0249] More generally, since the vector or DNA sequence encoding a monomer subunit of the Pseudomonas anti-Psl I give. PcrV binding molecule, for example, antibody or fragment, variant or derivative thereof, was prepared, the expression vector can be introduced into an appropriate host cell. The introduction of the plasmid into the host cell can be achieved through several sets of procedures well known to those skilled in the art. These include, but are not limited to, 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., Butterworths, Boston, Mass., Chapter 24.2, pages 470 to 472 (1988). Typically, the introduction of plasmid into the host is by electroporation. The host cells that host the expression construct are cultured under conditions suitable for the production of light and heavy chains, and evaluated for heavy and / or light chain protein synthesis. Sets of exemplary assay procedures include enzyme-linked immunosorbent assay (ELISA), radio immunoassay (RIA) or fluorescence-activated cell classifier analysis (FACS), immunohistochemistry and the like.
[0250] The expression vector is transferred to a host cell using sets of traditional procedures and the transfected cells are then cultured using sets of conventional procedures to produce an antibody for use in the methods described in this document. Thus, the disclosure includes host cells that contain a polynucleotide that encodes a Pseudomonas anti-Psl and / or PcrV binding molecule, for example, antibody or fragment, variant or derivative thereof, or a heavy or light chain thereof, of operationally linked to a heterologous promoter. In some modalities, for the expression of double chain antibodies, vectors that encode as many heavy chains as light chains can be coexpressed in the host cell for expression for the entire immunoglobulin molecule, as detailed below.
[0251] Certain embodiments include an isolated polynucleotide that comprises a nucleic acid encoding the VH and VL described above, wherein a binding molecule or antigen binding fragment thereof expressed by the polynucleotide specifically binds to Psl of Pseudomonas and / or PcrV . In some embodiments, the polynucleotides as described encode a scFv molecule that includes 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.
[0252] Some modalities include vectors that comprise the polynucleotides described above. In additional embodiments, polynucleotides are operatively associated with a promoter. In additional embodiments, the disclosure provides host cells that comprise such vectors. In additional embodiments, the disclosure provides vectors in which the polynucleotide is operatively associated with a promoter, where the vectors can express a binding molecule that specifically binds to Psl of Pseudomonas and / or PcrV in a suitable host cell.
[0253] A method is also provided to produce a binding molecule or fragment thereof that specifically binds to Psl from Pseudomonas and / or PcrV, which comprises cultivating a host cell containing a vector comprising the polynucleotides described above, and which retrieves said antibody, or fragment thereof. In additional embodiments, the disclosure provides an isolated binding molecule or fragment thereof produced using the method described above.
[0254] As used herein, "host cells" refers to cells that host vectors constructed using sets of recombinant DNA procedures and that encode at least one heterologous gene. In descriptions of processes for the isolation of antibodies from recombinant hosts, the terms "cell" and "cell culture" are used interchangeably to represent the antibody source unless clearly specified otherwise. In other words, the recovery of polypeptide from the "cells" can mean from centrifuged whole cells, or from the cell culture that contains both the medium and the suspended cells.
[0255] A variety of host-vector expression systems can be used to express antibody molecules for use in the methods described in this document. Such host expression systems represent vehicles through which the coding sequences of interest can be produced and subsequently purified, but they also represent cells that can, when transformed or transfected with the appropriate nucleotide coding sequences, express a molecule of in situ staining antibody. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA expression vectors, plasmid DNA or cosmid DNA that contains antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors that contain antibody coding sequences; insect cell systems with recombinant virus expression vectors (e.g., baculovirus) that contain antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (for example, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (for example, Ti plasmid ) that contain antibody coding sequences; or mammalian cellular systems (e.g., COS, CHO, BLK, 293, 3T3 cells) that harbor recombinant expression constructs that contain promoters derived from the mammalian cell genome (e.g., metallothionein promoter) or from viruses mammalian (for example, the adenovirus late promoter; the 7.5K vaccinia virus promoter). Bacterial cells such as Escherichia coli, or eukaryotic cells, especially for the expression of a total recombinant antibody molecule, are used for the expression of a recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovarian cells (CHO), together with a vector such as the primary intermediate early gene promoter element from human cytomegalovirus is an effective antibody expression system (Foecking et al ., Gene 45: 101 (1986); Cockett et al., Bio / Technology 8: 2 (1990)).
[0256] The host cell line used for protein expression is often of mammalian origin; those skilled in the art are credited with the ability to determine the particular cell lines that are best suited for the desired gene product to be expressed therein. Exemplary host cell lines include, but are not limited to, CHO (Chinese Hamster Ovary), DG44 and DUXB11 (Chinese Hamster Ovary lines, DHFR minus), HELA (human cervical carcinoma), CVI (monkey kidney line) , COS (a derivative of CVI with SV40 T antigen), VERY, BHK (puppy hamster kidney), MDCK, 293, WI38, R1610 (Chinese hamster fibroblast) BALBC / 3T3 (mouse fibroblast), HAK (kidney lineage) of hamster), SP2 / O (mouse myeloma), P3x63-Ag3.653 (mouse myeloma), BFA-1clBPT (bovine endothelial cells), RAJI (human lymphocyte) and 293 (human kidney). Host cell lines are typically available from commercial services, from the American Fabric Culture Collection or from published literature.
[0257] In addition, it is possible to choose a host cell strain that modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific way desired. Such modifications (for example, glycosylation) and the processing (for example, cleavage) of protein products can be important for the function of the protein. Different host cells have specific characteristics and mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure correct modification and processing of the expressed foreign protein. To that end, eukaryotic host cells that have the cellular machinery for proper processing of the primary transcript, glycosylation and phosphorylation of the gene product can be used.
[0258] For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines that express the antibody molecule stably can be designed. Instead of using expression vectors that contain viral origins of replication, host cells can be transformed with controlled DNA through appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.). ) and a selectable marker. After the introduction of foreign DNA, the projected cells can be allowed to grow for 1 to 2 days in an enriched medium and are then moved to selective media. The selectable marker on the recombinant plasmid confers resistance to selection and allows the cells to integrate stably with the plasmid inside if their chromosomes and grow to form the foci which in turn can be cloned and expanded into cell lines. This method can advantageously be used to manipulate cell lines that stablely express the antibody molecule.
[0259] Various selection systems can be used, which includes, but is not limited to, herpes simplex virus thymidine kinase (Wigler et al., Cell 77: 223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski , Proc. Natl. Acad. Sci. USA 48: 202 (1992)) and the adenine phosphoribosyltransferase genes (Lowy et al., Cell 22:% V1 1980) can be employed tk-, hgprt- or aprt-cells, respectively . Also, antimetabolite resistance can be used as a basis for selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77: 357 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 75: 1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78: 2072 (1981)); neo, which confers resistance to aminoglycoside G-418 Clinical Pharmacy 12'A% & a 505; Wu and Wu, Biotherapy 3:87 to 95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32: 573 to 596 (1993); Mulligan, Science 260: 926 to 932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62: 191 to 217 (1993); TIB TECH 11 (5): 155 to 215 (May, 1993); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30: 147 (1984). The methods commonly known in the art of recombinant DNA technology that 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 in this document in their entirety.
[0260] The expression levels of the antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vector based on gene amplification for the expression of cloned genes in mammal cells in DNA cloning, Academic Press, New York, Vol. 3. (1987)). When a marker in the vector system that expresses the antibody is amplifiable, the increase in the level of inhibitor present in the host cell culture will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, antibody production will also increase (Crouse et al., Mol. Cell. Biol. 3: 257 (1983)).
[0261] In vitro production allows scaling up to provide large quantities of the desired polypeptides. Sets of procedures for culturing a mammalian cell under tissue culture conditions are known in the art and include homogeneous suspension culture, for example, in a pneumatically elevated reactor or a continuously agitated reactor, or immobilized cell culture or trapped, for example, in hollow fibers, microcapsules, in agarose microspheres or ceramic cartridges. If necessary and / or desired, polypeptide solutions can be purified using customary chromatography methods, for example, gel filtration, ion exchange chromatography, DEAE cellulose chromatography or (immuno-) affinity chromatography, for example, after biosynthesis preference of a polypeptide of synthetic hinge region or prior to or subsequent to the HIC chromatography step described in this document.
[0262] The constructs encoding Pseudomonas anti-Psl and / or PcrV binding molecules, for example, antibodies or fragments, variants or derivatives thereof, as disclosed herein can also be expressed in non-mammalian cells as bacteria or yeast or plant cells. Bacteria that readily acquire nucleic acids include members of enterobacteriaceae, such as strains of Escherichia coli or Salmonella ', Bacillaceae, such as Bacillus subtilis', Pneumococcus', Streptococcus, and Haemophilus influenzae. It will be further noted that, when expressed in bacteria, heterologous polypeptides typically become part of inclusion bodies. The heterologous polypeptides need to be isolated, purified and then assembled into functional molecules. In which tetravalent forms of the antibodies are desired, the subunits will then self-assemble into tetravalent antibodies (WO02 / 096948A2).
[0263] In bacterial systems, numerous expression vectors can be advantageously selected, depending on the use intended for the antibody molecule being expressed. For example, when a large amount of such a protein must be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors that direct the expression of high levels of fusion protein products that are readily purified may 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 individually linked to the vector in framed with the lacZ coding region, so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 73: 3,101 to 3,109 (1985); Van Heeke & Schuster, J. Biol. Chem. 24: 5,503 to 5,509 (1989)); and the like. PGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to matrix glutathione-agarose microspheres followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the chemical portion of GST.
[0264] In addition to prokaryotes, eukaryotic microbes can also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among eukaryotic microorganisms, although numerous other strains are commonly available, for example, Pichiapastoris.
[0265] For expression in Saccharomyces, the YRp7 plasmid, for example, (Stinchcomb 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 TRP1 gene that provides a selection marker for a mutant strain of yeast that lacks the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1 (Jones, Genetics <55:12 (1977) ). The presence of the trpl lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
[0266] In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is typically used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence can be cloned individually into non-essential regions (for example, the polyhedrin gene) of the virus and placed under the control of an AcNPV promoter (for example, the polyhedrin promoter).
[0267] Since the PcrV and / or Psl binding molecule of anti-Pseudomonas, for example, antibody or fragment, variation or derivative thereof, as disclosed herein, has been recombinantly expressed, it can be purified by any method known in the art to purify an immunoglobulin molecule, for example, by chromatography (for example, ion exchange, affinity, particularly affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or any other set of standard procedures for protein purification. Another method for increasing the antibody affinity of the disclosure is disclosed in US 2002 0123057 A1. VII.IDENTIFICATION OF BINDING MOLECULES INDIFFERENT TO SEROTYPE
[0268] The disclosure encompasses an indifferent target whole cell approach to identifying serotype-independent therapeutic binding molecules, for example, antibodies or fragments thereof with desired or greater therapeutic activities. The method can be used to identify binding molecules that can antagonize, neutralize, clean or block unwanted activity from an infectious agent, for example, a bacterial pathogen. As is known in the art, many infectious agents exhibit significant variation in their dominant surface antigens, which allows them to evade immune surveillance. The identification method described in this document can identify binding molecules that target antigens that are shared among many different species of Pseudomonas or other gram-negative pathogens, thus providing a therapeutic agent that can target multiple pathogens of multiple species. For example, the method was used to identify a series of binding molecules that bind to the surface of P. aeruginosa independently of serotype, and when linked to bacterial pathogens, they mediate, promote or enhance opsonophagocyte (OPK) activity against bacterial cells , such as bacterial pathogens, for example, opportunistic Pseudomonas species (e.g., Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida, and Pseudomonas alkali genes) and / or inhibit the attachment of such bacterial cells to epithelial cells.
[0269] Certain modalities disclose a method for identifying serotype-independent binding molecules comprising: (a) preparing virgin and / or convalescent antibody libraries in the phage, (b) removing serotype-specific antibodies from the library by depletion panorama, (c) screening the library for antibodies that specifically bind to serotype-independent whole cells and (d) screening the resulting antibodies for functional properties.
[0270] Certain modalities provide a whole cell phenotypic screening approach as disclosed herein with antibody phage libraries derived from convalescent patients infected with P. aeruginosa or virgins. Using a panoramic strategy that initially selected against specific serotype reactivity, different clones that bind whole cells of P. aeruginosa were isolated. The selected clones were converted to human IgGl antibodies and confirmed to react with clinical isolates of P. aeruginosa regardless of serotype classification or tissue isolation site (See Examples). The screens of functional activity described in this document indicated that the antibodies were effective in preventing the binding of P. aeruginosa to mammalian cells and opsonophagocytic-mediated extermination (OPK) in a concentration-dependent and serotype-independent manner.
[0271] In the additional embodiments, the binding molecules described above 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 at least 80%, at least 85%, at least 90% or at least 95% of strains of P. aeruginosa isolated from infected patients. In the additional modalities, strains of P. aeruginosa are isolated from one or more of knee fluid, bone, blood, skin, a wound, bone cavity, urine, feces, pus, eye, spit or lung. VIII. PHARMACEUTICAL COMPOSITIONS THAT UNDERSTAND THE PSL AND / OR PCRV BINDING MOLECULES OF ANTI-PSEUDOMONES
[0272] The pharmaceutical compositions used in this disclosure comprise pharmaceutically acceptable carriers well known to those skilled in the art. Liquid preparations for parenteral administration include sterile aqueous or non-aqueous solutions, emulsions, suspensions and solutions. Certain pharmaceutical compositions as disclosed herein can be administered orally in an acceptable dosage form including, for example, capsules, tablets, suspensions or aqueous solutions. Certain pharmaceutical compositions can also be administered by inhalation or nasal spray. Preservatives and other additives can also be present such as, for example, antimicrobials, antioxidants, chelating agents and inert gases and the like. Formulations suitable for use in the therapeutic methods disclosed herein are described in Remington's Pharmaceutical Sciences, Mack Publishing Co., 16th edition (1980).
[0273] The amount of Pseudomonas anti-Psl and / or PcrV binding molecule, for example, antibody or fragment, variant or derivative thereof, which can be combined with carrier materials to produce a single dosage form will vary depending on of the treated host and the particular mode of administration. Dosage regimens can also be adjusted to provide the optimal desired response (for example, a therapeutic or prophylactic response). The compositions may also comprise the anti-Psl and / or PcrV binding molecules of Pseudomonas, for example, antibodies or fragments, variants or derivatives thereof dispersed in a biocompatible carrier material that functions as an appropriate delivery or support system for the compounds . IX. TREATMENT METHODS THAT USE THERAPEUTIC BINDING MOLECULES
[0274] Methods for preparing and administering Pseudomonas anti-Psl and / or PcrV binding molecules, for example, an antibody or fragment, variant or derivative thereof, as disclosed herein to an individual in need thereof they are well known or are readily determined by those skilled in the art. The route of administration of the anti-Psl and / or PcrV binding molecules of Pseudomonas, for example, antibody or fragment, variant or derivative thereof, may be, for example, oral, parenteral, by inhalation or topical. The term parenteral as used herein includes, for example, intravenous, intraarterial, intraperitoneal, intramuscular or subcutaneous administration. A suitable form for administration would be a solution for injection, particularly for intravenous or intra-arterial injection or drip. However, in other methods compatible with the teachings in this document, an anti-Psl and / or PcrV binding molecule from Pseudomonas, for example, antibody or fragment, variant or derivative thereof, as disclosed in this document can be delivered directly to the site of the adverse cell population, for example, the infection thereby increasing the exposure of the diseased tissue to the therapeutic agent. For example, an anti-Psl and / or PcrV binding molecule from Pseudomonas can be administered directly to eye tissue, burn injury or lung tissue.
[0275] Anti-Psl of Pseudomonas and / or PcrV binding molecules, for example, antibodies or fragments, variants or derivatives thereof, as disclosed herein can be administered in a pharmaceutically effective amount for the in vivo treatment of infection of Pseudomonas. In this regard, it should be noted that the revealed binding molecules will be formulated in order to facilitate administration and promote the stability of the active agent. For the purposes of the current application, a pharmaceutically effective amount should be maintained in an amount sufficient to achieve effective binding to a target or to achieve a benefit, for example, to treat, improve, decrease, eliminate or prevent Pseudomonas infection.
[0276] Some modalities are directed to a method of preventing or treating a Pseudomonas infection in an individual in need of it, which comprises administering to the individual an effective amount of the binding molecule or fragment thereof, the antibody or fragment thereof, the composition , polynucleotide, vector or host cell described in this document. In additional modalities, Pseudomonas infection is an infection by P. aeruginosa. In some modalities, the individual is a human. In certain embodiments, the infection is an eye infection, a lung infection, a burn infection, a wound infection, a skin infection, a blood infection, a bone infection or a combination of two or more of said infections. In additional modalities, the individual suffers from acute pneumonia, burn injury, corneal infection, cystic fibrosis or a combination thereof.
[0277] Certain modalities are directed to a method to block or prevent the attachment of P. aeruginosa to epithelial cells which comprises putting a mixture of epithelial cells and P. aeruginosa in contact with the binding molecule or fragment thereof, the antibody or fragment in addition, the composition, polynucleotide, vector or host cell described herein.
[0278] Also disclosed is a method of improving P. aeruginosa OPK which comprises putting a mixture of phagocytic cells and P. aeruginosa in contact with the binding molecule or fragment thereof, the antibody or fragment thereof, the composition, the polynucleotide , the vector or the host cell described in this document. In additional embodiments, the phagocytic cells are differentiated HL-60 cells or human polymorphonuclear leukocytes (PMNs).
[0279] Keeping within the scope of the disclosure, Pseudomonas anti-Psl and / or PcrV binding molecules, for example, antibodies or fragments, variants or derivatives thereof, can be administered to a human or other animal in accordance with treatment methods mentioned above in an amount sufficient to produce a therapeutic effect. The Pseudomonas anti-Psl and / or PcrV binding molecules, for example, antibodies or fragments, variants or derivatives thereof, disclosed herein can be administered to such a human or other animal in a conventional dosage form prepared by combining the antibody of the disclosure as a conventional pharmaceutically acceptable carrier or diluent according to known sets of procedures.
[0280] Effective doses of the compositions of the present disclosure for treating Pseudomonas infection vary depending on many different factors, including means of administration, target location, physiological state of the patient, whether the patient is human or an animal, other medications administered , and whether it is prophylactic or therapeutic treatment. Generally, the patient is a human, but non-human mammals including transgenic mammals can be treated as well. Treatment dosages can be titrated using routine methods known to those skilled in the art to optimize safety and efficacy.
[0281] Anti-Psl of Pseudomonas and / or PcrV binding molecules, for example, antibodies or fragments, variants or derivatives thereof can be administered on multiple occasions at various frequencies depending on several factors known to those skilled in the art. Alternatively, Pseudomonas anti-Psl and / or PcrV binding molecules, for example, antibodies or fragments, variants or derivatives thereof can be administered as a continuous release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody in the patient.
[0282] The compositions of the disclosure can be administered by any suitable method, for example, parenterally, intraventricularly, orally, by inhalation spray, topically, rectally, nasal, buccally, vaginally or via an implanted reservoir. The term “parenteral” as used herein includes sets of subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial infusion or injection procedures. X.SINERGY
[0283] Chou and Talalay (Adv. Enzyme Regul., 22: 27-55 (1984)) developed a mathematical method to describe the experimental findings of combined drug effects in a qualitative and quantitative way. For mutually exclusive drugs, they have shown that the generalized isobol equation applies for any degree of effect (see page 52 in Chou and Talalay). An isobol or isobologram is the graphical representation of all these combinations of two drugs that have the same degree of effect. In isobolograms, a straight line indicates additive effects, a concave curve (curve below the straight line) represents synergistic effects, and the convex curve (curve above the straight line) represents antagonistic effects. These curves also show that a combination of two mutually exclusive drugs will show the same type of effect over the entire concentration range, the combination being additive, synergistic or antagonistic. Most drug combinations show an additive effect. In some cases, however, the combinations show less or more than an additive effect. These combinations are called antagonistic or synergistic, respectively. The combination manifests therapeutic synergy if it is therapeutically superior to the other of the constituents used in the favorable dose of the same. See, T. H. Corbett et al., Cancer Treatment Reports, 66, 1187 (1982). Tallarida RJ (J Pharmacol Exp Ther. 2001 Sep; 298 (3): 865-72) also notes that "Two drugs that produce overtly similar effects will sometimes produce exaggerated or reduced effects when used simultaneously. A quantitative assessment is necessary to distinguish these cases of simple additive action. "
[0284] A synergistic effect can be measured using the Chou and Talalay method of combination index (CI) (see Chang et al., Cancer Res. 45: 2434-2439, (1985)) which is based on the principle of medium effect. This method calculates the degree of synergy, additivity or antagonism between two drugs at various levels of cytotoxicity. Where the Cl value is less than 1, there is synergy between the two drugs. Where the Cl value is 1, there is an additive effect, but not a synergistic effect. Cl values greater than 1 indicate antagonism. The lower the Cl value, the greater the synergistic effect. In another embodiment, a synergistic effect is determined using the fractional inhibitory concentration (FIC). This fractional value is determined by expressing the IC 50 of a drug acting in combination, as a function of the IC 50 of the drug acting alone. For two drugs interacting, the sum of the FIC value for each drug represents the measure of synergistic interaction. Where the FIC is less than 1, there is synergy between the two drugs. A FIC value of 1 indicates an additive effect. The lower the FIC value, the greater the synergistic interaction.
[0285] In some embodiments, a synergistic effect is obtained in the treatment of Pseudomonas in which one or more of the binding agents are administered in a "low dose" (that is, using a dose or doses that would be considered non-therapeutic) if administered alone), where administration of the low dose binding agent in combination with other binding agents (administered in both a low and therapeutic dose) results in a synergistic effect that exceeds the additive effects that would otherwise result from individual administration of the binding agent alone. In some embodiments, the synergistic effect is achieved by administering one or more of the binding agents administered in a "low dose" in which the low dose is provided to reduce or prevent toxicity or other undesirable side effects. XI.IMUNOENSAIOS
[0286] Kxdx-Pseudomonas Psl and / or PcrV binding molecules, for example, antibodies or fragments, variants or derivatives thereof, can be assayed for immunospecific binding by any method known in the art. Immunoassays that can be used include, but are not limited to, competitive and non-competitive assay systems using sets of procedures such as western blots, radioimmunoassays, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassays, assays immunoprecipitation, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name a few. Such assays are routine and well known in the art (see, for example, Ausubel et al., Eds, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, Vol. 1 (1994), which is incorporated reference in this document in its entirety). Exemplary immunoassays are briefly described below (but are not intended by way of limitation).
[0287] There are a variety of methods available to measure the affinity of an antibody-antigen interaction, but relatively few to determine rate constants. Most methods consist of labeling both the antibody and antigen, which inevitably complicates routine measurements and presents uncertainties in the quantities measured. Antibody affinity can be measured by a number of methods, including OCTET®, BIACORE®, ELISA, and FACS.
[0288] The OCTET * system uses biosensors in a 96-well plate format to report kinetic analysis. Protein binding and dissociation events can be monitored by measuring the binding of a protein in solution to a second protein immobilized on the FortéBio biosensor. In the case of measuring binding of anti-Psl or PcrV antibodies to Psl or PcrV, Psl or PcrV is immobilized in parts of OCTET * followed by the binding analysis of the antibody, which is in solution. The association and dissociation of antibody to the immobilized Psl or PcrV is then detected by the instrument sensor. The data is then collected and exported to GraphPad Prism to adjust the affinity curve.
[0289] Plasmonic Surface Resonance (SPR) as performed in BIACORE * offers a number of advantages in conventional methods of measuring antibody-antigen interaction affinity: (i) no requirement to label both the antibody and antigen; (ii) antibodies do not need to be purified in advance, the cell culture supernatant can be used directly; (iii) real-time measurements, which allow for quick semi-quantitative comparison of different monoclonal antibody interactions, are allowed and are sufficient for many evaluation purposes; (iv) the biospecific surface can be regenerated so that a series of different monoclonal antibodies can be easily compared under identical conditions; (v) analytical procedures are completely automated, and extensive series of measurements can be performed without user intervention. BIAapplications Handbook, version AB (reprinted in 1998), BIACOREk code number BR-1001-86; BIAtechnology Handbook, AB version (reprinted in 1998), BIACOREk code number BR-1001-84.
[0290] SPR-based bonding studies require that a member of a bonding pair be immobilized on a surface of the sensor. The immobilized bonding partner is referred to as the binder. The solution binding partner is referred to as the analyte. In some cases, the ligand is attached indirectly to the surface by attaching it to another immobilized molecule, which is termed as the capture molecule. The SPR response reflects a change in the mass concentration on the detector surface as the analytes bind or dissociate.
[0291] Based on SPR, BIACORE® measurement monitoring interactions in real time directly as they happen. The set of procedures is suitable for the determination of kinetic parameters. The comparative affinity classification is extremely simple to perform, and both the kinetic constant and the affinity constant can be derived from the sensorgram data.
[0292] When the analyte is injected into a discrete pulse along a binding surface, the resulting sensorgram can be divided into three essential phases: (i) association of the analyte with the ligand during a sample injection; (ii) equilibrium or steady state during sample injection, where the rate of binding of the analyte is balanced through the dissociation of the complex; (iii) dissociation of the surface analyte during the buffer flow.
[0293] The association and dissociation phases provide information on the analyte-ligand interaction kinetics (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 (KD) interaction.
[0294] The BIAevaluation software provides comprehensive facilities for curve fitting using both numerical integration and global adjustment algorithms. With proper analysis of the data, the separate rate and affinity constants for interaction can be obtained from simple BIACOREk investigations. The rate of measurable affinities for this set of procedures is very wide, ranging from mM to pM.
[0295] Epitope specificity is an important characteristic of a monoclonal antibody. Epitope mapping with BIACORE *, in contrast to sets of conventional procedures that use radioimmunoassay, ELISA or other surface absorption methods, does not require labeling or purified antibodies, and allows multiple site specificity tests using a sequence of several monoclonal antibodies. In addition, large numbers of analyzes can be processed automatically.
[0296] Pairing experiments test the ability of the two MAbs to simultaneously bind to the same antigen. MAbs directed against separate epitopes will bind independently, while MAbs directed against closely related identical epitopes will interfere with the binding of the other. These connection experiments with BIACORE * are easy to carry out.
[0297] For example, a person can use a capture molecule to bind the first Mab, followed by the addition of antigen and the second MAb sequentially. The sensorgrams will reveal: 1. How much of the antigen binds to the first Mab, 2. How far the second MAb binds to the antigen fixed on the surface, 3. If the second MAb does not bind, reversing the test order in pairs changes results.
[0298] Peptide inhibition is another technique used for epitope mapping. This method can complement antibody binding studies in pairs, and can relate functional epitopes to structural features when the primary sequence of the antigen is known. The peptides or antigen fragments are tested for the inhibition of MAbs other than the immobilized antigen. It is assumed that peptides that interfere with the binding of a given MAb are structurally related to the epitope defined by that MAb. XII. ADMINISTRATION
[0299] A composition comprising either an anti-Psl binding domain or anti-PcrV binding domain, or a composition comprising both an anti-Psl and anti-PcrV binding domain are administered in such a way that they provide a synergistic effect in the treatment of Pseudomonas in a patient. Administration can be by any suitable means as long as the administration provides the desired therapeutic effect, i.e., synergism. In certain embodiments, antibodies are administered during the same therapy cycle, for example, during a therapy cycle for a prescribed period of time, both antibodies are administered to the individual. In some embodiments, the administration of the antibodies may be during sequential administration in separate therapy cycles, for example, the first cycle of therapy that involves administration of an anti-Psl antibody and the second cycle of therapy that involves administration of a anti-PcrV antibody. The dosage of binding domains administered to a patient will also depend on the frequency of administration and can be readily determined by a person of ordinary skill in the art.
[0300] In other embodiments, the binding domains are administered more than once during a treatment cycle. For example, in some modalities, the binding domains are administered weekly for three consecutive weeks in a three or four week treatment cycle.
[0301] Administration of the composition comprising one or more of the binding domains can be at noon or on different days provided that the administration provides the desired therapeutic effect.
[0302] It will be readily apparent to those skilled in the art that other doses or frequencies of administration that provide the desired therapeutic effect are suitable for use in the present invention. XII. KITS
[0303] In still other embodiments, the present invention provides kits that can be used to carry out the methods described in this document. In certain embodiments, a kit comprises a binding molecule disclosed herein in one or more containers. A person skilled in the art will readily recognize that the disclosed binding domains, polypeptides and antibodies of the present invention can be readily incorporated into one of the established kit formats that are well known in the art.
[0304] The practice of disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the technique. These techniques are explained completely in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Edition, 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 (1985); Oligonucleotide Synthesis, M. J. Gait ed., (1984); Mullis et al. Patent under U.S. U.S .: 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); Immobilized Cells And Enzymes, IRL Press, (1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology, Academic Press, Inc., N.Y .; Gene Transfer Vectors For Mammalian Cells, J. H. Miller and M. P. Calos eds., Cold Spring Harbor Laboratory (1987); Methods In Enzymology, Vols. 154 and 155 (Wu et al. Eds.); Immunochemical Methods In Cell And Molecular 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 Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989).
[0305] The general principles of antibody modification are presented in Antibody Engineering, 2nd Edition, C.A.K .. Borrebaeck, Ed., Oxford Univ. Press (1995). The general principles of protein modification are presented in Protein Engineering, A Practical Approach, Rickwood, D., et al., Eds., IRL Press at Oxford Univ. Press, Oxford, Eng. (1995). The general principles of antibody and antibody-hapten binding are presented in: Nisonoff, A., Molecular Immunology, 2nd Edition, Sinauer Associates, Sunderland, MA (1984); and Steward, M.W., Antibodies, Their Structure and Function, Chapman and Hall, New York, NY (1984). In addition, standard immunology methods known in the art and not specifically described are generally followed as in Current Protocols in Immunology, John Wiley & Sons, New York; Stites et al. (eds), Basic and Clinical-Immunology (8 ~ Edition), Appleton & Lange, Norwalk, CT (1994) and Mishell and Shiigi (eds), Selected Methods in Cellular Immunology, WH Freeman and Co., New York (1980) .
[0306] Standard reference papers presenting general principles of immunology include Current Protocols in Immunology, John Wiley & Sons, New York; Klein, J., Immunology: The Science of SelfNonself Discrimination, John Wiley & Sons, New York (1982); Kennett, R., et al., Eds., Monoclonal Antibodies, Hybridoma: A New Dimension in Biological Analyzes, Plenum Press, New York (1980); Campbell, A., “Monoclonal Antibody Technology” in Burden, R., et al., Eds., Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 13, Elsevere, Amsterdam (1984), Kuby Immunnology 4 ~ Edition Ed. Richard A. Goldsby, Thomas J. Kindt and Barbara A. Osborne, H. Freemand & Co. (2000); Roitt, I., Brostoff, J. and Male D., Immunology 6 ~ London Edition: Mosby (2001); Abbas A., Abul, A. and Lichtman, A., Cellular and Molecular Immunology Ed. 5, Elsevier Health Sciences Division (2005); Kontermann and Dubel, Antibody Engineering, Springer Verlan (2001); Sambrook and Russell, Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press (2001); Lewin, Genes VIII, Prentice Hall (2003); Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988); Dieffenbach and Dveksler, PCR Primer Cold Spring Harbor Press (2003). EXAMPLES Example 1: Construction and screening of human antibody phage display libraries
[0307] This example describes a full-cell, target-indifferent panoramic approach with human antibody phage libraries derived in both recovery patients infected with P. aeruginosa and virgins to identify innovative protective antigens against Pseudomonas infection (Figure IA). The assays included in the functional in vitro screens included opsonophagocytosis (OPK) elimination assays and cell attachment assays using the epithelial cell line A549. Leading candidates, based on superior in vitro activity, were tested in models of acute P. aeruginosa pneumonia, keratitis and burn infection.
[0308] Figure 1B shows the construction of the patient antibody fact display library. Complete blood was pooled from 6 recovering patients 7 to 10 days after diagnosis followed by RNA extraction and construction of a phage library as previously described (Vaughan, TJ, et al., Nat Biotechnol 14, 309 to 314 (1996) ; Wrammert, J., et al., Nature 453, 667 to 671 (2008)). Figure 1C shows that the final cloned scFv library contained 5.4 x 10 transformants and sequencing revealed that 79% of the scFv genes were full-length and in frame. The VH CDR3 bonds, often important in determining epitope specificity, were 84% diverse at the amino acid level prior to library selection.
[0309] In addition to the patient library, a phage display library of virgin human scFv containing up to 1x1011 binding members (Lloyd, C., et al., Protein Eng Des Sei 22, 159 to 168 (2009)) used for antibody isolation (Vaughan, TJ, et al., Nat Biotechnol 14, 309 to 314 (1996)). P. aeruginosa eliminated by heat (1x109) was immobilized in IMMUNO ™ tubes (Nunc; MAXISORP ™) followed by the phage display selections as described (Vaughan, TJ, et al., Nat Biotechnol 14, 309 to 314 (1996) ) with the exception of triethanolamine (100nM) being used as the elution buffer. For selection in P. aeruginosa in suspension, cells eliminated by heat were blocked followed by the addition of blocked phage to the cells. After washing, the eluted phage was used to infect E. coli cells as described (Vaughan, 1996). The rescue of E. coli phage and binding to P. aeruginosa eliminated by heat by ELISA was performed as described (Vaughan, 1996).
[0310] After the development and validation of the full cell affinity selection methodology, both the new recovering patient library and a previously constructed virgin library (Vaughan, TJ, et al., Nat Biotechnol 14, 309-314 (1996 )) passed was affinity selection in suspensions of P. aeruginosa 3064 strain that has a complete O-antigen as well as a mutant strain of isogenic wapR that lacked O-antigen surface expression. Figure 1D shows that the output titrants of consecutive patient library selections increase at a higher rate for the patient library than for the virgin library (1x10 vs 3x10 in cycle 3, respectively). In addition, it was also observed that the duplication of VH CDR3 loop sequences in libraries (a measure of clonal enrichment during selection), is higher in the patient library, reaching 88 to 92%, compared to 15 to 25 % in the virgin library in cycle 3 (Figure 1D). The individual scFv phage from affinity selections were then screened by ELISA for the reactivity of the heterologous P. aeruginosa serotype strains (Figure IE). ELISA plates (Nunc; MAXISORP ™) were coated with strains of P. aeruginosa from overnight cultures as described (DiGiandomenico, A., et al., Infect Immun 72, 7012-7021 (2004) ). Diluted antibodies were added to blocked plates for 1 hour, washed, and treated with HRP-conjugated secondary anti-human antibodies for 1 hour followed by development and analysis as described (Ulbrandt, ND, et al., J Virol 80, 7,799 a 7,806 (2006)). The dominant phage species obtained from complete cell selections with both libraries yielded specific reactivity by serotype (data not shown). Clones that exhibit serotype-independent binding in the absence of non-specific binding to E. coli or bovine serum albumin were selected for further evaluation.
[0311] For IgG expression, the selected antibody VH and VL chains were cloned into human IgGl expression vectors, coexpressed into HEK293 cells, and purified protein A affinity chromatography as described (Persic, L., et al., Gene 187, 9 to 18 (1997)). Human IgGl antibodies made with the region variables of these phage-independent phage selected were confirmed for specificity by P. aeruginosa and prioritized for subsequent analysis by complete cell binding to clinically relevant dominant serotypes by FACS analysis (Figure 1F), seen that this method is more rigorous than ELISA. For flow cytometry-based binding assays the intermediate log phase P. aeruginosa strains were concentrated in PBS to an OD650 of 2.0. After incubating the antibody (10 μg / ml) and bacteria (~ 1 x 10 cells) for 1 hour at 4 ° C with shaking, the washed cells were incubated with an ALEXA FLUOR 647® goat anti-human IgG antibody ( Invitrogen, Carlsbad, CA) for 0.5 hour at 4 ° C. The washed cells were stained with BACLIGHT ™ green bacterial stain as recommended (Invitrogen, Carlsbad, CA). The samples were placed in an LSR II flow cytometer (BD Biosciences) and analyzed using BD FacsDiva (v. 6.1.3) and FlowJo (v. 9.2; TreeStar). Antibodies that exhibit FACS binding were additionally prioritized by testing for functional activity in an opsonophagocytosis (OPK) elimination assay. Example 2: Evaluation of mAbs that promotes P. aeruginosa OPK
[0312] This example describes the evaluation of human IgGl antibodies prioritized to promote P. aeruginosa OPK. Figure 2A shows that with the exception of WapR-007 and the negative control antibody R347, all antibodies mediated the concentration-dependent elimination of the luminescent P. aeruginosa serogroup 05 strain (PAOl.lux). WapR-004 and Cam-003 exhibited superior OPK activity. OPK assays were performed as described in (DiGiandomenico, A., et al., Infect Immun 72, 7.012 to 7.021 (2004)), with modifications. Briefly, the tests were carried out in 96-well plates using 0.025 ml of each OPK component; strains of P. aeruginosa-, diluted rabbit pup 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 to 448 (2005)), were used. The luminescent OPK assays were performed as described above, but with the determination of relative luciferase units (RLUs) using a Perkin Elmer ENVISION multi-tag plate reader (Perkin Elmer).
[0313] The ability of the WapR-004 and Cam-003 antibodies to mediate OPK activity against another clinically relevant O-antigen serotype strain, 9882-80.lux, has been evaluated. Figure 2B shows that the enhanced OPK activity of WapR-004 and Cam-003 extends to the 9882-80 (OH) strain.
[0314] In addition, this example describes the evaluation of WapR-004 (W4) mutants in the scFv-Fc format to promote the P. aeruginosa OPK. A mutant, Wap-004RAD (W4-RAD), was specifically created through site-directed mutagenesis to remove an RGD motif in VH. Other W4 mutants were prepared as follows. Nested PCR was performed as described (Roux, KH, PCR Methods Appl 4, SI85 to 194 (1995)), to amplify the W4 variants (derived from somatic hypermutation) from the scFv library derived from the recovered P. aeruginosa infected patients , for analysis. This is the library from which WapR-004 was derived. The fragments of the W4 variant were subcloned and sequenced using standard procedures known in the art. The W4 mutant (LC) light chains were recombined with the WapR-004 (HC) heavy chain to produce scFv-Fc W4 mutants. In addition, the heavy chain mutants of WapR-004 RAD (HC) were recombined with parental LCs of M7 and M8 in the scFv-Fc format. The 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 mutants of WapR-004 (W4) mediated the concentration-dependent elimination of the serogroup 05 strain of P. aeruginosa luminescent (PAOl.lux).
[0315] The variable region of WapR-004-RAD region was organized in germ line to reduce potential immunogenicity, producing the germ line WapR-004 ("WapR-004-GL"), and was optimized by leader for mutagenesis directed to local. Clones with enhanced affinity for Psl were selected in screening based on competition. The upper clones were classified by affinity enhancement and analyzed in an in vitro functional assay. The 14 leader-optimized clones are: Psl0096, Psl0170, Psl0225, Psl0304, Psl0337, Psl348, Psl0567, Psl0573, Psl0574, Psl0582, Psl0584, Psl0585, PslO588 and Psl0589. Example 3: Arti-P antibodies. aeruginosa independent of serotype target the exopolysaccharide Psl
[0316] This example describes the target identification of anti-P antibodies. aeruginosa derived from phenotypic screening. Target analysis was performed to treat whether serotype-independent antibodies targeted carbohydrate or protein antigens. No loss of binding was observed in full cell extracts of toPAOl ELISA digested extensively with proteinase K, suggesting those reactive-targeted carbohydrate residues (data not shown). Isogenic mutants were built on genes responsible for O-antigen, alginate, and LPS nucleus biosynthesis; yvbpL (deficient in O-antigen); wbpL / algD (deficient in O-antigen and alginate); rmlC (deficient in O-antigen and truncated outer nucleus); and galU (deficient in O-antigen and truncated internal nucleus). The P. aeruginosa mutants were constructed based on the allele replacement strategy described by Schweizer (Schweizer, H.P., Mol Microbiol 6, 1,195 to 1,204 (1992); Schweizer, H.D., Biotechniques 15, 831 to 834 (1993)). The vectors were mobilized from the strain of E. coli SI7.1 to the strain of P. aeruginosa PAO1; recombinants were isolated as described in (Hoang, T.T., et al., Gene 212, 77 to 86 (1998)). The gene deletion was confirmed by PCR. The mutants of P. aeruginosa were supplemented with constructs based on pUCP30T nourishing wild-type genes. Antibody reactivity was determined by indirect ELISA on plates coated with the strains of P. aeruginosa indicated above: Figure 3A shows that the binding of Cam-003 to wbpL or the double mutant of wbpL / algD was not affected, however the binding to the rmlC and galU mutants was abolished. Although these results were consistent with binding to the LPS core, reactivity to PAO1 purified LPS was not observed. It was recently shown that the rmlC and galU genes were required for the biosynthesis of Psl exopolysaccharide, a repeating pentasaccharide polymer consisting of D-mannose, L-rhamnose, and D-glucose. The binding of Cam-003 to a pslA inactivation PA01Aps / zt has been tested, since pslA is required for Psl biosynthesis (Byrd, M.S., et al., Mol Microbiol 73, 622 to 638 (2009)). The binding of Cam-003 to PAOlA / zsZ / l was abolished when tested by ELISA (Figure 3B) and FACS (Figure 3C), although the LPS molecule in this mutant was unaffected (Figure 3D). The binding of Cam-003 was restored in a triple PAOWwbpLIalgDIpslA mutant complemented with pslA (Figure 3E) as the ability of Cam-003 to mediate opsonic elimination to PAOlΔpsM complemented in contrast to the mutant (Figure 3F and 3G). The binding of antibody Cam-003 to an exopolysaccharide mutant Pel was also unaffected, further confirming Psl as the target antibody (Figure 3E). The binding assays will confirm that the remaining antibodies are also bound to Psl (Figure 3H and 31). Example 4: Attachment of a block of P. aeruginosa anti-Psl mAbs to cultured epithelial cells.
[0317] This example shows that anti-Psl antibodies blocked the association of P. aeruginosa with epithelial cells. Anti-Psl antibodies were added to a confluent monolayer of A549 cells (a basal alveolar epithelial cell line of human adenocarcinoma) cultured in 96-opaque well plates (Nunc Nunclon Delta). The P. aeruginosa PAO1 luminescent log-phase strain (PAOl.lux) was added to an MOI out of 10. After incubating PAOl.lux with A549 cells at 37 ° C for 1 hour, the A549 cells were washed, followed by the addition LB + 0.5% glucose. The bacteria were quantified after a brief incubation at 37 ° C as performed in the OPK assay described in Example 2. The well measurements in A549 cells were used to correct non-specific binding. Figure 4 shows that with the exception of Cam-005 and WapR-007, all antibodies reduced the association of PAOl.lux with A549 cells in a dose-dependent manner. The mAbs that performed best in the OPK, WapR-004 and Cam-003 assays (see Figures 2A-B, and Example 2), were also the most active in inhibiting P. aeruginosa cell attachment to epithelial cells lung A549, providing up to -80% reduction compared to the negative control. WapR-016 was the third most active antibody, showing inhibitory activity similar to WapR-004 and Cam-003 but an antibody concentration 10 times higher. Example 5: P. aeruginosa strains passed alive maintain / increase Psl expression
[0318] To test whether Psl expression was maintained in vivo, mice were injected intraperitoneally with P. aeruginosa isolates followed by collection of bacteria by peritoneal lavage four hours after infection. The presence of Psl was analyzed with a control antibody and Cam-003 by flow cytometry as the conditions for binding are more stringent and allow the quantification of cells that are positive or negative for the expression of Psl. For ex vivo binding, bacterial inoculums (0.1 ml) were prepared from a TSA plate overnight and delivered intraperitoneally to the BALB / c mice. In 4 hours. After stimulation, the bacteria were harvested, RBCs lysed, sonicated and resuspended in PBS supplemented with 0.1% Tween-20 and 1% BS A. The samples were stained and analyzed as described previously in Example 1. Figure 5 shows that bacteria harvested after peritoneal lavage with three strains of wild-type P. aeruginosa showed strong Cam-003 staining, which was comparable to bacteria grown in the log phase (compare Figures 5A and 5C). Wild-type bacteria passed in vivo showed marked staining compared to the inoculum (compare Figures 5B and 5C). Within the inocula, Psl was not detected for strain 6077 and was minimally detected for strains PAO1 (05) and 6206 (OI 1-cytotoxic). The binding of Cam-003 to the bacteria increased compared to those indicating that Psl expression is maintained or increased in vivo. Strains of the wild type 6077, PAO1, and 6206 express Psl after passage in vivo, however the strain of PAO1 that nourishes a deletion of pslA (PΔOXΔpslA) does not have the capacity to react with Cam-003. These results further emphasize Psl as the target for monoclonal antibodies. Example 6: Survival Rates for animals treated with anti-Psl monoclonal antibodies from Cam-003 and WapR-004 in a model of acute P. aeruginosa pneumonia
[0319] Antibodies or PBS were administered 24 hours before infection in each model. The models of acute P. aeruginosa pneumonia, keratite, and thermal injury infection were performed as described (DiGiandomenico, A., et al., Proc Natl Acad Sei USA 104, 4,624 to 4,629 (2007)), with modifications. In the acute pneumonia model, the BALB / c mice (The Jackson Laboratory) were infected with strains of P. aeruginosa suspended in a 0.05 ml inoculum. In the thermal injury model, CF-1 mice (Charles River) received a 10% total body surface area burn with a metal mark heated to 92 ° C for 10 seconds. The animals were infected subcutaneously with P. aeruginosa 6077 strain at the indicated dose. For organ load experiments, acute pneumonia was induced in mice followed by the collection of lungs, spleens and kidneys 24 hours post-infection to determine CFU.
[0320] Monoclonal antibodies Cam-003 and WapR-004 were evaluated in a model of acute lethal pneumonia against strains of P. aeruginosa that represent the most frequent serotypes associated with clinical disease. Figures 6A and 6C show concentration-dependent survival in mice treated by Cam-003 with strains PAO1 and 6294 when compared to controls. Figures 6B and 6D show that complete protection of the stimulus with 33356 and cytotoxic strain 6077 was provided by Cam-003 at 45 and 15 mg / kg while a survival of 80 and 90% was observed at 5mg / kg for 33356 and 6077, respectively . Figures 6E and 6F show significant concentration-dependent survival in mice treated with WapR-004 in the model of acute pneumonia with strain 6077 (011) (8 x 105 CFU) (Figure 6E), or 6077 (011) (6 x 105 CFU) (Figure 6F).
[0321] Cam-003 and WapR-004 were examined below for their ability to reduce the organ load of P. aeruginosa in the lung and spread to distal organs, and later the animals were treated with varying concentrations of WapR-004, Cam -003, or control antibodies in several different concentrations. Cam-003 was effective in reducing the burden on the lung of P. aeruginosa against all four strains tested. Cam-003 was the most effective against the highly pathogenic cytotoxic strain, 6077, in which the low dose was as effective as the upper dose (Figures 7D). Cam-003 also had a significant effect in reducing spread to the spleen and kidneys in mice infected with PAO1 (Figure 7A), 6294 (Figure 7C), and 6077 (Figure 7D), although spread to these organs has not been observed in 33356 infected mice (Figure 7B). Figures 7E and 7F show that in a similar way, WapR-004 reduced the organ load after the induction of acute pneumonia with 6294 (06) and 6206 (011). Specifically, WapR-004 was effective in reducing the spread of P. aeruginosa to the spleen and infected kidneys and mice. Example 7: Construction of anti-PcrV V2L2 monoclonal antibody
[0322] Veloclmmune® mice (Regeneron Pharmaceuticals) were immunized by the ultrashort immunization method with r-PcrV and serum titers were followed by binding to PcrV and neutralizing the hemolytic activity of live P.aeruginosa. Mice showing anti-hemolytic activity in the serum were sacrificed and the lymph and spleen (axial, inguinal and popliteal) were harvested. Cell populations for these organs were adhered to biotinylated r-Pcrv to select specific anti-PcrV B cells. The selected cells were then fused with mouse myeloma partner P3X63-Ag8 and seeded in 25K cells / well in the hybridoma selection medium. After 10 days the medium of the hybridoma wells was completely changed with fresh medium and after another 3 to 4 days the hybridoma supernatants were tested for antihemolytic activity. Colonies showing anti-hemolytic activity were cloned with dilution limited to 0.2 cells / well of 96-well plates and the anti-hemolytic activity assay was repeated. Clones showing anti-hemolytic activity have been adapted for ultra low IgG containing hybridoma culture medium. The IgG from the conditioned media was purified and tested for anti-hemolytic activity in vitro and in vivo to protect against infection by P. aeruginosa. The antibodies were also categorized by the competition assay in different groups. The variable domains (V) of the antibodies of interest were subcloned from the cDNA derived from their respective different clones. The subcloned V segments were fused in frame with the cDNA for the corresponding constant domain in a mammalian expression plasmid. Recombinant IgGs were expressed and purified from HEK293 cells. In cases where more than one cDNA V sequence was obtained from a particular clone, all combinations of light to heavy chains were expressed and characterized to identify the functional IgG. Example 8: Survival rates for animals treated with anti-Psl monoclonal antibodies Cam-003, WapR-004 and monoclonal anti-PcrV antibody V2L2 in a model of corneal infection by P. aeruginosa
[0323] The efficacy of Cam-003 and WapR-004 was evaluated below in a model of P. aeruginosa infection of the chromium that emphasizes the ability of pathogens to attach to 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 ocular homogenates than was observed in animals treated by negative control. Figure 8E shows that Cam-003 was also effective when tested on a thermal injury model, providing significant protection at 15 and 5 mg / kg compared to the antibody treated control. Figure 8 (H): The activity of monoclonal anti-Psl and anti-PcrV V2L2 antibodies was tested in a model of P. aeruginosa mouse eye keratite. C3H / HeN mice were injected intraperitoneally (IP) with PBS or a control IgGl antibody (R347) at 45 mg / kg or WapR-004 (a-Psl) at 5mg / kg or V2L2 (a-PcrV) at 5mg / kg, 16 hours hours before infection with (Ml (011-cytotoxic - Ix106 CFU). Immediately before infection, the mice were anesthetized followed by the initiation of three 1 mm scratches on the cornea and superficial stroma of one eye in each mouse using a 27 gauge needle under a dissecting microscope, followed by topical application of P. aeruginosa 6077 strain in a 5 μl inoculum. visualization of eyes under a dissecting microscope The classification of infection of the chromium was performed as previously described by Preston et al. (Preston, MJ., 1995, Infect. Immun. 63: 3,497). Soon, the infected eyes were classified 48 hours after infection with strain 6077 by an inv stigger who is unaware of animal treatments. The next classification scheme was used: grade 0, eye macroscopically identical to an uninfected eye; grade 1, slight opacity that partially covers the pupil; grade 2, dense opacity covering the pupil; grade 3, dense opacity that covers the entire pupil; grade 4, perforation of the cornea (shrinking of the eyeball). The mice receiving Cam-003 or WapR-004RAD systematically dosed (PI) showed significantly less pathology and reduced bacterial colony forming units (CFU) in total eye homogenates than was observed in the animals treated with control mAb R347. Similar results were seen in animals treated with V2L2 compared to controls treated with R347. Example 9: A Cam-003 Fc mutant antibody, Cam-003-TM, decreased OPK and in vivo efficacy but maintains anti-cell attachment activity.
[0324] Given the potential for dual mechanisms of action, a Cam-003 Fc mutant, Cam-003-TM, was created that nurtures mutations in the Fc domain that reduces its interaction with Fey receptors (Oganesyan, V., et al ., Acta Crystallogr D Biol Crystallogr 64, 700 to 704 (2008)), to identify whether protection was more correlated with anti-cell attachment or OPK activity. The P. aeruginosa mutants were constructed based on the allele replacement strategy described by Schweizer (Schweizer, H.P., Mol Microbiol 6, 1,195 to 1,204 (1992); Schweizer, H.D., Biotechniques 15, 831 to 834 (1993)). The vectors were mobilized from the strain of E. coli SI7.1 in the strain of P. aeruginosa PAO1; recombinants from were isolated as described (Hoang, T.T., et al., Gene 212, 77 to 86 (1998)). The gene deletion was confirmed by PCR. The P. aeruginosa mutants were supplemented with constructs based on pUCP30T that nourish wild-type genes. Figures 9A show that Cam-003-TM exhibited a 4-fold drop in OPK activity compared to Cam-003 (EC50 of 0.24 and 0.06, respectively) but was just as effective in the cell attachment assay ( Figure 9B). Figure 9C shows that Cam-003-TM was also less effective against pneumonia, suggesting that optimal OPK activity is necessary for optimal protection. Cell attachment and OPK assays were performed as previously described in Examples 2 and 4, respectively. Example 10: Epitope mapping and relative affinity for anti-Psl antibodies
[0325] Epitope mapping was performed by ELISA by competition and confirmed using an OCTET * flow system with Psl derived from the supernatant of a culture of P. aeruginosa PAO1 overnight. For competition ELISA, antibodies were biotinylated using Sulfo-NHS-Biotin and EZ-Link Biotinylation Kit (Thermo Scientific). The antigen-coated plates were treated with the EC50 of biotinylated antibodies matched with unlabeled antibodies. After incubation with streptavidin conjugated to HRP (Thermo Scientific), the plates were developed as described above. Competition experiments between anti-Psl mAbs determined that the antibodies targeted at least three unique epitopes, called class 1, 2, and 3 antibodies (Figure 10A). Class 1 and 2 antibodies do not compete for binding, however class 3 antibody, WapR-016, partially inhibits binding of class 1 and 2 antibodies.
[0326] Antibody affinity was determined by OCTET * binding assays using Psl derived from PAO1 culture supernatant overnight. The antibody KD was determined by weighting the binding kinetics of seven concentrations for each antibody. Affinity measurements were taken with a FORTEBIO "OCTET® 384 instrument using plates with 384 inclined wells. Supernatant from PAO1 cultures overnight ± the pslA gene was used as the source of Psl. Samples were loaded onto OCTET * aminopropylsilane sensors (hydrated in PBS) and blocked, followed by measurement of anti-Psl mAb binding in various concentrations, and dissociation in PBS + 1% BS A. All procedures were performed as described (Wang , X., et al., J Immunol Methods 362, 151 to 160). Crude association and dissociation AnM data were fitted in a curve with GraphPad Prism. Figure 10A shows the relative binding affinities of characterized anti-Psl antibodies. Class 2 antibodies had the highest affinities of all anti-Psl antibodies. Figure 10A also shows a summary of cell attachment and OPK data experiments. Figure 10B shows the relative binding affinities and values. of the OPK EC50 of the Wap-004RAD mutant (W4RAD) as well as other leader-optimized W4 mutants by means of site-directed mutagenesis as described in Example 2. Figure 10C shows the relative binding affinities of Wap-004RAD (W4RAD) , germ line Wap-004RAD (W4RAD-GL) as well as leader-optimized anti-Psl monoclonal antibodies (Psl0096, Psl0170, Psl0225, Psl0304, Psl0337, Psl348, Psl0567, Psl0573, Psl0574, Psl0582, Psl0582, Psl0582, Psl0582, Psl0582, Psl0582, Psl0582, Psl0582, Psl0582, Psl0582, Psl0582 . The highlighted clones Psl0096, Psl0225, Psl0337, Psl0567 and Psl0588 were selected based on their enhanced OPK activity, as shown in Example 10 below. Example 11: Evaluation of leader-optimized WapR-004 (W4) mutant clones and leader-optimized anti-Psl monoclonal antibodies in the P. aeruginosa (OPK) opsonophagocytic elimination assay
[0327] This example describes the evaluation of leader-optimized WapR-004 (W4) mutant clones and leader-optimized anti-Psl monoclonal antibodies to promote P. aeruginosa OPK using the method described in Example 2. The Figures 11A-Q show that with the exception of the negative control antibody R347, all antibodies mediated the concentration-dependent elimination of luminescent P. aeruginosa serogroup 05 strain (PAOl.lux). Example 12: Anti-PcrV monoclonal antibody V2L2 reduces the lethality of acute pneumonia from multiple strains
[0328] PcrV epitope diversity was analyzed using three approaches: microsphere-based flow cytometry method, competition ELISA and fragmented western blotting of rPcrV. Competition experiments between anti-PcrV mAbs determined that the antibodies targeted at least six unique epitopes, called class 1, 2, 3, 4, 5 and 6 antibodies (Figure 12A). Class 2 and 3 antibodies partially compete for binding. The mAbs that represent additional epitope classes: class 1 (V2L7, 3G5, 4C3 and 11A6), class 2 (1E6 and 1F3), class 3 (29D2, 4A8 and 2H3), class 4 (V2L2) and class 5 (21F1, LEIO and SH3) were staged for in vivo protection as described below.
[0329] The innovative anti-PcrV mAbs were isolated using hybridoma technology and the most potent T3SS inhibitors were selected using a rabbit red blood cell lysis inhibition assay. The percentage of inhibition of cytotoxicity analysis was analyzed for V2L2 parental mAb, mAbl66 (positive control) and R347 (negative control), in which antibodies were administered to the cultured bronchial epithelial cell line A549 combined with phase P. aeruginosa 6077 strain log (exoU +) at a MOI of approximately 10. The lysis of A549 was tested by measuring the activity of released lactate dehydrogenase (LDH) and the lysis in the presence of mAbs was compared to wells without mAb to determine the percentage of inhibition. V2L2 mAb, mAb 166 (positive control) and R347 (negative control) were evaluated for their ability to prevent lysis of RBCs, in which antibodies were mixed with P. aeruginosa 6077 log phase (exoU) and washed blood cells red rabbits (RBCs) and incubated for 2 hours at 37 °. The intact RBCs were pelleted and the extent of lysis determined by measuring the OD405 of the cell free supernatant. The present lysis of anti-PcrV mAbs was compared to wells without mAb to determine the percentage of inhibition. The positive control antibody, mAb 166, is an anti-PcrV antibody previously characterized (JInfect Dis. 186: 64 to 73 (2002), Crit Care Med. 40: 2,320 to 2,326 (2012)). (B) The mAb with Parental V2L2 demonstrated inhibition of cytotoxicity with an IC50 of 0.10 μg / ml and exhibited an IC50 concentration 28 times lower than 166 mAb (IC50 of 2.8 μg / ml). (C) V2L2 also demonstrated prevention of RBC lysis with an IC50 of 0.37 μg / ml and exhibited an IC50 concentration 10 times lower than 166 mAb (IC50 of 3.7 μg / ml).
[0330] The V2L2 variable region has been fully germinated to reduce potential immunogenicity. V2L2 was affinity matured using the parsimony mutagenesis approach to randomize each position with 20 amino acids for all six CDRs, identifying single affinity-enhanced mutations. A combinatorial library was then used, encoding all possible combinations of single affinity-enhanced mutations. Clones with enhanced PcrV affinity were selected with the use of ELISA binding in IgG format. The upper clones were classified by affinity enhancement and analyzed in a functional in vitro assay. V2L2 CDRs were systematically mutagenized and clones with enhanced PcrV affinity were selected in competition-based screens. The clones were classified by increases in affinity and analyzed in a functional assay. As shown in Figure 12D, RBC lysis was analyzed for MAb with V2L2 (V2L2-GL) germline, V2L2-GL-optimized mAbs (V2L2-P4M, V2L2-MFS, V2L2-MD and V2L2-MR), and one negative control antibody R347 using A549 cells infected by Pseudomonas 6077 strain. V2L2-GL, V2L2-P4M, V2L2-MFS, V2L2-MD and V2L2-MR demonstrated prevention of RBC lysis. As shown in Figure 12E, mAbs 1E6, 1F3, 11A6, 29D2, PCRV02 and V2L7 demonstrated prevention of RBC lysis. As shown in Figure 12F, V2L2 was more potent at preventing RBC lysis than 29D2.
[0331] V2L2-GL and V2L2-MD binding kinetics were measured using a Bio-Rad ProteOn ™ XPR36 instrument. The antibodies were captured on a GLC biosensor chip using anti-human IgG reagents. The rPcrV protein was injected in multiple concentrations and the dissociation phase continued for 600 seconds. The data were captured and analyzed using ProteOn Manager software. Figure 12 (G-H) shows the relative binding affinities of the (G) V2L2-GL and (H) V2L2-MD antibodies. The clone V2L2-MD increased Kd by 2 to 3 times over V2L2-GL.
[0332] The in vivo effect of administering an anti-PcrV antibody was studied in mice using an acute pneumonia model. The groups of mice were treated with either increasing concentrations of the V2L2 antibody, an anti-PcrV positive control antibody (mAb 166), or a negative control (R347), as shown in Figure 13 (A-B). The groups of mice were also treated with either increasing concentrations of the V2L2 antibody, the PcrV antibody PcrV-02, or a negative control (R347), as shown in Figure 13 (C-D). Twenty-four hours after treatment, all mice were infected with 5x10 CFU (C) Pseudomonas aeruginosa 6294 (06) or (D) PA103A (011). As shown in Figure 13, almost all animals treated for control succumbed to infection within 48 hours post infection. However, V2L2 showed a dose-dependent effect on improved survival even at 168 hours post infection. In addition, V2L2 provided significantly more potent protection than mAbl66 at similar doses (P = 0.025, 5 mg / kg for strain 6077; P <0.0001, 1 mg / kg for strain 6294).
[0333] The groups of mice were treated with increasing concentrations of 11A6, 3G5 or V2L7, the same concentrations of 29D2, 1F3, 1E6, V2L2, LEIO, SH3, 4A8, 2H3, or 21F1, increasing concentrations of 29D2, increasing concentrations of V2L2, the PcrV antibody PcrV-02, or a negative control (R347), as shown in Figure 13 (EH). The mice were injected intraperitoneally (IP) with mAbs 24 hours before intranasal infection with Pseudomonas 6077 strain (1 x 106 CFU / animal). As shown in Figure 13E mAbs 11A6, 3G5 and V2L7 did not provide protection in vivo. As shown in Figure 13F, mAb 29D2 provides in vivo protection. As shown in Figure 13G, mAb V2L2 also provides in vivo protection. Figure 13H shows the in vivo comparison of 29D2 and V2L2. Figure 131 shows that mAb V2L2 protects against additional Pseudomonas strains (i.e., 6294 and PA 103A).
[0334] The lung load of mice infected with Pseudomonas has also been studied in response to the administration of V2L2. The mice in Figure 14 (A) were treated with 1 mg / kg of R347 (control), or 1 mg / kg, 0.2 mg / kg, or 0.07 mg / kg of V2L2 and were then infected intranasally with 1 , 2 x 106 cfu of Pseudomonas 6206. The mice in Figure 14 (B) were also treated with 15 mg / kg of R347 (negative control); 15.0 mg / kg, 5.0 mg / kg, or 1.0 mg / kg of mAb 166 (positive control); or 5.0 mg / kg, 1.0 mg / kg, or 0.2 mg / kg of V2L2 and then were intranasally infected with 5.5 x 106 cfu of Pseudomonas 6206. As shown in Figure 14 (AB), although V2L2 has little effect on cleansing in the kidney, it greatly reduced the spread both in the lung and the spleen in a dose-dependent manner. In addition, V2L2 provided a significantly greater reduction in organ CFU than mAb 166 at similar doses (P <0.0001, 1 mg / kg, lung). Example 13: In vivo combination therapy activity using WapR-004 (anti-Psl) and V2L2 (anti-PcrV) antibodies
[0335] The in vivo effect of administering the combination of anti-Psl and anti-PcrVs binding domain was further studied in mice using the antibodies V2L2 and WapR-004 (RAD). Groups of mice were treated with R347 (2.1 mg / kg - negative control), V2L2 (0.1 mg / kg), W4-RAD (0.5 mg / kg), or a combination of V2L2 / W4 (both 0 , 1, 0.5, 1.0 or 2.0 mg / kg each). Twenty-four hours after antibody administration, all mice were infected with an inoculum containing 5.25 x 103 cfu 6206 (Oll-ExoU +). Twenty-four hours after infection, lungs, spleens, and kidneys were collected, homogenized, and laminated to a colony-forming unit (CFU) identification per gram of tissue. As shown in Figure 15, at the concentrations tested, both V2L2 and W4 were effective in decreasing organ load, the combination of V2L2 / W4 showed an additive effect on tissue clarity. Histological examination of lung tissue revealed less hemorrhage, less edema, and less inflammatory infiltrate compared to mice receiving V2L2 or WapR-004 alone (Table 5).
[0336] Animals similarly immunized were also analyzed for survival from acute pneumonia infections. Table 5

[0337] Example 14: Survival rate for animals treated with V2L2 monoclonal anti-PcrV antibody in a model of acute P. aeruginosa pneumonia
[0338] V2L2-GL, V2L2-MD, V2L2-A, V2L2-C, V2L2-PM4 and V2L2-MFS monoclonal antibodies were evaluated in a model of acute lethal pneumonia in P. aeruginosa strain 6077 as previously described in Example 11 Figures 16 (A to F) show survival in all mice treated with V2L2 infected with the 6077 strain when compared to the control. However, no significant difference in survival is observed between V2L2 antibodies, either at the dose: 0.5 mg / kg or at the dose of 1 mg / kg (A to C) or 0.5 mg / kg and 0.1 mg / kg ( DF). Figures 16 (G to I) show survival in all mice treated with V2L2 infected with strain 6077 when compared to the control, no significant difference in survival is observed between V2L2 antibodies in both dose: 0.5mg / kg and dose 1 mg / kg (GI). (A to H)
[0339] All control mice succumbed to infection for approximately 48 hours after infection. Example 15: Construction of bispecific antibodies to WapR-004 / V2L2
[0340] Figure 17A shows TNFo. bispecific model constructs. For Bsl-TNFot / W4, the W4 scFv is fused to the amino terminal of TNFot VL via a linker (G4S) 2. For Bs2-TNFot / W4, the W4 scFv is fused to the amino terminal of TNFoc VH via a (G4S) 2 linker. For Bs3-TNFot / W4, the W4 scFv is fused to the CH3 carboxy-terminal through a (G4S) 2 linker.
[0341] Since the combination of WapR-004 + V2L2 provides protection against Pseudomonas challenge, bispecific constructs were generated that comprise a WapR-004 scFv (W4-RAD) and V2L2 IgG (Figure 17B). To generate Bs2-V2L2-2C, the W4-RAD scFv is fused to the N-terminal of VH V2L2 via ligand (G4S) 2. To generate Bs3-V2L2-2C, W4-RAD scFv was fused to the CH3 C-terminus via ligand (G4S) 2. To generate Bs4-V2L2-2C, the W4-RAD scFv was inserted in the hinge region, connected by the ligand (G4S) 2 in N-terminal and C-terminal of scFv. To generate Bs2-W4-RAD-2C, the V2L2 scFv was fused to the amino terminal of VH W4-RAD through a linker (G4S) 2.
[0342] To generate the W4-RAD scFv for the Bs3 construct, the W4-RAD VH and VL were amplified by PCR. The primers used to amplify the W4-RAD VH were: advanced VH primer W4-RAD: includes ligand (G4S) 2 and 22bp of VH N-terminal sequence (GTAAAGGCGGAGGGGGATCCGGCGGAGGGGGCTCTGAGGTGCAG CTGTTGGAGTCGGTGGTCGGT) and W4-RAD VH reverse primer: includes part of ligand (G4S) 4 and 22 bp of VH C-terminal sequence (GATCCTCCGCCGCCGCTGCCCCCTCCCCCAGAGCCCCCTCCGCCA CTCGAGACGGTGACCAGGGTC (SEQ ID NO: 225). by PCR using the primers: advanced VL primer W4-RAD: includes part of ligand (G4S) 2 and 22 bp N-terminal sequence VL (AGGGGGCAGCGGCGGCGGAGGATC TGGGGGAGGGGGCAGCGAAATTGTGTTGACACAGTCTCTCTGGACACAGTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCAGTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCT VL W4-RAD: includes part of vector sequence and 22 bp of VL C-terminal sequence (CAATGAATTCGCGGCCGCTCATTTGATCTCCAGCTTGGTCCCAC SEQ ID NO: 227)). The overlapping fragments were then fused together to form the W4-RAD scFv. W4-RAD sequence of the scFv vector BS3: G4S linker sequences are underlined GGGGSGGGGSEVQLLESGPGLVKPSETLSLTCNVAGGSISPYYWTWIR QPPGKCLELIGYIHSSGYTDYNPSLKSRVTISGDTSKKQFSLHVSSVTA ADTAVYFCARADWDLLHALDIWGQGTLVTVSSGGGGSGGGGSGGG GSGGGGSEIVLTQSPSSLSTSVGDRVTITCRASQSIRSHLNWYQQKPGK APKLLIYGASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQS YSFPLTFGCGTKLEIK (SEQ ID NO: 228)
[0343] After the scFv W4-RAD fragment was amplified, it was then purified with gel and ligated to the Bs3 vector which had been digested with BamHI / NotI. The connection was made using the InFusion system, followed by transformation into competent Stellar cells. The colonies were sequenced to confirm the correct insertion of W4-RAD scFv.
[0344] To generate Bs3-V2L2-2C, the IgG portion in the Bs3 vector was replaced by V2L2 IgG. Briefly, the Bs3 vector containing W4-RAD scFv was digested with BssHII / SalI and the resulting vector band was gel purified. Similarly, the vector containing V2L2 vector was digested with BssHII / SalI and the V2L2 insert was gel purified. The V2L2 insertion was then linked with the Bs3-W4-RAD scFv vector and colonies were sequenced to confirm correct V2L2 IgG insertion.
[0345] A similar approach was used to generate Bs2-V2L2-2C. Sequences of VH scFv-W4-RAD V2L2 the Bs2 vector: G4S linker sequences are underlined EVQLLESGPGLVKPSETLSLTCNVAGGSISPYYWTWIRQPPGKCLELIG YIHSSGYTDYNPSLKSRVTISGDTSKKQFSLHVSSVTAADTAVYFCAR ADWDLLHALDIWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVL TQSPSSLSTSVGDRVTITCRASQSIRSHLNWYQQKPGKAPKLLIYGASN LQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSFPLTFGCGT KLEIKGGGGSGGGGSEMQLLESGGGLVOPGGSLRLSCAASGFTFSSY AMNWVRQAPGEGLEWVSAITISGITAYYTDSVKGRFTISRDNSKNTL YLQMNSLRAGDTAVYYCAKEEFLPGTHYYYGMDVWGQGTTVTVSS (SEQ ID NO: 229)
[0346] The following primers were used to amplify W4-RAD scFv. (advanced primer) VH and (reverse primer) VL: advanced VH primer W4-RAD for vector Bs2 which includes some introns, signal peptide 3 and 22bp of VH N-terminal sequence W4-R.AD (TTCTCTCCACAGGTGTACACTCCGAGGTGCAGCTGTTGGAGTCGGTTGGAGTCG (SEQ ID NO: 230)) and the VL reverse primer W4-RAD for Bs2 vector: include ligand (G4S) 2 and 32 bp of VL C-terminal sequence (ccTCTCCGCCGGATCCCCCTCCGCCTTTGATCTCCAGCTTGGTCC CACAGCCGAAAG (SEQ ID NO: 231)
[0347] To amplify the VH V2L2 region, the following primers were used: advanced VH V2L2 primer: includes ligand (G4S) 2 and 22 bp of V2L2 VH N-terminal sequence (GGCGGAGGGGGATC CGGCGGAGGGGGTCTGGGGG 232)), and VH V2L2 reverse primer: includes some of the CH 1 N-terminal sequence and 22 bp of VH V2L2 C-terminal sequence (ATGGGCCCTTGGTCGACGCTGAGGAGACGGTGACCGTGGTC (SEQ ID NO: 233)).
[0348] These primers were then used to amplify VH V2L2, which was then joined by overlapping with W4-RAD scFv and VH V2L2 to obtain W4-RAD scFv-V2L2-VH. The W4-RAD scFv-VH V2L2 was then linked to the Bs2 vector by purification with gel W4-RAD scFv -VH V2L2 (from the superimposed PCR); digestion of the Bs2 vector with BsrGI / SalI, and gel purification vector band. The W4-RAD scFv-V2L2-VH was then linked to the Bs2 vector by the In-Fusion system and transformed into competent Stellar cells and the colonies were confirmed for the correct insertion of W4-RAD scFv-VH V2L2. To replace VL in the Bs2 vector with VL V2L2, the Bs2 vector containing W4-RAD scFv-V2L2-VH was digested with BssHII / BsiWI and the vector band was gel purified. The pOE-V2L2 vector was then digested with BssHII / BsiWI and the VL V2L2 insert was gel purified. The VL V2L2 insertion was then linked with the Bs2-W4-RAD scFv-V2L2-VH vectors and the colonies were sequenced for correct V2L2 IgG insertion.
[0349] Finally, a similar PCR-based approach was used to generate the Bs4-V2L2-2C construct. The hinge region with ligand sequence is shown below: Hinge region with ligand sequence: KVDKRVEPKSCGGGGSGGGGS - scFv N termination (SEQ ID NO: 329) CHI scFv C-terminal ligand hinge - GGGGSGGGGSDKTHTCPPCPAPELL (330) binder hinge CH2 Sequence W4-RAD scFvs in vector BS4: W4-RAD scFv is in italics and bold with G4S linker underlined in italics bold ', hinge regions are doubly underlined KVDKRV1EPKSCGGGGSGGGGSE VQLLESGPGL VKPSE TLSL TCNVA GGSISPYYWTWIRQPPGKCLELIGYIHSSGYTD YNPSLKSR VTISGD TSK KQFSLHVSSVTAADTA VYFCARADWDLLHALDIWGQGTL VTVSSGGG GSGGGGSGGGGSGGGGSEIVL TQSPSSLSTSVGDR VTITCRASQSIRSH LNWYQQKPGKAPKLLIYGASNLQSGVPSRFSGSGSGTDFTLTISSLQPE DF TYYCQQSYSFPL TFGCGTKLEIKGGGGSGGGGS DKTHTCPPCPkYYYUSYÁ) ID NO: 324) W4-RAD scFv is shown in italics and bold with G4S binders underlined in italics bold and VQLLESGPGL VKPSETLSL TCNVA GGSISPYYWTWIRQPPGKCLELIG YIHSSGYTDYNPSLKSRVTISGDTSKKQFSLHVSSVTAADTAVYF CARA D WDLLHALDIWGQGTL VTVSSGGGGGGGGGSGGGGSGGGGSEI VL T QSPSSLSTSVGDRVTITCRASQSIRSHLNWYQQKPGKAPKLLIYGASNL QSGVPSRFSGSGSGTQTFFTL LEIK
[0350] W4-RAD scFv was generated using PCR and the following primers: advanced VH primer W4-RAD for vector Bs4: includes some of the ligand sequences and 24 bp N-terminal sequence of VH W4- RAD (GAGGTGCAGCTGTTGGAGTCGGGC (SEQ ID NO: 236)); and VL reverse primer W4-RAD for vector Bs4: includes some hinge sequence, ligand and 21 bp of W4-RAD VL C-terminal sequence (GTGTGAGTTTTGTCggatccCCCTCCGCCAGAGCCACCTCCGCCTTTG ATCTCCAGCTTGGTCCC (SEQ ID NO: 23).
[0351] W4-RAD scFv was then ligated into the Bs4 vector to obtain Bs4-V2L2-2C by purification with W4-RAD scFv gel (from PCR); the Bs4-V2L2 vector was digested with BamHI and the vector band was gel purified. The W4-RAD scFv was ligated with Bs4 vector by the In-Fusion system and the vector transforms competent Stellar cells. The colonies were sequenced for the correct insertion of W4-RAD scFv.
[0352] The sequences for the light chain and heavy chain of the construct Bs4-V2L2-2C are provided in SEQ ID NOS: 327 and 328, respectively. Example 16: A bispecific Psl / PcrV antibody promotes survival in models of pneumonia
[0353] As an initial question, bispecific antibodies Bs2 and Bs3 were tested to see whether they retained their W4 or V2L2 activity in a bispecific format. For parental W4 scFv, a bispecific antibody having W4 and a TNF-alpha binding arm was generated. A cell attachment analysis was performed as described above with the use of the luminescent strain P. aeruginosa PAOl.lux. As shown in Figure 18, all bispecific constructs performed similarly to the parent W4-IgGl construct.
[0354] As shown in Figure 19 (A to C), the inhibition of cytotoxicity percentage was analyzed for both Bs2-V2L2 and Bs3-V2L2 with the use of both (A) 6206 and (B) 6206 cells infected with ΔpslA, and (C) inhibition of RBC lysis percentage was analyzed for Bs2-V2L2-2C, Bs3-V2L2-2C and Bs4-V2L2-2C using 6206 infected cells. As shown in Figure 19 (A to C), all bispecific antibodies retained anti-cytotoxicity activity and inhibited RBC lysis at levels similar to the parental V2L2 antibody using cells infected with ΔpslA 6206 and 6206.
[0355] The ability of bispecific antibodies Bs2 and Bs3 to mediate OPK of P. aeruginosa was analyzed using the method described in Example 2. Although the Bs2-V2L2 antibody has shown similar elimination compared to the parental W4-RAD antibody, elimination of Bs3-V2L2 antibody was decreased (Figure 20A). Although the Bs2-V2L2-2C and Bs4-V2L2-2C antibodies have shown similar elimination compared to the parental W4-RAD antibody, the elimination of the Bs3-V2L2-2C antibody has been decreased (Figure 20B). Figure 20C shows that different preparations of Bs4 antibodies (old portion vs. new portion) showed similar elimination compared to the parental W4-RAD antibody, however, Bs4-V2L2-2C-YTE antibodies had a 3-fold drop in the activity of OPK when compared to Bs4-V2L2-2C. A YTE mutant comprises a combination of three "YTE mutations": M252Y, S254T, and T256E, where the numbering is in accordance with the EU index as presented in Kabat, introduced into the heavy chain of an IgG. See U.S. Patent Document No. 7,658,921, which is incorporated by reference into this document. The YTE mutant has been shown to increase the serum half-life of antibodies by approximately four times as compared to wild-type versions of the same antibody. See, for example, DalfAcqua et al., J. Biol. Chem. 281: 23514-24 (2006) and U.S. Patent Document 7,083,784, which are hereby incorporated by reference in their entirety.
[0356] Following confirmation that both W4 and V2L2 retained activity in a bispecific format, the Bs2-V2L2, Bs3-V2L2 and Bs4-V2L2 constructs were analyzed for survival from acute pneumonia infections. As shown in Figure 21A, all control mice succumbed to infection for approximately 30 hours after infection. All Bs3-V2L2 animals survived, along with those that received V2L2 control. Approximately 90% of animals immunized with W4-RAD survived. In contrast, Figures B to F show that approximately 50% of the animals in Bs2-V2L2 succumbed to infection for 120 hours. All control mice succumbed to the infection for approximately 48 hours after infection. Figures G-H do not differ in survival between mice treated with Bs4-V2L2-2C and Bs4-V2L2-2C-YTE in both doses. These results suggest that both antibodies function equivalent in the 6206 acute pneumonia model. Figure 21 I shows that the Bs2-V2L2, Bs4-V2L2-2C, and W4-RAD + V2L2 antibody mixture is the most effective in protecting against lethal pneumonia in mice stimulated with the 6206 strain of P. aeruginosa (ExoU +).
[0357] The organ load was also analyzed for similar immunized mice as described above. Following the immunization as above, the mice were stimulated with 2.75 x 103 CFU 6206. As shown in Figure 22, in the tested concentration, both Bs2-V2L2 and Bs3-V2L2 significantly decreased the organ load in the lung. However, none of the bispecific constructs was able to significantly affect the human load in the spleen or kidney compared to parental antibodies due to the use of sub-ideal concentrations of the bispecific constructs. Sub-ideal concentrations were used to allow the ability to decipher antibody activity.
[0358] The survival and organ load effects of bispecific antibodies were also directed to the use of strain 6294. Using the model 6294 system, both BS2-V2L2 and BS3-V2L2 significantly decreased the organ load in all tissues at a level comparable to that of the parental V2L2 Antibody. The parental W4-RAD antibody had no effect in decreasing organ load (Figure 23A). As shown in Figure 23B, Bs2-V2L2, Bs3-V2L2, and the combination W4-RAD + V2L2 significantly decreased the organ load in all tissues to a level comparable to that of the parental V2L2 antibody.
[0359] Survival data for immunized mice was similar to mice stimulated with 6294 as before. As shown in Figure 24, BS3-V2L2 showed similar survival activity to mice treated with V2L2 alone, while BS2-mice treated with V2L2 showed a slightly lower level of stimulus protection.
[0360] Organ load was also analyzed in bispecific antibodies treated in comparison with animals treated with combination as described above. As shown in Figures 25 (A to C), both BS2-V2L2 and BS3-V2L2 decreased the organ load in the lung, spleen and kidneys to a level comparable to that of the W4 + V2L2 combination. In the lung, the combination significantly reduced bacterial CFUs Bs2- and Bs3- V2L2 and V2L2 with the use of Kruskal-Wallis with Dunn's post-test. Significant differences in bacterial load in the spleen and kidney were not observed, although a tendency towards reduction was noted. A study of organ load was also performed with Bs4-GLO with the use of 6206 in the pneumonia model. As shown in Figure 25 (D), when higher concentrations of antibody are used in the prophylaxis of mice, a significant level (Kruskal-Wallis with Dunn's Post test) of reduction in the bacterial load of the lung was observed. Significant reductions in bacterial spread to the spleen and kidneys were also observed when using higher concentrations of Bs4-GLO in this model.
[0361] These results were confirmed by histological examination of lung tissue from mice immunized with BALB / c stimulated with 1.33x10 CFU using Cepa 6294 from P. aeruginosa (Table 6A), 1.7x10 CFU using Cepa 6294 of P. aeruginosa (Table 6B) and 9.25 x 10 CFU with the use of P. aeruginosa 6206 strain (Table 7). Example 17: Therapeutic adjunctive therapy: Bs4-V2L2-2C + antibiotic
[0362] Survival effect of bispecific antibody Bs4 and adjunctive antibiotic therapy was evaluated in a model of acute lethal pneumonia against P. aeruginosa 6206 strain as previously described in Example 6 (Figure 26 (A to J)). (A to B) Mice were treated 24 hours before infection with 6206 with R347 (negative control) or Bs4-V2L2-2C or Ciprofloxacin (CIP) 1 hour after infection, or a combination of Bs4-V2L2-2C 24 hours before infection and Cipro 1 hour after infection. (C) The mice were treated 1 hour after infection with 6206 with R347 or CIP or Bs4-V2L2-2C, or a combination of Bs4-V2L2-2C and CIP. (D) Mice were treated 2 hours after infection with 6206 with R347 or CIP or Bs4-V2L2-2C, or a combination of Bs4-V2L2-2C and CIP. (E) The mice were treated 2 hours after infection with 6206 with R347 or Bs4-V2L2-2C or CIP 1 hour after infection, or a combination of Bs4-V2L2-2C 2 hours after infection and CIP 1 hour after infection. infection. (F) The mice were treated 1 hour after infection with 6206 with R347 or Meropenem (MEM) or Bs4-V2L2-2C, or a combination of Bs4-V2L2-2C and MEM. (G) Mice were treated 2 hours after infection with 6206 with R347 or Bs4-V2L2-2C or MEM 1 hour after infection, or a combination of Bs4-V2L2-2C 2 hours after infection and MEM 1 hour after infection. infection. (H) The mice were treated 2 hours after infection with 6206 with R347 or Bs4-V2L2-2C or MEM, or a combination of Bs4-V2L2-2C 2 and MEM. (I) The mice were treated 4 hours after infection with 6206 with R347 or Cipro or Bs4-V2L2-2C or a combination of Bs4-V2L2-2C and Cipro. All control mice succumbed to the infection for approximately 24 hours after infection. As shown in Figures 26 (A to I) the Bs4 antibody combined with both CIP and MEM increases the effectiveness of antibiotic therapy, indicating synergistic protection when the molecules are combined. Additional studies focused on the level of bacterial load in mice treated with Bs4 or CIP alone or in combination (Bs4 + CIP). As shown in Figure 26 (J), the level of bacterial load in all organs (lung, spleen and kidneys) was similar in R347 + CIP and Bs4 + CIP, however, only the mice in which Bs4 was included in the combination with CIP survive the infection (Figures 26 (A through E, I)). In general, these data indicate that antibiotics are important for reducing bacterial load in this animal model adjustment, however, the specific antibody is required to reduce bacterial pathogenicity, thereby protecting the normal host's immunity.
[0363] The survival effect of bispecific Bs4 antibody and adjunctive antibiotic therapy Tobramycin will be evaluated in a model of acute lethal pneumonia against P. aeruginosa 6206 strain as previously described in Example 6. Mice will be treated 24 hours before infection with 6206 with R347 (negative control) or Bs4-V2L2-2C or Tobramycin 1 hour after infection, or a combination of Bs4-V2L2-2C 24 hours before infection and Tobramycin 1 hour after infection. Mice will also be treated 1 hour after infection with 6206 with R347 or Tobramycin or Bs4-V2L2-2C, or a combination of Bs4-V2L2-2C and Tobramycin. In addition, mice will be treated 2 hours after infection with 6206 with R347 or Tobramycin or Bs4-V2L2-2C, or a combination of Bs4-V2L2-2C and Tobramycin. In addition, mice will be treated 2 hours after infection with 6206 with R347 or Bs4-V2L2-2C or Tobramycin 1 hour after infection, or a combination of Bs4-V2L2-2C 2 hours after infection and Tobramycin 1 hour after infection . The mice will be treated 4 hours after infection with 6206 with R347 or Tobramycin or Bs4-V2L2-2C or a combination of Bs4-V2L2-2C and Tobramycin.
[0364] The survival effect of bispecific Bs4 antibody and adjunctive antibiotic therapy Aztreonam will be evaluated in a model of acute lethal pneumonia against the 6206 strain of P. aeruginosa as previously described in Example 6. Mice will be treated 24 hours before infection with 6206 with R347 (negative control) or Bs4-V2L2-2C or Aztreonam 1 hour after infection, or a combination of Bs4-V2L2-2C 24 hours before infection and Aztreonam 1 hour after infection. Mice will also be treated 1 hour after infection with 6206 with R347 or Aztreonam or Bs4-V2L2-2C, or a combination of Bs4-V2L2-2C and Aztreonam. In addition, the mice will be treated 2 hours after infection with 6206 with R347 or Aztreonam or Bs4-V2L2-2C, or a combination of Bs4-V2L2-2C and Aztreonam. In addition, mice will be treated 2 hours after infection with 6206 with R347 or Bs4-V2L2-2C or Aztreonam 1 hour after infection, or a combination of Bs4-V2L2-2C 2 hours after infection and Aztreonam 1 hour after infection . The mice will be treated 4 hours after infection with 6206 with R347 or Aztreonam or Bs4-V2L2-2C or a combination of Bs4-V2L2-2C and Aztreonam. Example 18: Construction of the bispecific BS4-GLO antibody
[0365] The bispecific construct of BS4-GLO (Optimized Germ Orientation) was generated, which comprises anti-Psl scFv (Psl0096 scfv) and V2L2-MD (VH + VL) as shown in Figure 35A. The BS4-GLO light chain comprises a light chain variable region (i.e., V2L2-MD) of germ-oriented anti-PcrV antibody. The BS4-GLO heavy chain comprises the formula VH-CH1-H1-L1-S-L2-H2-CH2-CH3, where CHI is a heavy chain constant region domain-1, H1 is a first region fragment heavy chain hinge, LI is a first linker, S is an anti-PcrV ScFv molecule, L2 is a second linker, H2 is a second heavy chain hinge region fragment, CH2 is a constant region-2 domain of heavy chain, and CH3 is a heavy chain constant region domain-3. light chain Bs4-GLO: AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLI YSASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDYNYPW TFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: ...) light chain variable region V2L2 (ie V2L2-MD) of GLO (optimized germ orientation) is underlined heavy chain Bs4-GLO : EMQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGEGLE WVSAITISGITAYYTDSVKGRFTISRDNSKNTLYLQMNSLRAGDTAVY YCAKEEFLPGTHYYYGMDVWGQGTTVTVSS1ASTKGPSVFPLAPSSK STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSSLGTOTYICNVNHKPSNTKVDKRVIEPKSÇGGGG.SGG 'GGSEVQLLESGPGL VKPSETLSL TCNVA GGSISPYYWTWIRQPPGKCL ELIGYIHSSGYTDYNPSLKSR VTISGDTSKKQFSLHVSSVTAADTA VYF CARAD WDLLHALDIWGQGTL VTVSSGGGGSGGGGSGGGGSGGGGSE IVL TQSPSSLSTSVGDR VTITCRASQSIRSHLNWYQQKPGKAPKLLFYG ASNLQSG VPSRFSGSGSGTDFTL TISSLQPEDFA TYYCQQSYSFPL TFG CGR # £ E // fGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LM1SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK heavy chain variable region of V2L2 (ie V2L2- MD) of GLO (optimized germ orientation) is underlined; CHI is enclosed in square brackets []; W4-RAD (ie Psl0096) of GLO (optimized germination orientation) scFv is in bold italics with the G4S ligands underlined in bold italics, hinge regions are double underlined,
[0366] A bispecific alternative Bs4-GLO construct comprising an anti-PcrV ScFv and an anti-Psl (VH + VL) is shown in Figure 35B, and similarity is generated. Example 19: Evaluation of the functional activity and efficacy of the bispecific Bs4-GLO antibody
[0367] Bispecific Bs4-WT antibodies (also referred to herein as Bs4-V2L2-2C), Bs4-GL (comprising anti-PcrV and anti-Psl variable regions) and Bs4-GLO produced as described in Example 18 were tested for differences in functional activity in an opsonophagocytic elimination assay (Figure 27A), as previously described in Example 2, anti-cellular attachment analysis (Figure 27B), as previously described in Example 4, and an anti-cytotoxicity lysis assay RBC (Figure 27C), as previously described in Example 12. No in vitro difference in functional activities between antibodies was observed.
[0368] The in vivo efficacy of Bs4-GLO was examined as follows. For prophylactic evaluation, the mice were prophylactically treated with different concentrations of Bs4-GLO (ie 0.007mg / kg, 0.02mg / kg, 0.07mg / kg, 0.2mg / kg, 0.5mg / kg, 1 mg / kg, 3mg / kg, 5mg / kg, 10mg / kg or 15mg / kg) (Figure 28A), 24 hours before infection with the following P. aeruginosa strains (6206 (1.0 x 106), 6077 (1.0 x 106), 6294 (2.0 x 107) or PA 103 (1.0 x 106)). For therapeutic evaluation, the mice were therapeutically treated with different concentrations of Bs4-GLO (ie, 0.03mg / kg, 0.3mg / kg, 0.5mg / kg, 1mg / kg, 2mg / kg, 5mg / kg, 10mg / kg, 15mg / kg, or 45mg / kg) (Figure 28B), one hour after infection with the following P. aeruginosa strains (6206 (1.0 x 106), 6077 (1.0 x 106) ), 6294 (2.0 x 107) or PA103 (1.0 x 106)).
[0369] The survival effect of the bispecific Bs4-GLO antibody was evaluated in a model of acute lethal pneumonia against different strains of P. aeruginosa as previously described in Example 6. Figure 29 shows survival rate for animals treated with Bs4- GLO in a model of lethal bacteremia of P. aeruginosa. The aspects of the bacteremia model are revealed in detail in provisional application No. US 61 / 723,128, filed on November 6, 2012 (Attorney Dossier Number ATOX-500P1, entitled “METHODS OF TREATING S. AUREUS ASSOCIATED DISEASES”) , which is incorporated into this document as a reference in its entirety.
[0370] Animals were treated with Bs4-GLO or R347, 24 hours before intraperitoneal infection with (A) 6294 (06) or (B) 6206. BS4-GLO is effective at all concentrations tested for protection against lethal pneumonia in mice stimulated with strains of P. aeruginosa (A) 6294 and (B) 6206.
[0371] The survival effect of the bispecific Bs4-GLO antibody was evaluated in a model of thermal injury by P. aeruginosa against different strains of P. aeruginosa. Figure 30 shows the survival rate for animals prophylactically treated with Bs4-GLO in a model of thermal injury by P. aeruginosa. The animals were treated with Bs4-GLO or R347 hours before the induction of thermal injury and subcutaneous infection with P. aeruginosa (A) 6077 (OH-EXOLT) OR (B) 6206 (OII-EXOLT) OR (C) 6294 (06) directly under the wound. BS4-GLO is effective in all concentrations tested for prevention in a model of thermal injury by P. aeruginosa in mice stimulated with strains of P. aeruginosa (A) 6077, (B) 6206 and (C) 6294.
[0372] Figure 31 shows the survival rate for animals therapeutically treated with bispecific Bs4-GLO antibody in a P. aeruginosa thermal injury model. (A) The animals were treated with Bs4-GLO or R347 (A) 4 hours or (B) 12 hours after induction of thermal injury and subcutaneous infection with P. aeruginosa strain 6077 (OI 1-EXOLC) directly under the wound . Bs4-GLO is effective at all concentrations tested in treatment in a model of thermal injury by P. aeruginosa in mice treated with Bs4-GLO (B) 4 hours or (B) 12 hours after induction of thermal injury and subcutaneous infection with P. aeruginosa 6077 strain. Example 20: Therapeutic adjunctive therapy: Bs4-GLO + antibiotic
[0373] The survival effect of bispecific Bs4-GLO antibody and antibiotic adjunctive therapy were evaluated in a model of acute lethal pneumonia against P. aeruginosa 6206 strain as previously described in Example 6.
[0374] Figure 32 shows adjunctive therapy with ciprofloxacin (CIP). (A) Mice were treated 4 hours after infection with P. aeruginosa 6206 strain with R347 + CIP or Bs4-WT or a combination of Bs4-WT and CIP. (B) Mice were treated 4 hours after infection with strain 6206 of P. aeruginosa with R347 + CIP or Bs4-GLO or a combination of Bs4-GLO and CIP. (A-B) The Bs4-WT or BS4-GLO antibody combined with CIP increased the effectiveness of antibiotic therapy.
[0375] Figure 33 shows adjunctive therapeutic therapy with meropenem (MEM): (A) Mice were treated 4 hours after infection with P. aeruginosa 6206 strain with R347 + MEM or Bs4-WT or a combination of BS4-WT and MEM. (B) Mice were treated 4 hours after infection with strain 6206 of P. aeruginosa with R347 + MEM or BS4 or a combination of Bs4-GLO and MEM. (A-B) The Bs4-WT or Bs4-GLO antibody combined with MEM increases the effectiveness of antibiotic therapy.
[0376] Figure 34 shows therapeutic adjunctive therapy: Bs4-GLO plus antibiotic in a model of lethal bacteremia. Mice were treated 24 hours before intraperitoneal infection with strain 6294 of P. aeruginosa with Bs4-GLO at the indicated concentrations, which were previously determined as protective sub-therapeutic doses in this model and R347 (negative control). One hour after infection, the mice were treated subcutaneously with antibiotics in the indicated concentrations, which were previously determined as protective sub-therapeutic doses, (A) Ciprofloxacin (CIP), (B) Meropenem (MEM) or (C) Tobramycin (TOB). The animals were carefully monitored for survival up to 72 hours after infection. The Bs4-GLO antibody combined with either CIP, MEM or TOB, in sub-protective doses, increases the effectiveness of antibiotic therapy.
[0377] The disclosure should not be limited in scope by the specific modalities described, which are intended for single illustrations of individual aspects of the disclosure and any compositions or methods that are functionally equivalent are within the scope of this disclosure. In fact, various modifications of the disclosure in addition to those shown and described in the present document will become apparent to those skilled in the art from the previous description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.
[0378] All publications and patent applications mentioned in this specification are incorporated into this document for reference to the same extent as if each individual publication or patent application was specifically and individually indicated as incorporated by reference. Additionally, Provisional Requests No. 61 / 556,645 filed on November 7, 2011; 61 / 624,651 deposited on April 16, 2012; 61 / 625,299 deposited on April 17, 2012; 61/697 filed in 585 filed on September 6, 2012 and International Application No. PCT / US2012 / 63639, filed on November 6, 2012 (dossier number AEMS-115WO1, named “MULTISPECIFIC AND MULTIVALENT BINDING PROTEINS AND USES THEREOF”) are incorporated by reference in their entirety for all purposes. Table 6A
Table 6B
Table 7

权利要求:
Claims (12)
[0001]
1. Bispecific antibody, characterized by the fact that it comprises a binding domain that binds to Ps1 of PSEUDOMONAS and a binding domain that binds to PcrV of PSEUDOMONAS, in which the binding domain of Ps1 comprises: (a) a CDR1 of heavy chain comprising PYYWT (SEQ ID NO: 47); a heavy chain CDR2 comprising YIHSSGYTDYNPSLKS (SEQ ID NO: 48); and a heavy chain CDR3 comprising ADWDRLRALDI (SEQ ID NO: 258); (b) a light chain CDR1 comprising RASQSIRSHLN (SEQ ID NO: 50); a light chain CDR2 comprising GASNLQS (SEQ ID NO: 51); and a light chain CDR3 comprising QQSTGAWNW (SEQ ID NO: 280); and wherein the PcrV binding domain comprises (c) a heavy chain CDR1 comprising SYAMN (SEQ ID NO: 218); a heavy chain CDR2 comprising AITMSGITAYYTDDVKG (amino acids 50-66 of SEQ ID NO: 264); and a heavy chain CDR3 comprising EEFLPGTHYYYGMDV (SEQ ID NO: 220); (d) a light chain CDR1 comprising RASQGIRNDLG (SEQ ID NO: 221); a light chain CDR2 comprising SASTLQS (SEQ ID NO: 222); and a light chain CDR3 comprising LQDYNYPWT (SEQ ID NO: 223).
[0002]
Bispecific antibody according to claim 1, characterized in that the Ps1 binding domain comprises a scFv fragment and the PcrV binding domain comprises an intact immunoglobulin.
[0003]
Bispecific antibody according to claim 1, characterized in that the Ps1 binding domain comprises an intact immunoglobulin and the PcrV binding domain comprises an scFv fragment.
[0004]
4. Bispecific antibody according to claim 2 or 3, characterized by the fact that (a) the scFv fuses at the amino terminal of the VH region of the intact immunoglobulin; or (b) the scFv fuses to the carboxy-terminal of the CH3 region of the intact immunoglobulin.
[0005]
Bispecific antibody according to claim 2 or 3, characterized in that the scFv is inserted into the hinge region of the intact immunoglobulin.
[0006]
Bispecific antibody according to any one of claims 1 to 5, characterized in that the anti-PcrV binding domain comprises a VH comprising amino acids 1-124 of SEQ ID NO: 264 and a VL comprising amino acids 1 -107 of SEQ ID NO: 263.
[0007]
Bispecific antibody according to any one of claims 1 to 6, characterized in that the anti-Psl binding domain comprises an scFv fragment comprising the amino acid sequence SEQ ID NO: 262.
[0008]
Bispecific antibody according to any one of claims 1 to 7, characterized in that the anti-Ps1 binding domain comprises an scFv fragment comprising the amino acid sequence SEQ ID NO: 262 and wherein the binding domain of anti-PcrV comprises a VH comprising amino acids 1-124 of SEQ ID NO: 264, and a VL comprising amino acids 1-107 of SEQ ID NO: 263.
[0009]
Bispecific antibody according to claim 1, characterized in that the bispecific antibody comprises a heavy chain comprising SEQ ID NO: 264 and a light chain comprising SEQ ID NO: 263.
[0010]
10. Composition, characterized by the fact that the bispecific antibody or fragment thereof as defined in any one of claims 1 to 9, and a pharmaceutically acceptable carrier.
[0011]
11. Use of the composition as defined in claim 10, characterized by the fact that it is for the manufacture of a medicine for the prevention of an infection by PSEUDOMONAS in an individual.
[0012]
12. Use of the composition as defined in claim 10, characterized by the fact that it is for the manufacture of a medicine for the treatment of an infection by PSEUDOMONAS in an individual.

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DK2776065T3|2020-10-12|

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CN104136042B|2017-08-18|

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US20150023966A1|2015-01-22|

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JP2015504421A|2015-02-12|

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HK1201453A1|2015-09-04|

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US20200317757A1|2020-10-08|

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KR20140091736A|2014-07-22|

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AU2012336028A1|2014-06-26|

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JP6182152B2|2017-08-16|

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NZ624072A|2016-09-30|

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ES2859323T3|2021-10-01|

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LT2776065T|2020-10-12|

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US10597439B2|2020-03-24|

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AU2017219081B9|2019-07-04|

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HRP20201370T1|2020-11-27|

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PL2776065T3|2020-12-14|

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法律状态:
2018-03-06| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-03-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-05-21| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-08-20| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-07-07| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2020-12-15| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-03-02| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 06/11/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201161556645P| true| 2011-11-07|2011-11-07|

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US61/556,645|2011-11-07|

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US201261625299P| true| 2012-04-17|2012-04-17|

---

US61/625,299|2012-04-17|

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US201261697585P| true| 2012-09-06|2012-09-06|

---

US61/697,585|2012-09-06|

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PCT/US2012/063722|WO2013070615A1|2011-11-07|2012-11-06|Combination therapies using anti- pseudomonas psl and pcrv binding molecules|

---