ANTIBODY AGAINST THE OPRF PROTEIN OF PSEUDOMONAS AERUGINOSA, USE THEREOF AS A MEDICAMENT AND PHARMACEUTICAL COMPOSITION CONTAINING SAME

Abstract

The invention relates to a monoclonal antibody against the OprF protein of Pseudomonas aeruginosa or to a functional fragment of this antibody. This antibody or antibody fragment is particularly useful for the preventive or curative treatment of infections with Pseudomonas aeruginosa.

Claims

1. A monoclonal antibody against the OprF protein of Pseudomonas aeruginosa or functional fragment of said antibody, comprising: a heavy chain variable region having the three complementarity determining regions (CDR) having the following amino acid sequences, or sequences having at least 80% identity with these sequences: VH-CDR1: GYXaa.sub.1FXaa.sub.2Xaa.sub.3Xaa.sub.4G (SEQ ID NO: 1) wherein Xaa.sub.1 is a threonine residue or a serine residue, Xaa.sub.2 is a serine residue or an asparagine residue, Xaa.sub.3 is an arginine residue, a serine residue or a threonine residue and Xaa.sub.4 is a phenylalanine residue or a tyrosine residue, VH-CDR2: INAXaa.sub.5TGKXaa.sub.6 (SEQ ID NO: 2) wherein Xaa.sub.5 is a glutamic acid residue or an aspartic acid residue and Xaa.sub.6 is an alanine residue or a serine residue, VH-CDR3: VR, and a light chain variable region having the three CDRs having the following amino acid sequences, or sequences having at least 80% identity with these sequences: VL-CDR1: SSVXaa.sub.7TXaa.sub.8Xaa.sub.9 (SEQ ID NO: 3) wherein Xaa.sub.7 is a threonine residue, an asparagine residue, a serine residue, an alanine residue or an arginine residue, Xaa.sub.8 is an asparagine residue, a glycine residue or a serine residue and Xaa.sub.9 is a tyrosine residue or a phenylalanine residue, VL-CDR2: Xaa.sub.10TS wherein Xaa.sub.10 is a glycine residue, an arginine residue or an alanine residue, VL-CDR3: QQGXaa.sub.11Xaa.sub.12Xaa.sub.13 (SEQ ID NO: 4) wherein Xaa.sub.11 is a histidine residue or an asparagine residue, Xaa.sub.12 is a serine residue or a threonine residue and Xaa.sub.13 is a valine residue or an isoleucine residue.

2. The monoclonal antibody or functional fragment of said antibody according to claim 1, wherein Xaa.sub.3 is an arginine residue and Xaa.sub.6 is an alanine residue.

3. The monoclonal antibody or functional fragment of said antibody according to claim 1, wherein Xaa.sub.1 is a threonine residue, Xaa.sub.5 is a glutamic acid residue and Xaa.sub.13 is a valine residue.

4. The monoclonal antibody or functional fragment of said antibody according to claim 1, wherein the complementarity determining regions (CDR) of the heavy chain variable region have the following respective amino acid sequences, or sequences having at least 80% identity with these sequences: TABLE-US-00015 VH-CDR1: (SEQ ID NO: 5) GYTFSRFG, VH-CDR2: (SEQ ID NO: 12) INAETGKA, VH-CDR3: VR and/or the complementarity determining regions (CDR) of the light chain variable region have the following respective amino acid sequences, or sequences having at least 80% identity with these sequences: TABLE-US-00016 VL-CDR1: (SEQ ID NO: 16) SSVTTNY, VL-CDR2: GTS VL-CDR3: (SEQ ID NO: 24) QQGHSV.

5. The monoclonal antibody or functional fragment of said antibody according to claim 1, wherein: the complementarity determining regions (CDR) of the heavy chain variable region have the following respective amino acid sequences, or sequences having at least 80% identity with these sequences: TABLE-US-00017 VH-CDR1: (SEQ ID NO: 6) GYSFSSYG VH-CDR2: (SEQ ID NO: 13) INADTGKS, VH-CDR3: VR and/or the complementarity determining regions (CDR) of the light chain variable region have the following respective amino acid sequences, or sequences having at least 80% identity with these sequences: TABLE-US-00018 VL-CDR1: (SEQ ID NO: 17) SSVTTGY or (SEQ ID NO: 16) SSVTTNY VL-CDR2: GTS VL-CDR3: (SEQ ID NO: 25) QQGHTI or (SEQ ID NO: 26) QQGNTI.

6. The monoclonal antibody or functional fragment of said antibody according to claim 1, wherein: the complementarity determining regions (CDR) of the heavy chain variable region have the following respective amino acid sequences, or sequences having at least 80% identity with these sequences: TABLE-US-00019 VH-CDR1: (SEQ ID NO: 7) GYSFSTYG or (SEQ ID NO: 8) GYSFSRYG VH-CDR2: (SEQ ID NO: 13) INADTGKS or (SEQ ID NO: 14) INADTGKA VH-CDR3: VR and/or the complementarity determining regions (CDR) of the light chain variable region have the following respective amino acid sequences, or sequences having at least 80% identity with these sequences: TABLE-US-00020 VL-CDR1: (SEQ ID NO: 18) SSVNTNY or (SEQ ID NO: 17) SSVTTGY or (SEQ ID NO: 16) SSVTTNY, VL-CDR2: GTS VL-CDR3: (SEQ ID NO: 25) QQGHTI or (SEQ ID NO: 26) QQGNTI.

7. The monoclonal antibody or functional fragment of said antibody according to claim 1, consisting of a single-chain variable fragment (scFv).

8. The monoclonal antibody or functional fragment of said antibody according to claim 7, wherein the heavy chain variable part and the light chain variable part are bound by a binding peptide.

9. The monoclonal antibody or functional fragment of said antibody according to claim 8, comprising a pair of sequences selected from the group consisting of the following pairs of sequences, SEQ ID NO: 28 and SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 31, SEQ ID NO: 32 and SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 45, SEQ ID NO: 46 and SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 49, and pairs of sequences having at least 80% identity with one of these pairs of sequences.

10. The monoclonal antibody or functional fragment of said antibody according to claim 1, consisting of a chimeric or humanized antibody or antibody fragment.

11. A nucleic acid molecule coding for a monoclonal antibody or functional fragment of said antibody according to claim 1.

12. An expression vector comprising a nucleic acid molecule according to claim 11.

13. A host cell comprising a nucleic acid molecule according to claim 11.

14. A method for preparing the monoclonal antibody or functional fragment of said antibody according to claim 1, the method comprising: culturing host cells comprising a nucleic acid molecule coding for the monoclonal antibody or functional fragment of said antibody under conditions enabling the expression of said monoclonal antibody or fragment of said antibody; and recovering said antibody or functional fragment of said antibody thus produced.

15. A method for preparing the monoclonal antibody or functional fragment of said antibody according to claim 1, comprising successive steps of: producing proteoliposomes containing the OprF protein of Pseudomonas aeruginosa, inoculating a non-human mammal with said proteoliposomes, constructing a bank of antibodies or antibody fragments from RNA extracted from cells of said mammal, screening said bank with respect to said proteoliposomes, by an expression technique, and selecting the clones which are reactive with respect to said proteoliposomes.

16. A pharmaceutical composition for combatting bacterial infections, comprising the monoclonal antibody or functional fragment of said antibody according to claim 1 as an active substance, in a pharmaceutically acceptable vehicle.

17. A method of preventively or curatively treating an infection in an individual in need thereof, comprising administering to said individual a therapeutically effective dose of the monoclonal antibody or functional fragment of said antibody according to claim 1.

18. The method of claim 17, wherein the infection is a bacterial Pseudomonas aeruginosa infections.

19. The method of claim 18, wherein the infection is a lung infection.

20. A method for in vitro or ex vivo detection of the bacterium Pseudomonas aeruginosa in a body fluid from an individual, the method comprising binding the monoclonal antibody or functional fragment of said antibody according to claim 1 to the bacterium.

21. A kit for the in vitro or ex vivo detection of the bacterium Pseudomonas aeruginosa in a body fluid from an individual, comprising the monoclonal antibody or functional fragment of said antibody according to claim 1, reagents, and instructions for implementing a method for detecting, in vitro or ex vivo, the bacterium Pseudomonas aeruginosa in a body fluid from an individual, by means of said monoclonal antibody or functional fragment of said antibody.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0112] The features and advantages of the invention will emerge more clearly in the light of the examples of implementation hereinafter, provided merely by way of illustration and not restriction of the invention, with reference to FIGS. 1 to 15, wherein:

[0113] FIG. 1 represents a graph showing the optical density at 450 nm, as a function of the serum dilution rate, for an ELISA test (affinity relative to the OprF protein of Pseudomonas aeruginosa) carried out on sera sampled from a macaque, on days 0, 24 and 38 after immunizing this macaque with proteoliposomes containing the OprF protein of Pseudomonas aeruginosa (OPRF D0, OPRF D24, OPRF D38) and on a serum sampled from a macaque, on day 38 after immunizing the macaque with bovine serum albumin (BSA D38).

[0114] FIG. 2 shows the sequence of 6 scFv fragments directed against the OprF protein of Pseudomonas aeruginosa according to the invention, wherein the sequences corresponding to the 6 CDRs, as well as the sequence of the binding peptide binding the heavy chain variable region to the light chain variable region, are underlined—in these sequences, the N-terminus is on the left, and the C-terminus is on the right, conventionally.

[0115] FIG. 3 shows the sequence of 5 other scFv fragments directed against the OprF protein of Pseudomonas aeruginosa according to the invention, wherein the sequences corresponding to the 6 CDRs, as well as the sequence of the binding peptide binding the heavy chain variable region to the light chain variable region, are underlined—in these sequences, the N-terminus is on the left, and the C-terminus is on the right, conventionally.

[0116] FIG. 4 represents a graph showing the optical density at 450 nm, as a function of the dilution rate, for ELISA tests (affinity relative to the OprF protein of Pseudomonas aeruginosa) carried out for different dilutions of an scFv fragment according to the invention (E2) and for bovine serum albumin (BSA) as a negative control.

[0117] FIG. 5 represents a graph showing the optical density at 450 nm, as a function of the dilution rate, for ELISA tests (affinity relative to the OprF protein of Pseudomonas aeruginosa) carried out for different dilutions of an scFv fragment according to the invention (E5) and for bovine serum albumin (BSA) as a negative control.

[0118] FIG. 6 represents a graph showing the optical density at 450 nm, as a function of the dilution rate, for ELISA tests (affinity relative to the OprF protein of Pseudomonas aeruginosa) carried out for different dilutions of an scFv fragment according to the invention (F8) and for bovine serum albumin (BSA) as a negative control.

[0119] FIG. 7 represents a graph showing the optical density at 450 nm, as a function of the dilution rate, for ELISA tests (affinity relative to the OprF protein of Pseudomonas aeruginosa) carried out for different dilutions of an scFv fragment according to the invention (G9) and for bovine serum albumin (BSA) as a negative control.

[0120] FIG. 8 represents a graph showing the optical density at 450 nm, as a function of the dilution rate, for ELISA tests (affinity relative to the OprF protein of Pseudomonas aeruginosa) carried out for different dilutions of an scFv fragment according to the invention (E3) and for bovine serum albumin (BSA) as a negative control.

[0121] FIG. 9 represents a graph showing the optical density at 450 nm, as a function of the dilution rate, for ELISA tests (affinity relative to the OprF protein of Pseudomonas aeruginosa) carried out for different dilutions of an scFv fragment according to the invention (E7) and for bovine serum albumin (BSA) as a negative control.

[0122] FIG. 10 represents a graph showing the optical density at 450 nm, as a function of the dilution rate, for ELISA tests (affinity relative to the OprF protein of Pseudomonas aeruginosa) carried out for different dilutions of an scFv fragment according to the invention (F10) and for bovine serum albumin (BSA) as a negative control.

[0123] FIG. 11 represents a graph showing the optical density at 450 nm, as a function of the dilution rate, for ELISA tests (affinity relative to the OprF protein of Pseudomonas aeruginosa) carried out for different dilutions of an scFv fragment according to the invention (F3) and for bovine serum albumin (BSA) as a negative control.

[0124] FIG. 12 represents a graph showing the optical density at 450 nm, as a function of the dilution rate, for ELISA tests (affinity relative to the OprF protein of Pseudomonas aeruginosa) carried out for different dilutions of an scFv fragment according to the invention (F4) and for bovine serum albumin (BSA) as a negative control.

[0125] FIG. 13 represents a graph showing the optical density at 450 nm, as a function of the dilution rate, for ELISA tests (affinity relative to the OprF protein of Pseudomonas aeruginosa) carried out for different dilutions of an scFv fragment according to the invention (A1) and for bovine serum albumin (BSA) as a negative control.

[0126] FIG. 14 represents a graph showing the optical density at 450 nm, as a function of the dilution rate, for ELISA tests (affinity relative to the OprF protein of Pseudomonas aeruginosa) carried out for different dilutions of an scFv fragment according to the invention (A8) and for bovine serum albumin (BSA) as a negative control.

[0127] FIG. 15 represents graphs showing the optical density (OD) at 450 nm as a function of the concentration, for ELISA tests (affinity relative to the OprF protein of Pseudomonas aeruginosa) carried out respectively on four scFv fragments according to the invention, named E7, F8, F10 and G9.

[0128] FIG. 16 shows a graph representing the value obtained by subtracting the absorbance at 680 nm from the absorbance at 490 nm (“A490-A680 nm”), during an in cellulo test for determining the neutralizing power, against the infection of macrophages (“Ma”) by Pseudomonas aeruginosa (“Pa”), of scFv fragments according to the invention (F8, G9, E7), by assaying the lactate dehydrogenase activity.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

A/ Production of Proteoliposomes Containing the OprF Protein of Pseudomonas aeruginosa

Construction of a Recombinant Vector Expressing OprF

[0129] The recombinant vector pIVEX2.4-OprF, wherein OprF comprises an N-terminal polyhistidine tag, is constructed by cloning the OprF gene amplified from genomic DNA of Pseudomonas aeruginosa amplified by polymerase chain reaction (PCR), by means of the following primers:

TABLE-US-00010 Sense (SEQ ID NO: 76) 5′-GGAATTCCATATGAAACTGAAGAACACCTTAG-3′ Antisense (SEQ ID NO: 77) 5′-TAGAAGCTGAAGCCAAGTAACTCGAGTAACGC-3′
in the expression vector pIVEX2.4d (Roche Diagnostics).

[0130] For this purpose, 30 PCR cycles are implemented using a high-fidelity DNA polymerase. The PCR product thus obtained is then purified by means of the QIAquick gel kit (Qiagen) then digested with the restriction enzymes Ndel, Xhol (Roche Diagnostics), purified once again then bound by means of the Rapid DNA ligation kit (Roche Diagnostics) in the plasmid vector pIVEX2.4d (Roche Diagnostics) previously digested by the enzymes Ndel and Xhol. The resulting recombinant plasmid pIVEX2.4-OprF is verified by sequencing (LGC Genomics) in order to validate the insertion of the gene coding for OprF in phase with the polyhistidine tag of the vector pIVEX2.4d.

Liposome Preparation

[0131] Liposomes are prepared by drying a lipid composition previously solubilized in chloroform, for the following different lipid compositions (LC): [0132] Lipid Composition 1 (LC1): cholesterol, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dimyristoyl-sn-glycero-3-phosphate (sodium salt) (DMPA), molar ratio [2-4-2-2]; [0133] LC1′: LC1+1 mg/mL monophosphoryl lipid A (MPLA); [0134] LC2: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt) (POPG), E. coli cardiolipin (CL), molar ratio [6-2-2]; [0135] LC2′: LC2+1 mg/mL MPLA; [0136] LC3: POPE, POPG, E. coli CL, DMPA, molar ratio [6-2-1-1]; [0137] LC 3′: LC 3+1 mg/mL MPLA (Avanti Polar Lipids).

[0138] Drying is carried out by evaporation under nitrogen. The residual traces of chloroform are removed using a vacuum pump. The lipid film is then hydrated in 500 μl of a Tris solution (50 mM, pH 7.5) by vortexing, then subjected to 4 freezing/thawing cycles in liquid nitrogen. The lipid mixture is extruded using an extruder (Avanti Polar Lipids) to produce liposomes of an average size of approximately 200 nm. The liposomes thus obtained are stored at 4° C.

Production and Purification of Proteoliposomes Containing OprF in Acellular System

[0139] The OprF membrane protein of Pseudomonas aeruginosa is synthesized in the presence of the different liposome compositions (LC1, or LC2, or LC3 or LC1′, or LC2′ or LC3′) using the RTS 500 ProteoMaster E. coli HY acellular protein synthesis kit, from Biotechrabbit. For this purpose, the recombinant plasmid pIVEX2.4-OprF containing the gene coding for the OprF protein fused with a polyhistidine tag (6xHis) is added at a concentration of 15 μg/ml to the cellular lysate of the kit in the presence of a quantity of 1 to 4 mg/ml of liposomes from one of the 6 lipid compositions LC1 to LC3′. Proteoliposomes containing the OprF recombinant protein are produced at 25° C. for 16 h under stirring (300 rpm) in a ratio of 1:30 of reaction volume/container volume. The resulting recombinant proteoliposomes are then purified in 2 steps: [0140] firstly in a sucrose gradient of 0-40% in 50 mM pH 7.5 Tris buffer whereon the reaction mixture is deposited on the top of the gradient then centrifuged at 287.660 g for 2 h using a TH-641 rotor. Fractions of 1 ml are collected from the top of the gradient and analyzed by Western Blot using an anti-histidine antibody coupled with HRP horseradish peroxidase (Sigma), [0141] then, 1 ml of a 50 mM pH 7.5 Tris buffer is added to each fraction containing the proteoliposomes containing OprF, and the solution is centrifuged at 30,000 g for 30 min at 4° C., to form a proteoliposome pellet. The pellet is washed twice for 30 min at 4° C. with a 5M NaCl solution and then resuspended in a 50 mM pH 7.5 Tris solution at the desired concentration. The purity of the samples is analyzed on an SDS-PAGE gel stained with Coomassie blue. Proteoliposomes containing the OprF protein of Pseudomonas aeruginosa are obtained.

B/ Preparation and Screening of a Bank of scFv Fragments

Immunization of Animals

[0142] scFv fragments targeted against the Pseudomonas aeruginosa OprF bacterial membrane antigenic target are obtained by an immunization with the proteoliposomes obtained from the lipid composition LC1, performed on days D0, D14, D28 and D50, of a macaque (Macaca fascicularis). The macaque is kept under sterile conditions in the presence of another similar animal and with no other species. Before the first injection, a blood test is performed to ascertain the physiological status of the animal. 100 μg of the composition LC1 in sterile phosphate buffered saline (PBS), mixed 50/50 with a Freund's adjuvant (complete for the first injection, incomplete for the subsequent injections) are injected in the animal subcutaneously at 2 points (at a rate of 250 μl per point) in the animal's scapular area, according to the following profile: administrations on days 0, 14, 28, 50.

[0143] The immune response is analyzed by immunoenzyme assay (ELISA) on sera sampled on days D0, D24, D38 to perform a titration of the antibodies targeted against the OprF membrane antigenic target. Bone marrow samples are taken after the final injection (D50) on D53, D60, D67 and D74 on the anesthetized animal.

Serum Titration

[0144] The post-immunization humoral response is analyzed by the indirect ELISA method using a series of dilutions of the pre-immune and immune sera (dilutions of 100, 1000, 10,000, 1,000,000, 10,000,000 and 100,000,000) following the protocol described in the book “Phage Display”, Methods and Protocols, Springer Protocols, “Construction of Macaque Immune Libraries” chapter, Arnaud Avril et al., Methods Mol Biol. 2018; 1701: 83-112. doi: 10.1007/978-1-4939-7447-4_5. Briefly, the OprF membrane antigen in proteoliposome form or a negative control such as bovine serum albumin (BSA) is first deposited at the bottom of the ELISA plates and incubated for 16 h at 4° C. After a saturation step (2% of dried milk resuspended in 200 μl of PBS phosphate buffered saline), each diluted serum (1:100 initially then 1:10, in PBS/Tween® 0.05%/BSA 0.5%) is then tested in parallel against the OprF antigen or the negative control (BSA) for 2 h at 37° C. The specific antibodies against OprF are then detected using a secondary anti-macaque Fc antibody conjugated with HRP horseradish peroxidase and by adding tetramethylbenzidine (TMB) until a color appears in the wells. The results are analyzed by reading the optical density at 450 nm.

[0145] The results obtained are shown in FIG. 1, for the sera sampled on days D0, D24 and D38 post-immunization with the proteoliposomes containing OprF and for the sera obtained on day D38 for the immunization with BSA.

[0146] On day D38, a titer of 1:200000 is observed, which is compatible with the remainder of the method.

Bone Marrow Samples and B Lymphocyte Isolation

[0147] Bone marrow samples are taken from the anesthetized animal. The samples are taken at the trochanteric fossa of the femur and at the tubercle of the humerus, by means of a Mallarme trocar. Each sample is collected in a 50 ml Falcon® tube containing a 10-15% citrate solution. Approximately 5 ml of sample are obtained on days D53, D60, D67 and D74. Each sample is then centrifuged for 10 min at 500 g (1500 rpm) at 4° C. The supernatant is removed and placed in a cryotube, then stored at −20° C. The total RNA of each of the bone marrows is then extracted with the Trizol/Chloroform technique and quantified by reading the optical density (OD) at 260 nm and 280 nm using a spectrophotometer.

[0148] The results obtained are shown in Table 3 hereinafter.

TABLE-US-00011 TABLE 3 OD at OD at OD 260 [RNA] Day 260 nm 280 nm nm/280 nm μg/ml D53 6.239 3.390 1.840 249.557 D60 16.215 8.446 1.920 648.609 D67 3.460 1.921 1.801 138.395 D74 5.313 2.911 1.825 212.535

RT-PCR Amplification of the RNA Coding for the Variable Parts VLκ, VLλ and VH

[0149] The messenger RNA (mRNA) coding for the variable domains of the heavy and light chains G and κ/λ for each of the bone marrow samples are retro-amplified to obtain a bank of complementary DNA (cDNA) using specific primers, described in the publication by Avril et al., 2018, Methods Mol. Biol., 1701: 83-112.

[0150] The amplification quality is controlled by agarose gel electrophoresis.

[0151] The PCR products amplified from the cDNA bank obtained on days D53 to D74 are cloned in the plasmid pGemT (Promega), according to the supplier's protocol, in order to obtain a bank of secured clones.

Construction of a Bank of scFv Fragments

[0152] From the DNA obtained on days D53 and D60, 2 banks (one for each day) are constructed by sequential cloning by inserting, according to the supplier's protocol, first the VL fragments in the phagemid vector pTh1 (Addgene) then the VH fragments, to obtain a construction in the format VH-[(G.sub.4S)×3 (SEQ ID NO: 11)]-VL-6xHistidine-EQKLISEEDL (SEQ ID NO: MM), wherein the VH and VL fragments are bound by the binding peptide GGGGSGGGGSGGGGS (SEQ ID NO: 11) binding the C-terminus of the VH fragment to the N-terminus of the VL fragment, and comprising at its C-terminus a polyhistidine tag (SEQ ID NO: 79) and a c-myc tag (SEQ ID NO: 78).

[0153] For the DNA obtained on day D53, a bank of 1.5.Math.10.sup.7 CFU (75% full size inserts) is obtained. For the DNA obtained on the day D60, a bank of 1.Math.10.sup.7 CFU (100% full size inserts) is obtained.

Screening of scFv Fragment Banks with the Phage Display Technique—Identification of Fragments Having an Affinity for the OprF Protein of Pseudomonas aeruginosa

[0154] The bank is encapsidated and amplified in phage M13Ko7 (Nebb), according to the supplier's protocol.

[0155] The scFv banks contained in the phagemids are subjected to 4 rounds of selection against the proteoliposomes containing the OprF membrane antigen fixed on 96-well plates.

[0156] The screening protocol is as follows: a microtitration plate is coated overnight with the targeted antigen at a concentration of 10 μg/ml in PBS at 4° C. Then, the plate is blocked with 3% BSA in PBS for 2 h at 37° C.; after washing, the bank is incubated for 2 further hours at 37° C. During the first round, the plate is washed twice using PBS containing 0.1% Tween® 20 with a 5-min interval between each wash. Finally, the plate is washed, rinsed with sterile PBS and the phage is eluted with trypsin (10 mg/ml in PBS) for 30 min at 37° C. The eluted phages are used for Escherichia coli infection (SURE strain, Stratagene, cultured in an SB (Super Broth) medium supplemented with tetracycline (10 μg/ml) and carbenicillin (50 μg/ml). For the production of new phage particles, the infected strain is co-infected with an auxiliary phage and cultured overnight at 30° C. in an SB medium supplemented with tetracycline (10 μg/mL), carbenicillin (50 μg/mL) and kanamycin (70 μg/mL). The phage particles are precipitated using PEG/NaCl (4% (w/v) PEG-8000, 3% (w/v) NaCl) and used for the next cycle. The second round is carried out as described above. The infected strains from the third round are cultured on SB media in Petri dishes and are used for the screening.

[0157] After each round, only the phages having interacted with OprF are eluted. The reactivity of the phages after each round of selection against the OprF target is tested by ELISA assay. The phages show a 30-fold signal increase between the first selection round and the 4.sup.th round, indicating an enrichment of the scFvs which are reactive against OprF.

[0158] 96 clones isolated from the second, third and fourth selection rounds are collected and used to produce soluble scFvs. From these clones, 57 positive clones are selected and 15 of these are retained. An analysis of the nucleotide and peptide sequence of the 57 clones is carried out to determine the potential redundancy of some sequences. 43 sequences are identified as non-redundant and non-recombined. Of these 43 sequences, 11 are produced in Escherichia coli bacteria, according to the following protocol: the phagemid DNA isolated after the selection process is used to transform the non-suppressive E. Coli strain such that it expresses the soluble scFv fragment. Single colonies of transformants selected at random are used to inoculate 5 ml of SB medium supplemented with carbenicillin. The cultures are incubated overnight at 37° C. under vigorous stirring (250 rpm). 500 ml of SB medium supplemented with carbenicillin are then inoculated with 500 μl of each culture. The cultures are cultured at 30° C. until the optical density at 600 nm reaches 1.5. IPTG (1 mM) is then added overnight to induce genic expression at 22° C. The cells are collected by centrifugation at 2500 g for 15 min at 4° C. The scFvs are extracted with polymyxin B sulfate and purified on a nickel column (Ni-NTA column, Qiagen) according to the manufacturer's instructions, then dialyzed against PBS.

[0159] The corresponding scFvs produced are then purified to confirm their affinity with respect to the OprF target with the ELISA method.

[0160] These 11 scFv fragments comprise the sequences indicated in Table 4 hereinafter.

TABLE-US-00012 TABLE 4 scFv Amino acid sequence A1 SEQ ID NO: 50 A8 SEQ ID NO: 51 E2 SEQ ID NO: 52 E3 SEQ ID NO: 53 E5 SEQ ID NO: 54 E7 SEQ ID NO: 55 F3 SEQ ID NO: 56 F4 SEQ ID NO: 57 F8 SEQ ID NO: 58 F10 SEQ ID NO: 59 G9 SEQ ID NO: 60

[0161] These sequences are prolonged therein, at their C-terminus, by a polyhistidine tag (SEQ ID NO: 79) and a c-myc tag (SEQ ID NO: 78).

[0162] These scFv fragments all comprise: [0163] a heavy chain variable region having the three complementarity determining regions (CDR) having the following amino acid sequences:

[0164] VH-CDR1: GYXaa.sub.1FXaa.sub.2Xaa.sub.3Xaa.sub.4G (SEQ ID NO: 1) wherein Xaa.sub.1 is a threonine residue or a serine residue, Xaa.sub.2 is a serine residue or an asparagine residue, Xaa.sub.3 is an arginine residue, a serine residue or a threonine residue and Xaa.sub.4 is a phenylalanine residue or a tyrosine residue,

[0165] VH-CDR2: INAXaa.sub.5TGKXaa.sub.6 (SEQ ID NO: 2) wherein Xaa.sub.5 is a glutamic acid residue or an aspartic acid residue and Xaa.sub.6 is an alanine residue or a serine residue,

[0166] VH-CDR3: VR, [0167] and a light chain variable region having the three CDRs having the following amino acid sequences:

[0168] VL-CDR1: SSVXaa.sub.7TXaa.sub.8Xaa.sub.9 (SEQ ID NO: 3) wherein Xaa.sub.7 is a threonine residue, an asparagine residue, a serine residue, an alanine residue or an arginine residue, Xaa.sub.8 is an asparagine residue, a glycine residue or a serine residue and Xaa.sub.9 is a tyrosine residue or a phenylalanine residue,

[0169] VL-CDR2: Xaa.sub.10TS wherein Xaa.sub.10 is a glycine residue, an arginine residue or an alanine residue,

[0170] VL-CDR3: QQGXaa.sub.11Xaa.sub.12Xaa.sub.13 (SEQ ID NO: 4) wherein Xaa.sub.11 is a histidine residue or an asparagine residue, Xaa.sub.12 is a serine residue or a threonine residue and Xaa.sub.13 is a valine residue or an isoleucine residue.

[0171] These scFv fragments are all according to the present invention.

[0172] Each of the sequences of these scFv fragments is shown in FIG. 2 for A1, A8, E2, E3, E5, E7 and in FIG. 3 for F3, F4, F8, F10, G9. The CDR sequences are underlined therein. From left to right, the successive sequences of the CDRs of the heavy chain variable region are thus visible (VH-CDR1, followed by VH-CDR2, followed by VH-CDR3), followed by the light chain variable region (VL-CDR1, followed by VL-CDR2, followed by VL-CDR3). The binding peptide sequence is also underlined, between the three CDR triplets.

C/ Analysis of the Affinity of scFv Fragments According to the Invention for the OprF Protein of Pseudomonas aeruginosa

[0173] The 11 scFv fragments produced above are purified.

[0174] The following quantities of each scFv fragment according to the invention are obtained: 0.346 mg/ml E2, 0.401 mg/ml E3, 0.559 mg/ml E5, 0.453 mg/ml E7, 0.387 mg/ml F4, 0.436 mg/ml F3, 0.333 mg/ml F8, 0.403 mg/ml F10, 0.570 mg/ml G9, 0.385 mg/ml A1 and 0.626 mg/ml A8.

[0175] An ELISA assay is carried out on MaxiSorp® plates, to confirm the affinity of these scFv fragments with respect to the OprF target in proteoliposome form, as follows.

[0176] The plate is saturated with 2.5% of dried milk resuspended in 200 μl of PBS phosphate buffered saline.

[0177] The scFv fragments are incubated at different dilution rates: 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280, in PBS/Tween®-20 0.05%/BSA 0.5%. Detection is performed using an anti-c-myc tag secondary antibody coupled with horseradish peroxidase. The optical density at 450 nm is recorded.

[0178] The results obtained, for the fragment according to the invention and for a negative control (BSA) are shown in FIG. 4 for E2, FIG. 5 for E5, FIG. 6 for F8, FIG. 7 for G9, FIG. 8 for E3, FIG. 9 for E7, FIG. 10 for F10, FIG. 11 for F3, FIG. 12 for F4, FIG. 13 for A1 and FIG. 14 for A8.

[0179] It can be seen that all of the scFv fragments according to the invention have a high affinity for the OprF protein of Pseudomonas aeruginosa.

[0180] By way of example, the dissociation constant Kd is determined for the scFv fragments E7, F8, F10 and G9, with an ELISA method.

[0181] For this purpose, 100 μl of OprF proteoliposomes (containing an OprF concentration of 1 μg/ml) contained in fixation buffer (0.1 M sodium carbonate, 0.1 M sodium bicarbonate) are fixed at the bottom of the wells of a 96-well plate (Thermo Scientific®) overnight at 4° C. and under stirring. The wells are then blocked for 1 h at 21° C. with 100 μl of TBS Tween (TBST) buffer containing 5% milk. After washing the wells with 100 μl of TBST buffer, 100 μl of scFv fragment (E7, F8, F10 or G9), at 1:50; 1:200; 1:400; 1:800; 1:1600 and 1:3200 dilutions in TBST buffer, are added to the corresponding wells and incubated for 1 h at 37° C. under stirring. After washing the wells, 100 μl of anti-c-myc-Peroxidase antibody (Roche) (1:10000 dilution in TBST buffer containing 5% milk) is added to the wells and incubated for 1 h at 37° C. under stirring. After washing the wells 3 times, 50 μl of TMB is added to the wells and the plate is incubated at ambient temperature and protected from light for approximately 15 min. 50 μl of 1 M HCl is then added and the absorbance of each well is measured at 450 nm. These absorbance data are then analyzed using the GraphPad Prism software (non-linear regression analysis, “one site-specific binding” equation) in order to calculate the dissociation constant Kd of each scFv fragment tested. By way of comparison, a measurement is also made on the buffer only.

[0182] The results obtained, for each of the fragments E7, F8, F10 and G9, are shown in FIG. 15.

[0183] The values of the dissociation constants Kd thus determined are specified in Table 5 hereinafter.

TABLE-US-00013 TABLE 5 scFv fragment E7 F8 F10 G9 Kd (nM) 213.4 211.4 1493 295.3

[0184] These results show a very good affinity of the fragments according to the invention for the proteoliposome target containing the OprF protein of Pseudomonas aeruginosa.

D/ Determination of the Neutralizing Power of scFv Fragments According to the Invention in cellulo

[0185] The fragments E7, F8 and G9 were tested in this experiment, to determine their neutralizing power with respect to macrophage infection with Pseudomonas aeruginosa (CHA strain) at an MOI (multiplicity of infection) of 10.

[0186] The following protocol was implemented: [0187] Differentiation of THP-1 cells (human monocytic line) into macrophages following the addition of phorbol myristate acetate (PMA): addition of 15 μl of PMA (0.1 mg/ml stock solution) to 10 ml of THP-1 cells suspended in RPMI medium containing 10% decomplemented fetal calf serum (dFCS) (440,000 cells/ml); incubation of the culture dish T25 in an oven at 37° C. (atmosphere containing 5% CO.sub.2) for at least 48 h in order to enable the differentiation of the THP-1 cells into adherent macrophages; [0188] Culture and preparation of P. aeruginosa (CHA strain): initiation of a culture of P. aeruginosa bacteria (CHA strain) from a small quantity of bacterial glycerol stock placed in 10 ml of LB culture medium; incubation of the bacterial culture at 37° C. under stirring overnight; dilution of the culture in LB medium until an optical density measurement at 600 nm equal to 0.5 in 1 ml of culture (6×10.sup.8 CFU/ml) is obtained; centrifugation at 4000 g for 5 min; removal of the supernatant and resuspension of the pellet in 1 ml of RPMI—10% dFCS culture medium; 2.sup.nd centrifugation at 4000 g for 5 min; removal of the supernatant and resuspension of the pellet in 1 ml of RPMI—10% dFCS culture medium; dilution in the same medium in order to obtain a suspension containing 15,000,000 bacteria/ml; incubation at 37° C. for 2 h of: 35 μl of the P. aeruginosa suspension+35 μl of scFv E7 in PBS (0.453 mg/ml), 35 μl of the P. aeruginosa suspension+35 μl of scFv F8 in PBS (0.333 mg/ml), 35 μl of the P. aeruginosa suspension+35 μl of scFv G9 in PBS (0.570 mg/ml), 35 μl of the P. aeruginosa suspension; [0189] Preparation of the macrophages: removal of the culture supernatant; addition of 2 ml of Versene in order to detach the adherent cells; after detaching the cell lawn, addition of 2 ml of medium and recovery of the cells; centrifugation at 400 g for 5 min; resuspension of the cell pellet in 2 ml of medium and enumeration of the cells; filling of the cell suspension in the wells of a 96-well plate so as to obtain 15,000 cells/well and addition of the necessary quantity of RPMI-10% dFCS medium in order to obtain the final volumes specified in Table 6 hereinafter:

TABLE-US-00014 TABLE 6 M represents macrophages 1 2 3 4 5 A 90 μl of 90 μl of 90 μl of 100 μl of 100 μl of medium + M medium + M medium medium medium B 90 μl of 90 μl of 90 μl of 100 μl of 100 μl of medium + M medium + M medium medium medium C 90 μl of 90 μl of 90 μl of 100 μl of 100 μl of medium + M medium + M medium medium medium D 80 μl of 80 μl of 80 μl of 80 μl of medium + M medium + M medium + M medium + M E 80 μl of 80 μl of 80 μl of 80 μl of medium + M medium + M medium + M medium + M F 80 μl of 80 μl of 80 μl of 80 μl of medium + M medium + M medium + M medium + M [0190] Completion of the cytotoxicity test for quantifying the release of cellular LDH (Lactate Dehydrogenase) in the culture medium, according to the protocol defined in the instructions of the lactate dehydrogenase (LDH) assay kit, “Pierce® LDH cytotoxicity assay kit”: addition of 10 μl of sterile PBS to wells No. 1 to 5 of rows A, B, C and to wells No. 1 of rows D, E, F; addition of 10 μl of P. aeruginosa (Pa) to wells No. 3 of rows A, B, C and to wells No. 1 of rows D, E, F; addition of 20 μl of the mixture: Pa+F8 to wells No. 2 of rows D, E, F, Pa+G9 to wells No. 3 of rows D, E, F, Pa+E7 to wells No. 4 of rows D, E, F; addition of 10 μl of ultrapure sterile water to wells No. 1 and 3 of rows A, B, C; incubation of the plate in an oven at 37° C. (atmosphere containing 5% CO.sub.2) for 16 h; addition of 10 μl of lysis buffer (10×) to wells No. 2 and 5 of rows A, B, C; incubation for 45 min in an oven at 37° C. (atmosphere containing 5% CO.sub.2); centrifugation of the plate at 250 g for 3 min; transfer of 50 μl from each well into a new 96-well plate; addition to each well of 50 μl of the reaction mixture; incubation of the plate at ambient temperature and protected from light for 30 min; addition to each well of 50 μl of the stop solution; measurement of the absorbance of each well at 490 and 680 nm; subtraction of the absorbance values: A.sub.490 nm-A.sub.680 nm.

[0191] The results obtained are shown in FIG. 16. A reduction of approximately ⅔ in the cytotoxicity of P. aeruginosa with respect to the macrophages is observed in the presence of the ScFv fragments according to the invention. This demonstrates a neutralizing action of these fragments against P. aeruginosa.

E/ Analysis of the Proteoliposomes Containing the OprF Protein of Pseudomonas aeruginosa Having Been Used to Obtain ScFvs According to the Invention

[0192] The proteoliposomes obtained in the experiment described in A/ above were subjected to the following analyses.

E.1/ Materials and Methods

[0193] Digestion with trypsin—The OprF proteoliposomes (LC1) purified by centrifugation in sucrose gradient were proteolyzed using a trypsin:protein mass ratio of 1:10 at ambient temperature (RT). The samples were retrieved at different times and loaded on an SDS-PAGE gel for a subsequent Western Blot analysis.

[0194] Negative staining electron microscopy—The samples were prepared using the negative staining on grid (SOG) technique. 10 μl of OprF proteoliposomes (LC1, [OprF]: 0.1 mg/mL) or 10 μl of liposomes (4 mg/mL) incubated in the reaction mixture without DNA (=negative control) were added to a glow discharge grid coated with a carbon film for 3 min and the grid was stained with 50 μl of phosphotungstite acid (PTA, 1% in distilled water) for 2 min. The excess solution was absorbed by filter paper and the grid was air-dried. The images were taken under low-dose conditions (<10 e−/Å2) with defocus values between 1.2 and 2.5 μm on a Tecnai 12 LaB6 electron microscope at an acceleration voltage of 120 kV using the CCD Gatan Orius® 1000 camera. The mean pore size was determined using the open source image processing program ImageJ.

[0195] AFM tip functionalization—The golden tips (NPG-10, Bruker Nano AXS) were coated with NTA-SAM after incubating overnight in 0.1 mM NTA-SAM (Prochimia) solution in ethanol. Then, the tips were rinsed with plenty of ethanol, dried under nitrogen and incubated for 1 h in 40 mM NiSO.sub.4 in a PBS solution and stored at 0-5° C.

[0196] AFM based on force/distance (FD)—A Resolve® AFM (Bruker) was used in “PeakForceTapping” mode. Rectangular cantilevers with nominal spring constants of approximately 0.06-0.12 N.Math.m.sup.−1 and a resonance frequency of approximately 18 kHz in water were chosen. All the AFM experiments were conducted in an imaging buffer solution at ambient temperature (approximately 24° C.). The adhesion charts were obtained by oscillating the functionalized tip at 0.25 kHz, with an amplitude of 25 nm, and by applying an imaging force of 100 pN. Topographies of 128×128 or 256×256 pixels were performed by digitizing 0.125 line per sec. The retraction speed was 1500 nm/sec and the contact time between the tip and the sample was 500 ms.

Data Analysis

[0197] The force/distance (FD) curves from each interaction recognition experiment were saved and exported in text file format. NanoScope Analysis v1.9 and BiomecaAnalysis were used to convert the force/time curves into FD curves showing specific adhesion events. The force/distance curves obtained were then analyzed based on the Worm-Like Chain (WLC) model. This model is the most suitable and the most frequently used to describe the extension of polypeptides. The extension z of the macromolecule is linked with the retraction force F.sub.adh by the equation:

[00001]$F_{\mathrm{adh}}\left(z\right)=-\frac{K_{B}T}{I_p}\left(\frac{z}{I_c}+4{\left(1-\frac{z}{I_c}\right)}^{-2}-\frac{1}{4}\right)$

wherein the persistence length l.sub.p is a direct measurement of the chain rigidity, l.sub.c is the total contour length of the biomacromolecule and K.sub.B is the Boltzmann constant.

[0198] The number of monomers in the polypeptide chains was then derived from the following equation:

[00002]$N=\frac{I_c}{I_p}$

E.2/ Determination of the Orientation of the OprF Proteins in the Liposomal Membrane by AFM (Atomic Force Microscopy)

[0199] The OprF proteoliposome samples were adsorbed on a mica surface and analyzed by AFM using a probe functionalized by a Tris-Ni.sup.+-NTA group binding the N-terminal polyhistidine tag of OprF. The AFM analysis was performed with and without Triton® X-100 detergent. Triton® 1x solution was used to solubilize the OprF proteins of the liposomal membrane, thus exposing all the polyhistidine tags located inside the liposome and enabling them to be bound by the functionalized probe. The topographic images, acquired with and without Triton® X-100, showed OprF proteoliposomes on the sample surface. In the absence of Triton®, very few specific adhesion phenomena between the functionalized probe and the N-terminal polyhistidine tag of OprF occurred on the surface of the proteoliposomes, as shown by the corresponding adhesion charts illustrating the adhesion forces between 80 and 150 pN. On the other hand, in the presence of Triton®, numerous specific adhesion events were detected. On average, the functionalized probe has bound the polyhistidine tag of one OprF out of 6 without Triton®, and of 5 OprF proteins out of 6 with Triton®, demonstrating that the N-terminal polyhistidine tag of OprF was primarily located inside the liposome.

E.3/ Determination of the Topology of OprF Proteins in the Liposomal Membrane by Trypsin Digestion and AFM

[0200] The OprF proteoliposomes purified by ultracentrifugation in a sucrose gradient were subjected to a limited proteolysis experiment in order to determine the topology of OprF in the liposomal membrane. The sequence of the OprF protein contains 32 trypsin cleavage sites. Without OprF membrane protection, trypsin generates peptides of a mass ranging from 146 to 4649 Da (PeptideCutter program). The result of trypsin digestion of the OprF proteoliposomes, visualized by Western Blot using an anti-histidine antibody, demonstrated that OprF adopts at least two different membrane topologies in the liposomes: a first topology wherein OprF is entirely inserted in the membrane and therefore protected from proteolysis, as the signal corresponding to the polyhistidine tag of the complete OprF protein did not disappear over time; and a second topology wherein about only half of the 6xHis-OprF protein is integrated in the membrane, as a smaller fragment of protein located between the molecular weight 20 and 25 kDa was generated over time.

[0201] These first observations were then corroborated and refined by AFM. The analysis of the force/distance (FD) curves showing specific adhesion phenomena in Triton® 1 x solution indicated that OprF adopts two different transmembrane topologies in the liposomal membrane, corresponding to its closed and open channel conformations. Based on the WLC model, 64% of the specific adhesion phenomena corresponded to 8 transmembrane domains (closed channel conformation) and 36% of the specific adhesion phenomena corresponded to 16 transmembrane domains (open channel conformation).

E.4/ Study of the Pore-Forming Activity of OprF in the Proteoliposomes Using Negative Staining Electron Microscopy and AFM

[0202] The negative staining electron microscopy and the AFM analysis of the OprF proteoliposomes made it possible to visualize the pore-forming activity of OprF in the liposomal membrane. In the electron microscopy images, a series of “holes” of an average size of 9.5±4 nm corresponding to pores were observed through the membranes of the liposomes wherein OprF was reconstituted. Such a perforation of the liposomal membrane was not observed in the images of the control liposomes, incubated with the cell lysate and the reaction mixture of the acellular system without DNA (negative control). Moreover, the topographic AFM images of the surface of the OprF proteoliposomes also revealed the presence of pores surrounded by OprF proteins and having a mean diameter of 10 nm. Pore formation was therefore attributed to the activity of the OprF protein in the liposomal membrane.