VHH antibody

Abstract

Provided is an excellent drug capable of increasing antimicrobial resistance against multi-drug resistant Pseudomonas aeruginosa.　The present invention relates to a VHH antibody capable of recognizing loop 1 or loop 2 in OprM of a RND-type multi-drug efflux pump of Pseudomonas aeruginosa, or a dimer or a trimer of the VHH antibody.

Description

VHH antibody

[0001] The present invention relates to antibodies that enhance the antimicrobial susceptibility of multidrug-resistant Pseudomonas aeruginosa.

[0002] Multidrug-resistant Gram-negative bacteria are a major cause of hospital-acquired infections, and their emergence and spread pose a serious public health problem worldwide. In 2017, the World Health Organization (WHO) published a list of drug-resistant pathogens for which the development of new antibiotics is urgently needed, identifying carbapenem-resistant Acinetobacter baumannii, Enterobacteriaceae, and Pseudomonas aeruginosa as particularly critical pathogens. In Japan, infections caused by these pathogens are designated as sentinel disease surveillance diseases under the Infectious Diseases Control Act, and designated designated medical institutions are required to report the number of cases every month. While infections caused by these pathogens are rare in healthy adults, pneumonia and bacteremia caused by bacterial infection can be fatal in immunocompromised individuals and those with serious underlying diseases. In particular, the mortality rate of bacteremia caused by multidrug-resistant Pseudomonas aeruginosa (MDRP) has been reported to be higher than that of other bacteria. According to 2019 statistics compiled by the inpatient specimen testing department of the Japan Nosocomial Infections Surveillance (JANIS), MDRP was isolated in 22.8% of the 2,075 medical institutions surveyed.

[0003] MDRP is resistant to all carbapenem, aminoglycoside, and fluoroquinolone antibiotics, which are generally effective against Pseudomonas aeruginosa infections. Pseudomonas aeruginosa acquires drug resistance through mechanisms such as the expression of drug efflux pumps, β-lactamase production, decreased antibiotic permeability, and DNA gyrase mutations. Drug efflux pumps, in particular, use the proton motive force to efflux various antibiotics taken up by the bacteria, independently causing multidrug resistance. Pseudomonas aeruginosa is a Gram-negative bacterium with an inner membrane, a lipid bilayer, and an outer membrane composed of peptidoglycan. Gram-negative bacteria express a complex called the Resistance Nodulation Cell Division (RND)-type multidrug efflux pump complex, which penetrates both the inner and outer membranes and effluxes drugs taken up by the bacteria, resulting in multidrug resistance. Twelve types of RND-type multidrug efflux pumps are known in Pseudomonas aeruginosa, of which MexAB-OprM is the most highly expressed and extrudes a wide range of drugs. MexAB-OprM consists of OprM, which penetrates the outer membrane and serves as a drug efflux pore; MexB, a transporter present in the inner membrane that utilizes the proton gradient to efflux drugs; and MexA, which connects OprM and MexB. OprM forms a tubular channel in the outer membrane, with two types of loops (loop 1 and loop 2) each with three protruding from the extracellular surface at the drug efflux port. It has been reported that MexAB-OprM-deficient Pseudomonas aeruginosa strains exhibit reduced minimum inhibitory concentrations (MICs) of antibiotics compared to wild-type strains. It has also been reported that the MICs of norfloxacin and aztreonam were reduced to 1 / 7 and 1 / 15, respectively, in Pseudomonas aeruginosa strains lacking only OprM. Although companies and research institutes have been developing efflux pump inhibitors, no clinically applicable inhibitors have been developed yet. PaβN (phenyl-arginine-β-naphthylamide) is a competitive efflux pump inhibitor that is excreted by MexB of Pseudomonas aeruginosa and increases the intracellular concentration of antibiotics (Non-Patent Document 1), but its toxicity has become an issue (Non-Patent Documents 2 and 3).It has been reported that PaβN,1-(1-naphthalmethyl)-piperazine (NMP) and phenothiazine reduced the MIC values ​​of antibacterial drugs in Pseudomonas aeruginosa to 1 / 8 or less, but neither of them has been used clinically as efflux pump inhibitors (Non-Patent Documents 4 and 5).

[0004] OprM is a trimer that forms a tubular channel in the outer membrane, with two types of loops (loop 1 and loop 2), each consisting of 10 amino acids, protruding three times from the extracellular surface of the drug efflux port. Yoshihara et al. of Tokai University produced mouse monoclonal antibodies against loop 1 and loop 2, aiming to suppress the function of the drug efflux pump MexAB-OprM of Pseudomonas aeruginosa and increase its susceptibility to antibacterial drugs, and reported that these antibodies enhanced the anti-Pseudomonas aeruginosa activity of aztreonam (Patent Document 1).

[0005] WO 2007 / 091395

[0006] J. Med. Chem. 1999, 42(24), 4928-4931. Antimicrob. Agents Chemother. 2001, 45(1), 105-116. J. Med. 2011, 6(1), 5870. Antimicrob. Agents Chemother. 2008, 52(10), 3604-3611. Antimicrob. Agents Chemother. 2014, 58(11), 6870-6878.

[0007] However, although the mouse monoclonal antibody described in Patent Document 1 enhanced the anti-Pseudomonas aeruginosa activity of aztreonam, the effect was insufficient, and there is a need for the development of a more effective agent that enhances the antibacterial sensitivity of multidrug-resistant Pseudomonas aeruginosa. Therefore, an object of the present invention is to provide an agent that enhances the antibacterial sensitivity of multidrug-resistant Pseudomonas aeruginosa.

[0008] Therefore, in order to develop a new antibody that increases the antimicrobial susceptibility of multidrug-resistant Pseudomonas aeruginosa, the present inventors prepared a VHH antibody, a dimer thereof, and a trimer thereof that recognizes loop 1 or loop 2 in OprM, an RND-type multidrug efflux pump of Pseudomonas aeruginosa, and examined their functions. Surprisingly, they found that although this VHH antibody does not have the effect of increasing susceptibility to antimicrobial drugs such as aztreonam, to which the antibody described in Patent Document 1 showed antimicrobial susceptibility, it has the effect of significantly increasing susceptibility to peptide antimicrobial drugs such as colistin, which are said to be a trump card against multidrug-resistant Pseudomonas aeruginosa, and that the mechanism of action is based on destabilizing the outer membrane of Pseudomonas aeruginosa, thereby completing the present invention.

[0009] That is, the present invention provides the following inventions [1] to

[16] . [1] A VHH antibody, a dimer or a trimer thereof that recognizes loop 1 or loop 2 in OprM of the RND-type multidrug efflux pump of Pseudomonas aeruginosa. [2] The VHH antibody, a dimer or a trimer thereof according to [1], wherein the CDR1 of the VHH antibody that recognizes loop 1 has the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having 90% or more homology to said amino acid sequence, the CDR2 has the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having 90% or more homology to said amino acid sequence, and the CDR3 has the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having 90% or more homology to said amino acid sequence. [3] The VHH antibody or dimer or trimer thereof according to [1], wherein the CDR1 of the VHH antibody recognizing loop 2 has the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having 90% or more homology to said amino acid sequence, the CDR2 has the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having 90% or more homology to said amino acid sequence, and the CDR3 has the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having 90% or more homology to said amino acid sequence. [4] The VHH antibody or dimer or trimer thereof according to [1] or [2], wherein the VHH antibody recognizing loop 1 has the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having 90% or more homology to said amino acid sequence. [5] The VHH antibody or dimer or trimer thereof according to [1] or [3], wherein the VHH antibody recognizing loop 2 has the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having 90% or more homology to said amino acid sequence. [6] The VHH antibody, dimer or trimer thereof according to any of [1] to [5], which is a humanized antibody. [7] The humanized VHH antibody, dimer or trimer thereof according to [6], wherein the humanized VHH antibody recognizing loop 1 has the amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence having 90% or more homology to said amino acid sequence. [8] The humanized VHH antibody, dimer or trimer thereof according to [6], wherein the humanized VHH antibody recognizing loop 2 has the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having 90% or more homology to said amino acid sequence.[9] A pharmaceutical composition comprising the VHH antibody, dimer, or trimer thereof according to any one of [1] to [8].

[10] The pharmaceutical composition according to [9], which is a pharmaceutical composition for enhancing the susceptibility of Pseudomonas aeruginosa to a peptide antibacterial agent.

[11] The pharmaceutical composition according to [9] or

[10] , which is used in combination with a peptide antibacterial agent.

[12] The pharmaceutical composition according to

[10] or

[11] , wherein the peptide antibacterial agent is colistin or a salt thereof.

[13] A Pseudomonas aeruginosa outer membrane destabilizer comprising the VHH antibody, dimer, or trimer thereof according to any one of [1] to [8] as an active ingredient.

[14] The VHH antibody, dimer, or trimer thereof according to any one of [1] to [8], used to enhance the susceptibility of Pseudomonas aeruginosa to a peptide antibacterial agent.

[15] Use of the VHH antibody, dimer, or trimer thereof according to any one of [1] to [8] for the manufacture of a medicament for enhancing the susceptibility of Pseudomonas aeruginosa to a peptide antibacterial agent.

[16] A method for increasing the susceptibility of Pseudomonas aeruginosa to a peptide antibacterial agent, comprising administering the VHH antibody, its dimer, or its trimer according to any one of [1] to [8].

[0010] The VHH antibodies of the present invention have the effect of significantly enhancing the sensitivity of Pseudomonas aeruginosa to peptide antibacterial drugs such as colistin, which are considered to be the ultimate weapon against multidrug-resistant Pseudomonas aeruginosa, and their mechanism of action is to destabilize the outer membrane of Pseudomonas aeruginosa. Furthermore, the effect of the VHH antibodies of the present invention to enhance the sensitivity of Pseudomonas aeruginosa to peptide antibacterial drugs has also been confirmed in vivo, and the dose of peptide antibacterial drugs such as colistin can be reduced.

[0011] The structure of MexAB-OprM and the secondary structure of OprM are shown. A. Schematic diagram showing the structure of MexAB-OprM. MexAB-OprM, an RND-type multidrug efflux pump of Pseudomonas aeruginosa, is composed of OprM, which penetrates the outer membrane and serves as a drug efflux pore; MexB, a transporter present in the inner membrane that utilizes the proton concentration gradient to efflux drugs; and MexA, which connects OprM and MexB. B. Secondary structure of OprM. OprM has a large α-helix domain that interacts with MexA and MexB, while the outer membrane-spanning domain is composed of β-sheets that form a barrel-shaped structure. There are two types of loops (loop 1 and loop 2) that protrude outside the cell, each consisting of 10 amino acids. The amino acid sequence of OprM and the antigen design are shown. Peptides were synthesized by adding 10 amino acids from OprM's extracellular protrusion loop 1 or loop 2, plus five amino acids from each of the sequences before and after, and adding a cysteine ​​to the C-terminus. Thyroglobulin was added to the loop 1 peptide as a carrier protein, and keyhole limpet hemocyanin (KLH) was added to the loop 2 peptide as a carrier protein to serve as antigens. The amino acid sequence of OprM is shown in SEQ ID NO: 15. The following shows the results of antibody titer measurements of alpaca serum. ELISA was performed using serum collected 5 weeks after immunization with loop 1 antigen or 7 weeks after immunization with loop 2 antigen. A. After immunization with loop 1 antigen, serum was reacted with immobilized thyroglobulin-loop 1 peptide or ovalbumin-loop 1 peptide, and bound antibodies were detected with anti-llama IgG-HRP. B. After immunization with loop 2 antigen, serum was reacted with immobilized loop 2 peptide, and bound antibodies were detected with anti-llama IgG-HRP. The results of enrichment of antigen-specific antibodies by biopanning are shown. Before panning, ELISA was performed using the phage library from rounds 1 to 4. A. Thyroglobulin, thyroglobulin-loop 1 peptide, and ovalbumin-loop 1 peptide were used as immobilized antigens, and detection was performed with anti-M13 monoclonal antibody and anti-mouse IgG-HRP. B. NaHCO3 (blank), KLH, and KLH-loop 2 peptide were used as immobilized antigens, and detection was performed with anti-M13 monoclonal antibody and anti-mouse IgG-HRP.96 phages were cloned from the loop 1 library after the fourth round of panning, showing the results of screening for phages that specifically bind to the loop 1 antigen, and ELISA was performed. Thyroglobulin and thyroglobulin-loop 1 peptide were used as immobilized antigens, and detection was performed with an anti-M13 monoclonal antibody and anti-mouse IgG-HRP. ELISA results for isolated phage clones are shown. A. ELISA was performed on phages monocloned after four rounds of biopanning against the loop 1 antigen. Thyroglobulin and thyroglobulin-loop 1 peptide were used as immobilized antigens, and detection was performed with an anti-M13 monoclonal antibody and anti-mouse IgG-HRP. B. ELISA was performed on phages monocloned after four rounds of biopanning against the loop 2 antigen. KLH and KLH-loop 2 peptide were used as immobilized antigens, and detection was performed with an anti-M13 monoclonal antibody and anti-mouse IgG-HRP. The amino acid sequences of VHHs that specifically bound to the antigens are shown. The amino acid sequences of 4P94, a VHH that specifically binds to loop 1 antigen, and 4P41, a VHH that specifically binds to loop 2 antigen, are shown. The antibody structures are shown. Based on the amino acid sequence information of 4P94 and 4P41, genes for the E. coli expression of 4P94 monomer, 4P94 LZ trimer, 4P41 monomer, 4P41 dimer, 4P41 trimer, and 4P41 LZ trimer were designed. Trimers were produced by linking three VHHs in tandem and by associating three VHHs via a leucine zipper. The results of SDS-PAGE of purified antibodies are shown. DNA of genes for the E. coli expression of 4P94 monomer, 4P94 LZ trimer, 4P41 monomer, 4P41 dimer, 4P41 trimer, and 4P41 LZ trimer were synthesized and transformed into E. coli RosettaGami. TM2 (DE3) (Novagen). Expression was induced by adding IPTG, and the prepared soluble fraction was purified by column chromatography as described in the methods section, followed by SDS-PAGE. 4P94 LZ trimer was eluted using a HisTrap column (A), and the eluted sample was purified by ion exchange chromatography (B). M: marker, SN: soluble fraction, PT: precipitated fraction, FT: flow-through fraction, E: eluted fraction. The antigen-binding activity of 4P94 monomer and 4P41 monomer was shown. A. The binding activity of 4P94 monomer to the loop 1 antigen was measured by ELISA. Thyroglobulin, thyroglobulin-loop 1 peptide, and ovalbumin-loop 1 peptide were immobilized as antigens, and detection was performed with anti-M13 monoclonal antibody and anti-mouse IgG-HRP. B. The binding activity of 4P41 monomer to the loop 2 antigen was measured by ELISA. The immobilized antigens were PBS, KLH, and KLH-loop 2 peptide, and detection was performed with anti-M13 monoclonal antibody and anti-mouse IgG-HRP. The binding affinity between the 4P41 monomer and loop 2 antigen is shown. The binding affinity between the 4P41 monomer and KLH-loop 2 was analyzed by surface plasmon resonance using a Biacore X-100 (Cytiva). Multi-cycle kinetics was performed, and the 4P41 monomer was measured at 4.4 nM, 89 nM, 178 nM, 355 nM, and 710 nM. The results of gel filtration chromatography of the 4P41 LZ trimer are shown. A purified sample of the 4P41 LZ trimer was analyzed using a HiLoad 16 / 60 Superdex 200 pg gel filtration column. The molecular weight was estimated from a calibration curve using a globular protein of known molecular weight as an index. The bacterial strains used are shown. The strains used in the experiment were Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli, and Staphylococcus aureus. The effects of 4P94 and 4P41 on antibiotics are shown. A. Minimum inhibitory concentrations (MICs) of antibiotics when 4P41 LZ trimer was added. The MICs of each antibiotic when 4P41 LZ trimer was added in NCGM2.S1 were measured by broth microdilution according to the CLSI guidelines. B. MICs of colistin when antibodies were added. The MICs of colistin when each antibody was added in NCGM2.S1 were measured by broth microdilution according to the CLSI guidelines.Antibodies were added at 10 μg / mL, and 4P94 LZ trimer + 4P41 LZ trimer were added at 15 μg / mL. 4P6 trimer, a human TRAIL-R1 VHH trimer, was used as a negative control. C. Minimum inhibitory concentration (MIC) of antibiotics when 4P94 LZ trimer was added. The MIC of each antibiotic when 4P94 LZ trimer was added to NCGM2.S1 was measured by broth microdilution according to CLSI guidelines. The colistin susceptibility of each bacterium when 4P41 LZ trimer was added is shown. Pseudomonas aeruginosa NCGM2.S1 The MICs of colistin in S1, OCR1, PAO1, TNP0721ΔOprM, Acinetobacter baumannii ATCC15308, Escherichia coli ATCC25922, and Staphylococcus aureus ATCC25923 were measured by broth microdilution according to CLSI guidelines, without and with 10 μg / mL 4P41 LZ trimer. Cytotoxicity of 4P41 LZ trimer in HepG2 cells was shown. Human liver cancer HepG2 cells were treated with 4P41 LZ trimer and solvent extraction buffer (50 mM Na Phosphate pH 7.8, 300 mM NaCl), and after 48 hours of incubation, Cell Counting Kit-8 was added and the absorbance at 450 nm was measured. The data were relative values, with the value for cells alone taken as 100%. Each concentration is shown as the mean value + standard error of five wells. The figures show the NCGM2.S1 administration concentration and survival rate in silkworms. An overnight culture of NCGM2.S1 was diluted 100-fold and cultured for 16 hours, then injected into the hemolymph of silkworms at 5th instar and 2 days old. The survival rate was measured 24 hours later. The figures show the therapeutically effective dose of colistin in a silkworm NCGM2.S1 infection model. A. An overnight culture of NCGM2.S1 was diluted 100-fold and cultured for 16 hours, then injected into the hemolymph of silkworms at 5th instar and 2 days old. Then, 0.05 mL of colistin was injected into the blood, and the survival rate was measured up to 96 hours later. B. NCGM2.S1 administration concentration and survival rate in silkworms at 5th instar and 2 days old. The overnight culture of S1 was diluted 100-fold and cultured for 16 hours. The diluted solution was then injected into the hemolymph of 2-day-old silkworms at the age of 5 years. Then, 0.05 mL of a mixture of colistin and 4P41 LZ trimer was injected into the hemolymph, and the survival rate was measured up to 96 hours later.C. An overnight culture of NCGM2.S1 was diluted 100-fold and cultured for 16 hours. The diluted solution was then injected into the hemolymph of 5-day-old silkworms. Then, 0.05 mL of a mixture of colistin and 4P94 LZ trimer was injected into the hemolymph, and survival rates were measured up to 96 hours later. The results of toxicity tests of 4P41 LZ trimer and 4P94 LZ trimer in silkworms are shown. Various concentrations of A. 4P41 LZ trimer or B. 4P94 LZ trimer were injected into the hemolymph of 5-day-old silkworms, and survival rates were measured up to 96 hours later. The results of FACS analysis of 4P41 LZ trimer and each bacterium are shown. 1 x 10 8 cfu / mL of each bacterium was incubated with 4P41 LZ trimer as the primary antibody or 4P6 VHH trimer as a negative control, Anti-His-tag mAb as the secondary antibody, and Alexa Fluor as the tertiary antibody. TM Antibody binding to bacteria was examined by FACS analysis using 488-conjugated AffiniPure Goat Anti-Mouse IgG. The effect of antibodies on the membrane of Pseudomonas aeruginosa was shown. The effect of antibodies on the membrane of NCGM2.S1 was examined using DiBAC4. NCGM2.S1 was added with DiBAC4, and the fluorescence intensity was measured when the pump inhibitor PaβN, 4P94 LZ trimer, 4P41 LZ trimer, and the anti-human TRAIL-R2 VHH antibody 4P6 VHH trimer were added. The fluorescence intensity ratio is shown, with the fluorescence intensity in the group without antibody set to 1. The amino acid sequences of humanized 4P94 and humanized 4P41 are shown. The framework sequences of 4P94 and 4P41 were humanized. The regions examined during humanization are shown for human 4P94 and human 4P41, and the parts whose sequences differ from the original antibodies due to humanization are shown in bold. The results of SDS-PAGE of the purified antibody and Western blotting using anti-His-Tag mouse monoclonal antibody HRP are shown. DNA was synthesized for the gene for expression of the humanized 4P41 LZ trimer in E. coli, and transformed into E. coli RosettaGami. TMThe vector was introduced into 2(DE3) (Novagen). Expression was induced by adding IPTG, and the prepared soluble fraction was purified on a HisTrap column (Cytiva) and then subjected to SDS-PAGE (A). Western blotting was also performed using anti-His-Tag mouse monoclonal antibody HRP (Thermo Fisher Scientific) (B). M: marker, SN: soluble fraction, PT: precipitate fraction, FT: flow-through fraction, E: elution fraction.

[0012] The present invention relates to a VHH antibody, a dimer thereof, or a trimer thereof that recognizes loop 1 or loop 2 in OprM of the RND-type multidrug efflux pump of Pseudomonas aeruginosa.

[0013] Among the RND-type multidrug efflux pumps of Pseudomonas aeruginosa, OprM is the drug efflux pump with the highest expression level and capable of effluxing a wide range of drugs. OprM is a trimer that forms a tubular channel in the outer membrane, with two types of loops (loop 1 and loop 2) consisting of 10 amino acids, each with three loops protruding from the extracellular side of the drug efflux port (see Figure 1). The VHH antibodies of the present invention include VHH antibodies that recognize loop 1 and VHH antibodies that recognize loop 2.

[0014] The VHH antibody recognizing loop 1 preferably has CDR1 having the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 90% or more homology thereto, CDR2 having the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having 90% or more homology thereto, and CDR3 having the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having 90% or more homology thereto. Here, homology refers to the percentage of identity of the amino acid sequences. The homology between these amino acid sequences is preferably 95% or more, more preferably 96% or more, even more preferably 98% or more, and even more preferably 99% or more.

[0015] The VHH antibody recognizing loop 1 preferably has the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having 90% or more homology to said amino acid sequence. The homology of these amino acid sequences is preferably 95% or more, more preferably 96% or more, even more preferably 98% or more, and even more preferably 99% or more.

[0016] The VHH antibody recognizing loop 2 preferably has CDR1 having the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having 90% or more homology thereto, CDR2 having the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having 90% or more homology thereto, and CDR3 having the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having 90% or more homology thereto. The homology between these amino acid sequences is preferably 95% or more, more preferably 96% or more, even more preferably 98% or more, and even more preferably 99% or more.

[0017] The VHH antibody recognizing loop 2 preferably has the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having 90% or more homology to said amino acid sequence. The homology of these amino acid sequences is preferably 95% or more, more preferably 96% or more, even more preferably 98% or more, and even more preferably 99% or more.

[0018] The VHH antibodies of the present invention can be produced by immunizing a camelid with a peptide having the amino acid sequence of the loop 1 region or a peptide having the amino acid sequence of the loop 2 region, and the resulting VHH antibodies can also be humanized. The humanized VHH antibody that recognizes loop 1 preferably has the amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence having 90% or more identity to said amino acid sequence. The homology between these amino acid sequences is preferably 95% or more, more preferably 96% or more, even more preferably 98% or more, and even more preferably 99% or more.

[0019] The humanized VHH antibody recognizing loop 2 preferably has the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having 90% or more homology to said amino acid sequence. The homology of these amino acid sequences is preferably 95% or more, more preferably 96% or more, even more preferably 98% or more, and even more preferably 99% or more.

[0020] The antibodies of the present invention are VHH antibodies, i.e., single-chain heavy-chain small molecule antibodies, and include monomers, dimers, and trimers. Of these, trimers are particularly preferred from the viewpoint of their ability to enhance sensitivity to peptide antibacterial agents.

[0021] The VHH antibodies of the present invention can be produced by immunizing a camelid with a peptide having the amino acid sequence of the extracellular protruding loop 1 region of OprM or a peptide having the amino acid sequence of the loop 2 region. However, because they are highly expressible in bacteria such as Escherichia coli, they can also be produced by designing a VHH antibody monomer, dimer, or trimer, artificially synthesizing the VHH antibody gene, and expressing and purifying it in recombinant Escherichia coli. Alpacas can be used as camelids. While peptides having a partial amino acid sequence of loop 1 or loop 2 can be used as antigens, it is preferable to use peptides having 10 to 20 amino acid residues to which a carrier protein is attached. Examples of carrier proteins that can be used include thyroglobulin and keyhole limpet hemoyanin (KLH). Mononuclear cells are prepared from the blood obtained by immunization of a camelid, and RNA is extracted from the mononuclear cells, followed by cDNA synthesis. The VHH region genes are amplified from the obtained cDNA by PCR, and the VHH genes are introduced into E. coli or the like to obtain a phage library. From the constructed phage library, cloned phages displaying VHH antibodies are obtained by panning, and clones that react with loop 1 or loop 2 antigens are selected from these, allowing the sequence of the antibody gene to be determined. Furthermore, once the sequence of the antibody gene has been determined, VHH antibody monomers, dimers, and trimers can be designed, and VHH antibody genes can be artificially synthesized, which can then be expressed in recombinant E. coli and purified for production.

[0022] The VHH antibodies of the present invention can also be humanized. Humanized VHH antibodies of the present invention can be obtained, for example, by synthesizing a DNA sequence designed to link the CDRs of an alpaca VHH antibody with the framework region (FR) of a human antibody by PCR from several oligonucleotides prepared so as to have overlapping portions at the termini, incorporating the resulting DNA into a vector, and introducing this into a host such as Escherichia coli for expression.

[0023] The VHH antibodies of the present invention did not exhibit the ability to lower the MIC (minimum inhibitory concentration) of various antibacterial agents, including aztreonam (fluoroquinolones, penicillins, carbapenems, aminoglycosides, and macrolides). However, they did exhibit in vitro activity to enhance the antibacterial activity of peptide antibacterial agents (colistin and polymyxin). Furthermore, a silkworm Pseudomonas aeruginosa infection model was created to examine the efficacy of the VHH antibodies of the present invention in vivo, demonstrating their ability to enhance the antibacterial activity of colistin. Furthermore, when the effect of the VHH antibodies of the present invention on the bacterial outer membrane was examined using a membrane potential-sensitive dye, the potential of the P. aeruginosa outer membrane changed in an antibody dose-dependent manner, suggesting that the VHH antibodies of the present invention destabilize the P. aeruginosa outer membrane, thereby enhancing the effect of colistin. Therefore, the VHH antibodies of the present invention, or their dimers or trimers, are useful as active ingredients in pharmaceutical compositions, particularly pharmaceutical compositions that enhance the susceptibility of P. aeruginosa to peptide antibacterial agents. Furthermore, the VHH antibody, dimer or trimer thereof of the present invention is also useful as an agent for destabilizing the outer membrane of Pseudomonas aeruginosa.

[0024] Pharmaceutical compositions containing the VHH antibodies, dimers, or trimers of the present invention can be prepared into oral or parenteral formulations by mixing, dissolving, emulsifying, encapsulating, lyophilizing, or the like, the VHH antibodies, dimers, or trimers of the present invention together with a pharmaceutically acceptable carrier.

[0025] Suitable formulations for oral administration include liquid preparations in which an effective amount of the VHH antibody of the present invention is dissolved in a diluent such as water or physiological saline; capsules, granules, powders, or tablets containing an effective amount as a solid or granule; suspensions in which an effective amount is suspended in a suitable dispersion medium; and emulsions in which a solution in which an effective amount is dissolved is dispersed and emulsified in a suitable dispersion medium.

[0026] For parenteral administration, the VHH antibodies of the present invention can be formulated into dosage forms such as injectable solutions, suspensions, emulsions, creams, ointments, inhalants, and suppositories, along with pharmaceutically acceptable solvents, excipients, binders, stabilizers, dispersants, etc. For injection formulations, the VHH antibodies of the present invention can be dissolved in an aqueous solution, preferably a physiologically compatible buffer such as Hank's solution, Ringer's solution, or physiological saline buffer. Furthermore, the medicaments of the present invention can take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle. Alternatively, the VHH antibodies of the present invention can be prepared in powder form, and an aqueous solution or suspension can be prepared using sterile water or the like before use. For inhalation administration, the VHH antibodies of the present invention can be powdered and mixed with a suitable base such as lactose or starch to form a powder mixture. Suppository formulations can be prepared by mixing the VHH antibodies of the present invention with a conventional suppository base such as cocoa butter. Furthermore, the VHH antibodies of the present invention can be encapsulated in a polymer matrix or the like and formulated as sustained-release preparations.

[0027] The pharmaceutical composition of the present invention is useful as a pharmaceutical composition that enhances the susceptibility of Pseudomonas aeruginosa to peptide antibacterial agents, and is therefore preferably used in combination with a peptide antibacterial agent. Examples of peptide antibacterial agents include colistin or a salt thereof, and polymyxin B. Of these, colistin or a salt thereof is preferred, with colistin or colistin methanesulfonate being more preferred. Colistin is considered a "trump card" against multidrug-resistant Gram-negative bacilli, but it is highly nephrotoxic and has a narrow efficacy and safety margin. Combination use with the VHH antibody of the present invention can reduce the colistin dosage, potentially leading to a safer treatment strategy. When using the pharmaceutical composition of the present invention in combination with a peptide antibacterial agent, these agents may be administered simultaneously, or one may be administered first and the other may be administered thereafter.

[0028] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0029] [Method] (1) Preparation of antigens. Peptides were synthesized by adding 10 amino acids from the extracellular protruding loop 1 or loop 2 of OprM and 5 amino acids from the adjacent sequences, and adding cysteine ​​to the C-terminus. Thyroglobulin was added to the loop 1 peptide as a carrier protein, and keyhole limpet hemoyanin (KLH) was added to the loop 2 peptide as a carrier protein to prepare antigens (Figure 2).

[0030] (2) Immunization of alpacas A 5-year-old female alpaca was given an emulsion of 500 μg / mL of antigen (loop 1 peptide-thyroglobulin or loop 2 peptide-KLH) mixed with an equal amount of adjuvant (Titer Max for the first time, Freund's adjuvant thereafter), which was then dispersed and administered into 10 sites in the thigh muscles. Immunization was carried out a total of 6 times, every other week. 10 mL of blood was collected before immunization, 5 weeks after immunization, and 7 weeks after immunization. 30 mL of blood was collected 10 weeks after immunization with loop 1 antigen and 14 weeks after immunization with loop 2 antigen.

[0031] (3) Antibody titer measurement. Loop 1 peptide-ovalbumin (5 μg / mL) or loop 2 peptide (5 μg / mL) was dispensed into a 96-well plate at 50 μL per well and immobilized. After blocking with Block Ace solution, 100 μL of serially diluted serum samples were added and incubated for 1 hour. After washing the wells, 100 μL of 10,000-fold diluted anti-Llama goat IgG-HRP (Bethyl Laboratories) was added as a secondary antibody and incubated for 1 hour. After washing the wells, the plates were incubated with ABTS and HO substrate solution (Thermo Fisher Scientific) for 15 minutes, and the absorbance at 415 nm was measured using a microplate reader (Corona).

[0032] (4) Preparation of mononuclear cells, RNA extraction, and cDNA synthesis. Heparinized blood was added to Leucosep (Greiner) filled with Lymphoprep (VERITAS), and the mixture was centrifuged at 20°C, 800 x g, for 30 minutes to collect the fraction containing mononuclear cells. RNAlater (QIGEN) was added and the mixture was suspended, then frozen and stored at -80°C. The frozen sample was thawed by shaking at 37°C, Buffer RLT was added, and the mixture was applied to a QIAshredder spin column (QIAGEN). After centrifugation at 18,000 x g for 2 minutes, RNA was extracted from the mononuclear cells using RNeasy Mini Kit (QIAGEN). PrimeScript II 1 st Using a strand cDNA synthesis kit (Takara Bio), single-strand cDNA was synthesized using an oligo dT primer and a random 6-mer primer (approximately 2.5 μg of total RNA was used in a 20 μL reaction solution).

[0033] (5) Preparation of Alpaca Library The VHH region genes were amplified from cDNA by PCR. The VHH antibody genes were amplified by PCR using PrimeSTAR GXL DNA polymerase (Takara Bio) with 25 cycles of 95°C for 1 minute, 98°C for 10 seconds, 55°C for 15 seconds, and 68°C for 1 minute (0.5 μL of cDNA reaction solution was used for 25 μL of PCR reaction solution). st The PCR product was subjected to PCR in the same manner, with one cycle of 95°C for 1 minute, followed by 25 cycles of 98°C for 10 seconds, 60°C for 15 seconds, and 68°C for 1 minute, to amplify the antibody gene (25 μL of PCR reaction solution was used, 1 st(5 pg-10 pg of PCR product was used.) The PCR primer DNA sequences are as follows: F: 5'-GGTGGTCCTGGCTGCTCTT-3' (SEQ ID NO: 11) R: 5'-AGGAGACGGTGACCTGGGT-3' (SEQ ID NO: 12) Second PCR primer F: 5'-GGTAAAGGCCCAGCCGGCCATGGCCGAGGTGCAGCTCGTGGAGTCTGGG-3' (SEQ ID NO: 13) SfiI R: 5'-TTGCAAGCAATTGCGGCCGCTGAGGAGACGGTGACCTGGGT-3' (SEQ ID NO: 14) NotI 2 nd The PCR products (VHH genes) were digested with SfiI and NotI and inserted into the SfiI and NotI sites of the pCANTAB6 vector (provided by Professor Yuji Ito, Faculty of Science and Technology, Kagoshima University). The vector was then introduced into Escherichia coli TG1 competent cells (Lucigen) by electroporation. TG1 cells were infected with M13K07 helper phage (provided by Professor Yuji Ito, Faculty of Science and Technology, Kagoshima University) to obtain a phage library expressing the VHH region. The Amersham Bioscience Expression Module / Recombinant Phage Antibody System instruction manual was used as a reference.

[0034] (6) Library Screening: Biopanning was performed to select phages expressing VHH antibodies against the target antigen from the prepared phage library. After immobilizing thyroglobulin-loop 1 peptide or KLH-loop 2 peptide on an immunotube for blocking, the phage library containing 200 μg / mL thyroglobulin or KLH was incubated overnight at 4°C. Unbound phages were removed by washing 20 times with PBS (137 mmol / L NaCl, 8.1 mmol / L NaHPO, 2.68 mmol / L KCl, 1.47 mmol / L KHPO, pH 7.4) and PBS-0.1% (v / v) Tween 20. 2 mL of 0.1 M Glycine HCl (pH 2.8) was added to the immunotube, followed by pipetting and neutralization with 200 μL of 1 M Tris HCl (pH 9.0), to elute the phage bound to the tube. The eluted phage were reinfected into E. coli and propagated. This procedure was repeated four times to enrich for phages specific to the target antigen. After reinfecting E. coli TG1 with the panned phage, TG1 clones containing the VHH-inserted pCANTAB phagemid vector were isolated, and individual TG1 clones were infected with KO7 helper phage to obtain cloned phages displaying VHH antibodies. Clones reactive to loop 1 or loop 2 antigens were selected from these, and the antibody gene sequences were determined.

[0035] (7) Confirmation of binding of VHH antibody monomer to loop 1 or loop 2 antigen by ELISA For anti-loop 1 VHH antibodies, 40 μL / well of 0.1 M NaHCO3 (Blank), 0.1 M NaHCO3 containing 10 μg / mL thyroglobulin, 0.1 M NaHCO3 containing 10 μg / mL thyroglobulin-loop 1 peptide, or 0.1 M NaHCO3 containing 10 μg / mL ovalbumin-loop 1 peptide was added to a 96-well immunoplate (Nunc) and allowed to stand overnight at 4°C. For anti-loop 2 VHH antibodies, 40 μL / well of 0.1 M NaHCO3 (Blank), 0.1 M NaHCO3 containing 10 μg / mL KLH, or 0.1 M NaHCO3 containing 10 μg / mL KLH-loop 2 peptide was added to a 96-well immunoplate (Nunc) and incubated overnight at 4°C. 350 μL / well of SuperBlock-PBS (ThermoFisher Scientific) was added and incubated at room temperature for 1 hour. After washing with 400 μL / well of PBS-T (phosphate-buffered saline containing 0.05% Tween 20), 40 μL / well of SuperBlock-TBS containing 0.2 μg / mL VHH antibody was added and incubated at room temperature for 1 hour. The plate was washed three times with 400 μL / well of PBS-T, and 40 μL / well of anti-Myc mouse monoclonal antibody 9E10 (Santa Cruz Biotechnology) diluted 500-fold with SuperBlock-TBS was added and allowed to react for 1 hour at room temperature. The plate was washed three times with 400 μL / well of PBS-T, and 40 μL / well of anti-mouse IgG goat antibody HRP was added and allowed to react for 1 hour at room temperature. The plate was then washed three times with 400 μL / well of PBS-T, and 50 μL / well of TMB reagent (Wako Pure Chemical Industries) was added. The reaction was allowed to proceed for approximately 10 minutes, after which the reaction was stopped with 0.5 M sulfuric acid. The absorbance at 450 nm was then measured, and the mean and standard deviation of triplicate or quadruplicate measurements were calculated.

[0036] (8) Expression and purification of recombinant 4P94 or 4P41 monomer protein in E. coli BL21(DE3) Based on the amino acid sequence information of 4P94 or 4P41, DNA synthesis of a gene for expression in E. coli was performed. Insertion of an NdeI-NotI DNA fragment into the NdeI and NotI sites of pET22b(+) (Novagen) constructs a gene encoding a protein with a cMyc-tag and His-tag attached to the C-terminus of a VHH antibody. The pET22b(+) plasmid DNA into which the VHH antibody DNA fragment was inserted was transformed into E. coli RosettaGami. TM The recombinant E. coli was cultured at 37°C in 200 mL of 2YT medium supplemented with 100 μg / mL ampicillin (SIGMA). 600 When the pH reached 0.4-0.5, 1 mM IPTG (isopropyl-β-thiogalactopyranoside, Takara Bio) was added and the mixture was cultured at 30°C for 3 hours. After the culture was completed, the E. coli cells were collected and resuspended in 20 mL of extraction buffer (50 mM Na phosphate pH 7.8, 300 mM NaCl, EDTA-free protease inhibitor cocktail (Roche)). The cells were disrupted by sonication twice for 1 minute on ice using a Sonifier 250 (Branson) under conditions of Output Control 2 and Duty cycle 80%. The suspension after the treatment was centrifuged at 20,000 x g at 4°C for 20 minutes, and the supernatant was collected. Protein purification was performed using a HisTrap column (GE Healthcare). The centrifuged supernatant of the sonicated suspension was directly applied to the HisTrap column. After washing the column with binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole pH 7.4), the fusion protein was eluted with an elution solution containing 500 mM imidazole. The purified sample was electrophoresed on 15% acrylamide SDS and stained with TaKaRa CBB Protein Safe Stain (Takara Bio), confirming the presence of 4P94 or 4P41 monomer protein at approximately 20 kDa. Western blotting was also performed using an anti-His-Tag mouse monoclonal antibody HRP.

[0037] (9) 4P41 dimer, 4P41 trimer NdeI site, Strep Tag 25 Two or three 4P41 monomer sequences were linked together with a (GGSG) linker peptide, and a HindIII site, Myc-Tag, and NotI site were attached to chemically synthesize a gene encoding a 4P41 dimeric or trimeric antibody.

[0038] (10) 4P94 Trimer A gene encoding a 4P94 trimeric antibody was chemically synthesized, comprising an NdeI site, a Strep Tag, and three 4P94 monomer sequences linked together with a (GGSG) linker peptide, and a HindIII site, a Myc-Tag, and a NotI site.

[0039] (11) 4P94 LZ trimer, 4P41 LZ trimer Genes encoding 4P94 LZ trimer and 4P41 LZ trimer were chemically synthesized, each containing an NdeI site, a 4P94 monomer sequence or a 4P41 monomer sequence, a HindIII site, a (GGSG) linker, a trimerization leucine zipper motif, a Myc-tag, and a NotI site. Insertion of an NdeI-NotI DNA fragment into the NdeI and NotI sites of pET22b(+) (Novagen) constructs a gene encoding a protein with a cMyc-tag and His-tag attached to the C-terminus of a VHH antibody. The pET22b(+) plasmid DNA containing the VHH antibody DNA fragment was transformed into E. coli. The recombinant E. coli was cultured at 37°C in 200 mL of 2YT medium supplemented with 100 μg / mL ampicillin (SIGMA). 600When the pH reached 0.3, 0.1 mM IPTG (Takara Bio) was added and the mixture was cultured at 20°C for 16 hours. After the culture was completed, the E. coli bacteria were collected and resuspended in 20 mL of extraction buffer (50 mM Na Phosphate pH 7.8, 300 mM NaCl, EDTA-Free protease inhibitor cocktail (Roche)). MicroSmash (Tomy Seiko) was used to disrupt the cells. 3 g of 0.1 mm diameter zirconia beads (Tomy Seiko) were added to 16 5 mL sample tubes (Tomy Seiko), and 5 mL of the cell suspension was added to each tube. This cycle was repeated three times at 4°C, 4200 rpm, and 5 minutes. After disruption, the beads were centrifuged at 20,000 x g and 4°C for 15 minutes, and the supernatant was collected and filtered through a 0.45 μm filter (Merck Millipore). Protein purification for 4P94 trimer and 4P94 LZ trimer was performed using a HisTrap column (Cytiva). The supernatant from the bead disruption was passed through a HisTrap column, washed with binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, pH 7.4), and the fusion protein was eluted with an elution solution containing 500 mM imidazole. The eluate for 4P94 LZ trimer was further purified by ion exchange chromatography using AKTA Pure 25 (Cytiva). A Resource S (Cytiva) column was used, and the fusion protein was eluted with a salt gradient of 25 mM MES pH 5.6 and 25 mM MES pH 5.6, 0.5 M NaCl. A Protein A column (Cytiva) was used to purify the 4P41 dimer, 4P41 trimer, and 4P41 LZ trimer. The supernatant of the bead disruption solution was loaded onto the Protein A column and washed with binding buffer (1.5 M glycine / NaOH pH 9.0, 0.5 M NaCl), after which the fusion protein was eluted with elution buffer (0.1 M glycine / HCl pH 2.5). The eluted fusion protein was subjected to endotoxin removal treatment using a NoEndo HC spin column kit (ProteinArk), and then the endotoxin concentration was confirmed to be less than 1 EU / mL using EndoLISA (J.K. International).The fusion protein was dialyzed against extraction buffer (50 mM Na Phosphate pH 7.8, 300 mM NaCl).

[0040] (12) Measurement of the dissociation constant (KD) between the 4P41 monomer and the KLH-loop 2 peptide. The binding affinity between the 4P41 monomer and the KLH-loop 2 peptide was analyzed by surface plasmon resonance using a Biacore X-100 (Cytiva). Measurements were performed using the multi-cycle kinetics method according to the instructions provided with the Biacore X-100, and the 4P41 monomer was measured at 4.4 nM, 89 nM, 178 nM, 355 nM, and 710 nM.

[0041] (13) Gel filtration chromatography of 4P41 LZ trimer. A purified sample of 4P41 LZ trimer was loaded onto a gel filtration column, HiLoad 16 / 60 Superdex 200 pg (Cytiva), equilibrated with SEC Running buffer (50 mM Tris pH 8.0, 150 mM NaCl), and separated at a flow rate of 1 mL / min. An AKTA Primeplus (Cytiva) was used for delivery and detection. The molecular weight was estimated from a calibration curve using a globular protein of known molecular weight as an index. The calibration curve was prepared using an LMW Gel filtration calibration kit (Cytiva).

[0042] (14) Cytotoxicity Test of 4P41 LZ Trimer Human liver cancer HepG2 cells (American Type Culture Collection) were suspended in DMEM medium (SIGMA) supplemented with 10% fetal bovine serum (Tissue Culture Biologicals) and plated at 3 × 10 cells / well in a Falcon 96-well culture plate (Becton Dickinson). 3Cells were added at a concentration of 100 μg / mL / 50 μL medium per well. After two nights of incubation, 50 μL of medium containing extraction buffer (50 mM Na Phosphate pH 7.8, 300 mM NaCl), a solvent for the 4P41 LZ trimer or antibody, was added at final concentrations of 10 μg / mL-160 μg / mL. After two more nights of incubation, 10 μL of Cell Counting Kit-8 (Dojindo Laboratories) was added, and after one hour of incubation, the absorbance at 450 nm was measured. The data were relative values, with the cell-only wells considered as 100%. The values ​​are shown as the mean ± standard error of five wells for each concentration.

[0043] (15) Antimicrobial susceptibility testing The minimum inhibitory concentration (MIC) of antimicrobial agents was measured by the broth microdilution method according to the Clinical Laboratory Standards Institute (CLSI) guidelines. Serially diluted antimicrobial agents were added to a 96-well round-bottom microtiter plate (Watson) at 100 μL / well, and each bacterial strain was diluted at 5 × 10 5 The plates were inoculated at CFU / mL / well. The antibiotics used were aztreonam (SIGMA), amikacin (SIGMA), arbekacin (Meiji Seika Pharma), ofloxacin (SIGMA), colistin (SIGMA), and polymyxin B (SIGMA). The synergistic effect of antibodies and antibiotics was evaluated using the checkerboard dilution method. Antibiotics were serially diluted two-fold vertically in a 96-well round-bottom titer plate to concentrations from 0.125 to 2 times the MIC. The antibodies were then serially diluted two-fold horizontally across the plate in the range of 0-10 μg / mL or 0-20 μg / mL. Each bacterial strain was inoculated at 5 x 10 5 The inoculation was at CFU / mL / well.

[0044] (16) Strains Used Multidrug-resistant Pseudomonas aeruginosa NCGM2.S1 and drug-susceptible Pseudomonas aeruginosa strains PAO1 and OCR1 were cultured at 37°C in Luria Bertani (LB) medium (BD) or on LB plates containing 15 g / L agar. OprM-deficient Pseudomonas aeruginosa TN0721ΔOprM (provided by Associate Professor Eisaku Yoshihara, Tokai University School of Medicine) was cultured in LB medium supplemented with 50 μg / mL sulbenicillin (MP Biomedicals). Drug-susceptible Escherichia coli ATCC25922 and Staphylococcus aureus ATCC25923 were cultured in tryptic soy broth at 37°C. Drug-susceptible Acinetobacter baumannii ATCC15308 was cultured in Difco TM The cells were cultured at 37°C using Nutrient broth (BD).

[0045] (17) Pseudomonas aeruginosa infection experiment using silkworms. Fertilized eggs of silkworm larvae (Shunreishougetsu, Ehime Silkworm Seeds) were disinfected with 70% ethanol (Wako), 4% formalin (Wako), and 99.5% ethanol (Wako) and reared in an incubator at 27°C on an artificial diet of Silkmate 2S (Nosan Corporation). One day before molting to 5 instars, the larvae were transferred to a new rearing box and fed an antibiotic-free artificial diet, Morus for 2 instars (Sysmex), for one day. An overnight culture of multidrug-resistant Pseudomonas aeruginosa NCGM2.S1 was diluted 100-fold and further cultured overnight. The bacterial solution was diluted with saline (Otsuka Pharmaceutical). 0.05 mL was injected into the hemolymph via the dorsal surface using a 1 mL syringe (Terumo) and a 27-gauge needle (Terumo). The larvae were then reared at 27°C. The median lethal dose (LD50) was determined from the survival rate after 24 hours, and 0.05 mL of bacteria four times the LD50 was injected to create a silkworm Pseudomonas aeruginosa infection model. 0.05 mL of colistin or colistin and 4P41 LZ trimer was injected into the hemolymph of the silkworm Pseudomonas aeruginosa infection model, and the number of survivors was counted for 4 days after injection.

[0046] (18) Membrane potential measurement 1 x 10 8Multidrug-resistant Pseudomonas aeruginosa NCGM2.S1 (cfu / mL) was washed with PBS and incubated with 1.1 M glucose at 37°C for 15 minutes. After adding 50 μM DiBAC4(3) (Dojindo Laboratories), 4P94 LZ trimer, 4P41 LZ trimer, or anti-human TRAIL-R1 VHH antibody 4P6 trimer or PaβN (Med ChemExpress), the plate was further incubated for 60 minutes and then measured using a microplate reader at an excitation wavelength of 490 nm and a maximum emission wavelength of 516 nm.

[0047] (19) FACS analysis: Pseudomonas aeruginosa NCGM2.S1, TNP0721ΔOprM, Acinetobacter baumannii ATCC15308, Escherichia coli ATCC25922, and Staphylococcus aureus ATCC25923 were each analyzed at 1 × 10 8 Cfu / mL of bacteria were incubated on ice for 60 minutes in 30 μL of PBS containing 10 μg / mL 4P41 LZ trimer or 4P6 trimer as a negative control. After washing with PBS, the cells were incubated on ice for 60 minutes in 30 μL of PBS containing 0.2 μg / mL Anti-His-tag mA (MBL). After washing with PBS, the cells were incubated on ice for 60 minutes in 30 μL of PBS containing 3 μg / mL Alexa Fluor. TM The cells were incubated on ice for 30 minutes in 30 μL of PBS containing 488-conjugated AffiniPure Goat Anti-Mouse IgG (Jackson ImmunoResearch), washed with PBS, incubated on ice for 10 minutes in 30 μL of PBS containing 0.5% paraformaldehyde, washed with PBS, and analyzed on a FACSVerse (BD Biosciences).

[0048] [Results] (1) Preparation of anti-loop 1 and anti-loop 2 VHH antibodies OprM is composed of 468 amino acids and has two extracellularly protruding loops ( Figure 1B ). Antigens against extracellularly protruding loops 1 and 2 of OprM were prepared as described in the Methods section above, and alpacas were immunized with these antibodies. Antibody titers were measured by ELISA using serum 5 weeks after immunization with loop 1 antigen and serum 7 weeks after immunization with loop 2 antigen ( Figure 3 ). Since an increase in antibody titer was confirmed, 30 mL of blood was collected 10 weeks after immunization with loop 1 antigen and 14 weeks after immunization with loop 2 antigen, and mononuclear cell fractions were prepared. RNA was extracted from the mononuclear cell fraction, cDNA was synthesized, and the VHH region genes were amplified by PCR. 1.2 x 10 ribosomal RNA was extracted from the mononuclear cell fraction of an alpaca immunized with loop 1 antigen. 5 Total RNA extracted from mononuclear cells of alpacas immunized with the loop 2 antigen was 6.3 x 10 5 The VHH genes were integrated into pCANTAB6 and introduced into E. coli TG1 competent cells by electroporation, and the TG1 cells were infected with M13K07 helper phage. 3 x 10 mAbs were extracted from total RNA derived from mononuclear cell fractions immunized with loop 1 antigen or loop 2 antigen. 7A phage library derived from TG1 colonies was obtained. Phages expressing VHH antibodies against loop 1 or loop 2 antigens were enriched in the phage library by biopanning. The phage pools obtained after four rounds of panning showed the highest reactivity with loop 1 or loop 2 peptides (Figure 4). After reinfection of E. coli TG1 with phages from the anti-loop 1 library and anti-loop 2 library after four rounds of panning, TG1 E. coli colonies containing VHH-inserted pCANTAB phagemid vectors were isolated, and individual TG1 E. coli clones were infected with K07 helper phage. Phage clones displaying anti-loop 1 VHH antibodies and anti-loop 2 VHH antibodies were screened by ELISA (Figure 5). Several phage clones showed specific binding to each antigen peptide (Figures 6A and 6B). VHH antibodies are classified into VHH type [37Phe / Tyr, 44Glu, 45Arg, 47Gly] and VH type [37Val, 44Gly, 45Leu, 47Trp] based on the amino acid sequence of the FR2 region. Phage clones that showed specific binding to loop 1 or loop 2 peptides were subjected to amino acid sequence analysis. As a result, 4P94 was identified as an anti-loop 1 VHH antibody, and 4P41 was identified as an anti-loop 2 VHH antibody (Figure 7). Based on the amino acid sequences of 4P94 and 4P41, genes for expression in E. coli were designed, and the genes for 4P94 monomer, 4P94 LZ trimer, 4P41 dimer, 4P41 trimer, and 4P41 LZ trimer were synthesized to prepare E. coli expression vectors (Figure 8). The expression vectors were inserted into pET22b(+) and transformed into E. coli. coli RosettaGami TM 2(DE3) (Novagen). The fusion proteins were expressed in the recombinant E. coli by adding IPTG, and after column purification, the proteins were confirmed by SDS-PAGE (Figure 9). The purified 4P94 monomer protein specifically bound to the ovalbumin-loop 1 peptide, and the purified 4P41 monomer protein specifically bound to the KLH-loop 2 peptide (Figures 10A and 10B). The binding affinity of the 4P41 monomer to the antigen, KLH-loop 2, was analyzed by surface plasmon resonance, revealing a KD value of 2.841 x 10 -7The 4P41 LZ trimer was detected as a monomer by SDS-PAGE, so it was analyzed by gel filtration column analysis to confirm the formation of a trimer. The gel filtration column analysis showed one monodisperse main peak, and the molecular weight estimated from a calibration curve using a globular protein of known molecular weight as an index was 55.5 kDa, suggesting the formation of a trimer (Figure 12). No peak corresponding to the monomer was observed, suggesting that all molecular species formed multimers.

[0049] (2) Antimicrobial susceptibility enhancement effect of antibodies against multidrug-resistant Pseudomonas aeruginosa 4P41 LZ trimer was added to the multidrug-resistant Pseudomonas aeruginosa strain NCGM2.S1, and the changes in the minimum inhibitory concentrations (MICs) of aztreonam, amikacin, arbekacin, ofloxacin, colistin, and polymyxin B were examined using the broth microdilution method (Figure 14-A). In the 4P41 LZ trimer-added group, the MICs of colistin and polymyxin B decreased to one-quarter, but the MICs of aztreonam, amikacin, arbekacin, and ofloxacin remained unchanged. The MICs of colistin were measured by broth microdilution when 4P94 monomer, 4P94 LZ trimer, 4P41 monomer, 4P41 dimer, 4P41 trimer, and 4P41 LZ trimer were added to multidrug-resistant P. aeruginosa at 10 μg / mL (Figure 14-B). The MICs of colistin were reduced to 1 / 4 with the addition of 4P94 LZ trimer, 4P41 trimer, and 4P41 LZ trimer. The MICs of colistin were reduced to 1 / 4 with the addition of 4P94 monomer, 4P41 monomer, and 4P41 dimer. The MICs of colistin were reduced to 1 / 4 with the addition of 15 μg / mL each of 4P94 LZ and 4P41 LZ. The MICs of colistin remained unchanged with the addition of 4P6 trimer, a human TRAIL-R1 VHH antibody trimer. For both 4P94 and 4P41 antibodies, trimers were more effective at enhancing colistin susceptibility against multidrug-resistant P. aeruginosa than monomers or dimers. The 4P94 LZ trimer was added to the multidrug-resistant P. aeruginosa strain NCGM2.S1, and the changes in the minimum inhibitory concentrations (MICs) of aztreonam, amikacin, arbekacin, ofloxacin, colistin, and polymyxin B were examined using the broth microdilution method (Fig. 14-C). The MICs of colistin and polymyxin B decreased to one-quarter when 4P94 LZ trimer was added.

[0050] (3) Enhancement of colistin susceptibility by antibodies against each bacterium. Wild-type P. aeruginosa strains PAO-1, ​​OCR1, and OprM-deficient P. aeruginosa strain TNP0721ΔOprM, Acinetobacter baumannii ATCC 15308, Escherichia coli ATCC 25922, and Staphylococcus aureus ATCC 25923 were treated with 10 μg / mL 4P94 LZ trimer or 4P41 LZ trimer, and the change in colistin MIC was examined using the broth microdilution method ( FIG. 15 ). The addition of 4P41 LZ trimer reduced the colistin MIC to one-quarter in P. aeruginosa, Acinetobacter baumannii, and E. coli, whereas the colistin MIC in S. aureus remained unchanged at >128 μg / mL.

[0051] (4) Cytotoxicity Test of 4P41 LZ Trimer The cytotoxicity of 4P41 LZ trimer against human liver cancer HepG2 cells (American Type Culture Collection) was examined (Figure 16). Cell viability when 4P41 LZ trimer was added to HepG2 cells at concentrations up to 160 μg / mL was similar to that observed when an extraction buffer, the antibody solvent, was added. This indicates that 4P41 LZ trimer is not toxic to human cells.

[0052] (5) Creation of a silkworm model for infection with multidrug-resistant Pseudomonas aeruginosa and the effect of antibodies on enhancing colistin susceptibility. NCGM2.S1 culture medium was injected into the hemolymph of silkworms, and the concentration at which 50% of the silkworms died after 24 hours was determined (Figure 17). The LD of NCGM2.S1 in silkworms was calculated using the probit method. 50 The result of the calculation was 0.4 x 10 7 The concentration at which 100% of the bacteria died after 24 hours was estimated to be 7 x 10 cfu / mL. 7 cfu / mL of NCGM2.S1 was injected into the silkworm Pseudomonas aeruginosa infection model. Colistin alone, or colistin with 4P41 LZ trimer or 4P94 LZ trimer was administered to the silkworm Pseudomonas aeruginosa infection model, and the therapeutically effective dose (ED) of colistin in each group was calculated. 50 The EDTA values ​​of colistin in each group were compared using the probit method (Figure 18). 50The calculated values ​​were 124 μg / g body weight for the colistin monotherapy group, 71 μg / g body weight for the colistin and 4P41 LZ trimer therapy group, and 85 μg / g body weight for the colistin and 4P94 LZ trimer therapy group. The therapeutically effective dose of colistin was lower in the 4P41 LZ trimer and 4P94 LZ trimer therapy groups compared with the colistin monotherapy group, demonstrating that the colistin susceptibility enhancing effect of 4P41 LZ trimer and 4P94 LZ trimer is also observed in vivo. Furthermore, 4P41 LZ trimer and 4P94 LZ trimer showed no toxicity to silkworms even at a dose of 22 μg / bw (Figures 19A and 19B). In a silkworm Pseudomonas aeruginosa infection model, the therapeutically effective dose of colistin was reduced by administration of 1.2-2.4 μg of 4P41 LZ trimer or 4P94 LZ trimer per body weight, and administration of 9-18 times that amount of antibody did not show toxicity to silkworms.

[0053] (6) FACS Analysis of 4P41 LZ Trimer and Various Bacteria. The 4P41 LZ trimer's ability to enhance colistin susceptibility was also observed in OprM-deficient Pseudomonas aeruginosa strains, Acinetobacter baumannii, and Escherichia coli. Therefore, FACS analysis confirmed that the 4P41 LZ trimer binds to these bacteria (Figure 20). Multidrug-resistant Pseudomonas aeruginosa NCGM2.S1, OprM-deficient Pseudomonas aeruginosa strain TNP0721ΔOprM, Acinetobacter baumannii ATCC15308, and Escherichia coli ATCC25922 were confirmed to bind to the 4P41 LZ trimer. The 4P41 LZ trimer did not bind to Staphylococcus aureus, whose colistin susceptibility remained unchanged upon addition of the 4P41 LZ trimer. A BLAST homology search revealed that Escherichia coli and Acinetobacter baumannii had sequences that were 60% and 100% identical to the loop 1 antigen sequence, respectively, and 74% and 100% identical to the loop 2 antigen sequence. Furthermore, OprJ, an OprM subfamily protein, had sequences that were 40% identical to the loop 1 antigen sequence and 60% identical to the loop 2 sequence, OpmA had sequences that were 40% identical to the loop 2 antigen sequence, and OpmB had sequences that were 40% identical to the loop 1 antigen sequence, suggesting the possibility that the antibody may bind to an efflux pump other than OprM. Furthermore, the TolC family protein of Pseudomonas aeruginosa had 100% identical to the loop 2 antigen sequence, suggesting the possibility that the antibody may bind to it.

[0054] (7) Effect of antibodies on P. aeruginosa membrane potential. Colistin and polymyxin B, which antibodies enhance the susceptibility of P. aeruginosa to, are positively charged and bind to the bacterial outer membrane, disrupting the calcium-magnesium bridges, causing leakage of cellular contents and cell death. The effect of antibodies on the bacterial outer membrane was examined using a membrane potential-sensitive dye (Fig. 21). The addition of 4P94 LZ trimer and 4P41 LZ trimer resulted in a dose-dependent change in the potential of the P. aeruginosa outer membrane. This suggests that the antibody may enhance the effect of colistin by destabilizing the P. aeruginosa outer membrane.

[0055] (8) Humanization of 4P94 and 4P41 Antibodies Humanization of 4P94 and 4P41 was investigated. The amino acid sequence of the humanized antibody was designed based on the humanization design of a VHH antibody that binds to the death receptor DR5 (Figure 22), and a humanized 4P41LZ trimer was produced. Expression and purification of recombinant humanized 4P41LZ trimer protein in E. coli BL21(DE3) 1. A gene encoding a humanized 4P41 VHH LZ trimer was chemically synthesized, combining an NdeI site, a humanized 4P41VHH monomer sequence, a (GGSG) linker, a trimerization leucine zipper motif, a Myc-Tag, and a NotI site. 2. Inserting the NdeI-NotI DNA fragment into the NdeI and NotI sites of pET22b(+) (Novagen) constructs a gene encoding a protein with a cMyc-Tag and His-Tag attached to the C-terminus of a VHH antibody. E. coli RosettaGami (DE3)2 ​​competent cells (Novagen) were transformed with the pET22b(+) plasmid DNA containing the VHH antibody DNA fragment. 3. The recombinant E. coli was cultured at 37°C in 200 mL of 2YT medium supplemented with 100 μg / mL ampicillin (Sigma-Aldrich) until the OD 600When the pH reached 0.3, 0.1 mM IPTG (Takara Bio) was added and the mixture was cultured at 30°C for 3 hours. After the culture was completed, the E. coli bacteria were collected and resuspended in 20 mL of extraction buffer (50 mM Na Phosphate pH 7.8, 300 mM NaCl, EDTA-Free protease inhibitor cocktail (Roche)). MicroSmash (Tomy Seiko) was used to disrupt the cells. 3 g of 0.1 mm diameter zirconia beads (Tomy Seiko) were added to 16 5 mL sample tubes (Tomy Seiko), and 5 mL of the cell suspension was added to each tube. This cycle was repeated three times at 4°C, 4200 rpm, and 5 minutes. After disruption, the beads were centrifuged at 20,000 x g and 4°C for 15 minutes, and the supernatant was collected and filtered through a 0.45 μm filter (Merck Millipore). 4. Protein purification was performed using a HisTrap column (Cytiva). The supernatant filtrate from the centrifuged bead disruption solution was passed through a HisTrap column, and after washing with binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole pH 7.4), the fusion protein was eluted with an elution solution containing 500 mM imidazole. 5. The purified sample was electrophoresed on 15% acrylamide SDS and stained with TaKaRa CBB Protein Safe Stain (Takara Bio). Western blotting was also performed using anti-His-Tag mouse monoclonal antibody HRP (Thermo Fisher Scientific). As a result, a protein reactive with the anti-His-Tag mouse monoclonal antibody was confirmed in the His-Tag column eluate sample at the predicted molecular weight of the humanized 4P41 LZ monomer protein (approximately 21 kDa) (Figure 23).

Claims

1. A VHH antibody, dimer or trimer thereof that recognizes loop 1 or loop 2 in OprM of the RND-type multidrug efflux pump of Pseudomonas aeruginosa, and that increases the susceptibility of Pseudomonas aeruginosa, Acinetobacter and Escherichia coli to peptide antibacterial drugs.

2. A VHH antibody, dimer or trimer thereof according to claim 1, wherein CDR1 of the VHH antibody recognizing loop 1 has the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having 90% or more homology to said amino acid sequence, CDR2 has the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having 90% or more homology to said amino acid sequence, and CDR3 has the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having 90% or more homology to said amino acid sequence.

3. A VHH antibody, dimer or trimer thereof according to claim 1, wherein the CDR1 of the VHH antibody recognizing loop 2 has the amino acid sequence shown in SEQ ID NO: 5 or an amino acid sequence having 90% or more homology to said amino acid sequence, the CDR2 has the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence having 90% or more homology to said amino acid sequence, and the CDR3 has the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having 90% or more homology to said amino acid sequence.

4. A VHH antibody, its dimer or its trimer as described in claim 1, wherein the VHH antibody recognizing loop 1 has the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having 90% or more homology to said amino acid sequence.

5. A VHH antibody, its dimer or its trimer as described in claim 1, wherein the VHH antibody recognizing loop 2 has the amino acid sequence shown in sequence number 8 or an amino acid sequence having 90% or more homology to said amino acid sequence.

6. The VHH antibody, dimer or trimer thereof according to claim 1, which is a humanized antibody.

7. A humanized VHH antibody, its dimer or its trimer described in claim 6, wherein the humanized VHH antibody recognizing loop 1 has the amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence having 90% or more homology to said amino acid sequence.

8. A humanized VHH antibody, its dimer or its trimer described in claim 6, wherein the humanized VHH antibody recognizing loop 2 has the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having 90% or more homology to said amino acid sequence.

9. A pharmaceutical composition comprising the VHH antibody, its dimer, or its trimer according to any one of claims 1 to 8.

10. The pharmaceutical composition according to claim 9, which is a pharmaceutical composition that enhances the susceptibility of Pseudomonas aeruginosa, Acinetobacter and Escherichia coli to a peptide antibacterial agent.

11. The pharmaceutical composition according to claim 9, which is used in combination with a peptide antibacterial agent.

12. The pharmaceutical composition according to claim 10, wherein the peptide antibacterial agent is colistin.

13. A Pseudomonas aeruginosa outer membrane destabilizing agent comprising the VHH antibody, its dimer, or its trimer according to any one of claims 1 to 8 as an active ingredient.

14. A VHH antibody, its dimer or its trimer according to claim 1, wherein the CDR1 of the VHH antibody recognizing loop 1 has the amino acid sequence shown in SEQ ID NO: 1, the CDR2 has the amino acid sequence shown in SEQ ID NO: 2, and the CDR3 has the amino acid sequence shown in SEQ ID NO:

3.

15. A VHH antibody, its dimer or its trimer according to claim 1, wherein the CDR1 of the VHH antibody recognizing loop 2 has the amino acid sequence shown in SEQ ID NO: 5, the CDR2 has the amino acid sequence shown in SEQ ID NO: 6, and the CDR3 has the amino acid sequence shown in SEQ ID NO:

7.

16. The VHH antibody, its dimer or its trimer according to claim 1, wherein the VHH antibody recognizing loop 1 has the amino acid sequence shown in SEQ ID NO:

4.

17. The VHH antibody, its dimer or its trimer according to claim 1, wherein the VHH antibody recognizing loop 2 has the amino acid sequence shown in SEQ ID NO:

8.

18. The VHH antibody, dimer or trimer thereof according to claim 14, which is a humanized antibody.

19. The VHH antibody, dimer or trimer thereof according to claim 15, which is a humanized antibody.

20. The humanized VHH antibody, its dimer or its trimer according to claim 14, wherein the humanized VHH antibody recognizing loop 1 has the amino acid sequence shown in SEQ ID NO:

9.

21. The humanized VHH antibody, its dimer or its trimer according to claim 15, wherein the humanized VHH antibody recognizing loop 2 has the amino acid sequence shown in SEQ ID NO:

10.

22. A pharmaceutical composition comprising the VHH antibody, its dimer, or its trimer according to any one of claims 14 to 21.

23. The pharmaceutical composition according to claim 22, which is a pharmaceutical composition that enhances the susceptibility of Pseudomonas aeruginosa, Acinetobacter and Escherichia coli to a peptide antibacterial agent.

24. The pharmaceutical composition according to claim 22, which is used in combination with a peptide antibacterial agent.

25. 24. The pharmaceutical composition according to claim 23, wherein the peptide antibacterial agent is colistin.

26. A Pseudomonas aeruginosa outer membrane destabilizing agent comprising the VHH antibody, its dimer, or its trimer according to any one of claims 14 to 21 as an active ingredient.