Patent Application
20250136669
The present invention relates to an antibody that increases the antimicrobial susceptibility of multidrug-resistant Pseudomonas aeruginosa.
Multidrug-resistant gram-negative bacteria are main pathogenic bacteria of hospital infection, and appearance and spread of these bacteria are serious public health problems all over the world. The World Health Organization (WHO) announced in 2017 a list of drug-resistant pathogens with high urgency for new antimicrobial agent development, and carbapenem-resistant Acinetobacter baumannii, Enterobacteriaceae, and Pseudomonas aeruginosa were listed as pathogens in particularly critical phases. In Japan, infectious diseases caused by these pathogens are all designated as sentinel diseases under the Infectious Diseases Law, and medical facilities designated as a sentinel site are required to report the number of patients every month. Healthy adults rarely develop the infectious diseases caused by these pathogens, but for patients with immunodeficiency or serious underlying diseases, pneumonia and bacteremia caused by the bacterial infection are potentially fatal. It has been reported that in particular, the mortality rate of bacteremia caused by multidrug-resistant Pseudomonas aeruginosa (MDRP) is higher than that of other bacteria. According to the 2019 tally in the inpatient specimen testing department of Japan Nosocomial Infections Surveillance (JANIS), MDRP was isolated at 22.8% of the 2,075 target medical facilities.
MDRP is resistant to all of carbapenem, aminoglycoside, and fluoroquinolone antimicrobial agents which are generally effective against Pseudomonas aeruginosa infectious diseases. Pseudomonas aeruginosa acquires drug resistance through mechanisms such as expression of a drug efflux pump, production of β-lactamase, a decrease in permeability of an antimicrobial agent, and a mutation of DNA gyrase. In particular, the drug efflux pump uses a proton motive force to excrete various antimicrobial agents taken into a bacterial cell and causes multidrug resistance independently.
Pseudomonas aeruginosa is a gram-negative bacterium and has inner membrane, and outer membrane composed of a lipid bilayer and peptide glycan. Gram-negative bacteria express a complex called resistance-nodulation-cell division (RND) type multidrug efflux pump complex that penetrates both the inner membrane and the outer membrane, which excretes the drug taken into a bacterial cell to cause multidrug resistance. Twelve types of RND-type multidrug efflux pumps are known in Pseudomonas aeruginosa. In particular, MexAB-OprM has the highest expression level and excretes a wide range of drugs. MexAB-OprM is composed of OprM that penetrates the outer membrane and functions as a through-hole for drug efflux, MexB that is a transporter present in the inner membrane and performs drug efflux utilizing the proton concentration gradient, and MexA that connects between OprM and MexB. OprM forms a tubular channel in the outer membrane, and 2 types of loops (loop 1 and loop 2), three each, protrude to the outside the cell at the outlet for drug efflux. It has been reported that the minimum growth inhibitory concentration (MIC) of an antimicrobial agent decreased in an MexAB-OprM-deficient Pseudomonas aeruginosa strain, compared to the wild-type strain. There is a report that in also a Pseudomonas aeruginosa strain in which only OprM has been deleted, the MICs of norfloxacin and Aztreonam decreased to 1/7 and 1/15, respectively. Until now, companies and research institutes have developed efflux pump inhibitors, but no inhibitor that can be used clinically has been obtained yet. PaβN (phenyl-arginine-β-naphthylamide) is a competitive efflux pump inhibitor that is excreted by MexB of Pseudomonas aeruginosa and increases the concentration of an antimicrobial agent in the bacterial cell (Non Patent Literature 1), but the toxicity thereof was a problem (Non Patent Literatures 2 and 3). It has been reported that PaβN, 1-(1-naphthylmethyl)-piperazine (NMP), and phenothiazine decreased the MIC value of an antimicrobial agent in Pseudomonas aeruginosa to ⅛ or less, but none of them is used clinically as an efflux pump inhibitor (Non Patent Literatures 4 and 5).
OprM is a trimer, forms a tubular channel in the outer membrane, and 2 types of loops (loop 1 and loop 2) constituted of 10 amino acids, three each, protrude to the outside the cell at the drug outlet. Yoshihara et al, Tokai University, produced a mouse monoclonal antibody to loop 1 and loop 2 for the purpose of suppressing the function of drug efflux pump MexAB-OprM of Pseudomonas aeruginosa and increasing the susceptibility to antimicrobial agents, and reported that this antibody enhances the anti-Pseudomonas aeruginosa activity of Aztreonam (Patent Literature 1).
However, the mouse monoclonal antibody described in Patent Literature 1 enhances the anti-Pseudomonas aeruginosa activity of Aztreonam, but the effect is not sufficient, and development of a further excellent drug that increases the antimicrobial susceptibility of multidrug-resistant Pseudomonas aeruginosa is being desired.
Accordingly, an object of the present invention is to provide an excellent drug that increases the antimicrobial susceptibility of multidrug-resistant Pseudomonas aeruginosa.
Accordingly, the present inventors produced a VHH antibody and a dimer and trimer thereof that recognize loop 1 or loop 2 in OprM of the RND-type multidrug efflux pump of Pseudomonas aeruginosa in order to develop a new antibody that increases the antimicrobial susceptibility of multidrug-resistant Pseudomonas aeruginosa and studied their functions. As a result, they found that this VHH antibody unexpectedly does not have an effect of increasing the susceptibility to antimicrobial agents such as Aztreonam to which the antibody described in Patent Literature 1 showed antimicrobial susceptibility, nevertheless, has an effect of significantly increasing the susceptibility to peptide antimicrobial agents such as colistin which are said to be a trump to multidrug-resistant Pseudomonas aeruginosa, and the effect mechanism is based on destabilization of the Pseudomonas aeruginosa outer membrane, and the present invention was accomplished.
That is, the present invention provides the following inventions [1] to:
The VHH antibody of the present invention has an effect of significantly increasing susceptibility to peptide antimicrobial agents such as colistin, which is said to be a potential trump card against multidrug-resistant Pseudomonas aeruginosa, and the effect mechanism is based on destabilization of the Pseudomonas aeruginosa outer membrane. The effect of increasing the susceptibility to peptide antimicrobial agents by the VHH antibody of the present invention is also confirmed in vivo, and the dose of peptide antimicrobial agents such as colistin can also be decreased.
The present invention is a VHH antibody or a dimer or trimer thereof that recognizes loop 1 or loop 2 in OprM of an RND-type multidrug efflux pump of Pseudomonas aeruginosa.
Among RND-type multidrug efflux pumps of Pseudomonas aeruginosa, OprM is a drug efflux pump that has the highest expression level and excretes a wide range of drugs. OprM is a trimer, forms a tubular channel in the outer membrane, and 2 types of loops (loop 1 and loop 2) each constituted of 10 amino acids, three each, protrude to the outside the cell at the drug outlet (see
The VHH antibody of the present invention includes a VHH antibody that recognizes loop 1 and a VHH antibody that recognizes loop 2.
In the VHH antibody that recognizes the loop 1, preferably, CDR1 has an amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least 90% homology to the amino acid sequence set forth in SEQ ID NO: 1, CDR2 has an amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence having at least 90% homology to the amino acid sequence set forth in SEQ ID NO: 2, and CDR3 has an amino acid sequence set forth in SEQ ID NO: 3 or an amino acid sequence having at least 90% homology to the amino acid sequence set forth in SEQ ID NO: 3.
Here, the term homology means the percentage identity of amino acid sequences. The homology of these amino acid sequences is preferably 95% or more, more preferably 96% or more, further more preferably 98% or more, and even more preferably 99% or more.
The VHH antibody that recognizes the loop 1 preferably has an amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having at least 90% homology to the amino acid sequence set forth in SEQ ID NO: 4.
The homology of these amino acid sequences is preferably 95% or more, more preferably 96% or more, further more preferably 98% or more, and even more preferably 99% or more.
As the VHH antibody that recognizes the loop 2, preferably, CDR1 has an amino acid sequence set forth in SEQ ID NO: 5 or an amino acid sequence having at least 90% homology to the amino acid sequence set forth in SEQ ID NO: 5, CDR2 has an amino acid sequence set forth in SEQ ID NO: 6 or an amino acid sequence having at least 90% homology to the amino acid sequence set forth in SEQ ID NO: 6, and CDR3 has an amino acid sequence set forth in SEQ ID NO: 7 or an amino acid sequence having at least 90% homology to the amino acid sequence set forth in SEQ ID NO: 7.
The homology of these amino acid sequences is preferably 95% or more, more preferably 96% or more, further more preferably 98% or more, and even more preferably 99% or more.
The VHH antibody that recognizes the loop 2 preferably has an amino acid sequence set forth in SEQ ID NO: 8 or an amino acid sequence having at least 90% homology to the amino acid sequence set forth in SEQ ID NO: 8.
The homology of these amino acid sequences is preferably 95% or more, more preferably 96% or more, further more preferably 98% or more, and even more preferably 99% or more.
The VHH antibody of the present invention can be produced by immunizing a Camelidae animal with a peptide including an amino acid sequence of a loop 1 region or a peptide including an amino acid sequence of a loop 2 region, and the obtained VHH antibody can be humanized.
The humanized VHH antibody that recognizes the loop 1 preferably has an amino acid sequence set forth in SEQ ID NO: 9 or an amino acid sequence having at least 90% homology to the amino acid sequence set forth in SEQ ID NO: 9. The homology of these amino acid sequences is preferably 95% or more, more preferably 96% or more, further more preferably 98% or more, and even more preferably 99% or more.
The humanized VHH antibody that recognizes the loop 2 preferably has an amino acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence having at least 90% homology to the amino acid sequence set forth in SEQ ID NO: 10. The homology of these amino acid sequences is preferably 958 or more, more preferably 968 or more, further more preferably 98% or more, and even more preferably 99% or more.
The antibody of the present invention is a VHH antibody, i.e., a single chain heavy chain small molecular antibody, and includes a monomer, a dimer, and a trimer. Among them, from the viewpoint of the effect of increasing the susceptibility to peptide antimicrobial agents, a trimer is particularly preferable.
The VHH antibody of the present invention can be produced by immunizing a Camelidae animal with a peptide including an amino acid sequence of the extracellularly protruding loop 1 region of OprM or a peptide including an amino acid sequence of loop 2 region, but since the VHH antibody is highly expressed in bacteria such as Escherichia coli, the VHH antibody can be manufactured by designing a monomer, dimer, or trimer of the VHH antibody, artificially synthesizing a VHH antibody gene, and performing expression in a recombinant Escherichia coli and purification.
As the Camelidae animal, alpaca can be used.
As the antigen, a peptide including an amino acid sequence of a part of loop 1 or loop 2 can be used, and a peptide in which a carrier protein is added to a peptide including 10 to 20 amino acid residues is preferably used. As the carrier protein, for example, thyroglobulin or Keyhole limpet hemocyanin (KLH) can be used.
Monocytes are prepared from the blood obtained by immunization of a Camelidae animal, and RNA extraction from the monocyte and cDNA synthesis are performed. The gene of a VHH region is amplified from the obtained cDNA by PCR, and the VHH gene is introduced into Escherichia coli or the like to obtain a phage library. Cloned phages displaying VHH antibodies are obtained from the produced phage library by panning, clones that react with a loop 1 or loop 2 antigen are selected from the phages, and sequences of antibody genes can be determined.
Furthermore, once the sequence of an antibody gene has been determined, the VHH antibody can be manufactured by designing a monomer, dimer, or trimer of the VHH antibody, artificially synthesizing a VHH antibody gene, and performing expression in a recombinant Escherichia coli and purification.
The VHH antibody of the present invention can also be humanized. The humanized VHH antibody of the present invention is obtained by, for example, designing a DNA sequence such that the CDR of an alpaca VHH antibody is linked to the framework region (FR) of a human antibody, synthesizing the DNA sequence by PCR from several oligonucleotides produced so as to have an overlapping portion at the end, inserting the obtained DNA into a vector, and introducing the vector into a host such as Escherichia coli for expression.
The VHH antibody of the present invention showed no function of decreasing the MIC (minimum bacterial growth inhibitory concentration) of various antimicrobial agents (new quinolones, penicillins, carbapenems, aminoglycosides, and macrolides) including Aztreonam. However, in vitro activity of enhancing the antibacterial activity of peptide antimicrobial agents (colistin and polymyxin) was recognized. In addition, a silkworm model of Pseudomonas aeruginosa infection was produced, and the in vivo effectiveness of the VHH antibody of the present invention was investigated. As a result, the activity of enhancing the antibacterial activity of colistin was also recognized in vivo. Furthermore, the influence of the VHH antibody of the present invention on the bacterial outer membrane was investigated using a membrane potential-sensitive dye, and it was suggested that the potential of the Pseudomonas aeruginosa outer membrane changes antibody dose-dependently and that the effect of colistin is enhanced by destabilizing the Pseudomonas aeruginosa outer membrane by the VHH antibody of the present invention.
Accordingly, the VHH antibody or a dimer or trimer thereof of the present invention is useful as an active ingredient of a pharmaceutical composition, in particular, a pharmaceutical composition that increases the peptide antimicrobial susceptibility of Pseudomonas aeruginosa. The VHH antibody or a dimer or trimer thereof of the present invention is also useful as a Pseudomonas aeruginosa outer membrane-destabilizing agent.
A pharmaceutical composition containing the VHH antibody or a dimer or trimer thereof of the present invention can be formulated into a preparation for oral administration or for parenteral administration by, for example, mixing, dissolving, emulsifying, capsulating, or lyophilizing the VHH antibody or a dimer or trimer thereof of the present invention together with a pharmaceutically acceptable carrier.
Examples of the preparation suitable for oral administration include a liquid in which an effective dose of the present inventive VHH antibody is dissolved in water or a diluent such as physiological saline; a capsule, granule, powder, or tablet containing an effective dose of the inventive VHH antibody as a solid or granule; a suspension in which an effective dose of the inventive VHH antibody is suspended in an appropriate dispersion medium; and an emulsion obtained by dispersing and emulsifying a solution in which an effective dose of the inventive VHH antibody is dissolved in an appropriate dispersion medium.
In the preparation for parenteral administration, the present inventive VHH antibody can be formulated together with a pharmaceutically acceptable solvent, excipient, binder, stabilizer, dispersant, or the like into a dosage form such as injectable solution, suspension, emulsion, cream, ointment, inhalant, and suppository. In a prescription for injection, the present inventive VHH antibody can be dissolved in an aqueous solution, preferably in a physiologically compatible buffer such as Hanks' solution, Ringel's solution, or a physiological saline buffer. Furthermore, the drug of the present invention can take a form of suspension, solution, emulsion, or the like in an oil-based or water-based vehicle. Alternatively, the present inventive VHH antibody may be produced in a powder form and may be prepared into an aqueous solution or suspension using sterile water or the like before use. For administration by inhalation, the present inventive VHH antibody is powdered and can be formed into a powder mixture with an appropriate base such as lactose or starch. In a prescription for suppository, the suppository can be produced by mixing the present inventive VHH antibody with a common suppository base such as cacao butter.
Furthermore, the present inventive VHH antibody can be prescribed as a sustained-release preparation by encapsulation in a polymer matrix or the like.
The pharmaceutical composition of the present invention is useful as a pharmaceutical composition for increasing the peptide antimicrobial susceptibility of Pseudomonas aeruginosa and is therefore preferably used in combination with a peptide antimicrobial agent. Examples of the peptide antimicrobial agent include colistin or its salt and polymyxin B.
Among them, colistin or its salt is preferable, and colistin or colistin methanesulfonate is more preferable.
Colistin is thought to be a “trump” to multidrug-resistant gram-negative bacilli, but is highly nephrotoxic and has a narrow efficacy and safety margins. It is inferred that a combination use with the present inventive VHH antibody can decrease the dose of colistin and leads to safer therapeutic strategy.
In the combination use of the pharmaceutical composition of the present invention and a peptide antimicrobial agent, these drugs may be administered simultaneously, or one may be administered first followed by the other.
The present invention will now be described in more detail with reference to Examples, but is not limited to these Examples.
Peptides that are each composed of 10 amino acids of the extracellularly protruding loop 1 or loop 2 of OprM, sequences of 5 amino acids added to before and after the 10 amino acids, and cysteine added to the C-terminal thereof were synthesized, and thyroglobulin and Keyhole limpet hemocyanin (KLH) were added as carrier proteins to the loop 1 peptide and the loop 2 peptide, respectively, to obtain antigens (
An emulsion prepared by mixing 500 μg/mL of an antigen (loop 1 peptide-thyroglobulin or loop 2 peptide-KLH) and an equal amount of adjuvant (Titer Max for the first time and Freund's adjuvant thereafter) was dispersively administered at 10 sites in the thigh muscles of a 5-year-old female alpaca. The immunization was carried out 6 times in total at one-week intervals. Ten milliliter of blood was collected before immunization, 5 weeks after immunization, and 7 weeks after immunization. Thirty milliliter of blood was collected 10 weeks after immunization with the loop 1 antigen and was collected 14 weeks after immunization with the loop 2 antigen.
Loop 1 peptide-ovalbumin (5 μg/mL) or loop 2 peptide (5 μg/mL) was dispensed in a 96-well plate at 50 μL per well and was immobilized. Block Ace solution was added thereto for blocking, and 100 μL of serially diluted serum samples were then added thereto, followed by reaction for 1 hour. The wells were then washed, and 100 μL of 10 000-times diluted anti-llama goat IgG-HRP (Bethyl Laboratories, Inc.) was added to each well as a secondary antibody, followed by reaction for 1 hour. The wells were washed, and ABTS and H2O2 substrate solution (Thermo Fisher Scientific) were applied thereto for 15 minutes. Absorbance at 415 nm was measured with a microplate reader (Corona Electric Co., Ltd.).
(4) Monocyte Preparation, RNA Extraction, and cDNA Synthesis
Heparin-treated blood was added to Leucosep (Greiner Bio-One) filled with Lymphoprep (VERITAS Corporation) and centrifugated at 20° C. at 800×g for 30 minutes, and a fraction containing monocytes was collected. RNAlater (QIAGEN) was added thereto to obtain suspension, which was then cryopreserved at −80° C. The frozen samples were shaken and thawed at 37° C., and Buffer RLT was added thereto. The mixture was applied to QIAshredder spin column (QIAGEN) and centrifugated at 18 000×g for 2 minutes, and RNA was extracted from monocytes using RNeasy Mini Kit (QIAGEN). Single strand CDNA was synthesized with PrimeScript II 1st strand cDNA synthesis kit (Takara Bio Inc.) using Oligo dT primer and Random 6mer primer (about 2.5 μg of total RNA was used for 20 μL of reaction solution).
A gene of a VHH region was amplified from cDNA by PCR. The VHH antibody gene was amplified using PrimeSTAR GXL DNA polymerase (Takara Bio Inc.) by PCR at 95° C. for 1 minute: 1 cycle, and 98° C. for 10 seconds, 55° C. for 15 seconds, and 68° C. for 1 minute: 25 cycles (0.5 μL of cDNA reaction solution was used for 25 μL of PCR reaction solution).
PCR of the 1st PCR product was similarly performed at 95° C. for 1 minute: 1 cycle, and 98° C. for 10 seconds, 60° C. for 15 seconds, and 68° C. for 1 minute: 25 cycles to amplify the antibody gene (5 pg to 10 μg of the 1st PCR product was used for 25 μL of PCR reaction solution). The primer DNA sequences for PCR were as follows:
SfiI
NotI.
The 2nd PCR product (VHH gene) was digested with SfiI and NotI, inserted into the SfiI and NotI sites of pCANTAB6 vector (provided by Professor Yuji Ito, Faculty of Science and Engineering, Kagoshima University), and introduced into Escherichia coli TG1 competent cells (Lucigen Corporation) by electroporation, and TG1 was infected with M13K07 helper phage (provided by Professor Yuji Ito, Faculty of Science and Engineering, Kagoshima University) to obtain a phage library expressing VHH region. Amersham Bioscience Expression Module/Recombinant Phage Antibody System instruction manual was referred to.
Biopanning of the produced phage library was performed for selecting a phage expressing a VHH antibody to a target antigen. Thyroglobulin-loop 1 peptide or KLH-loop 2 peptide was immobilized on an immune tube and blocked, and a phage library containing 200 μg/mL of thyroglobulin or KLH was reacted thereto at 4° C. overnight, followed by washing with PBS (137 mmol/L of NaCl, 8.1 mmol/L of Na2HPO4, 2.68 mmol/L of KCl, 1.47 mmol/L of KH2PO4, pH 7.4) and PBS-0.1% (v/v) Tween 20 20 times each to remove phages that did not bind. Two milliliter of 0.1 M Glycine HCl (pH 2.8) was added to the immune tube, and after pipetting, 200 μL of 1 M Tris HCl (pH 9.0) was put in the tube for neutralization, and the phage bound to the tube was eluted.
The eluted phage was re-infected to Escherichia coli and amplified. The above procedure was repeated 4 times to concentrate the phage specific to the target antigen. The panned phage was re-infected to Escherichia coli TG1, TG1 clone containing VHH-inserted pCANTAB phagemid vector was then isolated, and KO7 helper phage was infected to individual TG1 clones to obtain cloned phages displaying VHH antibodies. From these cloned phages, clones that react with loop 1 or loop 2 antigen were respectively selected to determine the sequences of the antibody genes.
In the anti-loop 1 VHH antibody, 0.1 M NaHCO3 (Blank), 0.1 M NaHCO3 containing 10 mg/ml of thyroglobulin, 0.1 M NaHCO3 containing 10 μg/mL of thyroglobulin-loop 1 peptide, or 0.1 M NaHCO3 containing 10 μg/mL of ovalbumin-loop 1 peptide was added to a 96-well immuno plate (Nunc) at 40 μL/well and was left to stand at 4° C. overnight.
In the anti-loop 2 VHH antibody, 0.1 M NaHCO3 (Blank), 0.1 M NaHCO3 containing 10 mg/ml of KLH, or 0.1 M NaHCO3 containing 10 mg/ml of KLH-loop 2 peptide was added to a 96-well immuno plate (Nunc) at 40 μL/well and was left to stand at 4° C. overnight. SuperBlock-PBS (Thermo Fisher Scientific) was added to the plate at 350 μL/well and left to stand at room temperature for 1 hour. After washing with 400 μL/well of PBS-T (phosphate buffer saline containing 0.05% Tween 20), 40 L/well of SuperBlock-TBS containing 0.2 μg/mL of the VHH antibody was added thereto, followed by reaction at room temperature for 1 hour. The wells were washed with 400 μL/well of PBS-T again three times, and 40 μL/well of anti-Myc mouse monoclonal antibody 9E10 (Santa Cruz Biotechnology Inc.) diluted 500 times with SuperBlock-TBS was added thereto, followed by reaction at room temperature for 1 hour. After washing with 400 μL/well of PBS-T three times, 40 μL/well of anti-mouse IgG goat antibody HRP was added thereto, followed by reaction at room temperature for 1 hour. Subsequently, the wells were washed with 400 μL/well of PBS-T three times, 50 μL/well of TMB reagent (FUJIFILM Wako Pure Chemical Corporation) was added thereto, reaction was performed for about 10 minutes, and the reaction was stopped with 0.5 M sulfuric acid. Subsequently, absorbance at 450 nm was measured, and the average and standard deviation were calculated of triplicate or quadruplicate measurement.
(8) Expression and Purification of Recombinant 4P94 Monomer Protein or 4P41 Monomer Protein in E. coli BL21 (DE3)
Genes for Escherichia coli expression were DNA-synthesized based on the amino acid sequence information on 4P94 or 4P41. An NdeI-NotI DNA fragment was inserted into the NdeI and NotI sites of pET22b(+) (Novagen) to construct a gene encoding a protein in which cMyc-Tag and His-Tag were added to the C-terminal side of a VHH antibody. An E. coli RosettaGami™ 2 (DE3) competent cell (Novagen) was transformed with pET22b(+) plasmid DNA into which the VHH antibody DNA fragment had been inserted. The recombinant Escherichia coli was cultured at 37° C. using 200 mL of a 2YT medium containing 100 μg/mL of ampicillin (Sigma-Aldrich), and 1 mM IPTG (isopropyl-β-thiogalactopyranoside, Takara Bio Inc.) was added thereto at the time when OD600 reached 0.4 to 0.5, followed by culturing at 30° C. for 3 hours. After completion of the culturing, Escherichia coli was 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 Diagnostics)). Ultrasonication treatment was performed in ice using Sonifier 250 (Branson Ultrasonics) twice for 1 minute under conditions of Output Control 2 and Duty cycle 80% to disrupt the bacterial cells. The suspension after the treatment was centrifugated at 20 000×g at 4° C. for 20 minutes, and the supernatant was collected. The protein was purified using a His-Trap column (GE Healthcare). The centrifuge supernatant of ultrasonicated suspension was directly applied to a His-Trap column, the column was washed with a binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, pH 7.4), and the fusion protein was then eluted with an eluate containing 500 mM imidazole. The purified sample was subjected to SDS electrophoresis and then stained with TakaRa CBB Protein Safe Stain (Takara Bio Inc.) to confirm 4P94 or 4P41 monomer protein at about 20 kDa. In addition, western blotting was performed using anti-His-Tag mouse monoclonal antibody HRP.
A gene encoding a 4P41 dimeric antibody or 4P41 trimeric antibody in which NdeI site, Strep Tag25, two or three 4P41 monomer sequences linked with a linker peptide (GGSG), HindIII site, Myc-Tag, and NotI site were bound was chemically synthesized.
A gene encoding a 4P94 trimeric antibody in which NdeI site, Strep Tag, three 4P94 monomer sequences linked with a linker peptide (GGSG), HindIII site, Myc-Tag, and NotI site were bound was chemically synthesized.
A gene encoding a 4P94 LZ trimer or 4P41 LZ trimer in which NdeI site, a 4P94 monomer sequence or 4P41 monomer sequence, HindIII site, a (GGSG) linker, a trimer-forming leucine zipper motif, Myc-Tag, and NotI site were bound was chemically synthesized.
An NdeI-NotI DNA fragment was inserted into the NdeI and NotI sites of pET22b(+) (Novagen) to construct a gene encoding a protein in which cMyc-Tag and His-Tag were added to the C-terminal side of a VHH antibody. An E. coli RosettaGami 2 (DE3) competent cell (Novagen) was transformed with pET22b(+) plasmid DNA into which the VHH antibody DNA fragment had been inserted. The recombinant Escherichia coli was cultured at 37° C. using 200 mL of a 2YT medium containing 100 μg/mL of ampicillin (Sigma-Aldrich), and 0.1 mM IPTG (Takara Bio Inc.) was added thereto at the time when OD600 reached 0.3, followed by culturing at 20° C. for 16 hours. After completion of the culturing, Escherichia coli was 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 Diagnostics)). Bacterial cell disruption used Micro Smash (Tomy Seiko Co., Ltd.). Three gram of 0.1 mm diameter zirconia beads (Tomy Seiko Co., Ltd.) was added to each of 16 5-mL sample tubes (Tomy Seiko Co., Ltd.), 5 mL of a bacterial cell suspension was added to each of the tubes, and disruption at 4° C. at 4 200 rpm for 5 minutes was repeated three times. After the disruption, centrifugation was performed at 20 000×g at 4° C. for 15 minutes, and the supernatant was collected and filtered through a 0.45 μm diameter filter (Merck Millipore). Purification of protein was performed using a His-Trap column (Cytiva) for the 4P94 trimer and the 4P94 LZ trimer. The centrifuge supernatant of the bead disruption solution was applied to a His-Trap column, the column was washed with a binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, pH 7.4), and the fusion protein was then eluted with an eluate containing 500 mM imidazole. The 4P94 LZ trimer was purified again by ion exchange chromatography using AKTA pure 25 (Cytiva) as the eluate. The fusion protein was eluted using Resource S (Cytiva) as the column with a salt concentration gradient from 25 mM MES pH 5.6 to 25 mM MES pH 5.6, 0.5 M NaCl. Purification of 4P41 dimer, 4P41 trimer, and 4P41 LZ trimer used Protein A column (Cytiva). The centrifuge supernatant of the bead disruption solution was applied to the Protein A column and washed with a binding buffer (1.5 M glycine/NaOH, pH 9.0, 0.5 M NaCl), and the fusion protein was eluted with an eluate (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 (Protein Ark), and it was then verified using EndoLISA (J. K. International) that the endotoxin concentration was less than 1 EU/mL. The fusion protein was dialyzed using an extraction buffer (50 mM Na Phosphate, pH 7.8, 300 mM NaCl).
The binding affinity between 4P41 monomer and KLH-loop 2 was analyzed by surface plasmon resonance using Biacore X-100 (Cytiva). The measurement was performed by a Multicycle kinetics method in accordance with the manual attached to Biacore X-100, and 4P41 monomer was measured at 4.4 nM, 89 nM, 178 nM, 355 nM, and 710 nM.
The purification sample of the 4P41 LZ trimer was added to 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 was separated at a flow rate of 1 mL/min. Liquid delivering and detection used AKTA Prime Plus (Cytiva). The molecular weight was estimated from a calibration curve using globular proteins of known molecular weights as an indicator. The calibration curve was produced using LMW Gel filtration calibration kit (Cytiva).
Human liver cancer HepG2 cells (American Type Culture Collection) were suspended in a DMEM medium (Sigma-Aldrich) containing 10% fetal bovine serum (Tissue Culture Biologicals) and were added to a Falcon 96-well culture plate (Becton, Dickinson and Company) at 3×103 cells/50 μL medium/well. After culturing for two nights, 50 μL of a medium containing the 4P41 LZ trimer or an antibody solvent extraction buffer (50 mM Na phosphate, pH 7.8, 300 mM NaCl) was added to each well at a final concentration of 10 μg/mL to 160 μg/mL. After further culturing for two nights, 10 μL Cell counting kit-8 (Dojindo Laboratories) was added thereto. After culturing for 1 hour, absorbance at 450 nm was measured. The well containing only cells was set to 100%, and the relative values thereto were used as data, which are shown as the average±standard error of 5 wells of each concentration.
The minimum growth inhibitory concentration (MIC) of an antimicrobial agent was measured by a microbroth dilution method in accordance with the Clinical Laboratory Standards Institute (CLSI) guideline. The serially diluted antimicrobial agent was added to a 96-well round bottom microtiter plate (Watson Co., Ltd.) at 100 μL/well, and each bacterial strain was inoculated at 5×105 CFU/mL/well. As the antimicrobial agent, Aztreonam (Sigma-Aldrich), Amikacin (Sigma-Aldrich), Arbekacin (Meiji Seika Pharma Co., Ltd.), Ofloxacin (Sigma-Aldrich), colistin (Sigma-Aldrich), and Polymyxin B (Sigma-Aldrich) were used. Synergistic effect of an antibody and an antimicrobial agent was evaluated using a checkerboard dilution method. Each of the antimicrobial agents was serially two-fold diluted in the vertical direction in a 96-well round bottom titer plate to concentrations between 0.125 to 2 times the MIC. Subsequently, an antibody was serially two-fold diluted in the horizontal direction of the plate in a range of 0 to 10 μg/mL or 0 to 20 μg/mL. Each bacterial strain was inoculated at 5×105 CFU/mL/well.
A multidrug-resistant Pseudomonas aeruginosa NCGM2.S1 and drug-susceptible Pseudomonas aeruginosa PAO1 and OCR1 were cultured using Luria Bertani (LB) medium (BD) or an LB plate containing 15 g/L of agar at 37° C. OprM-deficient Pseudomonas aeruginosa TN0721ΔOprM (provided by Associated Professor Eisaku Yoshihara, Tokai University School of Medicine) was cultured in an LB medium containing 50 μg/mL of Sulbenicillin (MP Biomedicals). Drug-susceptible Escherichia coli ATCC25922 and Staphylococcus aureus ATCC25923 were cultured using a tryptic soy broth at 37° C. Drug-susceptible Acinetobacter baumannii ATCC15308 was cultured using Difco™ Nutrient broth (BD) at 37° C.
(17) Pseudomonas aeruginosa Infection Experiment with Silkworm
Fertilized eggs of silkworm larvae (Shunrei Shogetsu, Ehime silkworm species) were disinfected with 70% ethanol (FUJIFILM Wako Pure Chemical Corporation), 4% formalin (FUJIFILM Wako Pure Chemical Corporation), and 99.5% ethanol (FUJIFILM Wako Pure Chemical Corporation) and were bred with artificial feed Silkmate 2S (Nosan Corporation) in an incubator of 27° C. The silkworm larvae were transferred to a new feeding box one day before shedding to 5th instar, and were fed with artificial feed not containing antibiotics Morus for 2nd instar (Sysmex Corporation) for 1 day. Overnight culture solution of multidrug-resistant Pseudomonas aeruginosa NCGM2.S1 was diluted 100 times and was further cultured overnight. The bacterial culture solution was diluted with physiological saline (Otsuka Pharmaceutical Co., Ltd.), and 0.05 mL thereof was injected into hemolymph from the dorsal surface using a 1-mL injection syringe (Terumo Corporation) and 27-gauge syringe needle (Terumo Corporation), and the silkworm larvae were fed at 27° C. The median lethal dose was measured from the survival rate after 24 hours, and 0.05 mL of the bacteria in an amount 4 times the median lethal dose was injected to make a silkworm Pseudomonas aeruginosa infection model. To the silkworm Pseudomonas aeruginosa infection model, 0.05 mL of colistin or colistin and the 4P41 LZ trimer was injected into hemolymph, and the number of surviving was counted over 4 days after the injection.
1×108 CFU/mL of multidrug-resistant Pseudomonas aeruginosa NCGM2.S1 was washed with PBS, and 1.1 M glucose was added thereto, followed by incubation at 37° C. for 15 minutes. 50 μM DiBAC4 (3) (Dojindo Laboratories) and the 4P94 LZ trimer or the 4P41 LZ trimer or an anti-human TRAIL-R1 VHH antibody 4P6 trimer or PaBN (Med Chemexpress) were added thereto, and further incubation was then performed for 60 minutes, and the absorbance was measured with a microplate reader at an excitation wavelength of 490 nm and a maximum fluorescence wavelength of 516 nm.
Regarding Pseudomonas aeruginosa NCGM2.S1, TNP0721ΔOprM, Acinetobacter baumannii ATCC15308, Escherichia coli ATCC25922, and Staphylococcus aureus ATCC25923, 1×108 CFU/mL of each of the bacteria was reacted in 30 μL of PBS containing 10 μg/mL of the 4P41 LZ trimer or the 4P6 trimer as a negative control on ice for 60 minutes. After washing with PBS, the bacteria were reacted in 30 μL of PBS containing 0.2 μg/mL of anti-His-tag mA (Medical & Biological Laboratories Co., Ltd.) on ice for 60 minutes. After washing with PBS, the bacteria were reacted in 30 μL of PBS containing 3 μg/mL of Alexa Fluor™ 488-conjugated AffiniPure Goat Anti-Mouse IgG (Jackson Immuno Research Inc.) on ice for 30 minutes. After washing with PBS, the bacteria were reacted in 30 μL of PBS containing 0.5% paraformaldehyde on ice for 10 minutes. After washing with PBS, analysis with FACSVerse (BD Biosciences) was performed.
OprM is composed of 468 amino acids and includes two loops protruding to the outside the cell (
The phage pool panned four times had the highest reactivity with the loop 1 or loop 2 peptide (
VHH antibodies are classified into a VHH type [37Phe/Tyr, 44Glu, 45Arg, and 47Gly] and a VH type [37Val, 44Gly, 45Leu, and 47Trp] depending on the FR2 region amino acid sequences. The amino acid sequences of the phage clones that showed specific binding to the loop 1 or loop 2 peptide were analyzed. As a result, 4P94 was obtained as an anti-loop 1 VHH antibody, and 4P41 was obtained as an anti-loop 2 VHH antibody (
Since the 4P41 LZ trimer is detected as a monomer by SDS-PAGE, in order to verify the formation of a trimer, analysis with a gel filtration column was performed. As a result of the gel filtration column analysis, one monodisperse main peak was observed and the molecular weight estimated from a calibration curve using spherical proteins of known molecular weights as an indicator was 55.5 kDa, which suggested that a trimer was formed (
(2) Antimicrobial Susceptibility-Increasing Effect of Antibody on Multidrug-Resistant Pseudomonas aeruginosa
The 4P41 LZ trimer was added to multidrug-resistant Pseudomonas aeruginosa strain NCGM2.S1, and changes in the minimum growth inhibitory concentration (MIC) of Aztreonam, Amikacin, Arbekacin, Ofloxacin, colistin, or Polymyxin B were investigated by a microbroth dilution method (
The MIC of colistin when the 4P94 monomer, 4P94 LZ trimer, 4P41 monomer, 4P41 dimer, 4P41 trimer, or 4P41 LZ trimer was added at a concentration of 10 μg/mL to multidrug-resistant Pseudomonas aeruginosa was measured using a microbroth dilution method (
The changes in the minimum growth inhibitory concentration (MIC) of Aztreonam, Amikacin, Arbekacin, Ofloxacin, colistin, and Polymyxin B when the 4P94 LZ trimer was added to multidrug-resistant Pseudomonas aeruginosa strain NCGM2.S1 were investigated by a microbroth dilution method (
The 4P94 LZ trimer or 4P41 LZ trimer was added at 10 μg/mL to wild-type Pseudomonas aeruginosa PAO-1 and OCR1, OprM deficiency Pseudomonas aeruginosa strain TNP0721ΔOprM, Acinetobacter baumannii ATCC15308, Escherichia coli ATCC25922, and Staphylococcus aureus ATCC25923, the change in the MIC of colistin was investigated by a microbroth dilution method (
The cytotoxicity of the 4P41 LZ trimer on human liver cancer HepG2 cells (American Type Culture Collection) was investigated (
(5) Production of Silkworm Multidrug-Resistant Pseudomonas aeruginosa Infection Model and Colistin Susceptibility-Increasing Effect of Antibody
NCGM2.S1 culture solution was injected into hemolymph of silkworms, and the concentration that causes 50% death after 24 hours was investigated (
Since the colistin susceptibility-increasing effect of the 4P41 LZ trimer was observed in OprM deficiency Pseudomonas aeruginosa strain, Acinetobacter baumannii, and also Escherichia coli, it was confirmed by FACS analysis that the 4P41 LZ trimer binds to these bacteria (
(7) Influence of Antibody on Membrane Potential of Pseudomonas aeruginosa
Colistin and Polymyxin B whose susceptibility of Pseudomonas aeruginosa was increased by an antibody shows a positive charge and binds to the outer membrane of a bacterium to collapse the cross-linked structure of calcium and magnesium to leak cell contents, leading to cell death. Influence of an antibody on the bacterial outer membrane was investigated using a membrane potential-sensitive dye (
Humanization of 4P94 and 4P41 was investigated. The amino acid sequence of a humanized antibody was designed based on the humanization model of the VHH antibody that binds to cell death receptor DR5 (
Expression and purification of recombinant humanized 4P41LZ trimer protein in E. coli BL21 (DE3)
1. A gene encoding a humanized 4P41 VHH LZ trimer in which NdeI site, a humanized 4P41 VHH monomer sequence, a (GGSG) linker, a trimer-forming leucine zipper motif, Myc-Tag, and NotI site were bound was chemically synthesized.
2. An NdeI-NotI DNA fragment was inserted into the NdeI and NotI sites of pET22b(+) (Novagen) to construct a gene encoding a protein in which cMyc-Tag and His-Tag were added to the C-terminal side of a VHH antibody. An E. coli RosettaGami 2 (DE3) competent cell (Novagen) was transformed with pET22b(+) plasmid DNA into which the VHH antibody DNA fragment had been inserted.
3. The recombinant Escherichia coli was cultured at 37° C. using 200 mL of a 2YT medium containing 100 μg/mL of ampicillin (Sigma-Aldrich), and 0.1 mM IPTG (Takara Bio Inc.) was added thereto at the time when OD600 reached 0.3, followed by culturing at 30° C. for 3 hours. After completion of the culturing, Escherichia coli was 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 Diagnostics)). Bacterial cell disruption used Micro Smash (Tomy Seiko Co., Ltd.). Three gram of 0.1 mm diameter zirconia beads (Tomy Seiko Co., Ltd.) was added to each of 16 5-mL sample tubes (Tomy Seiko Co., Ltd.), 5 mL of a bacterial cell suspension was added to each of the tubes, and disruption at 4° C. at 4 200 rpm for 5 minutes was repeated three times. After the disruption, centrifugation was performed at 20 000×g at 4° C. for 15 minutes, and the supernatant was collected and filtered through a 0.45 μm diameter filter (Merck Millipore).
4. Purification of protein was performed using a His-Trap column (Cytiva). The filtrate of centrifuge supernatant of the bead disruption solution was applied to a His-Trap column, the column was washed with a binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, pH 7.4), and the fusion protein was then eluted with an eluate containing 500 mM imidazole.
5. The purified sample was subjected to 158 acrylamide SDS electrophoresis and then stained with TakaRa CBB Protein Safe Stain (Takara Bio Inc.). In addition, western blotting was performed using anti-His-Tag mouse monoclonal antibody HRP (Thermo Fisher Scientific).
As a result, in the His-Tag column elution sample, a protein that reacts with an anti-His-Tag mouse monoclonal antibody was recognized at the position of a predicted molecular weight (about 21 kDa) of a humanized 4P41 LZ monomer protein.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2021-203699 | Dec 2021 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2022/046069 | 12/14/2022 | WO |
