WO2011017791A1 - ANTICORPS MONOCLONAUX POUR LA PROTÉINE PBP2a ET SÉQUENCES HOMOLOGUES POUR LE TRAITEMENT D'INFECTIONS ET L'IMMUNODIAGNOSTIC DANS DES BACTÉRIES DU PHYLUM FIRMICUTES - Google Patents

ANTICORPS MONOCLONAUX POUR LA PROTÉINE PBP2a ET SÉQUENCES HOMOLOGUES POUR LE TRAITEMENT D'INFECTIONS ET L'IMMUNODIAGNOSTIC DANS DES BACTÉRIES DU PHYLUM FIRMICUTES Download PDF

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WO2011017791A1
WO2011017791A1 PCT/BR2010/000263 BR2010000263W WO2011017791A1 WO 2011017791 A1 WO2011017791 A1 WO 2011017791A1 BR 2010000263 W BR2010000263 W BR 2010000263W WO 2011017791 A1 WO2011017791 A1 WO 2011017791A1
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seq
sequence
pbp2a
mrsa
bacteria
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PCT/BR2010/000263
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Portuguese (pt)
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WO2011017791A9 (fr
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José Procópio Moreno SENNA
João Luiz Sampaio QUEIROZ
Nadia Maria Batoreau
Maria da Glória Martins TEIXEIRA
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Fundação Oswaldo Cruz
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Priority to ES10807822T priority Critical patent/ES2773920T3/es
Priority to RU2012108941/10A priority patent/RU2575070C2/ru
Priority to KR1020127006442A priority patent/KR101868160B1/ko
Priority to SG2012009205A priority patent/SG178346A1/en
Priority to PCT/BR2010/000263 priority patent/WO2011017791A1/fr
Priority to US13/389,860 priority patent/US8940304B2/en
Priority to CU2012000026A priority patent/CU24095B1/es
Priority to CA2770771A priority patent/CA2770771C/fr
Application filed by Fundação Oswaldo Cruz filed Critical Fundação Oswaldo Cruz
Priority to CU20140101A priority patent/CU24180B1/es
Priority to CN201080043628.XA priority patent/CN102741281B/zh
Priority to MX2012001790A priority patent/MX2012001790A/es
Priority to AU2010282162A priority patent/AU2010282162B2/en
Priority to JP2012524064A priority patent/JP5785544B2/ja
Priority to EP10807822.1A priority patent/EP2476702B1/fr
Publication of WO2011017791A1 publication Critical patent/WO2011017791A1/fr
Publication of WO2011017791A9 publication Critical patent/WO2011017791A9/fr
Priority to HK13103458.4A priority patent/HK1176077A1/xx

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1271—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Micrococcaceae (F), e.g. Staphylococcus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]

Definitions

  • the present invention relates to monoclonal antibodies capable of recognizing and binding to PBP2a protein and other proteins having sequences homologous to PBP2a, including methicillin-resistant Staphylococcus aureus pathogens - MRSA, Coagulase negative Staphylococcus sciuri, Enterococcus spp. and any other bacteria that may possess the PBP2a or sequences homologous to this protein.
  • the invention further relates to the use of monoclonal antibodies capable of recognizing and binding to protein PBP2a and other homologous sequence proteins to PBP2a in complementary immunodiagnosis for the detection of beta-lactam resistance.
  • Methicillin-resistant Staphylococcus aureus (MRSA) infections are a major cause of concern for clinicians, with higher mortality and morbidity rates than methicillin-sensitive Staphylococci infections (1).
  • MRSA Methicillin-resistant Staphylococcus aureus
  • these infections lead to longer hospitalization and antimicrobial spending, leading to a higher cost of treating patients infected with this pathogen (1).
  • Vancomycin has been the antimicrobial of choice for the treatment of MRSA infections.
  • MRSA has been the antimicrobial of choice for the treatment of MRSA infections.
  • the increasing isolation of MRSA strains in communities in the United States and Australia (2, 3, 4) allied to the identification of MRSA strains with intermediate vancomycin resistance in Japan, the United States (5) and Brazil (6), make the current situation even more serious.
  • the description in 2004 of fully vancomycin-resistant MRSA strains (7) has caused enormous concern in the medical-scientific community. MRSA today represents a strong candidate for becoming the fearsome "superbug or superbug" - a pathogen resistant to all currently available drugs.
  • MRSA is considered the main pathogen that causes epidemic outbreaks in Brazilian hospitals (14).
  • S. aureus isolated from patients at university hospitals in S ⁇ o Paulo were methicillin resistant, and in 1993, the incidence of RSA at the Paulista Medical School Pediatric Hospital was 79% ( 15).
  • Resende et. al. (16) reported a 71% prevalence of MRSA.
  • MRSA strains have a very low affinity penicillin-binding protein for the beta-lactam antimicrobials, PBP2a (17).
  • this enzyme encoded by the mecA gene, the bacteria can synthesize peptideoglycan, even in the presence of beta-lactams.
  • This enzyme can also be found in coagulase negative Staphylococcus and Staphylococcus sciuri - a bacterium present in normal dog flora.
  • hospital MRSA strains are resistant to most other classes of antimicrobials available, leaving the first choice treatment as glycopeptides (vancomycin and teicoplanin).
  • PBP2a is a class II multimodular enzyme according to Goffin and Ghuysen classification (40). This 76 kilodalton enzyme is composed of a membrane-binding region, a non-transpeptidase domain, and the transpeptidase domain, containing a 4-amino acid active center (STQK), responsible for the bacterial transpeptidation reactions (20 bis Ryfell, 1990). ).
  • STQK 4-amino acid active center
  • a vaccine cannot generate protective antibodies in a timely manner to control a bacterial infection.
  • administration of anti-PBP2a monoclonal antibodies is the most appropriate therapy for the treatment of these infections.
  • the main object of the present invention is to provide monoclonal antibodies capable of recognizing and binding to the PBP2a protein (SEQ ID NO: 1) and other proteins having PBP2a homologous sequences, including those pathogens Methicillin resistant Staphylococcus aureus - MRSA, Coagulase negative Staphylococcus, Staphylococcus sciuri, Enterococcus spp. and any other bacteria that may possess the PBP2a or sequences homologous to this protein.
  • a further object of the invention is the use of monoclonal antibodies capable of recognizing and binding to PBP2a protein and other proteins having PBP2a homologous sequences in complementary immunodiagnosis for the detection of beta-lactam resistance.
  • the monoclonal antibodies of the present invention are represented by the sequences SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17.
  • FIG 1 shows the enzyme immunoassay (ELISA) of sera from immunized animals for the production of anti-PBP2a antibodies.
  • Figure 2 shows the result of a polyacrylamide gel with the crude samples and after purification.
  • Figure 3 shows the Immunoblotting assay of MRSA and MSSA lysates against the supernatant containing anti-PBP2a monoclonal antibodies.
  • Figure 4 is a representation of the flow cytometry results of the MRSA (CEB) and MSSA bacteria incubated with phycoerythrin (PE) labeled monoclonal antibody.
  • Figure 5 shows the in vitro protection test (MIC - minimum inhibitory concentration) conferred by purified anti-PBP2a monoclonal antibody and vancomycin against an inoculum of different MRSA strains.
  • Figure 6A shows the results of renal quantification in animals treated and untreated with the anti-PBP2a monoclonal antibody and subjected to sublethal dose systemic infection of the CEB MRSA strain.
  • Figure 6B shows the results of renal quantification in animals treated and untreated with the anti-PBP2a monoclonal antibody and subjected to sublethal dose systemic infection of the Iberian MRSA (European epidemic clone) strain.
  • Figure 6C shows the results of renal quantification in animals treated and untreated with the anti-PBP2a monoclonal antibody and subjected to sublethal dose CA-MRSA WB79 strain (Brazilian Community strain).
  • Figure 7A shows the survival curve of treated (protected) and untreated (control) animals after lethal dose bacterial infection (CEB MRSA).
  • Figure 7B shows the survival curve of treated (protected) and untreated (control) animals after lethal dose bacterial infection (Iberian MRSA).
  • Figure 7C shows the survival curve of treated (protected) and untreated (control) animals after lethal dose bacterial infection (CA-MRSA WB79).
  • Figure 8A shows bacterial quantification in kidneys of animals treated with anti-PBP2a monoclonal antibody, vancomycin, antibody + vancomycin combination and untreated animals after bacterial infection (CEB-MRSA).
  • Figure 8B shows bacterial quantification in kidneys of animals treated with anti-PBP2a monoclonal antibody.
  • Figures 9 show the interaction between recombinant PBP2a (antigen) with clone 38 ( Figure 9A) and clone 10 ( Figure 9B) monoclonal antibodies.
  • Figure 10 is a graph of the second flow cytometric analysis of MRSA samples in the presence of FITC-labeled anti-PBP2a antibody.
  • Figure 11 shows the result of in vitro protection conferred by the antibody against Enterococcus VRE strain.
  • Figure 12 refers to the determination of DL-50 and lethal dose intra-peritoneal infection.
  • Figure 13 shows the result of the efficacy of in vivo protection of enterococcal anti-PBP2a and PBP5 monoclonal antibody against systemic infection.
  • PBP2a had its structure elucidated in 2002 (29), being deposited in PDB (Protein Data Bank) under the code lwmr.
  • the inventors worked with an internal region of the molecule, 76 amino acids, which flanks the active enzyme center (SxxK) in the transpeptidase domain. Identification of epitopes in the molecule and their presentation to different MHC class II alleles (Tepitope program) were performed, with subsequent verification of the molecule's location (SPDB-Viewer program). This approach allows us to evaluate the accessibility of antibodies to these targets in the native molecule. This computational analysis demonstrated the presence of epitopes near the enzyme active center, with excellent recognition by MHC class II alleles located on the surface of PBP2a (data not shown).
  • MRSA infections show the presence of predominant clonal types, responsible for outbreaks and epidemics in hospitals worldwide. These clones have a higher capacity for colonization and virulence when compared to non-epidemic MRSA strains (30).
  • CEB Ceramic swine fever
  • CA-MRSA WB79 Due to the growth of cases of community infections caused by MRSA, one of these strains (CA-MRSA WB79) isolated in Brazil was included in the present experiments. These strains have different characteristics from those found in hospitals, such as higher virulence (presence of Panton-Valentine leucocidin) and different antimicrobial resistance profile than hospital MRSA strains (36). Recently, an outbreak of CA-MRSA infections has been identified among the population of men who have sex with men in the United States (37). From this date we can say that infections caused by MRSA assume an STD (sexually transmitted disease) character.
  • STD sexually transmitted disease
  • Staphylococcus aureus is known to have protein A on its surface. This molecule has the characteristic of binding to the Fc region of immunoglobulins, preventing the opsonizing activity of the immune system (38). Due to the results obtained in our experiments, we believe that the antibody concentration administered was able to saturate the protein A on the bacterial surface, not preventing the blockage of PBP2a by the administered antibodies.
  • these antibodies may have applicability of PBP2a identification by immunodiagnostic assays.
  • the use of antibody-based latex particle agglutination immunotests, for example, may give a result that predicts resistance to all beta-lactam antibiotics within a few hours. In conventional antimicrobial susceptibility testing - antibiogram - these results are released only after 12 to 24 hours.
  • DNA vaccine animal immunizations with constructs corresponding to the jnecA gene (SEQ ID NO: 2) without membrane attachment region and immunizations with an internal region of the 76 amino acid transpeptidase domain (SED ID NO: 3) comprising the active center of the enzyme.
  • Immunizations were performed under the same conditions as those presented by Ohwada et. al. and Senna et. al., who developed a DNA vaccine against PBP2a using the complete sequence (minus the membrane-binding region) of the mecA gene and an internal fragment of the transpeptidase domain. Immunizations were performed at 4 doses, and the immunized animals and an unimmunized control group were challenged by systemic MRSA infection and determination of the number of bacteria present in the animals' kidneys at two different times. The results showed that immunization with the transpeptidase fragment conferred a greater reduction in the number of bacteria present in the kidney of animals than in those immunized with the jnecA gene.
  • transpeptidase fragment covers the active center STQK enzyme (SEQ ID NO: 4).
  • the inventors have directed the invention to the production of monoclonal antibodies capable of recognizing and binding to the PBP2a protein and other proteins having sequences homologous to the PBP2a protein, and utilizing the center-spanning transpeptidase fragment. enzyme active.
  • methicillin resistant Staphyloccus aureus strains were used: Iberian-MRSA, COL-MRSA (provided by the Unotti des Agents Antimicrobiens - Institut Pasteur, Dr. Patrice Courvalin), CA-MRSA B79 and CEB-MRSA (provided by Dr. Agnes Figueiredo, UFRJ Institute of Microbiology); one strain of vancomycin-resistant Enterococcus faecalis and one of methicillin-sensitive Staphylococcus aureus (MSSA). Escherichia coli BL21 DE3 (Novagen) and TOP10 (Invitrogen) strains were also used as controls.
  • mice Female Balb / C mice, 4 to 8 weeks old, obtained from CECAL-FIOCRUZ and housed in LAEAN-BioManguinhos, were used in immunization assays and in vivo protection assays.
  • mice received an initial 100 microgram dose of plasmid pCI-Neo: MRSA mecA gene fragment (18), followed 14 days after a 10 microgram dose of purified recombinant protein corresponding to an internal region of the MRSA PBP2a (21), emulsified in complete Freund's adjuvant, followed 14 days later by another dose, emulsified in incomplete Freund's adjuvant.
  • the best immune response animal assessed by enzyme immunoassay - ELISA
  • PBS phosphate buffered saline
  • Lymphocytes removed from the spleen were fused with SP2 / 0-Agl4 myeloma cells (ATCC 1581) using polyethylene glycol for fusion and cultured in hypoxanthine-aminopterin-thymidine medium at 37 ° C in a 10% CO atmosphere. 2 , according to protocol for production of monoclonal antibodies in Current Protocols in Immunology (22).
  • the resulting hybridomas were evaluated after 14 days by ELISA using the purified recombinant protein as antigen as described below. The best hybridomas were cloned, with ELISA selection of the best clones, which were stored in liquid nitrogen.
  • the plate was again washed three times with PBS tween 20 (0.05%) and anti-IGs conjugate (anti-mouse HRP IGs SIGMA A 0412) at 1: 5000 dilution was added, followed by incubation at 37 ° C for 90 ° C. minutes After this At this time the plate was washed three times with PBS tween 20 (0.05%), the TB peroxidase color developer (BioRad) was added and light incubated for 15 minutes. The reaction was stopped by the addition of 0.5 N H 2 S0 4 and the reading was taken at 450 nm. A 1: 200 diluted hyperimmune polyclonal serum was used as a positive control.
  • the protocol is similar to that of immunoassay (5), with the following modifications: after incubation of the samples (100 ng clone 10 purified monoclonal antibody and 2.0 nanograms of clone 38 purified monoclonal antibody) for two hours at 37 ° C, they were submitted to three washes with 8M urea in PBS tween 20 (0.05%), followed by four washes in PBS tween 20 (0.05%), having as control the same sample processed normally (without treatment with urea). After reading the optical densities of the samples, the avidity index was calculated by the ratio between reading with urea divided by reading without urea, multiplied by 100 (result in percentage).
  • the protocol is similar to that of immunoassay (5), with the following modifications: after incubation of the samples (100 ng clone 10 purified monoclonal antibody and 2.0 nanograms of clone 38 purified monoclonal antibody) for two hours at 37 ° C, they were treated with ammonium thiocyanate for 30 minutes at 37 ° C in the following concentrations: 3; 1.5M; 1.0M; 0.75M; 0.50M; 0.25M and 0.125M. An untreated ammonium thiocyanate sample was used as a control for each clone.
  • A is the lowest concentration of ammonium thiocyanate giving an absorbance reduction of less than 50% and B is the largest concentration of ammonium thiocyanate giving an absorbance reduction of greater than 50%.
  • a sample of the previously selected clone 10 and clone 38 was grown in serum free medium (GIBCO VP-SFM) added with 1% BSA in 100 mL 10% CO 2 atmosphere oven flasks. The supernatants were centrifuged, followed by filtration through 0.22 micrometer filters and purified by high performance chromatography (HPLC) with a MAB SelectSure (GE) protein A resin. Antibodies were neutralized to pH 7.0 with 1M Tris, pH 10.0, dialyzed against 0.5x PBS in deionized water. The samples were subjected to lyophilization process, resuspended in deionized water, quantified by the Lowry method and evaluated by polyacrylamide gel electrophoresis.
  • MRSA methicillin-sensitive Staphylococcus aureus
  • MSSA methicillin-sensitive Staphylococcus aureus
  • vancomycin-resistant Enterococcus faeci strain and one Escherichia coli BL-21 strain DE3 were grown in exponential phase.
  • One mL of each sample was centrifuged and lysed by shaking on glass beads in a mini-Bead Beater apparatus (Biospect Products) 3 times for 30 seconds.
  • One aliquot of each sample was subjected to 12% denaturing polyacrylamide gel electrophoresis (SDS-PAGE), and the proteins were subsequently transferred to a nylon membrane (Hybond N-BioRad).
  • the membrane was blocked by gentle shaking for two hours in PBS buffer containing 10% skim milk and 1% BSA (bovine serum albumin). The membrane was washed three times in 0.05% PBS tween 20 and three times in PBS. It was then incubated for two hours with the supernatant of the anti-PBP2a monoclonal antibody diluted in PBS 1: 1. After incubation, the membrane was washed as described above, and alkaline phosphatase conjugate (anti murine IgG antibody - Sigma A3688) was added at a ratio of 1: 15000 and incubated for ninety minutes. After this time the PBS was washed again and developed with the Western Blue alkaline phosphatase substrate (Promega).
  • BSA bovine serum albumin
  • the samples were again washed as before and resuspended in 100 microliters of 0.5% BSA PBS and a 1: 1000 dilution of the anti IG mouse PE conjugate (phycoerythrin), and then incubated in the dark for 30 min at 4 ° C. ° C. Then the samples were again washed and fixed 15 minutes at 4 ° C in PBS containing 2% paraformaldehyde. After this preparation the samples were analyzed using a flow cytometer (Becton and Dickinson - FACScalibur).
  • MRSA strains (CEB, Iberian, COL and CA) were grown exponentially to an optical density of 600 nm equivalent to 0.5. The applied inoculum was adjusted to contain approximately 100,000 bacteria. In test tubes or 24-well plates, Muller Hinton broth, bacterial inoculum and increasing concentrations of purified anti-PBP2a monoclonal antibody were added. The plates or tubes were incubated at 37 ° C for 12 hours. After this period, the presence of turbidity or not of the samples was observed. The minimum inhibitory concentration was considered the smallest amount of antibody capable of inhibiting bacterial inoculum growth (100,000 bacteria).
  • Lethal and DL-50 doses were determined according to the Reeduench method (23) for MRSA strains (CEB, Iberian, CA WB79 and COL).
  • Groups of 8-week-old female Balb / C mice were inoculated intra-peritoneally with increasing doses of bacteria and observed for 7 days. Surviving animals after this period were euthanized according to established animal welfare standards.
  • MRSA strains (CEB, - Iberian and CA WB79) were grown exponentially (DC> 60 ⁇ 0.6), washed and resuspended in sterile 1x PBS at a DC> 60 ⁇ 0.5, corresponding to approximately 2x10 8. bacteria. This concentration was calculated by dilution and seeding in BHI Agar plates containing 10 micrograms oxacillin / mL. 8 week old female Balb / C mice received an intraperitoneal dose of 400 micrograms purified anti-PBP2a monoclonal antibody on the first day. On the sixth day, the animals were euthanized, the kidneys were aseptically removed.
  • kidneys were then homogenized in 1mL sterile Luria broth and successively diluted in 10 ⁇ L series. Each microliter of each dilution was seeded on BHI Agar plates containing 10 micrograms / ml oxacillin and incubated 24 hours at 37 ° C. The resulting colonies were counted and the total dilutions calculated.
  • mice Four groups of 8 week old female Balb / C mice (4 animals per group) received an infective dose of 6.0x10 7 bacteria (CEB-MRSA) intraperitoneally. The animals received doses of purified monoclonal antibody (MAB), vancomycin, MAB + vancomycin and negative control, according to the groups below:
  • group 1 MAB 400 micrograms (first day)
  • group 3 vancomycin + MAB (350 micrograms) (1 day after infection)
  • the first doses of antibody and vancomycin were administered 4 hours after administration of the infective dose.
  • the animals were euthanized in the On the fourth day, the kidneys were aseptically removed and subjected to renal quantification as described above.
  • This assay was performed in the same manner as the previous one, but with a lower infecting dose (7.0x10 6 bacteria), with group 1 treated with 500 micrograms of purified monoclonal antibody; group 2 with vancomycin (150 micrograms 12-12 hour intramuscular route; 5 doses); group 3 with vancomycin + 500 micrograms of monoclonal antibody and group 4 control (untreated animals).
  • a 10 ml pellet of a monoclonal antibody producing hybridoma cell culture was processed for mRNA extraction using the RNeasy Minikit kit (Qiagen).
  • M-MLV reverse transcriptase Reaction The M-MLV reverse transcriptase kit (Invitrogen) was used to obtain complementary DNA following the manufacturer's instructions.
  • the ABI Prism 3100 Genetic Analyzer (Hitachi) was used.
  • DNA sequences obtained were analyzed with the help of the DNA Star program, and were translated into amino acid sequences (Site ExPASy - Translate program) for further analysis by Kabat (24) and Chotia (25) algorithms for the identification of the CDRs of the DNA. light and heavy chains.
  • HBS-EP binding reagents and buffers (10mM hepes, 150mM NaCl, 3mM EDTA, 0.005% P20 (Tween 0, pH 7.4) were purchased from GE Healthcare.
  • 96-well supernatant (hybridomas) was analyzed by ELISA. From this total, the top five were selected, the cells were expanded (cloning). And again, the resulting supernatants analyzed by ELISA. Positive samples were validated by immunoblotting against purified recombinant protein (PBP2a) for validation of ELISA results. The final result is shown in Table I.
  • Figure 1 shows the result of the enzyme immunoassay (ELISA) of sera from animals immunized for the production of anti-PBP2a antibodies.
  • Each stroke corresponds to the 1: 100 diluted serum of the immunized animals.
  • First bar of each stroke preimmune serum; second bar: serum after fourth immunization and third bar: serum after fifth immunization.
  • clone 77-38 was subjected to the recloning process to verify the stability of cells secreting monoclonal antibodies. Of the 50 wells analyzed, all were positive by ELISA. These clones have been expanded and are stored in liquid nitrogen at the LATAM (Monoclonal Antibodies Technology Laboratory) facility.
  • the process was standardized as previously described.
  • the yield obtained is approximately 4 milligrams of monoclonal antibody per 100 ml of supernatant subjected to the purification process. The results obtained can be seen below.
  • FIG. 2 we can see a polyacrylamide gel with the crude samples and after purification.
  • This Figure 2 shows non-denaturing polyacrylamide gel with supernatant samples before (column 1) and after purification (2) on HPLC column with MAb SelectSure resin. Columns 3 to 9 correspond to fractions obtained from the purified sample. The arrow indicates the approximate size of 150 kDa.
  • Figure 3 shows the result of the MRSA and MSSA lysate immunoblotting assay against the supernatant containing anti-PBP2a monoclonal antibodies.
  • the purpose of the flow cytometry test is to validate the target recognition ability of the bacterium in its native form by the monoclonal antibody.
  • target recognition in proteins undergoing a denaturation process, which occurs during protein separation by denaturing polyacrylamide gel electrophoresis.
  • MSSA negative control strain
  • CEB MRSA strain
  • FIG 4 shows the flow cytometric results of the MRSA (CEB) and MSSA bacteria incubated with phycoerythrin (PE) labeled anti-PBP2a monoclonal antibody.
  • MSSA bacteria MSSA bacteria
  • PE phycoerythrin
  • MSSA populations show a right shift, corresponding to an increase in cells marked by the fluorescent conjugate.
  • Figure 5 shows the in vitro protection test (MIC - minimum inhibitory concentration) conferred by purified anti-PBP2a and vancomycin monoclonal antibody against an inoculum of 10 ° cells of different MRSA strains. The absence of turbidity indicates that there was no bacterial growth under the conditions analyzed.
  • IA MRSA CEB (Brazilian epidemic clone) + 250 ⁇ g of antibody;
  • a and 6A negative controls of the MRSA CEB strain.
  • Iberian MRSA European Epidemic Clone - EEC + 250 ⁇ g of antibody
  • 4C and 6C negative controls of the MRSA CEE strain.
  • 1D MRSA CEB + 150 ⁇ g vancomycin,
  • the MRSA COL strain the first MRSA clone to have its genome sequenced, is used as a reference strain for studies of this pathogen. However, it was not very virulent, requiring high infective doses compared to the other MRSA clones to cause infection in animals. Because of this it was not used in the protection tests. 4.2.2. Renal protection trials following sublethal dose systemic infection
  • Figure 6A shows the results of renal quantification in animals treated and untreated with the anti-PBP2a monoclonal antibody and subjected to sublethal dose systemic infection of the CEB MRSA strain.
  • Horizontal striped log of the concentration of bacteria isolated from the kidneys of each untreated animal.
  • Checkered Log the amount of bacteria isolated from the kidneys of each antibody-treated animal.
  • Bacterial Quantification Controls: Cl: 2000 Bacteria; C2: 29,000 bacteria; C3: 220,000 bacteria; C4: 52,000 bacteria (average 75,750 bacteria).
  • Figure 6B shows the results of renal quantification in animals treated and untreated with the anti-PBP2a monoclonal antibody and subjected to sublethal dose systemic infection of the Iberian RSA (European epidemic clone) strain.
  • Horizontal striped log of the concentration of bacteria isolated from the kidneys of each untreated animal.
  • Large grid Log the amount of bacteria isolated from the kidneys of each antibody treated animal. The small checkered bars indicate the respective averages obtained.
  • Bacterial Quantification Controls: Cl: 210,000 Bacteria; C2: 44,000 bacteria; C3: 300,000 bacteria; C: 290,000 bacteria (average 211,000 bacteria).
  • Figure 6C shows the results of renal quantification in animals treated and untreated with the anti-PBP2a monoclonal antibody and subjected to systemic infection with CA-MRSA B79 sublethal dose (Brazilian community strain).
  • the first five bars (in xx, horizontal dashed and large grid): log of the concentration of bacteria isolated from the kidneys of each untreated animal.
  • the first bar (in xx): estimate for a dead animal before euthanasia.
  • Bars 6, 7, 8 and 9 in horizontal and checkered dashes: log the amount of bacteria isolated from the kidneys of animals treated with 250 ⁇ g anti-PBP2a monoclonal antibody.
  • Bars 10, 11, 12 and 13 log of the amount of bacteria isolated kidneys of each animal treated with 500 ⁇ g of antibody.
  • the checkered bars (5a, and 9 to 13 bars) indicate the respective averages.
  • Bacterial Quantification Controls: Cl: 650,000 Bacteria; C2: 26,000 bacteria; C3: 17,000 bacteria; C4: 500,000 bacteria (dead animal estimate) (average 231,000 bacteria). Animals treated with 250 g of antibody: PI: zero; P2: 5,400 bacteria, P3: 830 bacteria and P4: 10 bacteria (average 1,560 bacteria).
  • Figure 7A shows the survival curve of treated (protected) and untreated (control) animals following infection by dose of 2.3x10 8 bacteria (CEB MRSA) administered intraperitoneally (DL-50).
  • Figure 7B shows the survival curve of treated (protected) and untreated (control) animals following dose infection of 4.2x10 8 bacteria (Iberian MRSA) administered intraperitoneally ( ⁇ DL-50).
  • Figure 7C shows the survival curve of treated (protected) and untreated (control) animals following infection by dose, ⁇ 9 bacteria (CA-MRSA WB79) administered intraperitoneally ( ⁇ DL-50).
  • vancomycin is the antimicrobial of choice for the treatment of severe MRSA infections.
  • a comparative protection study was performed on a different model from the previous ones. In this study, the animals were infected and only after four hours of infection did the antimicrobial or monoclonal antibodies be administered. The study was conducted with three distinct groups, one treated with vancomycin, another with monoclonal antibodies and a third with simultaneous administration of antimicrobial + antibodies. Vancomycin doses were adjusted and administered similarly to human infections (500 mg every 12 hours). The results indicated that there was a reduction of about 15 times in the amount of bacteria present in the kidneys of animals treated with antimicrobial or antibodies three days after infection. However, in the group receiving antimicrobial and antibody treatment, a 4,617-fold reduction was observed.
  • Figure 8A shows the bacterial count in kidneys of animals treated with monoclonal antibody anti-PBP2a, vancomycin, vancomycin + antibody and association untreated animals after infection with bacteria 6,0xl0 7 (CEB MRSA). Initiation of treatment occurred 4 hours after infection. Vancomycin administered 12/12 hours (5 doses). Bars 1, 2, 3, 4 and 5 (dashed): log of concentration of bacteria recovered from untreated animals (controls). Cl: 7,000,000; C2: 295,000; C3: 380,000; C4: 3.2,000 (average: 2,718,750 bacteria). Bars 6, 7, 8, 9 and 10 (checkered): log concentration of bacteria recovered from animals treated with 400 ⁇ g anti-PBP2a monoclonal antibody.
  • PI 4,200; P2: 310,000; P3: 330,000; P4: 90,000 (average 183,550 bacteria). Bars 11, 12, 13, 14 and 15 (balls): vancomycin treated animals. PI: 110,000, P2: 58,000, P3: 500,000, P4: 21,000 (average 172,250 bacteria). Bars 16, 17, 18, 19 and 20 (triangle): log of concentration of bacteria recovered from antibody treated animals (300 ⁇ g) + vancomycin. PI: 1.100, P2: 700, P3: 450, P: 90 (average 585 bacteria).
  • Figure 8B shows bacterial quantification in kidneys of animals treated with anti-PBP2a monoclonal antibody (bars 7 to 12), vancomycin (bars 13 to 18), antibody + vancomycin combination (19 to 24)) and untreated animals (bars 1 to 6) after infection with 7.0x10 6 bacteria (CEB-RSA). Start of treatment 4 hours after infection. Vancomycin administered 12/12 hours (5 doses). Bars 1 through 6: log of concentration of bacteria recovered from untreated animals (controls). Cl: 6,000; C2: 1,000; C3: 500; C4: 118,000 and C5: 1,000 (average: 25,220 bacteria). Bars 7 to 12: log concentration of bacteria recovered from animals treated with 500 ⁇ g of anti-PBP2a monoclonal antibody.
  • MB1 450; MB2: 200; MB3: 100; MB4: 20; MB5: zero (average 284 bacteria). Bars 13 through 18: Treated animals with vancomycin.
  • CV1 100, CV2: 700, CV3: zero, CV4: zero;
  • CV5 2800 (average 720 bacteria). Bars 19 to 24: log concentration of bacteria recovered from antibody-treated animals (500 ⁇ g) + vancomycin. MBV1: 130, MBV2: 20, MBV3: 10, MBV4: 80; MBV5: 20 (average 56 bacteria).
  • Figure 9 shows the interaction between recombinant PBP2a (antigen) with clone 38 ( Figure 9A) and clone 10 (Figure 9B) monoclonal antibodies.
  • Smoky dashed curves represent SPR data at concentrations according to the legend on the right. All samples were analyzed in duplicate and the Langmuir 1: 1 theoretical model for each curve is shown in black under each curve.
  • the vertical axis shows the response units and the horizontal line the time in seconds. At lines closest to the horizontal axis represent the baseline of each sample (negative control).
  • cDNA was obtained and PCR material was performed for the different light and heavy chain alleles.
  • the obtained materials were subjected to sequencing employing the same primer sequences (defined by SEQ ID NO: 18 through SEQ ID NO: 39) used in PCR reactions. They were identified in three distinct sequences, the light chain 391 and the heavy chain 310. Applying the Kabat and Chotia algorithms, we came to identify the light and heavy chain CDRs, which are the subject of the appended claims.
  • RSSQSIGHSNGNTYLE SEQ ID NO: 7 - CDR 2 light chain amino acids.
  • PQGSYVPLT SEQ ID NO: 9 - CDR 1 light chain DNA.
  • Example 2 A second study was carried out using the CEB MRSA strain, following the same protocol described in Example 1, item 7.2, with the addition of two vortexing pulses of 15 seconds each between each wash; aiming to disaggregate the Staphylococcus aureus clusters and increase the amount of PBP2a exposed to antibodies.
  • FITC fluorescein isothiocyanate
  • PE phytoerythrin
  • Figure 10 is a graph of flow cytometric analysis of MRSA samples in the presence of FITC-labeled anti-PBP2a antibody.
  • the curve (x) corresponding to the unlabeled sample and the curve (y) corresponding to the labeled sample.
  • the inventors further investigated the protection conferred by the methicillin resistant Staphylococcus aureus monoclonal anti-PBP2a antibody against Enterococci.
  • the antibody recognizes proteins present in Enterococcus sp. probably PBP5 - a low-affinity beta-lactam transpeptidase, present in all enterococcal strains and with a molecular weight of approximately 76 kDa (237 amino acids).
  • This enzyme shows homology with the MRSA PBP2a, as aligned (ClustalW), listed below:
  • DSFVPITVASEPVTELPTG AATKDTESRYYPLGEACAINR-VYGTITAEDIEKN — PE 292 DYFVPLKIIDGATPELPAG — ATIQEVDGRYYPLGEAAAQLIGYVGDI AEDIDK — PE 286
  • PBP5Eas EILLADTGYGQGQLLISPIQQATMYSVFQNNGTLVYPKLVLDKETKK-KDNVISANAANT 589
  • PBP2a EILLADSGYGQGEILINPVQILSIYSALEN GNINAPHLLKDTKNKVWKKNIISKENINL 570
  • the labeled sequences (bolded PBP5, underlined PBP2a) correspond to the region employed of PBP2a to generate monoclonal antibodies. In italics, the amino acids corresponding to the active center of the enzymes are marked.
  • the aim of this assay was to evaluate the in vitro protection conferred by the antibody against Enterococcus VRE strain.
  • Sample purified from supernatant by HPLC, lyophilized, dialyzed, SelecSure MAB resin.
  • Pre-inoculum 1 VRE colony in 20 mL Lb vanco (10 mg / mL), ON 37 " C 160 RPM Inoculum: 400 mL pre-inoculum in 20 mL Lb, erlen 200 mL, 37 ° C 160 RPM
  • Antibody concentrations 300, 400, 500, 600 and 700 mg of antibody
  • Figure 11 shows the result of the antibody protection assessment.
  • Figure 11 we have:
  • VRE Enterococcus faecium
  • Antibody purified from serum cell culture supernatant
  • Murine model animals balb / C, 8 weeks, female, weight 23 to 25 grams
  • group A (6 animals): 650 mg of antibody, (350 mg + 300 mg)
  • group B (6 animals): control (adm saline)
  • IP antibody inoculation 300 mg
  • IP afternoon systemic infection
  • IP 250 ml sol bacteria - 2.2x109 bacteria
  • VRE E. faecium strain
  • Antibody purified from serum cell culture supernatant
  • Murine model Balb / C animals, 8 weeks, female, weight 19 to 23 grams
  • group A (4 animals): 500 micrograms antibody (in 2 doses, dOl, d02)
  • Day 01 Inoculation 250 micrograms antibody intraperitoneally
  • Day 02 250 micrograms intraperitoneal antibody inoculation and systemic infection (intraperitoneal, 500 microliters sol bacter.)
  • 700 micrograms of the antibody is able to block the growth of 550,000 bacteria. These values are higher than the MIC obtained for the MRSA strains, which was approximately 500 micrograms.
  • PI intraperitoneal
  • mice received 500 micrograms of intraperitoneal (PI) monoclonal antibody and underwent systemic IP infection with 2.4x10 8 bacteria. Four days later they were euthanized and excised the kidneys for bacterial quantification. Treated animals had an average of 87.5 bacteria / animal, while controls (infected, untreated animals) had an average of 211,000 bacteria / animal.
  • the present invention described herein - anti-PBP2a monoclonal antibodies capable of specifically binding to PBP2a and homologous sequences has applicability in infections caused by bacteria bearing this protein or the like (MRSA, MRSE and Enterococcus spp. And any other pathogen). which has a protein homologous to PBP2a).
  • FIGUEIREDO AM BY LENCASTRE H, TOMASZ A. Geographic spread of epidemic multiresistant Staphylococcus aureus clone in Brazil. J Clin Microbiol. 33 (9): 2400-4.1995.
  • Gly Pro lie Asn Be Glu Glu Read Lys Gln Lys Glu Tyr Lys Gly Tyr
  • Lys Asp Asp Wing Val lie Gly Lys Lys Gly Leu Glu Lys Leu Tyr Asp
  • Lys Lys Read Gln His Glu Asp Gly Tyr Arg Go Thr Lie Go Asp Asp
  • Gly Lys Asp lie Gln Leu Thr lie Asp Ala Lys Val Gln Lys Ser lie

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Abstract

La présente invention concerne des anticorps monoclonaux capables de reconnaître la protéine PBP2a et d'autres protéines présentant des séquences homologues à PBP2a, et de se lier à celles-ci, y compris les pathogènes Staphylococcus aureus résistants à la méticilline - MRSA, Staphylococcus à coagulase négative, Staphylococcus sciuri, Enterococcus spp. et toute autre bactérie contenant la PBP2a ou des séquences homologues à cette protéine. L'invention concerne également l'utilisation de ces anticorps monoclonaux capables de reconnaître la protéine PBP2a et d'autres protéines présentant des séquences homologues à PBP2a, et de se lier à celles-ci, dans un immunodiagnostic complémentaire pour la détection de la résistance aux bêta-lactamines.
PCT/BR2010/000263 2009-08-10 2010-08-10 ANTICORPS MONOCLONAUX POUR LA PROTÉINE PBP2a ET SÉQUENCES HOMOLOGUES POUR LE TRAITEMENT D'INFECTIONS ET L'IMMUNODIAGNOSTIC DANS DES BACTÉRIES DU PHYLUM FIRMICUTES WO2011017791A1 (fr)

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CU20140101A CU24180B1 (es) 2009-08-10 2010-08-10 Anticuerpo monoclonal aislado para proteina pbp5
RU2012108941/10A RU2575070C2 (ru) 2009-08-10 2010-08-10 МОНОКЛОНАЛЬНЫЕ АНТИТЕЛА ПРОТИВ БЕЛКА РВР2-А И ГОМОЛОГИЧНЫХ ПОСЛЕДОВАТЕЛЬНОСТЕЙ ДЛЯ ЛЕЧЕНИЯ ЗАРАЖЕНИЯ БАКТЕРИЯМИ И ИММУНОДИАГНОСТИКИ БАКТЕРИЙ ТИПА Firmicutes
CN201080043628.XA CN102741281B (zh) 2009-08-10 2010-08-10 PBP2-a蛋白的单克隆抗体和同源序列用于由厚壁菌门的细菌引起的免疫诊断和感染的处理
PCT/BR2010/000263 WO2011017791A1 (fr) 2009-08-10 2010-08-10 ANTICORPS MONOCLONAUX POUR LA PROTÉINE PBP2a ET SÉQUENCES HOMOLOGUES POUR LE TRAITEMENT D'INFECTIONS ET L'IMMUNODIAGNOSTIC DANS DES BACTÉRIES DU PHYLUM FIRMICUTES
US13/389,860 US8940304B2 (en) 2009-08-10 2010-08-10 Monoclonal antibodies against the PBP2-A protein and homologous sequences for the treatment of infections by and immunodiagnostics of bacteria of the firmicutes phylum
CU2012000026A CU24095B1 (es) 2009-08-10 2010-08-10 ANTICUERPO MONOCLONAL AISLADO PARA PROTEÍNAS PBP2a
CA2770771A CA2770771C (fr) 2009-08-10 2010-08-10 Anticorps monoclonaux pour la proteine pbp2a et sequences homologues pour le traitement d'infections et l'immunodiagnostic dans des bacteries du phylum firmicutes
ES10807822T ES2773920T3 (es) 2009-08-10 2010-08-10 Anticuerpos monoclonales contra la proteína PBP2a y secuencias homólogas para el tratamiento de infecciones e inmunodiagnóstico de bacterias del filo Firmicutes
KR1020127006442A KR101868160B1 (ko) 2009-08-10 2010-08-10 Ｐｂｐ２－ａ 단백질용 단클론 항체 및 문 퍼미큐트 세균에 대한 면역진단 및 감염 치료를 위한 상동 서열
SG2012009205A SG178346A1 (en) 2009-08-10 2010-08-10 Monoclonal antibodies against the pbp2-a protein and homologous sequences for the treatment of infections by and immunodiagnostics of bacteria of the firmicutes phylum
MX2012001790A MX2012001790A (es) 2009-08-10 2010-08-10 Anticuerpos monoclonales para proteina pbp2-a y secuencias homologas para el tratamiento de infecciones e inmunodiagnostico en bacterias del filum firmicutes.
AU2010282162A AU2010282162B2 (en) 2009-08-10 2010-08-10 Monoclonal antibodies against the PBP2-a protein and homologous sequences for the treatment of infections by and immunodiagnostics of bacteria of the firmicutes phylum
JP2012524064A JP5785544B2 (ja) 2009-08-10 2010-08-10 ファーミキューテス門に属する細菌による感染症の治療及び免疫学的診断のための、ＰＢＰ２ａまたはそれに相同な配列を持つタンパク質に対するモノクローナル抗体
EP10807822.1A EP2476702B1 (fr) 2009-08-10 2010-08-10 Anticorps monoclonaux pour la protéine pbp2a et séquences homologues pour le traitement d'infections et l'immunodiagnostic dans des bactéries du phylum firmicutes
HK13103458.4A HK1176077A1 (en) 2009-08-10 2013-03-20 Monoclonal antibodies against the pbp2-a protein and homologous sequences for the treatment of infections by and immunodiagnostics of bacteria of the firmicutes phylum pbp2-a

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