Classifications

• • 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
  • C07K16/1282—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Clostridium (G)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
  • A61K2039/507—Comprising a combination of two or more separate antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

• the present invention relates to neutralizing tetanus human monoclonal antibodies for C. tetani infection, method of obtaining said monoclonal antibodies and their use in immunotherapy for accidents susceptible to tetanus bacilli infection.
• Tetanus is a neurological disease caused by the bacterial toxin Clostridium tetani, a sporulated gram-positive bacillus present in large amounts in soil, rusty instruments and dust (Collingridge, 1982).
• the active tetanus toxin (TxT) is a protein composed of a light (about 50 kDa) and a heavy (100 kDa) chain linked by disulfide bridges. Its synthesis in the bacterial cytoplasm occurs as a single polypeptide chain. When transported abroad, chain breakage occurs by enzymes present in the C. tetani cell wall generating the heavy and light chains mentioned above (Bizzini et al., 1970; Matsuda et al., 1974).
• TxT The major action of TxT involves blocking the release of inhibitory neurotransmitters (glycine and GABA-Y-aminobutyric acid) in spinal cord neurons (Humeau et al., 2000). As a result, it causes sustained muscle hypertonia, hyperreflexia and spasms.
• the light chain (domain A) contains zinc-binding motif that is responsible for the proteolytic action on a complex present in the vesicles. synaptic, called VAMP / synaptobrevine.
• VAMP when intact, has the function of favoring the approach and fusion of the vesicle to the presynaptic membrane, ensuring the release of inhibitory neurotransmitters in the synapse (Schiavo et al., 1992a; Schiavo et al., 1992b).
• the toxin heavy chain plays a role prior to this event.
• the domain present in the C-terminal portion of the heavy chain (known as the C fragment) is responsible for binding to the neuron through interaction with membrane gangliosides.
• Gangliosides are glycosphingolipids complexed with sialic acid (Chen et al., 2009).
• domain B Another domain (domain B) of the heavy chain plays a role in translocation, which helps the toxin cross the neuronal membrane.
• TxT is retrograde transported to spinal cord neurons, where it causes its toxic effects.
• the toxic lethal dose (LD) of the toxin in mice is between 0.1 and 1 ng / kg body weight.
• the sensitivity in humans to this toxin is similar to that of mice. It is one of the most toxic substances known (Schiavo et al., 2000). Protection against toxin is ensured by vaccination with tetanus toxoid, which corresponds to tetanus anatoxin (AnT) adsorbed by aluminum hydroxide (Brasil, 2010).
• the toxin is obtained from bacterial cultivation by tangential filtration and ammonium sulfate precipitation, then detoxified by treatment with formaldehyde and glycine for 30 days, generating AnT (Sonobe et al., 2007).
• AnT Nonobe et al., 2007
• a study in S ⁇ o Paulo showed that 3% of the population has no protection against tetanus (antibody level ⁇ 0.01 IU / mL) and at 18%, the amount of circulating antibodies in the blood is between 0.01 and 0.1 U / mL, which represents a baseline level of protection (Divono-Goes et al., 2007).
• Tetanus lethality reaches 30% and mainly affects under-fives and the elderly (Ministry of Health, 2005). In older individuals, a greater number of people without vaccine protection are observed.
• a study published in 2006 evaluated the immunity of 98 elderly people from an waiver in S ⁇ o Paulo, SP, with an average age of 84 years through the tetanus antibody (anti-TT) dosage. The result showed that 94% of subjects tested were susceptible to tetanus, and after a booster dose of tetanus vaccine, that number decreased to 79%, still considered worrying (Weckx et al., 2006).
• Table 1 shows data from confirmed cases of tetanus by region in Brazil.
• Table 2 shows a comparison between neonatal tetanus and total tetanus during the years 2010 to 2014.
• SAT equine tetanus serum
• This serum is obtained from the inoculation of tetanus toxoid in horses.
• a volume of plasma is collected by apheresis, from which the serum containing the anti-TT antibodies is processed.
• Immunoglobulins produced by the horse are administered to patients who have suffered a puncture accident and are at risk of contamination with C. tetani f and have the function of binding to and neutralizing the toxin.
• Immunity against toxin is provided by vaccination and the disease does not confer immunity to the patient as TxT is a weak immunogen. What guarantees the immunogenicity of the toxoid in the vaccine is the presence of the adjuvant, which may be aluminum hydroxide gel, as in the case of the Butantan Institute vaccine (Lindley-Jones et al., 2004). In cases where SAT is administered, protection lasts one week on average. In addition to serum there is tetanus hyperimmune human immunoglobulin (IGHAT). It is made from the plasma of newly immunized donors with high antibody titers.
• IGHAT tetanus hyperimmune human immunoglobulin
• IGHAT is indicated in cases of hypersensitivity to heterologous serum or past history of allergy or hypersensitivity reaction to the use of other heterologous sera (Funasa, 2001).
• human immunoglobulins although properly tested for contaminating viruses, is not completely without risk due to the potential to contain emerging or undetectable viruses.
• Mortality-causing infections by microorganisms are generally treated by passive immunotherapy by injecting neutralizing antibodies.
• serotherapy with hyperimmune serum produced in horses is used or, in cases of hypersensitivity, hyperimmune human immunoglobulin is used.
• Serotherapy has a crucial therapeutic role in combating accidents that are likely to be infected with Clostridium tetani, which causes tetanus.
• the Butantan Institute produces tetanus equine polyclonal serum that is effective but of heterologous origin.
• Tetanus heterologous serum was proposed over a hundred years ago. Today there are technologies that allow to obtain more defined products, with production consistency and less immunogenicity.
• Immunotherapy is proven effective when the antigen is known and antibodies are available that recognize it and perform some function, cytotoxic, blocking signaling pathways, adjuvant or neutralizing. Due to the proven therapeutic potential of antibodies, there is a continuing investment to make them more effective and safer. One challenge is the production of well-tolerated therapeutic molecules by patients. There is a great development in the area of monoclonal antibodies, to make them more human (humanized) when obtained from animals. There are companies that have genetically engineered mice, replacing their genes for immunoglobulin synthesis with human immunoglobulin genes, by the need to obtain antibodies with characteristics closer to humans.
• Antibodies are molecules secreted by B lymphocytes whose antigen recognition region is equal to that of the rearranged receptor present on the membrane. They are formed by two chains: heavy and light, each being encoded on different chromosomes.
• the heavy chain (50 kDa) is formed by four regions, CHI, C3 which correspond to the constant region and V which corresponds to the variable region.
• the constant region determines the class and subclass to which the antibody belongs.
• the classes known as isotypes, can be IgM (constant chain ⁇ ), IgG ( ⁇ ), IgA (a), IgE ( ⁇ ) and IgD (d), with each chain conferring different functions.
• the light chain (25 kDa) is composed of a constant region, CL, and a variable, VLH.
• CL constant region
• VLH variable
• kappa ⁇ -gene located on chromosome 2
• lambda ⁇ - gene located on chromosome 22
• AcMos Monoclonal antibodies
• AcMos Monoclonal antibodies
• Antibody studies gained encouragement in the 1970s, when it was developed the technique of producing hybridomas (Kohler; Milstein, 1976). Through this technique it was possible to immortalize an antibody-producing cell of interest by fusion with a murine myeloma cell.
• the hybridoma product can be cultured in vitro and yields the selected MAbs.
• Hybridoma technology has brought important advances in research related to the diagnosis of various pathologies.
• the diagnostic area has a dividing line before and after the introduction of AcMos, either alone or in kits.
• the target-oriented therapeutic approach provided by the AcMos required another wave of research innovation, creating chimeric or humanized antibodies.
• Peripheral blood B lymphocyte capture technique from individuals infected with HIV (human immunodeficiency virus) was used to obtain monoclonal antibody gene sequences for passive immunotherapy of AIDS patients (acquired immunodeficiency syndrome). that monoclonal antibodies are in experimental clinical use (Tiller et al, 2008; Scheid et al, 2009a, b; Mouquet et al, 2010, 2011; Wardermann et al, 2013; Barbian et al, 2015; Zhou et Al, 2015).
• the amino acid sequences of a heavy chain variable region and a light chain variable region of the monoclonal antibody are defined in SEQ ID NO: 3 and SEQ ID No. 4, respectively.
• the heavy chain and light chain amino acid sequences of the monoclonal antibody are defined in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
• Said Chinese patent application CN 105153305 further describes a method of preparing and applying the fully human monoclonal antibody against tetanus toxin and its derivative.
• the fully human monoclonal antibody against tetanus toxin provided by said document can eliminate the biological risks of anaphylactic reaction and viral contamination by having a sufficiently long half-life and high titer and in vivo activity that can be applied to large scale industrial production.
• a complementarity determining region (CDR) of a monoclonal antibody heavy chain variable region comprises CDR1 shown as SEQ ID NO: 6, CDR2 shown as SEQ ID NO: 8 and / or CDR3 shown as SEQ ID NO: 10; and a light chain variable region CDR of the monoclonal antibody comprises CDR1 shown as SEQ ID NO: 12, CDR2 shown as SEQ ID NO: 14 and CDR3 shown as SEQ ID NO: 16.
• the invention described in said patent application Chinese CN 102453091 also provides a deoxyribonucleic acid (DNA) molecule to encode the sequence, an expression vector and a host cell.
• DNA deoxyribonucleic acid
• Japanese Patent JPH0257195 published February 26, 1990, in the name of MORINAGA & CO LTD, entitled: "HUMAN TYPE MONOCLONAL ANTIBODY OF ANTI-TETANUS TOXIN” refers to a type of human monoclonal antibody. which has high neutralizing capacity of tetanus toxin.
• the subject matter described in said document is used for the prevention and treatment of tetanus and being produced stably in a large amount, wherein in the first preparation lymphocytes having high tetanus toxin neutralizing antibody value and forming an IgG-like antibody is collected from human immunized against tetanus toxin (TT).
• the human lymphocyte derivative is then challenged with an antigen and fused to a parent cell.
• a cell strain forming the above antibody is selected from the prepared hybridoma and the cell strain is produced.
• the monoclonal antibodies of said Japanese patent JPH0257195 are obtained from the hybridoma technique. Said document does not describe any sequence (SEQ ID NO) in the definition of said monoclonal antibodies. Accordingly, Japanese patent JPH0257195 and the present invention have totally different monoclonal antibodies.
• Japanese patent JPH1014570 published January 20, 1998, in the name of MATSUDA MORIHIRO and MORINAGA & CO LTD, entitled: "ANTIBODY DNA” refers to obtaining a cDNA containing a specific and useful nucleotide sequence. for antitoxic therapy and prevention of infectious diseases by Clostridium tetani and mass production of a human monoclonal antibody on an industrial scale by a genetic engineering technique.
• This cDNA which encodes a variable region of an antibody heavy chain, in particular, encodes a human monoclonal antibody that contains a nucleotide sequence of the formula, is obtained by cloning the cDNA produced from mRNA. taken from a hybridoma [e.g. TTG6 hybridoma (FERM P-15719)]
• the monoclonal antibodies of said Japanese patent JPH1014570 are obtained from the hybridoma technique. Said document does not describe any specific sequence (SEQ ID NO) in the definition of said monoclonal antibodies. Accordingly, Japanese patent JPH1014570 and the present invention have totally different monoclonal antibodies.
• European Patent EP 0562132 published September 29, 1993, in the name of MATSUDA MORIHIRO and MORINAGA & CO LTD, entitled: "MONOCLONAL ANTI-TETANUS TOXIN ANTIBODIES AND PHARMACEUTICAL CONTAINING THEM” refers to monoclonal antibodies against tetanus toxin that bind to the tetanus toxin light chain and neutralize the biological activity of tetanus toxin.
• binding antitetanus monoclonal antibodies are further disclosed for the tetanus toxin heavy chain fragment C.
• pharmaceutical compositions are described containing said monoclonal antibodies. In a preferred embodiment, these pharmaceutical compositions are for passive tetanus immunization.
• European patent EP 0562132 does not disclose a sequence listing relating to said antibody. Therefore, one of ordinary skill in the art based on the teachings disclosed in European patent EP 0562132 could not be motivated to achieve the defined monoclonal antibody through the sequence listing of the present invention.
• Obtaining human monoclonal tetanus antibodies represents a current trend of supplying biologics with consistency of production lots and zero or low immunogenicity.
• the present invention represents an innovation in the use of infectious disease immunotherapy, which began more than a century ago with the use of hyperimmune sera from immunized animals, and today can rely on biotechnology methods to obtain more consistent and safe immunobiologicals.
• the present invention will provide significant advantages over obtaining human monoclonal antibodies with neutralizing properties, enabling an increase in their performance and having a more favorable cost / benefit ratio.
• the present invention proposes to obtain human monoclonal antibodies with neutralizing properties for the treatment of persons likely to develop tetanus. These are products that can be obtained homogeneously, with batch consistency and without causing immunogenicity.
• the monoclonal antibodies of the present invention are obtained from engineering techniques. Genetics based on gene sequence information obtained from peripheral blood B lymphocytes from people immunized with tetanus toxoid. Genetic information is inserted into vectors used for mammalian cell transfection in order to generate cell lines that produce anti-tetanus monoclonal antibodies.
• the present invention proposes a novel product which can be obtained homogeneously and consistently from human origin to prevent hypersensitivity and immunogenicity reactions.
• the monoclonal antibodies of the present invention have unique nucleotide and amino acid sequences.
• the present invention relates to the use of said human monoclonal antibodies for immunotherapy of accidental tetanus infection.
• Vaccination although effective, shows decreased protection in the elderly, not recovered by revaccination. Also susceptible to tetanus infection are those injured who have not received a booster vaccine.
• the present invention in another aspect of the present invention it relates to neonatal tetanus, carried by the umbilical cord cut, where the human monoclonal antibodies of the present invention could be used in infants born with the infection, as other monoclonal antibodies to fight Infectious agents are used in babies.
• the neutralizing antibody gene sequences of the present invention will be used for generation of stable antibody producing cell lines, in productivity for staging and production. in bioreactors.
• the resulting product is an antibody with batch consistency, potency and effectiveness for use in accidents with potential tetanus infection.
• Figure 1 is a flowchart showing the experimental strategy for obtaining the monoclonal antibodies of the present invention
• Figure 2 shows the qualitative ELISA result of antibody tetanus binding, where the antibodies of the present invention were tested at concentrations of 0.4 to 50 ng / mL.
• High neutralizing donor serum was used to validate the test as a positive control at the initial 1:50 dilution and 1: 5 serial dilutions. Plate sensitized with 100 pL of 5 pg / mL tetanus toxin;
• Figure 3 shows the qualitative ELISA result of antibody binding to tetanus anatoxin.
• Antibodies of the present invention were tested at concentrations of 0.4 to 50 ng / mL.
• High neutralizing donor serum was used to validate the test as a positive control at the initial 1:50 dilution and 1: 5 serial dilutions.
• Figure 4 shows the result of in vivo tetanus toxin neutralization assay of the 243 + 143 + 120 monoclonal antibody mixture. ensured 100% survival of animals when 28 and 14 pg of said antibodies were used;
• Figure 5 shows the ELISA result of monoclonal antibodies 120, 143 and 243 of the present invention for diphtheria anatoxin binding assay.
• the present invention proposes to obtain human monoclonal antibodies with neutralizing properties for the treatment of persons likely to develop tetanus. These are products that can be obtained homogeneously, with batch consistency and without causing immunogenicity.
• the monoclonal antibodies of the present invention are obtained from genetic engineering techniques based on gene sequence information obtained from peripheral blood B lymphocytes from persons immunized with tetanus toxoid. Genetic information is inserted into vectors used for mammalian cell transfection to generate strains cells producing anti-tetanus monoclonal antibodies.
• the present invention proposes a novel product which can be obtained homogeneously and consistently from human origin to avoid hypersensitivity and immunogenicity reactions.
• the monoclonal antibodies of the present invention have unique nucleotide and amino acid sequences.
• the present invention relates to the use of said human monoclonal antibodies for immunotherapy of accidental tetanus infection.
• Vaccination although effective, shows diminished protection in the elderly, not recovered by revaccination. Also susceptible to tetanus infection are those injured who have not received a booster vaccine.
• the present invention in another aspect of the present invention it relates to neonatal tetanus, carried by the umbilical cord cut, where the human monoclonal antibodies of the present invention could be used in infants born with the infection, as other monoclonal antibodies to fight Infectious agents are used in babies.
• the neutralizing antibody gene sequences of the present invention will be used for generation of stable antibody-producing cell lines, in productivity for staging and production in bioreactors.
• the resulting product is a batch, potency, and effectiveness antibody for use in accidents with potential tetanus infection.
• the deployment of the human monoclonal antibody technology demonstrated in the present invention will open the way for the treatment of tetanus, as well as other infectious diseases such as diphtheria, rabies, zika, etc.
• the tetanus vaccine is always given together with diphtheria (DT).
• DT diphtheria
• the antibodies of the present invention have been tested against diphtheria toxin and are unique against tetanus toxin.
• the present invention is based on the cloning and expression of immunoglobulin producing genes obtained directly from human B lymphocytes.
• the immunoglobulin variable domain genes of a single human antibody-producing B lymphocyte are amplified and cloned into an expression vector upstream of the respective human constant region (heavy or light).
• Antibody production by this methodology requires the collection of blood from donors that have circulating B cells against a given antigen and the purified antigen for identification of these cells by immunophenotyping.
• the present invention has the objective of obtaining tetanus human monoclonal antibodies by capturing specific antibody-producing B lymphocytes using the antigen or by separating plasmablasts after vaccination enhancement.
• Cell separation from donors was performed by cell sorter equipment, which deposited a specific B cell in each well in 96 wells.
• the antibody variable regions were amplified and cloned into expression vectors that were used to transiently transfect HEK293-F cells. For the capture of rare specific B lymphocytes different strategies of labeling and cell sorting were used.
• PBMC Peripheral blood mononuclear cells
• the in vivo neutralization test was performed according to the method used by the Butantan Institute Quality Control and the Brazilian Pharmacopoeia (Brazil 2010), in a project approved by the Butantan Institute Animal Use Ethics Committee.
• Step 1 Separation of blood tetanus antibody-producing B lymphocytes from tetanus-vaccinated human volunteers
• PBMC donor mononuclear cells
• Isolated cells were used for PCR and gene sequencing of the light and heavy variable regions of the antibodies.
• Strategies for capturing B lymphocytes varied to obtain specific antibodies. The strategies have been refined for rare cell capture.
• Sorting 5 This sorting was performed with cells collected after the donor received the dT vaccine booster.
• Sorting 7 same condition as sorting 6.
• Table 2 shows the number of gene pairs obtained for the strategies used.
• Table 2 Antibody pairs obtained in the sortings of cells separated with tetanus toxin and plasmablasts.
• Step 2- Amplification of antibody variable regions of cells sorted by cell sorting
• Reverse transcription was performed on the same plate where cells were separated by cell sorting. Controls performed in wells where there was no cell were: two negative controls with 1 pL of water added, one reverse transcription control with purified HeLa RNA and one PCR control of the variable regions containing 1000 B lymphocytes that had been previously separated.
• the plate was sealed, rapidly centrifuged, incubated at 68 ° C for 60 seconds in the Mastercycler Nexus Gradient thermal cycler (Eppendorf, Hamburg, Germany) and immediately placed and kept on ice. They were added to each well 7 pL solution containing 3 ⁇ l of the enzyme reverse transcriptase SuperScript ® buffer III (Invitrogen) 5-fold concentrated with 15 mM MgCl2, 12.5 nmols of each dNTP (0.5 pL of 25 mM solution of each dNTP), 100 nmols DTT (1 pL 100 mM DTT), 8 U RNAsin® ( Promega) (0.2 pL to 40 U / pL), 50 U of SuperScript ® III reverse transcriptase enzyme (Invitrogen) (0.25 pL to 200 U / pL) and 2.05 pL of sterile type 1 water.
• the enzyme reverse transcriptase SuperScript ® buffer III Invitrogen
• the 7 pL volume was distributed to each well using homogenized multichannel micropipetator and nozzle changes to each column.
• the plate was sealed and centrifuged rapidly. The reaction occurred at 42 ° C for 5 min, 25 ° C for 10 min, 50 ° C for 60 min, 94 ° C for 5 min and finally held at 4 C.
• the obtained cDNA was aliquoted into four 96 - well plates new (3 pL per well), respecting the initial address of each well in the plate. Each plate was used for independent amplification of the variable regions of the heavy, light kappa, light lambda, and the ⁇ -actin control.
• the reactions were performed with a nested PCR protocol, with the first reaction with the cDNA aliquoted on each plate.
• the components (Oligonucleotide 5 '/ Oligonucleotide Mixture 5' 50 pM, Oligonucleotide 3 '50 pM, MgCl2, dNTP, HotStarTaq® Plus DNA Polymerase and Sterile Water Type 1 qsp) were added to each well (final volume of 40 pL), the plate was sealed and the reaction performed with enzyme activation for 5 min at 95 ° C, followed by 50 cycles of 94 ° C for 30 s, 58 ° C (IgD / Igic / p-actin) or 60 ° C (IgA) for 30 s, 72 ° C for 55 s, and final extension at 72 ° C for 10 min.
• the second reaction was performed in another 3.5 pL plate of the first PCR product transferred with multichannel micropipetator respecting the initial position of each well and the components described above, in a final volume of 40 pL.
• the condition was: 5 min at 95 ° C, followed by 50 cycles of 94 ° C for 30 s, 58 ° C (IgH / IgK) or 60 ° C (Ig ⁇ ) for 30 s, 72 ° C for 45 s, and final extension at 72 ° C for 10 min.
• Reactions were performed on GeneAmp ® PCR 9700 thermal cyclers (Applied Biosystems, Foster City, CA, USA) and Mastercycler Nexus Gradient (Eppendorf, Hamburg, Germany).
• the oligonucleotides used are described in the following table. Oligonucleotides used in amplifying immunoglobulin chain variable regions
• Control with ⁇ -actin was done in a single reaction with oligonucleotides.
• the heavy and light chains were paired as originally formed the corresponding pair.
• Step 3- Cloning of sequences in mammalian cell expression vectors.
• An aliquot of chemocompetent Escherichia coli DH5 bacteria (Invitrogen) was plated on LB / agar medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 1.5% agar) and incubated at 37 ° C for 16-18 hours.
• LB / agar medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 1.5% agar
• One colony was inoculated into 10 mL of liquid LB medium for 16-18 hours at 37 ° C under 200 RPM shaking.
• the culture was subdivided into tubes that were kept on ice for 20 min and then centrifuged at 2500 xg for 20 min at 4 ° C. The supernatant was discarded and the cells resuspended in 50 mL of ice-cold sterile 0.1 M MgCl2. It was incubated on ice for 20 min and centrifuged as indicated above. The pellet was resuspended in 25 mL of sterile ice-cold 0.1 M CaCl2, incubated on ice for 20 min and centrifuged as described.
• the bacteria were resuspended in 2.4 mL of sterile, ice-cold 0.1 M CaCl2 and the addition of 0.6 mL glycerol. After homogenization, the suspension was aliquoted in microtubes in an ice / ethanol bath and stored in the -80 ° C freezer. Transformation efficiency was determined using a control plasmid.
• Vectors used for cloning and expression of antibodies were kindly provided by Dr. Hedda Wardemann of the Max Plank Institute. Each vector contains the sequence coding for the constant region of one of the human immunoglobulin chains: heavy ⁇ , ⁇ or ⁇ . Upstream of the constant region is the cloning site for amplified variable chain insertion in the specific PCR (Tiller et al, 2008).
• the vectors were transformed into chemocompetent E. coli DH5 bacteria by mixing 2 ng of each vector (in 1 ⁇ l volume) independently into 10 ⁇ l of the bacterial suspension. The suspensions were incubated on ice for 30 min, followed by heat shock at 42 ° C for 45 min and again incubating on ice for 5 min.
• 100 ⁇ l SOC medium (20 g / L tryptone, 5 g / L yeast extract, 500 mg / L NaCl, 2.5 mM KCl, 10 mM MgCl 2 and 20 mM glucose) were added to the bacteria suspension and incubated. for one hour at 37 ° C while stirring at 180 RPM.
• the entire suspension was inoculated on a LB / agar / ampicillin plate (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 1.5% agar, 100 pg / mL ampicillin) and incubated. at 37 ° C for 16-18 hours.
• plgD - SalI-RF and Agel-HF enzymes (NEB - New England Biolabs)
• plg ⁇ - enzymes Xhol-EF and Agel (NEB)
• Digestions were sequential, in the order described above with each enzyme at a ratio of 2 U / pg DNA for Agel and BsiWI enzymes and 10 U / pg for Sall enzymes and Xhol. Digestion was carried out for four hours at 37 ° C for Agel, Sall and Xhol and 55 ° C for BsiWI.
• the digested vectors were purified by agarose gel electrophoresis and Wizard ® SV Gel and PCR Clean-Up System kit (Promega).
• RT-PCR samples from the selected wells after analysis of the variable chain sequences were subjected to PCR with specific oligonucleotides. This step served to amplify the fragment with an oligonucleotide at each end, avoiding the insertion of mutations, and adding restriction sites for cloning into expression vectors.
• the first PCR product was used as a template with 5 'and 3' oligonucleotides specific for the V and J genes, respectively, containing specific restriction site.
• the enzyme was partially digested with serial enzyme dilutions and incubation by 2 min at the specific enzyme temperature (CAMPEAU, 2009).
• PCR products were further purified with QIAquick ® 96 PCR Purification (Qiagen) and eluted with 60 ⁇ l of sterile type 1 water.
• Binding of the fragment to the vector was done in a total volume of 10 pL with 8 pL of the digested and purified specific PCR product, 0.5 pL of the respective linearized vector at
• the mixture was distributed into wells of a 96-well PCR plate. Using a sterile micropipette tip, an isolated colony was removed with the tip and transferred to a spare LB / agar / ampicillin petri dish. Then, the nozzle was immersed in the aliquoted PCR mixture in the 96-well plate. The petri dish was incubated at 37 ° C for 16-18 hours and stored in the refrigerator.
• the PCR reaction was as follows: 5 min 94 ° C; 27 cycles of 94 ° C for 30 s, 58 ° C for 30 s, 72 ° C for 60 s; and final extension at 72 ° C for 10 min.
• the PCR products were analyzed by 2% agarose electrophoresis, whose expected sizes were: 650 bp for IgD1, 700 bp for IgK and 590 bp for Ig ⁇ .
• the positive fragments were purified with ExoSAP-IT (Affymetrix) and sequenced as described above.
• the oligonucleotide used for sequencing was 5 'Absense.
• Sequences obtained from colony PCR were compared to those obtained from RT-PCR using the BLAST two or more sequence alignment tool (bl2seq - Align Sequence Nucleotide BLAST). The identity of the sequences was verified, disregarding the regions of oligonucleotide annealing and the colony containing the vector with the variable sequence identical to that obtained from RT-PCR was determined.
• a portion of the selected colony was collected from the spare plate and inoculated into 4 mL of TB medium.
• the vector was isolated from one of the 2 mL aliquots of the bacterial suspension and the other was frozen after centrifugation at 10,000 xg for 5 min and removal of the supernatant. Isolation was performed with Wizard ® Plus SV Miniprep DNA Purification kit (Promega) and concentration and purity determined by spectrophotometry at 260/280 nm.
• Antibody expression was done with transient cotransfection of the heavy and light chain vector corresponding to the pair obtained from the same well in RT-PCR.
• FreeStyle TM 293-F HEK 293f cells were cultured in suspension in FreeStyle TM 293 Expression Medium (Invitrogen) in a 37 ° C greenhouse with 8% CO2 and shaking at 120 RPM.
• the suspension was prepared in a volume of 30 mL of suspension for each antibody to be transfected at a concentration of approximately 0.5 x 106 cells / mL.
• the suspension was aliquoted in 30 mL volume in 125 mL conical flasks at a concentration between 0.9 and ⁇ , ⁇ 6 cells / mL for each antibody to be transfected.
• the transfection DNA solution was prepared in final volume of 1.5 ml PBS. Initially, 30 pg of the vectors [15 pg each (heavy and light chain)] were added to PBS volume and incubated for 5 minutes at room temperature. 103 ⁇ l of stock solution was added. Polyethylenimine - PEI (Sigma-Aldrich) at 0.45 mg / ml and vortexed for 15 seconds and incubated again for a further 10 min at room temperature protected from light before dripping onto the cell suspension that was maintained. in cultivation for 72 hours. The supernatant was collected after centrifugation at 800 xg for 10 min in tube centrifuge (Sorvai Legend RT, Newtown, CT, USA).
• Antibodies were purified by protein A affinity chromatography using the ⁇ KTA Purifier system (GE Healthcare, Sweden). The filtered supernatant was applied to the protein A-sepharose column (GE Healthcare, Sweden) equilibrated with 20 mM phosphate buffer pH 7. The column was washed with the same buffer. The first elution was made with 100 mM sodium citrate buffer pH 6 and the second with 100 mM sodium citrate buffer pH 3.2 for recovery of AcMos. The collected sample was neutralized with sufficient 1 M Tris solution. The purified and neutralized antibody was dialyzed against PBS and sterilized by filtration.
• Antibodies were tested for binding to available forms of the antigen: tetanus toxin and anatoxin, recombinant tetanus toxin fragment C.
• Figures 2 and 3 show the binding curves of antibodies against TxT and AnT.
• Figure 2 shows the result of qualitative ELISA binding of antibodies to tetanus toxin, where antibodies of the present invention were tested at concentrations of 0.4 to 50 ng / ml.
• Donor serum was used to validate the test as a positive control at the initial 1:50 dilution and 1: 5 serial dilutions.
• the plate was sensitized with 100 ⁇ l of 5 pg / ml tetanus toxin;
• Figure 3 shows the qualitative ELISA result of antibody binding to tetanus anatoxin.
• Antibodies of the present invention were tested at concentrations of 0.4 to 50 ng / mL.
• Donor serum was used to validate the test as a positive control at the initial 1:50 dilution and 1: 5 serial dilutions.
• the plate was sensitized with 100 pL of 5 pg / mL tetanus anatoxin.
• Composite mixtures of tetanus toxin were prepared at the same concentration and different amounts of the antibodies tested. After incubation at 37 ° C for one hour, 200 ⁇ l of each mixture was applied subcutaneously to 10 animals, which were kept under observation for 96 hours, during which time the number of healthy, dead or tetanus symptoms mice was verified.
• Figure 4 shows the result of tetanus toxin neutralization assay in vivo: In D, mixture of BUT-TT-120, BUT-TT-143 and BUT-TT-243 antibodies. Mixing of the AcMos ensured the survival of 100% of the animals when 28 and 14 pg of antibodies were used, being able to neutralize the toxic action of tetanus toxin, ensuring the survival of all animals in the groups with 28 and 14 pg of antibody.
• the strategy ensured obtaining clonally related sequences, suggestive of antigen selection.
• antibodies obtained from tetanus toxin labeling or from plasmablasts recognized the antigen in the assays performed.
• variable regions The amount of somatic mutations detected in the variable regions was also similar to that observed in human anti-HIV AcMos (Scheid et al., 2009a).
• the three mAbs obtained in the present invention (243, 143 and 120) and which mixed protected Swiss mice against the action of TxT interact at different sites on the toxin, according to the western blotting result: heavy chain, light chain to whole toxin, does not reduce going.
• two or more monoclonal antibodies when mixed, two or more monoclonal antibodies can have synergistic effect and prevent release of free toxin to exert its toxic function.
• the monoclonal antibodies of the present invention were obtained from human peripheral blood cells after immunization. It has been shown that in humans, the frequency of antitetanic antibody-producing memory cells is higher in secondary lymphoid organs such as tonsils rather than in peripheral blood (Cao et al., 2010). Obtaining the Monoclonal Antibodies of the present Invention
• SEQ ID NO 1 BUT-TT-120 light chain variable region nucleotide sequence
• SEQ ID NO 2 BUT-TT-120 antibody light chain variable region amino acid sequence Primers used:
• SEQ ID NO 4 BUT-TT-120 antibody heavy chain variable region amino acid sequence Primers used:
• SEQ ID NO 7 BUT-TT-143 antibody heavy chain variable region nucleotide sequence
• SEQ ID NO 8 BUT-TT-143 antibody heavy chain variable region amino acid sequence Primers used:
• SEQ ID NO 10 BUT-TT-243 antibody light chain variable region amino acid sequence Primer sequence:
• SEQ ID NO 12 BUT-TT-243 antibody heavy chain variable region amino acid sequence Primers used:
• the monoclonal antibodies of the present invention were obtained from human peripheral blood cells of immunized individuals. It has been shown that in humans, the frequency of anti-tetanus antibody-producing memory cells is higher in secondary lymphoid organs, such as tonsils, rather than in peripheral blood (Cao et al., 2010).
• the human tetanic antitoxin monoclonal antibody 1-BUT-TT-120 is a monoclonal antibody comprising a heavy chain variable region (VH) and light chain variable region (VL), wherein the VH heavy chain comprises a sequence of amino acids represented by SEQ ID NO: 4 and the light chain VL comprises an amino acid sequence represented by SEQ ID NO: 2.
• SEQ ID NO: 2 Human monoclonal antibody light chain variable region amino acid sequence # 1- BUT-TT-120-VL tetanus antitoxin
• Trp Ile Gly Be Thr His Phe Be Gly Be Thr Tyr Asn Pro Be Read 50 55 60
• the human tetanic antitoxin 2-BUT-TT-143 monoclonal antibody is a monoclonal antibody comprising a heavy chain variable region (VH) and light chain variable region (VL), wherein the VH heavy chain comprises a sequence of amino acids represented by SEQ ID NO: 8 and the light chain VL comprises an amino acid sequence represented by SEQ ID NO: 6.
• SEQ ID NO: 6 Human monoclonal antibody light chain variable region amino acid sequence # 2- BUT-TT-143 tetanus toxin
• Lys Gly Arg lie Thr Leu Be Arg Asp Asn Be Lys Asn Lie Met Tyr 65 70 75 80
• the 1-BUT-TT-243 tetanus human anti-toxin human monoclonal antibody is a monoclonal antibody comprising a heavy chain variable region (VH) and light chain variable region (VL), wherein the VH heavy chain comprises a sequence of amino acids represented by SEQ ID NO: 12 and the light chain VL comprises an amino acid sequence represented by SEQ ID NO: 10.
• SEQ ID NO: 10 Human monoclonal antibody light chain variable region amino acid sequence # 3- BUT-TT-243 tetanus toxin
• SEQ ID NO: 12 Human monoclonal antibody light chain variable region amino acid sequence # 1- BUT-TT-243 tetanus toxin
• Monoclonal antibodies of the present invention have been tested against diphtheric toxin and are exclusive against tetanus toxin
• BUT-TT-120, BUT-TT-143 and BUT-TT-243 monoclonal antibodies were also tested for binding to diphtheria anatoxin (AnD), as blood from some donors was collected a few days after booster dT vaccine, which is composed of tetanus and diphtheria toxoids. In addition, this antigen served as a binding test for a protein unrelated to tetanus toxin.
• Figure 5 shows the ELISA result of monoclonal antibodies 120, 143 and 243 of the present invention for diphtheria anatoxin binding assay.
• the monoclonal antibodies of the present invention may be incorporated into pharmaceutical / immunological compositions suitable for administration to a subject.
• the pharmaceutical / immunological composition of the present invention comprises a monoclonal antibody or monoclonal antibody portion of the present invention and a pharmaceutically acceptable carrier.
• pharmaceutically acceptable carrier includes any and all isotonic compatible solvents, dispersion media, coatings, absorption retarding agents and isotonic agents.
• pharmaceutically acceptable carriers include, but are not limited to, one or more of water, saline, phosphate buffered saline, dextrose, and the like, as well as combinations thereof. In many cases, it will be preferable to include amino acids or other salts.
• Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, which increase the half-life or effectiveness of the monoclonal antibody or monoclonal antibody portion of the present invention.
• the immunological compositions of the present invention may be in a variety of forms.
• Typical preferred compositions are in the form of injectable or infusion solutions, such as compositions similar to those used for passive immunization of humans with other antibodies.
• the preferred mode of administration is parenteral (e.g. intravenous, subcutaneous, intraperitoneal, intramuscular).
• the monoclonal antibody of the present invention is administered by infusion or intravenous injection. In another preferred embodiment, the monoclonal antibody of the present invention is administered by intramuscular or subcutaneous injection.
• Immunological compositions should typically be sterile and stable under the conditions of manufacture and storage.
• Sterile injectable solutions may be prepared by incorporating the active compound (i.e. the monoclonal antibody or monoclonal antibody moiety) in the required amount in an appropriate solvent with one or a combination of ingredients listed above as required, followed by filter sterilization.
• active compound i.e. the monoclonal antibody or monoclonal antibody moiety
• dispersions are prepared by incorporation of the active compound into a sterile vehicle containing a basic dispersion medium and the other necessary ingredients from those enumerated above.
• sterile powders for the preparation of sterile injectable solutions preferred methods of preparation are vacuum drying and lyophilization which produces a powder of the active ingredient plus any desired additional ingredient of a previously sterile filtration solution thereof.
• Monoclonal antibodies of the present invention may be administered by a variety of methods known in the art, although for many therapeutic applications, the preferred route / mode of administration is injection or infusion.
• additional therapeutic agents is an agent or agents for the treatment of a disease or condition involving the tetanus toxin.
• treat and “treatment” refer to therapeutic treatment, including prophylactic or preventative measures, wherein the purpose is to prevent or retard (decrease) an undesirable physiological change associated with a disease or disturb.
• beneficial or desired clinical outcomes include, but are not limited to, symptom relief, decreased extent. of a disease or disorder, stabilization of a disease or disorder (ie, where the disease or disorder does not get worse), slowing or slowing the progression of a disease or disorder, amelioration or palliation of the disease or disorder, and remission (whether partial or total) of the disease or disorder, whether detectable or undetectable.
• treatment may also mean prolonging survival compared to expected survival if not receiving treatment.
• Those in need of treatment include those already with the disease or disorder as well as those prone to have the disease or disorder or those in whom the disease or disorder is to be prevented.
• compositions of the present invention may include a "therapeutically effective amount” or a “prophylactically effective amount” of a human tetanus antitoxin human monoclonal antibody of the present invention.
• a “therapeutically effective amount” refers to an amount effective at the dosages and for periods of time necessary to achieve the desired therapeutic result.
• a therapeutically effective amount of the monoclonal antibody of the present invention may vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of the antibody or antibody moiety to induce a desired response. in the individual.
• a “therapeutically effective amount” is also one in which any toxic or detrimental effects of the antibody or antibody moiety are offset by the therapeutically beneficial effects.
• prophylactically effective amount refers to an amount effective, at dosages and for periods of time required, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects before or at an early stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.
• a non-limiting range for an effective therapeutically or prophylactically exemplary amount of the monoclonal antibody of the present invention is 0.4 to 50 ng / ml. Therefore, in the Swiss mouse trial this range proved sufficient.
• dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.
• Antibodies of the present invention may be used to treat any disease or disorder mediated by, associated with, or caused by action of the tetanus toxin.
• the monoclonal antibodies of the present invention may be used to treat a disease or disorder that results in tetanus.
• the monoclonal antibodies of the present invention may be used to treat a disease or disorder caused by localized tetanus, where onset of symptoms occurs with myalgia due to involuntary contractions of muscle groups near the injury and may be restricted to a particular limb.
• the monoclonal antibodies of the present invention may be used to treat a disease or disorder caused by cephalic tetanus, which occurs due to injuries to the scalp, face, oral cavity and ear, leading to ipsilateral facial paralysis to the lesion, trismus, cranial nerve dysphagia and impairment iii, iv, ix, x, xii.
• the monoclonal antibodies of the present invention may be used to treat a disease or disorder caused by generalized tetanus, which is characterized by trismus due to contraction of the facial mimic masseter and muscle causing sardonic laughter.
• Other muscle groups are affected, such as the rectus abdominis and paravertebral musculature, which may cause opisthotonus (characteristic of children).
• opisthotonus characteristic of children.
• Muscle contractures follow shortly and, depending on its intensity and frequency, tetanus may be less or more severe, worsening auditory, visual and tactile stimuli. Depending on their intensity, these spasms can progress even to vertebrae fractures or respiratory arrest.
• tetanus patient despite his severity, always remains lucid. Fever, when present, indicates poor prognosis or secondary infection.
• hyperactivity sympathetic we have: tachycardia, labile arterial hypertension, profuse sweating, peripheral vasoconstriction, cardiac arrhythmias and even hypotension.
• the monoclonal antibodies of the present invention may be used to treat a disease or disorder caused by neonatal tetanus, where it is caused by the application of contaminated substances to the cord wound or cut.
• the incubation period is approximately seven days and its main characteristic is the opistoton. At first, the child may only have difficulty eating. It usually occurs in children of unvaccinated or inadequately vaccinated prenatal mothers.
• the monoclonal antibodies of the present invention may be used in immunotherapy for accidents susceptible to tetanus bacilli infection.
• the monoclonal antibodies of the present invention may be used for the treatment of persons likely to develop tetanus. These are products that can be obtained homogeneously, with batch consistency and without causing immunogenicity.
• the present invention includes methods for treating a disease or disorder mediated by, associated with, or caused by action of the tetanus toxin.
• the methods comprise administering to a patient in need thereof a therapeutically or prophylactically amount of the monoclonal antibody as disclosed herein.
• monoclonal antibody as disclosed herein is intended to mean any monoclonal antibody comprising any of the VH regions and / or VL regions defined herein by their identifying sequences (SEQ ID), as well as any antibody. monoclonal comprising a variant of any of the VH regions set by the identifying sequences (SEQ ID) and / or a variant of any of the VL regions set forth herein.
• VZV Human Anti-Varicella-Zoster Virus
• Tetanus toxin is a zinc protein and its inhibition of neurotransmitter release and protease activity depends on zinc.

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