EP4110825A1 - Nouveaux anticorps monoclonaux therapeutiques humains et leurs utilisations - Google Patents
Nouveaux anticorps monoclonaux therapeutiques humains et leurs utilisationsInfo
- Publication number
- EP4110825A1 EP4110825A1 EP20792417.6A EP20792417A EP4110825A1 EP 4110825 A1 EP4110825 A1 EP 4110825A1 EP 20792417 A EP20792417 A EP 20792417A EP 4110825 A1 EP4110825 A1 EP 4110825A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- sequence seq
- monoclonal antibody
- identity
- fragment
- nucleic acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
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- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- 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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/52—Constant or Fc region; Isotype
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/52—Constant or Fc region; Isotype
- C07K2317/526—CH3 domain
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/55—Fab or Fab'
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- 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
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- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
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- G01N2333/914—Hydrolases (3)
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- G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
- G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
- G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
- G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
- G01N2333/96433—Serine endopeptidases (3.4.21)
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Definitions
- the invention relates to novel therapeutic human monoclonal antibodies and their uses.
- neutrophil proteinase 3 is the target of autoantibodies (ANC A) which leads to deleterious activation of polynuclear neutrophils (PMN), responsible for severe vasculitis, mainly affecting the kidneys, lungs and brain.
- Current treatments for GPA are based on immunosuppressants, corticosteroids, and more recently anti-CD20 antibodies.
- PMN polynuclear neutrophils
- Anti-CD20 more specifically target circulating B lymphocytes expressing the CD20 differentiation marker. This results in a deletion of B lymphocytes producing autoantibodies directed against PMN PR3 (ANC A).
- a first aim of the invention is to provide an antibody targeting neutrophil proteinase 3 (PR3).
- a second aim of the invention is to provide fragments of this antibody.
- a third aim of the invention is to provide the tools (nucleic acid, vector, etc.) making it possible to produce said antibody and / or said fragments.
- another aim of the invention is to provide pharmaceutical compositions and their uses.
- the present invention relates to the object as defined and described below. Furthermore, and unless otherwise indicated or if the context indicates otherwise, all terms have their ordinary meaning in the field (s) concerned.
- the object of the invention is a monoclonal antibody directed against neutrophil proteinase 3 represented by the sequence SEQ ID NO: 1, said monoclonal antibody:
- ⁇ being capable of inhibiting by at least 30% the production of reactive oxygen derivatives by neutrophils, said production of reactive oxygen derivatives being induced by the presence of autoantibodies directed against said neutrophil proteinase 3 .
- antibody refers to an immunoglobulin, a multimeric protein made up of 4 chains participating in the acquired immune response.
- Immunoglobulins are well known to those skilled in the art and consist of an assembly of two dimers each consisting of a heavy chain and a light chain.
- the multimeric complex assembled by the bonding of a light chain and a heavy chain through a disulfide bridge between two cysteines, the two heavy chains themselves also being linked together by two disulfide bridges.
- Each of the heavy chains and light chains consists of a constant region and a variable region.
- the assembly of the chains that make up an antibody makes it possible to define a characteristic three-dimensional structure in Y, where ⁇ the base of the Y corresponds to the constant region Fc which is recognized by the complement and the Fc receptors, and
- the ends of the arms of the Y correspond to the respective assembly of the variable regions of the light chain and the variable regions of the heavy chain.
- each light chain consists of a variable region (VL) and a constant region (CL).
- Each heavy chain consists of a variable region (VH) and a constant region consisting of three constant domains CH1, CH2 and CH3. The C H 2 and C H 3 domains make up the Fc domain.
- variable region of the light chain consists of three regions determining the recognition of the antigen (CDR) surrounded by four framework domains.
- the heavy chain variable region also consists of three antigen recognition determining (CDR) regions surrounded by four framework domains. The three-dimensional folding of these variable regions is such that the 6 CDRs are exposed on the same side of the protein and allow the formation of a specific structure recognizing a specific antigen.
- the antibodies described in the invention are isolated and purified, can be recombinant, can belong to any isotype / class (eg. IgG, IgE, IgM, IgD, IgA and Ig Y) or to a subclass (eg IgG1, IgG2, IgG3, IgG4, IgAl and IgA2) and are different from natural antibodies.
- IgG, IgE, IgM, IgD, IgA and Ig Y or to a subclass (eg IgG1, IgG2, IgG3, IgG4, IgAl and IgA2) and are different from natural antibodies.
- These antibodies are mature, that is to say they have an ad hoc three-dimensional structure allowing them to recognize the antigen, and have all the / s7-translational modifications essential for their antigen recognition, in particular glycosylation and formation. of intra and inter molecular disulfide bridges.
- the monoclonal antibodies of the invention specifically target a "conformational epitope" of PR3, which is formed / constituted by amino acids which are not contiguous in the sequence SEQ ID NO: 1. , some of said amino acids may be immediately next to each other and thus a conformational epitope according to the present invention may contain only one amino acid which is not contiguous with the others in the sequence SEQ ID NO: 1.
- ROS reactive oxygen species
- the expression "production of reactive oxygen species” refers in particular to the cellular mechanisms allowing neutrophils to produce oxygenated chemical species such as free radicals, oxygenated ions and peroxides, made chemically very reactive by the presence of unpaired valence electrons. It may be, for example, superoxide O2 flag, singlet oxygen O2 ' , hydrogen peroxide H2O2, or even ozone O3, which may, for example, be highlighted by the marking DHR123-FITC (detection of hydrogen peroxide H2O2) by flow cytometry (see Examples, point 14).
- the monoclonal antibodies of the invention are capable of "inhibiting by at least 30%” this production of reactive oxygen derivatives by neutrophils, which are activated in the presence of auto.
- -antibodies directed against said neutrophil proteinase 3.
- the expression “at least 30%” should be understood as being able to be at least 35%, at least 40%, at least 45%, d at least 50%, at least 55%, at least 60%, at least 65%, at least 70% or at least 75%.
- the capacity for inhibiting said production of reactive oxygen derivatives by the antibodies of the invention can be between 30% to 80% or from 36% to 71%.
- the monoclonal antibody (recombinant and / or isolated and purified) of the invention advantageously has this unexpected characteristic of particular inhibition, the invention responds to the issues raised. Indeed, the action of an antibody being immediate on circulating or endothelial cells, in GP A, where the target of PMNs is the endothelial cell, the monoclonal antibody of the invention:
- the subject of the invention is the monoclonal antibody (recombinant and / or isolated and purified) as defined above comprising:
- this sequence identity between an amino acid sequence of interest and a reference amino acid sequence being at least 80%, this means it can be at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86 %, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93% , at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%. In particular, it is at least 93%.
- the subject of the invention is the monoclonal antibody (recombinant and / or isolated and purified) as defined above comprising:
- ⁇ a heavy chain comprising its N-terminus to its C-terminus: - CDR1 of sequence SEQ ID NO: 15; CDR2 of sequence SEQ ID NO: 17; and
- the subject of the invention is the monoclonal antibody (recombinant and / or isolated and purified) as defined above comprising:
- ⁇ a heavy chain comprising a variable region having at least 80% identity with the sequence SEQ ID NO: 7, with the proviso that said heavy chain variable region comprises from its N-terminus to its C-terminus:
- ⁇ a light chain comprising a variable region having at least 80% identity with the sequence SEQ ID NO: 25, with the proviso that said light chain variable region comprises from its N-terminus to its C-terminus:
- the invention relates to the monoclonal antibody
- the subject of the invention is the monoclonal antibody (recombinant and / or isolated and purified) as defined above comprising: ⁇ a heavy chain comprising or consisting of a sequence having at least 80% identity with the sequence SEQ ID NO: 3, with the proviso that said heavy chain comprises the variable region sequence SEQ ID NO: 7; and a light chain comprising or consisting of a sequence having at least 80% identity with the sequence SEQ ID NO: 21, it being understood that said light chain comprises the variable region of sequence SEQ ID NO: 25.
- the subject of the invention is the monoclonal antibody as defined above comprising: a heavy chain comprising or consisting of the sequence SEQ ID NO: 3 or of the sequence SEQ ID NO: 37; and
- ⁇ a light chain comprising or consisting of the sequence SEQ ID NO: 21.
- this embodiment corresponds to the so-called “4C3” antibody of allotype Glm3 - 1 (SEQ ID NOs: 3 and 21) or to its recombinant form “r4C3” of allotype Glml7 - 1 (SEQ ID NOs: 37 and 21) and of which all the sequences are in Table 1 below, which differ only from a single mutation, namely, R> K in position 224 (or AGA> AAA in position 670-672) within the constant region of the heavy chain.
- the subject of the invention is the monoclonal antibody (recombinant and / or isolated and purified) as defined above comprising:
- ⁇ a heavy chain comprising a variable region having at least 80% identity with the sequence SEQ ID NO: 7, with the proviso that said heavy chain variable region comprises from its N-terminus to its C-terminus:
- ⁇ a light chain comprising a variable region having at least 80% identity with the sequence SEQ ID NO: 25, with the proviso that said light chain variable region comprises from its N-terminus to its C-terminus:
- a heavy chain comprising or consisting of a sequence having at least 80% identity with the sequence SEQ ID NO: 3, with the proviso that said heavy chain comprises the variable region sequence SEQ ID NO: 7;
- a light chain comprising or consisting of a sequence having at least 80% identity with the sequence SEQ ID NO: 21, with the proviso that said light chain comprises the variable region sequence SEQ ID NO: 25.
- the latter relates to a fragment of a monoclonal antibody (recombinant and / or isolated and purified) as described above.
- fragment denotes any part of an antibody which retains the ability to bind to the epitope recognized by the complete antibody.
- fragments include, but are not limited to, Fab, Fab 'and F (ab') 2, Fd, single chain Fvs (scFv), single chain antibodies, disulfide linked Fvs ( dsFv) and fragments comprising the V L or VH region.
- Fragments binding to the epitope, including single chain antibodies can comprise the variable region (s) alone or in combination with all or part of the following elements: hinge region, C H I, C H 2 and C H 3 domains.
- fragments may contain one or both Fab fragments or the F (ab ’) 2 fragment.
- fragments can be or can combine members of any of the following classes of immunoglobulins: IgG, IgM, IgA, IgD or IgE and their subclasses.
- Fab and F (ab ’) 2 fragments can be produced by proteolytic cleavage, using enzymes such as papain (Fab fragment) or pepsin (F (ab’) 2 fragment).
- Single-chain Fv ("scFv) fragments are epitope-binding fragments which contain at least one fragment of an antibody variable region (V H ) linked to at least one fragment of a variable region. (V L ) light chain antibody.
- the linker can be a short and flexible peptide chosen to ensure that the correct three-dimensional folding of the V L and V H regions occurs once they are linked, so as to maintain the specificity of binding to the target molecule of the. whole antibody from which the single chain antibody fragment is derived.
- the carboxyl terminus of the V L OR V H sequence can be covalently linked by a linker to the amino acid terminus of a complementary V L OR V H sequence.
- the subject of the invention is the above fragment of a monoclonal antibody (recombinant and / or isolated and purified) as defined above, said fragment being chosen from the group of fragments consisting of: Fv, Fab, F (ab ') 2, Fab', dsFv, scFv, sc (Fv) 2, “diabodies”.
- the latter relates to the above fragment of a monoclonal antibody (recombinant and / or isolated and purified) as defined above, said fragment comprising the combination of the six (6) CDR having at least 80% identity with the sequences SEQ ID NOs; 15, 17, 19, 31, 33 and 35.
- this sequence identity between an amino acid sequence of interest and a reference amino acid sequence being at least 80%, this means it can be at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86 %, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93% , at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%. More particularly, it is at least 93%.
- the subject of the invention is the above fragment of a monoclonal antibody (recombinant and / or isolated and purified) as defined above, said fragment comprising the combination of the six (6) CDR sequences SEQ ID NOs; 15, 17, 19, 31, 33 and 35.
- the subject of the invention is the above fragment of a monoclonal antibody (recombinant and / or isolated and purified) as defined above, said fragment comprising:
- variable region of a heavy chain having at least 80% identity with the sequence SEQ ID NO: 7, with the proviso that said heavy chain variable region comprises from its N-terminus to its C-terminus:
- variable region of a light chain having at least 80% identity with the sequence SEQ ID NO: 25, with the proviso that said light chain variable region comprises from its N-terminus to its C-terminus:
- the subject of the invention is the above fragment of a monoclonal antibody (recombinant and / or isolated and purified) as defined above, said fragment comprising:
- the latter relates to a nucleic acid comprising or consisting of a sequence encoding:
- the subject of the invention is nucleic acid as defined above comprising or consisting of a sequence encoding:
- this sequence identity between a nucleic acid sequence of interest and a reference nucleic acid sequence being at least 80%, this means it can be at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86 %, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93% , at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%. More particularly, it is at least 93%.
- the subject of the invention is nucleic acid as defined above comprising or consisting of a sequence encoding:
- the subject of the invention is nucleic acid as defined above comprising or consisting of a sequence encoding:
- a heavy chain comprising a variable region having at least 80% identity with the sequence SEQ ID NO: 6, with the proviso that said heavy chain variable region comprises from its N-terminus to its C-terminus:
- ⁇ a light chain comprising a variable region having at least 80% identity with the sequence SEQ ID NO: 24, with the proviso that said light chain variable region comprises from its N-terminus to its C-terminus:
- the subject of the invention is the nucleic acid as defined above comprising or consisting of a sequence encoding: ⁇ a heavy chain comprising the variable region sequence SEQ ID NO: 6; and or
- the subject of the invention is nucleic acid as defined above comprising or consisting of a sequence encoding:
- a heavy chain comprising or consisting of a sequence having at least 80% identity with the sequence SEQ ID NO: 2, with the proviso that said heavy chain comprises the variable region sequence SEQ ID NO: 6; and or
- ⁇ a light chain comprising or consisting of a sequence having at least 80% identity with the sequence SEQ ID NO: 20, with the proviso that said light chain comprises the variable region sequence SEQ ID NO: 24.
- nucleic acid as defined above comprising or consisting of a coding sequence:
- ⁇ a heavy chain comprising or consisting of SEQ ID NO: 2 or SEQ ID NO: 36; and or
- ⁇ a light chain comprising or consisting of the sequence SEQ ID NO: 20.
- this embodiment corresponds to the so-called “4C3” antibody of allotype Glm3 - 1 (SEQ ID NOs: 2 and 20) or to its recombinant form “r4C3” of allotype Glml7 - 1 (SEQ ID NOs: 36 and 20) and of which all the sequences are in Table 1 above, which differ only from a single mutation, namely, R> K at position 224 (or AGA> AAA at position 670-672) within the constant region of the heavy chain.
- the subject of the invention is an expression vector comprising at least one nucleic acid as defined above, said nucleic acid being under the control of elements allowing its expression.
- expression vector it is defined in the invention a DNA molecule (deoxyribonucleic acid) which has elements allowing its replication (duplication) in at least one living organism. These elements allowing replication are in particular the origins of replication of yeast or bacteria, or else elements of control of the replication of a virus.
- the vectors according to the invention are in particular plasmids, phages, artificial yeast chromosomes (YAC), artificial chromosomes of bacteria (BAC), the modified genomes of replicative viruses or of integrative viruses, etc.
- vectors are called "expression” because they have nucleotide sequences that allow the expression, that is to say the transcription into RNA (ribonucleic acid), of the nucleotide sequences that they control.
- said nucleic acid sequence contained in said vector is placed "under the control of the elements allowing its expression".
- said expression vector has at least one transcription initiation sequence such as a promoter of a virus such as the early promoter of the simian virus SV40, or of the Cytomegalovirus (CMV) or else the promoter sequences of the virus. Rous's sarcoma (RSV), and in particular a sequence or promoter comprising a TATAA box.
- said vector also has at least one transcription termination sequence and in particular a polyadenylation sequence derived from a mammalian gene, in particular a human.
- sequences which are essential for the expression of the nucleotide sequence contained in said vector, other sequences can be added which make it possible to regulate or modulate the expression of said sequence.
- a non-exhaustive list includes: introns of genes of mammals, and in particular human genes, enhancer-type transcription regulatory sequences (“enhancers”) or alternatively transcribed but untranslated sequences of genes of mammals, and in particular human genes.
- a particular embodiment of the invention therefore relates to an expression vector as defined above, comprising:
- ⁇ a first nucleic acid selected from those coding for all or part of the heavy chain of the monoclonal antibody as described above, said first nucleic acid being under the control of elements allowing its expression;
- a second nucleic acid selected from those coding for all or part of the light chain of the monoclonal antibody as described above, said second nucleic acid is under the control of elements allowing its expression.
- This expression vector therefore comprises two aforementioned nucleic acid sequences, and more particularly comprises a nucleic acid sequence encoding the heavy chain of the monoclonal antibody as described above, and a nucleic acid sequence encoding the light chain. of the monoclonal antibody as described above.
- said expression vector contains a first element allowing the expression of the nucleic acid sequence encoding the heavy chain of the monoclonal antibody (recombinant and / or isolated and purified) as described above and a second element allowing the 'expression of the nucleic acid sequence encoding the light chain of the monoclonal antibody (recombinant and / or isolated and purified) as described above, said first and said second element allowing the expression of said nucleic acid sequences being identical or different, and preferably identical.
- These control elements are in particular the long terminal repeated sequences (LTR) of the RSV virus.
- the subject of the invention is a host cell or cell line transformed with a nucleic acid as described above and / or an expression vector as described above.
- the subject of the invention is the monoclonal antibody (recombinant and / or isolated and purified) as described above and / or fragment as described above and / or nucleic acid as described above and / or expression vector as described above for its use as a medicament.
- the subject of the invention is a pharmaceutical composition
- a pharmaceutical composition comprising as active substance at least:
- the term “pharmaceutical composition” is understood to mean a particular packaging of the invention, which allows the pharmaceutical composition of the invention to be possibly administered to animals and humans by the oral, sublingual or sub- route. cutaneous, intramuscular, intravenous, transdermal, local, inhaled or rectal. Moreover, this packaging also allows the administration of the active principle (or active substance), alone or in combination with another active principle, in unit form or as a mixture with conventional pharmaceutical carriers. Suitable unit dosage forms include:
- oral administration forms such as tablets, capsules, powders, granules and oral suspensions or solutions;
- pharmaceutically acceptable vehicle is meant within the meaning of the invention a non-toxic material which is compatible with a biological system such as a cell, a cell culture, a tissue or an animal or human organism. These may include:
- ⁇ crystalloid solutes for example sodium chloride, bicarbonate, glucose
- ⁇ surfactants for example polysorbates.
- the subject of the invention is a pharmaceutical composition
- a pharmaceutical composition comprising as active substance at least:
- a monoclonal antibody (recombinant and / or isolated and purified) as described above, said monoclonal antibody being at a dose of 5 mg to 1000 mg, preferably of 100 mg to 800 mg; and or
- ⁇ a fragment as described above said fragment being at a dose of 5 mg to 1000 mg, preferably 100 mg to 800 mg; and or ⁇ a nucleic acid as described above, said nucleic acid being at a dose of 5 mg to 1000 mg, preferably of 100 mg to 800 mg; and or
- an expression vector as described above said expression vector being at a dose of 5 mg to 1000 mg, preferably 100 mg to 800 mg, in association with a pharmaceutically acceptable carrier.
- the subject of the invention is a pharmaceutical composition
- a pharmaceutical composition comprising as active substance at least:
- said monoclonal antibody (recombinant and / or isolated and purified) as described above being at a dose of from 5 mg to 1000 mg; and or
- said fragment as described above being at a dose of from 5 mg to 1000 mg; and or
- said nucleic acid as described above being at a dose of from 5 mg to 1000 mg; and or
- said expression vector as described above being at a dose of from 5 mg to 1000 mg.
- the expression “dose comprised from 5 mg to 1000 mg” means that the dose may be comprised from 50 mg to 900 mg, from 100 mg to 800 mg, from 200 mg to 700 mg, from 300 mg to 600 mg, 400 mg to 500 mg or 5 mg to 500 mg. It also means that the dose can be 5 mg, 10 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg , 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg or 1000 mg.
- the subject of the invention is a pharmaceutical composition as described above, for its use in the early treatment and / or prevention of relapse of outbreaks of granulomatosis with polyangiitis.
- the aim of the invention is to provide a tool for early treatment and / or prevention of relapse in the context of granulomatosis with polyangiitis, which is severe systemic necrotizing vasculitis, ie inflammation with necrosis of the vascular walls, the expression " early treatment and / or prevention of relapse ”is understood within the meaning of the invention as being the use of the antibody, of the antibody fragment, of the nucleic acid or of the expression vector of the.
- “early” within the meaning of the invention is understood to mean the use of the antibody, of the antibody fragment, of the nucleic acid or of the expression vector of the invention as described above from that the diagnosis of the pathology is made, said diagnosis being able in particular to be carried out by searching for biological markers such as ANCA autoantibodies in a blood test for example.
- a relapse within the meaning of the invention is understood to be the reappearance of the clinical signs of the pathology (see above) after the use of a suitable treatment (eg the antibody of the invention. ) and which worked following the first appearance of the pathology.
- a suitable treatment eg the antibody of the invention.
- the diagnosis of these relapses is therefore generally faster to make, the disease being already known.
- the expression “early treatment and / or prevention of relapse” can be understood as being the use (ie the establishment of a one-off or prolonged therapy) of the antibody, antibody fragment, nucleic acid or expression vector of the invention as described above in a patient in the the first two (2) to four (4) weeks following the appearance of the first clinical signs of the pathology and / or of a relapse.
- the subject of the invention is a pharmaceutical composition for its use as described above, said pharmaceutical composition further comprising anti-inflammatory drugs, such as corticosteroids, and / or immunosuppressants.
- anti-inflammatory drugs such as corticosteroids, and / or immunosuppressants.
- the subject of the invention is a pharmaceutical composition for its use as described above, in which:
- said active substance is a monoclonal antibody (recombinant and / or isolated and purified) as described above, said monoclonal antibody being at a dose of 1 mg / kg to 50 mg / kg, preferably of 5 mg / kg to 25 mg / kg; and or
- said active substance is a fragment as described above, said fragment being at a dose of 1 mg / kg to 50 mg / kg, preferably of 5 mg / kg to 25 mg / kg; and or
- said active substance is a nucleic acid as described above, said nucleic acid being at a dose of 1 mg / kg to 50 mg / kg, preferably of 5 mg / kg to 25 mg / kg; and or
- said active substance is an expression vector as described above, said expression vector being at a dose of 1 mg / kg to 50 mg / kg, preferably of 5 mg / kg to 25 mg / kg.
- the expression “dose comprised from 1 mg / kg to 50 mg / kg” means that the dose may be comprised from 5 mg / kg to 45 mg / kg, from 10 mg / kg to 40 mg. / kg, from 15 mg / kg to 35 mg / kg, from 20 mg / kg to 30 mg / kg, from 25 mg / kg to 50 mg / kg or from 1 mg / kg to 10 mg / kg.
- the dose can be 1 mg / kg, 2.5 mg / kg, 5 mg / kg, 7.5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg or 50 mg / kg.
- the subject of the invention is a unitary pharmaceutical composition for its use as described above, in which:
- said active substance is a monoclonal antibody (recombinant and / or isolated and purified) as described above, said monoclonal antibody being at a dose of 70 mg to 3500 mg per unit dose (based on a 70 kg man), preferably from 1000 mg to 2500 mg per unit dose (based on a 70 kg man); and or
- said active substance is a fragment as described above, said fragment being at a dose of 70 mg to 3500 mg per unit dose (based on a 70 kg man), preferably of 1000 mg to 2500 mg per unit dose (based on a 70 kg man); and or
- said active substance is a nucleic acid as described above, said nucleic acid being at a dose of 70 mg to 3500 mg per unit dose (based on a 70 kg man), preferably of 1000 mg to 2 500 mg per unit dose (based on a 70 kg man); and or
- said active substance is an expression vector as described above, said expression vector being at a dose of 70 mg to 3500 mg per unit dose (based on a 70 kg man), preferably of 1 000 mg to 2500 mg per unit dose (based on a 70 kg man).
- dose from 70 mg to 3500 mg per unit dose means that the dose may be from 100 mg to 3000 mg, from 500 mg to 2500 mg, from
- the dose can be 70 mg, 100 mg, 500 mg, 1000 mg, 1500 mg,
- the subject of the invention is a pharmaceutical composition for its use as described above, said composition being formulated to be administered by one of the following routes: oral, parenteral, injectable, topical , by inhalation, subcutaneous, nasal or pulmonary.
- the subject of the invention is a method of early treatment and / or prevention of early relapse of outbreaks of granulomatosis with polyangiitis of a patient suffering from granulomatosis with polyangiitis comprising administration to an effective dose of at least:
- nucleic acid as described above; and or an expression vector as described above.
- the latter relates to an antibody / antigen immune complex, in which:
- said antibody is the monoclonal antibody (recombinant and / or isolated and purified) as described above or a fragment as described above;
- neutrophil proteinase 3 represented by the sequence SEQ ID NO: 1.
- a subject of the invention is also a diagnostic kit for assaying the blood concentration of neutrophil proteinase 3, said diagnostic kit comprising a monoclonal antibody (recombinant and / or isolated and purified) as described above or a fragment such as as described above.
- the invention also relates to an in vitro diagnostic method comprising a step of determining the immune antibody / antigen complex as described above using a monoclonal antibody (recombinant and / or isolated and purified) as described above or of a fragment as described above, said dosage making it possible to determine the degree of severity and / or the risk of relapse of outbreaks of granulomatosis with polyangiitis in a patient.
- VAS B score i.e. score used to quantify the severity of the pathology
- MFI measurement of fluorescence
- Figure 1 Identification and characterization of the human anti-proteinase 3 (PR3) 4C3 monoclonal antibody.
- B. Verification of the specificity of 4C3 and 5D11 in response to different antigens indicated on the graph by ELISA (n 5).
- C. Determination of the IgG subclass of 4C3 by ELISA in comparison with a GPA patient serum (ANCA) containing different subclasses of anti-PR3 IgG (n 3).
- D. Determination of the kappa (in black) or lambda (in white) light chain of 4C3 in comparison with the serum of a GPA patient (n 3).
- B Production of clone 4C3 in the supernatants of high density flasks (PRO 01-17 and PRO 02-18) and change in the number of cells harvested during the PRO 01-17 (left graph) and PRO 02- productions. 18 (right graph).
- C Evaluation of the level of anti-PR3 IgG production by ELISA during the productions PRO 01-17 (left graph) and PRO 02-18 (right graph).
- D Purification of 4C3 by affinity chromatography (HiTrap TM Protein A). The purity of the different fractions was checked by SDS-PAGE and staining with Coomassie Blue.NR: Not retained; E: Elutions; Mq: Size marker; Avt: Sample before purification; Witness: Purified IgG1 Ab.
- E Purification of 4C3 by affinity chromatography (HiTrap TM Protein A). The purity of the different fractions was checked by SDS-PAGE and staining with Coomassie Blue.NR: Not retained; E: Elutions; Mq: Size marker; Avt
- Figure 4 Modeling and validation of the PR3 epitope recognized by 4C3.
- PR3 Recognition of PR3 by 4C3 by western blot: PR3 at different concentrations and in native or denatured form was deposited on 4-12% Bis-Tris polyacrylamide gel then transferred to a nitrocellulose membrane and saturated with 5% of BSA. Incubating the membrane with the 4C3 antibody diluted to 1/10 000 overnight at 4 ° C with stirring. Revelation of proteins with an anti-human Fc secondary antibody coupled to HRP.
- B In silico modeling of the PR3 epitope recognized by 4C3 by the MabTope method.
- C Amino acid sequence of PR3 (SEQ ID NO: 42) with the tested validation peptides underlined. The probability of the different residues of the epitope is indicated.
- D Recognition of PR3 by 4C3 by western blot: PR3 at different concentrations and in native or denatured form was deposited on 4-12% Bis-Tris polyacrylamide gel then transferred to a nitrocellulose membrane and saturated with 5% of BSA. Incubating the membrane with the 4C3 antibody
- IRSTLR N-terminal sequence of active native PR3
- PR3 (10nM) was or was not incubated with 4C3, 6H4 or GaIAT (50nM) for 30 minutes, ie a ratio of 5: 1, then the fluorescent substrate for PR3 was added. Enzymatic activity was measured during kinetics by spectrofluorimetry. The negative control (ctl-) corresponds to the substrate alone.
- B Representation of the difference in activity of PR3 expressed in ARFU. A representative experience of 5 is shown.
- Figure 6 Labeling of 4C3 on the surface of immune cells by flow cytometry.
- Figure 7 Cytoplasmic labeling of cANCA-type 4C3-AF488.
- Neutrophils purified from healthy donors were fixed with ethanol (top row) or formalin (bottom row) before being labeled with the nuclear marker DAPI ( 1st column) and 4C3-AF488 (1/100 th ) (2 nd column). Reading the slides under a fluorescence microscope with the objective x60. Overlay of fluorescence ( "Merged” 3rd column) using ImageJ software. The scale (10 microns) is shown on images in phase contrast ( "Brightfield", 4 th column). An experiment representative of the 3 carried out is shown. Figure 8: 4C3 specifically recognizes purified native PR3 and PR3 contained in neutrophils.
- Neutrophils from a GPA patient were purified and then pre-activated (TNFa +) or not (TNFa -) with TNFa at 2 ng / mL for 15 minutes at 37 ° C. 10 ⁇ g of neutrophils (wells 1 and 2) were deposited under reduced condition. HeLa cells having no PR3 were used as negative control (3rd well) and human native PR3 purified as a positive control (4 wells th). Incubation of the membranes with the antibody Hsc70 (1/10 000) or 4C3 antibody (1/10 000) overnight under stirring at 4 ° CB The 4C3 does not set the proteases elastase and cathepsin G (Cat G) but only PR3 at 5 pg and 2 pg. Incubation of 4C3 overnight at 4 ° C.
- Figure 9 4C3 induces the expression of PR3 on the surface of pre-activated neutrophils compared to an unrelated antibody.
- the membrane expression of PR3 (mbPR3) was analyzed by flow cytometry after labeling with the anti-PR3 antibody WGM2-FITC. The mean as well as the standard deviations for healthy donors are shown. The results were normalized against the TNFa condition and are expressed as a ratio of the mean fluorescence intensity (MFI). * p ⁇ 0.05.
- FIG. 10 4C3 does not induce the production of reactive oxygen species (ROS) by human neutrophils.
- ROS reactive oxygen species
- Neutrophils from healthy donors were incubated with cytochelasin B (5 pg / mL) 5 minutes at 37 ° C then sodium azide (2 mM) and DHR (2.5 pM) were added 5 minutes at 37 ° C before pre-activating the neutrophils with TNFa at 2 ng / mL at 37 ° C for 15 minutes (triangle).
- the production of ROS was analyzed by flow cytometry by measuring the MFI after labeling with DHR 123. The mean as well as the standard deviations for the healthy donors are shown. The results were normalized against the TNFa condition and are expressed as a ratio of MFI. ns: not significant; * p ⁇ 0.05; ** p ⁇ 0.005.
- Figure 12 4C3 and r4C3 do not induce an increase in cathepsin G activity by pre-activated neutrophils.
- the degranulation of the neutrophils was evaluated by the expression of CD63 represented as a percentage of positive cells. The results are expressed as the mean ⁇ SEM obtained in eight independent experiments, each circle representing one experiment.
- Neutrophils from healthy donors were pre-activated with TNF ⁇ at 2 ng / mL at 37 ° C for 15 minutes (left histogram), then were incubated for 90 minutes at room temperature with F AcMo 4C3 and F AcMo r4C3 at 2 and 20 pg / mL or with PMA-ICa.
- the activity of cathepsin G was measured in the neutrophil activation supernatants after addition of its substrate and reading by spectrofluorimetry. A representative experience of 3 is shown. The results are expressed in ARFU (Relative Fluorescence Units).
- Figure 13: 4C3 does not increase the adhesion phenotype of human neutrophils.
- a and B Purified neutrophils from eight independent healthy donors were primed with TNF ⁇ (2 ng / mL) for 15 minutes at 37 ° C (white columns) before being incubated for 45 minutes with 4C3 (gray columns) , separate (unmixed) IgG preparations from two healthy donors (hatched columns) or four GPA patients active at the time of diagnosis (gridded columns) or IgG from patient P2 (vertical lines).
- the neutrophil adhesion criterion was evaluated by measuring the surface expressions of CD 11b (A) and CD 18 (B) by flow cytometry. The results are expressed as the mean ⁇ SEM obtained in eight independent experiments, each circle representing one experiment.
- NS not significant; * p ⁇ 0.05; ** p ⁇ 0.005; *** p ⁇ 0.0005.
- Figure 14 SDS - PAGE analysis of the different forms of 4C3 obtained.
- Figure 15 4C3 derivatives do not increase PR3 expression and do not induce ROS production by pre-activated neutrophils.
- the membrane expression of PR3 (mbPR3) was analyzed by flow cytometry after labeling with the 4C3-AF488 antibody. An experiment representative of the 3 carried out is shown.
- n 1.
- the production of ROS was analyzed by flow cytometry by measuring the MFI after labeling with DHR 123. The mean as well as the standard deviations for the healthy donors are shown. The results were normalized against the TNFa condition and are expressed as a ratio of MFI.
- NS not significant.
- Figure 17 SDS electrophoresis - PAGE analysis of the different fractions obtained after purification of a GPA patient serum on G protein.
- the left (# 1) and right (# 15) wells correspond to the molecular weight (MW) marker in kDa. Representative image of the 6 experiments carried out.
- IgG purified from GPA patient serum contains anti-PR3 IgG unlike IgG purified from healthy donor serum.
- the IgGs contained in the serum of healthy donors and GPA patients were purified and then the anti-PR3 IgG assay was carried out by ELIS A using the Euroimmun TM kit. Internal kit controls (negative control / positive control / 20 UR / mL calibrator) were used in comparison with AcMo 4C3 at 50 pg / mL and unpurified sera. An experiment representative of the 6 carried out is shown.
- FIG. 19 The purified IgG induces an increase in the intracellular production of ROS by the neutrophils of healthy donors.
- the production of ROS was analyzed by flow cytometry by measuring the MFI after labeling with DHR 123. The mean as well as the standard deviations are shown. The results were normalized against the TNFa condition and are expressed as a ratio of MFI. ns: not significant; * p ⁇ 0.05; ** p ⁇ 0.005; *** p ⁇ 0.0005.
- Figure 20 4C3 is able to inhibit the intracellular production of ROS of human neutrophils induced by the presence of IgG GPA +.
- Neutrophils from healthy donors were incubated with cytochelasin B (5 pg / mL) 5 minutes at 37 ° C then sodium azide (2 mM) and DHR (2.5 pM) were added 5 minutes at 37 ° C before pre-activating neutrophils with TNF ⁇ at 2 ng / mL at 37 ° C for 15 minutes.
- the neutrophils were incubated for 15 minutes at 37 ° C with the mAb 4C3 at 20 pg / mL before adding for 35 minutes at 37 ° C IgG from patients with GPA to 200 pg / mL.
- the 4C3 clone comes from a patient suffering from granulomatosis with polyangiitis (GP A) and followed in consultation in the pneumology department of the Regional and University Hospital Center (CHRU) of Tours.
- This patient was diagnosed with GPA in 2011 following Otorhinolaryngological (ENT) and pulmonary disorders as well as alveolar hemorrhage.
- This patient received a first 6-month treatment with Endoxan TM followed by treatment with corticosteroids and methotrexate.
- the last biological examination at the time of collection indicated a level of circulating B lymphocytes of 72 / mm 3 and a level of autoantibodies directed against proteinase 3 (cANCA) greater than 177 IU / mL.
- cANCA proteinase 3
- the patient agreed to participate, after informed consent, in a collection of human biological samples declared to the Ministry of Research (No. DC-2012-1636) in accordance with decree No. 2007-1120 of August 10, 2007.
- ACD anti-coagulant dextrose citrate
- a kit marketed by the company Dendritics TM (DDXK-HuBBB TM kit) was used, which uses the EBV immortalization technique. (Epstein Barr Virus).
- EBV immortalization technique Epstein Barr Virus
- the protocol recommended by this company has been optimized by the inventors and is different from that described in the kit.
- the kit is used on fresh total Peripheral Blood Mononuclear Cell (PBMC) (from a daily blood sample) and requires a first immortalization step then subcloning steps to obtain monoclonal cells.
- PBMC Peripheral Blood Mononuclear Cell
- the first step made it possible to isolate the total B lymphocytes by negative selection (“B-cell isolation kit II” from Miltenyi Biotec TM, Ref. 130-091-151) then to sort specifically with the MoFlo TM Astrios sorter the CD19 + CD27 + memory B lymphocytes.
- B-cell isolation kit II from Miltenyi Biotec TM, Ref. 130-091-151
- MoFlo TM Astrios sorter the CD19 + CD27 + memory B lymphocytes.
- Different co-culture conditions were tested in P96 flat bottom: PBMC alone, PBMC enriched in LB memories, PBMC depleted in LB and enriched in LB memories with different quantities of cells
- the cells were stimulated and immortalized in the presence of EBV viral particles using the kit marketed by Dendritics TM (DDXK-HuBBB TM kit).
- D9 9 days
- the 4C3 clone was amplified by successive passages of the cells in wells P24 then in wells P6 and finally in flasks in order to increase the number of cells.
- the monoclonality of this clone was confirmed by Polymerase Chain Reaction (PCR) by studying the rearrangements of the heavy chain genes of immunoglobulins (IgH) (FIG. 2A).
- the 4C3 clone was then put into production in a high density flask (CELLine TM Wheaton TM) composed of two compartments separated by a semi-permeable membrane which allows proteins of less than 10 kDa to pass.
- the first compartment contains the nutritive extracellular medium composed of DMEM supplemented with penicillin / streptomycin and glutamine but without horse serum while the second compartment contains the cells in which the antibodies produced every three to four days over a period of time were harvested. 'at least 70 days.
- Two productions were carried out (experiments PRO 01-17 and PRO 02-18) during which the number of cells (FIG. 2B) as well as the level of production of anti-PR3 IgG (FIG. 2C) were verified.
- the sequences of the variable regions specific to PR3 were subcloned into recombinant IgG expression vectors (vectors marketed).
- the expression vectors thus constructed were transfected into mammalian cells HEK-293 (ATCC ® CRL-1573 TM) to produce monoclonal IgG isotype IgG form secreted into the culture supernatant.
- the r4C3 antibody was purified as previously described and made it possible to obtain a final r4C3 concentration of 3.5 mg / mL.
- sequences SEQ ID NOs: 6 to 19 and the same light chain differ only in the constant region of the heavy chain by the mutation
- the identification of the epitope recognized by the 4C3 antibody was carried out by the company MabSilico.
- the first step made it possible to model the epitope of PR3 by the method
- MabTope which consists of the computer prediction of an ordered list of peptides of the
- neutrophils were purified from the blood of healthy donors from the French Blood Establishment (EFS) using a commercial kit (Stem Cell kit, EasySep TM Direct Human Neutrophil Isolation; Kit reference 19666) ( Figure 6A). The unstimulated neutrophils were incubated for 30 minutes at 4 ° C. with the 4C3 antibody previously coupled with the fluorochrome AF488 (kit sold ThermoFisher TM).
- Neutrophils purified from healthy donors were previously fixed with formalin or ethanol and then labeled with 4C3 Ac coupled to FAF488 and DAPI (nuclear labeling). Analysis of the slides under a fluorescence microscope revealed diffuse cytoplasmic labeling of the cANCA type of 4C3 ( Figure 7).
- the 4C3 was used as primary antibody in Western blot ( Figure 8A and 8B) on protein neutrophil lysates of HeLa cells (ATCC CCL-2 ® TM) and human PR3.
- Figure 8A protein neutrophil lysates of HeLa cells
- Figure 8B protein neutrophil lysates of HeLa cells
- 4C3 did recognize human PR3 with labeling at the expected size, labeling also present with neutrophils and no labeling was detected in HeLa cells, which do not express PR3 ( Figure 8A).
- 4C3 did not recognize other proteases such as elastase and cathepsin G which are the same size as PR3 ( Figure 8B). This result therefore confirmed the fact that the 4C3 antibody is specific for PR3.
- Pepsin is a proteolytic enzyme which allows the cleavage of the antibody just below the disulfide bridges which connect the two heavy chains and to obtain an F (ab ') 2 fragment (Table 4 below).
- Papain is a cysteine protease capable of cleaving the antibody above the hinge region which will give two distinct Fab fragments on the one hand and Fc fragments on the other hand (Table 5 below). DIGESTION OF 4C3 BY PEPSIN
- Table 4 List of samples deposited by track
- IgG purified from unmixed sera were electrophoresed on a 4-12% Bis-tris gel before staining with Coomassie blue.
- the presence of PR3-ANCA in separate preparations of IgG from patients in active phase of GPA and from patient P2 was confirmed by ELISA using a Euroimmun anti-PR3 kit, whereas the separate preparations of GPA Purified IgGs from healthy donors did not contain PR3-ANCA.
- Table 6 Main characteristics of active GPA patients whose IgGs have been purified to induce autoimmune neutrophil activation
- Activation of neutrophils from healthy independent donors was assessed by production of ROS using a dihydrorhodamine 123 assay (DHR 123).
- DHR 123 dihydrorhodamine 123 assay
- Purified neutrophils were suspended in HBSS with 1 mM Ca 2+ and 1 mM Mg 2+ and incubated with 5 ug / ml of cytochalasin B (Cayman Chemical ®, USA) to increase the production of radicals of oxygen, for 5 minutes at 37 ° C.
- the cells were then loaded with 2 mM DHR 123 and 2 mM sodium azide (NaN3) for 5 minutes at 37 ° C with stirring.
- Primed neutrophils were incubated with 4C3 (2 to 100 pg / mL), r4C3 or separate IgG preparations from two healthy donors and four active GPA patients (200 pg / mL) for 45 minutes at 37 ° vs.
- An irrelevant antibody (6H4, IgGlK, anti-ovalbumin) was used as a negative control.
- the reaction was stopped with ice cold EDTA PB S (1 mM) before measuring the fluorescence of DHR 123 by flow cytometry.
- pretreated, award-winning neutrophils were first incubated with 4C3 (20 pg / mL) for 15 minutes at 37 ° C, followed by separate IgG preparations from five patients active at the time of diagnosis.
- GPA IgG GPA, 200 ⁇ g / mL
- Neutrophils were pre-activated with TNF ⁇ and stimulated with 4C3 (2 and 20 pg / mL), r4C3 (2 and 20 pg / mL), IgG from GPA patients (200 pg / mL) or PMA-ICa for 45 minutes at 37 ° C. After incubation, the cells were washed and labeled with CD63 coupled to FITC (degranulation) or labeled with CD 11b VioBlue / CD18 FITC antibodies (BD Biosciences ® , USA) (adhesion phenotype) for 20 minutes at 4 ° C. before being analyzed by flow cytometry.
- the percentage of positive cells and the mean fluorescence intensity (MFI) were determined using the FlowJo ® software. Neutrophil degranulation was also assessed by release of CatG. Supernatants were harvested and incubated with fluorescent cathepsin G substrate (ABZ-TPFSGQ-YN02 from GeneCust ® ) (Attucci S, et al. Measurement of free and membrane-bound cathepsin G in human neutrophils using new sensitive fluorogenic substrates. Biochem J. 2002 Sep 15; 366 (Pt 3): 965-70. Doi: 10.1042 / B J20020321.
- the first test consisted of evaluating the expression of membrane PR3 after incubation of the neutrophils with 4C3 (FIG. 9).
- the 6H4 antibody which is an unrelated antibody was used in comparison in the different tests and the PMA - Ionomycin calcium (PMA-ICa) solution was used as a positive control for the activation of neutrophils.
- the purified human neutrophils were pre-activated with TNF ⁇ for 15 minutes at 37 ° C before being incubated for 1 h 30 min with different concentrations of 6H4 and 4C3 antibodies (2, 20 and 100 pg / mL)
- the expression of membrane PR3 was analyzed by flow cytometry with the WGM2 antibody (murine anti-human PR3 antibody) ( Figure
- the third test consisted in analyzing the degranulation of neutrophils by measuring the expression of CD63 at the membrane of the neutrophil and the activity of cathepsin G in the supernatant of the neutrophils after incubation with the antibodies ( Figure 12). The result previously obtained was confirmed since it was reciprocally demonstrated an absence of the expression of CD63 ( Figure 12A) and an absence of release of cathepsin G in the presence of 4C3 or r4C3 at a dose of 2 and 20 pg / mL ( Figure 12B).
- the fourth test consisted of analyzing the expression of CD11b and CD18 (Macl complex) in order to explore the adhesion phenotype of the neutrophils after stimulation with 4C3 (FIG. 13).
- the deposition of the Fab fragment revealed several bands: two bands around 50 kDa, one corresponding to the Fab fragment and the other to the Fc fragment which was not completely removed after purification on resin coupled to protein A and one third band around 25 kDa corresponding to the reduced Fab (6 th pit, Figure 14).
- the deposition of the deglycosylated form revealed two bands: one a little below 150 kDa corresponding to an IgG that has lost its glycosylation site and the other about 50 kDa corresponding to the Fc fragment (5 th pit, Figure 14).
- the IgG purified from GPA patients and the IgG purified from healthy donors induced significantly greater ROS production than for the TNF ⁇ condition and the 4C3 condition ( Figure 19). This result therefore demonstrated that the IgG purified from GPA patients can be used as activator of neutrophils in order to test the potential neutralizing power of 4C3 and of the other derivatives.
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FR1911722A FR3102175B1 (fr) | 2019-10-18 | 2019-10-18 | Nouveaux anticorps monoclonaux therapeutiques humains et leurs utilisations |
PCT/EP2020/079208 WO2021074376A1 (fr) | 2019-10-18 | 2020-10-16 | Nouveaux anticorps monoclonaux therapeutiques humains et leurs utilisations |
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