Classifications

• • 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
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
  • C07K16/241—Tumor Necrosis Factors
• • 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
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/565—Complementarity determining region [CDR]
• • 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 invention relates to variants of adalimumab with reduced immunogenic potential and preserved or increased affinity and their therapeutic applications.
• Adalimumab is now widely used in the treatment of ankylosing spondylitis, rheumatoid arthritis, ulcerative colitis, psoriatic arthritis, Crohn's disease, skin psoriasis and juvenile arthritis (Feldmann , M. & Maini, Nat. Med. 9, 1245-1250 (2003)).
• Adalimumab is, however, immunogenic in a significant proportion of patients (up to 50% for certain conditions) who produce antibodies directed against the therapeutic protein (ADA for anti-drug antibody), which can be detected in the serum (Strand, V. et al., BioDrugs 31, 299-316 (2017)). ADAs cause the formation of immune complexes that accelerate the elimination of therapeutic antibodies.
• ADA inversely correlated with the response to treatment. Patients with a good response to treatment will be those in whom tests do not reveal ADA (van Schouwenburg, PA, Rispens, T. & Wolbink, GJ, Nat. Rev. Rheumatol. 9, 164-172 (2013)) .
• de-immunization The removal of T epitopes by disrupting the interaction with HLA II molecules, called de-immunization, has been shown as an effective method for reducing the immunogenicity of various proteins for therapeutic purposes, such as enzymes and immunotoxins (Mazor, R. et al. Oncotarget 7, 29916-29926 (2016); Cantor, JR et al., Proc. Natl. Acad. Sci. 108, 1272-1277 (2011); Ettinger, RA et al. , Blood Adv. 2, 309-322 (2016); Mazor, R. et al., Proc. Natl. Acad. Sci. USA 109, E3597-603 (2012)).
• T epitopes have previously been identified in the sequence of adalimumab (Meunier, S. et al., Cellular & Molecular Immunology, 2019, Oct 28. doi: 10.1038 / s41423-019-0304-3). These are mainly located on the heavy chain of the antibody on which they are divided into two regions. The first region concentrates the majority of T epitopes and is very overlapping with CDR-H3 (residues L82c to T107 according to Kabat numbering; Figure 1).
• the second region is located at CDR-H2 and has two T epitopes ( residues E46 to E64 according to the Kabat numbering; Figure 1) These T epitopes are potentially responsible for the immunogenicity of adalimumab. As this has significant repercussions for patients, the development of alternatives that are less immunogenic than adalimumab seems a necessity.
• variants of adalimumab in which the framework regions (FR) of the variable domains of the heavy and light chains have been replaced by less immunogenic framework regions of other human immunoglogulins G (IgG) are described in Application EP 3 178847. Such variants do not have a de- satisfactory immunization for therapeutic use since they comprise the majority of T epitopes present in CDRs.
• Adalimumab variants comprising mutations in a region containing putative CD4 T epitopes extending on either side of CDR1 of the light chain (CDR-L1; positions C23 to K45, according to the numbering of Kabat) are described in Application WO 2010/121140.
• CDR-L1 positions C23 to K45, according to the numbering of Kabat
• all the variants obtained have a reduced affinity for TNF-alpha compared to adalimumab, this reduction in affinity being drastic (at least 50%). for the majority (70%) of the variants obtained.
• the inventors have identified mutations in the immunogenic regions overlapping the CDR-H2 (or CDRH2) and CDR-H3 (or CDRH3) regions of adalimumab which reduce immunogenicity while retaining the affinity of binding to TNF -alpha. From combinatorial libraries, they isolated variants which surprisingly exhibit a reduced immunogenic potential and a TNF-alpha binding affinity greater than that of adalimumab. Some of the variants have an affinity 5 to 50 times that of adalimumab. Since the biological activity of anti-TNF antibodies - namely the neutralization of TNF-alpha - depends on their affinity for TNF, the variants of the invention can be expected to have a higher biological activity. to that of adalimumab.
• the present invention relates to a variant of an anti-TNF-alpha therapeutic antibody comprising variable domains VFI and VL of sequences SEQ ID NO: 1 and SEQ ID NO: 2, said variant comprising at least two amino acid substitutions in at least one sequence overlapping one of the CDRH2 or CDRH3 regions determining the complementarity of said VH variable domain; said at least two substitutions of amino acids in the sequence overlapping the CDRH2 region being selected from the group consisting of:
• T52 by another amino acid chosen from A, N or S; preferably N or S;
• T100 by another amino acid chosen from P or S; excluding variants comprising residues V89 or L89, V95, K96, Y97, L98, P99 and S100; V89, V95, A96, Y97, L98, P99 and S100; the positions of said amino acid residues being indicated with reference to the Kabat numbering; and said variant exhibiting a reduced immunogenic potential and a binding affinity for TNF-alpha at least equal to or greater, with respect to the anti-TNF-alpha therapeutic antibody from which it is derived.
• said variant comprises at least 3 substitutions in one of the sequences overlapping the CDRFI2 or CDRFI3 region; preferably in each of said sequences overlapping the CDRFI2 and CDRH3 regions.
• said variant comprises a combination of substitutions in the sequence overlapping the CDRFI2 region chosen from: - S54G and I57R;
• I57Q or I57N optionally A50T, A50G or A50S;
• said variant comprises a combination of substitutions in the sequence overlapping the CDRH2 region chosen from:
• said variant comprises a combination of substitutions in the sequence overlapping the CDRH3 region chosen from:
• said variant comprises a combination of substitutions in the sequence overlapping the CDRH3 region chosen from:
• V89L, V95T, S96K and S99P V95T, S96K, S99P and T100S; V95T, S96R L98T and S99P; V89L, V95T, S96R and S99P; V89L; V95T, S96T and S99P;
• V95T, S96K, S99P and T1 OOS V89L, V95T, S96R, S99P and T1 OOS.
• said variant comprises a combination of substitutions in the sequence overlapping the CDRH3 region chosen from: V95T, S96T and S99P; V95T, S96N and S99P; V95S, S96Q and S99P; V95A, S96H and S99P; V95T, S96G and S99P; S96T, L98T, S99P and T100S; V95T, S96R, L98T and S99P; S96K, S99P and T100P; V89L, V95T, S96T and S99P; preferably chosen from: V95T, S96T and S99P; V95T, S96N and S99P; V95S, S96Q and S99P; V95A, S96H and S99P; V89L, V95T, S96T and S99P.
• said variant comprises one of the following combinations of substitutions in the sequences overlapping the CDRH2 and CDRH3 regions:
• said variant further comprises the R90K substitution in the CDRL3 region determining the complementarity of the VL variable domain.
• said variant comprises a human IgG heavy chain and a human Kappa light chain.
• said variant is derived from adalimumab.
• said variant comprises a light chain of sequence SEQ ID NO: 2 or 32 and a heavy chain of sequence SEQ ID NO: 24 to 31.
• Another aspect of the invention relates to an expression vector comprising a polynucleotide encoding a variant according to the invention.
• Another aspect of the invention relates to a pharmaceutical composition
• a pharmaceutical composition comprising at least one variant according to the invention or a vector according to the invention, and a pharmaceutically acceptable vehicle and / or a carrier substance.
• Another aspect of the invention relates to a composition according to the invention, for use in the treatment of an inflammatory or autoimmune disease.
• the present invention relates to a variant of an anti-TNF-alpha therapeutic antibody comprising VH and VL variable domains of sequences SEQ ID NO: 1 and SEQ ID NO: 2, which variant comprising a reduced immunogenic potential and a binding affinity for TNF-alpha at least equal to or greater, relative to the anti-TNF-alpha therapeutic antibody from which it is derived. Due to its binding affinity at least equal or greater, one can expect that the variant according to the invention has a biological activity at least equal to or greater than that of the parent antibody.
• sequence SEQ ID NO: 1 corresponds to the sequence of the heavy chain variable domain (VH) of adalimumab and the sequence SEQ ID NO: 2 to the sequence of the light chain variable domain (VL) of the adalimumab ( Figure 1).
• sequence of the heavy chain of adalimumab corresponds to the sequence SEQ ID NO: 3
• sequence of the light chain of adalimumab corresponds to the sequence SEQ ID NO: 4.
• therapeutic antibody is understood to mean a human or humanized antibody.
• antibody means a whole antibody, an antibody fragment comprising at least the domain of binding to the antigen or a molecule derived from an antibody.
• TNF-alpha or TNFalpha (“Tumor Necrosis Factor Alpha”) is meant a multifunctional pro-inflammatory cytokine produced predominantly by monocytes and macrophages. Preferably, it is human TNF alpha.
• Human TNF alpha corresponds to the GenBank sequence QCI55793.1.
• immunogenic potential reduced relative to an antibody variant according to the invention means a decrease in the number of HLA II molecules which can be linked by at least one of the CD4 T epitopes of the variant compared to the parent antibody of which it is. issued.
• the number of HLA II molecules which can be bound by a variant according to the invention is evaluated according to standard techniques known to those skilled in the art such as those described in particular in the examples. These include in silico methods using MHC-II binding prediction tools, such as the netMHCIIpan 3.2 algorithm. (Jensen, K. K. et al., Immunology 154, 394-406 (2016)). Binding to HLA II is expressed as a score defined by the following formula:
• / is the first anchor position of the core and y is the allele for which the core is predicted.
• the variant according to the invention is characterized by an HLA II binding score reduced by at least 10% (1.1 times or factor 1.1) relative to the parent antibody from which it is derived.
• the binding affinity of the variant to TNF-alpha is evaluated according to standard techniques known to those skilled in the art such as those described in particular in the examples.
• Affinity can be evaluated by the value of the equilibrium dissociation constant KD of the variant, measured by conventional techniques as described in the examples.
• Affinity can also be evaluated by the relative enrichment factor of the variant with respect to the parent antibody which corresponds to the ratio between the enrichment values of the variant and of the parent antibody as defined in the examples.
• the variant according to the invention is characterized by a relative enrichment factor greater than or equal to 5 or a KD at least 1.1 times less (less by a factor of 1, 1 or 10%) relative to the antibody parent he is from.
• the term “individual” means a human or animal individual, preferably a human individual.
• Amino acids are shown in a one-letter code.
• a means at least one, and or means and / or.
• said variant comprises at least two substitutions of amino acids in at least one sequence overlapping one of the CDRH2 or CDRH3 regions determining the complementarity of said VH variable domain.
• the sequence overlapping the CDRH2 region extends from residues E46 to E64 according to the Kabat numbering (SEQ ID NO: 8).
• the sequence overlapping the CDRH3 region extends from residues V89 to G104 according to the Kabat numbering (SEQ ID NO: 9).
• Said variant advantageously comprises at least two substitutions in each of the two sequences overlapping the CDRH2 or CDRH3 region.
• said variant comprises at least 3 substitutions, generally 3, 4 or 5 substitutions in one of the overlapping sequences; preferably, in each of the two sequences overlapping the CDRH2 and CDRH3 regions.
• said at least two substitutions of amino acids in the sequence overlapping the CDRH2 region are selected from the group consisting of:
• the variants according to the invention are functional variants, that is to say they comprise a reduced immunogenic potential and a binding affinity to TNF-alpha at least equal or greater, relative to the antibody anti-TNF-alpha therapy from which it is derived.
• the invention excludes non-functional variants such as in particular variants comprising only two substitutions chosen from T52N and A50G or T52N and I57T.
• - T52 is substituted by N or S;
• - 157 is substituted by A, H, S, N, Q, R, T, preferably R, H, S or T.
• said substitutions in the sequence overlapping the CDRH2 region are chosen from the substitutions in positions S49, A50, T52, S54, H56 and I57.
• said substitutions are at positions S54 and I57; S49, T52 and I57; S49, S54 and I57; T52, S54 and I57; S49, A50, T52 and I57; S49, A50, S54 and I57; S49, T52, S54 and I57; A50, T52, S54 and I57; or S49, A50, T52, S54 and I57; preferably at positions S49, T52 and I57; S49, S54 and I57; T52, S54 and I57; S49, A50, S54 and I57; S49, T52, S54 and I57; A50, T52, S54 and I57; or S49, A50, T52, S54 and I57.
• said variant comprises a combination of substitutions in the sequence overlapping the CDRH2 region chosen from:
• I57Q or I57N optionally A50T, A50G or A50S;
• said variant comprises a combination of substitutions in the sequence overlapping the CDRH2 region chosen from:
• said variant comprises a combination of substitutions in the sequence overlapping the CDRH2 region chosen from:
• Particularly preferred variants according to the invention comprise one of the following combinations of substitutions in the sequence overlapping the CDRH2 region: S49G, T52N, S54G and I57R; S49G, A50T, T52N, S54G and I57S; S49G, T52N, S54G and I57H; S49G, T52N and 157H; S49G, A50D, T52S, S54G and I57T.
• said at least two substitutions of amino acids in the sequence overlapping the CDRH3 region are selected from the group consisting of:
• S96 is substituted by T, Q, N or H.
• said substitutions in the sequence overlapping the CDRH3 region are chosen from the substitutions in positions: V89, V95, S96, Y97, L98, S99 and T100.
• said substitutions are at positions V89, V95, S96 and S99 or V95, S96 and S99.
• said variant comprises a combination of substitutions in the sequence overlapping the CDRH3 region chosen from:
• Said variant advantageously comprises a combination of substitutions in the sequence overlapping the CDRH3 region chosen from:
• V89L, V95T, S96K and S99P V95T, S96K, S99P and T100S; V95T, S96R L98T and S99P; V89L, V95T, S96R and S99P; V89L; V95T, S96T and S99P;
• V95T, S96K, S99P and T100S V89L, V95T, S96R, S99P and T100S.
• said variant comprises a combination of substitutions in the sequence overlapping the CDRH3 region chosen from: V95T, S96T and S99P; V95T, S96N and S99P; V95S, S96Q and S99P; V95A, S96H and S99P; V95T, S96G and S99P; S96T, L98T, S99P and T100S; V95T, S96R, L98T and S99P; S96K, S99P and T1 OOP; V89L, V95T, S96T and S99P.
• Particularly preferred variants according to the invention comprise one of the following combinations of substitutions in the sequence overlapping the CDRH3 region: V95T, S96T and S99P; V95T, S96N and S99P; V95S, S96Q and S99P; V95A, S96H and S99P; V89L, V95T, S96T and S99P.
• said variant comprises at least two substitutions in each of the two sequences overlapping the CDRH2 or CDRH3 region as defined above.
• said variant comprises a combination of substitutions in the sequence overlapping the CDRH2 region and a combination of substitutions in the sequence overlapping the CDRH3 region chosen from the combinations as defined above.
• V95T, S96T and S99P V95T, S96N and S99P; V95S, S96Q and S99P; V95A, S96H and S99P; V89L, V95T, S96T and S99P.
• Examples of particularly preferred variants according to the invention comprise one of the following combinations of substitutions in the sequences overlapping the CDRH2 and CDRH3 regions:
• said variant further comprises the R90K substitution in the CDRL3 region determining the complementarity of the VL variable domain.
• the variant according to the invention comprises a heavy chain of human immunoglobulin of any isotype or class, preferably an IgG, preferably an IgG 1.
• the variant according to the invention also comprises a light chain d human immunoglobulin of any class, preferably human Kappa light chain.
• said variant is derived from adalimumab.
• said variant is selected from the group consisting of:
• a variant of adalimumab comprising a light chain of sequence SEQ ID NO: 2 or 32 and a heavy chain of sequence SEQ ID NO: 24, corresponding to mutant 1 of the examples;
• adalimumab comprising a light chain of sequence SEQ ID NO: 2 or 32 and a heavy chain of sequence SEQ ID NO: 25, corresponding to mutant 2 of the examples;
• a variant of adalimumab comprising a light chain of sequence SEQ ID NO: 2 or 32 and a heavy chain of sequence SEQ ID NO: 26, corresponding to mutant 3 of the examples;
• a variant of adalimumab comprising a light chain of sequence SEQ ID NO: 2 or 32 and a heavy chain of sequence SEQ ID NO: 27, corresponding to mutant 4 of the examples;
• a variant of adalimumab comprising a light chain of sequence SEQ ID NO: 2 or 32 and a heavy chain of sequence SEQ ID NO: 28, corresponding to mutant 5 of the examples;
• a variant of adalimumab comprising a light chain of sequence SEQ ID NO: 2 or 32 and a heavy chain of sequence SEQ ID NO: 29, corresponding to mutant 6 of the examples;
• a variant of adalimumab comprising a light chain of sequence SEQ ID NO: 2 or 32 and a heavy chain of sequence SEQ ID NO: 30, corresponding to mutant 7 of the examples; and a variant of adalimumab comprising a light chain of sequence SEQ ID NO: 2 or 32 and a heavy chain of sequence SEQ ID NO: 31, corresponding to mutant 8 of the examples.
• said variant is characterized by an HLA II binding score reduced by at least 1.5; 2; 2.5; 3; 3.5; 4; 4.5; 5 times or more of the parent antibody from which it is derived.
• said variant is characterized by a relative enrichment factor of at least 10 1 to 10 7 (10 1 , 5.10 1 , 10 2 , 5.10 2 , 10 3 , 5.10 3 , 10 4 , 5.10 4 , 10 5 , 5.10 5 , 10 6 , 5. 10 6 , 10 7 ) or a KD at least 1.2 to 40 times lower (2, 5, 10, 15, 20, 25, 30, 35) relative to the parent antibody from which it is derived.
• the present invention also encompasses variants further comprising at least one additional amino acid mutation (insertion, deletion, substitution) and / or at least one conservative modification of function, that is to say which preserve the reduced immunogenic potential and greater or equal affinity of the variant.
• additional mutations mention may be made in particular of:
• the variant can be modified by the introduction of any conservative modification of function at the level of amino acid residue (s), of the peptide bond or of the ends of the peptides.
• This or these modification (s), in particular one or more chemical modifications which are introduced into the peptides by conventional methods known to those skilled in the art include in particular; fusion of the sequence of said variant with that of a peptide (label useful for the purification of the variant, in particular in form cleavable by a protease) or of a protein of interest, and coupling to a molecule or a agent of interest.
• the antibody can be coupled to a PEG molecule by conventional methods known to those skilled in the art.
• the variant is in the form of a whole antibody, of an antibody fragment comprising at least the domain of binding to the antigen or of a molecule derived from an antibody.
• the whole antibodies can be of any isotype, in particular of human isotype (IgG (lgG1, lgG2, lgG3, lgG4), IgA (lgA1, lgA2), IgE, IgM, IgD).
• the antibody fragments include in particular the Fab, Fab ', F (ab') 2, Fv, scFv, Fabc or Fab fragments comprising a portion of the Fc region and the single chain antibody fragments derived from camelid immunoglobulins or shark (single domain antibody VHH and V-NAR).
• Derived antibody molecules include polyspecific or multivalent antibodies and immunoconjugates.
• multispecific scFvs dia, tria or tetrabodies
• diabodies of the scDb single-chain diabodies
• taFv tandem scFv fragments
• minibodies are in particular of the scFv-HLX type; scFv-ZIP; scFv-CH3; scFv -Fc or other.
• Another aspect of the present invention relates to an isolated polynucleotide encoding a variant in accordance with the invention as defined above.
• Said polynucleotide is DNA, RNA or a mixture of DNA and RNA, recombinant or synthetic.
• the DNA sequence can advantageously be modified so that the use of codons is optimal in the host in which it is expressed.
• Another aspect of the present invention relates to a vector comprising said polypeptide.
• Many vectors are known per se; the choice of an appropriate vector depends on the use envisaged for this vector (for example replication of the sequence of interest, expression of this sequence, maintenance of this sequence under extrachromosomal form, or integration into the chromosomal material of the host), as well as the nature of the host cell.
• naked nucleic acids DNA or RNA
• viral or bacterial vectors can be used.
• the viral vectors are in particular the adenoviruses, the retroviruses, the lentiviruses and the AAVs in which the sequence of interest has been inserted beforehand; it is also possible to combine said sequence (isolated or inserted into a plasmid vector) with a substance allowing it to cross the membrane of host cells, such as a transporter such as a nanotransporter or a preparation of liposomes, or of cationic polymers, or else the 'introducing into said host cell using physical methods such as electroporation or microinjection. In addition, one can advantageously combine these methods, for example by using electroporation associated with liposomes.
• said vector is an expression vector comprising all the elements necessary for the expression of the variant as defined above.
• said vector comprises an expression cassette including at least one polynucleotide as defined above, under the control of appropriate transcriptional and possibly translation regulatory sequences (promoter, activator, intron, initiation codon ( ATG), stop codon, polyadenylation signal, splice site), to allow expression of the variant according to the invention in a host cell.
• transcriptional and possibly translation regulatory sequences promoter, activator, intron, initiation codon ( ATG), stop codon, polyadenylation signal, splice site
• Another aspect of the present invention relates to a prokaryotic or eukaryotic host cell modified with a polynucleotide or a vector in accordance with the invention as defined above, the cell possibly being modified in a stable or transient manner.
• Another aspect of the present invention relates to a pharmaceutical composition
• a pharmaceutical composition comprising at least one derivative variant, polynucleotide, vector, and / or cell as defined above and a pharmaceutically acceptable vehicle and / or a carrier substance.
• Pharmaceutically acceptable vehicles and carrier substances are those conventionally used.
• the carrier substances are advantageously selected from the group consisting of: unilamellar or multilamellar liposomes, ISCOMS, virosomes, viral pseudoparticles, saponin micelles, microspheres solids of a saccharide (poly (lactide-co-glycolide)) or gold-bearing nature, and nanoparticles.
• the pharmaceutical composition may further comprise at least one other therapeutic agent, in particular an anti-inflammatory or immunomodulatory agent.
• the pharmaceutical composition comprises a therapeutically active amount of variant, polynucleotide, vector, cell.
• a therapeutically active amount means a dose sufficient to produce a therapeutic effect on the disease to be treated, that is to say to reduce the symptoms of this disease. This effective dose is determined and adjusted depending on factors such as the age, sex and weight of the subject.
• the pharmaceutical composition according to the invention is in a galenic form suitable for the administration chosen.
• the composition is generally administered according to the usual immunotherapy protocols at doses and for a period sufficient to induce an effective response against the pathology to be treated.
• the administration can be subcutaneous, intramuscular, intravenous, in particular by infusion; intradermal, intraperitoneal, oral, sublingual, rectal, vaginal, intranasal, by inhalation or by transdermal application.
• the composition is in a galenic form suitable for a chosen administration.
• the pharmaceutical composition according to the present invention is used in immunotherapy in the treatment of inflammatory or autoimmune pathologies. It can be used in combination with other therapeutic or surgical treatments, in particular in combination with other therapeutic agents as defined above, the composition according to the invention and the other therapeutic agents which can be administered simultaneously, separate or sequential.
• Inflammatory or autoimmune pathologies are those which are conventionally treated with anti-TNF-alpha.
• pathologies mention may be made of ankylosing spondylitis, rheumatoid arthritis, ulcerative colitis, psoriatic arthritis, Crohn's disease, skin psoriasis and juvenile arthritis.
• a subject of the present invention is also a variant, polynucleotide, vector, and / or cell derived as defined above for use as medicament, in particular in immunotherapy, in the treatment of inflammatory or autoimmune pathologies as defined above.
• a subject of the present invention is also an immunotherapy method, in particular intended for the treatment of inflammatory or autoimmune pathologies as defined above, characterized in that it comprises the administration of an effective dose of a variant, polynucleotide, vector, and / or cell derived in accordance with the invention as defined above, to an individual, by any suitable means as defined above.
• the method comprises the administration of a pharmaceutical composition according to the invention as defined above.
• the polynucleotides according to the invention are obtained by conventional methods, known in themselves. For example, they can be obtained by amplification of a nucleic acid sequence by PCR or RT-PCR or else by total or partial chemical synthesis.
• Recombinant eukaryotic or prokaryotic expression vectors are constructed and introduced into host cells by conventional recombinant DNA and genetic engineering methods, which are known per se. These are in particular the expression vectors conventionally used for the production of antibodies, in particular human or humanized therapeutic antibodies such as vectors of the tandem type allowing the simultaneous expression of the heavy and light chains of antibodies.
• the variants produced by the host cells modified by the recombinant vector are purified by conventional methods of purification of immunoglobulins, in particular by affinity chromatography.
• FIG. 1 shows the sequences of the variable parts of adalimumab.
• the sequences are numbered according to the Kabat nomenclature. Non-germinal residues denote mutations relative to alleles with the highest homology. The residues involved in the direct interaction with TNF ⁇ are derived from the structure published in Hu et al. J. Biol. Chem. 288, 27059-27067 (2013).
• the heavy chain variable domain (VH) corresponds to the sequence SEQ ID NO: 1 and the light chain variable domain (VL) corresponds to the sequence SEQ ID NO: 2.
• FIG. 2 presents the predicted adalimumab T epitopes and interaction cores.
• the T epitopes of adalimumab were identified in vitro by T lymphocyte activation test (data from Meunier et al.) They are mainly localized on the CDR2 and CDR3 of the heavy chain. An isolated epitope has also been identified on the light chain at CDR3.
• the possible interaction cores were predicted by the netMHCIIpan algorithm in gray. The major cores, because predicted by a large number of alleles, are shown in dark gray.
• FIG. 3 presents the prediction of substitutions reducing immunogenicity scores on the interaction with HLA II molecules.
• the netMHCIIpan prediction algorithm is used in a parallel fashion to predict the average effect that each substitution has on peptide binding for HLA class II molecules.
• Fig. 4 presents the prediction of substitutions reducing immunogenicity scores on the interaction with HLA II molecules.
• the netMHCIIpan prediction algorithm is used in a parallel fashion to predict the average effect that each substitution has on peptide binding for HLA class II molecules.
• FIG. 4 presents the expression and selection of Fab libraries by Yeast Surface Display.
• DMS libraries are sorted by rectangular windows based only on PE fluorescence.
• FIG. 5 presents the substitutions of CDRH2 and CDRH3 having no impact on the function of adalimumab by Deep Mutational Scanning. Permissivity matrices of CDRH2 and CDRH3 from positively sorted populations. Mutants having an enrichment greater than or equal to the parental sequence are indicated in shades of gray. The darker the gray, the stronger the enrichment, indicating a good affinity for TNF ⁇ . Sequence of positions E46 to E64 overlapping CDRH2 (SEQ ID NO: 8); Sequence of positions V89 to G104 overlapping CDRH3 (SEQ ID NO: 9).
• FIG. 6 shows the substitutions of CDRH2 and CDRH3 combining lower predicted immunogenicity and maintenance of functionality (DMS), corresponding to the combination of Figures 3 and 5. Sequence of positions E46 to E64 overlapping CDRH2 (SEQ ID NO: 8); Sequence of positions V89 to G104 overlapping CDRH3 (SEQ ID NO: 9).
• FIG. 7 represents combinatorial libraries for deimmunization.
• the major interaction cores and their anchor positions are shown in gray.
• the banks were designed to be located on these interaction cores.
• For the CDRH2 library the indicated degenerate codons were used.
• FIG. 8 presents the screening of combinatorial libraries by FACS.
• the two combinatorial libraries relating to CDRH2 and CDRH3 were screened independently.
• the first phase of equilibrium selection comprises three successive sorts at decreasing concentrations of biotinylated TNF ⁇ . The first sort at 3nM and the last at 500pM are shown in this figure.
• the second phase of selection consists in selecting the mutants on their dissociation rate. For this, the banks are incubated for 3 hours with biotinylated TNF ⁇ and then put into competition for 24 hours with non-biotinylated TNF ⁇ . At the end of the 24 hours, the banks are sorted to select the mutants for which the dissociation rate is the slowest.
• FIG. 9 presents the evolution of amino acid diversity during the various stages of selection.
• the amino acid diversity for the CDRH2 (A) region is shown before and after the equilibrium and kinetic selection steps.
• CDRH3 (B) the diversity is represented at the end of the first magnetic selection step then after the equilibrium and kinetic selection steps.
• Amino acids are represented as a function of their percentage in a sample of 1000 sequences. The native amino acid is shown in gray and the substitutions in black. Sequence of positions E46 to A60 including CDRH2 (SEQ ID NO: 10); Sequence of positions D86 to S100C including CDRH3 (SEQ ID NO: 11).
• FIG. 10 presents the screening of the CDRH2-CDRH3 combinatorial library.
• the first phase of equilibrium selection comprises three successive sorts at decreasing concentrations of biotinylated TNF ⁇ .
• the first sort at 3nM and the last at 500pM are shown in this figure.
• the second phase of selection consists in selecting the mutants on their dissociation rate.
• the bank is incubated for 3 hours with biotinylated TNF ⁇ and then put into competition for 24 hours with non-biotinylated TNF ⁇ .
• the bank is sorted to select the mutants for which the dissociation rate is the slowest.
• FIG. 11 presents the analysis of the immunogenic potential of the mutants of interest.
• A Prediction by netMHCIIpan of the probability of interaction with HLA II molecules at a threshold of 20% for the selected mutants and the native sequence. Predictions are given independently for the CDRH2 area and that of CDRH3.
• B Sequence of mutated cores (mutation in red) and effect of these different mutations on the prediction of these cores.
• ITWNSGHID (SEQ ID NO: 12); INWNGGHRD (SEQ ID NO: 13); INWNGGHSD (SEQ ID NO: 14); YYCAKVSYL (SEQ ID NO: 15); VSYLSTASS (SEQ ID NO: 16); YLSTASSLD (SEQ ID NO: 17); YYCAKSQYL (SEQ ID NO: 18); YYCAKTTYL (SEQ ID NO: 19); YYCAKAHYL (SEQ ID NO: 20); SQYLSTASS (SEQ ID NO: 21); TTYLSTASS (SEQ ID NO: 22); AHYLSTASS (SEQ ID NO: 23); YLPTASSLD (SEQ ID NO: 33).
• FIG. 12 presents the detail of the netMHCIIpan prediction for adalimumab and the 3 mutants of interest
• DMS libraries were constructed by PCR assembly. For each position, a forward primer comprising the degenerate codon NNS was used to randomize the amino acid concerned. At the end of the PCR assembly, the mutated genes are purified on gel independently and then pooled to form a library.
• PCR assembly was also used.
• CDRH2 diversity was introduced using degenerate codons chosen using the CodonCalculator tool (http://guinevere.otago.ac.nz/cgi- bin / aef / CodonCalculator.pl) and indicated in Figure 7.
• CDRH3 a trinucleotide synthesized primer (Ella Biotech GmbH, Martinsread, Germany) was used in order to insert the desired diversity.
• the final deimmunization bank was constructed by PCR assembly from the plasmids extracted from the CDRH2 and CDRH3 banks at the end of the selection.
• the libraries were cloned into a bicistronic plasmid derived from the plasmid pCT-L7.5.126 (Addgene plasmid # 429000) and described in Figure 4. Like pCT-L7.5.126, it has a CEN / ARS yeast origin of replication. , a TRP auxotrophy gene, a colE1 bacterial origin of replication, an ampicillin resistance gene and a galactose-inducible GLA1 / GAL10 bidirectional promoter.
• the cloning of the library in the YSD expression plasmid was carried out by homologous recombination during the transformation of EBY100 cells (ATCC ⁇ MYA- 4941 TM; a GAL1-AGA1 :: URA3 ura3-52 trp1 Ieu2 D1 his3 D200 pep4: : HIS2 prb1 D1 6R can1 GAL) as described in the previous part.
• 3 ⁇ g of linearized plasmid Nhel and SalI for VH, Ncol and Pfl23ll for VL
• 6 ⁇ g of insert were used for each of the transformations.
• the libraries thus cloned into the plasmid were cultured in SD-CAA medium [6.7 g / L yeast nitrogen base without casamino acids, 20 g / L dextrose, 5 g / L casamino acids, 100 mM sodium phosphate pH 6.0] and were run twice before inducing expression in SG-CAA medium [6.7 g / L yeast nitrogen base without casamino acids, 20 g / L galactose, 5 g / L casamino acids, 100 mM sodium phosphate, pH 6.0] in order to minimize double transformants.
• the culture and expression steps were carried out according to the description given in the previous part.
• the selection of the DMS libraries was made in a single step by FACS on an ARIA III device (Becton Dickinson, Franklin Lakes, United States). After induction of expression, the libraries were incubated for 3 hours at 20 ° C. with biotinylated TNF ⁇ (ACROBiosystems, Newark, United States) at a concentration of 80 pM. After washing cells with PBS 0.1% BSA these were labeled with an antibody directed against the APC coupled CK domain (Thermo Fisher Scientific, Waltham, USA; 1: 100 dilution) and PE-coupled streptavidin (Thermo Fisher Scientific, Waltham, USA; 1: 100 dilution).
• the selection of the libraries was made using a rectangular sorting window containing 5% of the clones of interest according to the optimal parameters described by Kowalski et al. PLoS One 10, 1-23 (2015)).
• the selection of the deimmunization banks was carried out in several stages. After optional magnetic sorting using anti-biotin magnetic beads (Miltenyi Biotec, Bergisch, Germany) after 3 hours of incubation at 20 ° C. at a concentration of 10 nM of biotinylated TNF ⁇ as described by Chao et al. Nat Protoc 1, 755-768 (2006).
• the banks have undergone different sorting steps by FACS.
• the plasmids were extracted from the cells by enzymatic lysis using the Zymoprep Yeast Plasmid Miniprep II kit (Zymo Research, Irvine, United States). The corresponding fragments were then amplified and the illumina adapters and multiplexing tags added by 2 steps of PCR as described by Kowalsky, C. A. et al. PLoS One 10, 1-23 (2015).
• the libraries were paired-end sequenced on a MiSeq using a 2x150 cycle V2 kit or on an iSeq still in 2x150 cycles (Illumina, San Diego, USA). For the DMS libraries a minimum sequencing depth of 50X was respected.
• sequences were demultiplexed and processed independently on the Galaxy platform (https://usegalaxy.org/) using the functions described by Blankenberg et al., Bioinformatics 26, 1783-1785 (2010).
• the sequences are paired (fasrq-join) and only the sequences having a quality score greater than or equal to 30 are kept (FASTQ Quality Trimmer).
• the sequences are then aligned (Align.seqs) and only the region of interest is kept (Chop.seqs).
• sequences are translated (transeq) and the identical sequences are counted and aggregated (Group).
• the evolution of diversity at each position was represented in the form of weblogo (http://weblogo.threeplusone.com/create.cgi) generated from a sample of one thousand sequences.
• mutants are represented by a selective value taking into account the enrichment of the native sequence:
• Or is the frequency of the native sequence before the selection e at the end of the selection.
• the scores are given in a relative manner compared to the native sequence, the negative scores reflect a decrease in the number of peptides below the 20% threshold.
• the panel of 46 alleles published by McKinney et al. , covering more than 80% of the phenotypes for each locus, which was used (McKinney, DM et al., Immunogenetics 65, 357-370 (2013)). ([Table 1]).
• Mutants 1 to 8 as well as native adalimumab were produced in Fab format, mutants of interest (1, 2 and 7) and adalimumab were also produced in IgG format.
• the heavy and light variable chains of the antibodies were cloned into the plasmids AbVec2.0-IGHG1 and AbVed .1 -IGKC respectively.
• 24 For the production of Fab the heavy chain was cloned into a plasmid derived from AbVec2.0-IGHG1 for which the CH2 and CH3 domains were removed and replaced by a 6His tag.
• Fab and IgG were carried out transiently with HEK293 Freestyle cells (Thermo Fisher Scientific, Waltham, USA) and cultured in the associated medium.
• the transfection was carried out at a density of 2.5 ⁇ 10 6 cells per ml of culture, a solution of PEI (Sigma-Aldrich, Saint-Louis, United States) was used as transfection agent.
• the plasmids were added to the culture at a 1: 1 ratio and at a final concentration in the culture of 1.5 ⁇ g / mL for each plasmid. After 5 minutes of stirring at 37 ° C. and 8% CO2, the PEI was added dropwise to a final concentration of 9 ⁇ g / mL of culture.
• the culture was diluted to half.
• the production was stopped 7 days after transfection and the supernatant was recovered by centrifugation at 4 ° C, 10 min at 3000G then 20 min at 20000G.
• the proteins were then purified on an AKTA system (GE Healthcare, Pittsburgh, USA).
• AKTA GE Healthcare, Pittsburgh, USA
• the purification was carried out using a HisTrap Excel column (GE Healthcare, Pittsburgh, United States) with elution in 20 mM imidazole buffer. Following the purification, the Fabs were dialyzed in order to decrease the concentration of imidazole.
• IgGs were purified using a HiTrap Protein A HP column (GE Healthcare, Pittsburgh, USA), followed by SEC a second time on a Sephacryl S-200 HR column (GE Healthcare, Pittsburgh, USA). United) in order to keep only the monomeric form of IgG. 6. Affinity measurement
• the affinity measurements were carried out kinetically with a Red96 octet (Molecular Devices, San Jose, United States) according to the protocol described by Schroter et al. (MAbs 7, 138-151 (2015)). Briefly, the biotinylated TNF ⁇ is immobilized on streptavidin sensors (Streptavidin (SA) Biosensor) at a concentration of 20nM. After saturating the sensors in a blocking solution containing biotin (Sigma-Aldrich, Saint-Louis, United States) at 10 ⁇ g / mL, the association and the dissociation are measured for 20 and 40 minutes respectively.
• SA streptavidin
• mutants 3 to 6 and mutant 8 the affinity measurements were made at three concentrations of Fab: 10nM, 5nM and 2.5nM, plus a reference to 10nM without TNF ⁇ .
• analyzes were carried out for six concentrations of Fab: 15nM, 10nM, 5nM, 2.5nM, 1, 25nM and 0.625nM, plus a reference to 15nM without TNFa.
• the reference is subtracted from each curve and a global 1: 1 Langmuir model is applied to obtain the affinity parameters.
• the masses of the IgG products (adalimumab, Mutants 1, 2 and 7) and of adalimumab in its commercial version (Humira) were determined by mass spectrometry.
• the analysis was carried out on a high resolution apparatus of the Q-Orbitrap type (Thermo Fisher Scientific, Waltham, United States) by UHPLC-MS as described by Contrepois et al. (J. Proteome Res. 9, 5501-5509 (2010)).
• the SEC-MALS analysis was carried out within the GIPSI platform of the University of Paris-Sud, on an HPLC (Shimadzu) with a Superdex 200 10/300 GL increase column (GE Healthcare, Pittsburgh, United States).
• T epitopes of adalimumab were identified in vitro by specific activation tests of CD4 T lymphocytes using overlapping peptides with a length of 15 or 20 amino acids.
• the regions comprising T epitopes are mainly located within the heavy chain, CDR2 on a zone between residues E46 and E64 and CDR3 between amino acids L82c and T107 (Meunier, S. et al., Cellular & Molecular Immunology, 2019, Oct 28. doi: 10.1038 / s41423-019-0304-3).
• a T epitope is also described on the light chain upstream of CDR3 (S76 to R90).
• peptides identified by MAPPS those corresponding to the T epitopes identified by cell test (in bold) are represented in the form of overlapping peptide clusters.
• the 9-mer “ITWNSGHID” (SEQ ID NO: 12) comprising residues 51 to 58 (in dark gray in FIG. 2) predicted by netMHCIIpan is probably the core of interaction of the T epitopes of CDRH2 among the predicted cores (in light gray in Figure 2).
• CDRH3 is more dense in T epitopes, a set of 6 overlapping T epitopes have been predicted on this region. The overlapping nature of these epitopes also suggests that these areas of overlap could be of particular interest for destabilizing the peptide / HLA II interaction of several T epitopes simultaneously.
• netMHCIIpan The modifications proposed by netMHCIIpan are generally substitutions towards a hydrophilic amino acid, with an effect which is all the stronger if the native residue is hydrophobic. netMHCIIpan taking into account neither the structure nor the functionality of the antibody, these predictions must be put into perspective with the tolerance to the mutation of these amino acids. Indeed, the deletion of all the hydrophobic amino acids on regions as important as the CDRs seems extremely risky for the functionality but also for the structure of the antibody. In addition, residues belonging to these two CDRs have been described for their contribution in the interaction with TNFa (Hu et al. J. Biol. Chem. 288, 27059-27067 (2013). In order to take these functional constraints into account and structural, a second systematic approach on the function this time was applied.
• DMS Deep Mutational Scanning
• the two immunogenic regions were processed in two independent libraries. They each relate to a region of twenty residues and were constructed by PCR assembly. For each of the twenty residues, the diversity was introduced using a degenerate NNS codon encoding all of the twenty natural amino acids. Degenerate codons were introduced by independent PCRs for each position. The twenty PCR products were mixed equimolarly in order to obtain a library containing all the single substitutions for each of the twenty positions. The libraries were then cloned into an expression plasmid for the Yeast Surface Display (YSD) allowing expression in the form by Fab. ( Figure 4A) The two libraries were cloned independently by homologous recombination during transformation into yeast.
• YSD Yeast Surface Display
• the libraries were then expressed in YSD and the mutants of interest selected by FACS.
• Figure 4B The screening was carried out according to the conditions described by Kowalsky et al. (PLoS One 10, 1-23 (2015)). Libraries were screened once after three hours of incubation at a TNF ⁇ concentration lower than the KD of adalimumab in order to be able to observe both gain and loss of function. The 5% of the mutants expressed having the weakest signal for the interaction were selected (cf. “Negative” sorting window, FIG. 4C), to select the mutations having a deleterious impact on the function. Similarly, the 5% of the mutants demonstrating the strongest interaction were selected (cf. “Positive” sorting window, FIG. 4C). The yeast population before any selection step as well as the two selected populations were then sequenced by NGS.
• the enrichments of the parental sequence (wild type) as well as those of each of the mutants were calculated. These made it possible to calculate for each of the variants a score called the selective value (fitness in English) which corresponds to the logarithm to base 2 of the relative enrichment of the variant with respect to the native sequence.
• the mutants are represented in a matrix by a color proportional to their selective value. For negative selection, the amplitude of the selective values is -8 to 3, ie an enrichment 256 times less than 8 times greater than the native sequence. For positive selection the amplitude of the selective values is substantially the same. Mutants having an enrichment greater than or equal to the parental sequence are indicated in shades of gray. The darker the gray, the stronger the enrichment, indicating a good affinity for TNF ⁇ . ( Figure 5).
• results obtained from the selection of mutants expressed but non-functional are particularly interesting for defining the residues participating in the interaction with TNF ⁇ .
• the population resulting from the positive selection is necessary for the identification of mutations making it possible to maintain or increase the affinity.
• the combination of these two result matrices allows to identify the residuals important for the good expression of the antibody (negative selective value in the two selection conditions).
• mutants of the N-terminal part are not very present in the positive selection so these residues seem rather involved in the folding and the expression of the antibody.
• Mutants belonging to the C-terminal part are somewhat more enriched in positive selection. ( Figure 5) Many mutations are allowed, including towards residues with different properties, while retaining a selective value equal to or greater than that of the native antibody.
• Example 2 Generation of mutants of adalimumab with reduced immunogenic potential
• CDRH2 has two overlapping T epitopes which probably share the “ITWNSGHID” interaction core (in dark gray in FIG. 2). Therefore the mutation strategy focused on this area.
• the cross-checking of the matrices obtained from the DMS and the prediction by netMHCIIpan made it possible to choose the mutations making it possible both to destabilize the interaction with the HLA II molecules while maintaining the functionality of the antibody (FIG. 6).
• Residues S49, A50, T52, S54, H56 and I57 were selected because they all have one or more mutations making it possible to destabilize the interaction with the HLA II molecules. For each of these residues, a degenerate codon making it possible to best represent the substitutions of interest was chosen. (FIG.
• CDRH3 comprises a set of 5 overlapping T epitopes for which we have defined a set of 3 probable interaction cores (in dark gray in FIG. 2).
• Each of the libraries was constructed by PCR assembly, with primers comprising degenerate codons for CDRH2 and primers synthesized from a mixture of trinucleotides for CDRH3. These banks respectively have a diversity of 1, 2.10 4 for CDRH2 and 3.8.10 6 for CDRH3. As previously, they were cloned by homologous recombination into a plasmid for expression and screening in YSD. After transformation of the yeasts and induction of expression, the FACS screening was carried out independently for the two libraries and each of the libraries underwent different selection steps.
• the banks were saturated for 3 h with 20 nM of biotinylated TNF ⁇ and then sorted at the end of a 24-hour competition against non-biotinylated TNF ⁇ .
• 2 to 5% of the cells with the strongest signal in PE were selected by FACS using diagonal sorting windows in order to normalize the binding signal by expression.
• Figure 8 To understand the evolution of the molecular diversity of the banks during the selection steps, we have chosen to perform NGS sequencing after each of the selection steps. The sequencing data from the CDRH2 library show that all the mutations were indeed present in the library before the start of the screening. (FIG.
• Adalimumab It is observed that the best mutants were enriched more than two hundred times, in total after screening there are 174 mutants having an enrichment greater than that of adalimumab. The netMHCIIpan algorithm was once again used to classify these mutants and 158 of them are predicted to be potentially less immunogenic.
• [0122] [Table 4]: Selection of the 30 most enriched mutants from the library relating to CDRH3 It is also observed that the enrichment factors are higher than for CDRH2 with values which can reach several millions. After screening, the library contains 310 mutants which are more enriched than the native sequence of adalimumab and, as for CDRH2, a large part of these (282) have a reduced immunogenic potential according to netMHCIIpan.
• this combinatorial library contains a minimum of 10 5 variants.
• This library also incorporates the R90K substitution described as preserving the affinity in the Humira® patent and making it possible to germinate the sequence in the region of CDRH3 and thus to remove a minor epitope (Salfed, JG et al. Human antibodies that bond human TNFa. (US Patent 6258562 B1)).
• This library was screened according to the same procedure applied to the previous libraries, namely three sorting at equilibrium with decreasing concentrations of TNF ⁇ followed by selection for the dissociation rate. ( Figure 10) As this library is derived from the recombination of sequences already selected, a strong initial enrichment in functional mutants is observed even before the first selection step. The population is also very homogeneous from the early stages of selection, the screening for the dissociation rate subsequently carried out, however, reveals a more heterogeneous population.
• mutants were evaluated according to their enrichment and 245 of them show a value greater than the native sequence. Among them, more than 200 mutants are predicted to have a lower number of interaction cores with HLA II molecules than adalimumab. Their sequences show, however, a redundancy in their sequences, particularly at the level of CDRH3. ([Table 5]).
• [0128] Selection of the 30 mutants best classified by netMHCII pan as well as of the 8 mutants of interest obtained from the recombinant CDRH2 and CDRH3 library [0129] At the end of the selection steps, a reduced number of variants of interest were chosen in order to carry out a complete biochemical characterization. The mutants were selected for their enrichment greater than the native sequence and for their reduced immunogenic potential according to netMHCIIpan but also by attaching particular importance to selecting mutants with various sequences. From these criteria, 8 mutants were chosen in order to be characterized. ([Table 6]).
• [0131] Mutants of interest selected from the CDRH2-CDRH3 combinatorial library For some, such as mutants 2 and 5, an affinity increased by more than ten times is measured. The increase in affinity for the different clones is mainly due to a decrease in the dissociation rate, a parameter selected during the screening. If all these mutants have a higher enrichment than the native antibody, these However, these values do not always seem to be strictly proportional to the affinities measured for these mutants.
• Mutants 1, 2 and 7 were selected for their reduced predicted immunogenicity in order to carry the characterization further. These three mutants as well as the native antibody were produced in HEK and purified in IgG format. Orbitrap mass spectrometry analysis confirmed that the antibodies have a mass corresponding to that expected, with a resolution of the order of the dalton. On the other hand, an analysis by SEC-MALS (Size Exclusion Chromatography - Multi-Angle Light Scattering) also made it possible to validate the monomeric character of these antibodies.
• SEC-MALS Size Exclusion Chromatography - Multi-Angle Light Scattering
• the mutants have a reduction in interaction with the HLA II molecules predicted according to the netMHCIIpan algorithm. As a result, they are less likely to be presented by HLA II molecules and recognized by T lymphocytes compared to adalimumab. These mutants thus constitute variants of adalimumab with reduced immunogenicity and make it possible to overcome the problems of immunogenicity encountered with anti-TNF ⁇ . These variants could represent a clinical improvement in to reduce the immunization rate of patients. In addition, the inventors have been able to demonstrate that they have an increased affinity for TNF ⁇ .
• mutants of the invention will have a higher biological activity. to that of adalimumab.
• the mutants obtained at the end of this work could thus be considered as potential drug candidates positioning themselves as an improved version of adalumimab.

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