Patent Application
20230142800
The invention relates to adalimumab variants with reduced immunogenic potential and retained or increased affinity and therapeutic applications thereof.
Autoimmune inflammatory diseases affect a very large part of the population, especially in developed countries. Rheumatoid arthritis, which is one of the most widespread conditions, affects over 1 million people in the United States (Hunter, T. M. et al., Rheumatol. Int. (2017). doi:10.1007/s00296-017-3726-1). The anti-TNF alpha such as Humira® (adalimumab), a human IgG1 antibody neutralizing TNF alpha, constitute an effective symptomatic treatment for very many of these autoimmune inflammatory diseases. For this reason, adalimumab is today widely used in the treatment of ankylosing spondylitis, rheumatoid arthritis, hemorrhagic colitis, psoriatic arthritis, Crohn's disease, cutaneous psoriasis and juvenile arthritis (Feldmann, M. & Maini, Nat. Med. 9, 1245-1250 (2003)). Adalimumab has however been seen to be immunogenic in a non-negligible number of patients (up to 50% for some conditions) who produce antibodies directed against the therapeutic protein (ADA, for anti-drug antibody), detectable in the serum (Strand, V. et al., BioDrugs 31, 299-316 (2017)). The ADA result in the formation of immune complexes accelerating the elimination of the therapeutic antibodies. Clinically, the presence of ADA is inversely correlated with the response to the treatment. Patients having a good response to the treatment are those in whom the tests do not reveal any ADA (van Schouwenburg, P. A., Rispens, T. & Wolbink, G. J., Nat. Rev. Rheumatol. 9, 164-172 (2013)).
Clinically, the ADA problem is treated by different approaches. It was observed that the effect of the ADA was reduced by increasing the doses administered. Thus the treatments are often done with doses that increase in step with the patients' immunity. On the other hand, the therapies are often co-administered with methotrexate, since this immunosuppressant inhibits the ADA production and distinctly improved results can be obtained. These measures are however unsatisfactory in so far as the patients continue just the same to get immunity against the protein (van Schouwenburg, P. A., Rispens, T. & Wolbink, G. J., Nat. Rev. Allergy Immunol. 38, 82-89 (2010); Radstake, T. R. D. J., Ann. Rheum. Dis., 68, 1739-1745 (2009)).
The suppression of T cell epitopes by disruption of the interaction with the HLA II molecules, called de-immunization, was shown to be an effective method for reducing the immunogenicity of proteins with therapeutic purpose, such as enzymes and immunotoxins (Mazor, R. et al. Oncotarget 7, 29916-29926 (2016); Cantor, J. R. et al., Proc. Natl. Acad. Sci. 108, 1272-1277 (2011); Ettinger, R. A. et al., Blood Adv. 2, 309-322 (2018); Mazor, R. et al., Proc. Natl. Acad. Sci. U.S.A. 109, E3597-603 (2012)).
Different T cell epitopes were previously identified in the adalimumab sequence (Meunier, S. et al., Cellular & Molecular Immunology, 2019, Oct. 28. doi: 10.1038/s41423-019-0304-3). They are mainly located on the heavy chain of the antibody on which they are distributed in two regions. The majority of the T cell epitopes are concentrated in the first region and overlap extensively with CDR-H3 (L82C to T107 residues using Kabat numbering;
However, this approach of de-immunization by elimination of T cell epitopes is not suited to therapeutic antibodies which are humanized or human antibodies, since T cell epitopes thereof are mainly present near the CDR which are essential for the biological activity of the therapeutic antibody (Harding et al., mAbs 2, 256-265 (2010)). In fact, the CDR are what determine the specificity and also the affinity of the antibody for the target antigen of the therapeutic antibody.
Adalimumab variants in which the framework regions (FR) from the variable domains of the heavy and light chains were replaced by less immunogenic framework regions from other human immunoglobulins G (IgG) are described in application EP 3,178,487. Such variants do not have a satisfactory de-immunization for therapeutic use since they comprise the majority of the T cell epitopes present in the CDR.
Adalimumab variants comprising mutations in a region containing suspected CD4 T cell epitopes extending on both sides of the CDR1 of the light chains (CDR-L1; positions C23 to K45, using Kabat numbering) are described in the application WO 2010/121140. Despite the choice of mutations for not significantly reducing the affinity of the variants, all the resulting variants had a reduced affinity for TNF alpha compared to adalimumab, where this reduction of affinity was drastic (at least 50%) for most (70%) of the resulting variants.
Consequently, there is a need to have new adalimumab variants which are better suited to their therapeutic use in that they have both a reduced immunogenic potential and an intact affinity.
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 the immunogenicity while maintaining the TNF alpha binding affinity. They isolated variants from combinatorial libraries which surprisingly have 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 greater than that of adalimumab. To the extent where the biological activity of the anti-TNF antibody—specifically the neutralization of the TNF alpha—depends on the affinity thereof for TNF, it can be expected that the variants from the invention will have a biological activity greater than that of adalimumab.
Consequently, the object of the present invention is a variant of a therapeutic anti-TNF alpha antibody comprising variable domains VH 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; where said at least two amino acid substitutions in the sequence overlapping the CDRH2 region are selected from the group consisting of:
According to an embodiment of the invention, said variant comprises at least three substitutions in one of the sequences overlapping the CDRH2 or CDRH3 region; preferably in each of said sequences overlapping the CDRH2 and CDRH3 regions.
According to an embodiment of the invention, said variant comprises a combination of substitutions in the sequence overlapping the CDRH2 region selected from:
According to a preferred embodiment of the invention, said variant comprises a combination of substitutions in the sequence overlapping the CDRH2 region selected from:
According to a preferred embodiment of the invention, said variant comprises a combination of substitutions in the sequence overlapping the CDRH3 region selected from:
According to a preferred embodiment of the invention, said variant comprises a combination of substitutions in the sequence overlapping the CDRH3 region selected from:
According to a more preferred embodiment of the invention, said variant comprises a combination of substitutions in the sequence overlapping the CDRH3 region selected 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 selected from: V95T, S96T and S99P; V95T, S96N and S99P; V95S, S96Q and S99P; V95A, S96H and S99P; V89L, V95T, S96T and S99P.
According to a preferred embodiment of the invention, said variant comprises one of the following combinations of substitutions in the sequences overlapping the CDRH2 and CDRH3 regions:
According to an embodiment of the invention, said variant further comprises the substitution R90K in the region CDRL3 determining the complementarity of the variable domain VL.
According to an embodiment of the invention, said variant comprises a human IgG heavy chain and a human Kappa light chain.
According to an embodiment of the invention, said variant is derived from adalimumab.
According to a preferred embodiment of the invention, 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 coding for a variant according to the invention.
Another aspect of the invention relates to 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 object of the present invention is an anti-TNF alpha therapeutic antibody comprising variable domains VH and VL of sequences SEQ ID NO: 1 and SEQ ID NO: 2, where the variant comprises a reduced immunogenic potential and a TNF alpha binding affinity at least equal to or superior compared to the therapeutic anti-TNF alpha antibody from which it is derived.
Because of the binding affinity that is at least equal or superior, it can be expected that the variant according to the invention has a biological activity at least equal or superior than that of the parent antibody.
The sequence SEQ ID NO: 1 correspondence to the heavy-chain variable domain (VH) of adalimumab and the sequence SEQ ID NO: 2 the sequence of the light-chain variable domain (VL) of adalimumab (
Therapeutic antibody is understood to mean a human or humanized antibody.
Antibody is understood to mean a whole antibody, an antibody fragment containing at least one antigen-binding domain or a molecule derived from an antibody.
TNF alpha or TNFalpha (“Tumor Necrosis Factor Alpha”) is understood to mean a multifunctional pro-inflammatory cytokine predominantly produced by monocytes and macrophages. Preferably, it means a human TNF alpha. Human TNF alpha corresponds to the GenBank QCI55793.1 sequence.
The terms “variant” and “mutant” are used interchangeably.
As it relates to an antibody variant according to the invention, reduced immunogenic potential is understood to mean a reduction of the number of HLA II molecules which can be bound by at least one of the CD4 T cell epitopes of the variant as compared to the parent antibody from which it is derived. The number of HLA II molecules which can be bound by a variant according to the invention is evaluated according to the standard techniques known to the person skilled in the art such as those described in particular in the examples. In particular it involves in silicone methods using CMH-II binding prediction tools such as the netMHCllpan 3.2 algorithm. (Jensen, K. K. et al., Immunology 154, 394-406 (2018)). HLA II binding is expressed in the form of the score defined by:
where the core is a nonhuman binding core predicted by netMHCllpan3.2; core>20%=0 and core<20%=1. i is the anchoring position of the core and j 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 of 1.1) relative to the parent antibody from which it is derived.
The TNF alpha binding affinity of the variant is evaluated according to the standard techniques known to the person skilled in the art such as those described in particular in the examples. The affinity may be evaluated by the value of the equilibrium dissociation constant KD of the variant, measured by conventional techniques such as described in the examples. The affinity may also be evaluated by the relative enrichment factor of the variant relative to the parent antibody which corresponds to the ratio between the enrichment values of the variant and the parent antibody such 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 by 10%) compared to the parent antibody from which it is derived.
An individual is understood to mean a human or animal individual, preferably a human individual.
Amino acids are indicated with the letter code.
The positions of the amino acid residues are indicated with reference to the Kabat numbering (
According to an embodiment of the invention, said variant comprises at least two amino acid substitutions in at least one sequence overlapping the CDRH2 or CDRH3 regions determining the complementarity of said variable domain VH. The sequence overlapping the CDRH2 region extends from the residues E46 to C64 according to the Kabat numbering (SEQ ID NO: 8). The sequence overlapping the CDRH3 region extends from the 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. Preferably, said variant comprises at least three substitutions, generally 3, 4 or 5 substitutions in one of the overlapping sequences; preferably, in each of the overlapping sequences of the CDRH2 and CDRH3 regions.
According to a preferred embodiment of the invention said at least two amino acid substitutions in the sequence overlapping the CDRH2 region are selected from the group consisting of:
The variants according to the invention are functional variants, meaning that they comprise a reduced immunogenic potential and a TNF alpha binding affinity at least equal or better, compared to the therapeutic anti-TNF alpha antibody from which it is derived. The invention excludes nonfunctional variants such as in particular variants comprising only two substitutions selected from T52N and A50G or T52N and I57T.
Preferably,
Advantageously, said substitutions in the sequence overlapping the CDRH2 region are selected from substitutions in positions S49, A50, T52, S54, H56 and I57. Preferably, said substitutions are in 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 in 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.
Preferably, said variant comprises a combination of substitutions in the sequence overlapping the CDRH2 region selected from:
Preferably, said variant comprises a combination of substitutions in the sequence overlapping the CDRH2 region selected from:
Even more preferably, said variant comprises a combination of substitutions in the sequence overlapping the CDRH2 region selected 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 I57H; S49G, A50D, T52S, S54G and I57T.
According to a preferred embodiment of the invention said at least two amino acid substitutions in the sequence overlapping the CDRH3 region are selected from the group consisting of:
Preferably, S96 is substituted by T, Q, N or H.
Advantageously, said substitutions in the sequence overlapping the CDRH3 region are selected from substitutions in positions: V89, V95, S96, Y97, L98, S99 and T100. Preferably, said substitutions are in the positions V89, V95, S96 and S99 or V95, S96 and S99.
Preferably, said variant comprises a combination of substitutions in the sequence overlapping the CDRH3 region selected from:
Advantageously, said variant comprises a combination of substitutions in the sequence overlapping the CDRH3 region selected from:
More preferably, said variant comprises a combination of substitutions in the sequence overlapping the CDRH3 region selected 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.
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.
According to a preferred embodiment of the invention, said variant comprises at least two substitutions in each of the two sequences overlapping the CDRH2 or CDRH3 region such as defined above. Preferably 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 selected from the combinations such as defined above.
Particularly preferred embodiments according to the invention comprise:
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 region:
According to an embodiment of the invention, said variant further comprises the substitution R90K in the region CDRL3 determining the complementarity of the variable domain VL.
The variant according to the invention comprises a human immunoglobulin heavy chain of any isotype or class, preferably an IgG, preferably an IgG1. The variant according to the invention also comprises a human immunoglobulin light chain of any class, preferably a human Kappa light chain.
According to a preferred embodiment of the invention, said variant is derived from adalimumab. Preferably, said variant is selected from the group consisting of:
According to a preferred embodiment of the invention, said variant is characterized by an HLA II binding score reduced at least 1.5; 2; 2.5; 3; 3.5; 4; 4.5; 5 times or more compared to the parent antibody from which it is derived.
According to a preferred embodiment of the invention, said variant is characterized by a relative enrichment factor of at least 101 á 107 (101, 5·101, 102, 5·102, 103, 5·103, 104, 5·104, 105, 5·105, 106, 5·106, 107) or a KD at least 1.2 to 40 times lower (2, 5, 10, 15, 20, 25, 30, 35) compared to the parent antibody from which it is derived.
The present invention also encompasses variants further comprising, at least one mutation (insertion, deletion, substitution) of additional amino acid and/or at least one function-conserving modification, meaning which preserves the reduced immunogenic potential and the greater or equal affinity of the variant. The following can be listed in particular among the additional mutations:
and;
The variant may be modified by the introduction of any function-conserving modification in the area of the amino acid residue(s), peptide bond or end peptides. This or these modification(s), in particular one or more chemical modifications are made in the peptides by conventional methods known to the person skilled in the art, in particular including: merging the sequence of the variant with that of a polypeptide (label useful for purification of the variant, in particular in form's political by a protease) or a protein of interest, and the coupling of a molecule or an agent of interest. For example, the antibody may be coupled to a PEG molecule by the conventional methods known to the person skilled in the art.
The variant is in the form of a whole antibody, an antibody fragment containing at least one antigen-binding domain or a molecule derived from an antibody. The whole antibodies may be of any isotype, in particular human isotype (IgG (IgG1, IgG2, IgG3, IgG4), IgA (IgA1, IgA2), IgE, IgM, IgD). The fragments of antibodies include in particular the fragments Fab, Fab′, F(ab′)2, Fv, scFv, Fabc or Fab comprising a portion of the Fc region and the single-chain antibody fragments derived from Camelidae or shark immunoglobulins (VHH and V-NAR domain simple antibodies). The derived antibody molecules include polyspecific or multivalent and immunoconjugated antibodies. The multi-specific scFv (dia, tris or tetrabodies), for example scDb (single-chain diabodies) or taFv (tandem scFv fragments) type diabodies, and the minibodies can be given as nonlimiting examples. The mini bodies are in particular scFv-HLX; scFv-ZIP; scFv-CH3, scFv-Fc or other type.
Another aspect of the present invention relates to an isolated polynucleotide coding for a variant conforming to the invention such as defined above. Said polynucleotide is DNA, RNA or a mixture of DNA and RNA, recombinant or synthetic. The DNA sequence may 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 as such; the choice of an appropriate vector depends on the intended use of this vector (for example replication of the sequence of interest, expression of that sequence, maintenance of that sequence in extra-chromosomic form, or else integration in the chromosomal material of the host), and also the nature of the host cell. For example, bare nucleic acids (DNA or RNA) can be used or viral or bacterial vectors. The viral vectors are in particular adenovirus, retrovirus, lentiviruses and the AAV in which the sequence of interest was previously inserted; said sequence (isolated or inserted in a plasmid vector) can also be associated with a substance allowing it to cross the membrane of the host cells, such as a transporter like a nanotransporter or liposome preparation, or cationic polymers or else inserted in said host cell by using physical methods such as electroporation or microinjection. Further, these methods can advantageously be combined, for example by using electroporation together with liposomes.
Preferably, said vector is an expression vector comprising all the elements necessary to the expression of the variant such as defined above. For example, said vector comprises an expression cassette including at least one polynucleotide such as defined above, under the control of appropriate transcription and possibly translation regulating sequences (promoter, activator, intron, initiation codon (ATG), stop codon, polyadenylation signal, splice site), in order to allow the expression of the variant conforming to the invention in a single host cell.
Another aspect of the present invention relates to a prokaryotic or eukaryotic host cell modified by a polynucleotide or a vector conforming to the invention is described above, where the cell can be stably or temporarily modified.
Another aspect of the present invention relates to a pharmaceutical composition comprising at least one variant, polynucleotide, vector and/or cell derived such as defined above and a pharmaceutically acceptable vehicle and/or a carrier substance.
The pharmaceutically acceptable vehicles and the carrier substances are those conventionally used.
The carrier substances are advantageously selected from the group consisting of: unilamellar or multilamellar liposomes, ISCOM, virosomes, viral pseudo-particles, saponin micelles, solid microspheres of saccharide (poly(lactide-co-glycolide)) or auriferous nature, and nanoparticles.
The pharmaceutical composition may further comprise at least one therapeutic agent, in particular anti-inflammatory or immunomodulating.
The pharmaceutical composition comprises a therapeutically active quantity of variant, polynucleotide, vector and cell. A therapeutically active quantity means a sufficient dose for producing a therapeutic effect on the disease to be treated, meaning reducing the symptoms of this illness. This effective dose is determined and adjusted as a function of factors such as age, gender and weight of the subject. The pharmaceutical composition according to the invention comes in a form for delivery suited to the chosen administration. The composition is generally administered according to the usual immunotherapy protocols at doses and for sufficient time in order to induce an effective response against the pathology to be treated. The administration may be subcutaneous, intramuscular, intravenous, in particular by infusion, intradermal, intraperitoneal, oral, sublingual, rectal, vaginal, intranasal, by inhalation or by transdermal application. The composition comes in a form for delivery suited to a selected administration.
The pharmaceutical composition according to the present invention is used in immunotherapy in the treatment of inflammatory or autoimmune pathologies. It may be used in combination with other therapeutic or surgical treatments, in particular in combination with other therapeutic agents such as defined above, where the composition according to the invention and the other therapeutic agents may be administered simultaneously, separately or sequentially.
The inflammatory or autoimmune pathologies are those which are conventionally treated with anti-TNF alpha. Ankylosing spondylitis, rheumatoid arthritis, hemorrhagic colitis, psoriatic arthritis, Crohn's disease, cutaneous psoriasis and juvenile arthritis can be given as nonlimiting examples of these pathologies.
The present invention also relates to a derived variant, polynucleotide, vector and/or cell such as defined above for use as medication, in particular in immunotherapy, in the treatment of autoimmune or inflammatory pathologies such as defined above.
An object of the present invention is also an immunotherapy method, in particular intended for the treatment of inflammatory or autoimmune pathologies such as defined above, characterized in that it comprises the administration to an individual of an effective dose of the derived variant, polynucleotide, vector and/or cell conforming to the invention such as defined above by any appropriate means such as defined above. Preferably, the method comprises the administration of a pharmaceutical composition according to the invention such as defined above.
The polynucleotides according to the invention are obtained by conventional methods, well known in themselves. For example, they can be obtained by amplification of a nucleic sequence by PCR or RT-PCR or else by complete or partial chemical synthesis. The eukaryotic or prokaryotic expression recombinant vectors are built and inserted in host cells by conventional methods of recombinant DNA or genetic engineering, which are well known in themselves. In particular it involves expression vectors conventionally used for the production of antibodies, in particular human or humanized therapeutic antibodies such as tandem type vectors allowing the simultaneous expression of heavy and light chain antibodies. The variants produced by the host cells modified by the recombinant vector are purified by conventional methods for purification of immunoglobulins, in particular by affinity chromatography.
The features disclosed in the preceding paragraphs may, optionally, be put into practice. They may be put into practice independently of each other or in combination with the others.
Other characteristics, details and advantages of the invention will appear to the reader of the following detailed description which refers to nonlimiting examples showing the identification and characterization of the variants according to the present invention, and also to the attached figures, on which:
The DMS libraries were built by PCR assembly. For each position, a direction primer comprising the NNS degenerate codon was used for randomizing the affected amino acid. At the outcome of the PCR assembly, the mutated genes are purified independently on gel and then regrouped to form a library.
For the de-immunization libraries, PCR assembly was also used. For CDRH2, diversity was inserted by means of degenerate codons chosen with the help of the CodonCalculator tool (http://guinevere.otago.ac.nz/cgi-bin/aef/CodonCalculator.pI) and indicated in
The final de-immunization library was constructed by PCR assembly with plasmids extracted from CDRH2 and CDRH3 libraries at the end of the selection.
The libraries were cloned in a bicistronic plasmid derived from the plasmid pCT-L7.5.126 (Addgene plasmid #429000) and described in
The selection of DMS libraries was done in a single step by FACS on an ARIA III device (Becton Dickinson, Franklin Lakes, United States). After induction of the expression, the libraries were incubated 3 hours at 20° C. with biotinylated TNF alpha (ACROBiosystems, Newark, United States) at an 80 pM concentration. After washing the cells with PBS 0.1% BSA, they were marked by using an APC coupled antibody directed against the OK domain (Thermo Fisher Scientific, Waltham, United States; dilution 1:100) and a PE coupled streptavidin (Thermo Fisher Scientific, Waltham, United States; dilution 1:100). The selection of libraries was done by means of 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 de-immunization libraries was done in several steps. After a possible magnetic sorting by using anti-biotin magnetic beads (Miltenyi Biotec, Bergisch, Germany) after 3 hours of incubation at 20° C. at a 10 nM concentration of biotinylated TNF alpha as described by Chao et al. Nat Protoc 1, 755-768 (2006). The libraries underwent different steps of sorting by FACS. First, three successive steps of equilibrium sorting at decreasing concentrations of biotinylated TNF alpha (3 nM, 1 nM then 500 pM) and then a step of selection by dissociation speed. For each of the sortings, Fab expression was induced and then the cells were incubated for 3 hours at 20° C. with biotinylated TNF alpha before being sorted by FACS. For kinetic sorting based on dissociation speed, the cells were incubated three hours with 20 nM of biotinylated TNF alpha, and then they were washed and incubated for 24 hours with non-biotinylated TNF alpha (Thermo Fisher Scientific, Waltham, United States) before being sorted 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 labels added by two PCR steps as described by Kowalsky, C. A. et al. PLoS One 10, 1-23 (2015). The libraries were sequenced in paired-end on a MiSeq using V2 kit 2×150 cycles or on a iSeq still with 2×150 cycles (Illumina, San Diego, United States). For the DMS libraries a minimum sequencing depth of 50× was followed.
The sequences were multiplexed and processed independently on the Galaxy platform (https://usegalaxy.org/) by means functions described by Blankenburg et al., Bioinformatics 26, 1783-1785 (2010). The sequences are unpaired (fasrq-join) and only the sequences having a quality score greater than equal to 30 were retained (FASTQ Quality Trimmer). The sequences were then aligned (Align.seqs) and only the region of interest is retained (Chop.seqs). Finally the sequences are translated (transeq) and the identical sequences are counted and aggregated (Group). The development of the diversity of each of the positions was shown in weblogo form (http://weblogo.threeplusone.com/create.cgi) generated from a thousand sequence sample.
These data were then processed with the R software in order to calculate the frequencies of the various mutants and thus determine their enrichment as follows:
Where Finputi is the frequency of the mutant i before selection and Foutputi at the outcome of the selection.
For the results of the DMS in matrix form, the mutants are represented by a selective value considering the enrichment of the native sequence:
where Finputwt is the frequency of the native sequence before selection and Foutputwt at the outcome of the selection.
The predictions of interaction with the HLA II molecules were done by means of the netMHCllpan 3.2 algorithm. (Jensen, K. K. et al., Immunology 154, 394-406 (2018)). Briefly this algorithm predicts the probability of interaction of a sequence with selected HLA II molecules and provides a result for each allele relative to a peptide set. A value of 1% for a peptide means that it is among the 1% of peptides having a high probability of interaction with HLA II molecules. For this study we used a 20% threshold value below which the peptides are considered as immunogenic, thus each nonhuman core below the 20% threshold for an allele counts for one unit in the score. For hitmaps, the scores are given relative to the native sequence; the negative score showing a reduction of the number of peptides below the 20% threshold. For each of the predictions made, it is the panel 46 alleles published by McKinney et al., covering over 80% of the phenotypes for each locus, which was used (McKinney, D. M. et al., Immunogenetics 65, 357-370 (2013)). ([Table 1]).
Further, a set of sequences covering the most frequent human immunoglobulin genes was used in order to evaluate the human or nonhuman character of each of the cores.
The mutants 1 to 8 and also native adalimumab were produced in Fab format, and the 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 in the plasmids respectively AbVec2.0-IGHG1 and AbVec1.1-IGKC.24 For the production of Fab, the heavy chain was cloned in a plasma derived from AbVec2.0-IGHG1 from which the domains CH2 and CH3 were withdrawn and replaced by a 6His tag. The production of Fab and IgG was done translationally with HEK293 Freestyle cells (Thermo Fisher Scientific, Waltham, United States) and cultivated in the associated medium. The transfection was done at a density of 2.5·106 cells/mL of culture; a PEI solution (Sigma-Aldrich, Saint-Louis, United States) was used as transfection agent. The plasmids were added to the cultures at a 1:1 ratio and at a final concentration in the culture of 1.5 μg/mL for each plasmid. After 5 minutes of stirring at 37° C. and 8% CO2, PEI was added drop by drop to a final concentration of 9 μg/mL of culture. After 24 hours under stirring at 37° C. and 8% CO2, the culture was diluted by half. The production was stopped 7 hours after transfection and the supernatant was recovered by centrifuging at 4° C. for 10 minutes at 3000 G and then 20 minutes at 20,000 G. The proteins were then purified on an AKTA system (GE Healthcare, Pittsburgh, United States). For the Fab, the purification was done by using a HisTrap Excel column (GE Healthcare, Pittsburgh, United States) with elution by an imidazole buffer. Following the purification, the Fab were dialyzed in order to reduce the imidazole concentration. The IgG were purified by means of a HiTrap Protein A HP column (GE Healthcare, Pittsburgh, United States), and then a second time by SEC on a Sephacryl S-200 HR column (GE Healthcare, Pittsburgh, United States), in order to keep the monomeric form of the IgG.
The affinity measurements were done kinetically with an Octet Red96 (Molecular Devices, San Jose, United States) according to the protocol described by Schroter et al. (MAbs 7, 138-151 (2015)). Briefly, biotinylated TNF alpha is immobilized on streptavidin sensors (Streptavidin (SA) Biosensor) at a 20 nM concentration. After saturation the sensors in a blocking solution containing 10 μg/mL biotin (Sigma-Aldrich, Saint-Louis, United States), the association and dissociation are measured over 20 and 40 minutes respectively. For the mutants 3 to 6 and the mutant 8, the affinity measurements were done at three Fab concentrations: 10 nM, 5 nM and 2.5 nM, plus a reference at 10 nM without TNFα. For adalimumab and the mutants 1, 2 and 7, the analyses were done for six concentrations of Fab: 15 nM, 10 nM, 5 nM, 2.5 nM, 1.25 nM et 0.625 nM, plus a reference at 15 nM without TNFα. During the analysis, the reference is subtracted from each curve and a 1:1 global Langmuir model is applied in order to get the affinity parameters.
The masses of the IgG products (adalimumab, Mutants 1, 2 and 7) and of the adalimumab in its commercial version (Humira) were determined by mass spectrometry. The analysis is done by a Q-Orbitrap type high-resolution device (Thermo Fisher Scientific, Waltham, United States) by UHPLC-MS as described by Contrepois et al. (J. Proteome Res. 9, 5501-5509 (2010)).
SEC-MALS was done on the GIPSI platform at Université Paris-Sud on an HPLC (Shimadzu) with a Superdex 200 10/300 GL increase column (GE Healthcare, Pittsburgh, United States).
Prior to this work, the adalimumab T cell epitopes were identified in vitro by specific activation tests of the CD4 T lymphocytes by means of peptides overlapping a length of 15 or 20 amino acids. The regions comprising the T cell epitopes are mostly located within the heavy chain, CDR2 over as zone included between the E46 and E64 residues and the CDR3 between the L82c and T107 amino acids (Meunier, S. et al., Cellular & Molecular Immunology, 2019, Oct. 28. doi: 10.1038/s41423-019-0304-3). A T cell epitope is also described on the light chain upstream from the CDR3 (S76 to R90). (
APGKGLEWVSAITWNSGHIDYADSVEGRFTI
ISRDNAKNSLYLQMNSLRAEDTAVYY
LRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGP
KPGKAPKLLIYAASTLQSGVPSR
indicates data missing or illegible when filed
Thus, the 9-mer “ITWNSGHID” (SEQ ID NO:12) comprising the residues 51 to 58 (dark gray in
Since the CDRH2 and CDRH3 are major immunogenicity zones found in several donors, action was focused on these two regions in order to get the greatest possible immunogenicity reduction.
In order to reduce the T cell epitope/HLA II affinity and thus define a de-immunization strategy, the inventors first sought to understand the influence of the substitutions on the prediction from the netMHCllpan algorithm. A systematic approach was adopted for evaluating the influence of all the substitutions possible in the area of the CDRH2 and the CDRH3 on the presentation by the HLA II molecules. The netMHCllpan algorithm was used in parallel in order to predict the average effect of each mutation on a panel of 46 HLA II alleles covering over 80% of the global population. These predictions, shown in matrix form, show the relative effect of the mutation compared to the native sequence (
The CDRH2 offers many mutations serving to reduce the probability of interactions with HLA II molecules (substitutions colored with gray shading (darker gray=lower predicted immunogenicity,
The results of the prediction for CDRH3 are fairly similar, the hydrophilic residues are preferred for destabilizing the peptide/HLA II interaction. (
The modifications proposed by netMHCIIpan are globally substitutions towards the hydrophilic amino acid, with an effect that is that much larger if the native residue is hydrophobic. netMHCIIpan does not consider either the structure or the functionality of the antibody; these predictions are to be put into perspective with the tolerance to the mutation of these amino acids. In fact, elimination of all the hydrophobic amino acids over such important regions as the CDR seems extremely risky for the functionality and also for the structure of the antibody. Further residues belonging to these two CDR have been described for their contribution to the interaction with TNFα (Hu et al. J. Biol. Chem. 288, 27059-27067 (2013). In order to take into account all these functional and structural constraints, a second systematic approach was applied, this time to the function.
In order to identify, among the substitutions proposed by netMHCIIpan, those which make it possible to get functional mutations, the inventors decided to perform a functional study of these two regions as a first step. An approach by systematic mutation, call Deep Mutational Scanning (DMS), was chosen for identifying permissive amino acids within these immunogenic zones. This strategy provides for the functional evaluation of all possible substitution at each position with each variant comprising only one mutation.
The two immunogenic regions were processed in two independent libraries. They each cover one region of 20 residues and they were built by PCR assembly. For each of the 20 residues, diversity was introduced by means of a degenerate NNS codon coding for the set of 20 natural amino acids. Degenerate codons were inserted by independent PCR for each position. The 20 PCR products were mixed in equal molar proportions in order to get a library containing all the single substitutions for each of the 20 positions. The libraries were then cloned in an expression plasmid for the Yeast Surface Display (YSD) allowing the expression in Fab form. (
Based on the sequencing data, the enrichments of the parental sequence (wild type) and also that of each of the mutants were calculated. With these it was possible to calculate for each of the variants a score called selective value (i.e. fitness) which corresponds to the base two logarithm of the relative enrichment of the variant compared to the native sequence. In order to get an overall view of the permissivity of each of the regions, the mutants are shown in a matrix with the color proportional to the selective value thereof. For the negative selection, the amplitude of the selective values is from −8 to 3, or enrichment 256 times lower to 8 times greater than the native sequence. For the positive selection, the amplitude of the selective values is substantially the same. The mutants that have an enrichment greater or equal to the parental sequence are indicated with gray shading. The darker the gray, the greater the enrichment, indicating a good affinity for TNF alpha. (
The results obtained from the selection of mutants expressed but not functional are particularly interesting in order to define the residues participating in the action with the TNF alpha. The population derived from the positive selection is necessary for identification of mutations with which to maintain or increase the affinity. Finally, the combination of these two matrices of results allows identification of the residues that are important for the proper expression of the antibody (negative selective value in the two selection conditions).
For the region of the CDRH2, the negative selection serves to identify fairly clearly a central region of six consecutive residues (T52 to H56) for which the selective values are globally higher. This patch of six residues is supplemented by alanine in position 50 and aspartic acid in position 58 on each side. These eight residues, particularly enriched in the negative selection, therefore seem particularly important for the interaction. With the positive selection however, some functional mutations for these residues can be identified, in particular for small size residues (alanine, lysine or serine) T52 and S54. (
The residues involved in the interaction in the area of CDRH3 seem to be broken into two groups. The residues K94 and V95 on one side and the residues S99 to S100c a little farther. Fairly surprisingly and the opposite of the CDRH2, some of these residues are permissive for nonconservative substitutions. (
These first libraries serve to identify substitutions allowing retention of the functionality for these two regions of interest. They also serve to show the permissivity difference of the residues from CDRH2 and CDRH3, in particular those directly involved in the interaction. These data were next used in order to design combinatorial libraries with the objective of eliminating the T cell epitopes identified in these zones, while also assuring that they contain functional mutants.
The CDRH2 comprises two overlapping T cell epitopes which most likely share the interaction core “ITWNSGHID” (in dark gray in
The CDRH3 comprises a set of five overlapping T cell epitopes for which we defined a set of three probable interaction cores (in dark gray in
Each of the libraries was built by PCR assembly, with primers comprising degenerate codons for the CDRH2 and primers synthesized from mixtures of trinucleotides for the CDRH3. These libraries respectively have a diversity of 1.2×104 for CDRH2 and 3.8×106 for CDRH3. As before, they were cloned by homologous recombination in a plasmid for expression and screening in YSD. After transformation of the yeasts and induction of expression, the screening by FACS was done independently for the two libraries and each of the libraries underwent various selection steps.
Because of the significant diversity thereof, the library covering the CDRH3 made a direct selection by FACS difficult. This was therefore enriched a first time by magnetic sorting (MACS) with a 10 nM concentration of biotinylated TNF alpha. Except for this enrichment, the screening steps are the same for the two libraries. A first phase of three successive selections at equilibrium was done at decreasing concentrations of biotinylated TNF alpha. (
In order to understand the progression of the molecular diversity of the libraries during the selection steps, we chose to do a NGS sequencing after each of the selection steps. The sequencing data from the library covering the CDRH2 show that all the mutations were present in the library before starting screening. (
The sequencing data allowed us to calculate the enrichment values for each of the mutants at the end of the selection process. The 30 most enriched mutants in this library are shown in [Table 3].
It can be seen that the best mutants were enriched over 200 times; in total after complete screening, 174 mutants had a greater enrichment than that of adalimumab. The netMHCllpan algorithm was once again used in order to rank these mutants and 158 of them are predicted for potentially being less immunogenic.
For the library covering the CDRH3, the sequencing of the population after the first selection by magnetic screening revealed a high diversity on the residues 95 to 100 validating the construction of the library. (
It is also observed that the enrichment factors are higher for CDRH2, with values which can reach several million. After screening, the library comprises 310 mutants more enriched then the adalimumab native sequence and, as for CDRH2, a major part of them (282) have a reduced immunogenic potential according to netMHCIIpan.
At the end of selection of these two libraries, a large number of alternative sequences, potentially less immunogenic, resulted both for CDRH2 and CDRH3. These were combined in order to get an entirely de-immunized heavy chain.
In order to avoid the combination of incompatible mutations on CDRH2 and CDRH3, the choice was made to recombine all of the sequences contained in the two libraries following screening thereof. The sequences of the mutants from each of the libraries were extracted by PCR on the final populations after kinetic sorting. The combination of sequences was then done by random recombination via an assembly by PCR. This combined CDRH2+CDRH3 library was then cloned in the expression plasmid in YSD described above by homologous recombination during the transformation in yeast. The CDRH2+CDRH3 libraries respectively comprising a minimum of 489 and 234 mutants (found at least 10 times during sequencing) at the end of selection thereof, this combinatorial library contains a minimum of 105 variants. This library also incorporates the R90K substitution described in the Humira® patent as preserving the affinity and serving to germinalize the sequence and the CDRH3 region and in that way to remove a minor epitope (Salfed, J. G. et al. Human antibodies that bond human TNFa. (U.S. Pat. No. 6,258,562 B1)).
This library was screened according to the same process applied to the previous libraries; specifically three equilibrium sortings with increasing TNF alpha concentrations followed by a selection on the dissociation speed. (
After sequencing, the selected mutants were evaluated according to the enrichment thereof and 245 of them showed a value greater than the native sequence. Among them, over 200 mutants are predicted to have fewer interaction cores with the HLA II molecules than adalimumab. Their sequences however show a redundancy in their sequences, particularly in the area of CDRH3. ([Table 5]).
At the outcome of the selection steps, a reduced number of variants of interest were selected in order to carry out a complete biochemical characterization. The mutants were selected for their enrichment better than the native sequence and for their reduced immunogenic potential according to netMHCII pan but also by giving specific importance to selecting mutants with diverse sequences. Based on these criteria, eight mutants were selected to be characterized. ([Table 6]).
These mutants and also adalimumab were produced in Fab form in HEK cells in order to be characterized. The affinity of these mutants for TNF alpha was evaluated by Bio Layer Interferometry, all showed a greater affinity than that of adalimumab. ([Table 6])
SAITWNSGHI
V-VSYLST
GAINWNGGHR
V-SQYLPT
GTINWNGGHS
V-TTYLPT
GAINWNGGHR
indicates data missing or illegible when filed
For some, like the mutants 2 and 5, an affinity increased more than 10 times was measured. The increase of the affinity for the various clones is mostly due to a reduction of the dissociation speed which was a parameter selected during screening. While all these mutants have an enrichment greater than the native antibody, these values do not however always seem rigorously proportional to the affinities measured for these mutants.
The mutants 1, 2 and 7 were selected for the predicted reduced immunogenicity thereof in order to move the characterization forward. These mutants and also the native antibody were produced in HEK and purified to IgG format. An analysis by Orbitrap mass spectrometry served to confirm that the antibodies have a mass corresponding to that expected, with a resolution of order one Dalton. Additionally, an analysis by SEC-MALS (Size Exclusion Chromatography—Multi-Angle Light Scattering) also serve to confirm the monomeric nature of these antibodies.
The inventors were next interested more specifically in the effects of various substitutions that the mutants had on their interaction potential with the HLA II molecules. These effects are presented globally for CDRH2 and CDRH3 in
With this work it was possible to more precisely understand the role of each of the residues from two zones in which the epitopes of adalimumab (CDRH2 and CDRH3) were identified. The YSD platform with Fab format implemented with the high throughput sequencing allowed the functional study of the immunogenic regions by DMS in a first step. Through this first step the inventors were able to observe that, as they had imagined, the strict reduction of immunogenicity by elimination of T cell epitopes does not tend towards functional solutions. The netMHCllpan algorithm mostly proposes substituting hydrophobic residues with small amino acids and/or hydrophilic amino acids. There are however many hydrophobic residues in the CDR and they are particularly important for the structure thereof on which the functionality of the antibody depends directly. It therefore seems difficult to imagine that the substitution of all the hydrophobic amino acids from the CDR can allow retention of the functionality. Having made this observation, the DMS turned out to be even more important for the identification of the functional substitutions. The libraries thus generated and screened based on the DMS data and predictions from the netMHCllpan algorithm made it possible to identify mutants having an increased affinity for TNF alpha and a potentially reduced immunogenicity. The problem of preservation of the functionality during suppression of T cell epitopes located on major regions for interaction with the target can be addressed by getting these mutants. In that way they show that the proposed de-immunization strategy served to reconcile the two non-convergent objectives which are the functionality of the antibody and the reduction of the immunogenicity thereof.
The mutants have a reduction of interaction with the HLA II molecules predicted according to the netMHCllpan algorithm. Because of this, they are less susceptible to being presented by HLA II molecules and recognized by the T cell lymphocytes compared to adalimumab. These mutants thus constitute variants of adalimumab with reduced immunogenic potential and allow overcoming immunogenicity problems encountered with the anti-TNF alpha. These variants could represent a clinical improvement by allowing reduction of the patient immunization rate. Additionally, the inventors were able to show that they have an increased affinity for TNF alpha. To the extent where the biological activity of the anti-TNF antibody—specifically the neutralization of TNF alpha—depends on the affinity thereof for TNF, it can be expected that the mutants from the invention will have a biological activity greater than that of adalimumab. The mutants obtained at the outcome of this work could be considered as potential medication candidates positioning them as an improved version of adalimumab.
| Number | Date | Country | Kind |
|---|---|---|---|
| FR19 14645 | Dec 2019 | FR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/FR2020/052485 | 12/17/2020 | WO |
