High affinity antibodies to human IL-6 receptor

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DESCRIPTION

High affinity antibodies against the human IL-6 receptor

Statement of Related Art

Interleukin 6 (IL-6) is a pleotropic cytokine produced by immune and non-immune cells that plays a crucial role in the regulation of the immune response, acute phase reactions, and hematopoiesis. It binds to soluble IL-6R bound to the cell membrane (chain a) forming a binary complex, and this complex is capable of interacting with gp130 bound to the cell membrane (β chain), induces the formation of a signaling complex that comprises two each of IL-6, IL-6R, and gp130.

Antibodies against hIL-6R are described in US 5,670,373, 5,795,965, 5,817,790, 6,410,691 and EP 409 607B1. Therapeutic methods are described in US 5,888,510 and 6,723,319.

Brief summary of the invention

In a first aspect, the invention discloses human antibodies, preferably recombinant human antibodies, that specifically bind to the human interleukin-6 receptor (hIL-6R). These antibodies are characterized by high affinity binding to hIL-6R and slow dissociation kinetics, and by the ability to neutralize IL-6 activity. Antibodies may be full length (eg, an IgG1 or IgG4 antibody) or may comprise only an antigen-binding portion (eg, a Fab, F (ab ') fragment₂ or scFv), and can be modified to affect functionality, for example, to eliminate residual effector functions (Reddy et al. (2000) J. Immunol. 164: 1925-1933). In a preferred embodiment, the invention provides an antibody or antigen-binding fragment thereof, which binds to the human IL-6 receptor (SEQ ID NO: 1) with a Kd of about 500 pM or less, as measured by surface plasmon resonance. In a more specific embodiment, the antibody or antigen-binding fragment has a Kd of less than 300 pM, or less than 200 pM, or even less than 100 pM. In various embodiments, the antibody or antigen-binding fragment thereof blocks hIL-6 activity with an IC50 of 250 pM or less, as measured by luciferase bioassay. In more specific embodiments, the antibody or antigen-binding fragment thereof exhibits an IC₅₀ 150 pM or less.

In related aspects, the antibody or antigen-binding fragment of the invention binds hIL-6R with an affinity at least 2 times higher than that with which it binds monkey IL-6R. In more preferred embodiments, the antibody or antigen-binding fragment binds to hIL-6R protein (SEQ ID NO: 1) with an affinity that is up to about 3-fold greater than its binding to monkey IL-6R (domain Macaca fascicularis extracellular cell shown in SEQ ID NO: 251).

Therefore, the invention provides an antibody or antigen-binding fragment thereof, which specifically binds to the human interleukin-6 receptor (hIL-6R) with a Kd of 500 pM or less, as measured by surface plasmon resonance. , in which:

(i) the heavy chain CDRs comprise SEQ ID NOs: 5, 7 and 9 as heavy chain CDR1, CDR2 and CDR3 respectively, and (ii) the light chain CDRs comprise SEQ ID NOs: 13 , 15 and 17 as light chain CDR1, CDR2 and CDR3 respectively

In specific embodiments, the antibody or antigen-binding fragment thereof comprises the HCVR / LCVR pair of SEQ ID NO: 3/11.

In a second aspect, the invention provides isolated nucleic acid molecules that encode an antibody or antigen-binding fragment of an antibody of the invention. In one embodiment, the nucleic acid molecule of the invention encodes an antibody or fragment thereof comprising an HCVR as described above. In specific embodiments, the nucleic acid molecule encoding HCVR is SEQ ID NO: 2. In a related aspect, the invention provides an isolated nucleic acid molecule encoding an LCVR as described above. In specific embodiments, the nucleic acid molecule encoding the LCVR is SEQ ID NO: 10.

The invention discloses anti-hIL-6R antibodies or antigen-binding fragments thereof having a modified glycosylation pattern. In some applications, a modification to remove undesirable glycosylation sites, or an antibody lacking a fucose residue on an oligosaccharide chain may be useful, for example, to increase antibody-dependent cellular cytotoxicity (ADCC) (see Shield et al. (2002) JBC 277: 26733). In other applications, a modification of a galactosylation can be performed to modify complement-dependent cytotoxicity (CDC).

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In additional aspects, the invention provides recombinant expression vectors carrying the nucleic acid molecules of the invention, and host cells into which said vectors have been introduced, as well as methods of generating the antibodies or antigen-binding fragments of the invention obtained by culturing the host cells of the invention. The host cell can be a prokaryotic or eukaryotic cell, preferably the host cell is an E. coli cell or a mammalian cell, such as a CHO cell.

In a further aspect, the invention features a pharmaceutical composition comprising a human antibody or antigen-binding fragment of an antibody that specifically binds hIL-6R and a pharmaceutically acceptable carrier.

In further aspects, the invention provides an antibody or antigen-binding fragment of an antibody, as defined above, for use in attenuating or inhibiting an IL-6-mediated disease or disorder in a human, in the that the IL-6-mediated disease or disorder is arthritis, an inflammatory bowel disease, or systemic lupus erythematosus. The disorder is therefore selected from arthritis, including chronic rheumatoid arthritis; an inflammatory bowel disease, including Crohn's disease and ulcerative colitis; or systemic lupus erythematosus

In additional aspects, the invention provides the use of an antibody or antigen-binding fragment of an antibody, as defined above, in the manufacture of a medicament for use to attenuate or inhibit an IL-6-mediated disease or disorder. in a human, wherein the IL-6 mediated disease or disorder is arthritis, an inflammatory bowel disease, or systemic lupus erythematosus.

Other objects and advantages will be apparent from a review of the subsequent detailed description.

Detailed description

Before describing the present methods, it should be understood that this invention is not limited to the particular experimental methods and conditions described, as such methods and conditions may vary. It should also be understood that the terminology used herein is solely for the purpose of describing particular embodiments, and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, the preferred methods and materials are described below.

The term human IL6R (hIL-6R), as used herein, is intended to refer to a human cytokine receptor that specifically binds to interleukin 6 (IL-6). The extracellular domain of hIL-6R is shown in SEQ ID NO: 1.

The term antibody, as used herein, is intended to refer to immunoglobulin molecules that comprise four polypeptide chains, two heavy (H) chains, and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains: CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is composed of a domain (CL1). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed flanking regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino-terminal end to the carboxy-terminal end in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

The term "antigen-binding portion of an antibody" (or simply antibody portion or antibody fragment), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen. (for example, hIL-6R). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments included within the term "antigen-binding portion of an antibody" include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL1, and CH1 domains; (ii) an F (ab ') fragment₂, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al. (1989) Nature 241: 544-546), consisting of a VH domain ; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined, using recombinant methods, by means of a synthetic linker that allows them to be generated as a single contiguous chain in which the VL and VH regions are paired to form monovalent molecules (known

ES 2 398 076 T3 as single chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single chain antibodies are also intended to be included within the term "antigen-binding portion of an antibody." Also included are other forms of single chain antibodies, such as diabodies (see, for example, Holliger et al. (1993) Proc. Natl. Acad Sci. USA 90: 6444-6448).

A neutralizing or blocking antibody, as used herein, is intended to refer to an antibody whose binding to hIL-6R results in inhibition of the biological activity of hIL-6. This inhibition of hIL-6 biological activity can be assessed by measuring one or more indicators of hIL6 biological activity known in the art, such as hIL-6-induced cell activation and hIL-6 binding to hIL-6R. (see examples below).

A CDR or complementarity determining region is a region of hypervariability sandwiched within regions that are more conserved, termed flanking regions (FRs). In different embodiments of the anti-hIL-6R antibody or fragment of the invention, the FRs may be identical to human germline sequences, or they may be naturally or artificially modified. A group of CDRs can be defined as a consensus amino acid sequence.

The term "surface plasmon resonance", as used herein, refers to an optical phenomenon that allows the analysis of interactions in real time by detecting alterations in protein concentrations within a biosensor matrix, for example using the BIAcore ™ system (Pharmacia Biosensor AB).

The term "epitope" is an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule known as a paratope. A single antigen can have more than one epitope. Epitopes can be conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. In certain circumstances, an epitope can include saccharide moieties, phosphoryl groups, or sulfonyl groups on the antigen.

The term "substantially or substantially identical identity", when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with the appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is an identity of nucleotide sequence at least about 95% and more preferably at least about 96%, 97%, 98%, or 99% of the nucleotide bases, as measured by any well-known sequence identity algorithm, such as FASTA, BLAST, or Gap, as discussed below.

As applied to polypeptides, the term "substantially similar or substantially similar" means that two peptide sequences, when optimally aligned, such as by the GAP or BESTFIT programs using the default gap weights, share a sequence identity of at least 95 %, even more preferably a sequence identity of at least 98% or 99%. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. A conservative amino acid substitution is one in which one amino acid residue is substituted for another amino acid residue that has a side chain (R group) with similar chemical properties (eg, charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity can be adjusted upward to correct for the conservative nature of the substitution. The means of making this adjustment are well known to those of skill in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331. Examples of amino acid groups that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine and tryptophan; 5) basic side chains: lysine, arginine and histidine; 6) acidic side chains: aspartate and glutamate, and 7) the sulfur-containing side chains are cysteine ​​and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alaninevaline, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix described in Gonnet et al. (1992) Science 256: 1443 45. A moderately conservative substitution is any change that has a non-negative value in the PAM250 log likelihood matrix.

Sequence similarity for polypeptides, which is also called sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software contains programs such as Gap and Bestfit that can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a protein from wild type and a mutein thereof. See, for example, GCG

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Version 6.1. Polypeptide sequences can also be compared using FASTA, using the default or recommended parameters, a program in GCG Version 6.1. FASTA (eg, FASTA2 and FASTA3) provides alignments and percentages of sequence identity of the regions of best overlap between the query and search sequences (Pearson (2000) above). Another preferred algorithm when comparing a sequence of the invention with a database containing a large number of sequences from different organisms is the BLAST computer program, especially blastp or tblastn, using the default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215: 403 410 and Altschul et al. (1997) Nucleic Acids Res. 25: 3389 402.

Preparation of human antibodies

Methods for generating human antibodies include, for example, Velocimmune ™ (Regeneran Pharmaceuticals), XenoMouse ™ technology (Green et al. (1994) Nature Genetics 7: 13-21; Abgenix), the minilocus strategy, and phage display ( and see, for example, US 5,545,807, US 6,787,637). Velocimmune ™ technology (US 6,596,541) includes a method of generating a fully human antibody of high specificity against a selected antigen. This technology involves the generation of a transgenic mouse having a genome comprising human heavy and light chain variable regions operably linked to endogenous mouse constant region loci, such that the mouse produces an antibody comprising a human variable region and a mouse constant region in response to antigenic stimulation. DNA encoding the antibody heavy and light chain variable regions is isolated and operably linked to DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in a cell capable of expressing the fully human antibody. In a specific embodiment, the cell is a CHO cell.

Antibodies may be therapeutically useful in blocking a ligand-receptor interaction or inhibiting receptor component interaction, rather than by killing cells through complement fixation (complement-dependent cytotoxicity) (CDC) and by the involvement of antibody-dependent cell-mediated cytotoxicity (ADCC). The constant region of an antibody is important in an antibody's ability to fix complement and mediate cell-dependent cytotoxicity. Therefore, the isotype of an antibody can be selected based on whether it is desirable for the antibody to mediate cytotoxicity.

Human immunoglobulins can exist in two forms that are associated with hinge heterogeneity. In one form, an immunoglobulin molecule comprises a stable four-chain construct of approximately 150-160 kDa in which the dimers are held together by an interchain heavy chain disulfide bond. In a second form, the dimers are not linked by interchain disulfide bonds and a molecule of approximately 75-80 kDa is formed composed of a covalently coupled heavy and light chain (half antibody). These forms have been extremely difficult to separate, even after affinity purification. The frequency of occurrence of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the isotype of the antibody hinge region. In fact, a single amino acid substitution in the hinge region of human IgG4 can significantly reduce the appearance of the second form (Angal et al. (1993) Molecular Immunology 30: 105) to levels typically seen using a hinge. of human lgG1. The present invention includes antibodies that have one or more mutations in the hinge, CH2 or CH3 region that may be desirable, eg, in production, to improve the performance of the desired antibody form.

The antibodies of the invention are preferably prepared with the use of Velocimmune ™ technology. A transgenic mouse in which the endogenous immunoglobulin heavy and light chain variable regions are replaced with the corresponding human variable regions is exposed to the antigen of interest, and lymphatic cells (such as B cells) are recovered from the antibody-expressing mice. . Lymphatic cells can be fused with a myeloma cell line to prepare immortal hybridoma cell lines, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce specific antibodies against the antigen of interest. DNA encoding the heavy chain and light chain variable regions can be isolated and joined to desirable isotypic heavy chain and light chain constant regions. Such an antibody protein can be produced in a cell, such as a CHO cell. Alternatively, DNA encoding antigen-specific chimeric antibodies or light and heavy chain variable domains can be isolated directly from antigen-specific lymphocytes.

In one aspect, the transgenic mouse comprises up to 18 functional human variable heavy chain genes and 12 functional human variable kappa light chain genes. In another aspect, the transgenic mouse comprises up to 39 human variable heavy chain genes and 30 human variable kappa light chain genes. In yet another aspect, the transgenic mouse comprises up to 80 human variable heavy chain genes and 40 human variable kappa light chain genes.

In general, the antibodies of the present invention possess very high affinities, typically possessing Kd values ​​of about 10^-9 to about 10^-12 M, when measured by binding to antigen immobilized on a solid phase or in solution phase.

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Initially, high affinity chimeric antibodies having a human variable region and a mouse constant region are isolated. As described below, antibodies are characterized and selected for their desirable characteristics, including binding affinity to hIL-6R, ability to block hIL-6, and / or selectivity for human protein. The mouse constant regions are substituted with a desired human constant region to generate the fully human antibody of the invention, for example wild-type or modified IgG4 or IgG1 (eg, SEQ ID NO: 242, 243, 244). Although the constant region selected may vary according to a specific use, the high affinity antigen-binding and target specificity characteristics reside in the variable region.

Epitope mapping and related technologies

To screen for antibodies that bind to a particular epitope, a routine cross-blocking assay can be performed such as that described in Antibodies: A Laboratory Manual 1988 Cold Spring Harbor Laboratory, Harlow and Lane, eds. Other methods include alanine scanning mutants, peptide blots (Reineke (2004) Methods Mol Biol 248: 443-63), or peptide cleavage analysis as described in the examples below. Furthermore, methods such as epitope cleavage, epitope extraction and chemical modification of antigens can be employed (Tomer (2000) Protein Science: 9: 487-496).

Modification Assisted Profiling (MAP), also known as Antigen Structure Based Antibody Profiling (ASAP), is a method that classifies large amounts of monoclonal antibodies (mAbs) directed against the same antigen according to the similarities of the binding profile of each antibody to chemically or enzymatically modified antigen surfaces (United States Patent Application Publication No. 2004/0101920). Each category may reflect a unique epitope distinctly different from, or partially overlapping with, an epitope represented by another category. This technology enables the rapid screening of genetically identical antibodies, so that characterization can focus on genetically distinct antibodies. When applied to hybridoma screening, MAP can facilitate the identification of rare hybridoma clones with desired characteristics. MAP can be used to separate the hIL-6R antibodies of the invention into groups of antibodies that bind to different epitopes.

Agents useful for altering the structure of the immobilized antigen are enzymes, such as, for example, proteolytic enzymes and chemical agents. The antigenic protein can be immobilized on the surfaces of biodetector microplates or polystyrene beads. The latter can be processed, for example, with an assay such as a Luminex ™ multiple detection assay (Luminex Corp., TX). Due to the ability of Luminex ™ to handle a multiple assay with up to 100 different types of beads, the Luminex ™ provides nearly unlimited antigenic surfaces with various modifications, resulting in improved resolution in profiling antibody epitopes over one assay. biosensor.

Therapeutic administration and formulations

The administration of therapeutic entities according to the invention will be administered with suitable vehicles, excipients and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance and the like. A multitude of suitable formulations can be found in the list of pharmaceutical specialties known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15^to ed, Mack Publishing Company, Easton, Pa., 1975), particularly Chapter 87 by Blaug, Seymour, therein. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oily lipids, lipids (cationic or anionic) containing vesicles (such as Lipofectin ™), DNA conjugates, anhydrous absorption pastes, oil-in-water emulsions. and water-in-oil, carbowax emulsions (polyethylene glycols of various molecular weights), semisolid gels, and semisolid mixtures containing carbowax. Any of the above mixtures may be appropriate in treatments and therapies according to the present invention, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerable with the route of administration. See also Powell et al. PDA (1998) J Pharm SciTechnol. 52: 238-311 and citations therein for additional information related to excipients and vehicles well known to pharmaceutical chemists.

Examples

The following examples are presented to provide those skilled in the art with a complete disclosure and description of how to prepare and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors consider their invention. Efforts have been made to ensure precision with respect to the numbers used (eg quantities, temperature, etc.), but some experimental errors and deviations should be assumed. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees centigrade, and pressure is or near atmospheric.

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Example 1. Generation of human antibodies against the human IL-6 receptor.

Rodent immunization can be performed by any method known in the art (see, for example, Harlow and Lane (1988) above; Malik and Lillehoj, Antibody techniques: Academic Press, 1994, CA). In a preferred embodiment, the hIL-6R antigen is administered directly to mice comprising DNA loci encoding both the heavy chain variable region and the kappa light chain variable region of human Ig (Velocimmune ™, Regeneron Pharmaceuticals, Inc .; US 6,596,541), with an adjuvant to stimulate the immune response. Such adjuvant includes Freund's complete and incomplete adjuvant, MPL + TDM adjuvant system (Sigma) or RIBI (muramil dipeptides) (see O'Hagan, Vaccine Adjuvant, by Human Press, 2000, NJ). Said adjuvant can prevent the rapid dispersal of the polypeptide by sequestration of the antigen in a depot of local prolonged administration, and can contain factors that can stimulate the immune response of the host. In one embodiment, hIL-6R is indirectly administered as a DNA plasmid containing the hIL-6R gene and expressing hIL-6R using the host's cellular protein expression machinery to produce antigenic polypeptide in vivo. In both strategies, the immunization schedule requires several administrations a few weeks apart. The antibody immune response is monitored by a standard antigen-specific immunoassay. When the animals reached their maximal immune response, the antibody-expressing B cells were harvested and fused with mouse myeloma cells to preserve their viability, forming hybridoma cells. To select for functionally desirable monoclonal antibodies, the conditioned media of the hybridoma cells or transfected cells were screened for their specificity, antigen-binding affinity, and potency in blocking the binding of hIL-6 to hIL-6R (described below ).

Example 2. Anti-hlL6R Antibodies Generated by Direct Isolation of Splenocytes

DNA encoding VH and VL domains can be isolated directly from a single antigen-positive B cell. Briefly, the transgenic mouse immunized with hIL-6Ra was sacrificed and its splenocytes were harvested. The erythrocytes were removed by lysis, followed by sedimentation of the collected splenocytes. The resuspended splenocytes were first incubated with a cocktail of human IgG, FITC-anti-mFc and biotin-IL6Ra for 1 hour. The stained cells were washed twice with PBS, then stained with a cocktail of human and rat IgG, APCanti-mlgM and SA-PE for one hour. The stained cells were washed once with PBS and analyzed by flow cytometry on a MoFlo (Cytomation). Each IgG positive, IgM negative and antigen positive B cell was separated and plated in a separate well of a 96-well plate. Antibody gene RT-PCR was performed from these B cells according to a method described by Wang et al. (2000) (J Immunol Methods 244: 217-225). Briefly, cDNAs for each individual B cell were synthesized by RT-PCR. Each resulting RT product was then partitioned and transferred to two corresponding wells in two 96-well plates. A set of the resulting RT products was first amplified by PCR using a 5 'degenerate primer specific for the leader sequence of the variable region of the human IgG heavy chain and a 3' primer specific for the constant region of the heavy chain of mouse, to form an amplicon. The amplicon was then amplified again by PCR using a 5 'degenerate primer set specific for flanking region 1 of the human IgG heavy chain variable region sequence and a nested 3' primer specific for the constant region of the mouse heavy chain. The other set of the resulting RT products was first amplified by PCR using a 5 'degenerate primer specific for the human kappa light chain variable region leader sequence and a 3' primer specific for the kappa light chain constant region. mouse to form an amplicon. The amplicon was then amplified again by PCR using a 5 'degenerate primer set specific for flanking region 1 of the human kappa light chain variable region sequence and a nested 3' primer specific for the constant region of the chain. slight mouse kappa. The heavy chain and light chain PCR products were cloned into Sap 1 linearized antibody vectors containing the IgG1 heavy chain constant region and the kappa light chain constant region, respectively. The heavy chain plasmid has a lox2272 site and a lox511 site flanking the heavy chain expression cassettes. Furthermore, immediately downstream of lox2272 in the heavy chain plasmid there is a hygromycin resistance gene lacking a promoter and initiator ATG. The hygromycin resistance gene is also transcriptionally linked to a downstream eGFP gene via an IRES sequence. The light chain plasmid has a loxP site and a lox2272 site flanking the light chain expression cassette. Furthermore, the light chain plasmid has an SV40 promoter immediately before an ATG at the lox2272 site, so that upon integration into an appropriate host cell the SV40 promoter proximal to lox2272 and the initiator ATG of the light chain plasmid are they place the hygromycin resistance gene on the heavy chain plasmid in the appropriate reading frame to allow transcription and translation of the hygromycin resistance and eGFP genes. Purified recombinant plasmids having a heavy chain variable region sequence and plasmids having a light chain variable region sequence from the same B cell were combined and then transfected together with a plasmid expressing Cre recombinase, into one line. modified CHO host cell. The modified CHO host cell line contains, 5 'to 3', a loxP site, an eCFP, a lox2272 site, DsRed, and a lox511 site at a transcriptionally active locus. Accordingly, the host CHO cell can be isolated by flow cytometry as a blue positive, red positive, and green negative cell. When recombinant plasmids expressing heavy chain and light chain genes are transfected together with a

ES 2 398 076 T3 plasmid expressing Cre recombinase, site-specific recombinase mediated by Cre recombinase results in integration of antibody plasmids at the chromosomal locus containing lox sites and replacement of eCFP genes and DSRed. The recombinants can then be isolated as blue negative, red negative and green positive cells by flow cytometry. Accordingly, CHO cells transfected with recombinant plasmids having a heavy chain variable region sequence and plasmids having a light chain variable region sequence from the same B cell were separated by flow cytometry, and appropriate recombinants showing the phenotype negative for blue, negative for red and positive for green were isolated, and CHO cell lines expressing recombinant antibody were established from the isolated clones.

Example 3. Determination of antigen-binding affinity

The Kd of antigen binding to selected antibodies described above was determined by surface kinetics in a real-time biosensor surface plasmon resonance assay (BIAcore ™). More specifically, the affinity of the antibodies for human IL-6R was measured using a BIAcore® 2000 or BIAcore® 3000. The antibody was captured on a surface with anti-mouse IgG and exposed to various concentrations of recombinant hIL-6R protein in monomeric or dimeric form. Kinetic analysis was performed using BIAevaluation ™ software to obtain rate constants of association and dissociation.

The binding affinities of the antibodies to hIL-6R were also measured for hybridoma conditioned media or purified proteins by plaque-based competition immunoassay. Antibody proteins were purified using Protein G affinity chromatography from hybridoma cell conditioning medium depleted of bovine IgG (Invitrogen). For the competition ELISA, in summary, constant amounts of antibody at different levels were premixed with serial dilutions of antigenic protein, hIL-6R-hFc, ranging from 0 to 10 pg / ml, and incubated for two hours at room temperature. to achieve a pseudo-binding equilibrium between the antibody and the antigen. These solutions were then transferred to 96-well plates previously coated with hIL-6R-hFc to allow free antibody in the mixtures to bind to the hIL-6R-hFc coated on the plate. Plates were typically coated with 1 to 2 pg / ml hIL-6R-hFc protein in PBS solution overnight at 4 ° C, followed by nonspecific blocking with BSA. After washing off excess antibody in solution, antibodies bound to the plate were detected with HRP-conjugated goat anti-mouse IgG or IgA polyclonal antibody reagent and developed using chemiluminescent or colorimetric substrates. The dependence of the signals on the antigen concentrations in solution was analyzed with a 4 parameter fit analysis using Prism software (Graph Pad) and was reported as IC50. A competition immunoassay was also carried out using a solution phase stable Kinexa ™ instrument (Sapidyne Inc.).

Results are shown in Table 1 (control: humanized monoclonal antibody against human IL-6R (US Patent No. 5,817,790 SEQ ID Nos: 69 and 71). Antibody (HCVR and LCVR amino acid sequences): VQ8A9-6 (3, 11); VQ8F11-21 (19, 27); VV7G4-1 (35, 43); VV7G4-10 (51, 59), VV6C10-1 (67, 75); VV6C10-3 (83, 91); VV6C10-4 (99, 107); W6F12-11 (115, 123); VV9A6-11 (131, 139); W6A9-5 (147, 155), VV3D8-4 (163, 171); VV1G4-7 (179, 187); 248982-13-1-E5 (195, 203); 248982-13-2-A9 (211, 219). The Kd of monomer and dimer was determined by BIAcore ™; the Kd in solution by Kinexa ™; IC50 by ELISA assays (nd = not determined).

Table 1. Antigen binding affinity

<td>Antibody</td><td>K_d Monomer (nM)</td><td>K_d Dimer (nM)</td><td>K_d Monomer in solution (nM)</td><td>CI ELISA₅₀ Dimer (nM)</td>

<td>VQ8A9-6</td><td> 0,222</td><td> 0,101</td><td> 0,120</td><td> 0,004</td>

<td>VQ8F11-21</td><td> 0,067</td><td> 0,023</td><td> 0,009</td><td> 0,008</td>

<td>VV3D8-4</td><td> 2,410</td><td> 0,172</td><td> 1,910</td><td> 0,013</td>

<td>VV6A9-5</td><td> 0,097</td><td> 0,146</td><td> 0,032</td><td> 0,005</td>

<td>VV1G4-7</td><td> 0,225</td><td> 0,070</td><td> 0,197</td><td> 0,041</td>

<td>VV6C10-1</td><td> 0,267</td><td> 0,032</td><td> 2,050</td><td> 0,010</td>

<td>VV6F12-11</td><td>nd</td><td>nd</td><td>nd</td><td> 0,033</td>

<td>VV7G4.10</td><td>nd</td><td>nd</td><td>nd</td><td> 1,980</td>

<td>VV9A6-11</td><td>nd</td><td>nd</td><td>nd</td><td> 0,347</td>

<td>VV6C10-3</td><td>nd</td><td>nd</td><td>nd</td><td> 0,009</td>

<td>248982-13-1-E5</td><td> 0,987</td><td> 0,785</td><td>nd</td><td> 0,360</td>

<td>248982-13-2-A9</td><td> 2,870</td><td>nd</td><td>nd</td><td> 0,054</td>

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<td>Antibody</td><td>Kd Monomer (nM)</td><td>Kd Dimer (nM)</td><td>Kd Monomer in solution (nM)</td><td>ELISA IC50 Dimer (nM)</td>

<td>Control</td><td> 1,790</td><td>nd</td><td> 1,960</td><td>nd</td>

Example 4. Neutralization of hIL-6 activity

The hIL-6 blocking activities of the anti-hIL-6R antibodies of the invention were explored by hIL-6 blocking immunoassays, in vitro hIL-6 dependent cell growth bioassays, and surface plasmon resonance (BIAcore ™) . The immunoassay was used to explore the ability of the tested antibody to block the binding of hIL-6 to hIL-6R, and the in vitro bioassay was used to determine the potency of the antibodies in neutralizing hIL-mediated cellular signal transduction. -6R.

For immunoassay, recombinant protein hIL-6 was coated on a 96-well plate in PBS buffer overnight at 4 ° C. This plate was used to capture free hIL-6R-hFc from antibody sample solutions, and the amount of captured hIL-6R-hFc was quantified according to the standard curve. The sample solutions were composed of a constant amount of recombinant hIL-6R-hFc protein (100 pM) and varying amounts of antibody, in crude hybridoma conditioned medium or as purified antibody protein, ranging from 0 to about 50 nM in serial dilutions. Antigen-antibody mixtures were incubated at room temperature for ~ 2 hours to allow antigen-antibody binding to reach equilibrium. The equilibrated sample solutions were then transferred to the hIL-6 coated plates for measurement of free hIL-6R-hFc. After 1 hour of binding, the plate was washed and bound hIL-6R-hFc was detected using HRP-conjugated goat anti-hFc polyclonal antibodies (Jackson Immuno Research), and developed using TMB substrate (BD Pharmigen). IC50's were determined as the amount of antibody required to reduce 50% of the detectable IL-6R-hFc against plaque-bound ligand hIL-6. The results are shown in the first column of Table 2.

In addition, the ability of the test antibody to block the binding of hIL-6 to the hIL-6R receptor was determined using surface plasmon resonance. Purified hIL-6R-hFc antigen molecules were captured by polyclonal goat anti-human IgG antibodies immobilized on the surface of CM-5 via amine coupling at a density of 250 UR. The hIL-6 solution (0.25 ml, 50 nM) was injected onto the receptor surface and bound hIL-6 was recorded (first IL-6 injection). The bound hIL-6 was then removed with a 3M MgCl2 stimulation, followed by conditioning buffer. Anti-hIL6R antibody in hybridoma conditioned medium was injected onto the captured receptor surface, followed by a second injection of hIL-6. The percent reduction in hIL-6 binding resulting from preformed receptor-antibody complex was used as a score to define hIL-6 blockers from non-blockers (second column, Table 2).

Table 2. Neutralization of hlL-6 binding

<td>Antibody</td><td>IC50 Inhibition of hlL6R / hlL6 binding (nM)</td><td>Inhibition of hlL6 / hlL6R binding (%)</td><td>IC50 Inhibition of cell proliferation of XG-1 (nM)</td><td>IC50 luciferase activity of HepG2 / Stat3 (nM)</td>

<td>VQ8A9-6</td><td> 0,39</td><td> 68</td><td> 0,40</td><td> 0,097</td>

<td>VQ8F11-21</td><td> 0,12</td><td> 98</td><td> 0,62</td><td> 0,135</td>

<td>VV3D8-4</td><td> 0,61</td><td> 93</td><td> &gt;100</td><td>nd</td>

<td>VV6A9-5</td><td> 0,35</td><td> 100</td><td> 1,10</td><td> 0,188</td>

<td>VV1G4-7</td><td> 1,10</td><td> 34</td><td> 1,80</td><td> 0,578</td>

<td>VV6C10-1</td><td> 4,60</td><td> 61</td><td> &gt;6,90</td><td>nd</td>

<td>VV6F12-11</td><td> 2,20</td><td>nd</td><td>nd</td><td>nd</td>

<td>VV7G4-10</td><td> 13,00</td><td>nd</td><td>nd</td><td>nd</td>

<td>VV9A6-11</td><td> 0,50</td><td>nd</td><td>nd</td><td>nd</td>

<td>VV6C10-3</td><td> 0,06</td><td>nd</td><td>nd</td><td>nd</td>

<td>Control</td><td> 2,20</td><td> 91</td><td> 1,50</td><td> 0,854</td>

The ability of hIL-6R antibodies to block hIL-6 activity in vitro was measured in the hIL-6 XG-1 dependent myeloma line. XG-1 cells maintained in medium containing hIL-6 were washed twice with medium without hIL-6 and cultured for ~ 24 hours in medium without hIL-6 to reduce residual hIL-6. The serum-deprived cells were then centrifuged and resuspended in the medium at 4 x 10⁵ cells per ml, and

ES 2 398 076 T3 plated 20,000 cells per well in a 96-well tissue culture plate. The purified antibody proteins were serially diluted in medium and added to the plated cells at concentrations ranging from 0 to 50 nM. Subsequently, recombinant hIL-6 was added to the wells at a final concentration of 8 pM. Cells were allowed to grow for ~ 72 hours at 37 ° C in a humidified CO incubator.₂ at 5%. At the end of the growth period, live cells were measured using the CCK-8 kit (Dojindo, Japan). IC50s were determined as described above, and are described in the third column of Table 2.

The ability of hIL-6R antibodies to block hIL-6 activity was also measured in vitro in the hIL-6 sensitive human hepatoma cell line, HepG2. HepG2 cells were transfected with a reporter plasmid containing a STAT3 response element (signal transducer and activator of transcription 3) linked to a luciferase gene. Transfected cells were trypsinized, centrifuged, and resuspended in medium at approximately 2.5 x 10⁵ cells per ml and plated at 20,000 cells per well in a 96-well tissue culture plate. The purified antibody proteins were serially diluted in medium and added to the plated cells at concentrations ranging from 0 to 100 nM. Subsequently, recombinant hIL-6 was added to the wells at a final concentration of 50 pM. Response was measured after incubating cells for 6 hours at 37 ° C in a humidified 5% CO2 incubator. Luciferase activity was measured with the Steady-Glo ™ Luciferase Assay System (Promega). IC50s were determined as described above, and are described in the fourth column of Table 2.

Example 5. Diversity of binding epitopes

An antibody binding competition immunoassay was performed using humanized antibody against human IL-6R as a control. Briefly, a 96-well immunosorbent plate was coated with 20 ng per well of recombinant protein hIL-6R overnight at 4 ° C. After blocking nonspecific binding with BSA, the hIL-6R binding sites on one half of the plate were saturated with binding of the control antibody by adding 500 ng of the control per well, and the other half of the plate was added binding buffer only. After binding for three hours at room temperature, the purified antibodies were added to a final concentration of 50 ng / ml with and without the pre-existing control antibody in the well. After an additional hour of binding, the free antibody was washed away and the antibody bound to the plate was detected with HRP-conjugated goat anti-IgG or mouse IgA polyclonal antibody and the plate was developed using HRP chromatic substrates, and the absorbance at 450 nm was recorded. Percentage deductions of anti-hIL6R antibody binding by the presence of the control antibody are listed in Table 3 below. A similar experiment was performed using surface plasmon resonance technology (Table 3). Both methods produced consistent results. Antibodies VQ8F11, VV3D8, VV6A9, VV6C10-1 bound epitopes that overlapped with the control antibody; whereas the antibodies VQ8A9, VV1G4, VV6F12, VV7G4, VV9A6 and VV6C10-3 appeared to bind to different epitopes since antigen binding was not blocked by the control antibody. Partial competition may be the result of steric hindrance of the first bound antibody, even though the epitopes may not be overlapping.

Table 3. Antigen-binding competition with control antibody

<td>Antibody</td><td>BIAcore ™ (% reduction)</td><td>Immunoassay (% reduction)</td>

<td>VQ8A9-6</td><td> 26</td><td> 3</td>

<td>VQ8F11-21</td><td> 96</td><td> 79</td>

<td>VV3D8-4</td><td> 97</td><td> 84</td>

<td>VV6A9-5</td><td> 96</td><td> 84</td>

<td>VV1G4-7</td><td> 12</td><td> 3</td>

<td>VV6C10-1</td><td> 90</td><td> 80</td>

<td>VV6F12-11</td><td>nd</td><td> 3</td>

<td>VV7G4-10</td><td>nd</td><td> 26</td>

<td>VV9A6-11</td><td>nd</td><td> 18</td>

<td>VV6C10-3</td><td>nd</td><td> 1</td>

Example 6. Cross-linking property between species

Four antibodies were tested for cross-reactivity with recombinant monkey IL-6R protein using BIAcore ™ technology. Briefly, a biosensor microplate on which polyclonal goat anti-mouse Fc was immobilized was used to present anti-hIL-6R monoclonal antibodies at a density

ES 2 398 076 T3 of approximately 75 UR. Monomeric or recombinant human IL-6R protein (Macaca fascicularis, extracellular domain; sEc ID No: 251), at a concentration range between 1.25-40 nM, was injected onto the antibody surface. Binding of the receptor to the antibody and the dissociation of the bound complex were monitored in real time. Both the association rate constant (ka) and the dissociation rate constant (kd) were obtained and the Kd was calculated (Table 4).

Table 4. Comparison of human and monkey IL-6R binding affinity

<td>Antibody</td><td>Antigen</td><td>ka (M^-1S^-1)</td><td>kd (S^-1)</td><td>Kd (nM)</td>

<td rowspan="2">Control</td><td>Human IL6R</td><td>1.74E + 05</td><td>1.67E-04</td><td> 0,963</td>

<td>Monkey IL6R</td><td>1.44E + 05</td><td>1.68E-04</td><td> 1,170</td>

<td rowspan="2">VQ8F11-21</td><td>Human IL6R</td><td>8.51 E + 05</td><td>4.38E-05</td><td> 0,051</td>

<td>Monkey IL6R</td><td>3.39E + 05</td><td>4.86E-05</td><td> 0,143</td>

<td rowspan="2">VV1G4-7</td><td>Human IL6R</td><td>2.57E + 05</td><td>6.18E-05</td><td> 0,240</td>

<td>Monkey IL6R</td><td>no union</td><td></td><td></td>

<td rowspan="2">VV6A9-5</td><td>Human IL6R</td><td>5.18E + 05</td><td>8.41 E-05</td><td> 0,162</td>

<td>Monkey IL6R</td><td>5.00E + 05</td><td>7.70E-05</td><td> 0,154</td>

<td rowspan="2">VQ8A9-6</td><td>Human IL6R</td><td>7.32E + 05</td><td>2.76E-04</td><td> 0,377</td>

<td>Monkey IL6R</td><td>7.31E + 05</td><td>4.16E-04</td><td> 0,569</td>

Among the four antibodies tested, VQ8F11, VV6A9, and VQ8A9 reacted strongly with the monkey receptor with Kd values ​​differing by up to about 1.5 to about 3-fold from human receptor binding, respectively. VV1G4, which was not blocked by the control antibody (Table 3), did not show binding to the monkey receptor despite strong binding to the human receptor with a K_d 241 pM.

Example 7. Effect of constant region on binding affinity

The monomeric hIL-6R binding affinity of four antibodies having mouse IgG, human IgG1, or human IgG4 (wild-type and modified) was determined using BIAcore ™ as described above, except that an antibody surface was used. goat anti-human Fc polyclonal to capture hlgG antibodies. Monomeric hIL-6R was injected at concentrations of 12.5, 6.25, 3.12 and 1.56 nM. The ability of antibodies to neutralize hIL-6 dependent HepG2 / STAT3 signal transduction was also determined in a luciferase assay (CI₅₀). The ci₅₀ for different IgG isotypes they were similar, suggesting the absence of an effect of the isotype on the affinity of the antibody for the antigen.

Table 5. Comparison of IgG isotypes

<td>Antibody</td><td>IgG</td><td>ka (M^-1S^-1)</td><td>kd (S^-1)</td><td>Kd (nM)</td><td>IC50 (nM)</td>

<td rowspan="4">VQ8F11-21</td><td>hlgG1</td><td>6.22E + 05</td><td>4.54E-05</td><td> 0,073</td><td> 0,150</td>

<td>hlgG4</td><td>7.17E + 05</td><td>5.22E-05</td><td> 0,073</td><td> 0,228</td>

<td>mlgG2a</td><td>7.86E + 05</td><td>5.27E-05</td><td> 0,067</td><td> 0,135</td>

<td>modhlgG4</td><td>8.81E + 05</td><td> 4,705-05</td><td> 0,053</td><td> 0,249</td>

<td rowspan="3">VQ8A9-6</td><td>hlgG1</td><td>1.09E + 06</td><td>2.60E-04</td><td> 0,238</td><td> 0,130</td>

<td>hlgG4</td><td>1.17E + 06</td><td>2.35E-04</td><td> 0,201</td><td> 0,185</td>

<td>mlgG1</td><td>9.95E + 05</td><td>2.21 E-04</td><td> 0,222</td><td> 0,097</td>

<td rowspan="3">VV6A9-5</td><td>hlgG1</td><td>7.12E + 05</td><td>8.87E-05</td><td> 0,125</td><td> 0,204</td>

<td>hlgG4</td><td>5.67E + 05</td><td>7.64E-05</td><td> 0,135</td><td> 0,343</td>

<td>mlgG2a</td><td>7.72E + 05</td><td>7.52E-05</td><td> 0,097</td><td> 0,188</td>

<td rowspan="3">VQ1G4-21</td><td>hlgG1</td><td>3.34E + 05</td><td>7.92E-05</td><td> 0,237</td><td> 0,767</td>

<td>hlgG4</td><td>2.73E + 05</td><td>9.18E-05</td><td> 0,336</td><td> 0,528</td>

<td>mlgG2a</td><td>3.41E + 05</td><td>7.66E-05</td><td> 0,225</td><td> 0,578</td>

ES 2 398 076 T3

Disclosure aspects

The following are aspects of the disclosure:

(1) An antibody or antigen-binding fragment thereof, which specifically binds to the human interleukin-6 receptor (hIL-6R) with a Kd of approximately 500 pM or less, and 300 pM or less, as measured by resonance surface plasmon.

(2) An antibody or antigen-binding fragment according to (1) that binds hIL-6R with an affinity at least 2-fold over its binding to monkey IL-6R.

(3) An antibody or antigen-binding fragment according to (1) or (2), comprising a heavy chain complementarity determining region 3 (CDR3) domain and a light chain CDR3 domain, wherein the heavy chain CDR3 domain comprises an amino acid sequence of formula X¹ - X²- X³ - X⁴ X⁵ - X⁶ - X⁷ -X⁸ - X⁹ - X¹⁰ - X¹¹ - X¹² - X¹³ - X¹⁴ - X¹⁵ - X¹⁶ - X¹⁷- X¹⁸- X¹⁹ (SEQ ID NO: 247) where X¹ = X wing² = Lys, X³ = Gly, X⁴ = Arg, X⁵ = Asp, X⁶ = Ser or Wing, X⁷ = Phe, X⁸ = Asp; X⁹ = Ile, X¹⁰ = Pro or absent, X¹¹= Phe or absent, X = Val or absent, X = Tyr or absent, X = Tyr or absent, X = Tyr or absent, X = 17 18 19

Gly or absent, X = Met or absent, X = Asp or absent and X = Val or absent; and the CDR3 light chain domain comprises an amino acid sequence of formula X¹ - X² - X³ - X⁴ X⁵ - X⁶ - X⁷ - X⁵ - X⁹ (SEQ ID NO: 250) where X¹ = Gln, X² = Gln or His, X³ = Wing, X⁴ = Asn or Tyr, X⁵ = Ser, X⁶ = Phe, X⁷ = Pro, X⁸ = Pro and X⁹ = Thr.

(4) An antibody or antigen-binding fragment according to (3), additionally comprising a heavy chain CDR1 domain comprising an amino acid sequence of formula X¹ - X² - X³

- X⁴- X⁵ - X⁶ - X⁷ - X⁸ (SEQ ID NO: 245) where X¹ = Gly or Arg, X² = Phe, X³ = Thr, X⁴ = Phe, X⁵ = Asp, X⁶= Asp, X⁷ = Tyr and X⁸ = Ala; a heavy chain CDR2 domain comprising an amino acid sequence of formula X¹ - X² - X³ - X⁴ - X⁵ - X⁶ - X⁷ - X⁸ (SEQ ID NO: 246) where X¹ = Ile or Val, X² = Ser, X³ = Trp, X⁴ = Asn, X⁵ = Ser, X⁶ = Gly, X⁷ = Ser and X⁸ = Ile;

a light chain CDR1 domain comprising an amino acid sequence of formula X¹ - X² - X³ X⁴ - X⁵ - X⁶ - X⁷ - X⁸ (SEQ ID NO: 248), where X¹ = Gln, X² = Gly, X³ = Ile, X⁴ = Ser, X⁵ = Ser and X⁶ = Trp; and a heavy chain CDR2 domain comprising an amino acid sequence of formula X¹ -X² -X³(SEQ ID NO: 249), where X¹ = Gly or Ala, X² = Wing and X³ = Be.

(5) An antibody or antigen-binding fragment according to (1) or (2), comprising:

a heavy chain CDR3 domain selected from the group consisting of SEQ ID NOs: 25, 153, 9, 185, 41, 57, 73, 89, 105, 121, 137, 169, 201, and 217; and a light chain CDR3 domain selected from the group consisting of SEQ ID NOs: 33, 161, 17, 193, 49, 65, 81, 97, 113, 129, 145, 177, 209, and 225.

(6) An antibody or antigen-binding fragment according to (5), further comprising:

a heavy chain CDR1 domain selected from the group consisting of SEQ ID NOs: 21, 149, 5,181, 37, 53, 69, 85, 101, 117, 133, 165, 197, and 213;

a heavy chain CDR2 domain selected from the group consisting of SEQ ID NOs: 23, 151, 7,183, 39, 55, 71, 87, 103, 119, 135, 167, 199 and 215;

a light chain CDR1 domain selected from the group consisting of SEQ ID NOs: 29, 157, 13, 189, 45, 61, 77, 93, 109, 125, 141, 173, 205, and 221; and a light chain CDR2 domain selected from the group consisting of SEQ ID NOs: 31, 159, 15, 191, 47, 63, 79, 95, 111, 127, 143, 175, 207, and 223.

(7) An antibody or antigen-binding fragment according to (5) or (6), wherein the heavy chain CDRs and the light chain CDRs comprise SEQ ID NOs: 21, 23, 25 and SEQ ID NO: 29, 31, 33;

SEQ ID NO: 149, 151, 153 and SEQ ID NO: 157, 159, 161;

SEQ ID NO: 5, 7, 9 and SEQ ID NO: 13, 15, 17; and SEQ ID NO: 181, 183, 185 and SEQ ID NO: 189, 191, 193, respectively.

(8)