HK1240945B - Antibodies to il-6 and use thereof - Google Patents

Description

BACKGROUND OF THE INVENTION Field of the Invention

This invention pertains to an IL-6 antibody. The invention also pertains to methods of screening for diseases and disorders associated with IL-6, and methods of preventing or treating diseases or disorders associated with IL-6 by administering said antibody.

Description of Related Art

Interleukin-6 (hereinafter "IL-6") (also known as interferon-β₂; B-cell differentiation factor; B-cell stimulatory factor-2; hepatocyte stimulatory factor; hybridoma growth factor; and plasmacytoma growth factor) is a multifunctional cytokine involved in numerous biological processes such as the regulation of the acute inflammatory response, the modulation of specific immune responses including B- and T-cell differentiation, bone metabolism, thrombopoiesis, epidermal proliferation, menses, neuronal cell differentiation, neuroprotection, aging, cancer, and the inflammatory reaction occurring in Alzheimer's disease. See A. Papassotiropoulos, et al, Neurobiology of Aging, 22:863-871 (2001).

IL-6 is a member of a family of cytokines that promote cellular responses through a receptor complex consisting of at least one subunit of the signal-transducing glycoprotein gp130 and the IL-6 receptor ("IL-6R")(also known as gp80). The IL-6R may also be present in a soluble form ("sIL-6R"). IL-6 binds to IL-6R, which then dimerizes the signal-transducing receptor gp130. See Jones, SA, J. Immunology, 175:3463-3468 (2005).

In humans, the gene encoding for IL-6 is organized in five exons and four introns, and maps to the short arm of chromosome 7 at 7p21. Translation of IL-6 RNA and post-translational processing result in the formation of a 21 to 28 kDa protein with 184 amino acids in its mature form. See A. Papassotiropoulos, et al, Neurobiology of Aging, 22:863-871 (2001).

As set forth in greater detail below, IL-6 is believed to play a role in the development of a multitude of diseases and disorders, including but not limited to fatigue, cachexia, autoimmune diseases, diseases of the skeletal system, cancer, heart disease, obesity, diabetes, asthma, Alzheimer's disease and multiple sclerosis. Due to the perceived involvement of IL-6 in a wide range of diseases and disorders, there remains a need in the art for compositions and methods useful for preventing or treating diseases associated with IL-6, as well as methods of screening to identify patients having diseases or disorders associated with IL-6. Particularly preferred anti-IL-6 compositions are those having minimal or minimizing adverse reactions when administered to the patient. Compositions or methods that reduce or inhibit diseases or disorders associated with IL-6 are beneficial to the patient in need thereof.

WO2004/039826 describes chimeric, humanized or CDR-grafted anti-IL-6 antibodies derived from the murine CLB-8 antibody, including isolated nucleic acids that encode at least one such anti-IL-6 antibody, vectors, host cells, transgenic animals or plants, methods of making and using thereof, including therapeutic compositions, methods and devices. WO2006/l19115 describes an anti-IL-6 antibody, including isolated nucleic acids that encode at least one anti-IL-6 antibody, vectors, host cells, transgenic animals or plants, and methods of making and using thereof have applications in diagnostic and/or therapeutic compositions, methods and devices.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to an anti-IL-6 antibody comprising: (a) a light chain comprising (i) a variable light (V_L) domain comprising a CDR1 polypeptide consisting of the amino acid sequence of SEQ ID NO:4, a CDR2 polypeptide consisting of the amino acid sequence of SEQ ID NO:5, and CDR3 polypeptide consisting of the amino acid sequence of SEQ ID NO:6, and (ii) a constant light (C_L) domain comprising a polypeptide consisting of the amino acid sequence of SEQ ID NO:586; and (b) a heavy chain comprising (i) a variable heavy (V_H) domain comprising a CDR1 polypeptide consisting of the amino acid sequence of SEQ ID NO:7, a CDR2 polypeptide consisting of the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:120, and a CDR3 polypeptide consisting of the amino acid sequence of SEQ ID NO:9, and (ii) a constant heavy (C_H) domain comprising a polypeptide consisting of the amino acid sequence of SEQ ID NO:588, wherein the antibody has a dissociation constant (KD) of less than 50 picomolar as assessed by BIAcore. This antibody may bind soluble IL-6 or cell surface expressed IL-6. Also, this antibody may inhibit the formation or the biological effects of one or more of IL-6, IL-6/IL-6R complexes, IL-6/IL-6R/gp130 complexes and/or multimers of IL-6/IL-6R/gp130.

There are described the antibodies described herein, comprising the sequences of the V_H, V_L and CDR polypeptides described herein, and the polynucleotides encoding them. These antibodies may block gp130 activation and/or possess binding affinities (K_Ds) less than 50 picomolar and/or K_off values less than or equal to 10^-4 S^-1.

There are described antibodies and humanized versions derived from rabbit immune cells (B lymphocytes), which may be selected based on their homology (sequence identity) to human germ line sequences. These antibodies may require minimal or no sequence modifications, thereby facilitating retention of functional properties after humanization.

The described antibodies may be selected based on their activity in functional assays such as IL-6 driven T1165 proliferation assays, and IL-6 simulated HepG2 haptoglobin production assays. There are described fragments from anti-IL-6 antibodies encompassing V_H, V_L and CDR polypeptides, e.g., derived from rabbit immune cells and the polynucleotides encoding the same, as well as the use of these antibody fragments and the polynucleotides encoding them in the creation of novel antibodies and polypeptide compositions capable of recognizing IL-6 and/or IL-6/IL-6R complexes or IL-6/IL-6R/gp130 complexes and/or multimers thereof.

There are described conjugates of anti-IL-6 antibodies and binding fragments thereof conjugated to one or more functional or detectable moieties. There are also described methods of making said humanized anti-IL-6 or anti-IL-6/IL-6R complex antibodies and binding fragments thereof. Binding fragments may include, but are not limited to, Fab, Fab', F(ab')₂, Fv and scFv fragments.

The antibody of the invention may be used for the diagnosis, assessment and treatment of diseases and disorders associated with IL-6 or aberrant expression thereof. There is described the use of fragments of anti-IL-6 antibodies for the diagnosis, assessment and treatment of diseases and disorders associated with IL-6 or aberrant expression thereof. The described antibodies may be used for the treatment and prevention of cancer associated fatigue, and/or cachexia and rheumatoid arthritis.

There is described the production of anti-IL-6 antibodies in recombinant host cells, such as diploid yeast, diploid Pichia and other yeast strains.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

• Fig. 1 shows that a variety of unique epitopes were recognized by the collection of anti-IL-6 antibodies prepared by the antibody selection protocol. Epitope variability was confirmed by antibody-IL-6 binding competition studies (ForteBio Octet).
• Fig. 2 shows alignments of variable light and variable heavy sequences between rabbit antibody variable light and variable heavy sequences and homologous human sequences and the final humanized sequences. Framework regions are identified FR1-FR4. Complementarity determining regions are identified as CDR1-CDR3. Amino acid residues are numbered as shown. The initial rabbit sequences are called RbtVL and RbtVH for the variable light and variable heavy sequences respectively. Three of the most similar human germline antibody sequences, spanning from Framework 1 through to the end of Framework 3, are aligned below the rabbit sequences. The human sequence that is considered the most similar to the rabbit sequence is shown first. In this example those most similar sequences are L12A for the light chain and 3-64-04 for the heavy chain. Human CDR3 sequences are not shown. The closest human Framework 4 sequence is aligned below the rabbit Framework 4 sequence. The vertical dashes indicate a residue where the rabbit residue is identical with one or more of the human residues at the same position. The bold residues indicate that the human residue at that position is identical to the rabbit residue at the same position. The final humanized sequences are called VLh and VHh for the variable light and variable heavy sequences respectively. The underlined residues indicate that the residue is the same as the rabbit residue at that position but different than the human residues at that position in the three aligned human sequences.
• Fig. 3 demonstrates the high correlation between the IgG produced and antigen specificity for an exemplary IL-6 protocol. 9 of 11 wells showed specific IgG correlation with antigen recognition.
• Fig. 4 provides the α-2-macroglobulin (A2M) dose response curve for antibody Ab1 administered intravenously at different doses one hour after a 100µg/kg s.c. dose of human IL-6.
• Fig. 5 provides survival data for the antibody Ab1 progression groups versus control groups.
• Fig. 6 provides additional survival data for the antibody Ab1 regression groups versus control groups.
• Fig. 7 provides survival data for polyclonal human IgG at 10mg/kg i.v. every three days (270-320mg tumor size) versus antibody Ab1 at 10mg/kg i.v. every three days (270-320mg tumor size).
• Fig. 8 provides survival data for polyclonal human IgG at 10mg/kg i.v. every three days (400-527mg tumor size) versus antibody Ab1 at 10mg/kg i.v. every three days (400-527mg tumor size).
• Fig. 9 provides a pharmacokinetic profile of antibody Ab1. Plasma levels of antibody Ab1 were quantitated through antigen capture ELISA. This protein displays a half life of between 12 and 17 days consistent with other full length humanized antibodies.
• Figs. 10 A-D provide binding data for antibodies Ab4, Ab3, Ab8 and Ab2, respectively. Fig. 10 E provides binding data for antibodies Ab1, Ab6 and Ab7.
• Fig. 11 summarizes the binding data of Figures 10 A-E in tabular form.
• Fig. 12 presents the sequences of the 15 amino acid peptides used in the peptide mapping experiment of Example 14.
• Fig. 13 presents the results of the blots prepared in Example 14.
• Fig. 14 presents the results of the blots prepared in Example 14.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Definitions

As used herein the singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the protein" includes reference to one or more proteins and equivalents thereof known to those skilled in the art, and so forth. All technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs unless clearly indicated otherwise.

Interleukin-6 (IL-6): As used herein, interleukin-6 (IL-6) encompasses not only the following 212 amino acid sequence available as GenBank Protein Accession No. NP_000591: MNSFSTSAFGPVAFSLGLLLVLPAAFPAPVPPGEDSKDVAAPHRQPLTSSERIDKQI RYILDGISALRKETCNKSNMCESSKEALAENNLNLPKMAEKDGCFQSGFNEETCLV KIITGLLEFEVYLEYLQNRFESSEEQARAVQMSTKVLIQFLQKKAKNLDAITTPDPT TNASLLTKLQAQNQWLQDMTTHLILRSFKEFLQSSLRALRQM (SEQ ID NO: 1), but also any pre-pro, pro- and mature forms of this IL-6 amino acid sequence, as well as mutants and variants including allelic variants of this sequence.

Mating competent yeast species: This is intended to broadly encompass any diploid or tetraploid yeast which can be grown in culture. Such species of yeast may exist in a haploid, diploid, or tetraploid form. The cells of a given ploidy may, under appropriate conditions, proliferate for indefinite number of generations in that form. Diploid cells can also sporulate to form haploid cells. Sequential mating can result in tetraploid strains through further mating or fusion of diploid strains. Diploid or polyploidal yeast cells may be produced by mating or spheroplast fusion.

The mating competent yeast may be a member of the Saccharomycetaceae family, which includes the genera Arxiozyma; Ascobotryozyma; Citeromyces; Debaryomyces; Dekkera; Eremothecium; Issatchenkia; Kazachstania; Kluyveromyces; Kodamaea; Lodderomyces; Pachysolen; Pichia; Saccharomyces; Saturnispora;Tetrapisispora; Torulaspora; Williopsis; and Zygosaccharomyces. Other types of yeast include Yarrowia, Rhodosporidium, Candida, Hansenula, Filobasium, Filobasidellla, Sporidiobolus, Bullera, Leucosporidium and Filobasidella.

The mating competent yeast may be a member of the genus Pichia. The mating competent yeast of the genus Pichia may be one of the following species: Pichia pastoris, Pichia methanolica, and Hansenula polymorpha (Pichia angusta). The mating competent yeast of the genus Pichia may be the species Pichia pastoris.

Haploid Yeast Cell: A cell having a single copy of each gene of its normal genomic (chromosomal) complement.

Polyploid Yeast Cell: A cell having more than one copy of its normal genomic (chromosomal) complement.

Diploid Yeast Cell: A cell having two copies (alleles) of essentially every gene of its normal genomic complement, typically formed by the process of fusion (mating) of two haploid cells.

Tetraploid Yeast Cell: A cell having four copies (alleles) of essentially every gene of its normal genomic complement, typically formed by the process of fusion (mating) of two haploid cells. Tetraploids may carry two, three, four, or more different expression cassettes. Such tetraploids might be obtained in S. cerevisiae by selective mating homozygotic heterothallic a/a and alpha/alpha diploids and in Pichia by sequential mating of haploids to obtain auxotrophic diploids. For example, a [met his] haploid can be mated with [ade his] haploid to obtain diploid [his]; and a [met arg] haploid can be mated with [ade arg] haploid to obtain diploid [arg]; then the diploid [his] x diploid [arg] to obtain a tetraploid prototroph. It will be understood by those of skill in the art that reference to the benefits and uses of diploid cells may also apply to tetraploid cells.

Yeast Mating: The process by which two haploid yeast cells naturally fuse to form one diploid yeast cell.

Meiosis: The process by which a diploid yeast cell undergoes reductive division to form four haploid spore products. Each spore may then germinate and form a haploid vegetatively growing cell line.

Selectable Marker: A selectable marker is a gene or gene fragment that confers a growth phenotype (physical growth characteristic) on a cell receiving that gene as, for example through a transformation event. The selectable marker allows that cell to survive and grow in a selective growth medium under conditions in which cells that do not receive that selectable marker gene cannot grow. Selectable marker genes generally fall into several types, including positive selectable marker genes such as a gene that confers on a cell resistance to an antibiotic or other drug, temperature when two ts mutants are crossed or a ts mutant is transformed; and negative selectable marker genes such as a biosynthetic gene that confers on a cell the ability to grow in a medium without a specific nutrient needed by all cells that do not have that biosynthetic gene, or a mutagenized biosynthetic gene that confers on a cell inability to grow by cells that do not have the wild type gene. Suitable markers include but are not limited to: ZEO; G418; LYS3; MET1; MET3a; ADE1; ADE3 and URA3.

Expression Vector: These DNA vectors contain elements that facilitate manipulation for the expression of a foreign protein within the target host cell. Conveniently, manipulation of sequences and production of DNA for transformation is first performed in a bacterial host, e.g. E. coli, and usually vectors will include sequences to facilitate such manipulations, including a bacterial origin of replication and appropriate bacterial selection marker. Selection markers encode proteins necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media. Exemplary vectors and methods for transformation of yeast are described, for example, in Burke, D., Dawson, D., & Stearns, T. (2000). Methods in yeast genetics: a Cold Spring Harbor Laboratory course manual. Plainview, N.Y.: Cold Spring Harbor Laboratory Press.

Expression vectors may further include yeast specific sequences, including a selectable auxotrophic or drug marker for identifying transformed yeast strains. A drug marker may further be used to amplify copy number of the vector in a yeast host cell.

The polypeptide coding sequence of interest is operably linked to transcriptional and translational regulatory sequences that provide for expression of the polypeptide in yeast cells. These vector components may include, but are not limited to, one or more of the following: an enhancer element, a promoter, and a transcription termination sequence. Sequences for the secretion of the polypeptide may also be included, e.g. a signal sequence,. A yeast origin of replication is optional, as expression vectors are often integrated into the yeast genome.

The polypeptide of interest may be operably linked, or fused, to sequences providing for optimized secretion of the polypeptide from yeast diploid cells.

Nucleic acids are "operably linked" when placed into a functional relationship with another nucleic acid sequence. For example, DNA for a signal sequence is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites or alternatively via a PCR/recombination method familiar to those skilled in the art (Gateway^R Technology; Invitrogen, Carlsbad California). If such sites do not exist, the synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice.

Promoters are untranslated sequences located upstream (5') to the start codon of a structural gene (generally within about 100 to 1000 bp) that control the transcription and translation of particular nucleic acid sequences to which they are operably linked. Such promoters fall into several classes: inducible, constitutive, and repressible promoters (that increase levels of transcription in response to absence of a repressor). Inducible promoters may initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, e.g., the presence or absence of a nutrient or a change in temperature.

The yeast promoter fragment may also serve as the site for homologous recombination and integration of the expression vector into the same site in the yeast genome; alternatively a selectable marker is used as the site for homologous recombination. Pichia transformation is described in Cregg et al. (1985) Mol. Cell. Biol. 5:3376-3385.

Examples of suitable promoters from Pichia include the AOX1 and promoter (Cregg et al. (1989) Mol. Cell. Biol. 9:1316-1323); ICL1 promoter (Menendez et al. (2003) Yeast 20(13):1097-108); glyceraldehyde-3-phosphate dehydrogenase promoter (GAP) (Waterham et al. (1997) Gene 186(1):37-44); and FLD1 promoter (Shen et al. (1998) Gene 216(1):93-102). The GAP promoter is a strong constitutive promoter and the AOX and FLD1 promoters are inducible.

Other yeast promoters include ADH1, alcohol dehydrogenase II, GAL4, PHO3, PHO5, Pyk, and chimeric promoters derived therefrom. Additionally, non-yeast promoters may be used such as mammalian, insect, plant, reptile, amphibian, viral, and avian promoters. Most typically the promoter will comprise a mammalian promoter (potentially endogenous to the expressed genes) or will comprise a yeast or viral promoter that provides for efficient transcription in yeast systems.

The polypeptides of interest may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, e.g. a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. In general, the signal sequence may be a component of the vector, or it may be a part of the polypeptide coding sequence that is inserted into the vector. The heterologous signal sequence selected preferably is one that is recognized and processed through one of the standard pathways available within the host cell. The S. cerevisiae alpha factor pre-pro signal has proven effective in the secretion of a variety of recombinant proteins from P. pastoris. Other yeast signal sequences include the alpha mating factor signal sequence, the invertase signal sequence, and signal sequences derived from other secreted yeast polypeptides. Additionally, these signal peptide sequences may be engineered to provide for enhanced secretion in diploid yeast expression systems. Other secretion signals of interest also include mammalian signal sequences, which may be heterologous to the protein being secreted, or may be a native sequence for the protein being secreted. Signal sequences include pre-peptide sequences, and in some instances may include propeptide sequences. Many such signal sequences are known in the art, including the signal sequences found on immunoglobulin chains, e.g., K28 preprotoxin sequence, PHA-E, FACE, human MCP-1, human serum albumin signal sequences, human Ig heavy chain, and human Ig light chain. For example, see Hashimoto et. al. Protein Eng 11(2) 75 (1998); and Kobayashi et. al. Therapeutic Apheresis 2(4) 257 (1998).

Transcription may be increased by inserting a transcriptional activator sequence into the vector. These activators are cis-acting elements of DNA, usually about from 10 to 300 bp, which act on a promoter to increase its transcription. Transcriptional enhancers are relatively orientation and position independent, having been found 5' and 3' to the transcription unit, within an intron, as well as within the coding sequence itself. The enhancer may be spliced into the expression vector at a position 5' or 3' to the coding sequence, but is preferably located at a site 5' from the promoter.

Expression vectors used in eukaryotic host cells may also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from 3' to the translation termination codon, in untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA.

Construction of suitable vectors containing one or more of the above-listed components employs standard ligation techniques or PCR/recombination methods. Isolated plasmids or DNA fragments are cleaved, tailored, and re-ligated in the form desired to generate the plasmids required or via recombination methods. For analysis to confirm correct sequences in plasmids constructed, the ligation mixtures are used to transform host cells, and successful transformants selected by antibiotic resistance (e.g. ampicillin or Zeocin) where appropriate. Plasmids from the transformants are prepared, analyzed by restriction endonuclease digestion and/or sequenced.

As an alternative to restriction and ligation of fragments, recombination methods based on att sites and recombination enzymes may be used to insert DNA sequences into a vector. Such methods are described, for example, by Landy (1989) Ann.Rev.Biochem. 58:913-949; and are known to those of skill in the art. Such methods utilize intermolecular DNA recombination that is mediated by a mixture of lambda and E.coli -encoded recombination proteins. Recombination occurs between specific attachment (att) sites on the interacting DNA molecules. For a description of att sites see Weisberg and Landy (1983) Site-Specific Recombination in Phage Lambda, in Lambda II, Weisberg, ed.(Cold Spring Harbor, NY:Cold Spring Harbor Press), pp.211-250. The DNA segments flanking the recombination sites are switched, such that after recombination, the att sites are hybrid sequences comprised of sequences donated by each parental vector. The recombination can occur between DNAs of any topology.

Att sites may be introduced into a sequence of interest by ligating the sequence of interest into an appropriate vector; generating a PCR product containing att B sites through the use of specific primers; or generating a cDNA library cloned into an appropriate vector containing att sites.

Folding, as used herein, refers to the three-dimensional structure of polypeptides and proteins, where interactions between amino acid residues act to stabilize the structure. While non-covalent interactions are important in determining structure, usually the proteins of interest will have intra- and/or intermolecular covalent disulfide bonds formed by two cysteine residues. For naturally occurring proteins and polypeptides or derivatives and variants thereof, the proper folding is typically the arrangement that results in optimal biological activity, and can conveniently be monitored by assays for activity, e.g. ligand binding, enzymatic activity, etc.

In some instances, for example where the desired product is of synthetic origin, assays based on biological activity will be less meaningful. The proper folding of such molecules may be determined on the basis of physical properties, energetic considerations, and modeling studies.

The expression host may be further modified by the introduction of sequences encoding one or more enzymes that enhance folding and disulfide bond formation, i.e. foldases, chaperonins, etc. Such sequences may be constitutively or inducibly expressed in the yeast host cell, using vectors, markers, etc. as known in the art. The sequences, including transcriptional regulatory elements sufficient for the desired pattern of expression, may be stably integrated in the yeast genome through a targeted methodology.

For example, the eukaryotic PDI is not only an efficient catalyst of protein cysteine oxidation and disulfide bond isomerization, but also exhibits chaperone activity. Co-expression of PDI can facilitate the production of active proteins having multiple disulfide bonds. Also of interest is the expression of BIP (immunoglobulin heavy chain binding protein) and cyclophilin. Each of the haploid parental strains may express a distinct folding enzyme, e.g. one strain may express BIP, and the other strain may express PDI or combinations thereof.

The terms "desired protein" or "target protein" are used interchangeably and refer generally to a humanized antibody or a binding portion thereof described herein. The term "antibody" is intended to include any polypeptide chain-containing molecular structure with a specific shape that fits to and recognizes an epitope, where one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope. The archetypal antibody molecule is the immunoglobulin, and all types of immunoglobulins, IgG, IgM, IgA, IgE, IgD, etc., from all sources, e.g. human, rodent, rabbit, cow, sheep, pig, dog, other mammals, chicken, other avians, etc., are considered to be "antibodies." A source for producing antibodies useful as starting material is rabbits. Numerous antibody coding sequences have been described; and others may be raised by methods well-known in the art. Examples thereof include chimeric antibodies, human antibodies and other non-human mammalian antibodies, humanized antibodies, single chain antibodies such as scFvs, camelbodies, nanobodies, IgNAR (single-chain antibodies derived from sharks), small-modular immunopharmaceuticals (SMIPs), and antibody fragments such as Fabs, Fab', and F(ab')₂. See Streltsov VA, et al., Structure of a shark IgNAR antibody variable domain and modeling of an early-developmental isotype, Protein Sci. 2005 Nov;14(11):2901-9. Epub 2005 Sep 30; Greenberg AS, et al, A new antigen receptor gene family that undergoes rearrangement and extensive somatic diversification in sharks, Nature. 1995 Mar 9;374(6518):168-73; Nuttall SD, et al., Isolation of the new antigen receptor from wobbegong sharks, and use as a scaffold for the display of protein loop libraries, Mol Immunol. 2001 Aug;38(4):313-26; Hamers-Casterman C, et al., Naturally occurring antibodies devoid of light chains, Nature. 1993 Jun 3;363(6428):446-8;Gill DS, et al., Biopharmaceutical drug discovery using novel protein scaffolds, Curr Opin Biotechnol. 2006 Dec;17(6):653-8. Epub 2006 Oct 19.

For example, antibodies or antigen binding fragments may be produced by genetic engineering. In this technique, as with other methods, antibody-producing cells are sensitized to the desired antigen or immunogen. The messenger RNA isolated from antibody producing cells is used as a template to make cDNA using PCR amplification. A library of vectors, each containing one heavy chain gene and one light chain gene retaining the initial antigen specificity, is produced by insertion of appropriate sections of the amplified immunoglobulin cDNA into the expression vectors. A combinatorial library is constructed by combining the heavy chain gene library with the light chain gene library. This results in a library of clones which co-express a heavy and light chain (resembling the Fab fragment or antigen binding fragment of an antibody molecule). The vectors that carry these genes are co-transfected into a host cell. When antibody gene synthesis is induced in the transfected host, the heavy and light chain proteins self-assemble to produce active antibodies that can be detected by screening with the antigen or immunogen.

Antibody coding sequences of interest include those encoded by native sequences, as well as nucleic acids that, by virtue of the degeneracy of the genetic code, are not identical in sequence to the disclosed nucleic acids, and variants thereof. Variant polypeptides can include amino acid (aa) substitutions, additions or deletions. The amino acid substitutions can be conservative amino acid substitutions or substitutions to eliminate non-essential amino acids, such as to alter a glycosylation site, or to minimize misfolding by substitution or deletion of one or more cysteine residues that are not necessary for function. Variants can be designed so as to retain or have enhanced biological activity of a particular region of the protein (e.g., a functional domain, catalytic amino acid residues, etc). Variants also include fragments of the polypeptides disclosed herein, particularly biologically active fragments and/or fragments corresponding to functional domains. Techniques for in vitro mutagenesis of cloned genes are known. Also described are polypeptides that have been modified using ordinary molecular biological techniques so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent.

Chimeric antibodies may be made by recombinant means by combining the variable light and heavy chain regions (V_L and V_H), obtained from antibody producing cells of one species with the constant light and heavy chain regions from another. Typically chimeric antibodies utilize rodent or rabbit variable regions and human constant regions, in order to produce an antibody with predominantly human domains. The production of such chimeric antibodies is well known in the art, and may be achieved by standard means (as described, e.g., in U.S. Patent No. 5,624,659 ). The human constant regions of chimeric antibodies may be selected from IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgG8, IgG9, IgG10, IgG11, IgG12, IgG13, IgG14, IgG15, IgG16, IgG17, IgG18 or IgG19 constant regions.

Humanized antibodies are engineered to contain even more human-like immunoglobulin domains, and incorporate only the complementarity-determining regions of the animal-derived antibody. This is accomplished by carefully examining the sequence of the hyper-variable loops of the variable regions of the monoclonal antibody, and fitting them to the structure of the human antibody chains. Although facially complex, the process is straightforward in practice. See, e.g., U.S. Patent No. 6,187,287 .

In addition to entire immunoglobulins (or their recombinant counterparts), immunoglobulin fragments comprising the epitope binding site (e.g., Fab', F(ab')₂, or other fragments) may be synthesized. "Fragment," or minimal immunoglobulins may be designed utilizing recombinant immunoglobulin techniques. For instance "Fv" immunoglobulins may be produced by synthesizing a fused variable light chain region and a variable heavy chain region. Combinations of antibodies are also of interest, e.g. diabodies, which comprise two distinct Fv specificities. SMIPs (small molecule immunopharmaceuticals), camelbodies, nanobodies, and IgNAR are also encompassed by immunoglobulin fragments.

Immunoglobulins and fragments thereof may be modified post-translationally, e.g. to add effector moieties such as chemical linkers, detectable moieties, such as fluorescent dyes, enzymes, toxins, substrates, bioluminescent materials, radioactive materials, or chemiluminescent moieties, or specific binding moieties, such as streptavidin, avidin, or biotin, may be utilized in the methods and compositions described herein. Examples of additional effector molecules are provided infra.

The term "polyploid yeast that stably expresses or expresses a desired secreted heterologous polypeptide for prolonged time" refers to a yeast culture that secretes said polypeptide for at least several days to a week, such as at least a month, at least 1-6 months, or for more than a year at threshold expression levels, typically at least 10-25 mg/liter and preferably substantially greater.

The term "polyploidal yeast culture that secretes desired amounts of recombinant polypeptide" refers to cultures that stably or for prolonged periods secrete at least 10-25 mg/liter of heterologous polypeptide, such as at least 50-500 mg/liter, or 500-1000 mg/liter or more.

A polynucleotide sequence "corresponds" to a polypeptide sequence if translation of the polynucleotide sequence in accordance with the genetic code yields the polypeptide sequence (i.e., the polynucleotide sequence "encodes" the polypeptide sequence), one polynucleotide sequence "corresponds" to another polynucleotide sequence if the two sequences encode the same polypeptide sequence.

A "heterologous" region or domain of a DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature. Thus, when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism. Another example of a heterologous region is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.

A "coding sequence" is an in-frame sequence of codons that (in view of the genetic code) correspond to or encode a protein or peptide sequence. Two coding sequences correspond to each other if the sequences or their complementary sequences encode the same amino acid sequences. A coding sequence in association with appropriate regulatory sequences may be transcribed and translated into a polypeptide. A polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence. A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. Promoter sequences typically contain additional sites for binding of regulatory molecules (e.g., transcription factors) which affect the transcription of the coding sequence. A coding sequence is "under the control" of the promoter sequence or "operatively linked" to the promoter when RNA polymerase binds the promoter sequence in a cell and transcribes the coding sequence into mRNA, which is then in turn translated into the protein encoded by the coding sequence.

Vectors are used to introduce a foreign substance, such as DNA, RNA or protein, into an organism or host cell. Typical vectors include recombinant viruses (for polynucleotides) and liposomes (for polypeptides). A "DNA vector" is a replicon, such as plasmid, phage or cosmid, to which another polynucleotide segment may be attached so as to bring about the replication of the attached segment. An "expression vector" is a DNA vector which contains regulatory sequences which will direct polypeptide synthesis by an appropriate host cell. This usually means a promoter to bind RNA polymerase and initiate transcription of mRNA, as well as ribosome binding sites and initiation signals to direct translation of the mRNA into a polypeptide(s). Incorporation of a polynucleotide sequence into an expression vector at the proper site and in correct reading frame, followed by transformation of an appropriate host cell by the vector, enables the production of a polypeptide encoded by said polynucleotide sequence.

"Amplification" of polynucleotide sequences is the in vitro production of multiple copies of a particular nucleic acid sequence. The amplified sequence is usually in the form of DNA. A variety of techniques for carrying out such amplification are described in a review article by Van Brunt (1990, Bio/Technol., 8(4):291-294). Polymerase chain reaction or PCR is a prototype of nucleic acid amplification, and use of PCR herein should be considered exemplary of other suitable amplification techniques.

The general structure of antibodies in vertebrates now is well understood (Edelman, G. M., Ann. N.Y. Acad. Sci., 190: 5 (1971)). Antibodies consist of two identical light polypeptide chains of molecular weight approximately 23,000 daltons (the "light chain"), and two identical heavy chains of molecular weight 53,000-70,000 (the "heavy chain"). The four chains are joined by disulfide bonds in a "Y" configuration wherein the light chains bracket the heavy chains starting at the mouth of the "Y" configuration. The "branch" portion of the "Y" configuration is designated the F_ab region; the stem portion of the "Y" configuration is designated the F_C region. The amino acid sequence orientation runs from the N-terminal end at the top of the "Y" configuration to the C-terminal end at the bottom of each chain. The N-terminal end possesses the variable region having specificity for the antigen that elicited it, and is approximately 100 amino acids in length, there being slight variations between light and heavy chain and from antibody to antibody.

The variable region is linked in each chain to a constant region that extends the remaining length of the chain and that within a particular class of antibody does not vary with the specificity of the antibody (i.e., the antigen eliciting it). There are five known major classes of constant regions that determine the class of the immunoglobulin molecule (IgG, IgM, IgA, IgD, and IgE corresponding to γ, µ, α, δ, and ε (gamma, mu, alpha, delta, or epsilon) heavy chain constant regions). The constant region or class determines subsequent effector function of the antibody, including activation of complement (Kabat, E. A., Structural Concepts in Immunology and Immunochemistry, 2nd Ed., p. 413-436, Holt, Rinehart, Winston (1976)), and other cellular responses (Andrews, D. W., et al., Clinical Immunobiology, pp 1-18, W. B. Sanders (1980); Kohl, S., et al., Immunology, 48: 187 (1983)); while the variable region determines the antigen with which it will react. Light chains are classified as either κ (kappa) or λ (lambda). Each heavy chain class can be paired with either kappa or lambda light chain. The light and heavy chains are covalently bonded to each other, and the "tail" portions of the two heavy chains are bonded to each other by covalent disulfide linkages when the immunoglobulins are generated either by hybridomas or by B cells.

The expression "variable region" or "VR" refers to the domains within each pair of light and heavy chains in an antibody that are involved directly in binding the antibody to the antigen. Each heavy chain has at one end a variable domain (V_H) followed by a number of constant domains. Each light chain has a variable domain (V_L) at one end and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.

The expressions "complementarity determining region," "hypervariable region," or "CDR" refer to one or more of the hyper-variable or complementarity determining regions (CDRs) found in the variable regions of light or heavy chains of an antibody (See Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., (1987)). These expressions include the hypervariable regions as defined by Kabat et al. ("Sequences of Proteins of Immunological Interest ," Kabat E., et al., US Dept. of Health and Human Services, 1983) or the hypervariable loops in 3-dimensional structures of antibodies (Chothia and Lesk, J Mol. Biol. 196 901-917 (1987)). The CDRs in each chain are held in close proximity by framework regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site. Within the CDRs there are select amino acids that have been described as the selectivity determining regions (SDRs) which represent the critical contact residues used by the CDR in the antibody-antigen interaction (Kashmiri, S., Methods, 36:25-34 (2005)).

The expressions "framework region" or "FR" refer to one or more of the framework regions within the variable regions of the light and heavy chains of an antibody (See Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., (1987)). These expressions include those amino acid sequence regions interposed between the CDRs within the variable regions of the light and heavy chains of an antibody.

Anti-IL-6 Antibodies and Binding Fragments Thereof

The invention relates to an antibody having binding specificity to IL-6 and comprising: (a) a light chain comprising (i) a variable light (V_L) domain comprising a CDR1 polypeptide consisting of the amino acid sequence of SEQ ID NO:4, a CDR2 polypeptide consisting of the amino acid sequence of SEQ ID NO:5, and CDR3 polypeptide consisting of the amino acid sequence of SEQ ID NO:6, and (ii) a constant light (C_L) domain comprising a polypeptide consisting of the amino acid sequence of SEQ ID NO:586; and (b) a heavy chain comprising (i) a variable heavy (V_H) domain comprising a CDR1 polypeptide consisting of the amino acid sequence of SEQ ID NO:7, a CDR2 polypeptide consisting of the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:120, and a CDR3 polypeptide consisting of the amino acid sequence of SEQ ID NO:9, and (ii) a constant heavy (C_H) domain comprising a polypeptide consisting of the amino acid sequence of SEQ ID NO:588, wherein the antibody has a dissociation constant (KD) of less than 50 picomolar as assessed by BIAcore. The invention includes antibodies possessing a variable light chain sequence comprising the sequence set forth below:

The invention also includes antibodies having binding specificity to IL-6 and possessing a variable heavy chain sequence comprising the sequence set forth below:

The antibody of the invention comprises the polypeptide sequences of SEQ ID NO: 4; SEQ ID NO: 5; and SEQ ID NO: 6 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 2, and the polypeptide sequences of SEQ ID NO: 7; SEQ ID NO: 8 or SEQ ID NO: 120; and SEQ ID NO: 9 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 3.

There are described other antibodies, such as for example chimeric antibodies, comprising one or more of the polypeptide sequences of SEQ ID NO: 4; SEQ ID NO: 5; and SEQ ID NO: 6 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 2, and/or one or more of the polypeptide sequences of SEQ ID NO: 7; SEQ ID NO: 8; and SEQ ID NO: 9 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 3, or combinations of these polypeptide sequences.

There are described fragments of the antibody having binding specificity to IL-6. Antibody fragments may comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 2. Antibody fragments may comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 3.

Fragments of antibody having binding specificity to IL-6 may comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 4; SEQ ID NO: 5; and SEQ ID NO: 6 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 2.

Fragments of antibody having binding specificity to IL-6 may comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 7; SEQ ID NO: 8; and SEQ ID NO: 9 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 3.

There are described antibody fragments which include one or more of the antibody fragments described herein. Fragments of the antibodies having binding specificity to IL-6 may comprise, or alternatively consist of, one, two, three or more, including all of the following antibody fragments: the variable light chain region of SEQ ID NO: 2; the variable heavy chain region of SEQ ID NO: 3; the complementarity-determining regions (SEQ ID NO: 4; SEQ ID NO: 5; and SEQ ID NO: 6) of the variable light chain region of SEQ ID NO: 2; and the complementarity-determining regions (SEQ ID NO: 7; SEQ ID NO: 8; and SEQ ID NO: 9) of the variable heavy chain region of SEQ ID NO: 3.

The invention also contemplates variants wherein either of the heavy chain polypeptide sequences of SEQ ID NO: 18 or SEQ ID NO: 19 is substituted for the heavy chain polypeptide sequence of SEQ ID NO: 3; the light chain polypeptide sequence of SEQ ID NO: 20 is substituted for the light chain polypeptide sequence of SEQ ID NO: 2; and the heavy chain CDR sequence of SEQ ID NO: 120 is substituted for the heavy chain CDR sequence of SEQ ID NO: 8.

In a preferred embodiment of the invention, the anti-IL-6 antibody is Ab1, comprising SEQ ID NO: 2 and SEQ ID NO: 3, and having at least one of the biological activities set forth herein.

There are described an anti-IL-6 antibody which is Ab2, comprising SEQ ID NO: 21 and SEQ ID NO: 22, and having at least one of the biological activities set forth herein.

There are described an anti-IL-6 antibody which is Ab3, comprising SEQ ID NO: 37 and SEQ ID NO: 38, and having at least one of the biological activities set forth herein.

There are described an anti-IL-6 antibody which is Ab4, comprising SEQ ID NO: 53 and SEQ ID NO: 54, and having at least one of the biological activities set forth herein.

There are described an anti-IL-6 antibody which is Ab5, comprising SEQ ID NO: 69 and SEQ ID NO: 70, and having at least one of the biological activities set forth herein.

There are described an anti-IL-6 antibody which is Ab6, comprising SEQ ID NO: 85 and SEQ ID NO: 86, and having at least one of the biological activities set forth herein. There are described an anti-IL-6 antibody which is Ab7, comprising SEQ ID NO: 101 and SEQ ID NO: 102, and having at least one of the biological activities set forth herein.

There are described an anti-IL-6 antibody which is Ab8, comprising SEQ ID NO: 122 and SEQ ID NO: 123, and having at least one of the biological activities set forth herein.

There are described an anti-IL-6 antibody which is Ab9, comprising SEQ ID NO: 138 and SEQ ID NO: 139, and having at least one of the biological activities set forth herein.

There are described an anti-IL-6 antibody which is Ab10, comprising SEQ ID NO: 154 and SEQ ID NO: 155, and having at least one of the biological activities set forth herein.

There are described an anti-IL-6 antibody which is Ab11, comprising SEQ ID NO: 170 and SEQ ID NO: 171, and having at least one of the biological activities set forth herein.

There are described an anti-IL-6 antibody which is Ab12, comprising SEQ ID NO: 186 and SEQ ID NO: 187, and having at least one of the biological activities set forth herein.

There are described an anti-IL-6 antibody which is Ab13, comprising SEQ ID NO: 202 and SEQ ID NO: 203, and having at least one of the biological activities set forth herein.

There are described an anti-IL-6 antibody which is Ab14, comprising SEQ ID NO: 218 and SEQ ID NO: 219, and having at least one of the biological activities set forth herein.

There are described an anti-IL-6 antibody which is Ab15, comprising SEQ ID NO: 234 and SEQ ID NO: 235, and having at least one of the biological activities set forth herein.

There are described an anti-IL-6 antibody which is Ab16, comprising SEQ ID NO: 250 and SEQ ID NO: 251, and having at least one of the biological activities set forth herein.

There are described ananti-IL-6 antibody which is Ab17, comprising SEQ ID NO: 266 and SEQ ID NO: 267, and having at least one of the biological activities set forth herein.

There are described an anti-IL-6 antibody which is Ab18, comprising SEQ ID NO: 282 and SEQ ID NO: 283, and having at least one of the biological activities set forth herein.

There are described an anti-IL-6 antibody which is Ab19, comprising SEQ ID NO: 298 and SEQ ID NO: 299, and having at least one of the biological activities set forth herein.

There are described an anti-IL-6 antibody which is Ab20, comprising SEQ ID NO: 314 and SEQ ID NO: 315, and having at least one of the biological activities set forth herein.

There are described an anti-IL-6 antibody which is Ab21, comprising SEQ ID NO: 330 and SEQ ID NO: 331, and having at least one of the biological activities set forth herein.

There are described an anti-IL-6 antibody which is Ab22, comprising SEQ ID NO: 346 and SEQ ID NO: 347, and having at least one of the biological activities set forth herein.

There are described an anti-IL-6 antibody which is Ab23, comprising SEQ ID NO: 362 and SEQ ID NO: 363, and having at least one of the biological activities set forth herein.

There are described an anti-IL-6 antibody which is Ab24, comprising SEQ ID NO: 378 and SEQ ID NO: 379, and having at least one of the biological activities set forth herein.

There are described an anti-IL-6 antibody which is Ab25, comprising SEQ ID NO: 394 and SEQ ID NO: 395, and having at least one of the biological activities set forth herein.

There are described ananti-IL-6 antibody which is Ab26, comprising SEQ ID NO: 410 and SEQ ID NO: 411, and having at least one of the biological activities set forth herein.

There are described ananti-IL-6 antibody which is Ab27, comprising SEQ ID NO: 426 and SEQ ID NO: 427, and having at least one of the biological activities set forth herein.

There are described an anti-IL-6 antibody which is Ab28, comprising SEQ ID NO: 442 and SEQ ID NO: 443, and having at least one of the biological activities set forth herein.

There are described an anti-IL-6 antibody which is Ab29, comprising SEQ ID NO: 458 and SEQ ID NO: 459, and having at least one of the biological activities set forth herein.

There are described an anti-IL-6 antibody which is Ab30, comprising SEQ ID NO: 474 and SEQ ID NO: 475, and having at least one of the biological activities set forth herein.

There are described an anti-IL-6 antibody which is Ab31, comprising SEQ ID NO: 490 and SEQ ID NO: 491, and having at least one of the biological activities set forth herein.

There are described an anti-IL-6 antibody which is Ab32, comprising SEQ ID NO: 506 and SEQ ID NO: 507, and having at least one of the biological activities set forth herein.

There are described an anti-IL-6 antibody which is Ab33, comprising SEQ ID NO: 522 and SEQ ID NO: 523, and having at least one of the biological activities set forth herein.

There are described an anti-IL-6 antibody which is Ab34, comprising SEQ ID NO: 538 and SEQ ID NO: 539, and having at least one of the biological activities set forth herein.

There are described an anti-IL-6 antibody which is Ab35, comprising SEQ ID NO: 554 and SEQ ID NO: 555, and having at least one of the biological activities set forth herein.

There are described an anti-IL-6 antibody which is Ab36, comprising SEQ ID NO: 570 and SEQ ID NO: 571, and having at least one of the biological activities set forth herein.

Antibody fragments described herein may be present in one or more of the following non-limiting forms: Fab, Fab', F(ab')₂, Fv and single chain Fv antibody forms. Within the invention, the anti-IL-6 antibody further comprises the kappa constant light chain sequence comprising the sequence set forth below:

Within the invention, the anti-IL-6 antibody further comprises and the gamma-1 constant heavy chain polypeptide sequence comprising the sequence set forth below:

There is described an isolated anti-IL-6 antibody comprising a V_H polypeptide sequence selected from the group consisting of: SEQ ID NO: 3, 18, 19, 22, 38, 54, 70, 86, 102, 117, 118, 123, 139, 155, 171, 187, 203, 219, 235, 251, 267, 283, 299, 315, 331, 347, 363, 379, 395, 411, 427, 443, 459, 475, 491, 507, 523, 539, 555 and SEQ ID NO: 571; and further comprising a V_L polypeptide sequence selected from the group consisting of: SEQ ID NO: 2, 20, 21, 37, 53, 69, 85, 101, 119, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346, 362, 378, 394, 410, 426, 442, 458, 474, 490, 506, 522, 538, 554 and SEQ ID NO: 570 or a variant thereof wherein one or more of the framework residues (FR residues) in said V_H or V_L polypeptide has been substituted with another amino acid residue resulting in an anti-IL-6 antibody that specifically binds IL-6. The invention contemplates humanized and chimeric forms of the claimed antibodies. The chimeric antibodies may include an Fc derived from IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgG8, IgG9, IgG10, IgG11, IgG12, IgG13, IgG14, IgG15, IgG16, IgG17, IgG18 or IgG19 constant regions.

In one embodiment of the invention, the antibodies or V_H or V_L polypeptides originate or are selected from one or more rabbit B cell populations prior to initiation of the humanization process referenced herein.

In another embodiment of the invention, the anti-IL-6 antibodies may have binding specificity for primate homologs of the human IL-6 protein. Non-limiting examples of primate homologs of the human IL-6 protein are IL-6 obtained from Macaca fascicularis (also known as the cynomolgus monkey) and the Rhesus monkey. In another embodiment of the invention, the anti-IL-6 antibodies may inhibit the association of IL-6 with IL-6R, and/or the production of IL-6/IL-6R/gp130 complexes and/or the production of IL-6/IL-6R/gp130 multimers and/or antagonizes the biological effects of one or more of the foregoing.

As stated in paragraph [0062] herein, antibodies may be modified post-translationally to add effector moieties such as chemical linkers, detectable moieties such as for example fluorescent dyes, enzymes, substrates, bioluminescent materials, radioactive materials, and chemiluminescent moieties, or functional moieties such as for example streptavidin, avidin, biotin, a cytotoxin, a cytotoxic agent, and radioactive materials.

Regarding detectable moieties, further exemplary enzymes include, but are not limited to, horseradish peroxidase, acetylcholinesterase, alkaline phosphatase, beta-galactosidase and luciferase. Further exemplary fluorescent materials include, but are not limited to, rhodamine, fluorescein, fluorescein isothiocyanate, umbelliferone, dichlorotriazinylamine, phycoerythrin and dansyl chloride. Further exemplary chemiluminescent moieties include, but are not limited to, luminol. Further exemplary bioluminescent materials include, but are not limited to, luciferin and aequorin. Further exemplary radioactive materials include, but are not limited to, Iodine 125 (¹²⁵I), Carbon 14 (¹⁴C), Sulfur 35 (³⁵S), Tritium (³H) and Phosphorus 32 (³²P).

Regarding functional moieties, exemplary cytotoxic agents include, but are not limited to, methotrexate, aminopterin, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine; alkylating agents such as mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU), mitomycin C, lomustine (CCNU), 1-methylnitrosourea, cyclothosphamide, mechlorethamine, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamine platinum (II) (DDP) cisplatin and carboplatin (paraplatin); anthracyclines include daunorubicin (formerly daunomycin), doxorubicin (adriamycin), detorubicin, carminomycin, idarubicin, epirubicin, mitoxantrone and bisantrene; antibiotics include dactinomycin (actinomycin D), bleomycin, calicheamicin, mithramycin, and anthramycin (AMC); and antimytotic agents such as the vinca alkaloids, vincristine and vinblastine. Other cytotoxic agents include paclitaxel (taxol), ricin, pseudomonas exotoxin, gemcitabine, cytochalasin B, gramicidin D, ethidium bromide, emetine, etoposide, tenoposide, colchicin, dihydroxy anthracin dione, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, procarbazine, hydroxyurea, asparaginase, corticosteroids, mytotane (O,P'-(DDD)), interferons, and mixtures of these cytotoxic agents.

Further cytotoxic agents include, but are not limited to, chemotherapeutic agents such as carboplatin, cisplatin, paclitaxel, gemcitabine, calicheamicin, doxorubicin, 5-fluorouracil, mitomycin C, actinomycin D, cyclophosphamide, vincristine and bleomycin. Toxic enzymes from plants and bacteria such as ricin, diphtheria toxin and Pseudomonas toxin may be conjugated to the humanized antibodies, or binding fragments thereof, to generate cell-type-specific-killing reagents (Youle, et al., Proc. Nat'l Acad. Sci. USA 77:5483 (1980); Gilliland, et al., Proc. Nat'l Acad. Sci. USA 77:4539 (1980); Krolick, et al., Proc. Nat'l Acad. Sci. USA 77:5419 (1980)).

Other cytotoxic agents include cytotoxic ribonucleases as described by Goldenberg in U.S. Pat. No. 6,653,104 . Embodiments of the invention also relate to radioimmunoconjugates where a radionuclide that emits alpha or beta particles is stably coupled to the antibody, with or without the use of a complex-forming agent. Such radionuclides include beta-emitters such as Phosphorus-32 (³²P), Scandium-47 (⁴⁷Sc), Copper-67 (⁶⁷Cu), Gallium-67 (⁶⁷Ga), Yttrium-88 (⁸⁸Y), Yttrium-90 (⁹⁰Y), Iodine-125 (¹²⁵I), Iodine-131 (¹³¹I), Samarium-153 (¹⁵³Sm), Lutetium-177 (¹⁷⁷Lu), Rhenium-186 (¹⁸⁶Re) or Rhenium-188 (¹⁸⁸Re), and alpha-emitters such as Astatine-211 (²¹¹At), Lead-212 (²¹²Pb), Bismuth-212 (²¹²Bi) or -213 (²¹³Bi) or Actinium-225 (²²⁵Ac).

Methods are known in the art for conjugating an antibody or binding fragment thereof to a detectable moiety, such as for example those methods described by Hunter et al, Nature 144:945 (1962); David et al, Biochemistry 13:1014 (1974); Pain et al, J. Immunol. Meth. 40:219 (1981); and Nygren, J., Histochem. and Cytochem. 30:407 (1982).

There are described polypeptide sequences having at least 90% or greater sequence homology to any one or more of the polypeptide sequences of antibody fragments, variable regions and CDRs set forth herein. There are described polypeptide sequences having at least 95% or greater sequence homology, such as at least 98% or greater sequence homology, or at least 99% or greater sequence homology to any one or more of the polypeptide sequences of antibody fragments, variable regions and CDRs set forth herein. Methods for determining homology between nucleic acid and amino acid sequences are well known to those of ordinary skill in the art.

The above-recited polypeptide homologs of the antibody fragments, variable regions and CDRs set forth herein may further have anti-IL-6 activity. Non-limiting examples of anti-IL-6 activity are set forth herein.

There is described the generation and use of anti-idiotypic antibodies that bind any of the foregoing sequences. Such an anti-idiotypic antibody could be administered to a subject who has received an anti-IL-6 antibody to modulate, reduce, or neutralize, the effect of the anti-IL-6 antibody. Such anti-idiotypic antibodies could also be useful for treatment of an autoimmune disease characterized by the presence of anti-IL-6 antibodies. A further exemplary use of such anti-idiotypic antibodies is for detection of the anti-IL-6 antibodies described herein, for example to monitor the levels of the anti-IL-6 antibodies present in a subject's blood or other bodily fluids.

Additional Described Antibodies

There are described one or more anti-human IL-6 antibodies or antibody fragment which specifically bind to the same linear or conformational epitope(s) and/or competes for binding to the same linear or conformational epitope(s) on an intact human IL-6 polypeptide or fragment thereof as an anti-human IL-6 antibody selected from the group consisting of Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab20, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, Ab35, and Ab36. The anti-human IL-6 antibody or fragment may specifically bind to the same linear or conformational epitope(s) and/or competes for binding to the same linear or conformational epitope(s) on an intact human IL-6 polypeptide or a fragment thereof as Ab1.

The anti-human IL-6 antibody which specifically binds to the same linear or conformational epitopes on an intact IL-6 polypeptide or fragment thereof that is (are) specifically bound by Ab1 may bind to a IL-6 epitope(s) ascertained by epitopic mapping using overlapping linear peptide fragments which span the full length of the native human IL-6 polypeptide. The IL-6 epitope may comprise, or alternatively consist of, one or more residues comprised in IL-6 fragments selected from those respectively encompassing amino acid residues 37-51, amino acid residues 70-84, amino acid residues 169-183, amino acid residues 31-45 and/or amino acid residues 58-72.

There is described an anti-IL-6 antibody that binds with the same IL-6 epitope and/or competes with an anti-IL-6 antibody for binding to IL-6 as an antibody or antibody fragment disclosed herein, including but not limited to an anti-IL-6 antibody selected from Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab20, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, Ab35, and Ab36.

There is described an isolated anti-IL-6 antibody or antibody fragment comprising one or more of the CDRs contained in the V_H polypeptide sequences selected from the group consisting of: SEQ ID NO: 3, 18, 19, 22, 38, 54, 70, 86, 102, 117, 118, 123, 139, 155, 171, 187, 203, 219, 235, 251, 267, 283, 299, 315, 331, 347, 363, 379, 395, 411, 427, 443, 459, 475, 491, 507, 523, 539, 555 and SEQ ID NO: 571 and/or one or more of the CDRs contained in the V_L polypeptide sequence consisting of: 2, 20, 21, 37, 53, 69, 85, 101, 119, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346, 362, 378, 394, 410, 426, 442, 458, 474, 490, 506, 522, 538, 554 and SEQ ID NO: 570.

The anti-human IL-6 antibody discussed in the two prior paragraphs may comprise at least 2 complementarity determining regions (CDRs) in each the variable light and the variable heavy regions which are identical to those contained in an anti-human IL-6 antibody selected from the group consisting of Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab20, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, Ab35, and Ab36.

The anti-human IL-6 antibody discussed may above comprises at least 2 complementarity determining regions (CDRs) in each the variable light and the variable heavy regions which are identical to those contained in Ab1. It is also described that all of the CDRs of the anti-human IL-6 antibody discussed above are identical to the CDRs contained in an anti-human IL-6 antibody selected from the group consisting of Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab20, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, Ab27, Ab28, Ab29, Ab30, Ab31, Ab32, Ab33, Ab34, Ab35, and Ab36. Within the invention, all of the CDRs of the anti-human IL-6 antibody discussed above are identical to the CDRs contained in Ab1.

The invention further contemplates that the anti-human IL-6 antibody is aglycosylated; are humanized or chimeric; and are a humanized antibody derived from a rabbit (parent) anti-human IL-6 antibody.

The framework regions (FRs) in the variable light region and the variable heavy regions may be human FRs which are unmodified or which have been modified by the substitution of at most 2 or 3 human FR residues in the variable light or heavy chain region with the corresponding FR residues of the parent rabbit antibody, and wherein said human FRs have been derived from human variable heavy and light chain antibody sequences which have been selected from a library of human germline antibody sequences based on their high level of homology to the corresponding rabbit variable heavy or light chain regions relative to other human germline antibody sequences contained in the library.

In one embodiment of the invention, the anti-human IL-6 antibody specifically binds to IL-6 expressing human cells and/or to circulating soluble IL-6 molecules in vivo, including IL-6 expressed on or by human cells in a patient with a disease associated with cells that express IL-6.

In another embodiment, the disease is selected from general fatigue, exercise-induced fatigue, cancer-related fatigue, inflammatory disease-related fatigue, chronic fatigue syndrome, cancer-related cachexia, cardiac-related cachexia, respiratory-related cachexia, renal-related cachexia, age-related cachexia, rheumatoid arthritis, systemic lupus erythematosus (SLE), systemic juvenile idiopathic arthritis, psoriasis, psoriatic arthropathy, ankylosing spondylitis, inflammatory bowel disease (IBD), polymyalgia rheumatica, giant cell arteritis, autoimmune vasculitis, graft versus host disease (GVHD), Sjögren's syndrome, adult onset Still's disease, rheumatoid arthritis, systemic juvenile idiopathic arthritis, osteoarthritis, osteoporosis, Paget's disease of bone, osteoarthritis, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, prostate cancer, leukemia, renal cell cancer, multicentric Castleman's disease, ovarian cancer, drug resistance in cancer chemotherapy, cancer chemotherapy toxicity, ischemic heart disease, atherosclerosis, obesity, diabetes, asthma, multiple sclerosis, Alzheimer's disease, cerebrovascular disease, fever, acute phase response, allergies, anemia, anemia of inflammation (anemia of chronic disease), hypertension, depression, depression associated with a chronic illness, thrombosis, thrombocytosis, acute heart failure, metabolic syndrome, miscarriage, obesity, chronic prostatitis, glomerulonephritis, pelvic inflammatory disease, reperfusion injury, transplant rejection, graft versus host disease (GVHD), avian influenza, smallpox, pandemic influenza, adult respiratory distress syndrome (ARDS), severe acute respiratory syndrome (SARS), sepsis, and systemic inflammatory response syndrome (SIRS). In a preferred embodiment, the disease is selected from a cancer, inflammatory disorder, viral disorder, or autoimmune disorder. In a particularly preferred embodiment, the disease is arthritis, cachexia, and wasting syndrome

The invention further contemplates anti-human IL-6 antibodies directly or indirectly attached to a detectable label or therapeutic agent.

The invention also contemplates one or more nucleic acid sequences which result in the expression of an anti-human IL-6 antibody of the invention, including those comprising, or alternatively consisting of, yeast or human preferred codons. The invention also contemplates vectors (including plasmids or recombinant viral vectors) comprising said nucleic acid sequence(s). There are also described host cells or recombinant host cells expressing at least one of the antibodies set forth above, including mammalian, yeast, bacterial, and insect cells. The host cell may be a yeast cell. The yeast cell may be a diploidal yeast cell. The yeast cell may be a Pichia yeast.

The invention also contemplates the antibody of the invention for use in a method of treatment comprising administering to a patient with a disease or condition associated with IL-6 expressing cells a therapeutically effective amount of the anti-human IL-6 antibody of the invention. Diseases that may be treated are presented in the non-limiting list set forth above. In a preferred embodiment, the disease is selected from a cancer, autoimmune disease, or inflammatory condition. In a particularly preferred embodiment, the disease is cancer or viral infection. In another embodiment the treatment further includes the administration of another therapeutic agent or regimen selected from chemotherapy, radiotherapy, cytokine administration or gene therapy.

The invention further contemplates a method of in vivo imaging which detects the presence of cells which express IL-6 comprising administering a diagnostically effective amount of at least one anti-human IL-6 antibody of the invention. In one embodiment, said administration further includes the administration of a radionuclide or fluorophore that facilitates detection of the antibody at IL-6 expressing disease sites. In another embodiment of the invention, the method of in vivo imaging is used to detect IL-6 expressing tumors or metastases or is used to detect the presence of sites of autoimmune disorders associated with IL-6 expressing cells. In a further embodiment, the results of said in vivo imaging method are used to facilitate design of an appropriate therapeutic regimen, including therapeutic regimens including radiotherapy, chemotherapy or a combination thereof.

Polynucleotides Encoding Anti-IL-6 Antibody Polypeptides

The invention is further directed to polynucleotides encoding the antibodies having binding specificity to IL-6 of the invention. The polynucleotide may comprise the following polynucleotide sequence encoding the variable light chain polypeptide sequence of SEQ ID NO: 2:

The polynucleotide may comprise the following polynucleotide sequence encoding the variable heavy chain polypeptide sequence of SEQ ID NO: 3:

There are described polynucleotides encoding fragments of the antibody having binding specificity to IL-6 comprising, or alternatively consisting of, one or more of the polynucleotide sequences of SEQ ID NO: 12; SEQ ID NO: 13; and SEQ ID NO: 14 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the light chain variable sequence of SEQ ID NO: 2.

There are described polynucleotides encoding fragments of the antibody having binding specificity to IL-6 comprising, or alternatively consisting of, one or more of the polynucleotide sequences of SEQ ID NO: 15; SEQ ID NO: 16; and SEQ ID NO: 17 which correspond to polynucleotides encoding the complementarity-determining regions (CDRs, or hypervariable regions) of the heavy chain variable sequence of SEQ ID NO: 3.

There are described polynucleotide sequences including one or more of the polynucleotide sequences encoding antibody fragments described herein. Polynucleotides encoding fragments of the antibody having binding specificity to IL-6 may comprise, or alternatively consist of, one, two, three or more, including all of the following polynucleotides encoding antibody fragments: the polynucleotide SEQ ID NO: 10 encoding the light chain variable region of SEQ ID NO: 2; the polynucleotide SEQ ID NO: 11 encoding the heavy chain variable region of SEQ ID NO: 3; polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 12; SEQ ID NO: 13; and SEQ ID NO: 14) of the light chain variable region of SEQ ID NO: 10; and polynucleotides encoding the complementarity-determining regions (SEQ ID NO: 15; SEQ ID NO: 16; and SEQ ID NO: 17) of the heavy chain variable region of SEQ ID NO: 11.

Polynucleotides of the invention may further comprise, the following polynucleotide sequence encoding the kappa constant light chain sequence of SEQ ID NO: 586:

Polynucleotides of the invention may further comprise, the following polynucleotide sequence encoding the gamma-1 constant heavy chain polypeptide sequence of SEQ ID NO: 588:

There is described an isolated polynucleotide comprising a polynucleotide encoding an anti-IL-6 V_H antibody amino acid sequence selected from SEQ ID NO: 3, 18, 19, 22, 38, 54, 70, 86, 102, 117, 118, 123, 139, 155, 171, 187, 203, 219, 235, 251, 267, 283, 299, 315, 331, 347, 363, 379, 395, 411, 427, 443, 459, 475, 491, 507, 523, 539, 555 and SEQ ID NO: 571 or encoding a variant thereof wherein at least one framework residue (FR residue) has been substituted with an amino acid present at the corresponding position in a rabbit anti-IL-6 antibody V_H polypeptide or a conservative amino acid substitution.

There is described an isolated polynucleotide comprising the polynucleotide sequence encoding an anti-IL-6 V_L antibody amino acid sequence of 2, 20, 21, 37, 53, 69, 85, 101, 119, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346, 362, 378, 394, 410, 426, 442, 458, 474, 490, 506, 522, 538, 554 and SEQ ID NO: 570 or encoding a variant thereof wherein at least one framework residue (FR residue) has been substituted with an amino acid present at the corresponding position in a rabbit anti-IL-6 antibody V_L polypeptide or a conservative amino acid substitution.

In yet another embodiment, the invention is directed to one or more heterologous polynucleotides comprising a sequence encoding the polypeptides contained in SEQ ID NO:2 and SEQ ID NO:3; SEQ ID NO:2 and SEQ ID NO:18; SEQ ID NO:2 and SEQ ID NO:19; SEQ ID NO:20 and SEQ ID NO:3; SEQ ID NO:20 and SEQ ID NO:18; or SEQ ID NO:20 and SEQ ID NO:19;.

There is described an isolated polynucleotide that expresses a polypeptide containing at least one CDR polypeptide derived from an anti-IL-6 antibody wherein said expressed polypeptide alone specifically binds IL-6 or specifically binds IL-6 when expressed in association with another polynucleotide sequence that expresses a polypeptide containing at least one CDR polypeptide derived from an anti-IL-6 antibody wherein said at least one CDR is selected from those contained in the V_L or V_H polypeptides contained in SEQ ID NO: 3, 18, 19, 22, 38, 54, 70, 86, 102, 117, 118, 123, 139, 155, 171, 187, 203, 219, 235, 251, 267, 283, 299, 315, 331, 347, 363, 379, 395, 411, 427, 443, 459, 475, 491, 507, 523, 539, 555; 571; 2, 20, 21, 37, 53, 69, 85, 101, 119, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330, 346, 362, 378, 394, 410, 426, 442, 458, 474, 490, 506, 522, 538, 554 and SEQ ID NO: 570.

Host cells and vectors comprising said polynucleotides are also described.

The invention further contemplates vectors comprising the polynucleotide sequences encoding the variable heavy and light chain polypeptide sequences of the invention, as well as host cells comprising said sequences. The host cell may be a yeast cell. The yeast host cell may belong to the genus Pichia.

Anti-IL-6 Activity

As stated previously, IL-6 is a member of a family of cytokines that promote cellular responses through a receptor complex consisting of at least one subunit of the signal-transducing glycoprotein gp130 and the IL-6 receptor (IL-6R). The IL-6R may also be present in a soluble form (sIL-6R). IL-6 binds to IL-6R, which then dimerizes the signal-transducing receptor gp130.

It is believed that the anti-IL-6 antibodies of the invention are useful by exhibiting anti-IL-6 activity. In one non-limiting embodiment of the invention, the anti-IL-6 antibodies of the invention exhibit anti-IL-6 activity by binding to IL-6 which may be soluble IL-6 or cell surface expressed IL-6 and/or may prevent or inhibit the binding of IL-6 to IL-6R and/or activation (dimerization) of the gp130 signal-transducing glycoprotein and the formation of IL-6/IL-6R/gp130 multimers and the biological effects of any of the foregoing. The described IL-6 antibodies may possess different antagonistic activities based on where (i.e., epitope) the particular antibody binds IL-6 and/or how it affects the formation of the foregoing IL-6 complexes and/or multimers and the biological effects thereof. Consequently, different IL-6 antibodies e.g., may be better suited for preventing or treating conditions involving the formation and accumulation of substantial soluble IL-6 such as rheumatoid arthritis whereas other antibodies may be favored in treatments wherein the prevention of IL-6/IL-6R/gp130 or IL-6/IL-6R/gp130 multimers is a desired therapeutic outcome. This can be determined in binding and other assays.

The anti-IL-6 activity of the anti-IL-6 antibody of the present invention may also be described by their strength of binding or their affinity for IL-6. This also may affect their therapeutic properties. The anti-IL-6 antibody of the present invention has a dissociation constant (KD) of less than 50 picomolar as assessed by BIAcore.

The anti-IL-6 activity of the anti-IL-6 antibody of the present invention may have binding specificity to IL-6, bind to IL-6 with an off-rate of less than or equal to 10^-4 S^-1, 5x10^-5 S^-1, 10^-5 S^-1, 5x10^-6 S^-1, 10^-6 S^-1, 5x10^-7 S^-1, or 10^-7 S^-1. The anti-IL-6 antibody of the invention may have binding specificity to IL-6, and may bind to a linear or conformational IL-6 epitope.

The anti-IL-6 activity of the anti-IL-6 antibody of the present invention may exhibit anti-IL-6 activity by ameliorating or reducing the symptoms of, or alternatively treating, or preventing, diseases and disorders associated with IL-6. Non-limiting examples of diseases and disorders associated with IL-6 are set forth infra. As noted cancer-related fatigue, cachexia and rheumatoid arthritis are indications for the subject IL-6 antibodies.

In another embodiment of the invention, the anti-IL-6 antibody of the invention does not have binding specificity for IL-6R or the gp-130 signal-transducing glycoprotein.

B-cell Screening and Isolation

There are described methods of isolating a clonal population of antigen-specific B cells that may be used for isolating at least one antigen-specific cell. As described and exemplified infra, these methods contain a series of culture and selection steps that can be used separately, in combination, sequentially, repetitively, or periodically. These methods may be used for isolating at least one antigen-specific cell, which can be used to produce a monoclonal antibody, which is specific to a desired antigen, or a nucleic acid sequence corresponding to such an antibody.

There is described a method comprising the steps of:

1. a. preparing a cell population comprising at least one antigen-specific B cell;
2. b. enriching the cell population, e.g., by chromatography, to form an enriched cell population comprising at least one antigen-specific B cell;
3. c. isolating a single B cell from the enriched B cell population; and
4. d. determining whether the single B cell produces an antibody specific to the antigen.

There is described an improvement to a method of isolating a single, antibody-producing B cell, the improvement comprising enriching a B cell population obtained from a host that has been immunized or naturally exposed to an antigen, wherein the enriching step precedes any selection steps, comprises at least one culturing step, and results in a clonal population of B cells that produces a single monoclonal antibody specific to said antigen.

Throughout this application, a "clonal population of B cells" refers to a population of B cells that only secrete a single antibody specific to a desired antigen. That is to say that these cells produce only one type of monoclonal antibody specific to the desired antigen.

In the present application, "enriching" a cell population cells means increasing the frequency of desired cells, typically antigen-specific cells, contained in a mixed cell population, e.g., a B cell-containing isolate derived from a host that is immunized against a desired antigen. Thus, an enriched cell population encompasses a cell population having a higher frequency of antigen-specific cells as a result of an enrichment step, but this population of cells may contain and produce different antibodies.

The general term "cell population" encompasses pre- and a post-enrichment cell populations, keeping in mind that when multiple enrichment steps are performed, a cell population can be both pre- and post-enrichment. For example, there is described a method:

1. a. harvesting a cell population from an immunized host to obtain a harvested cell population;
2. b. creating at least one single cell suspension from the harvested cell population;
3. c. enriching at least one single cell suspension to form a first enriched cell population;
4. d. enriching the first enriched cell population to form a second enriched cell population;
5. e. enriching the second enriched cell population to form a third enriched cell population; and
6. f. selecting an antibody produced by an antigen-specific cell of the third enriched cell population.

Each cell population may be used directly in the next step, or it can be partially or wholly frozen for long- or short- term storage or for later steps. Also, cells from a cell population can be individually suspended to yield single cell suspensions. The single cell suspension can be enriched, such that a single cell suspension serves as the pre-enrichment cell population. Then, one or more antigen-specific single cell suspensions together form the enriched cell population; the antigen-specific single cell suspensions can be grouped together, e.g., re-plated for further analysis and/or antibody production.

There is described a method of enriching a cell population to yield an enriched cell population having an antigen-specific cell frequency that is about 50% to about 100%, or increments therein. The enriched cell population may have an antigen-specific cell frequency greater than or equal to about 50%, 60%, 70%, 75%, 80%, 90%, 95%, 99%, or 100%.

There is described a method of enriching a cell population whereby the frequency of antigen-specific cells is increased by at least about 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or increments therein.

Throughout this application, the term "increment" is used to define a numerical value in varying degrees of precision, e.g., to the nearest 10, 1, 0.1, 0.01, etc. The increment can be rounded to any measurable degree of precision, and the increment need not be rounded to the same degree of precision on both sides of a range. For example, the range 1 to 100 or increments therein includes ranges such as 20 to 80, 5 to 50, and 0.4 to 98. When a range is open-ended, e.g., a range of less than 100, increments therein means increments between 100 and the measurable limit. For example, less than 100 or increments therein means 0 to 100 or increments therein unless the feature, e.g., temperature, is not limited by 0.

Antigen-specificity can be measured with respect to any antigen. The antigen can be any substance to which an antibody can bind including, but not limited to, peptides, proteins or fragments thereof; carbohydrates; organic and inorganic molecules; receptors produced by animal cells, bacterial cells, and viruses; enzymes; agonists and antagonists of biological pathways; hormones; and cytokines. Exemplary antigens include, but are not limited to, IL-2, IL-4, IL-6, IL-10, IL-12, IL-13, IL-18, IFN-α, IFN-γ, BAFF, CXCL13, IP-10, VEGF, EPO, EGF, HRG, Hepatocyte Growth Factor (HGF) and Hepcidin. Described antigens include IL-6, IL-13, TNF-α, VEGF-α, Hepatocyte Growth Factor (HGF) and Hepcidin. In a method utilizing more than one enrichment step, the antigen used in each enrichment step can be the same as or different from one another. Multiple enrichment steps with the same antigen may yield a large and/or diverse population of antigen-specific cells; multiple enrichment steps with different antigens may yield an enriched cell population with cross-specificity to the different antigens.

Enriching a cell population can be performed by any cell-selection means known in the art for isolating antigen-specific cells. For example, a cell population can be enriched by chromatographic techniques, e.g., Miltenyi bead or magnetic bead technology. The beads can be directly or indirectly attached to the antigen of interest. In a preferred embodiment, the method of enriching a cell population includes at least one chromatographic enrichment step.

A cell population can also be enriched by performed by any antigen-specificity assay technique known in the art, e.g., an ELISA assay or a halo assay. ELISA assays include, but are not limited to, selective antigen immobilization (e.g., biotinylated antigen capture by streptavidin, avidin, or neutravidin coated plate), non-specific antigen plate coating, and through an antigen build-up strategy (e.g., selective antigen capture followed by binding partner addition to generate a heteromeric protein-antigen complex). The antigen can be directly or indirectly attached to a solid matrix or support, e.g., a column. A halo assay comprises contacting the cells with antigen-loaded beads and labeled anti-host antibody specific to the host used to harvest the B cells. The label can be, e.g., a fluorophore. At least one assay enrichment step may be performed on at least one single cell suspension. The method of enriching a cell population may include at least one chromatographic enrichment step and at least one assay enrichment step.

Methods of "enriching" a cell population by size or density are known in the art. See, e.g., U.S. Patent 5,627,052 . These steps can be used in the present method in addition to enriching the cell population by antigen-specificity.

The cell populations described herein contain at least one cell capable of recognizing an antigen. Antigen-recognizing cells include, but are not limited to, B cells, plasma cells, and progeny thereof. There is described a clonal cell population containing a single type of antigen-specific B-cell, i.e., the cell population produces a single monoclonal antibody specific to a desired antigen.

It is believed that the clonal antigen-specific population of B cells may consist predominantly of antigen-specific, antibody-secreting cells, which are obtained by the novel culture and selection protocol provided herein. Accordingly, there are described methods for obtaining an enriched cell population containing at least one antigen-specific, antibody-secreting cell. There is described an enriched cell population containing about 50% to about 100%, or increments therein, or greater than or equal to about 60%, 70%, 80%, 90%, or 100% of antigen-specific, antibody-secreting cells.

There is described a method of isolating a single B cell by enriching a cell population obtained from a host before any selection steps, e.g., selecting a particular B cell from a cell population and/or selecting an antibody produced by a particular cell. The enrichment step can be performed as one, two, three, or more steps. A single B cell may be isolated from an enriched cell population before confirming whether the single B cell secretes an antibody with antigen-specificity and/or a desired property.

A method of enriching a cell population may be used in a method for antibody production and/or selection. Thus, there is described a method comprising enriching a cell population before selecting an antibody. The method can include the steps of: preparing a cell population comprising at least one antigen-specific cell, enriching the cell population by isolating at least one antigen-specific cell to form an enriched cell population, and inducing antibody production from at least one antigen-specific cell. The enriched cell population may contain more than one antigen-specific cell. Each antigen-specific cell of the enriched population may be cultured under conditions that yield a clonal antigen-specific B cell population before isolating an antibody producing cell therefrom and/or producing an antibody using said B cell, or a nucleic acid sequence corresponding to such an antibody. In contrast to prior techniques where antibodies are produced from a cell population with a low frequency of antigen-specific cells, the described method allows antibody selection from among a high frequency of antigen-specific cells. Because an enrichment step is used prior to antibody selection, the majority of the cells, such as virtually all of the cells, used for antibody production are antigen-specific. By producing antibodies from a population of cells with an increased frequency of antigen specificity, the quantity and variety of antibodies are increased.

In the antibody selection methods described herein, an antibody may be selected after an enrichment step and a culture step that results in a clonal population of antigen-specific B cells. The methods can further comprise a step of sequencing a selected antibody or portions thereof from one or more isolated, antigen-specific cells. Any method known in the art for sequencing can be employed and can include sequencing the heavy chain, light chain, variable region(s), and/or complementarity determining region(s) (CDR).

In addition to the enrichment step, the method for antibody selection can also include one or more steps of screening a cell population for antigen recognition and/or antibody functionality. For example, the desired antibodies may have specific structural features, such as binding to a particular epitope or mimicry of a particular structure; antagonist or agonist activity; or neutralizing activity, e.g., inhibiting binding between the antigen and a ligand. In one embodiment, the antibody functionality screen is ligand-dependent. Screening for antibody functionality includes, but is not limited to, an in vitro protein-protein interaction assay that recreates the natural interaction of the antigen ligand with recombinant receptor protein; and a cell-based response that is ligand dependent and easily monitored (e.g., proliferation response). The method for antibody selection may include a step of screening the cell population for antibody functionality by measuring the inhibitory concentration (IC50). At least one of the isolated, antigen-specific cells may produce an antibody having an IC50 of less than about 100, 50, 30, 25, 10 µg/mL, or increments therein.

In addition to the enrichment step, the method for antibody selection can also include one or more steps of screening a cell population for antibody binding strength. Antibody binding strength can be measured by any method known in the art (e.g., Biacore). At least one of the isolated, antigen-specific cells may produce an antibody having a high antigen affinity, e.g., a dissociation constant (Kd) of less than about 5x10^-10 M-1, such as about 1x10^-13 to 5x10^-10, 1x10^-12 to 1x10^-10, 1x10^-12 to 7.5x10^-11, 1x10^-11 to 2x10^-11, about 1.5x10^-11 or less, or increments therein. Herein, the antibodies are said to be affinity mature. The affinity of the antibodies may be comparable to or higher than the affinity of any one of Panorex® (edrecolomab), Rituxan® (rituximab), Herceptin® (traztuzumab), Mylotarg® (gentuzumab), Campath® (alemtuzumab), Zevalin™ (ibritumomab), Erbitux™ (cetuximab), Avastin™ (bevicizumab), Raptiva™ (efalizumab), Remicade® (infliximab), Humira™ (adalimumab), and Xolair™ (omalizumab). The affinity of the antibodies may be comparable to or higher than the affinity of Humira™. The affinity of an antibody can also be increased by known affinity maturation techniques. At least one cell population may be screened for at least one of, such as both, antibody functionality and antibody binding strength.

In addition to the enrichment step, the method for antibody selection can also include one or more steps of screening a cell population for antibody sequence homology, especially human homology. At least one of the isolated, antigen-specific cells may produce an antibody that has a homology to a human antibody of about 50% to about 100%, or increments therein, or greater than about 60%, 70%, 80%, 85%, 90%, or 95% homologous. The antibodies can be humanized to increase the homology to a human sequence by techniques known in the art such as CDR grafting or selectivity determining residue grafting (SDR).

There are described the antibodies themselves produced according to any of the method described above in terms of IC50, Kd, and/or homology.

The B cell selection protocol disclosed herein has a number of intrinsic advantages versus other methods for obtaining antibody-secreting B cells and monoclonal antibodies specific to desired target antigens. These advantages include, but are not restricted to, the following:

• First, it has been found that when these selection procedures are utilized with a desired antigen such as IL-6 or TNF-α, the methods reproducibly result in antigen-specific B cells capable of generating what appears to be a substantially comprehensive complement of antibodies, i.e., antibodies that bind to the various different epitopes of the antigen. Without being bound by theory, it is hypothesized that the comprehensive complement is attributable to the antigen enrichment step that is performed prior to initial B cell recovery. Moreover, this advantage allows for the isolation and selection of antibodies with different properties as these properties may vary depending on the epitopic specificity of the particular antibody.
• Second, it has been found that the B cell selection protocol reproducibly yields a clonal B cell culture containing a single B cell, or its progeny, secreting a single monoclonal antibody that generally binds to the desired antigen with a relatively high binding affinity, i.e. picomolar or better antigen binding affinities. By contrast, prior antibody selection methods tend to yield relatively few high affinity antibodies and therefore require extensive screening procedures to isolate an antibody with therapeutic potential. Without being bound by theory, it is hypothesized that the protocol results in both in vivo B cell immunization of the host (primary immunization) followed by a second in vitro B cell stimulation (secondary antigen priming step) that may enhance the ability and propensity of the recovered clonal B cells to secrete a single high affinity monoclonal antibody specific to the antigen target.
• Third, it has been observed (as shown herein with IL-6 specific B cells) that the B cell selection protocol reproducibly yields enriched B cells producing IgG's that are, on average, highly selective (antigen specific) to the desired target. Antigen-enriched B cells recovered by these methods are believed to contain B cells capable of yielding the desired full complement of epitopic specificities as discussed above.
• Fourth, it has been observed that the B cell selection protocols, even when used with small antigens, i.e., peptides of 100 amino acids or less, e.g., 5-50 amino acids long, reproducibly give rise to a clonal B cell culture that secretes a single high affinity antibody to the small antigen, e.g., a peptide. This is highly surprising as it is generally quite difficult, labor intensive, and sometimes not even feasible to produce high affinity antibodies to small peptides. Accordingly, the described methods can be used to produce therapeutic antibodies to desired peptide targets, e.g., viral, bacterial or autoantigen peptides, thereby allowing for the production of monoclonal antibodies with very discrete binding properties or even the production of a cocktail of monoclonal antibodies to different peptide targets, e.g., different viral strains. This advantage may especially be useful in the context of the production of a therapeutic or prophylactic vaccine having a desired valency, such as an HPV vaccine that induces protective immunity to different HPV strains.
• Fifth, the B cell selection protocol, particularly when used with B cells derived from rabbits, tends to reproducibly yield antigen-specific antibody sequences that are very similar to endogenous human immunoglobulins (around 90% similar at the amino acid level) and that contain CDRs that possess a length very analogous to human immunoglobulins and therefore require little or no sequence modification (typically at most only a few CDR residues may be modified in the parent antibody sequence and no framework exogenous residues introduced) in order to eliminate potential immunogenicity concerns. In particular, the recombinant antibody may contain only the host (rabbit) CDR1 and CDR2 residues required for antigen recognition and the entire CDR3. Thereby, the high antigen binding affinity of the recovered antibody sequences produced according to the B cell and antibody selection protocol remains intact or substantially intact even with humanization.

In sum, these methods can be used to produce antibodies exhibiting higher binding affinities to more distinct epitopes by the use of a more efficient protocol than was previously known.

There is described a method for identifying a single B cell that secretes an antibody specific to a desired antigen and that optionally possesses at least one desired functional property such as affinity, avidity or cytolytic activity, by a process including the following steps:

1. a. immunizing a host against an antigen;
2. b. harvesting B cells from the host;
3. c. enriching the harvested B cells to increase the frequency of antigen-specific cells;
4. d. creating at least one single cell suspension;
5. e. culturing a sub-population from the single cell suspension under conditions that favor the survival of a single antigen-specific B cell per culture well;
6. f. isolating B cells from the sub-population; and
7. g. determining whether the single B cell produces an antibody specific to the antigen.

Typically, these methods will further comprise an additional step of isolating and sequencing, in whole or in part, the polypeptide and nucleic acid sequences encoding the desired antibody. These sequences or modified versions or portions thereof can be expressed in desired host cells in order to produce recombinant antibodies to a desired antigen.

As noted previously, it is believed that the clonal population of B cells predominantly comprises antibody-secreting B cells producing antibody against the desired antigen. It is also believed based on experimental results obtained with several antigens and with different B cell populations that the clonally produced B cells and the isolated antigen-specific B cells derived therefrom produced according to the methods described herein secrete a monoclonal antibody that is typically of relatively high affinity and moreover is capable of efficiently and reproducibly producing a selection of monoclonal antibodies of greater epitopic variability as compared to other methods of deriving monoclonal antibodies from cultured antigen-specific B cells. The population of immune cells used in such B cell selection methods may be derived from a rabbit. However, other hosts that produce antibodies, including non-human and human hosts, can alternatively be used as a source of immune B cells. It is believed that the use of rabbits as a source of B cells may enhance the diversity of monoclonal antibodies that may be derived by the methods. Also, the antibody sequences derived from rabbits typically possess sequences having a high degree of sequence identity to human antibody sequences making them favored for use in humans since they should possess little antigenicity. In the course of humanization, the final humanized antibody contains a much lower foreign/host residue content, usually restricted to a subset of the host CDR residues that differ dramatically due to their nature versus the human target sequence used in the grafting. This enhances the probability of complete activity recovery in the humanized antibody protein.

The methods of antibody selection using an enrichment step disclosed herein include a step of obtaining a immune cell-containing cell population from an immunized host. Methods of obtaining an immune cell-containing cell population from an immunized host are known in the art and generally include inducing an immune response in a host and harvesting cells from the host to obtain one or more cell populations. The response can be elicited by immunizing the host against a desired antigen. Alternatively, the host used as a source of such immune cells can be naturally exposed to the desired antigen such as an individual who has been infected with a particular pathogen such as a bacterium or virus or alternatively has mounted a specific antibody response to a cancer that the individual is afflicted with.

Host animals are well-known in the art and include, but are not limited to, guinea pig, rabbit, mouse, rat, non-human primate, human, as well as other mammals and rodents, chicken, cow, pig, goat, and sheep. The host may be a mammal, such as a rabbit, mouse, rat, or human. When exposed to an antigen, the host produces antibodies as part of the native immune response to the antigen. As mentioned, the immune response can occur naturally, as a result of disease, or it can be induced by immunization with the antigen. Immunization can be performed by any method known in the art, such as, by one or more injections of the antigen with or without an agent to enhance immune response, such as complete or incomplete Freund's adjuvant. There is also described intrasplenic immunization. As an alternative to immunizing a host animal in vivo, the method can comprise immunizing a host cell culture in vitro.

After allowing time for the immune response (e.g., as measured by serum antibody detection), host animal cells are harvested to obtain one or more cell populations. A harvested cell population is screened for antibody binding strength and/or antibody functionality. A harvested cell population may be from at least one of the spleen, lymph nodes, bone marrow, and/or peripheral blood mononuclear cells (PBMCs). The cells can be harvested from more than one source and pooled. Certain sources may be preferred for certain antigens. For example, the spleen, lymph nodes, and PBMCs are preferred for IL-6; and the lymph nodes are preferred for TNF. The cell population is harvested about 20 to about 90 days or increments therein after immunization, preferably about 50 to about 60 days. A harvested cell population and/or a single cell suspension therefrom can be enriched, screened, and/or cultured for antibody selection. The frequency of antigen-specific cells within a harvested cell population is usually about 1% to about 5%, or increments therein.

A single cell suspension from a harvested cell population may be enriched, such as by using Miltenyi beads. From the harvested cell population having a frequency of antigen-specific cells of about 1% to about 5%, an enriched cell population is thus derived having a frequency of antigen-specific cells approaching 100%.

The method of antibody selection using an enrichment step includes a step of producing antibodies from at least one antigen-specific cell from an enriched cell population. Methods of producing antibodies in vitro are well known in the art, and any suitable method can be employed. An enriched cell population, such as an antigen-specific single cell suspension from a harvested cell population, may be plated at various cell densities, such as 50, 100, 250, 500, or other increments between 1 and 1000 cells per well. The sub-population may comprise no more than about 10,000 antigen-specific, antibody-secreting cells, such as about 50-10,000, about 50-5,000, about 50-1,000, about 50-500, about 50-250 antigen-specific, antibody-secreting cells, or increments therein. Then, these sub-populations are cultured with suitable medium (e.g., an activated T cell conditioned medium, particularly 1-5% activated rabbit T cell conditioned medium) on a feeder layer, such as under conditions that favor the survival of a single proliferating antibody-secreting cell per culture well. The feeder layer, generally comprised of irradiated cell matter, e.g., EL4B cells, does not constitute part of the cell population. The cells are cultured in a suitable media for a time sufficient for antibody production, for example about 1 day to about 2 weeks, about 1 day to about 10 days, at least about 3 days, about 3 to about 5 days, about 5 days to about 7 days, at least about 7 days, or other increments therein. More than one sub-population may be cultured simultaneously. A single antibody-producing cell and progeny thereof may survive in each well, thereby providing a clonal population of antigen-specific B cells in each well. At this stage, the immunoglobulin G (IgG) produced by the clonal population is highly correlative with antigen specificity. The IgGs may exhibit a correlation with antigen specificity that is greater than about 50%, such as greater than 70%, 85%, 90%, 95%, 99%, or increments therein. See Fig. 3, which demonstrates an exemplary correlation for IL-6. The correlations were demonstrated by setting up B cell cultures under limiting conditions to establish single antigen-specific antibody products per well. Antigen-specific versus general IgG synthesis was compared. Three populations were observed: IgG that recognized a single format of antigen (biotinylated and direct coating), detectable IgG and antigen recognition irrespective of immobilization, and IgG production alone. IgG production was highly correlated with antigen-specificity.

A supernatant containing the antibodies is optionally collected, which can be enriched, screened, and/or cultured for antibody selection according to the steps described above. The supernatant may be enriched (such as by an antigen-specificity assay, especially an ELISA assay) and/or screened for antibody functionality.

The enriched, optionally clonal, antigen-specific B cell population from which a supernatant described above is optionally screened in order to detect the presence of the desired secreted monoclonal antibody is used for the isolation of a few B cells, such as a single B cell, which is then tested in an appropriate assay in order to confirm the presence of a single antibody-producing B cell in the clonal B cell population. About 1 to about 20 cells may be isolated from the clonal B cell population, such as less than about 15, 12, 10, 5, or 3 cells, or increments therein, such as a single cell. The screen may be effected by an antigen-specificity assay, especially a halo assay. The halo assay can be performed with the full length protein, or a fragment thereof. The antibody-containing supernatant can also be screened for at least one of: antigen binding affinity; agonism or antagonism of antigen-ligand binding, induction or inhibition of the proliferation of a specific target cell type; induction or inhibition of lysis of a target cell, and induction or inhibition of a biological pathway involving the antigen.

The identified antigen-specific cell can be used to derive the corresponding nucleic acid sequences encoding the desired monoclonal antibody. (An AluI digest can confirm that only a single monoclonal antibody type is produced per well.) As mentioned above, these sequences can be mutated, such as by humanization, in order to render them suitable for use in human medicaments.

As mentioned, the enriched B cell population used in the process can also be further enriched, screened, and/or cultured for antibody selection according to the steps described above which can be repeated or performed in a different order. At least one cell of an enriched, such as a clonal, antigen-specific cell population is isolated, cultured, and used for antibody selection.

Thus, there is described a method comprising:

1. a. harvesting a cell population from an immunized host to obtain a harvested cell population;
2. b. creating at least one single cell suspension from a harvested cell population;
3. c. enriching at least one single cell suspension, such as by chromatography, to form a first enriched cell population;
4. d. enriching the first enriched cell population, such as by ELISA assay, to form a second enriched cell population which may be clonal, i.e., it contains only a single type of antigen-specific B cell;
5. e. enriching the second enriched cell population, such as by halo assay, to form a third enriched cell population containing a single or a few number of B cells that produce an antibody specific to a desired antigen; and
6. f. selecting an antibody produced by an antigen-specific cell isolated from the third enriched cell population.

The method can further include one or more steps of screening the harvested cell population for antibody binding strength (affinity, avidity) and/or antibody functionality. Suitable screening steps include, but are not limited to, assay methods that detect: whether the antibody produced by the identified antigen-specific B cell produces an antibody possessing a minimal antigen binding affinity, whether the antibody agonizes or antagonizes the binding of a desired antigen to a ligand; whether the antibody induces or inhibits the proliferation of a specific cell type; whether the antibody induces or elicits a cytolytic reaction against target cells; whether the antibody binds to a specific epitope; and whether the antibody modulates (inhibits or agonizes) a specific biological pathway or pathways involving the antigen.

Similarly, the method can include one or more steps of screening the second enriched cell population for antibody binding strength and/or antibody functionality.

The method can further include a step of sequencing the polypeptide sequence or the corresponding nucleic acid sequence of the selected antibody. The method can also include a step of producing a recombinant antibody using the sequence, a fragment thereof, or a genetically modified version of the selected antibody. Methods for mutating antibody sequences in order to retain desired properties are well known to those skilled in the art and include humanization, chimerisation, production of single chain antibodies; these mutation methods can yield recombinant antibodies possessing desired effector function, immunogenicity, stability, and removal or addition of glycosylation. The recombinant antibody can be produced by any suitable recombinant cell, including, but not limited to mammalian cells such as CHO, COS, BHK, HEK-293, bacterial cells, yeast cells, plant cells, insect cells, and amphibian cells. In one embodiment, the antibodies are expressed in polyploidal yeast cells, i.e., diploid yeast cells, particularly Pichia.

The method may comprise:

1. a. immunizing a host against an antigen to yield host antibodies;
2. b. screening the host antibodies for antigen specificity and neutralization;
3. c. harvesting B cells from the host;
4. d. enriching the harvested B cells to create an enriched cell population having an increased frequency of antigen-specific cells;
5. e. culturing one or more sub-populations from the enriched cell population under conditions that favor the survival of a single B cell to produce a clonal population in at least one culture well;
6. f. determining whether the clonal population produces an antibody specific to the antigen;
7. g. isolating a single B cell; and
8. h. sequencing the nucleic acid sequence of the antibody produced by the single B cell.

Methods of Humanizing Antibodies

There is described a method for humanizing antibody heavy and light chains. The following method is described for the humanization of the heavy and light chains:

Light Chain

1. 1. Identify the amino acid that is the first one following the signal peptide sequence. This is the start of Framework 1. The signal peptide starts at the first initiation methionine and is typically, but not necessarily 22 amino acids in length for rabbit light chain protein sequences. The start of the mature polypeptide can also be determined experimentally by N-terminal protein sequencing, or can be predicted using a prediction algorithm. This is also the start of Framework 1 as classically defined by those in the field. Example: RbtVL Amino acid residue 1 in Figure 2, starting 'AYDM...'
2. 2. Identify the end of Framework 3. This is typically 86-90 amino acids following the start of Framework 1 and is typically a cysteine residue preceded by two tyrosine residues. This is the end of the Framework 3 as classically defined by those in the field. Example: RbtVL amino acid residue 88 in Figure 2, ending as 'TYYC'
3. 3. Use the rabbit light chain sequence of the polypeptide starting from the beginning of Framework 1 to the end of Framework 3 as defined above and perform a sequence homology search for the most similar human antibody protein sequences. This will typically be a search against human germline sequences prior to antibody maturation in order to reduce the possibility of immunogenicity, however any human sequences can be used. Typically a program like BLAST can be used to search a database of sequences for the most homologous. Databases of human antibody sequences can be found from various sources such as NCBI (National Center for Biotechnology Information). Example: RbtVL amino acid sequence from residues numbered 1 through 88 in Figure 2 is BLASTed against a human antibody germline database. The top three unique returned sequences are shown in Figure 2 as L12A, VI and Vx02.
4. 4. Generally the most homologous human germline variable light chain sequence is then used as the basis for humanization. However those skilled in the art may decide to use another sequence that wasn't the highest homology as determined by the homology algorithm, based on other factors including sequence gaps and framework similarities. Example: In Figure 2, L12A was the most homologous human germline variable light chain sequence and is used as the basis for the humanization of RbtVL.
5. 5. Determine the framework and CDR arrangement (FR1, FR2, FR3, CDR1 & CDR2) for the human homolog being used for the light chain humanization. This is using the traditional layout as described in the field. Align the rabbit variable light chain sequence with the human homolog, while maintaining the layout of the framework and CDR regions. Example: In Figure 2, the RbtVL sequence is aligned with the human homologous sequence L12A, and the framework and CDR domains are indicated.
6. 6. Replace the human homologous light chain sequence CDR1 and CDR2 regions with the CDR1 and CDR2 sequences from the rabbit sequence. If there are differences in length between the rabbit and human CDR sequences then use the entire rabbit CDR sequences and their lengths. It is possible that the specificity, affinity and/or immunogenicity of the resulting humanized antibody may be unaltered if smaller or larger sequence exchanges are performed, or if specific residue(s) are altered, however the exchanges as described have been used successfully, but do not exclude the possibility that other changes may be permitted. Example: In Figure 2, the CDR1 and CDR2 amino acid residues of the human homologous variable light chain L12A are replaced with the CDR1 and CDR2 amino acid sequences from the RbtVL rabbit antibody light chain sequence. The human L12A frameworks 1, 2 and 3 are unaltered. The resulting humanized sequence is shown below as VLh from residues numbered 1 through 88. Note that the only residues that are different from the L12A human sequence are underlined, and are thus rabbit-derived amino acid residues. In this example only 8 of the 88 residues are different than the human sequence.
7. 7. After framework 3 of the new hybrid sequence created in Step 6, attach the entire CDR3 of the rabbit light chain antibody sequence. The CDR3 sequence can be of various lengths, but is typically 9 to 15 amino acid residues in length. The CDR3 region and the beginning of the following framework 4 region are defined classically and identifiable by those skilled in the art. Typically the beginning of Framework 4, and thus after the end of CDR3 consists of the sequence 'FGGG...', however some variation may exist in these residues. Example: In Figure 2, the CDR3 of RbtVL (amino acid residues numbered 89-100) is added after the end of framework 3 in the humanized sequence indicated as VLh.
8. 8. The rabbit light chain framework 4, which is typically the final 11 amino acid residues of the variable light chain and begins as indicated in Step 7 above and typically ends with the amino acid sequence '...VVKR' is replaced with the nearest human light chain framework 4 homolog, usually from germline sequence. Frequently this human light chain framework 4 is of the sequence 'FGGGTKVEIKR'. It is possible that other human light chain framework 4 sequences that are not the most homologous or otherwise different may be used without affecting the specificity, affinity and/or immunogenicity of the resulting humanized antibody. This human light chain framework 4 sequence is added to the end of the variable light chain humanized sequence immediately following the CDR3 sequence from Step 7 above. This is now the end of the variable light chain humanized amino acid sequence. Example: In Figure 2, Framework 4 (FR4) of the RbtVL rabbit light chain sequence is shown above a homologous human FR4 sequence. The human FR4 sequence is added to the humanized variable light chain sequence (VLh) right after the end of the CD3 region added in Step 7 above.

Heavy Chain

1. 1. Identify the amino acid that is the first one following the signal peptide sequence. This is the start of Framework 1. The signal peptide starts at the first initiation methionine and is typically 19 amino acids in length for rabbit heavy chain protein sequences. Typically, but not necessarily always, the final 3 amino acid residues of a rabbit heavy chain signal peptide are '...VQC', followed by the start of Framework 1. The start of the mature polypeptide can also be determined experimentally by N-terminal protein sequencing, or can be predicted using a prediction algorithm. This is also the start of Framework 1 as classically defined by those in the field. Example: RbtVH Amino acid residue 1 in Figure 2, starting 'QEQL...'
2. 2. Identify the end of Framework 3. This is typically 95-100 amino acids following the start of Framework 1 and typically has the final sequence of '...CAR' (although the alanine can also be a valine). This is the end of the Framework 3 as classically defined by those in the field. Example: RbtVH amino acid residue 98 in Figure 2, ending as '...FCVR'.
3. 3. Use the rabbit heavy chain sequence of the polypeptide starting from the beginning of Framework 1 to the end of Framework 3 as defined above and perform a sequence homology search for the most similar human antibody protein sequences. This will typically be against a database of human germline sequences prior to antibody maturation in order to reduce the possibility of immunogenicity, however any human sequences can be used. Typically a program like BLAST can be used to search a database of sequences for the most homologous. Databases of human antibody sequences can be found from various sources such as NCBI (National Center for Biotechnology Information). Example: RbtVH amino acid sequence from residues numbered 1 through 98 in Figure 2 is BLASTed against a human antibody germline database. The top three unique returned sequences are shown in Figure 2 as 3-64-04, 3-66-04, and 3-53-02.
4. 4. Generally the most homologous human germline variable heavy chain sequence is then used as the basis for humanization. However those skilled in the art may decide to use another sequence that wasn't the most homologous as determined by the homology algorithm, based on other factors including sequence gaps and framework similarities. Example: 3-64-04 in Figure 2 was the most homologous human germline variable heavy chain sequence and is used as the basis for the humanization of RbtVH.
5. 5. Determine the framework and CDR arrangement (FR1, FR2, FR3, CDR1 & CDR2) for the human homolog being used for the heavy chain humanization. This is using the traditional layout as described in the field. Align the rabbit variable heavy chain sequence with the human homolog, while maintaining the layout of the framework and CDR regions. Example: In Figure 2, the RbtVH sequence is aligned with the human homologous sequence 3-64-04, and the framework and CDR domains are indicated.
6. 6. Replace the human homologous heavy chain sequence CDR1 and CDR2 regions with the CDR1 and CDR2 sequences from the rabbit sequence. If there are differences in length between the rabbit and human CDR sequences then use the entire rabbit CDR sequences and their lengths. In addition, it may be necessary to replace the final three amino acids of the human heavy chain Framework 1 region with the final three amino acids of the rabbit heavy chain Framework 1. Typically but not always, in rabbit heavy chain Framework 1 these three residues follow a Glycine residue preceded by a Serine residue. In addition, it may be necessary replace the final amino acid of the human heavy chain Framework 2 region with the final amino acid of the rabbit heavy chain Framework 2. Typically, but not necessarily always, this is a Glycine residue preceded by an Isoleucine residue in the rabbit heavy chain Framework 2. It is possible that the specificity, affinity and/or immunogenicity of the resulting humanized antibody may be unaltered if smaller or larger sequence exchanges are performed, or if specific residue(s) are altered, however the exchanges as described have been used successfully, but do not exclude the possibility that other changes may be permitted. For example, a tryptophan amino acid residue typically occurs four residues prior to the end of the rabbit heavy chain CDR2 region, whereas in human heavy chain CDR2 this residue is typically a Serine residue. Changing this rabbit tryptophan residue to the human Serine residue at this position has been demonstrated to have minimal to no effect on the humanized antibody's specificity or affinity, and thus further minimizes the content of rabbit sequence-derived amino acid residues in the humanized sequence. Example: In Figure 2, The CDR1 and CDR2 amino acid residues of the human homologous variable heavy chain are replaced with the CDR1 and CDR2 amino acid sequences from the RbtVH rabbit antibody light chain sequence, except for the boxed residue, which is tryptophan in the rabbit sequence (position number 63) and Serine at the same position in the human sequence, and is kept as the human Serine residue. In addition to the CDR1 and CDR2 changes, the final three amino acids of Framework 1 (positions 28-30) as well as the final residue of Framework 2 (position 49) are retained as rabbit amino acid residues instead of human. The resulting humanized sequence is shown below as VHh from residues numbered 1 through 98. Note that the only residues that are different from the 3-64-04 human sequence are underlined, and are thus rabbit-derived amino acid residues. In this example only 15 of the 98 residues are different than the human sequence.
7. 7. After framework 3 of the new hybrid sequence created in Step 6, attach the entire CDR3 of the rabbit heavy chain antibody sequence. The CDR3 sequence can be of various lengths, but is typically 5 to 19 amino acid residues in length. The CDR3 region and the beginning of the following framework 4 region are defined classically and are identifiable by those skilled in the art. Typically the beginning of framework 4, and thus after the end of CDR3 consists of the sequence WGXG...(where X is usually Q or P), however some variation may exist in these residues. Example: The CDR3 of RbtVH (amino acid residues numbered 99-110) is added after the end of framework 3 in the humanized sequence indicated as VHh.
8. 8. The rabbit heavy chain framework 4, which is typically the final 11 amino acid residues of the variable heavy chain and begins as indicated in Step 7 above and typically ends with the amino acid sequence '...TVSS' is replaced with the nearest human heavy chain framework 4 homolog, usually from germline sequence. Frequently this human heavy chain framework 4 is of the sequence 'WGQGTLVTVSS'. It is possible that other human heavy chain framework 4 sequences that are not the most homologous or otherwise different may be used without affecting the specificity, affinity and/or immunogenicity of the resulting humanized antibody. This human heavy chain framework 4 sequence is added to the end of the variable heavy chain humanized sequence immediately following the CDR3 sequence from Step 7 above. This is now the end of the variable heavy chain humanized amino acid sequence. Example: In Figure 2, framework 4 (FR4) of the RbtVH rabbit heavy chain sequence is shown above a homologous human heavy FR4 sequence. The human FR4 sequence is added to the humanized variable heavy chain sequence (VHh) right after the end of the CD3 region added in Step 7 above.

Methods of Producing Antibodies and Fragments thereof

There is described the production of the antibodies described herein or fragments thereof. Recombinant polypeptides corresponding to the antibodies described herein or fragments thereof are secreted from polyploidal, preferably diploid or tetraploid strains of mating competent yeast. There are described methods for producing these recombinant polypeptides in secreted form for prolonged periods using cultures comprising polyploid yeast, i.e., at least several days to a week, such as at least a month or several months, or at least 6 months to a year or longer. These polyploid yeast cultures will express at least 10-25 mg/liter of the polypeptide, such as at least 50-250 mg/liter, at least 500-1000 mg/liter, or a gram per liter or more of the recombinant polypeptide(s).

A pair of genetically marked yeast haploid cells may be transformed with expression vectors comprising subunits of a desired heteromultimeric protein. One haploid cell comprises a first expression vector, and a second haploid cell comprises a second expression vector. Diploid yeast cells may be transformed with one or more expression vectors that provide for the expression and secretion of one or more of the recombinant polypeptides. A single haploid cell may be transformed with one or more vectors and used to produce a polyploidal yeast by fusion or mating strategies. A diploid yeast culture may be transformed with one or more vectors providing for the expression and secretion of a desired polypeptide or polypeptides. These vectors may comprise vectors e.g., linearized plasmids or other linear DNA products that integrate into the yeast cell's genome randomly, through homologous recombination, or using a recombinase such as Cre/Lox or Flp/Frt. Optionally, additional expression vectors may be introduced into the haploid or diploid cells; or the first or second expression vectors may comprise additional coding sequences; for the synthesis of heterotrimers; heterotetramers; etc. The expression levels of the non-identical polypeptides may be individually calibrated, and adjusted through appropriate selection, vector copy number, promoter strength and/or induction. The transformed haploid cells are genetically crossed or fused. The resulting diploid or tetraploid strains are utilized to produce and secrete fully assembled and biologically functional proteins, humanized antibodies described herein or fragments thereof.

The use of diploid or tetraploid cells for protein production provides for unexpected benefits. The cells can be grown for production purposes, i.e. scaled up, and for extended periods of time, in conditions that can be deleterious to the growth of haploid cells, which conditions may include high cell density; growth in minimal media; growth at low temperatures; stable growth in the absence of selective pressure; and which may provide for maintenance of heterologous gene sequence integrity and maintenance of high level expression over time. Without wishing to be bound thereby, the inventors theorize that these benefits may arise, at least in part, from the creation of diploid strains from two distinct parental haploid strains. Such haploid strains can comprise numerous minor autotrophic mutations, which mutations are complemented in the diploid or tetraploid, enabling growth and enhanced production under highly selective conditions.

Transformed mating competent haploid yeast cells provide a genetic method that enables subunit pairing of a desired protein. Haploid yeast strains are transformed with each of two expression vectors, a first vector to direct the synthesis of one polypeptide chain and a second vector to direct the synthesis of a second, non-identical polypeptide chain. The two haploid strains are mated to provide a diploid host where optimized target protein production can be obtained.

Optionally, additional non-identical coding sequence(s) are provided. Such sequences may be present on additional expression vectors or in the first or the second expression vectors. As is known in the art, multiple coding sequences may be independently expressed from individual promoters; or may be coordinately expressed through the inclusion of an "internal ribosome entry site" or "IRES", which is an element that promotes direct internal ribosome entry to the initiation codon, such as ATG, of a cistron (a protein encoding region), thereby leading to the cap-independent translation of the gene. IRES elements functional in yeast are described by Thompson et al. (2001) P.N.A.S. 98:12866-12868.

Antibody sequences may be produced in combination with a secretory J chain, which provides for enhanced stability of IgA (see U.S. Patent Nos. 5,959,177 ; and 5,202,422 ).

The two haploid yeast strains may each be auxotrophic, and require supplementation of media for growth of the haploid cells. The pair of auxotrophs may be complementary, such that the diploid product will grow in the absence of the supplements required for the haploid cells. Many such genetic markers are known in yeast, including requirements for amino acids (e.g. met, lys, his, arg, etc.), and nucleosides (e.g. ura3, ade1, etc.). Amino acid markers may be used. Alternatively diploid cells which contain the desired vectors can be selected by other means, e.g., by use of other markers, such as green fluorescent protein, antibiotic resistance genes, and various dominant selectable markers.

Two transformed haploid cells may be genetically crossed and diploid strains arising from this mating event selected by their hybrid nutritional requirements and/or antibiotic resistance spectra. Alternatively, populations of the two transformed haploid strains are spheroplasted and fused, and diploid progeny regenerated and selected. By either method, diploid strains can be identified and selectively grown based on their ability to grow in different media than their parents. For example, the diploid cells may be grown in minimal medium that may include antibiotics. The diploid synthesis strategy has certain advantages. Diploid strains have the potential to produce enhanced levels of heterologous protein through broader complementation to underlying mutations, which may impact the production and/or secretion of recombinant protein. Furthermore, once stable strains have been obtained, any antibiotics used to select those strains do not necessarily need to be continuously present in the growth media.

As noted above, a haploid yeast may be transformed with a single or multiple vectors and mated or fused with a non-transformed cell to produce a diploid cell containing the vector or vectors. A diploid yeast cell may be transformed with one or more vectors that provide for the expression and secretion of a desired heterologous polypeptide by the diploid yeast cell.

Two haploid strains may be transformed with a library of polypeptides, e.g. a library of antibody heavy or light chains. Transformed haploid cells that synthesize the polypeptides are mated with the complementary haploid cells. The resulting diploid cells are screened for functional protein. The diploid cells provide a means of rapidly, conveniently and inexpensively bringing together a large number of combinations of polypeptides for functional testing. This technology is especially applicable for the generation of heterodimeric protein products, where optimized subunit synthesis levels are critical for functional protein expression and secretion.

The expression level ratio of the two subunits may be regulated in order to maximize product generation. Heterodimer subunit protein levels have been shown previously to impact the final product generation (Simmons LC, J Immunol Methods. 2002 May 1;263(1-2): 133-47). Regulation can be achieved prior to the mating step by selection for a marker present on the expression vector. By stably increasing the copy number of the vector, the expression level can be increased. In some cases, it may be desirable to increase the level of one chain relative to the other, so as to reach a balanced proportion between the subunits of the polypeptide. Antibiotic resistance markers are useful for this purpose, e.g. Zeocin resistance marker, G418 resistance, etc. and provide a means of enrichment for strains that contain multiple integrated copies of an expression vector in a strain by selecting for transformants that are resistant to higher levels of Zeocin or G418. The proper ratio, e.g. 1:1; 1:2; etc. of the subunit genes may be important for efficient protein production. Even when the same promoter is used to transcribe both subunits, many other factors contribute to the final level of protein expressed and therefore, it can be useful to increase the number of copies of one encoded gene relative to the other. Alternatively, diploid strains that produce higher levels of a polypeptide, relative to single copy vector strains, are created by mating two haploid strains, both of which have multiple copies of the expression vectors.

Host cells are transformed with the above-described expression vectors, mated to form diploid strains, and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants or amplifying the genes encoding the desired sequences. A number of minimal media suitable for the growth of yeast are known in the art. Any of these media may be supplemented as necessary with salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as phosphate, HEPES), nucleosides (such as adenosine and thymidine), antibiotics, trace elements, and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature and pH, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

Secreted proteins are recovered from the culture medium. A protease inhibitor, such as phenyl methyl sulfonyl fluoride (PMSF) may be useful to inhibit proteolytic degradation during purification, and antibiotics may be included to prevent the growth of adventitious contaminants. The composition may be concentrated, filtered, dialyzed, etc., using methods known in the art.

The diploid cells are grown for production purposes. Such production purposes desirably include growth in minimal media, which media lacks pre-formed amino acids and other complex biomolecules, e.g., media comprising ammonia as a nitrogen source, and glucose as an energy and carbon source, and salts as a source of phosphate and calcium. Such production media may lack selective agents such as antibiotics, amino acids, purines, pyrimidines, etc. The diploid cells can be grown to high cell density, for example at least about 50 g/L; more usually at least about 100 g/L; and may be at least about 300, about 400, about 500 g/L or more.

The growth of the subject cells for production purposes may be performed at low temperatures, which temperatures may be lowered during log phase, during stationary phase, or both. The term "low temperature" refers to temperatures of at least about 15°C, more usually at least about 17°C, and may be about 20°C, and is usually not more than about 25°C, more usually not more than about 22°C. The low temperature may usually not be more than about 28°C. Growth temperature can impact the production of full-length secreted proteins in production cultures, and decreasing the culture growth temperature can strongly enhance the intact product yield. The decreased temperature appears to assist intracellular trafficking through the folding and post-translational processing pathways used by the host to generate the target product, along with reduction of cellular protease degradation.

The described methods provide for expression of secreted, active protein, such as a mammalian protein. Secreted, "active antibodies", as used herein, refers to a correctly folded multimer of at least two properly paired chains, which accurately binds to its cognate antigen. Expression levels of active protein are usually at least about 10-50 mg/liter culture, more usually at least about 100 mg/liter, preferably at least about 500 mg/liter, and may be 1000 mg/liter or more.

The described methods can provide for increased stability of the host and heterologous coding sequences during production. The stability is evidenced, for example, by maintenance of high levels of expression of time, where the starting level of expression is decreased by not more than about 20%, usually not more than 10%, and may be decreased by not more than about 5% over about 20 doublings, 50 doublings, 100 doublings, or more.

The strain stability also provides for maintenance of heterologous gene sequence integrity over time, where the sequence of the active coding sequence and requisite transcriptional regulatory elements are maintained in at least about 99% of the diploid cells, usually in at least about 99.9% of the diploid cells, such as in at least about 99.99% of the diploid cells over about 20 doublings, 50 doublings, 100 doublings, or more. Substantially all of the diploid cells may maintain the sequence of the active coding sequence and requisite transcriptional regulatory elements.

Other methods of producing antibodies are well known to those of ordinary skill in the art. For example, methods of producing chimeric antibodies are now well known in the art (See, for example, U.S. Patent No. 4,816,567 to Cabilly et al .; Morrison et al., P.N.A.S., USA, 81:8651-55 (1984); Neuberger, M.S. et al., Nature, 314:268-270 (1985); Boulianne, G.L. et al., Nature, 312:643-46 (1984)

Likewise, other methods of producing humanized antibodies are now well known in the art (See, for example, U.S. Patent Nos. 5,530,101 , 5,585,089 , 5,693,762 , and 6,180,370 to Queen et al ; U.S. Patent Nos. 5,225,539 and 6,548,640 to Winter ; U.S. Patent Nos. 6,054,297 , 6,407,213 and 6,639,055 to Carter et al ; U.S. Patent No. 6,632,927 to Adair ; Jones, P.T. et al, Nature, 321:522-525 (1986); Reichmann, L., et al, Nature, 332:323-327 (1988); Verhoeyen, M, et al, Science, 239:1534-36 (1988)).

Antibody polypeptides having IL-6 binding specificity may be produced by constructing, using conventional techniques well known to those of ordinary skill in the art, an expression vector containing an operon and a DNA sequence encoding an antibody heavy chain in which the DNA sequence encoding the CDRs required for antibody specificity is derived from a non-human cell source, preferably a rabbit B-cell source, while the DNA sequence encoding the remaining parts of the antibody chain is derived from a human cell source.

A second expression vector is produced using the same conventional means well known to those of ordinary skill in the art, said expression vector containing an operon and a DNA sequence encoding an antibody light chain in which the DNA sequence encoding the CDRs required for antibody specificity is derived from a non-human cell source, preferably a rabbit B-cell source, while the DNA sequence encoding the remaining parts of the antibody chain is derived from a human cell source.

The expression vectors are transfected into a host cell by convention techniques well known to those of ordinary skill in the art to produce a transfected host cell, said transfected host cell cultured by conventional techniques well known to those of ordinary skill in the art to produce said antibody polypeptides.

The host cell may be co-transfected with the two expression vectors described above, the first expression vector containing DNA encoding an operon and a light chain-derived polypeptide and the second vector containing DNA encoding an operon and a heavy chain-derived polypeptide. The two vectors contain different selectable markers, but preferably achieve substantially equal expression of the heavy and light chain polypeptides. Alternatively, a single vector may be used, the vector including DNA encoding both the heavy and light chain polypeptides. The coding sequences for the heavy and light chains may comprise cDNA.

The host cells used to express the antibody polypeptides may be either a bacterial cell such as E. coli, or a eukaryotic cell. A mammalian cell of a well-defined type for this purpose, such as a myeloma cell or a Chinese hamster ovary (CHO) cell line may be used.

The general methods by which the vectors may be constructed, transfection methods required to produce the host cell and culturing methods required to produce the antibody polypeptides from said host cells all include conventional techniques. Although preferably the cell line used to produce the antibody is a mammalian cell line, any other suitable cell line, such as a bacterial cell line such as an E. coli-derived bacterial strain, or a yeast cell line, may alternatively be used.

Similarly, once produced the antibody polypeptides may be purified according to standard procedures in the art, such as for example cross-flow filtration, ammonium sulphate precipitation, and affinity column chromatography.

The antibody polypeptides described herein may also be used for the design and synthesis of either peptide or non-peptide mimetics that would be useful for the same therapeutic applications as the antibody polypeptides of the invention. See, for example, Saragobi et al, Science, 253:792-795 (1991).

Screening Assays

The invention also includes screening assays designed to assist in the identification of diseases and disorders associated with IL-6 in patients exhibiting symptoms of an IL-6 associated disease or disorder.

In one embodiment of the invention, the anti-IL-6 antibody of the invention is used to detect the presence of IL-6 in a biological sample obtained from a patient exhibiting symptoms of a disease or disorder associated with IL-6. The presence of IL-6, or elevated levels thereof when compared to pre-disease levels of IL-6 in a comparable biological sample, may be beneficial in diagnosing a disease or disorder associated with IL-6.

Another embodiment of the invention provides a diagnostic or screening assay to assist in diagnosis of diseases or disorders associated with IL-6 in patients exhibiting symptoms of an IL-6 associated disease or disorder identified herein, comprising assaying the level of IL-6 expression in a biological sample from said patient using a post-translationally modified anti-IL-6 antibody. The anti-IL-6 antibody may be post-translationally modified to include a detectable moiety such as set forth previously in the disclosure.

The IL-6 level in the biological sample is determined using a modified anti-IL-6 antibody as set forth herein, and comparing the level of IL-6 in the biological sample against a standard level of IL-6 (e.g., the level in normal biological samples). The skilled clinician would understand that some variability may exist between normal biological samples, and would take that into consideration when evaluating results.

The above-recited assay may also be useful in monitoring a disease or disorder, where the level of IL-6 obtained in a biological sample from a patient believed to have an IL-6 associated disease or disorder is compared with the level of IL-6 in prior biological samples from the same patient, in order to ascertain whether the IL-6 level in said patient has changed with, for example, a treatment regimen.

The invention is also directed to the antibody of the invention for use in a method of in vivo imaging which detects the presence of cells which express IL-6 comprising administering a diagnostically effective amount of a diagnostic composition. Said in vivo imaging is useful for the detection and imaging of IL-6 expressing tumors or metastases and IL-6 expressing inflammatory sites, for example, and can be used as part of a planning regimen for design of an effective cancer or arthritis treatment protocol. The treatment protocol may include, for example, one or more of radiation, chemotherapy, cytokine therapy, gene therapy, and antibody therapy, as well as an anti-IL-6 antibody.

A skilled clinician would understand that a biological sample includes, but is not limited to, sera, plasma, urine, saliva, mucous, pleural fluid, synovial fluid and spinal fluid.

Methods of Ameliorating or Reducing Symptoms of, or Treating, or Preventing, Diseases and Disorders Associated with, IL-6

In another embodiment of the invention, anti-IL-6 antibodies of the invention are useful for ameliorating or reducing the symptoms of, or treating, or preventing, diseases and disorders associated with IL-6. Anti-IL-6 antibodies described herein can also be administered in a therapeutically effective amount to patients in need of treatment of diseases and disorders associated with IL-6 in the form of a pharmaceutical composition as described in greater detail below.

In one embodiment of the invention, anti-IL-6 antibodies of the invention are useful for ameliorating or reducing the symptoms of, or treating, or preventing, diseases and disorders associated with fatigue. Diseases and disorders associated with fatigue include, but are not limited to, general fatigue, exercise-induced fatigue, cancer-related fatigue, inflammatory disease-related fatigue and chronic fatigue syndrome. See, for example, Esper DH, et al, The cancer cachexia syndrome: a review of metabolic and clinical manifestations, Nutr Clin Pract., 2005 Aug;20 (4):369-76; Vgontzas AN, et al, IL-6 and its circadian secretion in humans, Neuroimmunomodulation, 2005;12(3):131-40; Robson-Ansley, PJ, et al, Acute interleukin-6 administration impairs athletic performance in healthy, trained male runners, Can J Appl Physiol., 2004 Aug;29(4):411-8; Shephard RJ., Cytokine responses to physical activity, with particular reference to IL-6: sources, actions, and clinical implications, Crit Rev Immunol., 2002;22(3): 165-82; Arnold, MC, et al, Using an interleukin-6 challenge to evaluate neuropsychological performance in chronic fatigue syndrome, Psychol Med., 2002 Aug;32(6):1075-89; Kurzrock R., The role of cytokines in cancer-related fatigue, Cancer, 2001 Sep 15;92(6 Suppl):1684-8; Nishimoto N, et al, Improvement in Castleman's disease by humanized anti-interleukin-6 receptor antibody therapy, Blood, 2000 Jan 1; 95 (1):56-61; Vgontzas AN, et al, Circadian interleukin-6 secretion and quantity and depth of sleep, J Clin Endocrinol Metab., 1999 Aug;84(8):2603-7; and Spath-Schwalbe E, et al, Acute effects of recombinant human interleukin 6 on endocrine and central nervous sleep functions in healthy men, J Clin Endocrinol Metab., 1998 May;83(5):1573-9.

In a preferred embodiment of the invention, anti-IL-6 antibodies of the invention are useful for ameliorating or reducing the symptoms of, or treating, or preventing, cachexia. Diseases and disorders associated with cachexia include, but are not limited to, cancer-related cachexia, cardiac-related cachexia, respiratory-related cachexia, renal-related cachexia and age-related cachexia. See, for example, Barton, BE., Interleukin-6 and new strategies for the treatment of cancer, hyperproliferative diseases and paraneoplastic syndromes, Expert Opin Ther Targets, 2005 Aug;9(4):737-52; Zaki MH, et al, CNTO 328, a monoclonal antibody to IL-6, inhibits human tumor-induced cachexia in nude mice, Int J Cancer, 2004 Sep 10;111(4):592-5; Trikha M, et al, Targeted anti-interleukin-6 monoclonal antibody therapy for cancer: a review of the rationale and clinical evidence, Clin Cancer Res., 2003 Oct 15;9(13):4653-65; Lelli G, et al, Treatment of the cancer anorexia-cachexia syndrome: a critical reappraisal, J Chemother., 2003 Jun;15(3):220-5; Argiles JM, et al, Cytokines in the pathogenesis of cancer cachexia, Curr Opin Clin Nutr Metab Care, 2003 Jul;6(4):401-6; Barton BE., IL-6-like cytokines and cancer cachexia: consequences of chronic inflammation, Immunol Res., 2001;23(1):41-58; Yamashita JI, et al, Medroxyprogesterone acetate and cancer cachexia: interleukin-6 involvement, Breast Cancer, 2000;7(2):130-5; Yeh SS, et al, Geriatric cachexia: the role of cytokines, Am J Clin Nutr., 1999 Aug;70(2): 183-97; Strassmann G, et al, Inhibition of experimental cancer cachexia by anti-cytokine and anti-cytokine-receptor therapy, Cytokines Mol Ther., 1995 Jun;1(2):107-13; Fujita J, et al, Anti-interleukin-6 receptor antibody prevents muscle atrophy in colon-26 adenocarcinoma-bearing mice with modulation of lysosomal and ATP-ubiquitin-dependent proteolytic pathways, Int J Cancer, 1996 Nov 27;68(5):637-43; Tsujinaka T, et al, Interleukin 6 receptor antibody inhibits muscle atrophy and modulates proteolytic systems in interleukin 6 transgenic mice, J Clin Invest., 1996 Jan 1;97(1):244-9; Emilie D, et al, Administration of an anti-interleukin-6 monoclonal antibody to patients with acquired immunodeficiency syndrome and lymphoma: effect on lymphoma growth and on B clinical Symptoms, Blood, 1994 Oct 15;84 (8):2472-9; and Strassmann G, et al, Evidence for the involvement of interleukin 6 in experimental cancer cachexia, J Clin Invest., 1992 May;89(5):1681-4.

In another embodiment of the invention, anti-IL-6 antibodies of the invention are useful for ameliorating or reducing the symptoms of, or treating, or preventing, autoimmune diseases and disorders. Diseases and disorders associated with autoimmunity include, but are not limited to, rheumatoid arthritis, systemic lupus erythematosis (SLE), systemic juvenile idiopathic arthritis, psoriasis, psoriatic arthropathy, ankylosing spondylitis, inflammatory bowel disease (IBD), polymyalgia rheumatica, giant cell arteritis, autoimmune vasculitis, graft versus host disease (GVHD), Sjogren's syndrome, adult onset Still's disease. In a preferred embodiment of the invention, humanized anti-IL-6 antibodies of the invention are useful for ameliorating or reducing the symptoms of, or treating, or preventing, rheumatoid arthritis and systemic juvenile idiopathic arthritis. See, for example, Nishimoto N., Clinical studies in patients with Castleman's disease, Crohn's disease, and rheumatoid arthritis in Japan, Clin Rev Allergy Immunol., 2005 Jun;28(3):221-30; Nishimoto N, et al, Treatment of rheumatoid arthritis with humanized anti-interleukin-6 receptor antibody: a multicenter, double-blind, placebo-controlled trial, Arthritis Rheum., 2004 Jun;50(6):1761-9; Choy E., Interleukin 6 receptor as a target for the treatment of rheumatoid arthritis, Ann Rheum Dis., 2003 Nov;62 Suppl 2:ii68-9; Nishimoto N, et al, Toxicity, pharmacokinetics, and dose-finding study of repetitive treatment with the humanized anti-interleukin 6 receptor antibody MRA in rheumatoid arthritis. Phase I/II clinical study, J Rheumatol., 2003 Jul;30(7): 1426-35; Mihara M, et al, Humanized antibody to human interleukin-6 receptor inhibits the development of collagen arthritis in cynomolgus monkeys, Clin Immunol., 2001 Mar;98(3):319-26; Nishimoto N, et al, Anti-interleukin 6 receptor antibody treatment in rheumatic disease, Ann Rheum Dis., 2000 Nov;59 Suppl I:i21-7; Tackey E, et al, Rationale for interleukin-6 blockade in systemic lupus erythematosus, Lupus, 2004;13(5):339-43; Finck BK, et al, Interleukin 6 promotes murine lupus in NZB/NZW Fl mice, J Clin Invest., 1994 Aug;94 (2):585-91; Kitani A, et al, Autostimulatory effects of IL-6 on excessive B cell differentiation in patients with systemic lupus erythematosus: analysis of IL-6 production and IL-6R expression, Clin Exp Immunol., 1992 Apr;88(1):75-83; Stuart RA, et al, Elevated serum interleukin-6 levels associated with active disease in systemic connective tissue disorders, Clin Exp Rheumatol., 1995 Jan-Feb;13 (1):17-22; Mihara M, et al, IL-6 receptor blockage inhibits the onset of autoimmune kidney disease in NZB/W Fl mice, Clin Exp Immunol., 1998 Jun;12(3):397-402; Woo P, et al, Open label phase II trial of single, ascending doses of MRA in Caucasian children with severe systemic juvenile idiopathic arthritis: proof of principle of the efficacy of IL-6 receptor blockade in this type of arthritis and demonstration of prolonged clinical improvement, Arthritis Res Ther., 2005;7(6):RI281-8. Epub 2005 Sep 15; Yokota S, et al, Clinical study of tocilizumab in children with systemic-onset juvenile idiopathic arthritis, Clin Rev Allergy Immunol., 2005 Jun;28(3):231-8; Yokota S, et al, Therapeutic efficacy of humanized recombinant anti-interleukin-6 receptor antibody in children with systemic-onset juvenile idiopathic arthritis, Arthritis Rheum., 2005 Mar;52(3):818-25; de Benedetti F, et al, Targeting the interleukin-6 receptor: a new treatment for systemic juvenile idiopathic arthritis?, Arthritis Rheum., 2005 Mar;52(3):687-93; De Benedetti F, et al, Is systemic juvenile rheumatoid arthritis an interleukin 6 mediated disease?, J Rheumatol., 1998 Feb;25(2):203-7; Ishihara K, et al, IL-6 in autoimmune disease and chronic inflammatory proliferative disease, Cytokine Growth Factor Rev., 2002 Aug-Oct;13 (4-5):357- 68; Gilhar A, et al, In vivo effects of cytokines on psoriatic skin grafted on nude mice :involvement of the tumor necrosis factor (TNF) receptor, Clin Exp Immunol., 1996 Oct;106(1): 134-42; Spadaro A, et al, Interleukin-6 and soluble interleukin-2 receptor in psoriatic arthritis: correlations with clinical and laboratory parameters, Clin Exp Rheumatol., 1996 Jul-Aug;14 (4):413-6; Ameglio F, et al, Interleukin-6 and tumor necrosis factor levels decrease in the suction blister fluids of psoriatic patients during effective therapy, Dermatology, 1994;189(4):359-63; Wendling D, et al, Combination therapy of anti-CD4 and anti-IL-6 monoclonal antibodies in a case of severe spondylarthropathy, Br J Rheumatol., 1996 Dec;35(12):1330; Gratacos J, et al, Serum cytokines (IL-6, TNF-alpha, IL-1 beta and IFN-gamma) in ankylosing spondylitis: a close correlation between serum IL-6 and disease activity and severity, Br J Rheumatol., 1994 Oct;33(10):927-31; Ito H., Treatment of Crohn's disease with anti-IL-6 receptor antibody, J Gastroenterol., 2005 Mar;40 Suppl 16:32-4; Ito H, et al, A pilot randomized trial of a human anti-interleukin-6 receptor monoclonal antibody in active Crohn's disease, Gastroenterology, 2004 Apr;126(4):989-96; discussion 947; Ito H., IL-6 and Crohn's disease, Curr Drug Targets Inflamm Allergy, 2003 Jun;2(2):12530; Ito H, et al, Anti-IL-6 receptor monoclonal antibody inhibits leukocyte recruitment and promotes T-cell apoptosis in a murine model of Crohn's disease, J Gastroenterol., 2002 Nov;37 Suppl 14:56-61; Ito H., Anti-interleukin-6 therapy for Crohn's disease, Curr Pharm Des., 2003;9(4):295-305; Salvarani C, et al, Acute-phase reactants and the risk of relapse/recurrence in polymyalgia rheumatica: a prospective follow-up study, Arthritis Rheum., 2005 Feb 15;53(1):33-8; Roche NE, et al, Correlation of interleukin-6 production and disease activity in polymyalgia rheumatica and giant cell arteritis, Arthritis Rheum., 1993 Sep;36(9): 1286-94; Gupta M, et al, Cytokine modulation with immune gamma-globulin in peripheral blood of normal children and its implications in Kawasaki disease treatment, J Clin Immunol., 2001 May;21(3):193-9; Noris M, et al, Interleukin-6 and RANTES in Takayasu arteritis: a guide for therapeutic decisions?, Circulation, 1999 Jul 6;100(1):55-60; Besbas N, et al, The role of cytokines in Henoch Schonlein purpura, Scand J Rheumatol., 1997;26(6):456-60; Hirohata S, et al, Cerebrospinal fluid interleukin-6 in progressive Neuro-Behcet's syndrome, Clin Immunol Immunopathol., 1997 Jan;82(1):12-7; Yamakawa Y, et al, Interleukin-6 (IL-6) in patients with Behçet's disease, J Dermatol Sci., 1996 Mar;11(3):189-95; Kim DS., Serum interleukin-6 in Kawasaki disease, Yonsei Med J., 1992 Jun;33(2):183-8; Lange, A., et al, Cytokines, adhesion molecules (E-selectin and VCAM-1) and graft-versus-host disease, Arch. Immunol Ther Exp., 1995, 43(2):99-105; Tanaka, J., et al, Cytokine gene expression after allogeneic bone marrow transplantation, Leuk. Lymphoma, 1995 16(5-6):413-418; Dickenson, AM, et al, Predicting outcome in hematological stem cell transplantation, Arch Immunol Ther Exp., 2002 50(6):371-8; Zeiser, R, et al, Immunopathogenesis of acute graft-versus-host disease: implications for novel preventive and therapeutic strategies, Ann Hematol., 2004 83(9):551-65; Dickinson, AM, et al, Genetic polymorphisms predicting the outcome of bone marrow transplants, Br. J Haematol., 2004 127(5):479-90; and Scheinberg MA, et al, Interleukin 6: a possible marker of disease activity in adult onset Still's disease, Clin Exp Rheumatol., 1996 Nov-Dec;14 (6):653-5.

In another embodiment of the invention, anti-IL-6 antibodies of the invention are useful for ameliorating or reducing the symptoms of, or treating, or preventing, diseases and disorders associated with the skeletal system. Diseases and disorders associated with the skeletal system include, but are not limited to, osteoarthritis, osteoporosis and Paget's disease of bone. In a preferred embodiment of the invention, humanized anti-IL-6 antibodies of the invention are useful for ameliorating or reducing the symptoms of, or treating, or preventing, osteoarthritis. See, for example, Malemud CJ., Cytokines as therapeutic targets for osteoarthritis, BioDrugs, 2004;18(1):23-35; Westacott CI, et al, Cytokines in osteoarthritis: mediators or markers of joint destruction?, Semin Arthritis Rheum., 1996 Feb;25(4):254-72; Sugiyama T., Involvement of interleukin-6 and prostaglandin E2 in particular osteoporosis of postmenopausal women with rheumatoid arthritis, J Bone Miner Metab., 2001;19(2):89-96; Abrahamsen B, et al, Cytokines and bone loss in a 5-year longitudinal study - hormone replacement therapy suppresses serum soluble interleukin-6 receptor and increases interleukin-1-receptor antagonist: the Danish Osteoporosis Prevention Study, J Bone Miner Res., 2000 Aug;15(8):1545-54; Straub RH, et al, Hormone replacement therapy and interrelation between serum interleukin-6 and body mass index in postmenopausal women: a population-based study, J Clin Endocrinol Metab., 2000 Mar;85(3):1340-4; Manolagas SC, The role of IL-6 type cytokines and their receptors in bone, Ann N Y Acad Sci., 1998 May 1;840:194-204; Ershler WB, et al, Immunologic aspects of osteoporosis, Dev Comp Immunol., 1997 Nov-Dec;21(6):487-99; Jilka RL, et al, Increased osteoclast development after estrogen loss: mediation by interleukin-6, Science, 1992 Jul 3;257(5066):88-91; Kallen KJ, et al, New developments in IL-6 dependent biology and therapy: where do we stand and what are the options?, Expert Opin Investig Drugs, 1999 Sep;8(9):1327-49; Neale SD, et al, The influence of serum cytokines and growth factors on osteoclast formation in Paget's disease, QJM, 2002 Apr;95 (4):233 - 40; Roodman GD, Osteoclast function In Paget's disease and multiple myeloma, Bone, 1995 Aug;17(2 Suppl):57S-61S; Hoyland JA, et al, Interleukin-6, IL-6 receptor, and IL-6 nuclear factor gene expression in Paget's disease, J Bone Miner Res., 1994 Jan;9(1):75-80; and Roodman GD, et al, Interleukin 6. A potential autocrine/paracrine factor in Paget's disease of bone, J Clin Invest., 1992 Jan;89(1):46-52.

In another embodiment of the invention, anti-IL-6 antibodies of the invention are useful for ameliorating or reducing the symptoms of, or treating, or preventing, diseases and disorders associated with cancer. Diseases and disorders associated with cancer include, but are not limited to, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, prostate cancer, leukemia, renal cell cancer, multicentric Castleman's disease, ovarian cancer, drug resistance in cancer chemotherapy and cancer chemotherapy toxicity. See, for example, Hirata T, et al, Humanized anti-interleukin-6 receptor monoclonal antibody induced apoptosis of fresh and cloned human myeloma cells in vitro, Leuk Res., 2003 Apr;27(4):343-9, Bataille R, et al, Biologic effects of anti-interleukin-6 murine monoclonal antibody in advanced multiple myeloma, Blood, 1995 Jul 15;86 (2):685-91; Goto H, et al, Mouse anti-human interleukin-6 receptor monoclonal antibody inhibits proliferation of fresh human myeloma cells in vitro, Jpn J Cancer Res., 1994 Sep;85(9):958-65; Klein B, et al, Murine anti-interleukin-6 monoclonal antibody therapy for a patient with plasma cell leukemia, Blood, 1991 Sep 1;78(5):1198-204; Mauray S, et al, Epstein-Barr virus-dependent lymphoproliferative disease: critical role of IL-6, Eur J Immunol., 2000 Jul;30(7):2065-73; Tsunenari T, et al, New xenograft model of multiple myeloma and efficacy of a humanized antibody against human interleukin-6 receptor, Blood, 1997 Sep 15;90(6):2437-44; Emilie D, et al, Interleukin-6 production in high-grade B lymphomas: correlation with the presence of malignant immunoblasts in acquired immunodeficiency syndrome and in human immunodeficiency virus-seronegative patients, Blood, 1992 Jul 15;80(2):498-504; Emilie D, et al, Administration of an anti-interleukin-6 monoclonal antibody to patients with acquired immunodeficiency syndrome and lymphoma: effect on lymphoma growth and on B clinical Symptoms, Blood, 1994 Oct 15; 84(8):2472-9; Smith PC, et al, Anti-interleukin-6 monoclonal antibody induces regression of human prostate cancer xenografts in nude mice, Prostate, 2001 Jun 15;48(1):47-53; Smith PC, et al, Interleukin-6 and prostate cancer progression, Cytokine Growth Factor Rev., 2001 Mar;12(1):33-40; Chung TD, et al, Characterization of the role of IL-6 in the progression of prostate cancer, Prostate, 1999 Feb 15;38(3):199-207; Okamoto M, et al, Interleukin-6 as a paracrine and autocrine growth factor in human prostatic carcinoma cells in vitro, Cancer Res., 1997 Jan 1;57(1):141-6; Reittie JE, et al, Interleukin-6 inhibits apoptosis and tumor necrosis factor induced proliferation of B-chronic lymphocytic leukemia, Leuk Lymphoma, 1996 Jun;22(1-2):83-90, follow 186, color plate VI; Sugiyama H, et al, The expression of IL-6 and its related genes in acute leukemia, Leuk Lymphoma, 1996 Mar;21(1-2):49-52; Bataille R, et al, Effects of an anti-interleukin-6 (IL-6) murine monoclonal antibody in a patient with acute monoblastic leukemia, Med Oncol Tumor Pharmacother., 1993;10(4):185-8; Kedar I, et al, Thalidomide reduces serum C-reactive protein and interleukin-6 and induces response to IL-2 in a fraction of metastatic renal cell cancer patients who failed IL-2-based therapy, Int J Cancer, 2004 Jun 10;110(2):260-5; Angelo LS, Talpaz M, Kurzrock R, Autocrine interleukin-6 production in renal cell carcinoma: evidence for the involvement of p53, Cancer Res., 2002 Feb 1;62(3):932-40; Nishimoto N, Humanized anti-interleukin-6 receptor antibody treatment of multicentric Castleman disease, Blood, 2005 Oct 15;106(8):2627-32, Epub 2005 Jul 5; Katsume A, et al, Anti-interleukin 6 (IL-6) receptor antibody suppresses Castleman's disease like symptoms emerged in IL-6 transgenic mice, Cytokine, 2002 Dec 21;20(6):304-11; Nishimoto N, et al, Improvement in Castleman's disease by humanized anti-interleukin-6 receptor antibody therapy, Blood, 2000 Jan 1;95(1):56-61; Screpanti I, Inactivation of the IL-6 gene prevents development of multicentric Castleman's disease in C/EBP beta-deficient mice, J Exp Med., 1996 Oct 1;184(4):1561-6; Hsu SM, et al, Expression of interleukin-6 in Castleman's disease, Hum Pathol., 1993 Aug;24(8):833-9; Yoshizaki K, et al, Pathogenic significance of interleukin-6 (IL 6/BSF-2) in Castleman's disease, Blood, 1989 Sep;74(4):1360-7; Nilsson MB, et al, Interleukin-6, secreted by human ovarian carcinoma cells, is a potent proangiogenic cytokine, Cancer Res., 2005 Dec 1;65(23):10794-800; Toutirais O, et al, Constitutive expression of TGF-betal, interleukin-6 and interleukin-8 by tumor cells as a major component of immune escape in human ovarian carcinoma, Eur Cytokine Netw., 2003 Oct-Dec;14(4):246-55; Obata NH, et al, Effects of interleukin 6 on in vitro cell attachment, migration and invasion of human ovarian carcinoma, Anticancer Res., 1997 Jan-Feb;17 (1A):337-42; Dedoussis GV, et al, Endogenous interleukin 6 conveys resistance to cis-diamminedichloroplatinum-mediated apoptosis of the K562 human leukemic cell line, Exp Cell Res., 1999 Jun 15;249(2):269-78; Borsellino N, et al, Blocking signaling through the Gp130 receptor chain by interleukin-6 and oncostatin M inhibits PC-3 cell growth and sensitizes the tumor cells to etoposide and cisplatin-mediated cytotoxicity, Cancer, 1999 Jan 1;85(1):134-44; Borsellino N, et al, Endogenous interleukin 6 is a resistance factor for cis-diamminedichloroplatinum and etoposide-mediated cytotoxicity of human prostate carcinoma cell lines, Cancer Res., 1995 Oct 15;55(20):4633-9; Mizutani Y, et al, Sensitization of human renal cell carcinoma cells to cis-diamminedichloroplatinum(II) by anti-interleukin 6 monoclonal antibody or anti-interleukin 6 receptor monoclonal antibody; Cancer Res., 1995 Feb 1;55(3):590-6; Yusuf RZ, et al, Paclitaxel resistance: molecular mechanisms and pharmacologic manipulation, Curr Cancer Drug Targets, 2003 Feb;3(1):1-19; Duan Z, et al, Overexpression of IL-6 but not IL-8 increases paclitaxel resistance of U-20S human osteosarcoma cells, Cytokine, 2002 Mar 7;17(5):234-42; Conze D, et al, Autocrine production of interleukin 6 causes multidrug resistance in breast cancer cells, Cancer Res., 2001 Dec 15;61(24):8851-8; Rossi JF, et al, Optimizing the use of anti-interleukin-6 monoclonal antibody with dexamethasone and 140 mg/m2 of melphalan in multiple myeloma: results of a pilot study including biological aspects, Bone Marrow Transplant, 2005 Nov;36(9):771-9; and Tonini G, et al, Oxaliplatin may induce cytokine-release syndrome in colorectal cancer patients, J Biol Regul Homeost Agents, 2002 Apr-Jun;16 (2):105-9.

In another embodiment of the invention, anti-IL-6 antibodies of the invention are useful for ameliorating or reducing the symptoms of, or treating, or preventing, ischemic heart disease, atherosclerosis, obesity, diabetes, asthma, multiple sclerosis, Alzheimer's disease, cerebrovascular disease, fever, acute phase response, allergies, anemia, anemia of inflammation (anemia of chronic disease), hypertension, depression, depression associated with a chronic illness, thrombosis, thrombocytosis, acute heart failure, metabolic syndrome, miscarriage, obesity, chronic prostatitis, glomerulonephritis, pelvic inflammatory disease, reperfusion injury, and transplant rejection. See, for example, Tzoulaki I, et al, C-reactive protein, interleukin-6, and soluble adhesion molecules as predictors of progressive peripheral atherosclerosis in the general population: Edinburgh Artery Study, Circulation, 2005 Aug 16;112(7):976-83, Epub 2005 Aug 8; Rattazzi M, et al, C-reactive protein and interleukin-6 in vascular disease: culprits or passive bystanders?, J Hypertens., 2003 Oct;21(10):1787-803; Ito T, et al, HMG-CoA reductase inhibitors reduce interleukin-6 synthesis in human vascular smooth muscle cells, Cardiovasc Drugs Ther., 2002 Mar;16(2):121-6; Stenvinkel P, et al, Mortality, malnutrition and atherosclerosis in ESRD: what is the role of interleukin-6?, Kidney Int Suppl., 2002 May;(80): 103-8; Yudkin JS, et al, Inflammation, obesity, stress and coronary heart disease: is interleukin-6 the link?, Atherosclerosis, 2000 Feb;148(2):209-14; Huber SA, et al, Interleukin-6 exacerbates early atherosclerosis in mice, Arterioscler Thromb Vasc Biol., 1999 Oct;19(10):2364-7; Kado S, et al, Circulating levels of interleukin-6, its soluble receptor and interleukin-6/interleukin-6 receptor complexes in patients with type 2 diabetes mellitus, Acta Diabetol.,1999 Jun;36(1-2):67-72; Sukovich DA, et al, Expression of interleukin-6 in atherosclerotic lesions of male ApoE-knockout mice: inhibition by 17beta-estradiol, Arterioscler Thromb Vasc Biol.,1998 Sept;8(9): 1498-505; Klover PJ, et al, Interleukin-6 depletion selectively improves hepatic insulin action in obesity, Endocrinology, 2005 Aug;146(8):3417-27, Epub 2005 Apr 21; Lee YH, et al, The evolving role of inflammation in obesity and the metabolic syndrome, Curr Diab Rep., 2005 Feb;5(1):70-5; Diamant M, et al, The association between abdominal visceral fat and carotid stiffness is mediated by circulating inflammatory markers in uncomplicated type 2 diabetes, J Clin Endocrinol Metab., 2005 Mar;90(3):1495-501, Epub 2004 Dec 21; Bray GA, Medical consequences of obesity, J Clin Endocrinol Metab., 2004 Jun;89(6):2583 9; Klover PJ, et al, Chronic exposure to interleukin-6 causes hepatic insulin resistance in mice, Diabetes, 2003 Nov;52 (11):2784-9; Yudkin JS, et al, Inflammation, obesity, stress and coronary heart disease: is interleukin-6 the link?, Atherosclerosis, 2000 Feb;148(2):209-14; Doganci A, et al, Pathological role of IL-6 in the experimental allergic bronchial asthma in mice, Clin Rev Allergy Immunol., 2005 Jun;28(3):257-70; Doganci A, et al, The IL-6R alpha chain controls lung CD4+CD25+ Treg development and function during allergic airway inflammation in vivo, J Clin Invest., 2005 Feb;115(2):313-25, (Erratum in: J Clin Invest., 2005 May;l15(5):1388, Lehr, Hans A); Stelmasiak Z, et al, IL 6 and sIL-6R concentration in the cerebrospinal fluid and serum of MS patients, Med Sci Monit., 2001 Sep-Oct;7(5):914-8; Tilgner J, et al, Continuous interleukin-6 application in vivo via macroencapsulation of interleukin-6-expressing COS-7 cells induces massive gliosis, Glia, 2001 Sep;35(3):234-45, Brunello AG, et al, Astrocytic alterations in interleukin-6 Soluble interleukin-6 receptor alpha double-transgenic mice, Am J Pathol., 2000 Nov;157(5):1485-93; Hampel H, et al, Pattern of interleukin-6 receptor complex immunoreactivity between cortical regions of rapid autopsy normal and Alzheimer's disease brain, Eur Arch Psychiatry Clin Neurosci., 2005 Aug;255(4):269-78, Epub 2004 Nov 26; Cacquevel M, et al, Cytokines in neuroinflammation and Alzheimer's disease, Curr Drug Targets, 2004 Aug;5(6):529-34; Quintanilla RA, et al, Interleukin 6 induces Alzheimer-type phosphorylation of tau protein by deregulating the cdk5/p35 pathway, Exp Cell Res., 2004 Apr 15; 295 (1):245-57; Gadient RA, et al, Interleukin-6 (IL-6)--a molecule with both beneficial and destructive potentials, Prog Neurobiol., 1997 Aug;52(5):379-90; Hull M, et al, Occurrence of interleukin-6 in cortical plaques of Alzheimer's disease patients may precede transformation of diffuse into neuritic plaques, Ann N Y Acad Sci., 1996 Jan 17;777:205-12; Rallidis LS, et al, Inflammatory markers and in-hospital mortality in acute ischaemic stroke, Atherosclerosis, 2005 Dec 30; Emsley HC, et al, Interleukin-6 and acute ischaemic stroke, Acta Neurol Scand., 2005 Oct;112(4):273-4; Smith CJ, et al, Peak plasma interleukin-6 and other peripheral markers of inflammation in the first week of ischaemic stroke correlate with brain infarct volume, stroke severity and long-term outcome, BMC Neurol., 2004 Jan 15;4:2; Vila N, et al, Proinflammatory cytokines and early neurological worsening in ischemic stroke, Stroke, 2000 Oct;31(10):2325-9; and Tarkowski E, et al, Early intrathecal production of interleukin-6 predicts the size of brain lesion in stroke, Stroke, 1995 Aug;26(8):1393-8.

In another embodiment of the invention, anti-IL-6 antibodies of the invention are useful for ameliorating or reducing the symptoms of, or treating, or preventing, diseases and disorders associated with cytokine storm. Diseases and disorders associated with cytokine storm include, but are not limited to, graft versus host disease (GVHD), avian influenza, smallpox, pandemic influenza, adult respiratory distress syndrome (ARDS), severe acute respiratory syndrome (SARS), sepsis, and systemic inflammatory response syndrome (SIRS). See, for example, Cecil, R. L., Goldman, L., & Bennett, J. C. (2000). Cecil textbook of medicine. Philadelphia: W.B. Saunders; Ferrara JL, et al., Cytokine storm of graft-versus-host disease: a critical effector role for interleukin-1, Transplant Proc. 1993 Feb;25(1 Pt 2):1216-7; Osterholm MT, Preparing for the Next Pandemic, N Engl J Med. 2005 May 5;352(18):1839-42; Huang KJ, et al., An interferon-gamma-related cytokine storm in SARS patients, J Med Virol. 2005 Feb;75(2):185-94; and Cheung CY, et al., Induction of proinflammatory cytokines in human macrophages by influenza A (H5N1) viruses: a mechanism for the unusual severity of human disease? Lancet. 2002 Dec 7;360(9348):1831-7.

In another embodiment of the invention, anti-IL-6 antibodies of the invention are are useful as a wakefulness aid.

Administration

The anti-IL-6 antibodies described herein, or IL-6 binding fragments thereof, as well as combinations of said antibody fragments, may be administered to a subject at a concentration of between about 0.1 and 20 mg/kg, such as about 0.4 mg/kg, about 0.8 mg/kg, about 1.6 mg/kg, or about 4 mg/kg, of body weight of recipient subject. The anti-IL-6 antibodies described herein, or IL-6 binding fragments thereof, as well as combinations of said antibody fragments, may be administered to a subject at a concentration of about 0.4 mg/kg of body weight of recipient subject. The anti-IL-6 antibodies described herein, or IL-6 binding fragments thereof, as well as combinations of said antibody fragments, are administered to a recipient subject with a frequency of once every twenty-six weeks or less, such as once every sixteen weeks or less, once every eight weeks or less, or once every four weeks, or less.

It is understood that the effective dosage may depend on recipient subject attributes, such as, for example, age, gender, pregnancy status, body mass index, lean body mass, condition or conditions for which the composition is given, other health conditions of the recipient subject that may affect metabolism or tolerance of the composition, levels of IL-6 in the recipient subject, and resistance to the composition (for example, arising from the patient developing antibodies against the composition). A person of skill in the art would be able to determine an effective dosage and frequency of administration through routine experimentation, for example guided by the disclosure herein and the teachings in Goodman, L. S., Gilman, A., Brunton, L. L., Lazo, J. S., & Parker, K. L. (2006). Goodman & Gilman's the pharmacological basis of therapeutics. New York: McGraw-Hill; Howland, R. D., Mycek, M. J., Harvey, R. A., Champe, P. C., & Mycek, M. J. (2006). Pharmacology. Lippincott's illustrated reviews. Philadelphia: Lippincott Williams & Wilkins; and Golan, D. E. (2008). Principles of pharmacology: the pathophysiologic basis of drug therapy. Philadelphia, Pa., [etc.]: Lippincott Williams & Wilkins.

The anti-IL-6 antibodies described herein, or IL-6 binding fragments thereof, as well as combinations of said antibody fragments, may be administered to a subject in a pharmaceutical formulation.

A "pharmaceutical composition" refers to a chemical or biological composition suitable for administration to a mammal. Such compositions may be specifically formulated for administration via one or more of a number of routes, including but not limited to buccal, epicutaneous, epidural, inhalation, intraarterial, intracardial, intracerebroventricular, intradermal, intramuscular, intranasal, intraocular, intraperitoneal, intraspinal, intrathecal, intravenous, oral, parenteral, rectally via an enema or suppository, subcutaneous, subdermal, sublingual, transdermal, and transmucosal. In addition, administration can occur by means of injection, powder, liquid, gel, drops, or other means of administration.

The anti-IL-6 antibodies described herein, or IL-6 binding fragments thereof, as well as combinations of said antibody fragments, may be optionally administered in combination with one or more active agents. Such active agents include analgesic, antipyretic, anti-inflammatory, antibiotic, antiviral, and anti-cytokine agents. Active agents include agonists, antagonists, and modulators of TNF-α, IL-2, IL-4, IL-6, IL-10, IL-12, IL-13, IL-18, IFN-α, IFN-γ, BAFF, CXCL13, IP-10, VEGF, EPO, EGF, HRG, Hepatocyte Growth Factor (HGF), Hepcidin, including antibodies reactive against any of the foregoing, and antibodies reactive against any of their receptors. Active agents also include 2-Arylpropionic acids, Aceclofenac, Acemetacin, Acetylsalicylic acid (Aspirin), Alclofenac, Alminoprofen, Amoxiprin, Ampyrone, Arylalkanoic acids, Azapropazone, Benorylate/Benorilate, Benoxaprofen, Bromfenac, Carprofen, Celecoxib, Choline magnesium salicylate, Clofezone, COX-2 inhibitors, Dexibuprofen, Dexketoprofen, Diclofenac, Diflunisal, Droxicam, Ethenzamide, Etodolac, Etoricoxib, Faislamine, fenamic acids, Fenbufen, Fenoprofen, Flufenamic acid, Flunoxaprofen, Flurbiprofen, Ibuprofen, Ibuproxam, Indometacin, Indoprofen, Kebuzone, Ketoprofen, Ketorolac, Lornoxicam, Loxoprofen, Lumiracoxib, Magnesium salicylate, Meclofenamic acid, Mefenamic acid, Meloxicam, Metamizole, Methyl salicylate, Mofebutazone, Nabumetone, Naproxen, N-Arylanthranilic acids, Oxametacin, Oxaprozin, Oxicams, Oxyphenbutazone, Parecoxib, Phenazone, Phenylbutazone, Phenylbutazone, Piroxicam, Pirprofen, profens, Proglumetacin, Pyrazolidine derivatives, Rofecoxib, Salicyl salicylate, Salicylamide, Salicylates, Sulfinpyrazone, Sulindac, Suprofen, Tenoxicam, Tiaprofenic acid, Tolfenamic acid, Tolmetin, and Valdecoxib. Antibiotics include Amikacin, Aminoglycosides, Amoxicillin, Ampicillin, Ansamycins, Arsphenamine, Azithromycin, Azlocillin, Aztreonam, Bacitracin, Carbacephem, Carbapenems, Carbenicillin, Cefaclor, Cefadroxil, Cefalexin, Cefalothin, Cefalotin, Cefamandole, Cefazolin, Cefdinir, Cefditoren, Cefepime, Cefixime, Cefoperazone, Cefotaxime, Cefoxitin, Cefpodoxime, Cefprozil, Ceftazidime, Ceftibuten, Ceftizoxime, Ceftobiprole, Ceftriaxone, Cefuroxime, Cephalosporins, Chloramphenicol, Cilastatin, Ciprofloxacin, Clarithromycin, Clindamycin, Cloxacillin, Colistin, Co-trimoxazole, Dalfopristin, Demeclocycline, Dicloxacillin, Dirithromycin, Doripenem, Doxycycline, Enoxacin, Ertapenem, Erythromycin, Ethambutol, Flucloxacillin, Fosfomycin, Furazolidone, Fusidic acid, Gatifloxacin, Geldanamycin, Gentamicin, Glycopeptides, Herbimycin, Imipenem, Isoniazid, Kanamycin, Levofloxacin, Lincomycin, Linezolid, Lomefloxacin, Loracarbef, Macrolides, Mafenide, Meropenem, Meticillin, Metronidazole, Mezlocillin, Minocycline, Monobactams, Moxifloxacin, Mupirocin, Nafcillin, Neomycin, Netilmicin, Nitrofurantoin, Norfloxacin, Ofloxacin, Oxacillin, Oxytetracycline, Paromomycin, Penicillin, Penicillins, Piperacillin, Platensimycin, Polymyxin B, Polypeptides, Prontosil, Pyrazinamide, Quinolones, Quinupristin, Rifampicin, Rifampin, Roxithromycin, Spectinomycin, Streptomycin, Sulfacetamide, Sulfamethizole, Sulfanilimide, Sulfasalazine, Sulfisoxazole, Sulfonamides, Teicoplanin, Telithromycin, Tetracycline, Tetracyclines, Ticarcillin, Tinidazole, Tobramycin, Trimethoprim, Trimethoprim-Sulfamethoxazole, Troleandomycin, Trovafloxacin, and Vancomycin. Active agents also include Aldosterone, Beclometasone, Betamethasone, Corticosteroids, Cortisol, Cortisone acetate, Deoxycorticosterone acetate, Dexamethasone, Fludrocortisone acetate, Glucocorticoids, Hydrocortisone, Methylprednisolone, Prednisolone, Prednisone, Steroids, and Triamcinolone. Antiviral agents include abacavir, acyclovir, acyclovir, adefovir, amantadine, amprenavir, an antiretroviral fixed dose combination, an antiretroviral synergistic enhancer, arbidol, atazanavir, atripla, brivudine, cidofovir, combivir, darunavir, delavirdine, didanosine, docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir, entry inhibitors, famciclovir, fomivirsen, fosamprenavir, foscarnet, fosfonet, fusion inhibitor, ganciclovir, gardasil, ibacitabine, idoxuridine, imiquimod, imunovir, indinavir, inosine, integrase inhibitor, interferon, interferon type I, interferon type II, interferon type III, lamivudine, lopinavir, loviride, maraviroc, MK-0518, moroxydine, nelfinavir, nevirapine, nexavir, nucleoside analogues, oseltamivir, penciclovir, peramivir, pleconaril, podophyllotoxin, protease inhibitor, reverse transcriptase inhibitor, ribavirin, rimantadine, ritonavir, saquinavir, stavudine, tenofovir, tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine, truvada, valaciclovir, valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine, zanamivir, and zidovudine. Any suitable combination of these active agents is also contemplated.

A "pharmaceutical excipient" or a "pharmaceutically acceptable excipient" is a carrier, usually a liquid, in which an active therapeutic agent is formulated. The active therapeutic agent may be a humanized antibody described herein, or one or more fragments thereof. The excipient generally does not provide any pharmacological activity to the formulation, though it may provide chemical and/or biological stability, and release characteristics. Exemplary formulations can be found, for example, in Remington's Pharmaceutical Sciences, 19th Ed., Grennaro, A., Ed., 1995.

As used herein "pharmaceutically acceptable carrier" or "excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents that are physiologically compatible. In one embodiment, the carrier is suitable for parenteral administration. Alternatively, the carrier can be suitable for intravenous, intraperitoneal, intramuscular, or sublingual administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.

Pharmaceutical compositions typically must be sterile and stable under the conditions of manufacture and storage. The pharmaceutical composition may be present in lyophilized form. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. The invention further contemplates the inclusion of a stabilizer in the pharmaceutical composition.

In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin. Moreover, the alkaline polypeptide can be formulated in a time release formulation, for example in a composition which includes a slow release polymer. The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers (PLG). Many methods for the preparation of such formulations are known to those skilled in the art.

The compounds can be administered by a variety of dosage forms. Any biologically-acceptable dosage form known to persons of ordinary skill in the art, and combinations thereof, are contemplated. Examples of such dosage forms include, without limitation, reconstitutable powders, elixirs, liquids, solutions, suspensions, emulsions, powders, granules, particles, microparticles, dispersible granules, cachets, inhalants, aerosol inhalants, patches, particle inhalants, implants, depot implants, injectables (including subcutaneous, intramuscular, intravenous, and intradermal), infusions, and combinations thereof.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the subject invention. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees centigrade; and pressure is at or near atmospheric.

EXAMPLES Example 1 Production of Enriched Antigen-Specific B Cell Antibody Culture

Panels of antibodies are derived by immunizing traditional antibody host animals to exploit the native immune response to a target antigen of interest. Typically, the host used for immunization is a rabbit or other host that produces antibodies using a similar maturation process and provides for a population of antigen-specific B cells producing antibodies of comparable diversity, e.g., epitopic diversity. The initial antigen immunization can be conducted using complete Freund's adjuvant (CFA), and the subsequent boosts effected with incomplete adjuvant. At about 50-60 days after immunization, preferably at day 55, antibody titers are tested, and the Antibody Selection (ABS) process is initiated if appropriate titers are established. The two key criteria for ABS initiation are potent antigen recognition and function-modifying activity in the polyclonal sera.

At the time positive antibody titers are established, animals are sacrificed and B cell sources isolated. These sources include: the spleen, lymph nodes, bone marrow, and peripheral blood mononuclear cells (PBMCs). Single cell suspensions are generated, and the cell suspensions are washed to make them compatible for low temperature long term storage. The cells are then typically frozen.

To initiate the antibody identification process, a small fraction of the frozen cell suspensions are thawed, washed, and placed in tissue culture media. These suspensions are then mixed with a biotinylated form of the antigen that was used to generate the animal immune response, and antigen-specific cells are recovered using the Miltenyi magnetic bead cell selection methodology. Specific enrichment is conducted using streptavidin beads. The enriched population is recovered and progressed in the next phase of specific B cell isolation.

Example 2 Production of Clonal, Antigen-Specific B Cell-Containing Culture

Enriched B cells produced according to Example 1 are then plated at varying cell densities per well in a 96 well microtiter plate. Generally, this is at 50, 100, 250, or 500 cells per well with 10 plates per group. The media is supplemented with 4% activated rabbit T cell conditioned media along with 50K frozen irradiated EL4B feeder cells. These cultures are left undisturbed for 5-7 days at which time supernatant-containing secreted antibody is collected and evaluated for target properties in a separate assay setting. The remaining supernatant is left intact, and the plate is frozen at -70°C. Under these conditions, the culture process typically results in wells containing a mixed cell population that comprises a clonal population of antigen-specific B cells, i.e., a single well will only contain a single monoclonal antibody specific to the desired antigen.

Example 3 Screening of Antibody Supernatants for Monoclonal Antibody of Desired Specificity and/or Functional Properties

Antibody-containing supernatants derived from the well containing a clonal antigen-specific B cell population produced according to Example 2 are initially screened for antigen recognition using ELISA methods. This includes selective antigen immobilization (e.g., biotinylated antigen capture by streptavidin coated plate), nonspecific antigen plate coating, or alternatively, through an antigen build-up strategy (e.g., selective antigen capture followed by binding partner addition to generate a heteromeric protein-antigen complex). Antigen-positive well supernatants are then optionally tested in a function-modifying assay that is strictly dependant on the ligand. One such example is an in vitro protein-protein interaction assay that recreates the natural interaction of the antigen ligand with recombinant receptor protein. Alternatively, a cell-based response that is ligand dependent and easily monitored (e.g., proliferation response) is utilized. Supernatant that displays significant antigen recognition and potency is deemed a positive well. Cells derived from the original positive well are then transitioned to the antibody recovery phase.

Example 4 Recovery of Single, Antibody-Producing B Cell of Desired Antigen Specificity

Cells are isolated from a well that contains a clonal population of antigen-specific B cells (produced according to Example 2 or 3), which secrete a single antibody sequence. The isolated cells are then assayed to isolate a single, antibody-secreting cell. Dynal streptavidin beads are coated with biotinylated target antigen under buffered medium to prepare antigen-containing microbeads compatible with cell viability. Next antigen-loaded beads, antibody-producing cells from the positive well, and a fluorescein isothiocyanate (FITC)-labeled anti-host H&L IgG antibody (as noted, the host can be any mammalian host, e.g., rabbit, mouse, rat, etc.) are incubated together at 37°C. This mixture is then re-pipetted in aliquots onto a glass slide such that each aliquot has on average a single, antibody-producing B-cell. The antigen-specific, antibody-secreting cells are then detected through fluorescence microscopy. Secreted antibody is locally concentrated onto the adjacent beads due to the bound antigen and provides localization information based on the strong fluorescent signal. Antibody-secreting cells are identified via FITC detection of antibody-antigen complexes formed adjacent to the secreting cell. The single cell found in the center of this complex is then recovered using a micromanipulator. The cell is snap-frozen in an eppendorf PCR tube for storage at -80°C until antibody sequence recovery is initiated.

Example 5 Isolation of Antibody Sequences From Antigen-Specific B Cell

Antibody sequences are recovered using a combined RT-PCR based method from a single isolated B-cell produced according to Example 4 or an antigenic specific B cell isolated from the clonal B cell population obtained according to Example 2. Primers are designed to anneal in conserved and constant regions of the target immunoglobulin genes (heavy and light), such as rabbit immunoglobulin sequences, and a two-step nested PCR recovery step is used to obtain the antibody sequence. Amplicons from each well are analyzed for recovery and size integrity. The resulting fragments are then digested with AluI to fingerprint the sequence clonality. Identical sequences display a common fragmentation pattern in their electrophoretic analysis. Significantly, this common fragmentation pattern which proves cell clonality is generally observed even in the wells originally plated up to 1000 cells/well. The original heavy and light chain amplicon fragments are then restriction enzyme digested with HindIII and XhoI or HindIII and BsiWI to prepare the respective pieces of DNA for cloning. The resulting digestions are then ligated into an expression vector and transformed into bacteria for plasmid propagation and production. Colonies are selected for sequence characterization.

Example 6 Recombinant Production of Monoclonal Antibody of Desired Antigen Specificity and/or Functional Properties

Correct full-length antibody sequences for each well containing a single monoclonal antibody is established and miniprep DNA is prepared using Qiagen solid-phase methodology. This DNA is then used to transfect mammalian cells to produce recombinant full-length antibody. Crude antibody product is tested for antigen recognition and functional properties to confirm the original characteristics are found in the recombinant antibody protein. Where appropriate, large-scale transient mammalian transfections are completed, and antibody is purified through Protein A affinity chromatography. Kd is assessed using standard methods (e.g., Biacore) as well as IC50 in a potency assay.

Example 7 Preparation of Antibodies that Bind Human IL-6

By using the antibody selection protocol described herein, one can generate an extensive panel of antibodies. The antibodies have high affinity towards IL-6 (single to double digit pM Kd) and demonstrate potent antagonism of IL-6 in multiple cell-based screening systems (T1165 and HepG2). Furthermore, the collection of antibodies displays distinct modes of antagonism toward IL-6-driven processes.

Immunization Strategy

Rabbits were immunized with huIL-6 (R&R). Immunization consisted of a first subcutaneous (sc) injection of 100 µg in complete Freund's adjuvant (CFA) (Sigma) followed by two boosts, two weeks apart, of 50 µg each in incomplete Freund's adjuvant (IFA) (Sigma). Animals were bled on day 55, and serum titers were determined by ELISA (antigen recognition) and by non-radioactive proliferation assay (Promega) using the T1165 cell line.

Antibody Selection Titer Assessment

Antigen recognition was determined by coating Immulon 4 plates (Thermo) with 1 µg/ml of huIL-6 (50 µl/well) in phosphate buffered saline (PBS, Hyclone) overnight at 4°C. On the day of the assay, plates were washed 3 times with PBS /Tween 20 (PBST tablets, Calbiochem). Plates were then blocked with 200 µl/well of 0.5% fish skin gelatin (FSG, Sigma) in PBS for 30 minutes at 37°C. Blocking solution was removed, and plates were blotted. Serum samples were made (bleeds and pre-bleeds) at a starting dilution of 1:100 (all dilutions were made in FSG 50 µl/well) followed by 1:10 dilutions across the plate (column 12 was left blank for background control). Plates were incubated for 30 minutes at 37°C. Plates were washed 3 times with PBS/Tween 20. Goat anti-rabbit FC-HRP (Pierce) diluted 1:5000 was added to all wells (50 µl/well), and plates were incubated for 30 minutes at 37°C. Plates were washed as described above. 50 µl/well of TMB-Stable stop (Fitzgerald Industries) was added to plates, and color was allowed to develop, generally for 3 to 5 minutes. The development reaction was stopped with 50 µl/well 0.5 M HCl. Plates were read at 450 nm. Optical density (OD) versus dilution was plotted using Graph Pad Prizm software, and titers were determined.

Functional Titer Assessment

The functional activity of the samples was determined by a T1165 proliferation assay. T1165 cells were routinely maintained in modified RPMI medium (Hyclone) supplemented with Hepes, sodium pyruvate, sodium bicarbonate, L-glutamine, high glucose, penicillin/streptomycin, 10% heat inactivated fetal bovine serum (FBS) (all supplements from Hyclone), 2-mercaptoethanol (Sigma), and 10 ng/ml of huIL-6 (R&D). On the day of the assay, cell viability was determined by trypan blue (Invitrogen), and cells were seeded at a fixed density of 20,000 cells/well. Prior to seeding, cells were washed twice in the medium described above without human-IL-6 (by centrifuging at 13000 rpm for 5 minutes and discarding the supernatant). After the last wash, cells were resuspended in the same medium used for washing in a volume equivalent to 50 µl/well. Cells were set aside at room temperature.

In a round-bottom, 96-well plate (Costar), serum samples were added starting at 1:100, followed by a 1:10 dilution across the plate (columns 2 to 10) at 30 µl/well in replicates of 5 (rows B to F: dilution made in the medium described above with no huIL-6). Column 11 was medium only for IL-6 control. 30 µl/well of huIL-6 at 4x concentration of the final EC50 (concentration previously determined) were added to all wells (huIL-6 was diluted in the medium described above). Wells were incubated for 1 hour at 37°C to allow antibody binding to occur. After 1 hour, 50 µl/well of antibody-antigen (Ab-Ag) complex were transferred to a flat-bottom, 96-well plate (Costar) following the plate map format laid out in the round-bottom plate. On Row G, 50 µl/well of medium were added to all wells (columns 2 to 11) for background control. 50 µl/well of the cell suspension set aside were added to all wells (columns 2 to 11, rows B to G). On Columns 1 and 12 and on rows A and H, 200 µl/well of medium was added to prevent evaporation of test wells and to minimize edge effect. Plates were incubated for 72 h at 37°C in 4% CO2. At 72 h, 20 µl/well of CellTiter96 (Promega) reagents was added to all test wells per manufacturer protocol, and plates were incubated for 2 h at 37°C. At 2 h, plates were gently mixed on an orbital shaker to disperse cells and to allow homogeneity in the test wells. Plates were read at 490 nm wavelength. Optical density (OD) versus dilution was plotted using Graph Pad Prizm software, and functional titer was determined. A positive assay control plate was conducted as described above using MAB2061 (R&D Systems) at a starting concentration of 1 µg/ml (final concentration) followed by 1:3 dilutions across the plate.

Tissue Harvesting

Once acceptable titers were established, the rabbit(s) were sacrificed. Spleen, lymph nodes, and whole blood were harvested and processed as follows: Spleen and lymph nodes were processed into a single cell suspension by disassociating the tissue and pushing through sterile wire mesh at 70 µm (Fisher) with a plunger of a 20 cc syringe. Cells were collected in the modified RPMI medium described above without huIL-6, but with low glucose. Cells were washed twice by centrifugation. After the last wash, cell density was determined by trypan blue. Cells were centrifuged at 1500 rpm for 10 minutes; the supernatant was discarded. Cells were resuspended in the appropriate volume of 10% dimethyl sulfoxide (DMSO, Sigma) in FBS (Hyclone) and dispensed at 1 ml/vial. Vials were then stored at -70°C for 24 h prior to being placed in a liquid nitrogen (LN2) tank for long-term storage.

Peripheral blood mononuclear cells (PBMCs) were isolated by mixing whole blood with equal parts of the low glucose medium described above without FBS. 35 ml of the whole blood mixture was carefully layered onto 8 ml of Lympholyte Rabbit (Cedarlane) into a 45 ml conical tube (Corning) and centrifuged 30 minutes at 2500 rpm at room temperature without brakes. After centrifugation, the PBMC layers were carefully removed using a glass Pasteur pipette (VWR), combined, and placed into a clean 50 ml vial. Cells were washed twice with the modified medium described above by centrifugation at 1500 rpm for 10 minutes at room temperature, and cell density was determined by trypan blue staining. After the last wash, cells were resuspended in an appropriate volume of 10% DMSO/FBS medium and frozen as described above.

B cell culture

On the day of setting up B cell culture, PBMC, splenocyte, or lymph node vials were thawed for use. Vials were removed from LN2 tank and placed in a 37°C water bath until thawed. Contents of vials were transferred into 15 ml conical centrifuge tube (Corning) and 10 ml of modified RPMI described above was slowly added to the tube. Cells were centrifuged for 5 minutes at 1.5K rpm, and the supernatant was discarded. Cells were resuspended in 10 ml of fresh media. Cell density and viability was determined by trypan blue. Cells were washed again and resuspended at 1E07 cells/80 ul medium. Biotinylated huIL-6 (B huIL-6) was added to the cell suspension at the final concentration of 3 ug/mL and incubated for 30 minutes at 4°C. Unbound B huIL-6 was removed with two 10 ml washes of phosphate-buffered (PBF):Ca/Mg free PBS (Hyclone), 2 mM ethylenediamine tetraacetic acid (EDTA), 0.5% bovine serum albumin (BSA) (Sigma-biotin free). After the second wash, cells were resuspended at 1E07 cells/80 µl PBF. 20 µl of MACS® streptavidin beads (Milteni)/10E7 cells were added to the cell suspension. Cells were incubated at 4°C for 15 minutes. Cells were washed once with 2 ml of PBF/10E7 cells. After washing, the cells were resuspended at 1E08 cells/500 µl of PBF and set aside. A MACS® MS column (Milteni) was pre-rinsed with 500 ml of PBF on a magnetic stand (Milteni). Cell suspension was applied to the column through a pre-filter, and unbound fraction was collected. The column was washed with 1.5 ml of PBF buffer. The column was removed from the magnet stand and placed onto a clean, sterile 5 ml Polypropylene Falcon tube. 1 ml of PBF buffer was added to the top of the column, and positive selected cells were collected. The yield and viability of positive and negative cell fraction was determined by trypan blue staining. Positive selection yielded an average of 1% of the starting cell concentration.

A pilot cell screen was established to provide information on seeding levels for the culture. Three 10-plate groups (a total of 30 plates) were seeded at 50, 100, and 200 enriched B cells/well. In addition, each well contained 50K cells/well of irradiated EL-4.B5 cells (5,000 Rads) and an appropriate level of T cell supernatant (ranging from 1-5% depending on preparation) in high glucose modified RPMI medium at a final volume of 250 µl/well. Cultures were incubated for 5 to 7 days at 37°C in 4% CO2.

Identification of Selective Antibody Secreting B Cells

Cultures were tested for antigen recognition and functional activity between days 5 and 7.

Antigen Recognition Screening

The ELISA format used is as described above except 50 µl of supernatant from the B cell cultures (BCC) wells (all 30 plates) was used as the source of the antibody. The conditioned medium was transferred to antigen-coated plates. After positive wells were identified, the supernatant was removed and transferred to a 96-well master plate(s). The original culture plates were then frozen by removing all the supernatant except 40 µl/well and adding 60 µl/well of 16% DMSO in FBS. Plates were wrapped in paper towels to slow freezing and placed at -70°C.

Functional Activity Screening

Master plates were then screened for functional activity in the T1165 proliferation assay as described before, except row B was media only for background control, row C was media + IL-6 for positive proliferation control, and rows D-G and columns 2-11 were the wells from the BCC (50 µl/well, single points). 40 µl of IL-6 was added to all wells except the media row at 2.5 times the EC50 concentration determined for the assay. After 1 h incubation, the Ab/Ag complex was transferred to a tissue culture (TC) treated, 96-well, flat-bottom plate. 20 µl of cell suspension in modified RPMI medium without huIL-6 (T1165 at 20,000 cells/well) was added to all wells (100 µl final volume per well). Background was subtracted, and observed OD values were transformed into % of inhibition.

B cell recovery

Plates containing wells of interest were removed from -70°C, and the cells from each well were recovered with 5-200 µl washes of medium/well. The washes were pooled in a 1.5 ml sterile centrifuge tube, and cells were pelleted for 2 minutes at 1500 rpm.

The tube was inverted, the spin repeated, and the supernatant carefully removed. Cells were resuspended in 100 µl/tube of medium. 100 µl biotinylated IL-6 coated streptavidin M280 dynabeads (Invitrogen) and 16 µl of goat anti-rabbit H&L IgG-FITC diluted 1:100 in medium was added to the cell suspension.

20 µl of cell/beads/FITC suspension was removed, and 5 µl droplets were prepared on a glass slide (Corning) previously treated with Sigmacote (Sigma), 35 to 40 droplets/slide. An impermeable barrier of parafin oil (JT Baker) was added to submerge the droplets, and the slide was incubated for 90 minutes at 37°C, 4% CO2 in the dark.

Specific B cells that produce antibody can be identified by the fluorescent ring around them due to antibody secretion, recognition of the bead-associated biotinylated antigen, and subsequent detection by the fluorescent-IgG detection reagent. Once a cell of interest was identified, the cell in the center of the fluorescent ring was recovered via a micromanipulator (Eppendorf). The single cell synthesizing and exporting the antibody was transferred into a 250 µl microcentrifuge tube and placed in dry ice. After recovering all cells of interest, these were transferred to -70°C for long-term storage.

Example 8 Yeast Cell Expression

Antibody genes: Genes were cloned and constructed that directed the synthesis of a chimeric humanized rabbit monoclonal antibody.

Expression vector: The vector contains the following functional components: 1) a mutant ColE1 origin of replication, which facilitates the replication of the plasmid vector in cells of the bacterium Escherichia coli; 2) a bacterial Sh ble gene, which confers resistance to the antibiotic Zeocin and serves as the selectable marker for transformations of both E. coli and P. pastoris; 3) an expression cassette composed of the glyceraldehyde dehydrogenase gene (GAP gene) promoter, fused to sequences encoding the Saccharomyces cerevisiae alpha mating factor pre pro secretion leader sequence, followed by sequences encoding a P. pastoris transcriptional termination signal from the P. pastoris alcohol oxidase I gene (AOX1). The Zeocin resistance marker gene provides a means of enrichment for strains that contain multiple integrated copies of an expression vector in a strain by selecting for transformants that are resistant to higher levels of Zeocin.

P. pastoris strains: P. pastoris strains met1, lys3, ura3 and ade1 may be used. Although any two complementing sets of auxotrophic strains could be used for the construction and maintenance of diploid strains, these two strains are especially suited for this method for two reasons. First, they grow more slowly than diploid strains that are the result of their mating or fusion. Thus, if a small number of haploid ade1 or ura3 cells remain present in a culture or arise through meiosis or other mechanism, the diploid strain should outgrow them in culture.

The second is that it is easy to monitor the sexual state of these strains since diploid Ade+ colonies arising from their mating are a normal white or cream color, whereas cells of any strains that are haploid ade1 mutants will form a colony with a distinct pink color. In addition, any strains that are haploid ura3 mutants are resistant to the drug 5-fluoro-orotic acid (FOA) and can be sensitively identified by plating samples of a culture on minimal medium + uracil plates with FOA. On these plates, only uracil-requiring ura3 mutant (presumably haploid) strains can grow and form colonies. Thus, with haploid parent strains marked with ade1 and ura3, one can readily monitor the sexual state of the resulting antibody-producing diploid strains (haploid versus diploid).

Methods

Construction of pGAPZ-alpha expression vectors for transcription of light and heavy chain antibody genes. The humanized light and heavy chain fragments were cloned into the pGAPZ expression vectors through a PCR directed process. The recovered humanized constructs were subjected to amplification under standard KOD polymerase (Novagen) kit conditions ((1) 94° C, 2 minutes; (2) 94° C, 30 seconds (3) 55° C, 30 seconds; (4) 72° C, 30 seconds-cycling through steps 2-4 for 35 times; (5) 72° C 2 minutes) employing the following primers (1) light chain forward AGCGCTTATTCCGCTATCCAGATGACCCAGTC-the AfeI site is single underlined. The end of the HSA signal sequence is double underlined, followed by the sequence for the mature variable light chain (not underlined); the reverse CGTACGTTTGATTTCCACCTTG.

Variable light chain reverse primer. BsiWI site is underlined, followed by the reverse complement for the 3' end of the variable light chain. Upon restriction enzyme digest with AfeI and BsiWI this enable insertion in-frame with the pGAPZ vector using the human HAS leader sequence in frame with the human kapp light chain constant region for export. (2) A similar strategy is performed for the heavy chain. The forward primer employed is AGCGCTTATTCCGAGGTGCAGCTGGTGGAGTC. The AfeI site is single underlined. The end of the HSA signal sequence is double underlined, followed by the sequence for the mature variable heavy chain (not underlined). The reverse heavy chain primer is CTCGAGACGGTGACGAGGGT. The XhoI site is underlined, followed by the reverse complement for the 3' end of the variable heavy chain. This enables cloning of the heavy chain in-frame with IgG-γ1 CH1-CH2-CH3 region previous inserted within pGAPZ using a comparable directional cloning strategy.

Transformation of expression vectors into haploid ade1 ura3, met1 and lys3 host strains of P. pastoris. All methods used for transformation of haploid P. pastoris strains and genetic manipulation of the P. pastoris sexual cycle are as described in Higgins, D. R., and Cregg, J. M., Eds. 1998. Pichia Protocols. Methods in Molecular Biology. Humana Press, Totowa, NJ.

Prior to transformation, each expression vector is linearized within the GAP promoter sequences with AvrII to direct the integration of the vectors into the GAP promoter locus of the P. pastoris genome. Samples of each vector are then individually transformed into electrocompetent cultures of the ade1, ura3, met1 and lys3 strains by electroporation and successful transformants are selected on YPD Zeocin plates by their resistance to this antibiotic. Resulting colonies are selected, streaked for single colonies on YPD Zeocin plates and then examined for the presence of the antibody gene insert by a PCR assay on genomic DNA extracted from each strain for the proper antibody gene insert and/or by the ability of each strain to synthesize an antibody chain by a colony lift/immunoblot method (Wung et al. Biotechniques 21 808-812 (1996). Haploid ade1, met1 and lys3 strains expressing one of the three heavy chain constructs are collected for diploid constructions along with haploid ura3 strain expressing light chain gene. The haploid expressing heavy chain genes are mated with the appropriate light chain haploid ura3 to generate diploid secreting protein.

Mating of haploid strains synthesizing a single antibody chain and selection of diploid derivatives synthesizing tetrameric functional antibodies. To mate P. pastoris haploid strains, each ade1 (or met1 or lys3) heavy chain producing strain to be crossed is streaked across a rich YPD plate and the ura3 light chain producing strain is streaked across a second YPD plate (-10 streaks per plate). After one or two days incubation at 30°C, cells from one plate containing heavy chain strains and one plate containing ura3 light chain strains are transferred to a sterile velvet cloth on a replica-plating block in a cross hatched pattern so that each heavy chain strain contain a patch of cells mixed with each light chain strain. The cross-streaked replica plated cells are then transferred to a mating plate and incubated at 25°C to stimulate the initiation of mating between strains. After two days, the cells on the mating plates are transferred again to a sterile velvet on a replica-plating block and then transferred to minimal medium plates. These plates are incubated at 30°C for three days to allow for the selective growth of colonies of prototrophic diploid strains. Colonies that arose are picked and streaked onto a second minimal medium plate to single colony isolate and purify each diploid strain. The resulting diploid cell lines are then examined for antibody production.

Putative diploid strains are tested to demonstrate that they are diploid and contain both expression vectors for antibody production. For diploidy, samples of a strain are spread on mating plates to stimulate them to go through meiosis and form spores. Haploid spore products are collected and tested for phenotype. If a significant percentage of the resulting spore products are single or double auxotrophs it may be concluded that the original strain must have been diploid. Diploid strains are examined for the presence of both antibody genes by extracting genomic DNA from each and utilizing this DNA in PCR reactions specific for each gene.

Fusion of haploid strains synthesizing a single antibody chain and selection of diploid derivatives synthesizing tetrameric functional antibodies. As an alternative to the mating procedure described above, individual cultures of single-chain antibody producing haploid ade1 and ura3 strains are spheroplasted and their resulting spheroplasts fused using polyethylene glycol/CaCl₂. The fused haploid strains are then embedded in agar containing 1 M sorbitol and minimal medium to allow diploid strains to regenerate their cell wall and grow into visible colonies. Resulting colonies are picked from the agar, streaked onto a minimal medium plate, and the plates are incubated for two days at 30°C to generate colonies from single cells of diploid cell lines. The resulting putative diploid cell lines are then examined for diploidy and antibody production as described above.

Purification and analysis of antibodies. A diploid strain for the production of full length antibody is derived through the mating of met1 light chain and lys3 heavy chain using the methods described above. Culture media from shake-flask or fermenter cultures of diploid P. pastoris expression strains are collected and examined for the presence of antibody protein via SDS-PAGE and immunoblotting using antibodies directed against heavy and light chains of human IgG, or specifically against the heavy chain of IgG.

To purify the yeast secreted antibodies, clarified media from antibody producing cultures are passed through a protein A column and after washing with 20 mM sodium phosphate, pH 7.0, binding buffer, protein A bound protein is eluted using 0.1 M glycine HCl buffer, pH 3.0. Fractions containing the most total protein are examined by Coomasie blue strained SDS-PAGE and immunoblotting for antibody protein. Antibody is characterized using the ELISA described above for IL-6 recognition.

Assay for antibody activity. The recombinant yeast-derived humanized antibody is evaluated for functional activity through the IL-6 driven T1165 cell proliferation assay and IL-6 stimulated HepG2 haptoglobin assay described above.

Example 9 Acute Phase Response Neutralization by Intravenous Administration of Anti-IL-6 Antibody Ab1.

Human IL-6 can provoke an acute phase response in rats, and one of the major acute phase proteins that is stimulated in the rat is α-2 macroglobulin (A2M). A study was designed to assess the dose of antibody Ab1 required to ablate the A2M response to a single s.c. injection of 100 µg of human IL-6 given one hour after different doses (0.03, 0.1, 0.3, 1, and 3 mg/kg) of antibody Ab1 administered intravenously (n=10 rats/dose level) or polyclonal human IgG1 as the control (n=10 rats). Plasma was recovered and the A2M was quantitated via a commercial sandwich ELISA kit (ICL Inc., Newberg OR; cat. no.- E-25A2M). The endpoint was the difference in the plasma concentration of A2M at the 24 hour time point (post-Abl). The results are presented in Figure 4.

The ID50 for antibody Ab1 was 0.1 mg/kg with complete suppression of the A2M response at the 0.3 mg/kg. This firmly establishes in vivo neutralization of human IL-6 can be accomplished by antibody Ab1.

Example 10 RXF393 Cachexia Model Study 1. Introduction

The human renal cell cancer cell line, RXF393 produces profound weight loss when transplanted into athymic nude mice. Weight loss begins around day 15 after transplantation with 80% of all animals losing at least 30% of their total body weight by day 18 - 20 after transplantation. RXF393 secretes human IL-6 and the plasma concentration of human IL-6 in these animals is very high at around 10ng/ml. Human IL-6 can bind murine soluble IL-6 receptor and activate IL-6 responses in the mouse. Human IL-6 is approximately 10 times less potent than murine IL-6 at activating IL-6 responses in the mouse. The objectives of this study were to determine the effect of antibody Ab1, on survival, body weight, serum amyloid A protein, and hematology parameters in athymic nude mice transplanted with the human renal cell cancer cell line, RXF393.

Methods

Eighty, 6 week old, male athymic nude mice were implanted with RXF393 tumor fragments (30-40mg) subcutaneously in the right flank. Animals were then divided into eight groups of ten mice. Three groups were given either antibody Ab1 at 3mg/kg, 10mg/kg, or 30mg/kg intravenously weekly on day 1, day 8, day 15 and day 22 after transplantation (progression groups). Another three groups were given either antibody Ab1 at 3mg/kg, or 10mg/kg, or 30mg/kg intravenously weekly on day 8, day 15 and day 22 after transplantation (regression groups). Finally, one control group was given polyclonal human IgG 30mg/kg and a second control group was given phosphate buffered saline intravenously weekly on day 1, day 8, day 15 and day 22 after transplantation.

Animals were euthanized at either day 28, when the tumor reached 4,000 mm³ or if they became debilitated (>30% loss of body weight). Animals were weighed on days 1, 6 and then daily from days 9 to 28 after transplantation. Mean Percent Body Weight (MPBW) was used as the primary parameter to monitor weight loss during the study. It was calculated as follows: (Body Weight - Tumor Weight)/Baseline Body Weight x 100. Tumor weight was measured on days 1, 6, 9, 12, 15, 18, 22, 25 and 28 after transplantation. Blood was taken under anesthesia from five mice in each group on days 5 and 13 and all ten mice in each group when euthanized (day 28 in most cases). Blood was analyzed for hematology and serum amyloid A protein (SAA) concentration. An additional group of 10 non-tumor bearing 6 week old, athymic nude male mice had blood samples taken for hematology and SAA concentration estimation to act as a baseline set of values.

Results - Survival

No animals were euthanized or died in any of the antibody Ab1 groups prior to the study termination date of day 28. In the two control groups, 15 animals (7/9 in the polyclonal human IgG group and 8/10 in the phosphate buffered saline group) were found dead or were euthanized because they were very debilitated (>30% loss of body weight). Median survival time in both control groups was 20 days.

The survival curves for the two control groups and the antibody Ab1 progression (dosed from day 1 of the study) groups are presented in Figure 5.

The survival curves for the two control groups and the anibody Ab1 regression (dosed from day 8 of the study) groups are presented in Figure 6.

There was a statistically significant difference between the survival curves for the polyclonal human IgG (p=0.0038) and phosphate buffered saline (p=0.0003) control groups and the survival curve for the six antibody Ab1 groups. There was no statistically significant difference between the two control groups (p=0.97).

Results - Plasma Serum Amyloid A

The mean (± SEM) plasma serum amyloid A concentration versus time for the two control groups and the antibody Ab1 progression (dosed from day 1 of the study) and regression (dosed from day 8 of the study) groups are presented in Table 1. Table 1: Mean Plasma SAA - antibody Ab1, all groups versus control groups

	Mean Plasma SAA±SEM Day 5 (µg/ml)	Mean Plasma SAA±SEM Day 13 (µg/ml)	Mean Plasma SAA±SEM Terminal Bleed (µg/ml)
Polyclonal IgG iv weekly from day 1	675 ± 240 (n=5)	3198 ± 628 (n=4)	13371 ± 2413 (n=4)
PBS iv weekly from day 1	355 ± 207 (n=5)	4844 ± 1126 (n=5)	15826 ± 802 (n=3)
Ab1 30mg/kg iv weekly from day 1	246 ± 100 (n=5)	2979 ± 170 (n=5)	841 ± 469 (n=10)
Ab1 10mg/kg iv weekly from day 1	3629 ± 624 (n=5)	3096 ± 690 (n=5)	996 ± 348 (n=10)
Ab1 3mg/kg iv weekly from day 1	106 ± 9 (n=5)	1623 ± 595 (n=4)	435 ± 70 (n=9)
Ab1 30mg/kg iv weekly from day 8	375 ± 177 (n=5)	1492 ± 418 (n=4)	498 ± 83 (n=9)
Ab1 10mg/kg iv weekly from day 8	487 ± 170 (n=5)	1403 ± 187 (n=5)	396 ± 58 (n=10)
Ab1 3mg/kg iv weekly from day 8	1255 ± 516 (n=5)	466 ± 157 (n=5)	685 ± 350 (n=5)

SAA is up-regulated via the stimulation of hIL-6 and this response is directly correlated with circulating levels of hIL-6 derived from the implanted tumor. The surrogate marker provides an indirect readout for active hIL-6. Thus in the two treatment groups described above there are significantly decreased levels of SAA due to the neutralization of tumor-derived hIL-6. This further supports the contention that antibody Ab1 displays in vivo efficacy.

Example 11 RXF393 Cachexia Model Study 2. Introduction

A second study was performed in the RXF-393 cachexia model where treatment with antibody Ab1 was started at a later stage (days 10 and 13 post-transplantation) and with a more prolonged treatment phase (out to 49 days post transplantation). The dosing interval with antibody Ab1 was shortened to 3 days from 7 and also daily food consumption was measured. There was also an attempt to standardize the tumor sizes at the time of initiating dosing with antibody Ab1.

Methods

Eighty, 6 week old, male athymic nude mice were implanted with RXF393 tumor fragments (30-40mg) subcutaneously in the right flank. 20 mice were selected whose tumors had reached between 270 - 320mg in size and divided into two groups. One group received antibody Ab1 at 10mg/kg i.v. every three days and the other group received polyclonal human IgG 10mg/kg every 3 days from that time-point (day 10 after transplantation). Another 20 mice were selected when their tumor size had reached 400 - 527mg in size and divided into two groups. One group received antibody Ab1 at 10mg/kg i.v. every three days and the other group received polyclonal human IgG 10mg/kg every 3 days from that time-point (day 13 after transplantation). The remaining 40 mice took no further part in the study and were euthanized at either day 49, when the tumor reached 4,000 mm³ or if they became very debilitated (>30% loss of body weight).

Animals were weighed every 3-4 days from day 1 to day 49 after transplantation. Mean Percent Body Weight (MPBW) was used as the primary parameter to monitor weight loss during the study. It was calculated as follows: ((Body Weight - Tumor Weight)/Baseline Body Weight) x 100. Tumor weight was measured every 3-4 days from day 5 to day 49 after transplantation. Food consumption was measured (amount consumed in 24 hours by weight (g) by each treatment group) every day from day 10 for the 270-320mg tumor groups and day 13 for the 400-527mg tumor groups.

Results -survival

The survival curves for antibody Ab1 at 10mg/kg i.v. every three days (270-320mg tumor size) and for the polyclonal human IgG 10mg/kg i.v. every three days (270-320mg tumor size) are presented in Figure 7.

Median survival for the antibody Ab1 at 10mg/kg i.v. every three days (270-320mg tumor size) was 46 days and for the polyclonal human IgG at 10mg/kg i.v. every three days (270-320mg tumor size) was 32.5 days (p=0.0071).

The survival curves for the antibody Ab1 at 10mg/kg i.v. every three days (400-527mg tumor size) and for the polyclonal human IgG at 10mg/kg i.v. every three days (400-527mg tumor size) are presented in Figure 8. Median survival for the antibody Ab1 at 10mg/kg i.v. every three days (400-527mg tumor size) was 46.5 days and for the polyclonal human IgG at 10mg/kg i.v. every three days (400-527mg tumor size) was 27 days (p=0.0481).

Example 12 Multi-dose Pharmacokinetic Evaluation of Antibody Ab1 in Non-human Primates.

Antibody Ab1 was dosed in a single bolus infusion to a single male and single female cynomologus monkey in phosphate buffered saline. Plasma samples were removed at fixed time intervals and the level of antibody Ab1 was quantitated through of the use of an antigen capture ELISA assay. Biotinylated IL-6 (50 µl of 3 µg/mL) was captured on Streptavidin coated 96 well microtiter plates. The plates were washed and blocked with 0.5% Fish skin gelatin. Appropriately diluted plasma samples were added and incubated for 1 hour at room temperature. The supernatants removed and an anti-hFc-HRP conjugated secondary antibody applied and left at room temperature.

The plates were then aspirated and TMB added to visualize the amount of antibody. The specific levels were then determined through the use of a standard curve. A second dose of antibody Ab1 was administered at day 35 to the same two cynomologus monkeys and the experiment replicated using an identical sampling plan. The resulting concentrations are then plot vs. time as show in Figure 9.

This humanized full length aglycosylated antibody expressed and purified Pichia pastoris displays comparable characteristics to mammalian expressed protein. In addition, multiple doses of this product display reproducible half-lives inferring that this production platform does not generate products that display enhanced immunogenicity.

Example 13 Octet Mechanistic Characterization of Antibody Proteins.

IL-6 signaling is dependent upon interactions between IL-6 and two receptors, IL-6R1 (CD126) and GP130 (IL-6 signal transducer). To determine the antibody mechanism of action, mechanistic studies were performed using bio-layer interferometry with an Octet QK instrument (ForteBio; Menlo Park, CA). Studies were performed in two different configurations. In the first orientation, biotinylated IL-6 (R&D systems part number 206-IL-001MG/CF, biotinylated using Pierce EZ-link sulfo-NHS-LC-LC-biotin product number 21338 according to manufacturer's protocols) was initially bound to a streptavidin coated biosensor (ForteBio part number 18-5006). Binding is monitored as an increase in signal.

The IL-6 bound to the sensor was then incubated either with the antibody in question or diluent solution alone. The sensor was then incubated with soluble IL-6R1 (R&D systems product number 227-SR-025/CF) molecule. If the IL-6R1 molecule failed to bind, the antibody was deemed to block IL-6/IL-6R1 interactions. These complexes were incubated with GP130 (R&D systems 228-GP-010/CF) in the presence of IL-6R1 for stability purposes. If GP130 did not bind, it was concluded that the antibody blocked GP130 interactions with IL-6.

In the second orientation, the antibody was bound to a biosensor coated with an anti-human IgG1 Fc-specific reagent (ForteBio part number 18-5001). The IL-6 was bound to the immobilized antibody and the sensor was incubated with IL-6R1. If the IL-6R1 did not interact with the IL-6, then it was concluded that the IL-6 binding antibody blocked IL-6/IL-6R1 interactions. In those situations where antibody/IL-6/IL-6R1 was observed, the complex was incubated with GP130 in the presence of IL-6R1. If GP130 did not interact, then it was concluded that the antibody blocked IL-6/GP130 interactions. All studies were performed in a 200 µL final volume, at 30C and 1000 rpms. For these studies, all proteins were diluted using ForteBio's sample diluent buffer (part number 18-5028).

Results are presented in Figures 10A-E and 11.

Example 14 Peptide Mapping.

In order to determine the epitope recognized by Ab1 on human IL-6, the antibody was employed in a western-blot based assay. The form of human IL-6 utilized in this example had a sequence of 183 amino acids in length (shown below). A 57-member library of overlapping 15 amino acid peptides encompassing this sequence was commercially synthesized and covalently bound to a PepSpots nitrocellulose membrane (JPT Peptide technologies, Berlin, Germany). The sequences of the overlapping 15 amino acid peptides is shown in Figure 12. Blots were prepared and probed according to the manufacturer's recommendations.

Briefly, blots were pre-wet in methanol, rinsed in PBS, and blocked for over 2 hours in 10% non-fat milk in PBS/0.05% Tween (Blocking Solution). The Ab1 antibody was used at 1 mg/ml final dilution, and the HRP-conjugated Mouse Anti-Human-Kappa secondary antibody (Southern BioTech #9220-05) was used at a 1:5000 dilution. Antibody dilutions/incubations were performed in blocking solution. Blots were developed using Amersham ECL advance reagents (GE# RPN2135) and chemiluminescent signal documented using a CCD camera (Alphalnnotec). The results of the blots is shown in Figures 13 and 14.

The sequence of the form of human IL-6 utilized to generate peptide library is set forth:

SEQUENCE LISTING

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• <150> PCT/US2008/064432 <151> 2008-05-21
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• <400> 16 atcatttatg gtagtgatga aacggcctac gcgacctggg cgataggc   48
• <210> 17 <211> 36 <212> DNA <213> Oryctolagus cuniculus
• <400> 17 gatgatagta gtgactggga tgcaaaattt aacttg   36
• <210> 18 <211> 109 <212> PRT <213> Oryctolagus cuniculus
• <400> 18
• <210> 19 <211> 109 <212> PRT <213> Oryctolagus cuniculus
• <400> 19
• <210> 20 <211> 99 <212> PRT <213> Oryctolagus cuniculus
• <400> 20
• <210> 21 <211> 122 <212> PRT <213> Oryctolagus cuniculus
• <400> 21
• <210> 22 <211> 126 <212> PRT <213> Oryctolagus cuniculus
• <400> 22
• <210> 23 <211> 11 <212> PRT <213> Oryctolagus cuniculus
• <400> 23
• <210> 24 <211> 7 <212> PRT <213> Oryctolagus cuniculus
• <400> 24
• <210> 25 <211> 12 <212> PRT <213> Oryctolagus cuniculus
• <400> 25
• <210> 26 <211> 5 <212> PRT <213> Oryctolagus cuniculus
• <400> 26
• <210> 27 <211> 16 <212> PRT <213> Oryctolagus cuniculus
• <400> 27
• <210> 28 <211> 12 <212> PRT <213> Oryctolagus cuniculus
• <400> 28
• <210> 29 <211> 366 <212> DNA <213> Oryctolagus cuniculus
• <400> 29
• <210> 30 <211> 378 <212> DNA <213> Oryctolagus cuniculus
• <400> 30
• <210> 31 <211> 33 <212> DNA <213> Oryctolagus cuniculus
• <400> 31 caggccagtg agaccattta cagttggtta tcc   33
• <210> 32 <211> 21 <212> DNA <213> Oryctolagus cuniculus
• <400> 32 caggcatccg atctggcatc t   21
• <210> 33 <211> 36 <212> DNA <213> Oryctolagus cuniculus
• <400> 33 caacagggtt atagtggtag taatgttgat aatgtt   36
• <210> 34 <211> 15 <212> DNA <213> Oryctolagus cuniculus
• <400> 34 gaccatgcaa tgggc   15
• <210> 35 <211> 48 <212> DNA <213> Oryctolagus cuniculus
• <400> 35 ttcattaata gtggtggtag cgcacgctac gcgagctggg cagaaggc   48
• <210> 36 <211> 36 <212> DNA <213> Oryctolagus cuniculus
• <400> 36 gggggtgctg tttggagtat tcatagtttt gatccc   36
• <210> 37 <211> 123 <212> PRT <213> Oryctolagus cuniculus
• <400> 37
• <210> 38 <211> 125 <212> PRT <213> Oryctolagus cuniculus
• <400> 38
• <210> 39 <211> 13 <212> PRT <213> Oryctolagus cuniculus
• <400> 39
• <210> 40 <211> 7 <212> PRT <213> Oryctolagus cuniculus
• <400> 40
• <210> 41 <211> 11 <212> PRT <213> Oryctolagus cuniculus
• <400> 41
• <210> 42 <211> 5 <212> PRT <213> Oryctolagus cuniculus
• <400> 42
• <210> 43 <211> 16 <212> PRT <213> Oryctolagus cuniculus
• <400> 43
• <210> 44 <211> 12 <212> PRT <213> Oryctolagus cuniculus
• <400> 44
• <210> 45 <211> 369 <212> DNA <213> Oryctolagus cuniculus
• <400> 45
• <210> 46 <211> 375 <212> DNA <213> Oryctolagus cuniculus
• <400> 46
• <210> 47 <211> 39 <212> DNA <213> Oryctolagus cuniculus
• <400> 47 caggccagtc agagtgttta tgacaacaac tacttatcc   39
• <210> 48 <211> 21 <212> DNA <213> Oryctolagus cuniculus
• <400> 48 ggtgcatcca ctctggcatc t   21
• <210> 49 <211> 33 <212> DNA <213> Oryctolagus cuniculus
• <400> 49 gcaggcgttt atgatgatga tagtgataat gcc   33
• <210> 50 <211> 15 <212> DNA <213> Oryctolagus cuniculus
• <400> 50 gtctactaca tgaac   15
• <210> 51 <211> 48 <212> DNA <213> Oryctolagus cuniculus
• <400> 51 ttcattacaa tgagtgataa tataaattac gcgagctggg cgaaaggc   48
• <210> 52 <211> 36 <212> DNA <213> Oryctolagus cuniculus
• <400> 52 agtcgtggct ggggtacaat gggtcggttg gatctc   36
• <210> 53 <211> 123 <212> PRT <213> Oryctolagus cuniculus
• <400> 53
• <210> 54 <211> 126 <212> PRT <213> Oryctolagus cuniculus
• <400> 54
• <210> 55 <211> 13 <212> PRT <213> Oryctolagus cuniculus
• <400> 55
• <210> 56 <211> 7 <212> PRT <213> Oryctolagus cuniculus
• <400> 56
• <210> 57 <211> 11 <212> PRT <213> Oryctolagus cuniculus
• <400> 57
• <210> 58 <211> 5 <212> PRT <213> Oryctolagus cuniculus
• <400> 58
• <210> 59 <211> 16 <212> PRT <213> Oryctolagus cuniculus
• <400> 59
• <210> 60 <211> 12 <212> PRT <213> Oryctolagus cuniculus
• <400> 60
• <210> 61 <211> 369 <212> DNA <213> Oryctolagus cuniculus
• <400> 61
• <210> 62 <211> 378 <212> DNA <213> Oryctolagus cuniculus
• <400> 62
• <210> 63 <211> 39 <212> DNA <213> Oryctolagus cuniculus
• <400> 63 caggccagtc agagtgttta tgagaacaac tatttatcc   39
• <210> 64 <211> 21 <212> DNA <213> Oryctolagus cuniculus
• <400> 64 ggtgcatcca ctctggattc t   21
• <210> 65 <211> 33 <212> DNA <213> Oryctolagus cuniculus
• <400> 65 gcaggcgttt atgatgatga tagtgatgat gcc   33
• <210> 66 <211> 15 <212> DNA <213> Oryctolagus cuniculus
• <400> 66 gcctactaca tgaac   15
• <210> 67 <211> 48 <212> DNA <213> Oryctolagus cuniculus
• <400> 67 ttcattactc tgaataataa tgtagcttac gcgaactggg cgaaaggc   48
• <210> 68 <211> 36 <212> DNA <213> Oryctolagus cuniculus
• <400> 68 agtcgtggct ggggtgcaat gggtcggttg gatctc   36
• <210> 69 <211> 122 <212> PRT <213> Oryctolagus cuniculus
• <400> 69
• <210> 70 <211> 122 <212> PRT <213> Oryctolagus cuniculus
• <400> 70
• <210> 71 <211> 13 <212> PRT <213> Oryctolagus cuniculus
• <400> 71
• <210> 72 <211> 7 <212> PRT <213> Oryctolagus cuniculus
• <400> 72
• <210> 73 <211> 10 <212> PRT <213> Oryctolagus cuniculus
• <400> 73
• <210> 74 <211> 5 <212> PRT <213> Oryctolagus cuniculus
• <400> 74
• <210> 75 <211> 16 <212> PRT <213> Oryctolagus cuniculus
• <400> 75
• <210> 76 <211> 9 <212> PRT <213> Oryctolagus cuniculus
• <400> 76
• <210> 77 <211> 366 <212> DNA <213> Oryctolagus cuniculus
• <400> 77
• <210> 78 <211> 366 <212> DNA <213> Oryctolagus cuniculus
• <400> 78
• <210> 79 <211> 39 <212> DNA <213> Oryctolagus cuniculus
• <400> 79 caggccagtc agagtgttga tgataacaac tggttaggc   39
• <210> 80 <211> 21 <212> DNA <213> Oryctolagus cuniculus
• <400> 80 tctgcatcca ctctggcatc t   21
• <210> 81 <211> 30 <212> DNA <213> Oryctolagus cuniculus
• <400> 81 gcaggcggtt ttagtggtaa tatctttgct   30
• <210> 82 <211> 15 <212> DNA <213> Oryctolagus cuniculus
• <400> 82 agctatgcaa tgagc   15
• <210> 83 <211> 48 <212> DNA <213> Oryctolagus cuniculus
• <400> 83 atcattggtg gttttggtac cacatactac gcgacctggg cgaaaggc   48
• <210> 84 <211> 27 <212> DNA <213> Oryctolagus cuniculus
• <400> 84 ggtggtcctg gtaatggtgg tgacatc   27
• <210> 85 <211> 122 <212> PRT <213> Oryctolagus cuniculus
• <400> 85
• <210> 86 <211> 128 <212> PRT <213> Oryctolagus cuniculus
• <400> 86
• <210> 87 <211> 12 <212> PRT <213> Oryctolagus cuniculus
• <400> 87
• <210> 88 <211> 7 <212> PRT <213> Oryctolagus cuniculus
• <400> 88
• <210> 89 <211> 11 <212> PRT <213> Oryctolagus cuniculus
• <400> 89
• <210> 90 <211> 5 <212> PRT <213> Oryctolagus cuniculus
• <400> 90
• <210> 91 <211> 17 <212> PRT <213> Oryctolagus cuniculus
• <400> 91
• <210> 92 <211> 14 <212> PRT <213> Oryctolagus cuniculus
• <400> 92
• <210> 93 <211> 366 <212> DNA <213> Oryctolagus cuniculus
• <400> 93
• <210> 94 <211> 384 <212> DNA <213> Oryctolagus cuniculus
• <400> 94
• <210> 95 <211> 36 <212> DNA <213> Oryctolagus cuniculus
• <400> 95 cagtccagtc agagtgttta taataatttc ttatcg   36
• <210> 96 <211> 21 <212> DNA <213> Oryctolagus cuniculus
• <400> 96 caggcatcca aactggcatc t   21
• <210> 97 <211> 33 <212> DNA <213> Oryctolagus cuniculus
• <400> 97 ctaggcggtt atgatgatga tgctgataat get   33
• <210> 98 <211> 15 <212> DNA <213> Oryctolagus cuniculus
• <400> 98 gactatgcaa tgagc   15
• <210> 99 <211> 51 <212> DNA <213> Oryctolagus cuniculus
• <400> 99 atcatttatg ctggtagtgg tagcacatgg tacgcgagct gggcgaaagg c   51
• <210> 100 <211> 42 <212> DNA <213> Oryctolagus cuniculus
• <400> 100 gatggatacg atgactatgg tgatttcgat cgattggatc tc   42
• <210> 101 <211> 122 <212> PRT <213> Oryctolagus cuniculus
• <400> 101
• <210> 102 <211> 125 <212> PRT <213> Oryctolagus cuniculus
• <400> 102
• <210> 103 <211> 11 <212> PRT <213> Oryctolagus cuniculus
• <400> 103
• <210> 104 <211> 7 <212> PRT <213> Oryctolagus cuniculus
• <400> 104
• <210> 105 <211> 12 <212> PRT <213> Oryctolagus cuniculus
• <400> 105
• <210> 106 <211> 5 <212> PRT <213> Oryctolagus cuniculus
• <400> 106
• <210> 107 <211> 16 <212> PRT <213> Oryctolagus cuniculus
• <400> 107
• <210> 108 <211> 12 <212> PRT <213> Oryctolagus cuniculus
• <400> 108
• <210> 109 <211> 366 <212> DNA <213> Oryctolagus cuniculus
• <400> 109
• <210> 110 <211> 375 <212> DNA <213> Oryctolagus cuniculus
• <400> 110
• <210> 111 <211> 33 <212> DNA <213> Oryctolagus cuniculus
• <400> 111 caggccagtc agagcattaa caatgaatta tcc   33
• <210> 112 <211> 21 <212> DNA <213> Oryctolagus cuniculus
• <400> 112 agggcatcca ctctggcatc t   21
• <210> 113 <211> 36 <212> DNA <213> Oryctolagus cuniculus
• <400> 113 caacagggtt atagtctgag gaatattgat aatgct   36
• <210> 114 <211> 15 <212> DNA <213> Oryctolagus cuniculus
• <400> 114 aactactaca tgacc   15
• <210> 115 <211> 48 <212> DNA <213> Oryctolagus cuniculus
• <400> 115 atgatttatg gtagtgatga aacagcctac gcgaactggg cgataggc   48
• <210> 116 <211> 36 <212> DNA <213> Oryctolagus cuniculus
• <400> 116 gatgatagta gtgactggga tgcaaaattt aacttg   36
• <210> 117 <211> 109 <212> PRT <213> Oryctolagus cuniculus
• <400> 117
• <210> 118 <211> 109 <212> PRT <213> Oryctolagus cuniculus
• <400> 118
• <210> 119 <211> 100 <212> PRT <213> Oryctolagus cuniculus
• <400> 119
• <210> 120 <211> 16 <212> PRT <213> Oryctolagus cuniculus
• <400> 120
• <210> 121 <211> 16 <212> PRT <213> Oryctolagus cuniculus
• <400> 121
• <210> 122 <211> 123 <212> PRT <213> Oryctolagus cuniculus
• <400> 122
• <210> 123 <211> 128 <212> PRT <213> Oryctolagus cuniculus
• <400> 123
• <210> 124 <211> 13 <212> PRT <213> Oryctolagus cuniculus
• <400> 124
• <210> 125 <211> 7 <212> PRT <213> Oryctolagus cuniculus
• <400> 125
• <210> 126 <211> 11 <212> PRT <213> Oryctolagus cuniculus
• <400> 126
• <210> 127 <211> 5 <212> PRT <213> Oryctolagus cuniculus
• <400> 127
• <210> 128 <211> 16 <212> PRT <213> Oryctolagus cuniculus
• <400> 128
• <210> 129 <211> 15 <212> PRT <213> Oryctolagus cuniculus
• <400> 129
• <210> 130 <211> 369 <212> DNA <213> Oryctolagus cuniculus
• <400> 130
• <210> 131 <211> 384 <212> DNA <213> Oryctolagus cuniculus
• <400> 131
• <210> 132 <211> 39 <212> DNA <213> Oryctolagus cuniculus
• <400> 132 cagtccagtc agagtgttgg taataaccag gacttatcc   39
• <210> 133 <211> 21 <212> DNA <213> Oryctolagus cuniculus
• <400> 133 gaaatatcca aactggaatc t   21
• <210> 134 <211> 33 <212> DNA <213> Oryctolagus cuniculus
• <400> 134 ctaggcggtt atgatgatga tgctgataat get   33
• <210> 135 <211> 15 <212> DNA <213> Oryctolagus cuniculus
• <400> 135 agtcgtacaa tgtcc   15
• <210> 136 <211> 48 <212> DNA <213> Oryctolagus cuniculus
• <400> 136 tacatttgga gtggtggtag cacatactac gcgacctggg cgaaaggc   48
• <210> 137 <211> 45 <212> DNA <213> Oryctolagus cuniculus
• <400> 137 ttgggcgata ctggtggtca cgcttatgct actcgcttaa atctc   45
• <210> 138 <211> 123 <212> PRT <213> Oryctolagus cuniculus
• <400> 138
• <210> 139 <211> 126 <212> PRT <213> Oryctolagus cuniculus
• <400> 139
• <210> 140 <211> 13 <212> PRT <213> Oryctolagus cuniculus
• <400> 140
• <210> 141 <211> 7 <212> PRT <213> Oryctolagus cuniculus
• <400> 141
• <210> 142 <211> 11 <212> PRT <213> Oryctolagus cuniculus
• <400> 142
• <210> 143 <211> 5 <212> PRT <213> Oryctolagus cuniculus
• <400> 143
• <210> 144 <211> 16 <212> PRT <213> Oryctolagus cuniculus
• <400> 144
• <210> 145 <211> 12 <212> PRT <213> Oryctolagus cuniculus
• <400> 145
• <210> 146 <211> 369 <212> DNA <213> Oryctolagus cuniculus
• <400> 146
• <210> 147 <211> 378 <212> DNA <213> Oryctolagus cuniculus
• <400> 147
• <210> 148 <211> 39 <212> DNA <213> Oryctolagus cuniculus
• <400> 148 cagtccagtc agagtgttta tagtaataag tacctagcc   39
• <210> 149 <211> 21 <212> DNA <213> Oryctolagus cuniculus
• <400> 149 tggacatcca aactggcatc t   21
• <210> 150 <211> 33 <212> DNA <213> Oryctolagus cuniculus
• <400> 150 ctaggcgctt atgatgatga tgctgataat gct   33
• <210> 151 <211> 15 <212> DNA <213> Oryctolagus cuniculus
• <400> 151 ggcggctaca tgacc   15
• <210> 152 <211> 48 <212> DNA <213> Oryctolagus cuniculus
• <400> 152 atcagttatg atagtggtag cacatactac gcgagctggg cgaaaggc   48
• <210> 153 <211> 36 <212> DNA <213> Oryctolagus cuniculus
• <400> 153 tcactaaaat atcctactgt tacttctgat gacttg   36
• <210> 154 <211> 123 <212> PRT <213> Oryctolagus cuniculus
• <400> 154
• <210> 155 <211> 129 <212> PRT <213> Oryctolagus cuniculus
• <400> 155
• <210> 156 <211> 13 <212> PRT <213> Oryctolagus cuniculus
• <400> 156
• <210> 157 <211> 7 <212> PRT <213> Oryctolagus cuniculus
• <400> 157
• <210> 158 <211> 11 <212> PRT <213> Oryctolagus cuniculus
• <400> 158
• <210> 159 <211> 5 <212> PRT <213> Oryctolagus cuniculus
• <400> 159
• <210> 160 <211> 16 <212> PRT <213> Oryctolagus cuniculus
• <400> 160
• <210> 161 <211> 16 <212> PRT <213> Oryctolagus cuniculus
• <400> 161
• <210> 162 <211> 369 <212> DNA <213> Oryctolagus cuniculus
• <400> 162
• <210> 163 <211> 387 <212> DNA <213> Oryctolagus cuniculus
• <400> 163
• <210> 164 <211> 39 <212> DNA <213> Oryctolagus cuniculus
• <400> 164 cagtccagtc agagtgttta taataataac gacttagcc   39
• <210> 165 <211> 21 <212> DNA <213> Oryctolagus cuniculus
• <400> 165 tatgcatcca ctctggcatc t   21
• <210> 166 <211> 33 <212> DNA <213> Oryctolagus cuniculus
• <400> 166 ctaggcggtt atgatgatga tgctgataat gct   33
• <210> 167 <211> 15 <212> DNA <213> Oryctolagus cuniculus
• <400> 167 agcaatacaa taaac   15
• <210> 168 <211> 48 <212> DNA <213> Oryctolagus cuniculus
• <400> 168 tacatttgga gtggtggtag tacatactac gcgagctggg tgaatggt   48
• <210> 169 <211> 48 <212> DNA <213> Oryctolagus cuniculus
• <400> 169 gggggttacg ctagtggtgg ttatccttat gccactcggt tggatctc   48
• <210> 170 <211> 123 <212> PRT <213> Oryctolagus cuniculus
• <400> 170
• <210> 171 <211> 123 <212> PRT <213> Oryctolagus cuniculus
• <400> 171
• <210> 172 <211> 13 <212> PRT <213> Oryctolagus cuniculus
• <400> 172
• <210> 173 <211> 7 <212> PRT <213> Oryctolagus cuniculus
• <400> 173
• <210> 174 <211> 11 <212> PRT <213> Oryctolagus cuniculus
• <400> 174
• <210> 175 <211> 6 <212> PRT <213> Oryctolagus cuniculus
• <400> 175
• <210> 176 <211> 16 <212> PRT <213> Oryctolagus cuniculus
• <400> 176
• <210> 177 <211> 8 <212> PRT <213> Oryctolagus cuniculus
• <400> 177
• <210> 178 <211> 369 <212> DNA <213> Oryctolagus cuniculus
• <400> 178
• <210> 179 <211> 369 <212> DNA <213> Oryctolagus cuniculus
• <400> 179
• <210> 180 <211> 39 <212> DNA <213> Oryctolagus cuniculus
• <400> 180 cagtccagtc agagtgttta taataacgac tacttatcc   39
• <210> 181 <211> 21 <212> DNA <213> Oryctolagus cuniculus
• <400> 181 ggtgcttcca aactggcatc t   21
• <210> 182 <211> 33 <212> DNA <213> Oryctolagus cuniculus
• <400> 182 ctgggcgatt atgatgatga tgctgataat act   33
• <210> 183 <211> 18 <212> DNA <213> Oryctolagus cuniculus
• <400> 183 accaactact acctgagc   18
• <210> 184 <211> 48 <212> DNA <213> Oryctolagus cuniculus
• <400> 184 atcatttatc ctagtggtaa cacatattgc gcgaagtggg cgaaaggc   48
• <210> 185 <211> 24 <212> DNA <213> Oryctolagus cuniculus
• <400> 185 aattatggtg gtgatgaaag tttg   24
• <210> 186 <211> 119 <212> PRT <213> Oryctolagus cuniculus
• <400> 186
• <210> 187 <211> 128 <212> PRT <213> Oryctolagus cuniculus
• <400> 187
• <210> 188 <211> 11 <212> PRT <213> Oryctolagus cuniculus
• <400> 188
• <210> 189 <211> 7 <212> PRT <213> Oryctolagus cuniculus
• <400> 189
• <210> 190 <211> 9 <212> PRT <213> Oryctolagus cuniculus
• <400> 190
• <210> 191 <211> 6 <212> PRT <213> Oryctolagus cuniculus
• <400> 191
• <210> 192 <211> 17 <212> PRT <213> Oryctolagus cuniculus
• <400> 192
• <210> 193 <211> 11 <212> PRT <213> Oryctolagus cuniculus
• <400> 193
• <210> 194 <211> 357 <212> DNA <213> Oryctolagus cuniculus
• <400> 194
• <210> 195 <211> 384 <212> DNA <213> Oryctolagus cuniculus
• <400> 195
• <210> 196 <211> 33 <212> DNA <213> Oryctolagus cuniculus
• <400> 196 caggccagtg agaccattgg caatgcatta gcc   33
• <210> 197 <211> 21 <212> DNA <213> Oryctolagus cuniculus
• <400> 197 aaggcatcca aactggcatc t   21
• <210> 198 <211> 27 <212> DNA <213> Oryctolagus cuniculus
• <400> 198 caatggtgtt attttggtga tagtgtt   27
• <210> 199 <211> 18 <212> DNA <213> Oryctolagus cuniculus
• <400> 199 agcggctact acatgtgc   18
• <210> 200 <211> 51 <212> DNA <213> Oryctolagus cuniculus
• <400> 200 tgtattttca ctattactac taacacttac tacgcgagct gggcgaaagg c   51
• <210> 201 <211> 33 <212> DNA <213> Oryctolagus cuniculus
• <400> 201 gggatttatt ctgataataa ttattatgcc ttg   33
• <210> 202 <211> 119 <212> PRT <213> Oryctolagus cuniculus
• <400> 202
• <210> 203 <211> 128 <212> PRT <213> Oryctolagus cuniculus
• <400> 203
• <210> 204 <211> 11 <212> PRT <213> Oryctolagus cuniculus
• <400> 204
• <210> 205 <211> 7 <212> PRT <213> Oryctolagus cuniculus
• <400> 205
• <210> 206 <211> 9 <212> PRT <213> Oryctolagus cuniculus
• <400> 206
• <210> 207 <211> 6 <212> PRT <213> Oryctolagus cuniculus
• <400> 207
• <210> 208 <211> 17 <212> PRT <213> Oryctolagus cuniculus
• <400> 208
• <210> 209 <211> 11 <212> PRT <213> Oryctolagus cuniculus
• <400> 209
• <210> 210 <211> 357 <212> DNA <213> Oryctolagus cuniculus
• <400> 210
• <210> 211 <211> 384 <212> DNA <213> Oryctolagus cuniculus
• <400> 211
• <210> 212 <211> 33 <212> DNA <213> Oryctolagus cuniculus
• <400> 212 caggccagtg agagcattgg caatgcatta gcc   33
• <210> 213 <211> 21 <212> DNA <213> Oryctolagus cuniculus
• <400> 213 aaggcatcca ctctggcatc t   21
• <210> 214 <211> 27 <212> DNA <213> Oryctolagus cuniculus
• <400> 214 caatggtgtt attttggtga tagtgtt   27
• <210> 215 <211> 18 <212> DNA <213> Oryctolagus cuniculus
• <400> 215 agcggctact acatgtgc   18
• <210> 216 <211> 51 <212> DNA <213> Oryctolagus cuniculus
• <400> 216 tgcattttta ctattactga taacacttac tacgcgaact gggcgaaagg c   51
• <210> 217 <211> 33 <212> DNA <213> Oryctolagus cuniculus
• <400> 217 gggatttatt ctactgataa ttattatgcc ttg   33
• <210> 218 <211> 123 <212> PRT <213> Oryctolagus cuniculus
• <400> 218
• <210> 219 <211> 133 <212> PRT <213> Oryctolagus cuniculus
• <400> 219
• <210> 220 <211> 11 <212> PRT <213> Oryctolagus cuniculus
• <400> 220
• <210> 221 <211> 7 <212> PRT <213> Oryctolagus cuniculus
• <400> 221
• <210> 222 <211> 13 <212> PRT <213> Oryctolagus cuniculus
• <400> 222
• <210> 223 <211> 5 <212> PRT <213> Oryctolagus cuniculus
• <400> 223
• <210> 224 <211> 16 <212> PRT <213> Oryctolagus cuniculus
• <400> 224
• <210> 225 <211> 19 <212> PRT <213> Oryctolagus cuniculus
• <400> 225
• <210> 226 <211> 369 <212> DNA <213> Oryctolagus cuniculus
• <400> 226
• <210> 227 <211> 399 <212> DNA <213> Oryctolagus cuniculus
• <400> 227
• <210> 228 <211> 33 <212> DNA <213> Oryctolagus cuniculus
• <400> 228 caggccagtc agagcgttag tagctactta aac   33
• <210> 229 <211> 21 <212> DNA <213> Oryctolagus cuniculus
• <400> 229 agggcatcca ctctggaatc t   21
• <210> 230 <211> 39 <212> DNA <213> Oryctolagus cuniculus
• <400> 230 caatgtactt atggtactag tagtagttat ggtgctgct   39
• <210> 231 <211> 15 <212> DNA <213> Oryctolagus cuniculus
• <400> 231 agcaatgcaa taagc   15
• <210> 232 <211> 48 <212> DNA <213> Oryctolagus cuniculus
• <400> 232 atcattagtt atagtggtac cacatactac gcgagctggg cgaaaggc   48
• <210> 233 <211> 57 <212> DNA <213> Oryctolagus cuniculus
• <400> 233 gatgacccta cgacagttat ggttatgttg ataccttttg gagccggcat ggacctc   57
• <210> 234 <211> 125 <212> PRT <213> Oryctolagus cuniculus
• <400> 234
• <210> 235 <211> 119 <212> PRT <213> Oryctolagus cuniculus
• <400> 235
• <210> 236 <211> 13 <212> PRT <213> Oryctolagus cuniculus
• <400> 236
• <210> 237 <211> 7 <212> PRT <213> Oryctolagus cuniculus
• <400> 237
• <210> 238 <211> 13 <212> PRT <213> Oryctolagus cuniculus
• <400> 238
• <210> 239 <211> 5 <212> PRT <213> Oryctolagus cuniculus
• <400> 239
• <210> 240 <211> 17 <212> PRT <213> Oryctolagus cuniculus
• <400> 240
• <210> 241 <211> 4 <212> PRT <213> Oryctolagus cuniculus
• <400> 241
• <210> 242 <211> 375 <212> DNA <213> Oryctolagus cuniculus
• <400> 242
• <210> 243 <211> 357 <212> DNA <213> Oryctolagus cuniculus
• <400> 243
• <210> 244 <211> 39 <212> DNA <213> Oryctolagus cuniculus
• <400> 244 caggccagtc agagtgttta taagaacaac tacttatcc   39
• <210> 245 <211> 21 <212> DNA <213> Oryctolagus cuniculus
• <400> 245 tctgcatcga ctctagattc t   21
• <210> 246 <211> 39 <212> DNA <213> Oryctolagus cuniculus
• <400> 246 ctaggcagtt atgattgtag tagtggtgat tgttatgct   39
• <210> 247 <211> 15 <212> DNA <213> Oryctolagus cuniculus
• <400> 247 agctactgga tgtgc   15
• <210> 248 <211> 51 <212> DNA <213> Oryctolagus cuniculus
• <400> 248 tgcattgtta ctggtaatgg taacacttac tacgcgaact gggcgaaagg c   51
• <210> 249 <211> 12 <212> DNA <213> Oryctolagus cuniculus
• <400> 249 gcctatgact tg   12
• <210> 250 <211> 123 <212> PRT <213> Oryctolagus cuniculus
• <400> 250
• <210> 251 <211> 125 <212> PRT <213> Oryctolagus cuniculus
• <400> 251
• <210> 252 <211> 13 <212> PRT <213> Oryctolagus cuniculus
• <400> 252
• <210> 253 <211> 7 <212> PRT <213> Oryctolagus cuniculus
• <400> 253
• <210> 254 <211> 11 <212> PRT <213> Oryctolagus cuniculus
• <400> 254
• <210> 255 <211> 5 <212> PRT <213> Oryctolagus cuniculus
• <400> 255
• <210> 256 <211> 16 <212> PRT <213> Oryctolagus cuniculus
• <400> 256
• <210> 257 <211> 12 <212> PRT <213> Oryctolagus cuniculus
• <400> 257
• <210> 258 <211> 369 <212> DNA <213> Oryctolagus cuniculus
• <400> 258
• <210> 259 <211> 375 <212> DNA <213> Oryctolagus cuniculus
• <400> 259
• <210> 260 <211> 39 <212> DNA <213> Oryctolagus cuniculus
• <400> 260 caggccagtc agagtgttta tgacaacaac tatttatcc   39
• <210> 261 <211> 21 <212> DNA <213> Oryctolagus cuniculus
• <400> 261 ggtgcatcca ctctggcatc t   21
• <210> 262 <211> 33 <212> DNA <213> Oryctolagus cuniculus
• <400> 262 gcaggcgttt ttaatgatga tagtgatgat gcc   33
• <210> 263 <211> 15 <212> DNA <213> Oryctolagus cuniculus
• <400> 263 gcatactata tgagc   15
• <210> 264 <211> 48 <212> DNA <213> Oryctolagus cuniculus
• <400> 264 ttcattactc tgagtgatca tatatcttac gcgaggtggg cgaaaggc   48
• <210> 265 <211> 36 <212> DNA <213> Oryctolagus cuniculus
• <400> 265 agtcgtggct ggggtgcaat gggtcggttg gatctc   36
• <210> 266 <211> 123 <212> PRT <213> Oryctolagus cuniculus
• <400> 266
• <210> 267 <211> 121 <212> PRT <213> Oryctolagus cuniculus
• <400> 267
• <210> 268 <211> 13 <212> PRT <213> Oryctolagus cuniculus
• <400> 268
• <210> 269 <211> 7 <212> PRT <213> Oryctolagus cuniculus
• <400> 269
• <210> 270 <211> 11 <212> PRT <213> Oryctolagus cuniculus
• <400> 270
• <210> 271 <211> 5 <212> PRT <213> Oryctolagus cuniculus
• <400> 271
• <210> 272 <211> 16 <212> PRT <213> Oryctolagus cuniculus
• <400> 272
• <210> 273 <211> 8 <212> PRT <213> Oryctolagus cuniculus
• <400> 273
• <210> 274 <211> 369 <212> DNA <213> Oryctolagus cuniculus
• <400> 274
• <210> 275 <211> 363 <212> DNA <213> Oryctolagus cuniculus
• <400> 275
• <210> 276 <211> 39 <212> DNA <213> Oryctolagus cuniculus
• <400> 276 caggccagtc agagtgttta taacaacaaa aatttagcc   39
• <210> 277 <211> 21 <212> DNA <213> Oryctolagus cuniculus
• <400> 277 tgggcatcca ctctggcatc t   21
• <210> 278 <211> 33 <212> DNA <213> Oryctolagus cuniculus
• <400> 278 ctaggcgttt ttgatgatga tgctgataat gct   33
• <210> 279 <211> 15 <212> DNA <213> Oryctolagus cuniculus
• <400> 279 agctactcca tgacc   15
• <210> 280 <211> 48 <212> DNA <213> Oryctolagus cuniculus
• <400> 280 gtcattggta ctagtggtag cacatactac gcgacctggg cgaaaggc   48
• <210> 281 <211> 24 <212> DNA <213> Oryctolagus cuniculus
• <400> 281 agtctttctt ctattacttt cttg   24
• <210> 282 <211> 120 <212> PRT <213> Oryctolagus cuniculus
• <400> 282
• <210> 283 <211> 128 <212> PRT <213> Oryctolagus cuniculus
• <400> 283
• <210> 284 <211> 11 <212> PRT <213> Oryctolagus cuniculus
• <400> 284
• <210> 285 <211> 7 <212> PRT <213> Oryctolagus cuniculus
• <400> 285
• <210> 286 <211> 10 <212> PRT <213> Oryctolagus cuniculus
• <400> 286
• <210> 287 <211> 6 <212> PRT <213> Oryctolagus cuniculus
• <400> 287
• <210> 288 <211> 18 <212> PRT <213> Oryctolagus cuniculus
• <400> 288
• <210> 289 <211> 10 <212> PRT <213> Oryctolagus cuniculus
• <400> 289
• <210> 290 <211> 360 <212> DNA <213> Oryctolagus cuniculus
• <400> 290
• <210> 291 <211> 384 <212> DNA <213> Oryctolagus cuniculus
• <400> 291
• <210> 292 <211> 33 <212> DNA <213> Oryctolagus cuniculus
• <400> 292 caggccagtc agaacattta tagatactta gcc   33
• <210> 293 <211> 21 <212> DNA <213> Oryctolagus cuniculus
• <400> 293 ctggcatcta ctctggcatc t   21
• <210> 294 <211> 30 <212> DNA <213> Oryctolagus cuniculus
• <400> 294 caaagttatt atagtagtaa tagtgtcgct   30
• <210> 295 <211> 18 <212> DNA <213> Oryctolagus cuniculus
• <400> 295 agcggctact ggatatgc   18
• <210> 296 <211> 54 <212> DNA <213> Oryctolagus cuniculus
• <400> 296 tgcatttata ctggtagtag tggtagcact ttttacgcga gttgggcgaa aggc   54
• <210> 297 <211> 30 <212> DNA <213> Oryctolagus cuniculus
• <400> 297 ggttatagtg gctttggtta ctttaagttg   30
• <210> 298 <211> 122 <212> PRT <213> Oryctolagus cuniculus
• <400> 298
• <210> 299 <211> 123 <212> PRT <213> Oryctolagus cuniculus
• <400> 299
• <210> 300 <211> 11 <212> PRT <213> Oryctolagus cuniculus
• <400> 300
• <210> 301 <211> 7 <212> PRT <213> Oryctolagus cuniculus
• <400> 301
• <210> 302 <211> 12 <212> PRT <213> Oryctolagus cuniculus
• <400> 302
• <210> 303 <211> 5 <212> PRT <213> Oryctolagus cuniculus
• <400> 303
• <210> 304 <211> 16 <212> PRT <213> Oryctolagus cuniculus
• <400> 304
• <210> 305 <211> 10 <212> PRT <213> Oryctolagus cuniculus
• <400> 305
• <210> 306 <211> 366 <212> DNA <213> Oryctolagus cuniculus
• <400> 306
• <210> 307 <211> 369 <212> DNA <213> Oryctolagus cuniculus
• <400> 307
• <210> 308 <211> 33 <212> DNA <213> Oryctolagus cuniculus
• <400> 308 caggccagtg aggacattta taggttattg gcc   33
• <210> 309 <211> 21 <212> DNA <213> Oryctolagus cuniculus
• <400> 309 gattcatccg atctggcatc t   21
• <210> 310 <211> 36 <212> DNA <213> Oryctolagus cuniculus
• <400> 310 caacaggctt ggagttatag tgatattgat aatgct   36
• <210> 311 <211> 15 <212> DNA <213> Oryctolagus cuniculus
• <400> 311 agctactaca tgagc   15
• <210> 312 <211> 48 <212> DNA <213> Oryctolagus cuniculus
• <400> 312 atcattacta ctagtggtaa tacattttac gcgagctggg cgaaaggc   48
• <210> 313 <211> 30 <212> DNA <213> Oryctolagus cuniculus
• <400> 313 acttctgata ttttttatta tcgtaacttg   30
• <210> 314 <211> 123 <212> PRT <213> Oryctolagus cuniculus
• <400> 314
• <210> 315 <211> 129 <212> PRT <213> Oryctolagus cuniculus
• <400> 315
• <210> 316 <211> 13 <212> PRT <213> Oryctolagus cuniculus
• <400> 316
• <210> 317 <211> 7 <212> PRT <213> Oryctolagus cuniculus
• <400> 317
• <210> 318 <211> 11 <212> PRT <213> Oryctolagus cuniculus
• <400> 318
• <210> 319 <211> 5 <212> PRT <213> Oryctolagus cuniculus
• <400> 319
• <210> 320 <211> 16 <212> PRT <213> Oryctolagus cuniculus
• <400> 320
• <210> 321 <211> 16 <212> PRT <213> Oryctolagus cuniculus
• <400> 321
• <210> 322 <211> 369 <212> DNA <213> Oryctolagus cuniculus
• <400> 322
• <210> 323 <211> 387 <212> DNA <213> Oryctolagus cuniculus
• <400> 323
• <210> 324 <211> 39 <212> DNA <213> Oryctolagus cuniculus
• <400> 324 cagtccagtc agagtgttta taatgacatg gacttagcc   39
• <210> 325 <211> 21 <212> DNA <213> Oryctolagus cuniculus
• <400> 325 tctgcatcca ctctggcatc t   21
• <210> 326 <211> 33 <212> DNA <213> Oryctolagus cuniculus
• <400> 326 ctaggcgctt ttgatgatga tgctgataat act   33
• <210> 327 <211> 15 <212> DNA <213> Oryctolagus cuniculus
• <400> 327 aggcatgcaa taacc   15
• <210> 328 <211> 48 <212> DNA <213> Oryctolagus cuniculus
• <400> 328 tgcatttgga gtggtggtag cacatactac gcgacctggg cgaaaggc   48
• <210> 329 <211> 48 <212> DNA <213> Oryctolagus cuniculus
• <400> 329 gtcattggcg atactgctgg ttatgcttat tttacggggc ttgacttg   48
• <210> 330 <211> 121 <212> PRT <213> Oryctolagus cuniculus
• <400> 330
• <210> 331 <211> 130 <212> PRT <213> Oryctolagus cuniculus
• <400> 331
• <210> 332 <211> 11 <212> PRT <213> Oryctolagus cuniculus
• <400> 332
• <210> 333 <211> 7 <212> PRT <213> Oryctolagus cuniculus
• <400> 333
• <210> 334 <211> 11 <212> PRT <213> Oryctolagus cuniculus
• <400> 334
• <210> 335 <211> 5 <212> PRT <213> Oryctolagus cuniculus
• <400> 335
• <210> 336 <211> 16 <212> PRT <213> Oryctolagus cuniculus
• <400> 336
• <210> 337 <211> 16 <212> PRT <213> Oryctolagus cuniculus
• <400> 337
• <210> 338 <211> 363 <212> DNA <213> Oryctolagus cuniculus
• <400> 338
• <210> 339 <211> 390 <212> DNA <213> Oryctolagus cuniculus
• <400> 339
• <210> 340 <211> 33 <212> DNA <213> Oryctolagus cuniculus
• <400> 340 caggccagtc agagtgttta taattggtta tcc   33
• <210> 341 <211> 21 <212> DNA <213> Oryctolagus cuniculus
• <400> 341 actgcatcca gtctggcatc t   21
• <210> 342 <211> 33 <212> DNA <213> Oryctolagus cuniculus
• <400> 342 caacagggtt atactagtga tgttgataat gtt   33
• <210> 343 <211> 15 <212> DNA <213> Oryctolagus cuniculus
• <400> 343 agctatgcaa tgggc   15
• <210> 344 <211> 48 <212> DNA <213> Oryctolagus cuniculus
• <400> 344 atcattagta gtagtggtag cacatactac gcgacctggg cgaaaggc   48
• <210> 345 <211> 48 <212> DNA <213> Oryctolagus cuniculus
• <400> 345 gggggtgctg gtagtggtgg tgtttggctg cttgatggtt ttgatccc   48
• <210> 346 <211> 123 <212> PRT <213> Oryctolagus cuniculus
• <400> 346
• <210> 347 <211> 130 <212> PRT <213> Oryctolagus cuniculus
• <400> 347
• <210> 348 <211> 11 <212> PRT <213> Oryctolagus cuniculus
• <400> 348
• <210> 349 <211> 7 <212> PRT <213> Oryctolagus cuniculus
• <400> 349
• <210> 350 <211> 12 <212> PRT <213> Oryctolagus cuniculus
• <400> 350
• <210> 351 <211> 5 <212> PRT <213> Oryctolagus cuniculus
• <400> 351
• <210> 352 <211> 16 <212> PRT <213> Oryctolagus cuniculus
• <400> 352
• <210> 353 <211> 16 <212> PRT <213> Oryctolagus cuniculus
• <400> 353
• <210> 354 <211> 369 <212> DNA <213> Oryctolagus cuniculus
• <400> 354
• <210> 355 <211> 390 <212> DNA <213> Oryctolagus cuniculus
• <400> 355
• <210> 356 <211> 33 <212> DNA <213> Oryctolagus cuniculus
• <400> 356 caggccagtg agaacattta taattggtta gcc   33
• <210> 357 <211> 21 <212> DNA <213> Oryctolagus cuniculus
• <400> 357 actgtaggcg atctggcatc t   21
• <210> 358 <211> 36 <212> DNA <213> Oryctolagus cuniculus
• <400> 358 caacagggtt atagtagtag ttatgttgat aatgtt   36
• <210> 359 <211> 15 <212> DNA <213> Oryctolagus cuniculus
• <400> 359 gactatgcag tgggc   15
• <210> 360 <211> 48 <212> DNA <213> Oryctolagus cuniculus
• <400> 360 tacattcgta gtagtggtac cacagcctac gcgacctggg cgaaaggc   48
• <210> 361 <211> 48 <212> DNA <213> Oryctolagus cuniculus
• <400> 361 gggggtgctg gtagtagtgg tgtgtggatc cttgatggtt ttgctccc   48
• <210> 362 <211> 121 <212> PRT <213> Oryctolagus cuniculus
• <400> 362
• <210> 363 <211> 130 <212> PRT <213> Oryctolagus cuniculus
• <400> 363
• <210> 364 <211> 13 <212> PRT <213> Oryctolagus cuniculus
• <400> 364
• <210> 365 <211> 7 <212> PRT <213> Oryctolagus cuniculus
• <400> 365
• <210> 366 <211> 9 <212> PRT <213> Oryctolagus cuniculus
• <400> 366
• <210> 367 <211> 6 <212> PRT <213> Oryctolagus cuniculus
• <400> 367
• <210> 368 <211> 18 <212> PRT <213> Oryctolagus cuniculus
• <400> 368
• <210> 369 <211> 13 <212> PRT <213> Oryctolagus cuniculus
• <400> 369
• <210> 370 <211> 363 <212> DNA <213> Oryctolagus cuniculus
• <400> 370
• <210> 371 <211> 390 <212> DNA <213> Oryctolagus cuniculus
• <400> 371
• <210> 372 <211> 39 <212> DNA <213> Oryctolagus cuniculus
• <400> 372 caggccagtc agagtgttta tcagaacaac tacttatcc   39
• <210> 373 <211> 21 <212> DNA <213> Oryctolagus cuniculus
• <400> 373 ggtgcggcca ctctggcatc t   21
• <210> 374 <211> 27 <212> DNA <213> Oryctolagus cuniculus
• <400> 374 gcaggcgctt atagggatgt ggattct   27
• <210> 375 <211> 18 <212> DNA <213> Oryctolagus cuniculus
• <400> 375 agtacctact acatctac 18
• <210> 376 <211> 54 <212> DNA <213> Oryctolagus cuniculus
• <400> 376 tgtattgatg ctggtagtag tggtagcact tactacgcga cctgggtgaa tggc   54
• <210> 377 <211> 39 <212> DNA <213> Oryctolagus cuniculus
• <400> 377 tgggattatg gtggtaatgt tggttggggt tatgacttg   39
• <210> 378 <211> 120 <212> PRT <213> Oryctolagus cuniculus
• <400> 378
• <210> 379 <211> 127 <212> PRT <213> Oryctolagus cuniculus
• <400> 379
• <210> 380 <211> 11 <212> PRT <213> Oryctolagus cuniculus
• <400> 380
• <210> 381 <211> 7 <212> PRT <213> Oryctolagus cuniculus
• <400> 381
• <210> 382 <211> 10 <212> PRT <213> Oryctolagus cuniculus
• <400> 382
• <210> 383 <211> 6 <212> PRT <213> Oryctolagus cuniculus
• <400> 383
• <210> 384 <211> 18 <212> PRT <213> Oryctolagus cuniculus
• <400> 384
• <210> 385 <211> 10 <212> PRT <213> Oryctolagus cuniculus
• <400> 385
• <210> 386 <211> 360 <212> DNA <213> Oryctolagus cuniculus
• <400> 386
• <210> 387 <211> 381 <212> DNA <213> Oryctolagus cuniculus
• <400> 387
• <210> 388 <211> 33 <212> DNA <213> Oryctolagus cuniculus
• <400> 388 caggccagtc agagcattag tagttactta gcc   33
• <210> 389 <211> 21 <212> DNA <213> Oryctolagus cuniculus
• <400> 389 agggcgtcca ctctggcatc t   21
• <210> 390 <211> 30 <212> DNA <213> Oryctolagus cuniculus
• <400> 390 caaagctatt atgatagtgt ttcaaatcct   30
• <210> 391 <211> 18 <212> DNA <213> Oryctolagus cuniculus
• <400> 391 acctactggt tcatgtgc   18
• <210> 392 <211> 54 <212> DNA <213> Oryctolagus cuniculus
• <400> 392 tgtatttata ctggtagtag tggttccact ttctacgcga gctgggtgaa tggc   54
• <210> 393 <211> 30 <212> DNA <213> Oryctolagus cuniculus
• <400> 393 ggttatagtg gttatggtta ttttaagttg   30
• <210> 394 <211> 124 <212> PRT <213> Oryctolagus cuniculus
• <400> 394
• <210> 395 <211> 130 <212> PRT <213> Oryctolagus cuniculus
• <400> 395
• <210> 396 <211> 13 <212> PRT <213> Oryctolagus cuniculus
• <400> 396
• <210> 397 <211> 7 <212> PRT <213> Oryctolagus cuniculus
• <400> 397
• <210> 398 <211> 12 <212> PRT <213> Oryctolagus cuniculus
• <400> 398
• <210> 399 <211> 6 <212> PRT <213> Oryctolagus cuniculus
• <400> 399
• <210> 400 <211> 17 <212> PRT <213> Oryctolagus cuniculus
• <400> 400
• <210> 401 <211> 14 <212> PRT <213> Oryctolagus cuniculus
• <400> 401
• <210> 402 <211> 372 <212> DNA <213> Oryctolagus cuniculus
• <400> 402
• <210> 403 <211> 390 <212> DNA <213> Oryctolagus cuniculus
• <400> 403
• <210> 404 <211> 39 <212> DNA <213> Oryctolagus cuniculus
• <400> 404 caggccagtc agagtgttta taagaacaac caattatcc   39
• <210> 405 <211> 21 <212> DNA <213> Oryctolagus cuniculus
• <400> 405 ggtgcatcgg ctctggcatc t   21
• <210> 406 <211> 36 <212> DNA <213> Oryctolagus cuniculus
• <400> 406 gcaggcgcta ttactggtag tattgatacg gatggt   36
• <210> 407 <211> 18 <212> DNA <213> Oryctolagus cuniculus
• <400> 407 agcagctact tcatttgc   18
• <210> 408 <211> 51 <212> DNA <213> Oryctolagus cuniculus
• <400> 408 tgcatttatg gtggtgatgg cagcacatac tacgcgagct gggcgaaagg c   51
• <210> 409 <211> 42 <212> DNA <213> Oryctolagus cuniculus
• <400> 409 gaatgggcat atagtcaagg ttattttggt gcttttgatc tc   42
• <210> 410 <211> 124 <212> PRT <213> Oryctolagus cuniculus
• <400> 410
• <210> 411 <211> 124 <212> PRT <213> Oryctolagus cuniculus
• <400> 411
• <210> 412 <211> 11 <212> PRT <213> Oryctolagus cuniculus
• <400> 412
• <210> 413 <211> 7 <212> PRT <213> Oryctolagus cuniculus
• <400> 413
• <210> 414 <211> 14 <212> PRT <213> Oryctolagus cuniculus
• <400> 414
• <210> 415 <211> 5 <212> PRT <213> Oryctolagus cuniculus
• <400> 415
• <210> 416 <211> 16 <212> PRT <213> Oryctolagus cuniculus
• <400> 416
• <210> 417 <211> 11 <212> PRT <213> Oryctolagus cuniculus
• <400> 417
• <210> 418 <211> 372 <212> DNA <213> Oryctolagus cuniculus
• <400> 418
• <210> 419 <211> 372 <212> DNA <213> Oryctolagus cuniculus
• <400> 419
• <210> 420 <211> 33 <212> DNA <213> Oryctolagus cuniculus
• <400> 420 caggccagtg aggatattag tagctactta gcc   33
• <210> 421 <211> 21 <212> DNA <213> Oryctolagus cuniculus
• <400> 421 gctgcatcca atctggaatc t   21
• <210> 422 <211> 42 <212> DNA <213> Oryctolagus cuniculus
• <400> 422 caatgtactt atggtactat ttctattagt gatggtaatg ct   42
• <210> 423 <211> 15 <212> DNA <213> Oryctolagus cuniculus
• <400> 423 agctacttca tgacc   15
• <210> 424 <211> 48 <212> DNA <213> Oryctolagus cuniculus
• <400> 424 ttcattaatc ctggtggtag cgcttactac gcgagctggg tgaaaggc   48
• <210> 425 <211> 33 <212> DNA <213> Oryctolagus cuniculus
• <400> 425 gttctgattg tttcttatgg agcctttacc atc   33
• <210> 426 <211> 124 <212> PRT <213> Oryctolagus cuniculus
• <400> 426
• <210> 427 <211> 124 <212> PRT <213> Oryctolagus cuniculus
• <400> 427
• <210> 428 <211> 11 <212> PRT <213> Oryctolagus cuniculus
• <400> 428
• <210> 429 <211> 7 <212> PRT <213> Oryctolagus cuniculus
• <400> 429
• <210> 430 <211> 14 <212> PRT <213> Oryctolagus cuniculus
• <400> 430
• <210> 431 <211> 5 <212> PRT <213> Oryctolagus cuniculus
• <400> 431
• <210> 432 <211> 16 <212> PRT <213> Oryctolagus cuniculus
• <400> 432
• <210> 433 <211> 11 <212> PRT <213> Oryctolagus cuniculus
• <400> 433
• <210> 434 <211> 372 <212> DNA <213> Oryctolagus cuniculus
• <400> 434
• <210> 435 <211> 372 <212> DNA <213> Oryctolagus cuniculus
• <400> 435
• <210> 436 <211> 33 <212> DNA <213> Oryctolagus cuniculus
• <400> 436 caggccagtg aggacattga aagctatcta gcc   33
• <210> 437 <211> 21 <212> DNA <213> Oryctolagus cuniculus
• <400> 437 ggtgcatcca atctggaatc t   21
• <210> 438 <211> 42 <212> DNA <213> Oryctolagus cuniculus
• <400> 438 caatgcactt atggtattat tagtattagt gatggtaatg ct   42
• <210> 439 <211> 15 <212> DNA <213> Oryctolagus cuniculus
• <400> 439 agctacttca tgacc   15
• <210> 440 <211> 48 <212> DNA <213> Oryctolagus cuniculus
• <400> 440 ttcatgaata ctggtgataa cgcatactac gcgagctggg cgaaaggc   48
• <210> 441 <211> 33 <212> DNA <213> Oryctolagus cuniculus
• <400> 441 gttcttgttg ttgcttatgg agcctttaac atc   33
• <210> 442 <211> 124 <212> PRT <213> Oryctolagus cuniculus
• <400> 442
• <210> 443 <211> 127 <212> PRT <213> Oryctolagus cuniculus
• <400> 443
• <210> 444 <211> 13 <212> PRT <213> Oryctolagus cuniculus
• <400> 444
• <210> 445 <211> 7 <212> PRT <213> Oryctolagus cuniculus
• <400> 445
• <210> 446 <211> 12 <212> PRT <213> Oryctolagus cuniculus
• <400> 446
• <210> 447 <211> 5 <212> PRT <213> Oryctolagus cuniculus
• <400> 447
• <210> 448 <211> 16 <212> PRT <213> Oryctolagus cuniculus
• <400> 448
• <210> 449 <211> 14 <212> PRT <213> Oryctolagus cuniculus
• <400> 449
• <210> 450 <211> 372 <212> DNA <213> Oryctolagus cuniculus
• <400> 450
• <210> 451 <211> 381 <212> DNA <213> Oryctolagus cuniculus
• <400> 451
• <210> 452 <211> 39 <212> DNA <213> Oryctolagus cuniculus
• <400> 452 cagtccagta agagtgttat gaataacaac tacttagcc   39
• <210> 453 <211> 21 <212> DNA <213> Oryctolagus cuniculus
• <400> 453 ggtgcatcca atctggcatc t   21
• <210> 454 <211> 36 <212> DNA <213> Oryctolagus cuniculus
• <400> 454 caaggcggtt atactggtta tagtgatcat gggact   36
• <210> 455 <211> 15 <212> DNA <213> Oryctolagus cuniculus
• <400> 455 agctatccaa tgaac   15
• <210> 456 <211> 48 <212> DNA <213> Oryctolagus cuniculus
• <400> 456 ttcattaata ctggtggtac catagtctac gcgagctggg caaaaggc   48
• <210> 457 <211> 42 <212> DNA <213> Oryctolagus cuniculus
• <400> 457 ggcagttatg tttcatctgg ttatgcctac tattttaatg tc   42
• <210> 458 <211> 121 <212> PRT <213> Oryctolagus cuniculus
• <400> 458
• <210> 459 <211> 126 <212> PRT <213> Oryctolagus cuniculus
• <400> 459
• <210> 460 <211> 13 <212> PRT <213> Oryctolagus cuniculus
• <400> 460
• <210> 461 <211> 7 <212> PRT <213> Oryctolagus cuniculus
• <400> 461
• <210> 462 <211> 9 <212> PRT <213> Oryctolagus cuniculus
• <400> 462
• <210> 463 <211> 5 <212> PRT <213> Oryctolagus cuniculus
• <400> 463
• <210> 464 <211> 16 <212> PRT <213> Oryctolagus cuniculus
• <400> 464
• <210> 465 <211> 13 <212> PRT <213> Oryctolagus cuniculus
• <400> 465
• <210> 466 <211> 363 <212> DNA <213> Oryctolagus cuniculus
• <400> 466
• <210> 467 <211> 378 <212> DNA <213> Oryctolagus cuniculus
• <400> 467
• <210> 468 <211> 39 <212> DNA <213> Oryctolagus cuniculus
• <400> 468 cagtccagtc agagtgttta taataacaac tggttatcc   39
• <210> 469 <211> 21 <212> DNA <213> Oryctolagus cuniculus
• <400> 469 aaggcatcca ctctggcatc t   21
• <210> 470 <211> 27 <212> DNA <213> Oryctolagus cuniculus
• <400> 470 gcgggcggtt atcttgatag tgttatt   27
• <210> 471 <211> 15 <212> DNA <213> Oryctolagus cuniculus
• <400> 471 acctattcaa taaac   15
• <210> 472 <211> 48 <212> DNA <213> Oryctolagus cuniculus
• <400> 472 atcattgcta atagtggtac cacattctac gcgaactggg cgaaaggc   48
• <210> 473 <211> 39 <212> DNA <213> Oryctolagus cuniculus
• <400> 473 gagagtggaa tgtacaatga atatggtaaa tttaacatc   39
• <210> 474 <211> 122 <212> PRT <213> Oryctolagus cuniculus
• <400> 474
• <210> 475 <211> 128 <212> PRT <213> Oryctolagus cuniculus
• <400> 475
• <210> 476 <211> 11 <212> PRT <213> Oryctolagus cuniculus
• <400> 476
• <210> 477 <211> 7 <212> PRT <213> Oryctolagus cuniculus
• <400> 477
• <210> 478 <211> 12 <212> PRT <213> Oryctolagus cuniculus
• <400> 478
• <210> 479 <211> 5 <212> PRT <213> Oryctolagus cuniculus
• <400> 479
• <210> 480 <211> 16 <212> PRT <213> Oryctolagus cuniculus
• <400> 480
• <210> 481 <211> 15 <212> PRT <213> Oryctolagus cuniculus
• <400> 481
• <210> 482 <211> 366 <212> DNA <213> Oryctolagus cuniculus
• <400> 482
• <210> 483 <211> 384 <212> DNA <213> Oryctolagus cuniculus
• <400> 483
• <210> 484 <211> 33 <212> DNA <213> Oryctolagus cuniculus
• <400> 484 caggccagtg agaacattta tagctttttg gcc   33
• <210> 485 <211> 21 <212> DNA <213> Oryctolagus cuniculus
• <400> 485 aaggcttcca ctctggcatc t   21
• <210> 486 <211> 36 <212> DNA <213> Oryctolagus cuniculus
• <400> 486 caacagggtg ctactgtgta tgatattgat aataat   36
• <210> 487 <211> 15 <212> DNA <213> Oryctolagus cuniculus
• <400> 487 gcctatgcaa tgatc   15
• <210> 488 <211> 48 <212> DNA <213> Oryctolagus cuniculus
• <400> 488 atcatttatc ctaatggtat cacatactac gcgaactggg cgaaaggc   48
• <210> 489 <211> 45 <212> DNA <213> Oryctolagus cuniculus
• <400> 489 gatgcagaaa gtagtaagaa tgcttattgg ggctacttta acgtc   45
• <210> 490 <211> 122 <212> PRT <213> Oryctolagus cuniculus
• <400> 490
• <210> 491 <211> 128 <212> PRT <213> Oryctolagus cuniculus
• <400> 491
• <210> 492 <211> 11 <212> PRT <213> Oryctolagus cuniculus
• <400> 492
• <210> 493 <211> 7 <212> PRT <213> Oryctolagus cuniculus
• <400> 493
• <210> 494 <211> 12 <212> PRT <213> Oryctolagus cuniculus
• <400> 494
• <210> 495 <211> 5 <212> PRT <213> Oryctolagus cuniculus
• <400> 495
• <210> 496 <211> 16 <212> PRT <213> Oryctolagus cuniculus
• <400> 496
• <210> 497 <211> 15 <212> PRT <213> Oryctolagus cuniculus
• <400> 497
• <210> 498 <211> 366 <212> DNA <213> Oryctolagus cuniculus
• <400> 498
• <210> 499 <211> 384 <212> DNA <213> Oryctolagus cuniculus
• <400> 499
• <210> 500 <211> 33 <212> DNA <213> Oryctolagus cuniculus
• <400> 500 caggccagtg agaacattta tagctttttg gcc   33
• <210> 501 <211> 21 <212> DNA <213> Oryctolagus cuniculus
• <400> 501 agggcttcca ctctggcatc t   21
• <210> 502 <211> 36 <212> DNA <213> Oryctolagus cuniculus
• <400> 502 caacagggtg ctactgtgta tgatattgat aataat   36
• <210> 503 <211> 15 <212> DNA <213> Oryctolagus cuniculus
• <400> 503 gcctatgcaa tgatc   15
• <210> 504 <211> 48 <212> DNA <213> Oryctolagus cuniculus
• <400> 504 atcatttatc ctaatggtat cacatactac gcgaactggg cgaaaggc   48
• <210> 505 <211> 45 <212> DNA <213> Oryctolagus cuniculus
• <400> 505 gatgcagaaa gtagtaagaa tgcttattgg ggctacttta acgtc   45
• <210> 506 <211> 124 <212> PRT <213> Oryctolagus cuniculus
• <400> 506
• <210> 507 <211> 123 <212> PRT <213> Oryctolagus cuniculus
• <400> 507
• <210> 508 <211> 12 <212> PRT <213> Oryctolagus cuniculus
• <400> 508
• <210> 509 <211> 7 <212> PRT <213> Oryctolagus cuniculus
• <400> 509
• <210> 510 <211> 13 <212> PRT <213> Oryctolagus cuniculus
• <400> 510
• <210> 511 <211> 5 <212> PRT <213> Oryctolagus cuniculus
• <400> 511
• <210> 512 <211> 16 <212> PRT <213> Oryctolagus cuniculus
• <400> 512
• <210> 513 <211> 10 <212> PRT <213> Oryctolagus cuniculus
• <400> 513
• <210> 514 <211> 372 <212> DNA <213> Oryctolagus cuniculus
• <400> 514
• <210> 515 <211> 369 <212> DNA <213> Oryctolagus cuniculus
• <400> 515
• <210> 516 <211> 36 <212> DNA <213> Oryctolagus cuniculus
• <400> 516 caggccagtg agagtgtttt taataatatg ttatcc   36
• <210> 517 <211> 21 <212> DNA <213> Oryctolagus cuniculus
• <400> 517 gatgcatccg atctggcatc t   21
• <210> 518 <211> 39 <212> DNA <213> Oryctolagus cuniculus
• <400> 518 gcagggtata aaagtgatag taatgatggc gataatgtt   39
• <210> 519 <211> 15 <212> DNA <213> Oryctolagus cuniculus
• <400> 519 aggaattcaa taacc   15
• <210> 520 <211> 48 <212> DNA <213> Oryctolagus cuniculus
• <400> 520 atcattactg gtagtggtag aacgtactac gcgaactggg caaaaggc   48
• <210> 521 <211> 30 <212> DNA <213> Oryctolagus cuniculus
• <400> 521 ggccatcctg gtcttggtag tggtaacatc   30
• <210> 522 <211> 121 <212> PRT <213> Oryctolagus cuniculus
• <400> 522
• <210> 523 <211> 122 <212> PRT <213> Oryctolagus cuniculus
• <400> 523
• <210> 524 <211> 12 <212> PRT <213> Oryctolagus cuniculus
• <400> 524
• <210> 525 <211> 7 <212> PRT <213> Oryctolagus cuniculus
• <400> 525
• <210> 526 <211> 10 <212> PRT <213> Oryctolagus cuniculus
• <400> 526
• <210> 527 <211> 5 <212> PRT <213> Oryctolagus cuniculus
• <400> 527
• <210> 528 <211> 16 <212> PRT <213> Oryctolagus cuniculus
• <400> 528
• <210> 529 <211> 8 <212> PRT <213> Oryctolagus cuniculus
• <400> 529
• <210> 530 <211> 363 <212> DNA <213> Oryctolagus cuniculus
• <400> 530
• <210> 531 <211> 366 <212> DNA <213> Oryctolagus cuniculus
• <400> 531
• <210> 532 <211> 36 <212> DNA <213> Oryctolagus cuniculus
• <400> 532 cagtccagtc agagtgttta taataactac ttatcc   36
• <210> 533 <211> 21 <212> DNA <213> Oryctolagus cuniculus
• <400> 533 actgcatcca gcctggcatc t   21
• <210> 534 <211> 30 <212> DNA <213> Oryctolagus cuniculus
• <400> 534 caaggctatt atagtggtcc tataattact   30
• <210> 535 <211> 15 <212> DNA <213> Oryctolagus cuniculus
• <400> 535 aactactaca tacaa   15
• <210> 536 <211> 48 <212> DNA <213> Oryctolagus cuniculus
• <400> 536 atcatttatg ctggtggtag cgcatactac gcgacctggg caaacggc   48
• <210> 537 <211> 24 <212> DNA <213> Oryctolagus cuniculus
• <400> 537 gggacatttg atggttatga gttg   24
• <210> 538 <211> 122 <212> PRT <213> Oryctolagus cuniculus
• <400> 538
• <210> 539 <211> 124 <212> PRT <213> Oryctolagus cuniculus
• <400> 539
• <210> 540 <211> 13 <212> PRT <213> Oryctolagus cuniculus
• <400> 540
• <210> 541 <211> 7 <212> PRT <213> Oryctolagus cuniculus
• <400> 541
• <210> 542 <211> 10 <212> PRT <213> Oryctolagus cuniculus
• <400> 542
• <210> 543 <211> 5 <212> PRT <213> Oryctolagus cuniculus
• <400> 543
• <210> 544 <211> 16 <212> PRT <213> Oryctolagus cuniculus
• <400> 544
• <210> 545 <211> 11 <212> PRT <213> Oryctolagus cuniculus
• <400> 545
• <210> 546 <211> 366 <212> DNA <213> Oryctolagus cuniculus
• <400> 546
• <210> 547 <211> 372 <212> DNA <213> Oryctolagus cuniculus
• <400> 547
• <210> 548 <211> 39 <212> DNA <213> Oryctolagus cuniculus
• <400> 548 cagtccagtg agagcgttta tagtaataac ctcttatcc   39
• <210> 549 <211> 21 <212> DNA <213> Oryctolagus cuniculus
• <400> 549 agggcatcca atctggcatc t   21
• <210> 550 <211> 30 <212> DNA <213> Oryctolagus cuniculus
• <400> 550 caaggctatt atagtggtgt cattaatagt   30
• <210> 551 <211> 15 <212> DNA <213> Oryctolagus cuniculus
• <400> 551 agctacttca tgagc   15
• <210> 552 <211> 48 <212> DNA <213> Oryctolagus cuniculus
• <400> 552 ttcattaatc ctggtggtag cgcatactac gcgagctggg cgagtggc   48
• <210> 553 <211> 33 <212> DNA <213> Oryctolagus cuniculus
• <400> 553 attcttattg tttcttatgg agcctttacc atc   33
• <210> 554 <211> 122 <212> PRT <213> Oryctolagus cuniculus
• <400> 554
• <210> 555 <211> 128 <212> PRT <213> Oryctolagus cuniculus
• <400> 555
• <210> 556 <211> 11 <212> PRT <213> Oryctolagus cuniculus
• <400> 556
• <210> 557 <211> 7 <212> PRT <213> Oryctolagus cuniculus
• <400> 557
• <210> 558 <211> 12 <212> PRT <213> Oryctolagus cuniculus
• <400> 558
• <210> 559 <211> 5 <212> PRT <213> Oryctolagus cuniculus
• <400> 559
• <210> 560 <211> 17 <212> PRT <213> Oryctolagus cuniculus
• <400> 560
• <210> 561 <211> 14 <212> PRT <213> Oryctolagus cuniculus
• <400> 561
• <210> 562 <211> 366 <212> DNA <213> Oryctolagus cuniculus
• <400> 562
• <210> 563 <211> 384 <212> DNA <213> Oryctolagus cuniculus
• <400> 563
• <210> 564 <211> 33 <212> DNA <213> Oryctolagus cuniculus
• <400> 564 caggccactg agagcattgg caatgagtta tcc   33
• <210> 565 <211> 21 <212> DNA <213> Oryctolagus cuniculus
• <400> 565 tctgcatcca ctctggcatc t   21
• <210> 566 <211> 36 <212> DNA <213> Oryctolagus cuniculus
• <400> 566 caacagggtt atagtagtgc taatattgat aatgct   36
• <210> 567 <211> 15 <212> DNA <213> Oryctolagus cuniculus
• <400> 567 aagtactaca tgagc   15
• <210> 568 <211> 51 <212> DNA <213> Oryctolagus cuniculus
• <400> 568 tacattgata gtactactgt taatacatac tacgcgacct gggcgagagg c   51
• <210> 569 <211> 42 <212> DNA <213> Oryctolagus cuniculus
• <400> 569 ggaagtactt attttactga tggaggccat cggttggatc tc   42
• <210> 570 <211> 122 <212> PRT <213> Oryctolagus cuniculus
• <400> 570
• <210> 571 <211> 124 <212> PRT <213> Oryctolagus cuniculus
• <400> 571
• <210> 572 <211> 11 <212> PRT <213> Oryctolagus cuniculus
• <400> 572
• <210> 573 <211> 7 <212> PRT <213> Oryctolagus cuniculus
• <400> 573
• <210> 574 <211> 12 <212> PRT <213> Oryctolagus cuniculus
• <400> 574
• <210> 575 <211> 5 <212> PRT <213> Oryctolagus cuniculus
• <400> 575
• <210> 576 <211> 16 <212> PRT <213> Oryctolagus cuniculus
• <400> 576
• <210> 577 <211> 11 <212> PRT <213> Oryctolagus cuniculus
• <400> 577
• <210> 578 <211> 366 <212> DNA <213> Oryctolagus cuniculus
• <400> 578
• <210> 579 <211> 372 <212> DNA <213> Oryctolagus cuniculus
• <400> 579
• <210> 580 <211> 33 <212> DNA <213> Oryctolagus cuniculus
• <400> 580 caggccactg agagcattgg caatgagtta tcc   33
• <210> 581 <211> 21 <212> DNA <213> Oryctolagus cuniculus
• <400> 581 tctgcatcca ctctggcatc t   21
• <210> 582 <211> 36 <212> DNA <213> Oryctolagus cuniculus
• <400> 582 caacagggtt atagtagtgc taatattgat aatgct   36
• <210> 583 <211> 15 <212> DNA <213> Oryctolagus cuniculus
• <400> 583 acctacaaca tgggc   15
• <210> 584 <211> 48 <212> DNA <213> Oryctolagus cuniculus
• <400> 584 agtattacta ttgatggtcg cacatactac gcgagctggg cgaaaggc   48
• <210> 585 <211> 33 <212> DNA <213> Oryctolagus cuniculus
• <400> 585 attcttattg tttcttatgg ggcctttacc atc   33
• <210> 586 <211> 105 <212> PRT <213> Artificial Sequence
• <220> <223> Kappa constant domain of Ab1
• <400> 586
• <210> 587 <211> 315 <212> DNA <213> Artificial Sequence
• <220> <223> Kappa constant domain of Ab1
• <400> 587
• <210> 588 <211> 330 <212> PRT <213> Artificial Sequence
• <220> <223> Gamma-1 constant domain of Ab1
• <400> 588
• <210> 589 <211> 990 <212> DNA <213> Artificial Sequence
• <220> <223> Gamma-1 constant domain of Ab1
• <400> 589
• <210> 590 <211> 15 <212> PRT <213> Homo sapiens
• <400> 590
• <210> 591 <211> 15 <212> PRT <213> Homo sapiens
• <400> 591
• <210> 592 <211> 15 <212> PRT <213> Homo sapiens
• <400> 592
• <210> 593 <211> 15 <212> PRT <213> Homo sapiens
• <400> 593
• <210> 594 <211> 15 <212> PRT <213> Homo sapiens
• <400> 594
• <210> 595 <211> 15 <212> PRT <213> Homo sapiens
• <400> 595
• <210> 596 <211> 15 <212> PRT <213> Homo sapiens
• <400> 596
• <210> 597 <211> 15 <212> PRT <213> Homo sapiens
• <400> 597
• <210> 598 <211> 15 <212> PRT <213> Homo sapiens
• <400> 598
• <210> 599 <211> 15 <212> PRT <213> Homo sapiens
• <400> 599
• <210> 600 <211> 15 <212> PRT <213> Homo sapiens
• <400> 600
• <210> 601 <211> 15 <212> PRT <213> Homo sapiens
• <400> 601
• <210> 602 <211> 15 <212> PRT <213> Homo sapiens
• <400> 602
• <210> 603 <211> 15 <212> PRT <213> Homo sapiens
• <400> 603
• <210> 604 <211> 15 <212> PRT <213> Homo sapiens
• <400> 604
• <210> 605 <211> 15 <212> PRT <213> Homo sapiens
• <400> 605
• <210> 606 <211> 15 <212> PRT <213> Homo sapiens
• <400> 606
• <210> 607 <211> 15 <212> PRT <213> Homo sapiens
• <400> 607
• <210> 608 <211> 15 <212> PRT <213> Homo sapiens
• <400> 608
• <210> 609 <211> 15 <212> PRT <213> Homo sapiens
• <400> 609
• <210> 610 <211> 15 <212> PRT <213> Homo sapiens
• <400> 610
• <210> 611 <211> 15 <212> PRT <213> Homo sapiens
• <400> 611
• <210> 612 <211> 15 <212> PRT <213> Homo sapiens
• <400> 612
• <210> 613 <211> 15 <212> PRT <213> Homo sapiens
• <400> 613
• <210> 614 <211> 15 <212> PRT <213> Homo sapiens
• <400> 614
• <210> 615 <211> 15 <212> PRT <213> Homo sapiens
• <400> 615
• <210> 616 <211> 15 <212> PRT <213> Homo sapiens
• <400> 616
• <210> 617 <211> 15 <212> PRT <213> Homo sapiens
• <400> 617
• <210> 618 <211> 15 <212> PRT <213> Homo sapiens
• <400> 618
• <210> 619 <211> 15 <212> PRT <213> Homo sapiens
• <400> 619
• <210> 620 <211> 15 <212> PRT <213> Homo sapiens
• <400> 620
• <210> 621 <211> 15 <212> PRT <213> Homo sapiens
• <400> 621
• <210> 622 <211> 15 <212> PRT <213> Homo sapiens
• <400> 622
• <210> 623 <211> 15 <212> PRT <213> Homo sapiens
• <400> 623
• <210> 624 <211> 15 <212> PRT <213> Homo sapiens
• <400> 624
• <210> 625 <211> 15 <212> PRT <213> Homo sapiens
• <400> 625
• <210> 626 <211> 15 <212> PRT <213> Homo sapiens
• <400> 626
• <210> 627 <211> 15 <212> PRT <213> Homo sapiens
• <400> 627
• <210> 628 <211> 15 <212> PRT <213> Homo sapiens
• <400> 628
• <210> 629 <211> 15 <212> PRT <213> Homo sapiens
• <400> 629
• <210> 630 <211> 15 <212> PRT <213> Homo sapiens
• <400> 630
• <210> 631 <211> 15 <212> PRT <213> Homo sapiens
• <400> 631
• <210> 632 <211> 15 <212> PRT <213> Homo sapiens
• <400> 632
• <210> 633 <211> 15 <212> PRT <213> Homo sapiens
• <400> 633
• <210> 634 <211> 15 <212> PRT <213> Homo sapiens
• <400> 634
• <210> 635 <211> 15 <212> PRT <213> Homo sapiens
• <400> 635
• <210> 636 <211> 15 <212> PRT <213> Homo sapiens
• <400> 636
• <210> 637 <211> 15 <212> PRT <213> Homo sapiens
• <400> 637
• <210> 638 <211> 15 <212> PRT <213> Homo sapiens
• <400> 638
• <210> 639 <211> 15 <212> PRT <213> Homo sapiens
• <400> 639
• <210> 640 <211> 15 <212> PRT <213> Homo sapiens
• <400> 640
• <210> 641 <211> 15 <212> PRT <213> Homo sapiens
• <400> 641
• <210> 642 <211> 15 <212> PRT <213> Homo sapiens
• <400> 642
• <210> 643 <211> 15 <212> PRT <213> Homo sapiens
• <400> 643
• <210> 644 <211> 15 <212> PRT <213> Homo sapiens
• <400> 644
• <210> 645 <211> 15 <212> PRT <213> Homo sapiens
• <400> 645
• <210> 646 <211> 15 <212> PRT <213> Homo sapiens
• <400> 646

Claims (13)

1. An anti-IL-6 antibody comprising:

   (a) a light chain comprising (i) a variable light (V_L) domain comprising a CDR1 polypeptide consisting of the amino acid sequence of SEQ ID NO:4, a CDR2 polypeptide consisting of the amino acid sequence of SEQ ID NO:5, and CDR3 polypeptide consisting of the amino acid sequence of SEQ ID NO:6, and (ii) a constant light (C_L) domain comprising a polypeptide consisting of the amino acid sequence of SEQ ID NO:586; and

   (b) a heavy chain comprising (i) a variable heavy (V_H) domain comprising a CDR1 polypeptide consisting of the amino acid sequence of SEQ ID NO:7, a CDR2 polypeptide consisting of the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:120, and a CDR3 polypeptide consisting of the amino acid sequence of SEQ ID NO:9, and (ii) a constant heavy (C_H) domain comprising a polypeptide consisting of the amino acid sequence of SEQ ID NO:588,

   wherein the antibody has a dissociation constant (KD) of less than 50 picomolar as assessed by Biacore.
2. The anti-IL-6 antibody of claim 1,

   a) which is aglycosylated,

   b) which is a humanized or chimeric antibody,

   c) which is a humanized antibody derived from a rabbit anti-human IL-6 antibody,

   d) which specifically binds to IL-6 expressing human cells and/or to circulating soluble IL-6 molecules in vivo,

   e) which specifically binds to IL-6 expressed on or by human cells in a patient with a disease wherein IL-6 levels are elevated, wherein the disease is selected from asthma, general fatigue, exercise-induced fatigue, cancer-related fatigue, inflammatory disease-related fatigue, chronic fatigue syndrome, cancer-related cachexia, cardiac-related cachexia, respiratory-related cachexia, renal-related cachexia, age-related cachexia, rheumatoid arthritis, systemic lupus erythematosis (SLE), systemic juvenile idiopathic arthritis, psoriasis, psoriatic arthropathy, ankylosing spondylitis, inflammatory bowel disease (IBD), polymyalgia rheumatica, giant cell arteritis, autoimmune vasculitis, graft versus host disease (GVHD), Sjogren's syndrome, adult onset Still's disease, rheumatoid arthritis, systemic juvenile idiopathic arthritis, osteoarthritis, osteoporosis, Paget's disease of bone, osteoarthritis, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, prostate cancer, leukemia, renal cell cancer, multicentric Castleman's disease, ovarian cancer, drug resistance in cancer chemotherapy, cancer chemotherapy toxicity, ischemic heart disease, atherosclerosis, obesity, diabetes, multiple sclerosis, Alzheimer's disease, cerebrovascular disease, fever, acute phase response, allergies, anemia, anemia of inflammation (anemia of chronic disease), hypertension, depression, depression associated with a chronic illness, thrombosis, thrombocytosis, acute heart failure, metabolic syndrome, miscarriage, obesity, chronic prostatitis, glomerulonephritis, pelvic inflammatory disease, reperfusion injury, transplant rejection, graft versus host disease (GVHD), avian influenza, smallpox, pandemic influenza, adult respiratory distress syndrome (ARDS), severe acute respiratory syndrome (SARS), sepsis, systemic inflammatory response syndrome (SIRS), a cancer, inflammatory disorder, viral disorder, or autoimmune disorder, arthritis, cachexia, and wasting syndrome,

   f) which is directly or indirectly attached to a detectable label or therapeutic agent,

   g) which binds at least one of soluble IL-6, cell-surface expressed IL-6, IL-6/IL-6R, IL-6/IL-6R/gp130 complexes and/or IL-6/IL-6R/gp130 complex multimers,

   h) which antagonizes the biological effects of one or more of soluble IL-6, cell-surface expressed IL-6, IL-6/IL-6R, IL-6/IL-6R/gp130 complexes and/or IL-6/IL-6R/gp130 complex multimers,

   i) wherein the V_H or V_L polypeptides contained in said antibody originated from one or more rabbit B cell populations,

   j) which does not have binding specificity for soluble IL-6R (sIL-6R) or gpl30,

   k) which inhibits the association of IL-6 with IL-6R, and/or the production of IL-6/IL-6R/gp130 complexes and/or the production of IL-6/IL-6R/gpl30 multimers, or

   l) which further comprises an effector moiety selected from:

   (i) a detectable moiety selected from a fluorescent dye, an enzyme, a substrate, a bioluminescent material, a radioactive material, and a chemiluminescent material, and

   (ii) a functional moiety selected from streptavidin, avidin, biotin, a cytotoxin, a cytotoxic agent, a radioactive material.
3. An anti-IL-6 antibody according to claim 1, comprising a V_L chain and a V_H chain, respectively, comprising the amino acid sequences of SEQ ID NO:2 and SEQ ID NO:3; SEQ ID NO:2 and SEQ ID NO:18; SEQ ID NO:2 and SEQ ID NO:19; SEQ ID NO:20 and SEQ ID NO:3; SEQ ID NO:20 and SEQ ID NO:18; or SEQ ID NO:20 and SEQ ID NO:19, or an antibody comprising a V_L chain and a V_H chain, respectively, comprising amino acid sequences having at least 90% or greater homology thereto.
4. A nucleic acid sequence which encodes an anti-IL-6 antibody according to any one of claims 1-3.
5. A vector comprising a nucleic acid sequence according to claim 4.
6. A recombinant cell which expresses an anti-IL-6 antibody according to any one of claims 1-3.
7. A pharmaceutical or diagnostic composition containing at least one anti-IL-6 antibody according to any one of claims 1-3 and a pharmaceutically acceptable carrier.
8. The pharmaceutical or diagnostic composition of claim 7,

   a) which further comprises at least one stabilizer,

   b) which is lyophilized, or

   c) which comprises one or more anti-IL-6 antibodies comprising (a) a V_H chain having the amino acid sequence of SEQ ID NO: 3, 18, or 19; and (b) a V_L chain having the amino acid sequence of SEQ ID NO: 2 or 20.
9. An anti-IL-6 antibody according to any one of claims 1-3 for use as a therapeutic or diagnostic agent.
10. Use of an anti-IL-6 antibody according to any one of claims 1-3 in the manufacture of a medicament or in the manufacture of an agent for use in a method of treatment.
11. An anti-IL-6 antibody for use as a therapeutic agent according to claim 9 or use according to claim 10:

    a) wherein the disease or condition to be treated is selected from asthma, general fatigue, exercise-induced fatigue, cancer-related fatigue, inflammatory disease-related fatigue, chronic fatigue syndrome, cancer-related cachexia, cardiac-related cachexia, respiratory-related cachexia, renal-related cachexia, age-related cachexia, rheumatoid arthritis, systemic lupus erythematosis (SLE), systemic juvenile idiopathic arthritis, psoriasis, psoriatic arthropathy, ankylosing spondylitis, inflammatory bowel disease (IBD), polymyalgia rheumatica, giant cell arteritis, autoimmune vasculitis, graft versus host disease (GVHD), Sjogren's syndrome, adult onset Still's disease, rheumatoid arthritis, systemic juvenile idiopathic arthritis, osteoarthritis, osteoporosis, Paget's disease of bone, osteoarthritis, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, prostate cancer, leukemia, renal cell cancer, multicentric Castleman's disease, ovarian cancer, drug resistance in cancer chemotherapy, cancer chemotherapy toxicity, ischemic heart disease, atherosclerosis, obesity, diabetes, multiple sclerosis, Alzheimer's disease, cerebrovascular disease, fever, acute phase response, allergies, anemia, anemia of inflammation (anemia of chronic disease), hypertension, depression, depression associated with a chronic illness, thrombosis, thrombocytosis, acute heart failure, metabolic syndrome, miscarriage, obesity, chronic prostatitis, glomerulonephritis, pelvic inflammatory disease, reperfusion injury, transplant rejection, graft versus host disease (GVHD), avian influenza, smallpox, pandemic influenza, adult respiratory distress syndrome (ARDS), severe acute respiratory syndrome (SARS), sepsis, and systemic inflammatory response syndrome (SIRS), arthritis, cancer, autoimmune disease, or inflammatory condition,

    b) wherein the treatment further includes the administration of another therapeutic agent or regimen selected from chemotherapy, radiotherapy, cytokine administration or gene therapy, or

    c) which is used to treat a side effect of cancer or viral infection, preferably wherein the side effect is fatigue or weight loss.
12. An anti-IL-6 antibody for use as a diagnostic agent according to claim 9, wherein the use is for diagnostic in vivo imaging

    a) wherein said use detects the presence of cells which express IL-6 comprising administering a diagnostically effective amount of at least one anti-IL-6 antibody according to claim 1,

    b) wherein said administration further includes the administration of a radionuclide or fluorophore that facilitates detection of the antibody at IL-6 expressing disease sites,

    c) which is used to detect IL-6 expressing tumors or metastases,

    d) which is used to detect the presence of sites of inflammation associated with IL-6 expressing cells, or

    e) wherein the results are used to facilitate design of an appropriate therapeutic regimen.
13. An anti-IL-6 antibody for use as a diagnostic agent according to claim 9, wherein the use is for diagnostic in vivo imaging wherein the use

    a) is to detect or image IL-6 expressing tumors or metastases, or

    b) is to detect or image IL-6 expressing inflammatory sites.