[go: up one dir, main page]

WO2011153592A1 - Molécules thérapeutiques - Google Patents

Molécules thérapeutiques Download PDF

Info

Publication number
WO2011153592A1
WO2011153592A1 PCT/AU2011/000718 AU2011000718W WO2011153592A1 WO 2011153592 A1 WO2011153592 A1 WO 2011153592A1 AU 2011000718 W AU2011000718 W AU 2011000718W WO 2011153592 A1 WO2011153592 A1 WO 2011153592A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
antibody
csf
set forth
hgm4
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/AU2011/000718
Other languages
English (en)
Inventor
Charles Mackay
David Zahra
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
CRC for Asthma and Airways Ltd
Original Assignee
CRC for Asthma and Airways Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from AU2010902567A external-priority patent/AU2010902567A0/en
Application filed by CRC for Asthma and Airways Ltd filed Critical CRC for Asthma and Airways Ltd
Publication of WO2011153592A1 publication Critical patent/WO2011153592A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/243—Colony Stimulating Factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation

Definitions

  • the present invention relates generally to antibodies that bind to granulocyte-macrophage colony stimulating factor (GM-CSF).
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • the invention also relates to high affinity, neutralizing humanized monoclonal antibodies specific for GM-CSF.
  • the invention also relates to uses of such antibodies in the treatment or prevention of GM-CSF-mediated and GM-CSF-associated diseases or conditions.
  • the invention further relates to methods for modulating GM-CSF-mediated and GM-CSF-associated diseases or conditions by the administration of antibodies of the invention.
  • Granulocyte-macrophage colony stimulating factor is a hematopoietic growth factor which regulates the differentiation, proliferation and function of granulocytes and monocytic cells such as macrophages.
  • GM-CSF is also a potent inflammatory cytokine, the activity or overexpression of which can have significant detrimental effects.
  • GM-CSF is implicated in a variety of autoimmune and inflammatory diseases including rheumatoid arthritis, asthma, multiple sclerosis and idiopathic thrombocytopenic purpura.
  • GM-CSF knockout mice are protected against the onset of the disease.
  • SNPs single nucleotide polymorphisms
  • One such example in the human GM-CSF gene results in a variant GM-CSF protein with an amino acid substitution ofthreonine for isoleucine at position 100 of the mature secreted human protein sequence.
  • the Asain population approximately 60% of individuals carry GM-CSF with threonine at this same position.
  • Antibodies against GM-CSF are suitable GM- CSF antagonists with therapeutic applications such as the treatment or prevention of autoimmune or inflammatory conditions.
  • the present invention relates generally to antibodies that bind, interact or otherwise associate with GM-CSF or a fragment, portion or part thereof and antagonize or neutralize GM-CSF activity.
  • the antibodies preferably are monoclonal antibodies or antigen-binding fragments thereof.
  • the antibodies are in an isolated, homogenous or fully or partially purified form.
  • the antibodies may be humanized or human antibodies suitable for administration to humans. These include humanized antibodies prepared, for example, from murine monoclonal antibodies, and human monoclonal antibodies which may be prepared, for example, using transgenic mice or by phage display.
  • humanized monoclonal antibodies are generated which bind to human GM-CSF with high affinity and which inhibit the activity of GM-CSF.
  • the antibodies bind with high affinity to multiple variants of GM-CSF.
  • the present invention provides a monoclonal antibody or antigen-binding fragment thereof that binds to human GM-CSF or a fragment thereof, the antibody comprising variable light chain CDR1 and CDR2 sequences as set forth in SEQ ID NOs: 43 and 44, respectively, variable heavy chain CDR1 and CDR2 sequences as set forth in SEQ ID NOs: 46 and 47, respectively; and (a) a variable light chain region CDR3 comprising the sequence as set forth in SEQ ID NO:45 with one or two amino acid substitutions therein; and/or
  • variable heavy chain region CDR3 comprising the sequence as set forth in SEQ ID NO:48 with one or two amino acid substitutions therein,
  • the present invention provides a monoclonal antibody or antigen-binding fragment thereof that binds to human GM-CSF or a fragment thereof, the antibody comprising:
  • variable light chain region CDR3 comprising the sequence as set forth in SEQ ID NO:45 with one or two amino acid substitutions therein;
  • variable heavy chain region CDR3 comprising the sequence as set forth in SEQ ID NO:48 with one or two amino acid substitutions therein,
  • the CDR1 and CDR2 sequences within the variable light chain of the monoclonal antibody or antigen-binding fragment thereof comprise the sequences set forth in SEQ ID NOs:43 and 44, respectively.
  • the CDR1 and CDR2 sequences within the variable heavy chain of the monoclonal antibody or antigen-binding fragment thereof comprise the sequences set forth in SEQ ID NOs:46 and 47, respectively.
  • the GM-CSF to which the monoclonal antibody or antigen-binding fragment thereof binds may comprise the amino acid sequence set forth in any one of SEQ ID NOs:1 to 4.
  • the monoclonal antibody or antigen-binding fragment thereof binds to each of the 1001 and 100T GM-CSF variants.
  • variable light chain region CDR3 comprises an amino acid substitution at position 4 and/or 5 of the sequence set forth in SEQ ID NO:45.
  • the threonine at position 4 may be substituted with a lysine residue.
  • the histidine at position 5 may be substituted with a tyrosine, glutamine, asparagine or lysine residue.
  • variable heavy chain region CDR3 comprises an amino acid substitution at position 5 of the sequence set forth in SEQ ID NO:48.
  • the aspartic acid at position 5 may be substituted with a glutamine or asparagine residue.
  • the monoclonal antibody or antigen-binding fragment thereof comprises a variable light chain region CDR3 and/or a variable heavy chain region CDR3 with the amino acid substitutions as shown in Table 2, with respect to the CDR3 sequences of SEQ ID NOs:45 and 48, respectively.
  • the monoclonal antibody or antigen-binding fragment thereof comprises:
  • variable heavy chain sequence as set forth in any one SEQ ID NOs: 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39 or 41 or a fragment or variant thereof;
  • variable light chain sequence as set forth in any one SEQ ID NOs: 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40 or 42 or a fragment or variant thereof.
  • the monoclonal antibody is selected from the antibodies hGM4/35 to hGM4/51 comprising the variable light chain and variable heavy chain sequences as shown Table 1.
  • the monoclonal antibody is selected from the antibodies hGM4 35, HGM4/38, hGM4/39, HGM4/40 and hGM4/41 comprising the variable light chain and variable heavy chain sequences as shown in Table 1.
  • the antibody is a humanized antibody.
  • the invention provides a monoclonal antibody or antigen-binding fragment thereof that binds to human GM-CSF or a fragment thereof, comprising a variable heavy chain sequence as set forth in any one SEQ ID NOs: 9, 11 , 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 , 33, 35, 37, 39 or 41 or a fragment or variant thereof.
  • the invention provides a monoclonal antibody or antigen-binding fragment thereof that binds to human GM-CSF or a fragment thereof, comprising a variable light chain sequence as set forth in any one SEQ ID NOs: 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40 or 42 or a fragment or variant thereof.
  • the invention also provides for monoclonal antibodies comprising an amino acid sequence having at least about 70% sequence identity to an amino acid sequence as set forth in any one of SEQ ID NOs: 9 to 42.
  • the invention also provides hybridomas producing the monoclonal antibodies disclosed herein.
  • the present invention provides a method for the treatment or prevention of a GM- CSF - mediated disease or condition or a disease or condition otherwise associated with elevated or aberrant GM-CSF expression and/or activity, the method comprising administering to a subject in need thereof an effective amount of at least one antibody disclosed herein or an antigen-binding fragment thereof.
  • the disease or condition is an autoimmune or inflammatory disease or condition.
  • the disease or condition may be selected from, for example, asthma, rheumatoid arthritis, chronic obstructive pulmonary disease, idiopathic thrombocytopenic purpura, acute respiratory distress syndrome, multiple sclerosis, Alzheimer's disease, Crohn's disease, irritable bowel syndrome, colitis, psoriasis, macular degeneration, uveitis, Wallerian degeneration, antiphospholipid syndrome, restinosis, atherosclerosis, idiopathic pulmonary fibrosis, relapsing polychondritis, hepatitis, glomerulonephritis, lupus and other metabolic diseases.
  • the present invention provides the use of an antibody disclosed herein or an antigen-binding fragment thereof for the manufacture of a medicament for treating or preventing a GM-CSF - mediated disease or condition or a disease or condition otherwise associated with elevated or aberrant GM-CSF expression and/or activity.
  • the invention further provides compositions comprising one or more antibodies disclosed herein or antigen-binding fragments thereof, optionally together with suitable pharmaceutically acceptable carriers and/or diluents.
  • Figure 1 Polypeptide sequences of human GM-CSF.
  • A Precursor or immature form with a 17 amino acid N-tenminal signal sequence for secretion and possessing isoleucine (I; bold and underlined) at position 117 (SEQ ID NO:1).
  • B Mature, secreted form possessing isoleucine (I; bold and underlined) at position 100 (SEQ ID NO:2).
  • C Precursor or immature form with 17 amino acid N-terminal signal sequence for secretion and possessing threonine (T; bold and underlined) at position 117 (SEQ ID NO:3).
  • FIG. 1 Mature, secreted form possessing threonine (T; bold and underlined) at position 100 (SEQ ID NO:4).
  • Figure 2. Schematic representation of the affinity maturation process to generate the antibodies exemplified herein.
  • Figure 3. Neutralisation of TF-1 cellular growth induced by human GM-CSF variant 1001 or 100T by 4K21 , hGM4/34 or hGM4/35. Each point calculated in triplicate for each experiment.
  • FIG. 5 Neutralisation of TF-1 cellular growth induced by (A) human GM-CSF variant 1001 or (B) human GM-CSF variant 100T by 4K21, hGM4/34, hGM4/35, hGM4/38, hGM4/39, hGM4/40, hGM4/41. Each point calculated in triplicate for each experiment. Amino acid sequences are referred to by a sequence identifier number (SEQ ID NO:). The SEQ ID NOs correspond numerically to the sequence identifiers ⁇ 400>1 (SEQ ID NO:1), ⁇ 400>2 (SEQ ID NO:2), etc. in the sequence listing provided at the end of the specification.
  • SEQ ID NO's 1-4 represent human GM-CSF sequences
  • SEQ ID NO's 5 and 6 represent the VL and VH chain sequences of antibody 4K21
  • SEQ ID NO's 7 and 8 represent the VL and VH chain sequences of antibody hGM4/34
  • SEQ ID NO's 9-42 represent the VH and VL chain sequences of antibodies hGM4/35-hGM4/51
  • SEQ ID NO's 43-48 represent VH and VL CDR sequences of antibody 4K21.
  • an element means one element or more than one element.
  • polypeptide means a polymer made up of amino acids linked together by peptide bonds.
  • polypeptide and protein are used interchangeably herein, although for the purposes of the present invention a “polypeptide” may constitute a portion of a full length protein.
  • an “antibody” or “antibodies” includes reference to all the various forms of antibodies, including but not limited to whole antibodies, antibody fragments, including, for example, Fv, Fab, Fab' and F(ab')2 fragments, humanized antibodies, human antibodies and immunoglobulin-derived polypeptides produced through genetic engineering techniques.
  • binding of an antibody means binding, interacting or associating with or to a target antigen such as GM-CSF. Consequently, reference to an antibody binding to GM-CSF includes within its scope the binding, interaction or association of the antibody or an antigen-binding portion thereof to part, fragment or epitope-containing region of GM- CSF.
  • binding means or includes the specific binding, interaction or association of the antibody to GM-CSF or a portion thereof.
  • GM-CSF in the context of the present disclosure includes GM-CSF polypeptides (and polynucleotides encoding said polypeptides) from any organism, typically murine or human.
  • the GM-CSF polypeptide may be in precursor form, such as the immature sequence prior to secretion from a cell or in a mature, secreted form.
  • exemplary polypeptide sequences of immature (nonsecreted or precursor) human GM-CSF and mature, secreted GM-CSF are shown in Figure 1 and set forth in SEQ ID NO's:1-4.
  • the term "GM-CSF” as used herein also refers to naturally occurring and synthetically-derived variants of native or wild-type GM-CSF sequences.
  • Such variants include single nucleotide polymorphisms (SNPs) and splice variants.
  • SNPs single nucleotide polymorphisms
  • Exemplary polypeptide sequences of naturally occurring human GM-CSF variants resulting from a SNP are shown in Figure 1 , in which the amino acid residue at position 117 of the nonsecreted precursor sequence, or position 100 of the mature, secreted sequence is either an isoleucine (I) (hereinafter also referred to as the "1001 variant”) or a threonine (hereinafter also referred to as the "100T variant”).
  • I isoleucine
  • T threonine
  • GM-CSF molecules, and the sequences associated therewith, to which the present disclosure relates are not limited to those exemplified herein.
  • GM-CSF as used herein also includes fragments or portions of full length GM-CSF molecules, which fragments or portions typically comprise the epitopes to which an antibody disclosed herein binds.
  • variant refers to substantially similar sequences.
  • polypeptide sequence variants possess qualitative biological activity in common.
  • a variant polypeptide sequence may be a derivative of a sequence as disclosed herein, which derivative comprises the addition, deletion, or substitution of one or more amino acids. Further, these polypeptide sequence variants may share at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • homologues of polypeptides of the disclosure are typically a polypeptide from a different species but sharing substantially the same biological function or activity as the corresponding polypeptide disclosed herein.
  • the terms “inhibits” and “inhibiting” as used herein as they relate to the activity of GM-CSF does not necessarily mean completely inhibiting activity. Rather, activity may be inhibited to an extent, and/or for a time, sufficient to produce the desired effect. Thus inhibition of GM-CSF activity may be partial or complete attenuation of one or more biological effects of GM-CSF and such inhibition may be temporally and/or spatially limited. By temporally and/or spatially limited is meant that the inhibition may be limited to particular physiological conditions or circumstances and/or to particular regions of the body.
  • GM-CSF activity means any cellular function, action, effect or influence exerted by the GM-CSF, either by the protein or polypeptide itself or any fragment or portion thereof.
  • treating refers to any and all uses which remedy a disease, disorder or condition or a symptom thereof, prevent the establishment of a disease, disorder or condition, or otherwise prevent, hinder, retard, or reverse the progression of a disease, disorder or condition or other undesirable symptom in any way whatsoever.
  • treating does not necessarily imply that a patient is treated until total recovery.
  • the term "effective amounf includes within its meaning a non-toxic but sufficient amount of an agent to provide the desired effect.
  • the exact amount or dose required will vary from subject to subject depending on factors such as the species being treated, the age and general condition of the subject, the severity of the condition being treated, the particular agent being administered and the mode of administration and so forth. Thus, it is not possible to specify an exact "effective amount”. However, for any given case, an appropriate "effective amounf may be determined by one of ordinary skill in the art using only routine experimentation .
  • subject typically refers to mammals including humans, primates, livestock animals (e.g. sheep, pigs, cattle, horses, donkeys), laboratory test animals (e.g. apes, monkeys, mice, rabbits, rats, guinea pigs), companion animals (e.g. dogs, cats) and captive wild animals (e.g. foxes, kangaroos, deer).
  • livestock animals e.g. sheep, pigs, cattle, horses, donkeys
  • laboratory test animals e.g. apes, monkeys, mice, rabbits, rats, guinea pigs
  • companion animals e.g. dogs, cats
  • captive wild animals e.g. foxes, kangaroos, deer.
  • the mammal is human or a laboratory test animal. Even more preferably, the mammal is a human.
  • the present invention provides antibodies that function as GM-CSF antagonists and may be used for treating certain diseases, disorders or conditions induced by or otherwise associated with elevated levels and/or activity of GM-CSF.
  • the present invention also provides methods for treating these diseases, disorders or conditions comprising administering an anti-GM-CSF antibody disclosed herein to a patient afflicted with such a disease, disorder or condition.
  • compositions for use in such methods the compositions comprising one or more anti-GM-CSF antibodies.
  • the antibodies of the present invention bind, interact or otherwise associate with GM-CSF, a variant or portion thereof.
  • the antibodies are typically specific for GM-CSF from a particular species, such as human GM-CSF, or, in view of the level of sequence similarity between GM-CSF from different species, the antibodies may show some cross-reactivity with GM-CSF from two or more species. In the case of antibodies directed towards human GM-CSF, some level of cross-reactivity with other mammalian forms of GM-CSF may be desirable in certain circumstances, such as for example, for testing antibodies in animal models of a particular disease and for conducting toxicology, safety and dosage studies.
  • Antibodies of the present invention may bind with high affinity to one or more GM-CSF variants, including SNP-derived and splice variants.
  • antibodies disclosed herein bind with high affinity to multiple GM-CSF variants such as the human 1001 and 100T variants disclosed herein.
  • antibodies of the invention are monoclonal antibodies or antigen-binding fragments thereof.
  • the antibodies are humanized or are human antibodies suitable for administration to humans. These include humanized antibodies prepared, for example, from murine monoclonal antibodies and human monoclonal antibodies which may be prepared, for example, using transgenic mice or by phage display.
  • Antibodies of the present invention may be prepared by a variety of procedures well known to those skilled in the art. For example, methods for the preparation of antibodies are described in Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.), Plenum Press, New York (1980); Antibodies: A Laboratory Manual, Harlow and Land (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (198B); and Monoclonal Antibodies: Principles and Practice, Goding, 3" 1 Edition, Academic Press (1996). The disclosures thereof are incorporated herein by reference in their entirety. Similarly, monoclonal antibodies secreted by hybridoma cell lines may be purified by conventional techniques.
  • one method for producing an antibody of the present invention comprises immunizing a non-human animal, such as a mouse or a transgenic mouse, with a GM-CSF polypeptide, or immunogenic portion or fragment thereof, whereby antibodies directed against the GM-CSF polypeptide are generated in said animal.
  • the GM-CSF polypeptide or immunogenic portion or fragment thereof used to immunize animals may be from any mammalian source.
  • the GM-CSF polypeptide or an immunogenic portion of fragment thereof is human GM- CSF or an immunogenic portion of fragment thereof.
  • Antigen-binding fragments of antibodies of the present invention may be produced by conventional techniques. Examples of such fragments include, but are not limited to, Fab, Fab", F(ab')2 and Fv fragments, including single chain Fv fragments (termed sFv or scFv). Antibody fragments and derivatives produced by genetic engineering techniques, such as disulphide stabilized Fv fragments (dsFv), single chain variable region domain (Abs) molecules and minibodies are also contemplated for use. Unless otherwise specified, the terms "antibody” and “monoclonal antibody” as used herein encompass both whole antibodies and antigen-binding fragments thereof.
  • Derivatives of monoclonal antibodies directed against GM-CSF may be prepared and screened for desired properties, by known techniques.
  • the techniques may involve, for example, isolating DNA encoding a polypeptide chain (or a portion thereof) of a monoclonal antibody of interest, and manipulating the DNA through recombinant DNA technology.
  • the DNA may be used to generate another DNA of interest, or altered (for example by mutagenesis or other conventional techniques) to add, delete, substitute or insert one or more amino acid residues.
  • DNA encoding antibody polypeptides e.g. heavy or light chain, variable region only or full length
  • the DNA may be isolated by conventional procedures including polymerase chain reaction (PCR).
  • Phage display is an alternative example of a suitable technique whereby derivatives of antibodies of the invention may be prepared.
  • polypeptides that are components of an antibody of interest are expressed in any suitable recombinant expression system, and the expressed polypeptides are allowed to assemble to form antibody molecules.
  • Single chain antibodies may be formed by linking heavy and light chain variable region (Fv region) fragments via an amino acid bridge (short peptide linker), resulting in a single polypeptide chain.
  • Such single-chain Fvs (scFvs) may be prepared by fusing DNA encoding a peptide linker between DNAs encoding the two variable region polypeptides (VL and VH).
  • the resulting antibody fragments can form dimers or trimers, depending on the length of a flexible linker between the two variable domains (see Kortt ef a/., Protein Engineering 10: 423, 1997).
  • Techniques developed for the production of single chain antibodies include those described in U.S. Pat. No. 4,946,778; Bird (Science 242: 423, 1988), Huston et al. (Proc. NatJ. Acad Sci USA 85: 5879, 1988) and Ward ef a/. (Nature 334: 544, 1989). The disclosures thereof are incorporated herein by reference in their entirety.
  • Single chain antibodies derived from antibodies provided herein are encompassed by the present invention.
  • monoclonal antibodies contemplated by the present invention are the affinity matured humanized monoclonal antibodies hGM4/35 to hGM4/51, the generation of which is described herein. These antibodies comprise the variable heavy chain sequences as set forth in SEQ ID NOs:9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 , 33, 35, 37, 39 or 41, respectively, and the variable light chain sequence as set forth in SEQ ID NOs: 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40 or 42, respectively.
  • amino acid sequences of monoclonal antibodies of the invention may include one or more amino acid substitutions such that although the primary sequence of the polypeptide is altered, the ability of the antibody to bind GM-CSF and the activity of the antibody is retained.
  • the substitution may be a conservative substitution.
  • conservative amino acid substitution refers to a substitution or replacement of one amino acid for another amino acid with similar properties within a polypeptide chain (primary sequence of a protein). For example, the substitution of the charged amino acid glutamic acid (Glu) for the similarly charged amino acid aspartic acid (Asp) would be a conservative amino acid substitution.
  • the present invention contemplates variants of the light chain and heavy chain sequences disclosed herein and such variants are encompassed within the scope of the present invention.
  • Variants may differ from the disclosed sequences within framework regions or within CDRs of either the light or heavy chain sequences.
  • monoclonal antibodies or antigen-binding fragments thereof comprising amino acid sequences having at least about 70% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 9 to 42 are contemplated.
  • the monoclonal antibody or antigen- binding fragment thereof may comprise amino acid sequences having at least about at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequences set forth in SEQ ID NOs: 9 to 42.
  • the term “variant” encompasses antibody sequences modified from those disclosed herein by any suitable means. For example, when used in the context of murine sequences, the term “variant” includes within its scope humanized forms of such sequences. When used in the context of humanized sequences disclosed herein the term “variant” includes within its scope modified sequences comprising one or more murine back mutations.
  • Antibodies derived from non-human animals are generally unsuitable for administration to humans as they may cause an immune response and result in the generation of anti-mouse antibodies (the so-called HAMA response).
  • HAMA response can neutralize the mouse antibodies by rapidly clearing them from the blood, thus preventing the mouse antibody from binding to its target.
  • one strategy is to "humanize" the mouse antibody by replacing as many foreign" residues in the non-epitope binding regions with human sequences.
  • the specificity of the interaction between an antibody and an antigen involves the hypervariable or complementarity-determining regions (CDRs) in the variable domain. These residues are generally not changed during the humanization process.
  • CDRs complementarity-determining regions
  • the remaining residues in the variable domain, referred to as the framework (FW) and the constant regions of the antibody, on both heavy and light chains are usually replaced with human sequences.
  • FW framework
  • certain mouse residues in the framework regions may need to be preserved.
  • Suitable humanization processes such as CDR grafting, are well known to those skilled in the art. A particularly suitable approach is exemplified herein. Procedures for the production of chimeric and humanized monoclonal antibodies also include those described in, for example, Riechmann et al., Nature 332: 323, 1988, Liu et al, Proc. Natl. Acad. Sci. USA 84: 3439, 1987, Larrick et al., Bio/Technology 7: 934, 1989 and Winter and Harris, VPS 14: 139, 1993.
  • the complementarity determining regions (CDRs) of a given antibody may be identified using the system described by Kabat et al. in Sequences of Proteins of Immunological Interest, 5th Ed., US Dept. of Health and Human Services, PHS, NIH, NIH Publication No. 91-3242, 1991).
  • the murine monoclonal antibody 4K21 was previously produced by the present inventors (the subject of international patent application no. PCT/AU2008/000728, the disclosure of which is incorporated herein by reference) by isolation from mice immunized with human GM-CSF 1001. 4K21 and a humanized variant thereof hGM4/34 (also the subject of international patent application no. PCT/AU2008/000728) bind and neutralise the function of human GMCSF 1001, with sub nanomolar EC50, but have a much higher EC50 for the 100T variant. To develop antibodies that bind both variants, affinity matured " humanized variants of hGM4/34 (humanized 4K21) were isolated.
  • these antibodies are able to bind and inhibit both the 1001 and 100T variants of human GM-CSF.
  • These exemplary affinity matured variants possess changes in either the CDR3 of the variable heavy chain and/ or. the CDR3 of the variable light chain with respect to both 4K21 and hGM4/34 (which share the same variable heavy chain and the same variable light chain CDR sequences).
  • the present invention provides a monoclonal antibody or antigen-binding fragment thereof that binds to human GM-CSF or a fragment thereof, the antibody comprising:
  • variable light chain region CDR3 comprising the sequence as set forth in SEQ ID NO:45 with one or two amino acid substitutions therein;
  • variable heavy chain region CDR3 comprising the sequence as set forth in SEQ ID NO:48 with oneor two amino acid substitutions therein,
  • variable light chain region CDR3 and/or variable heavy chain region CDR3 comprise at least one of the subsitutions shown in Table 2.
  • variable light chain region CDR3 comprises an amino acid substitution at position 4 and/or 5 of the sequence set forth in SEQ ID NO:45.
  • the threonine at position 4 (position 97 of the variable light chain sequence set forth in SEQ ID NO:7) may be substituted with a lysine residue.
  • the histidine at position 5 (position 98 of the variable light chain sequence set forth in SEQ ID NO:7) may be substituted with a tyrosine, glutamine, asparagine or lysine residue.
  • variable heavy chain region CDR3 comprises an amino acid substitution at position 5 of the sequence set forth in SEQ ID NO:48.
  • the aspartic acid at position 5 may be substituted with a glutamine or asparagine residue.
  • the monoclonal antibody or antigen-binding fragment thereof comprises a variable light chain region CDR3 and/or a variable heavy chain region CDR3 with the amino acid substitutions as shown in Table 1, with respect to the CDR3 sequences of SEQ ID Nos:45 and 48, respectively.
  • the monoclonal antibody or antigen-binding fragment thereof comprises:
  • variable heavy chain sequence as set forth in any one SEQ ID Nos: 9, 11, 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39 or 41 , or a fragment or variant thereof;
  • variable light chain sequence as set forth in any one SEQ ID Nos: 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40 or 42 or a fragment or variant thereof.
  • the monoclonal antibody is selected from the antibodies hGM4/35 to hGM4/51 comprising the variable light chain and variable heavy chain sequences as shown in Table 1.
  • humanized antibodies of the invention comprise, within the variable region of their light chain, CDR1 and CDR2 found in the light chain of murine antibody 4K21. Further, among the antibodies contemplated by the present invention are those that comprise, within the variable region of their heavy chain, CDR1 and CDR2 found in the heavy chain of murine antibody 4K21.
  • variable heavy and variable light chain sequences of affinity matured humanized antibodies in accordance with particular embodiments of the invention are described in Table 1.
  • the antibodies may be partially human, or completely human.
  • transgenic mice into which genetic material encoding one or more human immunoglobulin chains have been introduced may be used to produce antibodies.
  • the transgenic mice may be such that human immunoglobulin polypeptide chains replacing endogenous immunoglobulin chains are present in at least some antibodies produced by the animal upon immunization.
  • Another method for generating human antibodies is phage display. Phage display techniques for generating human antibodies are well known to those skilled in the art, and include the methods used by Cambridge Antibody Technology and MorphoSys, which are described in International Patent Publication Nos. WO 92/01047, WO 92/20791 , WO 93/06213 and WO 93/11236,the disclosures of which are incorporated herein by reference.
  • Antibodies of the present invention or hybridomas comprising such antibodies may be screened and manipulated further to identify monoclonal antibodies with particularly desirable properties, such as increased binding affinity, reduced immunogenicity and/or increased inhibitory activity against GM- CSF.
  • the present invention provides methods for treating or preventing GM-CSFr mediated diseases or conditions, diseases or conditions otherwise associated with elevated levels and/or activity of GM- CSF, and other diseases or conditions which may be beneficially treated by inhibiting or neutralizing GM-CSF activity by the administration of antibodies of the present invention.
  • Diseases and conditions which may be treated in accordance with the present invention include autoimmune and inflammatory diseases.
  • Such diseases include but are not limited to asthma, rheumatoid arthritis, chronic obstructive pulmonary disease, idiopathic thrombocytopenic purpura, acute respiratory distress syndrome, multiple sclerosis, Alzheimer's disease, Crohn's disease, irritable bowel syndrome, colitis, psoriasis, macular degeneration, uveitis, Wallerian degeneration, antiphospholipid syndrome, restenosis, atherosclerosis, idiopathic pulmonary fibrosis, relapsing polychondritis, hepatitis, glomerulonephritis, lupus and other metabolic diseases.
  • autoimmune diseases which may be treated in accordance with the invention include systemic sclerosis, scleroderma, Sjogren syndrome, spondyloarthritis, Sapho syndrome, juvenile idiopathic arthritis, lyme disease, polymyositis, dermatomyositis, autoimmune thyroditis, Grave's disease, Type 1 diabetes, adrenalitis, autoimmune Addison's disease, polyendocrine syndromes, gastritis, pernicious anemia, hypophysitis, hemolytic anemia, neutropenia, aplastic anemia, clotting disorders including acquired von Willebrand syndrome, Guillain-Barre Syndrome, chronic inflammatory demyelinating polyradiculoneuropathy, myasthenia gravis, Lambert-Eaton myasthenic syndrome, acquired neuromyotonia, stiff-person syndrome, cerebellar ataxia, Rasmussen's encephalitis, Morvan's syndrome, limbic encephalitis, ocular disease, inner ear
  • Antibodies disclosed herein also find application in the treatment of failed or rejected implants for example cells such as islet cells, and prostheses and failed or rejected organ transplants, such as for example lung, kidney, heart, heart valve, pancreas, vein, bone, tendon, skin, cornea, thymus, intestine, and liver. Additional applications, both in vivo and in vitro, of antibodies of the invention are contemplated.
  • antibodies of the invention may be employed in assays designed to detect the presence of GM-CSF and/or to purify GM-CSF.
  • antibodies of the invention may be used in the diagnosis of diseases and conditions associated with aberrant or dysregulated GM-CSF expression.
  • Such diagnostic embodiments may comprise exposing one or more cells or tissues from a subject, either in vivo or ex vivo, to an antibody disclosed herein and determining the level of GM-CSF expression , optionally in comparison with a reference sample, wherein the level of GM-CSF expression is indicative of the subject having, or being predisposed to, a disease or condition associated with aberrant or dysregulated GM-CSF expression .
  • Antibodies may also be tested in animal models of particular diseases and for conducting toxicology, safety and dosage studies.
  • the antibodies may be conjugated or otherwise linked to molecules or compounds facilitating their detection when bound to the antibody ligand. Suitable detectable markers include but are not limited to luminescent, fluorescent and phosphorescent tags.
  • antibodies of the invention are administered to a subject in need thereof in an amount effective to obtain the desired therapeutic or prophylactic effect.
  • the specific effective amount or dose for any particular subject will depend upon a variety of factors including, for example, the activity of the specific antibody(ies) employed, the age, body weight, general health and diet of the individual to be treated, the time of administration, rate of excretion, and combination with any other treatment or therapy. Single or multiple administrations can be carried out with dose levels and pattern being selected by the treating physician. In treating or preventing autoimmune and inflammatory conditions, the present invention contemplates the administration of multiple antibodies if required or desirable.
  • Whether it is suitable or desirable to administer one or more antibodies can be determined by those skilled in the art on a case-by-case basis.
  • the invention also contemplates combination therapies, wherein antibodies as described herein are coadministered with other suitable agents which may facilitate the desired therapeutic or prophylactic outcome.
  • suitable agents which may facilitate the desired therapeutic or prophylactic outcome.
  • coadministered is meant simultaneous administration in the same formulation or in two different formulations via the same or different routes or sequential administration by the same or different routes.
  • sequential administration is meant a time difference of from seconds, minutes, hours, days, weeks, months or years between the administration of the two agents.
  • antibodies may be administered in any suitable form.
  • antibodies are typically administered in the form of pharmaceutical compositions, which compositions may comprise one or more pharmaceutically acceptable carriers, excipients or diluents.
  • Such compositions may be administered systemically, regionally or locally and via any suitable route such as by parenteral (including intravenous, intraarterial or intramuscular), oral, nasal, topical and subcutaneous routes.
  • Examples of pharmaceutically acceptable carriers or diluents are demineralised or distilled water; saline solution; vegetable based oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oil, arachis oil or coconut oil; silicone oils, including polysildxanes, such as methyl polysiloxane, phenyl polysiloxane and methylphenyl polysolpoxane; volatile silicones; mineral oils such as liquid paraffin, soft paraffin or squalane; cellulose derivatives such as methyl cellulose, ethyl cellulose, carboxymethylcellulose, sodium carboxymethylcellulose or hydroxypropylmethylcellulose; lower alkanols, for example ethanol or iso-propanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glycol, polypropylene glycol, ethylene glycol, 5 propylene glycol, 1 ,3-but
  • suitable carriers, diluents, excipients and adjuvants for oral use include peanut oil, liquid paraffin, sodium carboxymethylcellulose, methylcellulose, sodium alginate, gum acacia, gum tragacanth, dextrose, sucrose, sorbitol, mannitol, gelatine and lecithin.
  • these oral formulations may contain suitable flavouring and colourings agents.
  • the capsules When used in capsule form the capsules may be coated with compounds such as glyceryl monostearate or glyceryl distearate which
  • Adjuvants typically include emollients, emulsifiers, thickening agents, preservatives, bactericides and buffering agents.
  • non-toxic parenteral ⁇ acceptable diluents or carriers can include, Ringer's solution, medium chain triglyceride (MCT), isotonic saline, 20 phosphate buffered saline, ethanol and 1 ,2 propylene glycol.
  • MCT medium chain triglyceride
  • isotonic saline 20 phosphate buffered saline
  • ethanol 1 ,2 propylene glycol
  • hybridoma containing 4K21 has been deposited on 17 May 2007 at the European Collection of Cell Cultures (ECACC), Centre for Applied Microbiology and Research, Porton Down, Salisbury, United Kingdom, under Accession No. 07051601.
  • variable light chain and heavy chain region amino acid sequences of mAb 4K21 are set forth in SEQ ID No's 5 and 6, respectively.
  • variable light chain and heavy chain region amino acid sequences of the humanized 4K21 antibody hGM4/34 are set forth in SEQ ID No's 7 and 8, respectively.
  • the CDRs of the variable light chain region of both 4K21 and hGM4/34 are as follows: CDR1 : RSSQSLVNSNGNTYLH (SEQ ID NO:43); CDR2: KVSNRFS (SEQ ID NO:44); and CDR3: SQSTHVPPT (SEQ ID NO:45).
  • the CDRs of the variable heavy chain region of both 4K21 and hGM4/34 are as follows: CDR1 : GFAFSAYDMS (SEQ ID NO:46); CDR2: YISSGGSSFYYPDTVKG (SEQ ID NO:47); and CDR3: HLGFDY (SEQ ID NO:48).
  • GM-CSF has a pi of 5.21.
  • the 'M5' region of GM-CSF is the epitope that 4K21 binds and this has a high number of acidic residues.
  • Initial results in a humanisation process demonstrated that the heavy chain is most important and that the light chain provides minimal assistance in antibody binding.
  • affinity maturation the light chain was targeted to attain additional affinity.
  • basic residues during CDR maturation soft randomisation because of the acidic nature of the 'M5' epitope.
  • hGM4/34 antibodies were screened by Biacore to identify antibodies with increased off-rates compared to hGM4 34.
  • hGM4/34 has been expressed in a scFv format and tested for binding to GM-CSF on the Biacore.
  • hGM4 34 has very high affinity for GM-CSF and the off-rate is measured in hours to days. This long off-rate made it very difficult to screen for antibodies with greater affinity for GM-CSF.
  • a number of lower affinity clones were isolated and then these clones used to mature other CDRs. In particular, a library of the CDRH3 clones was made and a low affinity clone was isolated.
  • SEQ ID Nos: 942 represent the variable light chain and variable heavy chain amino acid sequences of the affinity matured antibodies of the present invention, designated hGM4/35 through to hGM4/51 (see Table 1). These sequences differ from those of hGM4/34 and 4K21 only in either or both the variable light chain CDR3 and the variable heavy chain CDR3 as shown in Table 2.
  • Antibody hGM4/35 was demonstrated to be able to neutralize the growth promoting function of both the 1001 and 100T variants of human GM-CSF in a TF-1 cell growth bioassay (Figure 3).
  • TF-1 cells are dependant on the presence of GM-CSF for their growth and are maintained with 2 ng/ml recombinant GM-CSF (Peprotech).
  • Glycosylated human GM-CSF 1001 or 100T was produced and purified by MSSA.
  • To perform the neutralization assay TF-1 cells were stimulated with glycosylated hGM-CSF (0.25ng/ml) and incubated with the varying concentrations of neutralising antibody for 72hrs. Cellular growth was quantitated by pulsing each well for 4hrs with 0.5uCi of 3 H-thymidine. Each point was calculated in triplicate for each experiment.
  • hGM4/35 neutralizes TF-1 cellular growth induced by human GM-CSF variant 1001 or 100T, whereas the activities of antibodies 4K21 and hGM4/34 are significantly lower against the 100T variant.
  • the EC50 values calculated from Figure 3 are shown in Table 3.
  • Antibody hGM4/35 also neutralizes each of the 1001 and 100T variants of human GM-CSF in an IL-8 secretion assay in U937 cells.
  • Neutralization of IL-8 secretion from U937 cells stimulated with human GM-CSF 1001 or 100T by 4K21, hGM4/34 or hGM4/35 is shown in Figure 4.
  • Glycosylated human GM-CSF 1001 or 100T was produced and purified by MSSA.
  • IL-8 secretion from U937 cells was stimulated by either human GM-CSF 1001 or human GM-CSF 100T.
  • Glycosylated hGM-CSF (2.1ng/ml) was neutralized with the indicated concentrations of antibody for 16hrs at 37°C.
  • IL-8 secretion was quantitated by IL-8 specific ELISA. Each point was calculated in triplicate. As shown in Figure 4, each of 4K21 , hGM4/34 and hGM4/35 showed activity against the 1001 GM-CSF variant, but unlike 4K21 and hGM4/34, hGM4/35 also displayed potent activity in inhibiting IL-8 secretion from cells stimulated with GM-CSF 100T. The EC50 values calculated from Figure 4 are shown in Table 4.
  • Antibodies hGM4/34, hGM4/35, and hGM4/38 to hGM4/41 were demonstrated to be able to neutralize the growth promoting function of both the 1001 and 100T variants of human GM-CSF in a TF-1 cell growth bioassay.
  • TF-1 cells are dependant on the presence of GM-CSF for their growth and were maintained with 2 ng/ml recombinant GM-CSF (Peprotech).
  • Glycosylated human GM-CSF 1001 or 100T was produced and purified by MSSA.
  • TF-1 cells were stimulated with either glycosylated hGM-CSF 1001 or 100T (0.25ng/ml) and incubated with the varying concentrations of neutralizing antibody for 72hrs. Cellular growth was quantitated by pulsing each well for 4hrs with 0.5uCi of 3 H-thymidine. Each point was calculated in triplicate for each experiment.
  • hGM4/34, hGM4/35, and hGM4/38 to hGM4/41 neutralize TF-1 cellular growth induced by human GM-CSF variant 1001.
  • the EC50 values calculated from Figure 5A are shown in Table 5.
  • hGM4/41 0.075 1001 hGM4/34, hGM4 35, and hGM4/38 to hGM4/41 neutralize TF-1 cellular growth induced by human GM-CSF variant 100T.
  • the EC50 values calculated from Figure 5B are shown in Table 6.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Cette invention concerne des anticorps monoclonaux ou des fragments de liaison à l'antigène se liant au GM-CSF humain ou à un fragment de ce dernier. Les anticorps comprennent des séquences CDR1 et CDR2 à chaîne légère variable comme présentes dans SEQ ID NO : 43 et 44, respectivement, des séquences de CDR1 et CDR2 à chaîne lourde variable comme présentes dans SEQ ID NO 46 et 47, respectivement, et une région CDR3 à chaîne légère variable comme présente dans SEQ ID NO 45 renfermant une ou deux substitutions d'acides aminés; et un CDR3 à région de chaîne lourde comprenant la séquence présente dans SEQ ID NO; 48 et renfermant une ou deux substitutions d'acides aminés.
PCT/AU2011/000718 2010-06-10 2011-06-10 Molécules thérapeutiques Ceased WO2011153592A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AU2010902567A AU2010902567A0 (en) 2010-06-10 Therapeutic molecules
AU2010902567 2010-06-10

Publications (1)

Publication Number Publication Date
WO2011153592A1 true WO2011153592A1 (fr) 2011-12-15

Family

ID=45097407

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU2011/000718 Ceased WO2011153592A1 (fr) 2010-06-10 2011-06-10 Molécules thérapeutiques

Country Status (1)

Country Link
WO (1) WO2011153592A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018050111A1 (fr) 2016-09-19 2018-03-22 I-Mab Anticorps anti-gm-csf et leurs utilisations

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008141391A1 (fr) * 2007-05-23 2008-11-27 Crc For Asthma And Airways Ltd Anticorps neutralisants
WO2009062238A1 (fr) * 2007-11-12 2009-05-22 Crc For Asthma And Airways Ltd Epitope pour la neutralisation d'anticorps

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008141391A1 (fr) * 2007-05-23 2008-11-27 Crc For Asthma And Airways Ltd Anticorps neutralisants
WO2009062238A1 (fr) * 2007-11-12 2009-05-22 Crc For Asthma And Airways Ltd Epitope pour la neutralisation d'anticorps

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018050111A1 (fr) 2016-09-19 2018-03-22 I-Mab Anticorps anti-gm-csf et leurs utilisations
EP3328887A4 (fr) * 2016-09-19 2019-04-03 I-Mab Anticorps anti-gm-csf et leurs utilisations
US10647767B2 (en) 2016-09-19 2020-05-12 I-Mab Biopharma Co., Ltd. Anti-GM-CSF antibodies and uses thereof
US10889641B2 (en) 2016-09-19 2021-01-12 I-Mab Biopharma Co., Ltd. Anti-GM-CSF antibodies and uses thereof
US11926662B2 (en) 2016-09-19 2024-03-12 I-Mab Biopharma (Hangzhou) Co., Ltd. Anti-GM-CSF antibodies and uses thereof

Similar Documents

Publication Publication Date Title
US11254746B2 (en) Anti-PD-1 monoclonal antibody, and preparation method therefor and application thereof
US9469689B2 (en) Therapeutic DLL4 binding proteins
DK2260055T3 (en) Monoclonal antibodies against RGM A protein and uses thereof
US20110044996A1 (en) Neutralizing antibodies
US20210277097A1 (en) Monoclonal antibodies against the rgm a protein for use in the treatment of retinal nerve fiber layer degeneration
KR20120089659A (ko) 치료용 ｄｌｌ4 결합 단백질
CN114685655B (zh) Pd-1结合分子及其应用
US10556947B2 (en) Binding molecule that binds specifically to the precursor of brain-derived neurotrophic factor
CN109689687A (zh) 抗met抗体及其应用
US20100297135A1 (en) Epitope for neutralizing antibodies
US12258392B2 (en) Anti-IL-25 antibodies and use thereof
JP5733546B2 (ja) インテグリンα８β１の機能を阻害する事による線維化の抑制
WO2011153592A1 (fr) Molécules thérapeutiques
JP2025503202A (ja) 抗インターロイキン－１１抗体及びその使用
HK1229347A (en) Therapeutic dll4 binding proteins
HK1229347A1 (en) Therapeutic dll4 binding proteins
AU2015202980A1 (en) Therapeutic DLL4 binding proteins
AU2015200167A1 (en) Monoclonal antibodies against the RGM A protein and uses thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11791755

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11791755

Country of ref document: EP

Kind code of ref document: A1