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WO1995001997A1 - ANTICORPS RECOMBINANTS ET HUMANISES, DIRIGES CONTRE L'IL-1β ET DESTINES AU TRAITEMENT DE TROUBLES INFLAMMATOIRES INDUITS PAR IL-1 CHEZ L'HOMME - Google Patents

ANTICORPS RECOMBINANTS ET HUMANISES, DIRIGES CONTRE L'IL-1β ET DESTINES AU TRAITEMENT DE TROUBLES INFLAMMATOIRES INDUITS PAR IL-1 CHEZ L'HOMME Download PDF

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Publication number
WO1995001997A1
WO1995001997A1 PCT/US1994/007659 US9407659W WO9501997A1 WO 1995001997 A1 WO1995001997 A1 WO 1995001997A1 US 9407659 W US9407659 W US 9407659W WO 9501997 A1 WO9501997 A1 WO 9501997A1
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Prior art keywords
antibody
seq
sequence
light chain
amino acid
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PCT/US1994/007659
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English (en)
Inventor
Peter Ronald Young
Mitchell Stuart Gross
Zdenka Ludmila Jonak
Timothy Wayne Theisen
Mark Robert Hurle
Jeffrey Richard Jackson
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Smithkline Beecham Corporation
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Publication of WO1995001997A1 publication Critical patent/WO1995001997A1/fr

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    • 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
    • C07K16/244—Interleukins [IL]
    • C07K16/245—IL-1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/545—IL-1
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6869—Interleukin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • This invention relates generally to the field of monoclonal and recombinant, humanized antibodies, and more specifically, to antibodies directed to epitopes on the human protein, IL-l ⁇ , compositions employing these antibodies, and methods for their preparation and use.
  • Interleukin 1 is a protein mediator, or lymphokine, which is produced by macrophages, monocytes, lymphocytes and other cells, generally when such cells are themselves stimulated by various agents, e.g., endotoxins, cytokines, etc.
  • IL-1 when produced by these cells, stimulates the activities of many cells at the site of inflammation, resulting in the further activation of lymphocytes, granulocytes and fibroblasts.
  • the production of IL-1 acts as a mediator of the acute phase inflammatory response.
  • Two proteins which share IL- 1 activity are referred to as IL- 1 ⁇ and IL-l ⁇ .
  • IL-1 cytokine For a current review of the activities and uses of the IL-1 cytokine, see, e.g., C. A. Dinarello and R. C. Thompson, Immunol. Today.12:404-410 (1991); see also, March et al., Nature.115:641-647 (1985).
  • IL-l ⁇ has been cloned and found to have a precursor protein [P. Auron et al,
  • the mature IL-l ⁇ protein begins at amino acid residue 117
  • IL-1 When IL-1, and particularly IL-l ⁇ , is produced in excess quantities, it can contribute to the pathology of such disorders as septicemia, septic or endotoxic shock, chronic allergies, asthma, chronic rheumatoid arthritis, ischemia, stroke and other inflammatory disorders.
  • IL-l ⁇ antibodies have been described as useful in the treatment of such disorders. See, Wolfe Editorial, New Engl. J. Med..124(7):486-488 (1991); Wakabayashi, FASEBJ,, 5:338-343 (1991); Bone. JAMA.
  • inflammatory and other conditions in heterologous species, particularly in humans, is limited by the immune response of these species to the foreign, e.g., murine, antibody.
  • immune responses in humans against murine antibodies have been shown to both immunoglobulin constant and variable regions.
  • the invention provides a murine monoclonal antibody (mAb), SK48-E26, which is a neutralizing antibody specific for human recombinant interleukin-l ⁇ , a functional fragment such as a Fab fragment or a F(ab')2 fragment, an analog or modification thereof.
  • mAb murine monoclonal antibody
  • SK48-E26 which is a neutralizing antibody specific for human recombinant interleukin-l ⁇ , a functional fragment such as a Fab fragment or a F(ab')2 fragment, an analog or modification thereof.
  • CDR complementarity determining region
  • the present invention provides an isolated nucleic acid sequence encoding at least one CDR-encoding sequence of the present invention.
  • the nucleic acid sequence may encode an entire antibody sequence, or fragments, analogs or modifications thereof.
  • the nucleic acid sequence may be part of a recombinant plasmid and may be transformed or transfected into a host cell.
  • the invention provides the nucleotide and amino acid sequences [SEQ ID NOS: 1 and 2] of the VH and CHI regions (Fd) of the heavy chain of the cDNA clone of mAb SK48-E26 or fragments, such as the variable region and CDRs thereof, or analogs or modifications thereof.
  • the invention provides the nucleotide and amino acid sequences [SEQ ID NO: 3 and 4] of the light chain of the cDNA clone of mAb SK48- E26, fragments, such as the variable region and CDRs thereof, and analogs or modifications thereof.
  • the present invention provides a fusion protein comprising the amino acid sequence of a variable light chain and/or heavy chain of mAb SK48- E26, an anti IL-l ⁇ CDR, or a functional fragment or analog or modifications thereof, operatively linked to a selected fusion partner.
  • fusion proteins are characterized by the antigen binding specificity of the antibody from which the CDR or heavy or light chain variable region, or functional fragment or analog or modification is derived.
  • Particularly disclosed are the CDRs from the murine monoclonal antibody SK48-E26 and modifications to the heavy chain CDR region, for example, CDR3.
  • the invention provides nucleic acid sequences which encode all or a portion of the fusion proteins of this invention.
  • the present invention provides an engineered neutralizing antibody with specificity for IL-l ⁇ , in which at least parts of the CDRs in the light and/or heavy chain variable domains of an acceptor antibody have been replaced by analogous parts of CDRs from the antibody SK48-E26 described herein, which have specificity for human IL-l ⁇ .
  • the invention provides an IL-l ⁇ antibody, a Fab fragment or a F(ab')2 fragment thereof produced by screening an antibody library comprising hybridoma products and libraries from any species immunoglobulin repertoires, derived from mAb SK48-E26.
  • the invention also provides methods for producing the fusion proteins, engineered antibodies and Fab and F(ab')2 fragments described herein, including methods for producing them in selected host cells.
  • Such methods may employ other aspects of the invention, i.e., a plasmid vector containing nucleotide sequences encoding the fusion protein under the control of regulatory sequences capable of directing the replication and expression thereof in selected host cells, and host cells transfected with same.
  • Yet another aspect of this invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutic amount of at least one such fusion protein, engineered antibody, or Fab or F(ab')2 fragments described herein and a pharmaceutically acceptable carrier or diluent.
  • a method of prophylactically or therapeutically treating an IL-1 ⁇ -mediated related inflammatory condition in a human or animal in need thereof comprises administering an effective amount of at least one such fusion protein, monoclonal antibody, engineered antibody, Fab or F(ab')2 fragment or modification thereof, to such human or animal.
  • the present invention relates to a method of diagnosing IL- 1 ⁇ -mediated disorders comprising the step of contacting a biological sample from a human or animal suspected of having an IL- 1 ⁇ -mediated disorder with the protein(s) of this invention (e.g., fusion protein, monoclonal antibody, engineered antibody, Fab or F(ab')2 fragment or modification thereof)- Also embodied is a diagnostic kit for the diagnosis IL-1 ⁇ -mediated disorders.
  • the protein(s) of this invention e.g., fusion protein, monoclonal antibody, engineered antibody, Fab or F(ab')2 fragment or modification thereof.
  • Fig. 1 is a graph demonstrating the neutralizing effect of purified SK48-E26 vs. IL-l ⁇ (open circle) and IL-l ⁇ (open triangle) by bioassay. This figure shows the dose dependent inhibition of IL-l ⁇ induced IL-1 production (but not IL-l ⁇ ) by the purified antibody.
  • Fig. 2 is a graph demonstrating the results of an ELISA for human IL-l ⁇ using a rabbit anti-human IL-l ⁇ polyclonal antibody as the detection antibody and the neutralizing mAb SK48-E26 as the capture antibody.
  • the level of detection was between 0.1 to 0.2 ng/mL.
  • a linear relationship was observed between the concentration of IL-1 and the corresponding optical density reading at 405 nm in the ELISA for IL-l ⁇ .
  • Fig. 3 is a schematic drawing of plasmid pILlhzhcpCD employed to express a synthetic IL-l ⁇ heavy chain in mammalian cells.
  • the plasmid contains a beta lactamase gene (BETA LAC), an SV-40 origin of replication (S V40), a cytomegalovirus promoter sequence (CMV), a signal sequence, the synthetic variable heavy chain of SEQ ID NOS: 5 and 6, a human heavy chain constant region, a poly A signal from bovine growth hormone (BGH), a betaglobin promoter (beta glopro), a dihydrofolate reductase gene (DHFR), and another BGH sequence poly A signal in a pUC19 background.
  • BGH bovine growth hormone
  • beta glopro betaglobin promoter
  • DHFR dihydrofolate reductase gene
  • Fig. 4 is a schematic drawing of plasmid pILlhzlcpCDN employed to express the synthetic IL-l ⁇ light chain variable region of SEQ ID NOS: 7 and 8 in mammalian cells.
  • the plasmid differs from that of Fig. 3 by containing a synthetic humanized light chain variable region rather than the synthetic heavy chain, a human light chain constant region and a neomycin gene (Neo) in place of DHFR.
  • the present invention provides a variety of antibodies or fragments thereof, and fusion proteins, particularly humanized antibodies, which are characterized by IL-l ⁇ binding specificity and preferably the same or enhanced neutralizing activity as the murine monoclonal antibody (mAb) SK48-E26.
  • mAb murine monoclonal antibody
  • These products are useful in therapeutic and pharmaceutical compositions for treating IL-1 ⁇ -mediated inflammatory disorders.
  • These products are also useful in the diagnosis of an IL-l ⁇ mediated pathology by measurement, e.g., by ELISA, of circulating, endogenous IL-1 levels in vj'vo in humans.
  • first fusion partner refers to a nucleic acid sequence encoding an amino acid sequence, which can be all or pan of a heavy chain, a light chain, functional fragment thereof including the variable region from one or both chains and CDRs therefore, or analog thereof, having the antigen binding specificity of a selected antibody, preferably the IL-l ⁇ antibody, SK48-E26.
  • second fusion partner refers to another nucleotide sequence encoding a protein or peptide to which the first fusion partner is fused in frame or by means of an optional conventional linker sequence. Such second fusion partners may be heterologous to the first fusion partner.
  • a second fusion partner may include a nucleic acid sequence encoding a second antibody region of interest, e.g., all or part of an appropriate human constant region or framework region.
  • fusion molecule refers to the product of a first fusion partner operatively linked to a second fusion partner.
  • "Operative linkage" of the fusion partners is defined as an association which permits expression of the antigen specificity of the anti-IL-l ⁇ sequence (the first fusion partner) from the donor antibody as well as the desired characteristics of the second fusion partner.
  • a nucleic acid sequence encoding an amino acid linker may be optionally used, or linkage may be via fusion in frame to the second fusion partner.
  • fusion protein refers to the result of the expression of a fusion molecule in a selected host cell.
  • fusion proteins may be engineered antibodies, e.g., chimeric antibodies, humanized antibodies, or any of the antibody regions identified herein fused to other immunoglobulin or non-immunoglobulin proteins and the like.
  • donor antibody refers to an antibody (polyclonal, monoclonal, or recombinant) which contributes the nucleic acid sequences of its naturally-occurring or modified light and/or heavy chains, variable regions thereof, CDRs thereof or other functional fragments or analogs thereof to a first fusion partner, so as to provide the fusion molecule and resulting expressed fusion protein with the antigenic specificity or neutralizing activity characteristic of the donor antibody.
  • a donor antibody suitable for use in this invention is murine IL-l ⁇ antibody SK48-E26.
  • the murine IL-l ⁇ antibody SK48-E26 is defined as an antibody having the light chain DNA and amino acid sequences of SEQ ID NO: 3 and 4, and the heavy chain (VH and CHI) DNA and amino acid sequences of SEQ ID NO: 1 and 2 on a suitable murine IgG framework.
  • suitable framework regions may be identified through the literature, commercial sources, or from databases, e.g., the KABAT database, Los Alamos database, and Swiss Protein database, or isolated from an appropriate hybridoma as is described in Example 1 below.
  • acceptor antibody refers to an antibody (polyclonal, monoclonal, or recombinant) heterologous to the donor antibody, but homologous to the patient (human or animal) to be treated, which contributes all or any portion of the nucleic acid sequences encoding its heavy and/or light chain framework regions and/or its heavy and/or light chain constant regions to a second fusion partner.
  • a human antibody is an acceptor antibody.
  • CDRs are defined as the complementarity determining region amino acid sequences of an antibody which are the hypervariable regions of immunoglobulin heavy and light chains which provide the majority of contact residues for the binding of the antibody to the antigen or epitope.
  • CDRs of interest in this invention are derived from donor antibody variable heavy and light chain sequences, and include functional fragments and analogs of the naturally occurring CDRs, which fragments and analogs also share or retain the same antigen binding specificity and/or neutralizing ability as the donor antibody from which they were derived.
  • SK48-E26 may be characterized by a certain level of antigen affinity, and a CDR encoded by a nucleic acid sequence of SK48-E26 in an appropriate structural environment may have a lower or higher affinity, it is expected that CDRs of SK48-E26 in such environments will nevertheless recognize the same epitope(s) as SK48-E26.
  • a “functional fragment” is a partial CDR sequence or partial heavy or light chain variable sequence or Fab or F(ab')2 fragment which retains the same antigen binding specificity, and preferably the same neutralizing ability, as the antibody from which the fragment was derived.
  • an “analog” is an amino acid sequence altered by the deletion and/or replacement of at least one amino acid, or chemical modification of an amino acid, which alteration permits the amino acid sequence to retain the biological characteristics, e.g., antigen specificity, antigen avidity, etc., of the unmodified sequence.
  • an "allelic variation or modification” is an alteration in the nucleic acid sequence encoding the amino acid or peptide sequences of the invention. Such variations or modifications may be due to degeneracies in the genetic code or may be deliberately engineered (e.g., site-directed mutagenesis) to provide desired characteristics. When the variations or modifications do not result in the alteration of encoded amino acid sequence(s), the biological characteristics of the resultant antibody (or fusion protein or peptide), e.g., antigen specificity, antigen avidity, etc., are the same as the wild-type or unmodified sequence.
  • an “engineered antibody” describes a type of fusion protein, i.e., a synthetic antibody (e.g., a chimeric or humanized antibody) in which a portion of the light and/or heavy chain variable domains of a selected acceptor antibody are replaced by analogous parts of CDRs from one or more donor antibodies which have specificity for the selected epitope.
  • These engineered antibodies may also be characterized by alteration of the nucleic acid sequences encoding the acceptor antibody light and/or heavy variable domain framework regions in order to retain donor antibody binding specificity.
  • These antibodies can comprise immunoglobulin (Ig) constant regions and variable framework regions from the acceptor antibody, and one or more CDRs from the IL-l ⁇ donor antibody described herein.
  • a “chimeric antibody” refers to a type of engineered antibody which contains naturally-occurring variable region light chain and heavy chains (both CDR and framework regions) derived from a non-human donor antibody in association with light and heavy chain constant regions derived from a human (or other heterologous animal) acceptor antibody.
  • a “humanized antibody” refers to an engineered antibody having its CDRs and/or other portions of its light and/or heavy variable domain framework regions derived from a non-human donor immunoglobulin, the remaining immunoglobulin- derived parts of the molecule being derived from one or more human immunoglobulins.
  • Such antibodies can also include engineered antibodies characterized by a humanized heavy chain associated with a donor or acceptor unmodified light chain or a chimeric light chain, or vice versa.
  • effector agents refers to non-immunoglobulin carrier molecules to which the fusion proteins, and/or natural or synthetic light or heavy chain of the donor antibody or other fragments of the donor antibody may be associated by conventional means.
  • non-protein carriers can include conventional carriers used in the diagnostic field, e.g., polystyrene or other plastic beads, or other non-protein substances useful in the medical field and safe for administration to humans and animals.
  • Other effector agents may include a macrocycle, for chelating a heavy metal atom, or a toxin, such as ricin. Such effector agents are useful to increase the half-life of the anti-IL-l ⁇ derived amino acid sequences or to add to its properties.
  • a non-human species may be employed to generate a desirable immunoglobulin upon presentment with the active human IL-l ⁇ molecule or a peptide epitope therefrom.
  • Conventional hybridoma techniques are employed to provide a hybridoma cell line secreting a non-human monoclonal antibody (mAb) to the IL-l ⁇ protein.
  • mAb monoclonal antibody
  • the production of murine mAb SK48-E26 is described in detail in Example 1 below.
  • SK48-E26 is a desirable donor antibody for use in developing a chimeric or humanized antibody of this invention.
  • Other mAbs generated against a desired IL-l ⁇ epitope and produced by conventional techniques include without limitation, genes encoding murine mAbs, human mAbs, and combinatorial antibodies.
  • the characteristics of the neutralizing murine mAb SK48-E26 obtained as described in Example 1 include an antigen binding specificity for active, human recombinant IL-l ⁇ (hrlL-l ⁇ ) or naturally occurring IL-l ⁇ .
  • SK48-E26 binds to IL-l ⁇ at a conformational epitope of the hrlL-l ⁇ molecule which includes amino acids #95- 101 [SEQ ID NO: 11] of IL-l ⁇ , which is adjacent to the Type I receptor binding site of IL-l ⁇ [See, e.g., C. A. Dinarello and R. C. Thompson, Immunol. Today. 1.2:404- 410 (1991)]. It has been demonstrated that monoclonal antibodies which are directed against this epitope are neutralizing in vitro. The binding is illustrated by binding and functional activity (neutralization) in an in vitro assay (see Examples 2 and 3 below).
  • the isotype of the mAb SK48-E26 of Example 1 is IgG], and it has an affinity for IL-l ⁇ of between about 0.5 and 3 nM, depending on the assay employed.
  • the antibody recognizes a conformational epitope on EL-l ⁇ and does not recognize denatured IL-l ⁇ .
  • Several potential epitopes were suggested by Geysen et al, Proc. Natl. Acad. Sc ⁇ . (USA). £1:3998-4002 (1984), via scanning peptide analysis with the primary epitope from amino acids 95-101 [SEQ ID NO: 11]. See also, Geysen et al, Mol. Immunol.. 23:709-715 (1986).
  • SK48-E26 [SEQ ID NO: 3 and 4] and the heavy chain variable and the first portion of the constant region [SEQ ID NOS: 1 and 2]
  • one of skill in the art could obtain the remaining portions of the heavy chain using, for example, polymerase chain reaction, and thus obtain a complete mAb molecule.
  • a SK48-E26 molecule could be constructed using techniques analogous to those described below for the synthetic and recombinant mAbs of the invention and employing other murine IgG subtype heavy chains, e.g.,
  • IL-l ⁇ protein may elicit additional mAbs and
  • IL-l ⁇ epitope spanning amino acids #95-101 [SEQ ID NO: 11] to which SK48- E26 is responsive, and analogs thereof, is anticipated to be useful in the screening and development of additional IL-l ⁇ antibodies, which are anticipated to be similarly useful in this invention.
  • IL-l ⁇ antibodies may be developed by screening hybridomas or combinatorial libraries, or antibody phage displays [W. D. Huse et ⁇ /., Science. 24 ⁇ :1275-1281 (1988)] using the murine mAb described herein and its IL-l ⁇ epitope.
  • a collection of antibodies, including hybridoma products or antibodies derived from any species immunoglobulin repertoire may be screened in a conventional competition assay, such as described in Example 2 below, with one or more epitopes described herein.
  • the invention may provide an antibody, other than SK48-E26, which is capable of binding to and neutralizing the IL-l ⁇ molecule, more specifically to the conformational epitope containing amino acid #95- 101 [SEQ ID NO: 1 1 ] and analogs and modifications thereof.
  • This invention is not limited to the use of the SK48-E26 mAb or its hypervariable sequences. It is anticipated that any appropriate IL-l ⁇ neutralizing antibodies and corresponding anti-IL-l ⁇ CDRs described in the art may be substituted therefor. Wherever in the following description the donor antibody is identified as
  • the present invention also includes the use of Fab fragments or F(ab')2 fragments derived from mAbs directed against an epitope of IL-l ⁇ as agents protective in vivo against IL-l ⁇ mediated inflammatory diseases.
  • a Fab fragment contains the entire light chain and F , which consists of the amino terminal portion of the heavy chain (VH and CHI) and part of the hinge region.
  • a F(ab')2 fragment is the fragment formed by two Fab fragments bound by disulfide bonds.
  • MAb SK48-E26 or other similar IL-l ⁇ binding and neutralizing antibodies, provide a source of Fab fragments and F(ab')2 fragments which can be obtained by conventional means, e.g., cleavage of the mAb with the appropriate proteolytic enzymes, papain and/or pepsin, or by recombinant methods.
  • a Fab fragment of mAb SK48-E26 is provided by SEQ ID NO: 4 (light chain) and SEQ ID NO: 2 (VH + CH 1 ).
  • SEQ ID NO: 4 light chain
  • SEQ ID NO: 2 VH + CH 1 .
  • the mAb SK48-E26 or other antibodies described above may contribute sequences, such as variable heavy and/or light chain peptide sequences, framework sequences, CDR sequences, functional fragments, modifications and analogs thereof, and the nucleic acid sequences encoding them, useful in designing and obtaining various fusion proteins (including engineered antibodies) which are characterized by the antigen binding specificity of the donor antibody.
  • the present invention thus provides isolated naturally- occurring or synthetic variable light chain and variable heavy chain sequences derived from the IL-l ⁇ antibody mAb SK48-E26, as shown in SEQ ID NOS: 1-8.
  • the VH and CHI portions of the naturally-occurring heavy chain clone of SK48-E26 is characterized by the amino acid and encoding nucleic acid sequences illustrated in SEQ ID NOS: 2 and 1, respectively.
  • the nucleotide region of the heavy chain variable region spans nucleotide #226-582 of SEQ ID NO: 1.
  • the amino acid region of the heavy chain variable region spans amino acids #20 through 138 of SEQ ID NO: 2.
  • the remainder of the sequence includes some 5' untranslated sequence as well as the native signal sequence and only part of the conserved regions.
  • the variable heavy region is specifically described in Example 4, part B, below.
  • the naturally occurring light chain clone of SK48-E26 is characterized by the amino acid sequence and encoding nucleic acid sequence of SEQ ID NOS: 4 and 3, respectively. Its variable region spans nucleotides #93-413 of SEQ ID NO: 3. The amino acid region of the light chain variable region spans amino acids #21 through 127 of SEQ ID NO: 4. The remainder of the sequence includes the light chain conserved region and some 5' untranslated sequence as well as the native signal sequence. See, Example 4, part B, below. Synthetic, humanized heavy chain variable region nucleotide and amino acid sequences are illustrated in SEQ ID NOS: 5 and 6. An exemplary humanized light chain variable sequence is illustrated in SEQ LD NOS: 7 and 8.
  • the heavy chain and the light chain variable regions of SEQ ID NOS: 1-4 have three CDR sequences described in detail in Example 4.
  • the nucleic acid sequences of this invention, or fragments thereof, encoding the variable light chain and heavy chain peptide sequences or CDR peptides, or functional fragments thereof are used in unmodified form or are synthesized to introduce desirable modifications.
  • the isolated naturally-occurring or synthetic nucleic acid sequences which are derived from mAb SK48-E26 or from other desired ILl ⁇ antibodies may optionally contain restriction sites to facilitate insertion or ligation into a suitable nucleic acid sequence encoding a desired antibody framework region, ligation with mutagenized CDRs or fusion with a nucleic acid sequence encoding a selected second fusion partner.
  • variable heavy, and light chain amino acid sequences, and CDR sequences of the invention may be constructed which encode the variable heavy, and light chain amino acid sequences, and CDR sequences of the invention and functional fragments and analogs thereof which share the antigen specificity of the donor antibody.
  • the isolated or synthetic nucleic acid sequences of this invention, or fragments thereof, encoding the variable chain peptide sequences or CDRs or functional fragments thereof can be used to produce fusion proteins, chimeric or humanized antibodies, or other engineered antibodies of this invention when operatively combined with a second fusion partner.
  • sequences are also useful for mutagenic introduction of specific changes within the nucleic acid sequences encoding the CDRs or framework regions, and for incorporation of the resulting modified, or fusion, nucleic acid sequence iiito a vector for expression.
  • silent nucleotide substitutions were made in the nucleotide sequence encoding CDR2 of the human heavy and light chain variable regions described below to create restriction enzyme sites used to facilitate insertion of mutagenic framework regions. These regions were used in the construction of a humanized antibody of this invention.
  • Other modifications were made in the nucleotide sequence encoding CDR3 of the human heavy chain to create antibodies with increased affinity for ILl ⁇ . Positions 121 and 122 are the most tolerant of substitutions.
  • Positions 119-124 of SEQ ID NO: 2 were mutagenized. For antibodies with increased affinity for IL-l ⁇ , it is preferable that positions 119 and 123 are glycine, position 120 is valine and position 124 is tyrosine. Preferred modifications to position 121 include arginine (R), histidine (H) and threonine (T). Preferred modifications to position 122 include arginine (R) or lysine (K). V. Engineered Antibodies and Other Fusion Molecules
  • Fusion proteins can include engineered antibodies, chimeric antibodies, and humanized antibodies.
  • a desired fusion protein contains a first fusion partner sequence encoding a peptide having the antigen specificity and neutralizing activity of an IL- 1 ⁇ antibody such as SK48-E26 and analogs thereof, operatively linked to a second fusion partner.
  • Engineered antibodies directed against functional fragments or analogs of IL ⁇ l ⁇ may be designed to elicit enhanced binding with the same antibody.
  • the second fusion partners are defined above, and may include a sequence encoding a peptide or protein such as a second antibody region of interest. Second fusion partners may also include sequences encoding another protein or peptide to which the light or heavy chain is fused in frame or by means of a linker sequence.
  • the first fusion partners may also be associated with effector agents as defined above, including non-protein carrier molecules, to which the first fusion partner may be operatively linked by conventional means.
  • Fusion or linkage between the first fusion partners, e.g., the IL- 1 ⁇ antibody sequences, and the selected second fusion partner may be by any suitable means, e.g., by conventional covalent or ionic bonds, protein fusions, or hetero-bifunctional cross- linkers, e.g., carbodiimide, glutaraldehyde, and the like.
  • first fusion partner is associated with an effector agent, non-proteinaceous
  • conventional chemical linking agents may be used to fuse or join the anti-IL-l ⁇ sequences to the effector agent. Such techniques are known in the art and readily described in conventional chemistry and biochemistry texts.
  • linker sequences which simply provide for a desired amount of space between the fusion partners or between the first fusion partner and the effector agent may also be constructed into the fusion molecule.
  • the design of such linkers is well known to those of skill in the art.
  • fusion proteins of this invention results in fusion proteins of this invention.
  • a desired fusion protein contains an amino acid sequence of a naturally occurring heavy chain sequence of SEQ ID NO: 2, a functional fragment, modification or an analog thereof, a naturally occurring light chain sequence of SEQ ID NO: 4, a functional fragment, modification or analog thereof.
  • the functional fragments of these heavy and light chains are their respective variable regions.
  • Another exemplary fusion protein contains a synthetic variable heavy and/or light chain peptide or protein sequence having the antigen specificity and similar neutralizing activity of mAb SK48-E26, e.g., those of SEQ ID NOS: 6 and 8.
  • Still another desirable fusion protein of this invention is characterized by the amino acid .sequence containing at least one, and preferably all of the CDRs of the variable region of the heavy and/or light chains of the murine antibody molecule SK48-E26, or a functional fragment, modification or analog thereof (see Example 4, below).
  • the engineered antibody (or the other monoclonal antibodies) of the invention may have attached to it an effector agent.
  • the procedure of recombinant DNA technology may be used to produce an engineered antibody of the invention in which the F c fragment or CH3 domain of a complete antibody molecule has been replaced by an enzyme or toxin molecule.
  • An example of a fusion protein of this invention provides the anti-IL-l ⁇ sequence of the invention associated with a macrocycle, for chelating a heavy metal atom, or a toxin, such as ricin, attached to the SK48-E26 encoding nucleic acids by a covalent bridging structure.
  • the second fusion partner is a second peptide, protein or fragment thereof heterologous to the CDR-containing sequence having the antigen specificity of SK48-E26
  • the resulting fusion protein may exhibit anti-EL- 1 ⁇ antigen specificity, neutralizing activity, and the characteristic of the second fusion partner upon expression.
  • That fusion partner characteristic may be, e.g., a functional characteristic such as secretion from a recombinant host, or a therapeutic characteristic if the fusion partner is itself a therapeutic protein, or additional antigenic characteristics, if the fusion protein has its own antigen specificity.
  • Another fusion protein of this invention may comprise a complete antibody molecule, having full length heavy and light chains, or any fragment thereof, such as the Fab or F(ab')2 fragment, a heavy chain dimer, or any recombinant fragment thereof such as an F v or a single-chain antibody (SCA) or any other molecule with the same specificity, and preferably neutralizing activity, as the selected donor mAb, e.g., the mAb SK48-E26.
  • SCA single-chain antibody
  • one or more of these fragments may be used in an unfused form.
  • an engineered antibody results.
  • an engineered antibody at least fragments of the variable heavy and/or light domains of an acceptor antibody have been replaced by analogous parts of the variable light and/or heavy chains from one or more donor antibodies.
  • These engineered antibodies can comprise immunoglobulin (Ig) constant regions and variable framework regions from one source, e.g., the acceptor antibody, and one or more CDRs from the donor antibody, e.g., the IL-l ⁇ antibody described herein.
  • alteration e.g., deletions, substitutions, or additions, of the acceptor mAb light and/or heavy variable domain framework region, or the CDRs, at the nucleic acid or amino acid levels may be made in order to retain donor antibody antigen binding specificity.
  • Such engineered antibodies are designed to employ one or more of the variable heavy or light chains of the IL-l ⁇ mAb (optionally modified as described) or one or more of the below-identified heavy or light chain CDR amino acid and encoding nucleic acid sequences (see Example 4).
  • the engineered antibodies desirably block binding to the receptor of the IL-l ⁇ protein.
  • the engineered antibody is directed against a specific tertiary protein epitope of human IL-l ⁇ , which includes amino acid #95-101 [SEQ ID NO: 11], as described herein for SK48-E26.
  • Such engineered antibodies include a humanized antibody containing the framework regions of a selected human immunoglobulin or subtype, or a chimeric antibody containing the human heavy chain constant regions fused to the IL-l ⁇ antibody functional fragments.
  • a suitable human (or other animal) acceptor antibody may be one selected from a conventional database, e.g., the KABAT database, Los Alamos database, and Swiss Protein database, by homology to the nucleotide and amino acid sequences of the donor antibody.
  • a human antibody characterized by a homology to the framework regions of the donor antibody (on an amino acid basis) may be suitable to provide a heavy chain constant region and/or a heavy chain variable framework region for the insertion of the donor CDRs.
  • acceptor antibody chain A suitable acceptor antibody capable of donating light chain constant or variable framework regions (i.e., acceptor antibody chain) may be selected in a similar manner.
  • acceptor antibody heavy and light chains are not required to originate from the same acceptor antibody.
  • heterologous framework and constant regions are selected from human immunoglobulin classes and isotypes, such as IgG (subtypes 1 through 4), IgM, IgA, and IgE.
  • the acceptor antibody need not comprise only human immunoglobulin protein sequences. For instance a gene may be constructed in which a DNA sequence encoding part of a human immunoglobulin chain is fused to a DNA sequence encoding the amino acid sequence of a polypeptide effector or reporter molecule.
  • One example of a particularly desirable humanized antibody contains all or a portion of the variable domain amino acid sequences of SK48-E26 and some portions of the donor antibody framework regions, or CDRs therefrom inserted onto the framework regions of a selected human antibody.
  • This umanized antibody is directed against an IL-l ⁇ epitope, preferably the epitope containing amino acids 95-101 [SEQ ID NO: 11] of huIL-l ⁇ .
  • one, two or preferably three CDRs from the ILl ⁇ antibody heavy chain and/or light chain variable regions are inserted into the framework regions of a selected human antibody, replacing the native CDRs of that latter antibody.
  • variable domains in both human heavy and light chains have been engineered by one or more CDR replacements. It is possible to use all six CDRs, or various combinations of less than the six CDRs. For example, it is possible to replace the CDRs only in the human heavy chain, using as light chain the unmodified light chain from the human acceptor antibody. Still alternatively, a compatible light chain may be selected from another human antibody by recourse to the conventional antibody databases. The remainder of the engineered antibody may be derived from any suitable acceptor human immunoglobulin.
  • the engineered humanized antibody thus preferably has the structure of a natural human antibody or a fragment thereof, and possesses the combination of properties required for effective therapeutic use, e.g., treatment of IL- 1 ⁇ mediated inflammatory diseases in man.
  • an engineered antibody may contain three CDRs of the variable light chain region of SK48-E26 or a functional fragment thereof in place of at least a part of the light chain variable region of an acceptor mAb, and three CDRs of the variable heavy chain region of SK48-E26 or a functional fragment thereof in place of at least a part of the heavy chain variable region of an acceptor mAb, such as a human antibody.
  • the resulting humanized antibody is characterized by the antigen binding specificity of mAb SK48-E26. It will be understood by those skilled in the art that an engineered antibody may be further modified by changes in variable domain amino acids without necessarily affecting the specificity of the donor antibody.
  • a fusion protein which is a chimeric antibody differs from the humanized antibodies described above by providing the entire non-human donor antibody heavy chain and light chain variable regions, including framework regions, e.g., amino acids 20 to 138 of SEQ ID NO: 2 and amino acids 21 to 127 of SEQ ID NO: 4, in association with human immunoglobulin constant regions for both chains. It is anticipated that chimeric antibodies which retain additional non-human sequence in comparison to humanized antibodies of this invention may also elicit immune response in a human.
  • Such engineered antibodies are effective in the prevention and treatment of IL- 1 mediated inflammatory disorders in humans and other animals.
  • Humanized, monoclonal antibodies which neutralize the biological activity of endogenous IL- 1 ⁇ are useful therapeutically or prophylactically for such IL-l ⁇ mediated disorders.
  • Such disorders include those identified above, such as septic shock, arthritis and the like.
  • the fusion proteins and engineered antibodies of the invention will be produced by recombinant DNA technology using genetic engineering techniques.
  • the same or similar techniques may also be employed to generate other embodiments of this invention, e.g., to construct the chimeric or humanized antibodies, the synthetic light and heavy chains, the CDRs, the Fabs, and the nucleic acid sequences encoding them, as above mentioned.
  • compositions of this invention is set out in Example 1 below using the CDRs of murine SK48-E26 and one or more selected human antibody light and heavy chain framework regions.
  • This exemplary humanized antibody is characterized by the humanized heavy chain variable and light chain variable sequences reported in SEQ ID NOS: 5 through 8. Briefly described, a hybridoma producing the murine antibody SK48-E26 is conventionally cloned, and the cDNA of its heavy and light chain variable regions obtained by techniques known to one of skill in the art, e.g., the techniques described in Sambrook et al., Molecular Cloning (A Laboratory Manual).2nd edition, Cold Spring Harbor Laboratory (1989). The variable regions of the SK48-E26 are obtained using polynucleotide primers and reverse transcriptase. The CDRs are identified using a known database and by comparison to other antibodies.
  • Homologous framework regions of a heavy chain variable region from a human antibody were identified using computerized databases, e.g., KABAT, and a human antibody having homology to SK48-E26 was selected as the acceptor antibody.
  • the sequences of synthetic heavy chain variable regions containing the SK48-E26 CDRs within the human antibody frameworks are designed with optional nucleotide replacements in the framework regions to incorporate restriction sites [SEQ ID NOS: 5 and 6]. This designed sequence is then synthesized by overlapping oligonucleotides, amplified by polymerase chain reaction (PCR), and corrected for errors.
  • a suitable light chain variable framework region [SEQ ED NOS: 7 and 8] was designed in a similar manner.
  • variable light and/or heavy chain sequences and the CDRs of mAb SK48-E26, and their encoding nucleic acid sequences are utilized in the construction of fusion proteins and engineered antibodies, preferably humanized antibodies, of this invention, by the following process.
  • a DNA sequence is obtained which encodes the donor antibody variable heavy or light chain regions containing at least the CDRs and those minimal portions of the acceptor mAb light and/or heavy variable domain framework region required in order to retain donor mAb binding specificity, as well as the remaining immunoglobulin-derived parts of the antibody chain derived from a human immunoglobulin.
  • a conventional expression vector or recombinant plasmid is produced by placing these coding sequences for the fusion protein in operative association with conventional regulatory control sequences capable of controlling the replication and expression in, and/or secretion from, a host cell.
  • regulatory sequences may be readily selected by one of skill in the art and are not intended as a limitation of the present invention.
  • Regulatory sequences include promoter sequences, e.g., CMV promoter, and signal sequences (e.g., SEQ ID NOS: 9 and 10), which can be derived by one of skill in the art from antibodies.
  • a second expression vector is produced having a DNA sequence which encodes a complementary antibody light or heavy chain.
  • this second expression vector is identical to the first except in so far as the coding sequences and selectable markers are concerned so to ensure as much as possible that each polypeptide chain is functionally expressed.
  • a single vector of the invention may be used, the vector including the sequence encoding both light chain and heavy chain-derived polypeptides.
  • the DNA in the coding sequences for the light and heavy chains may comprise cDNA or genomic DNA or both.
  • a selected host cell is co-transfected by conventional techniques with both the first and second vectors or simply transfected by a single vector to create the transfected host cell of the invention comprising both the recombinant or synthetic light and heavy chains.
  • the transfected cell is then cultured by conventional techniques to produce the engineered antibody of the invention.
  • the humanized antibody which includes the association of both the recombinant heavy chain and/or light chain is screened from culture by appropriate assay, such as the ELISA assay described in Example 2 below. Similar conventional techniques may be employed to construct other fusion proteins and molecules of this invention.
  • Suitable vectors for the cloning and subcloning steps employed in the methods and construction of the compositions of this invention may be selected by one of skill in the art.
  • the conventional pUC series of cloning vectors may be used.
  • One vector used is pUC19, which is commercially available from supply houses, such as Amersham (Buckinghamshire, United Kingdom) or Pharmacia (Uppsala, Sweden).
  • any vector which is capable of replicating readily, has an abundance of cloning sites and marker genes, and is easily manipulated may be used for cloning.
  • the selection of the cloning vector is not a limiting factor in this invention.
  • the vectors employed for expression of the engineered antibodies according to this invention may be selected by one of skill in the art from any conventional vector.
  • the vectors also contain selected regulatory sequences which are in operative association with the DNA coding sequences of the immunoglobulin regions and capable of directing the replication and expression of heterologous DNA sequences in selected host cells, such as CMV promoters.
  • These vectors contain the above described DNA sequences which code for the engineered antibody or fusion molecule.
  • the vectors may incorporate the selected immunoglobulin sequences modified by the insertion of desirable restriction sites for ready manipulation.
  • the expression vectors may also be characterized by marker genes suitable for amplifying expression of the heterologous DNA sequences, e.g., the mammalian dihydrofolate reductase gene (DHFR) or neomycin resistance gene (neo ).
  • marker genes suitable for amplifying expression of the heterologous DNA sequences e.g., the mammalian dihydrofolate reductase gene (DHFR) or neomycin resistance gene (neo ).
  • Other preferable vector sequences include a poly A signal sequence, such as from bovine growth hormone (BGH) and the betaglobin promoter sequence (betaglopro).
  • BGH bovine growth hormone
  • betaglopro betaglobin promoter sequence
  • replicons e.g. replicons, selection genes, enhancers, promoters, signal sequences and the like
  • selection genes e.g. replicons, selection genes, enhancers, promoters, signal sequences and the like
  • Other appropriate expression vectors of which numerous types are known in the art for mammalian, bacterial, insect, yeast, and fungal expression may also be selected for this purpose.
  • Two exemplary expression vectors employed in the following examples for expression of the IL-l ⁇ fusion proteins of this invention are the mammalian vectors illustrated in Figs. 3 and 4. However, this invention is not limited to the use of these illustrative vectors.
  • the present invention also encompasses a cell line transfected with a recombinant plasmid containing the coding sequences of the engineered antibodies or fusion molecules hereof.
  • Host cells useful for the cloning and other manipulations of these cloning vectors are also conventional. However, most desirably, cells from various strains of E. coli are used for replication of the cloning vectors and other steps in the construction of fusion proteins of this invention.
  • Suitable host cells or cell lines for the expression of the engineered antibody or fusion protein of the invention are preferably a eukaryotic cell, and most preferably a mammalian cell, such as a CHO cell or a myeloid cell.
  • Other primate cells may be used as host cells and, most desirably, human cells are used, thus enabling the molecule to be modified with human glycosylation patterns.
  • other eukaryotic cell lines may be employed.
  • the selection of suitable mammalian host cells and methods for transformation, culture, amplification, screening and product production and purification are known in the art. See, e.g., Sambrook et al., cited above.
  • Bacterial cells may prove useful as host cells suitable for the expression of the recombinant mAbs of the present invention.
  • any recombinant mAb produced in a bacterial cell would have to be screened for retention of antigen binding ability. If the molecule expressed by the bacterial cell was produced in a properly folded form, that bacterial cell would be a desirable host.
  • various strains of E. coli used for expression are well- known as host cells in the field of biotechnology.
  • Various strains of B. subtilis, Streptomyces, other bacilli and the like may also be employed in this method.
  • strains of yeast cells known to those skilled in the art are also available as host cells, as well as insect cells (e.g., S2, Sf9) and viral expression systems. See, e.g. Miller et al., Genetic Engineering. £:277-298, Plenum Press (1986) and references cited therein.
  • the general methods by which the vectors of the invention may be constructed transfection methods required to produce the host cells of the invention, and culture methods necessary to produce the fusion protein or engineered antibody of the invention from such host cell are all conventional techniques.
  • the fusion proteins or engineered antibodies of the invention may be purified from the cell culture contents according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like. Such techniques are within the skill of the art and do not limit this invention.
  • Yet another method of expression of the humanized antibodies may utilize expression in a transgenic animal, such as described in U. S. Patent No. 4,873,316.
  • This relates to an expression system using the animal's casein promoter which when transgenically incorporated into a mammal permits the female to produce the desired recombinant protein in its milk.
  • the engineered antibody is then examined for in vitro activity by use of an appropriate assay.
  • an appropriate assay Presently conventional ELISA assay formats are employed to assess qualitative and quantitative binding of the engineered antibody to the IL-l ⁇ epitope.
  • variable region frameworks can be produced with variable region frameworks potentially recognized as "self by recipients of the engineered antibody. Modifications to the variable region frameworks can be implemented to effect large increases in antigen binding without appreciable increased immunogenicity for the recipient.
  • engineered antibodies can effectively treat a human for IL- 1 mediated inflammatory diseases. Such antibodies may also be useful in the diagnosis of such diseases.
  • This invention also relates to a method of treating humans experiencing an IL ⁇ l ⁇ mediated inflammatory disorder which comprises administering an effective dose of antibodies including one or more of the engineered antibodies or fusion proteins described herein, or fragments thereof.
  • the therapeutic response induced by the use of the engineered antibodies of this invention is produced by the binding of the immunoglobulin to the IL-l ⁇ molecule and the subsequent sequestering and/or clearance of this bound complex.
  • the engineered antibodies of the present invention when in preparations and formulations appropriate for therapeutic use, are highly desirable for those persons experiencing an IL-l ⁇ mediated inflammatory response, such as an allergy, rheumatoid arthritis, septic or endotoxic shock, septicemia, asthma, graft versus host disease, Crohn's disease, and other inflammatory bowel diseases.
  • the fusion proteins, antibodies, engineered antibodies, modifications or fragments thereof of this invention may also be used in conjunction with other antibodies, particularly human mAbs reactive with other markers (epitopes) responsible for the disease against which the engineered antibody of the invention is directed.
  • mAbs reactive with epitopes responsible for the disease in a selected animal against which the antibody of the invention is directed may also be employed in veterinary compositions. Any antibody that is capable of operating without interfering with the ILl ⁇ antibody of this invention, e.g., an antibody to other IL- l ⁇ epitopes, an antibody to IL-l ⁇ epitopes, etc. is useful in these compositions.
  • the therapeutic agents of this invention are believed to be desirable for treatment of inflammatory conditions for from about 2 days to about 3 weeks, or as needed. This represents a considerable advance over the currently used infusion protocol with prior art treatments of IL-l ⁇ mediated disorders, such as septic shock.
  • This duration of treatment relates to the relative duration of the recombinant antibodies of the present invention in the human circulation.
  • the mode of administration of the therapeutic agent of the invention may be any suitable route which delivers the agent to the host.
  • the fusion proteins, antibodies, engineered antibodies, and fragments thereof, and pharmaceutical compositions of the invention are particularly useful for parenteral administration, i.e., subcutaneously, intramuscularly or intravenously. However, the agent is preferably administered by i.v. injection, depending on the condition treated.
  • Therapeutic agents of the invention may be prepared as pharmaceutical compositions containing an effective amount of the engineered or chimeric antibody of the invention as an active ingredient in a nontoxic and sterile pharmaceutically acceptable carrier.
  • aqueous suspension or solution containing the engineered antibody preferably buffered at physiological pH, in a form ready for injection is preferred.
  • the compositions for parenteral administration will commonly comprise a solution of the engineered antibody of the invention or a cocktail thereof dissolved in an acceptable carrier, preferably an aqueous carrier.
  • aqueous carriers may be employed, e.g., water, buffered water, 0.4% saline, 0.3% glycine, and the like. These solutions are sterile and generally free of paniculate matter.
  • compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, etc.
  • concentration of the antibody of the invention in such pharmaceutical formulation can vary widely, i.e., from less than about 0.5%, usually at or at least about 1% to as much as 15 or 20% by weight and will be selected primarily based on fluid volumes, viscosities, etc., according to the particular mode of administration selected.
  • a pharmaceutical composition of the invention for intramuscular injection could be prepared to contain 1 mL sterile buffered water, and between about 50 to about 100 mg of an engineered antibody of the invention.
  • a pharmaceutical composition of the invention for intravenous infusion could be made up to contain about 250 ml of sterile Ringer's solution, and about 150 mg of an engineered antibody of the invention.
  • Actual methods for preparing parenterally administrable compositions are well known or will be apparent to those skilled in the art and are described in more detail in, for example, Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pennsylvania.
  • the therapeutic agent of the invention when in a pharmaceutical preparation, be present in unit dose forms.
  • the appropriate therapeutically effective dose can be determined readily by those of skill in the art.
  • one dose of approximately 1 mg/kg to approximately 20 mg/kg of a protein or an antibody of this invention should be administered parenterally, preferably i.v. (intravenously).
  • Such dose may, if necessary, be repeated at appropriate time intervals during the inflammatory response.
  • the invention also encompasses the administration of the IL-l ⁇ fusion proteins of this invention concurrently or sequentially with other antibodies or fusion proteins characterized by anti-EL-l ⁇ activity, anti-tumor necrosis factor activity or other pharmaceutical activities compatible with the IL-l ⁇ receptor binding ability of the fusion proteins of this invention.
  • other antibodies are available commercially or can be designed in a manner similar to that described herein.
  • the fusion proteins and engineered antibodies of this invention may also be used in diagnostic regimens, such as for the determination of IL-l ⁇ mediated disorders or teaching progress of treatment of such disorders.
  • diagnostic regimens such as for the determination of IL-l ⁇ mediated disorders or teaching progress of treatment of such disorders.
  • these fusion proteins may be conventionally labeled for use in ELISA's and other conventional assay formats for the measurement of IL- 1 levels in serum, plasma or other appropriate tissue.
  • the nature of the assay in which the fusion proteins are used are conventional and do not limit this disclosure.
  • the antibodies, engineered antibodies or fragments thereof described herein can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional immunoglobulins and art- known lyophilization and reconstitution techniques can be employed.
  • Single or multiple administrations of the pharmaceutical compositions can be carried out with dose levels and pattern being selected by the treating physician.
  • the pharmaceutical composition of the invention should provide a quantity of the engineered antibodies of the invention sufficient to effectively treat the patient.
  • mice Females were immunized with E. cr / -expressed human recombinant IL- 1 ⁇ protein [C. A. Meyers et al, J. Biol. Chem.. 262: 11176-11181
  • the immunization schedule is described in Table 1 below.
  • Various amounts of protein were injected into mice via i.p., s.c. and i.v. routes.
  • the antigen was presented in different types of adjuvants to maximize the immune stimulation.
  • the immunization protocol varied for each injection, since simple immunization procedures were not successful in generating neutralizing IL- 1 ⁇ monoclonal antibodies. Over 108 mAbs against hrlL-l ⁇ were generated with binding properties. However, they did not have neutralizing activities.
  • mice were injected with a combination of adjuvants, e.g., Incomplete Freunds Adjuvant, Complete Freunds Adjuvant, and monophosphoryl lipid A (MPL) + TDM emulsion (0.5 mg each in 2% oil-Tween-80- H2O) (Catalog #060-130; Ribi Immunochem, Hamilton, MT) and via different routes.
  • adjuvants e.g., Incomplete Freunds Adjuvant, Complete Freunds Adjuvant, and monophosphoryl lipid A (MPL) + TDM emulsion (0.5 mg each in 2% oil-Tween-80- H2O) (Catalog #060-130; Ribi Immunochem, Hamilton, MT)
  • MPL monophosphoryl lipid A
  • mice Three mice were sacrificed on Day 76 and single cell suspensions were o prepared from their spleens. A total of 1.7 x 10 spleen cells were fused with 1.7 x
  • Antibody-secretion was detected in supematants from the hybridomas of Example 1 in an ELISA measuring binding to hrIL- l ⁇ 1 -25 days after fusion.
  • a standard ELISA as essentially described by R. H. Kennett "Enzyme linked antibody assay with cells attached to polyvinyl chloride plates" in Monoclonal Antibodies. Hybridomas: A New Dimension in Biological Analyses. T. J. McKearn and K. B. Bechtol, eds., Plenum Press, NY, pp. 376-377 (1983) with the following modifications.
  • the ELISA plates were coated with hrIL- 1 ⁇ , 100 ⁇ L/well were suspended in 0.1 M sodium bicarbonate buffer (pH 9.6) at a concentration of 5 ⁇ g/mL and the o-phenylenediamine substrate was incubated for 10 minutes at room temperature.
  • hybridoma clone designated as SK48-E26 was identified as a clone which produced mAbs with positive binding to IL-l ⁇ in the ELISA assay of Example 2 and this neutralizing (EL-4 bioassay) activity (Fig. 1).
  • the original hybridoma was subcloned twice (by limited dilution method) in order to assure monoclonality and also shows the activity described above.
  • the characteristics of the neutralizing monoclonal antibody SK48-E26 include a specificity for human recombinant E -l ⁇ , as determined in these assays. Its isotype is IgGi, and it has an affinity of between about 1 and 3 nM, depending on the way that affinity is measured.
  • SK48-E26 presumably bound to the conformational epitope which includes amino acids #95-101 of the hrIL- 1 ⁇ molecule (MEKRFVF SEQ ID NO: 11 ), which was identified as the major epitope by Geysen mapping.
  • This hybridoma produced on average 60-70 ⁇ g/mL of specific IgGi antibodies from confluent cultures (T75 flasks, HY medium, 13% FCS). In serum-free medium the cells produced approximately 25 ⁇ g/mL.
  • a humanized version of the IL-l ⁇ specific mouse antibody, SK48-E26 was prepared by performing the following manipulations. Briefly, cDNA clones were made of the SK48-E26 heavy and light chains (Part A). DNA sequencing thereof determined that these clones were antibody genes (Part B). A mouse/human chimeric antibody was then prepared and assayed for binding ability (Part C). To prepare a humanized antibody, human framework regions were selected and CDRs from the SK48-E26 heavy and light chains were inserted appropriately within the selected heavy and light chain framework. There may be subsequent manipulation of framework residues (Part D). The resulting humanized antibody was expressed in mammalian cells, e.g., COS cells (Part E).
  • mammalian cells e.g., COS cells
  • cDNA Cloning cDNA clones were made of the SK48-E26 heavy and light chains from mRNA extracted out of the SK48-E26 hybridoma cell line [SmithKline Beecham] using a Boehringer Mannheim kit. Primers specific for either the mouse heavy chain hinge region or kappa constant region were used for first strand synthesis.
  • the kappa chain primer is [SEQ ID NO: 12]: 5' CTAACACTCATTCCTGTTGAAGCTCTTGACAATGGG 3'.
  • the heavy chain primer is [SEQ ID NO: 13]: 5' GTACATATGCAAGGCTTACAACCACAATC 3'.
  • the double stranded cDNA was cloned directly into plasmids pGEM7
  • a cloning adaptor spans nucleotides #1-14.
  • the 5' untranslated region spans nucleotides #15-168.
  • the naturally occurring variable heavy chain signal sequence spans nucleotides #169-225 and amino acids #1 to 19.
  • Framework region 1 spans nucleotides #226-315, and amino acids #20-49.
  • the first CDR spans nucleotides #316-330, amino acids #50-54 (SYDMS).
  • the second framework region spans nucleotides #331-372, amino acids #55-68.
  • the second CDR spans nucleotides #373-423, amino acids #69-85 (YISSGGGGTYYPDTVKG).
  • the third framework region spans nucleotide #424- 519, amino acids #86-117.
  • the third CDR spans nucleotides #520-549, amino acids #118-127 (GGVRRGYFDV).
  • the fourth framework region spans nucleotide #550- 582, amino acids #128-138.
  • a portion of the heavy chain constant region spans nucleotides #583-909, including amino acids #139-247.
  • a cloning adaptor spans nucleotides #910-923.
  • a cloning adaptor spans nucleotides #1-14.
  • the 5' untranslated region spans nucleotides #15-32.
  • the signal sequence spans nucleotides #33-92, and amino acids #1 to 20 thereof.
  • Framework region 1 spans nucleotides #93-161, amino acids #21-43.
  • the first CDR spans nucleotides #162-194, amino acids #44-54 (RASGNIHNYLT).
  • the second framework region spans nucleotides #195-239, amino acids #55-69.
  • the second CDR spans nucleotides #240-260, amino acids #70-76 (NAKTLAD).
  • the third framework region spans nucleotide #261-356, amino acids #77- 108.
  • the third CDR spans nucleotides #357-383, amino acids #109-117 (QHFWSIPYT).
  • the fourth framework region spans nucleotide #384-413, amino acids #118-127.
  • the light chain constant region spans nucleotides #414-737 and includes amino acids #128-234.
  • a cloning adaptor spans nucleotides #738-751.
  • DNA coding for the native signal sequence and variable region of the heavy and light chain of SK48-E26 was ligated to a human IgGi constant region or human kappa constant regions contained within plasmids pCD and pCDN (Figs. 3 and 4) designed for expression in mammalian cells. These plasmids were then co-transfected by conventional means and expressed in COS cells.
  • ELISA plates were coated with 0. l ⁇ g/well of a goat antibody specific for the Fc region of human antibodies. The media supernatants were added for one hour. A horseradish peroxidase conjugated goat antibody specific for an entire human IgG antibody was added. This was followed by addition of ABTS peroxidase substrate (Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD) for one hour. O.D. was measured using a THERMO MAX microplate reader (Molecular Devices Corporation, Menlo Park, California) at 405 nm.
  • SK48-E26 heavy and light chains For each of the SK48-E26 heavy and light chains, six to twelve human antibody chains with high overall amino acid sequence homology to the SK48-E26 heavy and light chains were selected from the KABAT® database. The best match of these was then detected using a method that weighs the impact that changes in the framework residues may have on the structure of a CDR.
  • a synthetic heavy chain variable region was designed to replace the naturally occurring CDRs of the human framework sequence with the CDRs of SK48-E26. In designing the sequence, conservative nucleotide replacements were placed in the framework regions to introduce selected restriction sites suitable for enzymatic cleavage.
  • the human heavy chain was described in the reference I. W. Schmidt, et al., "Die Primarfigured des kristallisierbaren monoklonalen Immunoglobulins IgG 1 KOL", Hoppe-Seyler's Z. Phvsioi. Chem.. Number 364, p. 713 (1983).
  • the light chain was published in W. Palm and H. Hilschmann, "Die Primar Modell für kristallinen monoklonalen Immunoglobulin-L-Kette vom k-Typ, Subteil I (Bence- Jones-Portein Rei.) ein Beitrag zur Aufklarung der dreidimensionalen Struktur der Immunoglobuline.”, Hoppe-Sever's Z. Phvsioi. Chem.. Number 354. p. 1651 (1973).
  • the framework regions of these human antibodies were determined to be approximately 74% and 76% identical in amino acid sequence to the SK48-E26 variable heavy chain and light chain framework regions, respectively.
  • the humanized variable regions were synthesized using a combination of overlapping oligonucleotides and PCR amplification. Any errors in the designed sequences which were inserted by PCR were corrected.
  • the humanized heavy chain and light chain variable region DNA and protein sequences are shown in SEQ ID NOS: 5 through 8.
  • Mutagenesis (random and site-directed) of SK48-E26 was performed as described below. Briefly, DNA encoding the antibody light chain and antibody Fd chain (comprising the heavy-chain variable region and heavy-chain constant region domain 1 (CHi)) of SK48-E26 was inserted into a phage display vector. The CDR3 region of the heavy chain of SK8-E26 was modified and antibodies with increased affinity for ILl ⁇ were further selected for characterization.
  • CHi heavy-chain variable region and heavy-chain constant region domain 1
  • the vector pMK or pComb 3 [Barbas et al., Proc. Natl. Acad. Sci. H£A ££:7978-7982 (1991)] can be used for cloning, periplasmic expression, and Ml 3 phage display of Fab antibody fragments.
  • the light chain DNA from SK48-E26 was PCR amplified using the following primers [SEQ ID NO: 14 and 15, respectively]:
  • the underlined region is a Sst I site.
  • the underlined region is a Xba I site.
  • the PCR fragment was digested with Sst I and Xba I, followed by isolation of the digested product from an acrylamide gel. The fragment was then ligated into Sst I,
  • Heavy chain The heavy chain DNA from SK48- ⁇ 26 was PCR amplified using the following primers [SEQ ID NO: 16 and 17, respectively]: 5' CTCCGCGTCGACCTCQAGTCTGGGGGAGGC 3' The underlined region is a Xho I site. 5' CTCCGCGTCGACACTAG1ACAATCCCTGGGCAC 3' The underlined region is an Spe I site.
  • the PCR fragment was digested with Xho I and Spe I, followed by isolation of the digested product from an acrylamide gel. The fragment was then ligated into Xho I, Spe I digested pMK vector DNA (already containing the SK48-E26 light chain), and the ligation reaction was used to transform E. coli XL-1 blue by electroporation. Insertion of the SK48-E26 heavy chain (Fd) fragment was confirmed by restriction digestion.
  • the resultant plasmid (pMK-SK48) codes for an SK48-E26 heavy chain in which the amino-terminal end is fused to the pel B leader sequence, and the carboxyl- terminal end is fused to the Ml 3 gene III anchor domain.
  • a library of CDR3H mutants was created using the following oligos as PCR primers. 5' GGTTGAGGCAGGTCAGACGATTGGC 3' [SEQ ID NO: 19] (anneals within the gene III region of pMK) and
  • the mutagenesis oligo was synthesized using a codon based strategy [Glaser et al., ⁇ Immunol..142:3903-3913 (1992)] in which there is a 50% chance that the targeted codon will not be wild-type.
  • the targeted codons were chosen on the basis of previous mutagenesis experiments (Table 2) demonstrating they were tolerant of substitutions. Table 2
  • the PCR fragment was digested with Xma I, and Spe I, ligated into pMK-SK48 vector DNA digested with the same enzymes and used to transform E. coli XL-1 blue. This was followed by expression of the Fab in the presence of VCSM13 helper phage which results in the library of Fabs being displayed on phage particles.
  • This library is random with respect to amino acid identity at positions 101, 102, 103, and 105 of the heavy chain (positions 120, 121, 122, and 124 of SEQ. ID NO: 1 or 2).
  • the library was subjected to a Bio-panning selection process [Parmley and Smith, Gene.22:305-318 (1988)] in 96 well microtiter plates coated with a range of 200 ng to 25 ng of IL-l ⁇ per well.
  • Several variations of the selection process were performed including elution with acid, elution with soluble IL-l ⁇ (2.4 mM), selection in the presence of a 10 fold molar excess of wild-type soluble SK48 Fab, and selection on IL-l ⁇ conjugated agarose beads. Each variation is designed to select those phage with the highest affinity for IL-l ⁇ .
  • Soluble expression is facilitated by the removal of the gene III fusion from the heavy chain [Barbas et al., Proc. Natl. Acad. Sci. USA. £8:7978-7982 (1991)].
  • Several mutants were characterized for their ability to bind IL-l ⁇ in solution via an ELISA assay [Friguet et al., J. Immunol. Methods. 77:305-319 (1985)].
  • Purified Fab was mixed with various concentrations of IL-l ⁇ and allowed to reach equilibrium. Subsequently the amount of free Fab remaining was measured via an IL-l ⁇ mediated capture ELISA, and this value was used to calculate the amount of Fab and IL-l ⁇ in complex at equilibrium.
  • MOLECULE TYPE DNA (genomic)
  • FEATURE FEATURE
  • CCCAGTTCCT CACGTTCAGT GATGAGCACT GAACACAGAC ACCTCACC ATG AAC TTT 177
  • GCA AGA GGG GGG GTA CGA CGA GGG TAC TTC GAT GTC TGG GGC GCA GGG 561 Ala Arg Gly Gly Val Arg Arg Gly Tyr Phe Asp Val Trp Gly Ala Gly 120 125 130
  • AGC ACC AAG GTG GAC AAG AAA ATT GTG CCC AGG GAT TGT GGT TGT AAG 897 Ser Thr Lys Val Asp Lys Lys lie Val Pro Arg Asp Cys Gly Cys Lys 230 235 240
  • MOLECULE TYPE DNA (genomic)
  • Trp Lys lie Asp Gly Ser Glu Arg Gin Asn Gly Val Leu Asn Ser Trp 170 175 180
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

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Abstract

L'invention se rapporte à des anticorps monoclonaux et recombinants, chimères et humanisés, dirigés contre des épitopes se trouvant sur la protéine humaine Interleukine-1β (IL-1β), à des compositions utilisant ces anticorps, ainsi qu'à des procédés destinés à leur préparation et à leur utilisation.
PCT/US1994/007659 1993-07-09 1994-07-07 ANTICORPS RECOMBINANTS ET HUMANISES, DIRIGES CONTRE L'IL-1β ET DESTINES AU TRAITEMENT DE TROUBLES INFLAMMATOIRES INDUITS PAR IL-1 CHEZ L'HOMME WO1995001997A1 (fr)

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US9053493A 1993-07-09 1993-07-09
US08/090,534 1993-07-09
US20619094A 1994-03-04 1994-03-04
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