WO2001012646A1 - Anticorps d'agonistes et d'antagonistes de sialoadhesine facteur-1 - Google Patents

Anticorps d'agonistes et d'antagonistes de sialoadhesine facteur-1 Download PDF

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Publication number
WO2001012646A1
WO2001012646A1 PCT/US2000/022663 US0022663W WO0112646A1 WO 2001012646 A1 WO2001012646 A1 WO 2001012646A1 US 0022663 W US0022663 W US 0022663W WO 0112646 A1 WO0112646 A1 WO 0112646A1
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antibody
immunoglobuhn
seq
cdr
polypeptide
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PCT/US2000/022663
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English (en)
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Connie L. Erickson-Miller
Stephen D. Holmes
Andrew G. King
Alexander H. Taylor
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Smithkline Beecham Corporation
Smithkline Beecham Plc
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Publication of WO2001012646A1 publication Critical patent/WO2001012646A1/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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates to agonist and antagonist monoclonal antibodies (mAbs) that bind to staloadhesin factor- 1 (SAF- 1) and to the use of such antibodies for therapeutic purposes
  • the staloadhesin family of proteins consisting of sialoadhesin, CD22, CD33 and myelin associated glycoprotein (MAG), are also known as the I-type lectins or siglecs (siahc acid-binding Ig-like lectins) These proteins mediate, among other things, sialic acid-dependent interactions with other cells and, at least in the case of CD22, signal through SH2 binding domains
  • Thev belong to the immunoglobuhn (Ig) supergene family and have an N-terminal V-set Ig domain followed by 1- 16 C2-set Ig domains
  • the V-set and C2-set Ig domains vary in the number and pairing of cysteines although the overall three dimensional structure is nearly the same
  • SAF-1 sialoadhesin family members
  • SAF-4 Several new sialoadhesin family members, called SAF-1 through SAF-4, have been disclosed in International Patent Application. Publication No WO 98/06733, published February 19, 1998 (SAF-1), EP Application No 98302537 1 (SAF-2), EP Application No 98302527 1 (SAF-3), and International Patent Application PCT US98/10791 , published December 3, 1998 (SAF-4)
  • the new clones have the highest amino acid simila ⁇ tv to CD33 (from 55-68%)
  • the cytoplasmic portion of the novel SAFs have the immunoreceptor tyrosine-based activation and inhibition motifs (IT AM and ITIM) therefore it is hkelv they participate in signaling and may regulate cellular responses to certain stimuli
  • SAF- 1 having the identifying characteristics of monoclonal antibody 13G3 or 1 1G4
  • Another aspect to the present invention includes a method for treating or preventing infectious disease states in a mammal comprising administering to a subject in need thereof an effective dose of a S AF- 1 agonist antibody having the identifying characte ⁇ stics of monoclonal antibody 13G3 or 1 1G4
  • Yet another aspect of the present invention includes a pharmaceutical composition comprising a monoclonal antibody against human SAF-1 having the identifying characteristics of monoclonal antibody 13G3 or 1 1G4
  • Figure 2 shows the V ⁇ cDNA sequence and the predicted amino acid sequence of the ant ⁇ -SAF- 1 mAb, 13G3 (SEQ ID NOs 6 and 7, respectively) The bolded residues indicate the three CDRs (SEQ ID NOs 8, 9, and 10)
  • Figure 3 shows the Vfj cDNA sequence and the predicted amino acid sequence of the ant ⁇ -SAF-1 mAb, 1 1G4 (SEQ ID NOs 11 and 12, respectively) The bolded residues indicate the three CDRs (SEQ ID NOs 13, 14, and 15)
  • Figure 4 shows the V ⁇ cDNA sequences and the predicted amino acid sequence of the ant ⁇ -SAF-1 mAb, 11G4 (SEQ ID NOs 16 and 17, respectively) The bolded residues indicate the three CDRs (SEQ ID NOs 18, 19, and 20)
  • acceptor antibody refers to monoclonal or recombinant antibodies heterologous to the donor antibody, that contributes all, or a 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 or V region subfamily consensus sequences to the first immunoglobuhn partner
  • a human antibody is the acceptor antibody
  • agonist activity refers to the activity of an antibody against SAF-1 to enhance the activation of neutrophils and/or monocytes
  • altered immunoglobuhn coding region refers to a nucleic acid sequence encoding an altered antibody of the invention
  • the altered antibody is a complementarity determining region-grafted (CDR-gratted) or humanized antibody
  • the sequences that encode the CDRs from a non-human immunoglobuhn are inserted into a first immunoglobuhn partner comprising human variable framework sequences
  • the first immunoglobuhn partner is operatively linked to a second immunoglobuhn partner
  • an “analog” is an amino acid sequence modified by at least one ammo acid, wherein said modification can be chemical or a substitution or a rearrangement of a few ammo acids (t e , no more than 10) and corresponding nucleic acid sequences Such a modification permits the ammo acid sequence to retain the biological characte ⁇ stics, e.g , antigen specificity and high affinity, of the unmodified sequence
  • Exemplary nucleic acid analogs include silent mutations that can be constructed, via substitutions, to create certain endonuclease restriction sites within or surrounding CDR-encoding regions Analogs can also arise as allehc va ⁇ ations
  • An "allehc variation or modification” is an alteration in the nucleic acid sequence encoding the amino acid or peptide sequences of the invention.
  • the term 'antagonist activity refers to the activity of an antibody against SAF- 1 to inhibit the activation of neutrophils and/or monocytes
  • antibody also includes “altered antibody” that refers to a protein encoded by an altered immunoglobuhn coding region, that can be obtained by expression in a selected host cell
  • altered antibodies are engineered antibodies (e g , chime ⁇ c or humanized antibodies) or antibody fragments lacking all or part of an immunoglobuhn constant region, e g , Fv, Fab, Fab' or F(ab " )2 and the like
  • CDRs are defined as the complementarity determining region amino acid sequences of an antibody that are the hyperva ⁇ able regions of immunoglobuhn heavy and light chains See. e g Kabat. et al , Sequences of Proteins of Immunological Interest 4th Ed . U S Department of Health and Human Services.
  • CDRs refers to all three heavy chain CDRs, or all three light chain CDRs or both all heavy and all light chain CDRs, if appropriate CDRs 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 analogs of the naturally occurring CDRs Such analogs share or retain the same antigen binding specificity and/or antagonist ability as the donor antibody from which they were derived, yet may exhibit increased affinity for the antigen
  • An exemplary process for obtaining analogs is affinity maturation by means of phage display technology as reviewed by Hoogenboom.
  • a “chime ⁇ c antibody” refers to a type of engineered antibody that contains a naturally-occur ⁇ ng variable region (light chain and heavy chains) de ⁇ ved from a donor antibody in association with light and heavy chain constant regions derived from an acceptor antibody.
  • donor antibody refers to a monoclonal or recombinant antibody that cont ⁇ butes the nucleic acid sequences of its variable regions, CDRs or other functional fragments or analogs thereof to a first immunoglobuhn partner, so as to provide the altered immunoglobuhn coding region and resulting expressed altered antibody with the antigenic specificity and neutralizing activity characteristic of the donor antibody.
  • effector agents refers to non-protein earner molecules to which the altered antibodies, and/or natural or synthetic light or heavy chains of the donor antibody or other fragments of the donor antibody may be associated by conventional means.
  • non-protein earners can include conventional carriers used in the diagnostic field, e.g., polystyrene or other plastic beads, polysaccha ⁇ des, e.g., as used in the BIAcore (Pharmacia) system, 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 radioisotopes Such effector agents may also be useful to increase the half-life of the altered antibodies, e g., polyethylene glycol
  • an "engineered antibody” describes a type of altered antibody, i.e.. a full-length synthetic antibody (e g., a chime ⁇ c or humanized antibody as opposed to an antibodv fragment) in which a portion of the light and/or heavy chain variable domains of a selected acceptor antibody are replaced by analogous parts from one or more donor antibodies that have specificity for the selected epitope
  • such molecules may include antibodies characterized by a humanized heavy chain associated with an unmodified light chain (or chime ⁇ c light chain), or vice versa
  • 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 replacement of one or more CDRs (preferably all) from the acceptor antibody with CDRs from a donor antibody described herein
  • First immunoglobuhn partner refers to a nucleic acid sequence encoding a human framework or human immunoglobuhn variable region in which the native (or naturally- occur ⁇ ng) CDR-encoding regions are replaced by the CDR-encod g regions of a donor antibody.
  • the human va ⁇ able region can be an immunoglobuhn heavy chain, a light chain (or both chains), an analog or functional fragments thereof
  • Such CDR regions, located within the variable region of antibodies (immunoglobulins) can be determined by known methods in the art For example Kabat, et al, SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST. 4th Ed , U.S . Department of Health and Human
  • Fv, Fc, Fd, Fab, Fab' or F(ab I )2 are used with their standard meanings. See, e.g., Harlow, et al, ANTIBODIES A LABORATORY MANUAL, Cold Spring Harbor Laboratory, (1988)
  • a “humanized antibody” refers to a type of engineered antibody having its CDRs de ⁇ ved from a non-human donor immunoglobuhn, the remaining lmmunoglobuhn-de ⁇ ved parts of the molecule being derived from one or more human immunoglobulins.
  • framework support residues may be altered to preserve binding affinity See. e g , Queen, et al, Proc. Natl Acad Sci USA, 86, 10029- 10032 ( 1989), Hodgson, et al,
  • a “functional fragment” is a partial heavy or light chain variable sequence (e.g., minor deletions at the amino or carboxyl terminus of the immunoglobuhn va ⁇ able region) that retains the same antigen binding specificity and/or antagonist ability as the antibody from which the fragment was de ⁇ ved
  • infectious disease state(s) diseases, including but not limited to, osteomyelitis, deep seated wound infections, sepsis, antibiotic resistant bacterial infections, viral infections and those involving intracellular pathogens and parasites, such as, but not limited to Listena moncytogenes. Salmonella, and Leishmania Moreover,
  • infectious disease state(s) also refers to any infectious diseases caused by, but not limited to, any one or a combination of the following organisms, a (l) prokaryote, including but not limited to, a member of the genus Streptococcus, Staphylococcus, Bordetella, Corvnebacteriiim, Mycobactenum, Neissena, Haemophilus, Actinomvcetes, Streptomvcetes, Nocardia, Enterobacter, Yersinia, Fancisella, Pasturella, Moraxella, Acinetobacter,
  • Erysipelothrix Branhamella, Actinobacillus, Streptobacillus, Listena, Calymmatobactenum, Brucella, Bacillus, Clostridium, Treponema, Eschenchia, Salmonella, Kleibsiella, Vibrio, Proteus, Er ⁇ inia, Borreha, Leptospira, Spirillum, Camp lobacter, Shigella, Legwnella.
  • Pseudomonas Aeromonas, Rickettsia, Chlamydia, Borreha and M ⁇ coplasma, and further including, but not limited to, a member of the species or group, Group A Streptococcus, Group B Streptococcus, Group C Streptococcus, Group D Streptococcus, Group G Streptococcus, Streptococcus pneumomae, Streptococcus pyogenes.
  • Streptococcus agalactiae Streptococcus faecahs, Streptococcus faecium, Streptococcus durans, Neisseria gonorrheae, Neissena meningttidis, Staphylococcus aureus, Staphylococcus epidermidis, Corynebacterium dipthenae, Gardnerella vaginalts, Mycobactenum tuberculosis, Mycobactenum bovis, Mycobactenum ulcerans, Mycobactenum leprae, Actinomyctes israelu, Listena monocytogenes, Bordetella pertusis.
  • Bordatella parapenusis Bordetella bronchiseptica, Eschenchia coh, Shigella dysentenae, Haemoph us influenzae, Haemoph ⁇ us aegyptius.
  • Haemophdus parainfluenzae Haemophdus ducreyi, Bordetella, Salmonella typhi, Citrobacter freundu, Proteus mirabihs, Proteus vulgans.
  • Yerstnia pestis Kletbsiella pneumomae, Serratia marcessens, Serratia liquefaciens, Vibrio cholera, Shigella dysenterii, Shigella flexner i, Pseudomonas aeruginosa, Franscisella tularensis, Brucella abortis, Bacillus anthracis, Bacillus cereus, Clostridium perfnngens, Clostndium tetant, Clostndium botu num. Treponema pallidum. Rickettsia rickettsu and Chlamydia trachomitis, (n) an archaeon. including but not limited to Archaebacter, (in) a unicellular or filamentous eukaryote, including but not limited to, a protozoan, a fungus, a member of the genus
  • Saccharomyces Kluveromyces. or Candida
  • Saccharomyces cenviseae Kluveromyces lactis
  • Candida a member of the species Saccharomyces cenviseae, Kluveromyces lactis, or Candida
  • a virus a virus
  • Isolated means altered “by the hand of man” from its natural state, i.e., if it occurs in nature, it has been changed or removed from its o ⁇ ginal environment, or both.
  • a polynucleotide or a polypeptide naturally present in a living organism is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated", as the term is employed herein
  • a polynucleotide or polypeptide that is introduced into an organism by transformation, genetic manipulation or by any other recombinant method is "isolated” even if it is still present in said organism, which organism may be living or non-living
  • “Monoclonal antibodies” refers to immunoglobulins that can be prepared by conventional hyb ⁇ doma techniques, phage display combinatorial libraries, immunoglobuhn chain shuffling and humamzation techniques Also included are fully human monoclonal antibodies
  • "operatively linked” means that the polynucleotide sequences being linked are contiguous, being present in a single larger polynucleotide sequence Certain operatively linked sequences shall be contiguous in reading phase, such as secretory leader open reading frames and membrane anchor sequences Other operatively linked polynucleotide sequences are not contiguous reading phase (.
  • the term “pharmaceutical” includes veterinary applications of the invention
  • the term "therapeutically effective amount refers to that amount of a receptor agonist or antagonist antibody that is useful for alleviating a selected condition
  • These therapeutic compositions of the invention may be administered to mimic the effect of the normal receptor hgand
  • “Second immunoglobuhn partner ' refers to another nucleotide sequence encoding a protein or peptide to which the first immunoglobuhn partner is fused in frame or by means of an optional conventional linker sequence (. e , operatively linked) Preferably, it is an immunoglobuhn gene
  • the second immunoglobuhn partner may include a nucleic acid sequence encoding the entire constant region for the same (i e , homologous, where the first and second altered antibodies are derived from the same source) or an additional (i e , heterologous) antibody of interest It may be an immunoglobuhn heavy chain or light chain (or both chains as part of a single polypeptide)
  • the second immunoglobuhn partner is not limited to a particular immunoglobuhn class or isotype
  • the second immunoglobuhn partner may comp ⁇ se part of an immunoglobuhn constant region, such as found in a Fab, or F(ab)2 e ,
  • a non-human species such as bovine, ovine, monkey, chicken, rodent (e g , mu ⁇ ne and rat) may be employed to generate a desirable immunoglobuhn upon presentment with human SAF-1 or a peptide epitope therefrom
  • rodent e g , mu ⁇ ne and rat
  • Conventional hyb ⁇ doma techniques are employed to provide a hyb ⁇ doma cell line secreting a non-human mAb to the human SAF- 1
  • Such hyb ⁇ domas are then screened for binding and antagonist activity, as described in the Examples section
  • fully human mAbs can be generated by techniques known to those skilled in the art and used in this invention
  • Exemplary agonist mAbs of the present invention are mu ⁇ ne mAbs 13G3 and
  • mAbs are characterized by agonist activity on human SAF- 1 and subsequent inhibition of the ability of SAF- 1 to activate neutrophils and/or monocytes
  • the present invention also includes the use of Fab fragments or F(ab * )2 fragments derived from mAbs directed against human SAF- 1 as bivalent fragments These fragments are useful as agents having agonist or antagonist activity at SAF- 1
  • a Fab fragment contains the entire light chain and amino terminal portion of the heavy chain.
  • An F(ab ⁇ >2 fragment is the fragment formed by two Fab fragments bound by disulfide bonds.
  • the mAbs 13G3, 1 1G4, and other similar high affinity antibodies provide sources of Fab fragments and F(ab')2 fragments that can be obtained by conventional means, e.g., cleavage of the mAb with the approp ⁇ ate proteolytic enzymes, papain and/or pepsin, or by recombinant methods
  • Fab and F(ab")2 fragments are useful themselves as therapeutic, prophylactic or diagnostic agents, and as donors of sequences including the va ⁇ able regions and CDR sequences useful m the formation of recombinant or humanized antibodies as described herein.
  • the Fab and F(ab')2 fragments can be constructed via a combmato ⁇ al phage library
  • the Fd or V j immunoglobuhn from a selected antibody e g , 13G3 or 1 1G4
  • the light chain immunoglobuhn from a selected antibody may be allowed to associate with a repertoire of heavy chain immunoglobulins, Vj j (or Fd), to form novel Fabs.
  • SAF- 1 agonist or antagonist Fabs can be obtained by allowing the Fd of the mABs of the present invention to associate with a repertoire of light chain immunoglobulins Hence, one is able to recover neutralizing Fabs with unique sequences (nucleotide and amino acid) from the chain shuffling technique
  • the mAbs of the present invention contribute sequences, such as variable heavy and/or light chain peptide sequences, framework sequences, CDR sequences, functional fragments, and analogs thereof, and the nucleic acid sequences encoding them, useful in designing and obtaining various altered antibodies that are characterized by the antigen binding specificity of the donor antibody
  • nucleic acid sequences of this invention, or fragments thereof, encoding the va ⁇ able light chain and heavy chain peptide 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 into a plasmid for expression.
  • va ⁇ ous coding sequences can be constructed that encode the va ⁇ able heavy and light chain am o acid sequences and CDR sequences of the invention as well as functional fragments and analogs thereof that share the antigen specificity of the donor antibody.
  • CDRs can be used to produce altered antibodies, e g , chime ⁇ c or humanized antibodies or other engineered antibodies of this invention when operatively combined with a second immunoglobuhn partner
  • nucleic acid sequences encoding portions of the altered antibody and antibodies described herein
  • other such nucleic acid sequences are encompassed by the present invention, such as those complementary to the native CDR-encoding sequences or complementary to the modified human framework regions surrounding the CDR-encoding regions
  • Useful DNA sequences include those sequences that hybridize under stringent hybridization conditions to the DNA sequences See T Maniatis, et al , MOLECULAR CLONING A LABORATORY MANUAL, Cold Sp ⁇ ng Harbor Laboratory (1982), pp 387-389
  • An example of one such stringent hyb ⁇ dization condition is hybridization at 4XSSC at 65°C, followed by a washing in 0 1XSSC at 65°C for one hour
  • an exemplary stnngent hybndization condition is 50% formamide, 4XSSC at 42°C
  • these hybridizing DNA sequences are at least about 18 nucleotides in length, i e , about the size
  • Altered immunoglobuhn molecules can encode altered antibodies that include engineered antibodies such as chimenc antibodies and humanized antibodies
  • a desired altered immunoglobuhn coding region contains CDR-encoding regions that encode peptides having the antigen specificity of a SAF- 1 antibody, preferably a high-affinity agonist or antagonist antibody, such as provided by the present invention, inserted into a first immunoglobuhn partner such as a human framework or human immunoglobuhn variable region
  • the first immunoglobuhn partner is operatively linked to a second immunoglobuhn partner
  • the second immunoglobuhn partner is defined above, and may include a sequence encoding a second antibody region of interest, for example an Fc region
  • Second immunoglobuhn partners may also include sequences encoding another immunoglobuhn to which the light or heavy chain constant region is fused in frame or by means of a linker sequence
  • engineered antibodies directed against functional fragments or analogs of SAF- 1 are designed to elicit enhanced binding with the same antibody
  • the second immunoglobuhn partner is associated with effector agents as defined above, including non-protein earner molecules, to which the second immunoglobuhn partner is operatively linked by conventional means
  • the invention provides that the fusion or linkage between the second immunoglobuhn partners, e g , antibody sequences, and the effector agent can be formed by any suitable means, e g , by conventional covalent or ionic bonds, protein fusion
  • signal sequences for the molecules of the invention are modified by techniques known to those skilled in the art to enhance expression
  • a preferred altered antibody contains a vanable heavy and/or light chain peptide or protein sequence having the antigen specificity of mAb 13G3 or 1 1 G4, e g , the VJJ and V ⁇ chains
  • Still another desirable altered antibody of this invention is characte ⁇ zed 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 mu ⁇ ne antibody molecule 13G3 or 11G4 with the remaining sequences being derived from a human source, or a functional fragment or analog thereof
  • the altered antibody of the invention is attached to an additional agent
  • an additional agent for example, recombinant DNA technology may be used to produce an altered antibody of the invention in which the Fc fragment or CH2 CH3 domain of a complete antibody molecule has been replaced by an enzyme or other detectable molecule, i e , a polypeptide effector or reporter molecule
  • additional agents include toxins and radionuchdes
  • the second immunoglobuhn partner is operatively linked to a non-immunoglobuhn peptide, protein or fragment thereof heterologous to the CDR-contaimng sequence having antigen specificity to SAF- 1
  • the resulting protein may exhibit both antigen specificity and characteristics of the non- lmmunoglobuhn upon expression
  • That fusion partner characteristic may be, e g , a functional characteristic such as another binding or receptor domain or a therapeutic characteristic if the fusion partner is itself a therapeutic protein or additional antigenic characteristics
  • Another preferred protein of this invention comp ⁇ ses a complete antibody molecule, having full length heavy and light chains or any discrete fragment thereof, such as the Fab or F(ab * )2 fragments, a heavy chain dimer or any minimal recombinant fragments thereof such as an F v or a single-chain antibody (SCA) or any other molecule with the same specificity as the selected donor mAb, e g , the 13Gs or 1 1G4 mAB
  • SCA single-chain antibody
  • Such a protein may be used in the form of an altered antibody or may be used in its unfused form Whenever the second immunoglobuhn partner is de ⁇ ved from an antibody different from the donor antibody, e g , any isotype or class of immunoglobuhn framework or constant regions, an engineered antibody results
  • Engineered antibodies can comp ⁇ se immunoglobuhn constant regions and variable framework regions from one source, e g , the acceptor antibody, and one or more (preferably all) CDRs from the donor
  • Such engineered antibodies are designed to employ one (or both) of the vanable heavy and/or light chains of the SAF-1 mAb (optionally modified as described) or one or more of the heavy or light chain CDRs
  • the engineered antibodies of the invention exhibit agonist activity
  • such engineered antibodies include a humanized antibody containing the framework regions of a selected human immunoglobuhn or subtype or a chime ⁇ c antibody containing the human heavy and light chain constant regions fused to the SAF- 1 mAb functional fragments
  • a suitable human (or other animal) acceptor antibodv may be one selected from a conventional database, e g , the KABAT® database.
  • the heterologous framework and constant regions are selected from human immunoglobuhn classes and isotypes, such as IgG (subtypes 1 through 4) IgM, IgA, and lgE IgG 1 , k and IgG4, k are preferred Particularly preferred is IgG 4, k.
  • IgG4 subtype variant containing the mutations S228P and L235E (PE mutation) in the heavy chain constant region that results in reduced effector function
  • This IgG4 subtype variant is known herein as IgG4PE See U S. Patent Nos. 5, 624,821 and 5,648,260
  • the acceptor antibody need not comprise only human immunoglobuhn protein sequences.
  • a gene may be constructed in which a DNA sequence encoding part of a human immunoglobuhn chain is fused to a DNA sequence encoding a non- lmmunoglobuhn amino acid sequence such as a polypeptide effector or reporter molecule.
  • a particularly preferred humanized antibody contains CDRs of 13G3 or 1 1G4 mAb inserted onto the framework regions of a selected human antibody sequence.
  • 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.
  • CDRs are replaced 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 is selected from another human antibody by recourse to conventional antibody databases The remainder of the engineered antibody is then derived from any suitable acceptor human immunoglobuhn
  • 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 infectious disease states, included, but not limited to osteomyelitis, deep seated wound infections, sepsis, antibiotic resistant bacterial infections, viral infections and those involving mtracellular pathogens and parasites, such as. but not limited to, Listena moncytogenes. Salmonella, and Leishmania
  • engineered humanized antibody of the invention preferably, has the structure of a natural human antibody or a fragment thereof, and possesses the combination of properties required for effective therapeutic use.
  • a prokaryote including but not limited to, a member of the genus Stieptococcus, Staphylococcus, Bordetella, Cor nebactenum Mycobactenum Neisse a, Haemophdus, Acttnomvcetes, Streptomvcetes Nocardia, Enterobacter, Yersima, Fancisella, Pasturella Moraxella, Acinetobacter, Erysipeloth x, Branhamella Actmobac lus, Streptobacillus, Listena, Calymmatobacterium, Brucella, Bacillus, Clostndium Treponema, Eschenchia, Salmonella, Kleibsiella, Vibrio, Proteus, Erwinia, Borreha, Leptospira, Spirillum, Campvlobacter, Shigella
  • Streptococcus pyogenes Streptococcus agalactiae, Streptococcus faecahs. Streptococcus faecium, Streptococcus durans, Neissena gonorrheae, Neisseria meningittdis, Staphylococcus aureus, Staphylococcus epidermidis, Corynebacterium dipthenae, Gardnerella vaginahs. Mycobactenum tuberculosis, Mycobactenum bovis, Mycobactenum ulcerans, Mycobactenum leprae
  • Actinomyctes israelu Listena monocytogenes, Bordetella pertusis, Bordatella parapertusis, Bordetella bronchiseptica, Eschenchia colt, Shigella dysenteriae, Haemophdus influenzae, Haemophdus aegyptius, Haemophdus parainfluenzae, Haemophdus ducreyi, Bordetella, Salmonella typhi Citrobacter freundu Proteus mirabihs, Proteus vulgans Yersima pestis Kleibsiella pneumomae, Serratia marcessens, Serratia liquefaciens, Vibrio cholera.
  • Saccharomyces Kluveromyces, or Candida, and a member of the species Saccharomyces cenviseae, Kluveromyces lactis, or Candida, and (IV) a virus
  • an engineered antibody can be further modified by changes in variable domain amino acids without necessarily affecting the specificity and high affinity of the donor antibody (; e an analog) It is anticipated that heavy and light chain amino acids can be substituted by other amino acids either in the va ⁇ able domain frameworks or CDRs or both These substitutions could be supplied by the donor antibody or consensus sequences from a particular subgroup
  • the constant region may be altered to enhance or decrease selective properties of the molecules of this invention For example, dime ⁇ zation, binding to Fc receptors, or the ability to bind and activate complement (see, e g , Angal, et al . Mol Immunol 30. 105- 108 ( 1993), Xu, et al , J Biol Chem 269. 3469-3474 ( 1994), Winter, et al , EP 307434-B)
  • variable light and/or heavy chain sequences and the CDRs of the mAbs of the invention or other suitable donor mAbs and their encoding nucleic acid sequences are utilized in the construction of altered antibodies, preferably humanized antibodies, of this invention, by the following process
  • a hyb ⁇ doma producing a selected donor mAb, e g , one of the mu ⁇ ne antibodies of the invention is conventionally cloned and the DNA of its heavy and light chain vanable regions obtained by
  • a mouse/human chime ⁇ c antibody may then be prepared and assayed for binding ability
  • Such a chime ⁇ c antibody contains the entire non-human donor antibody Vir ⁇ and VL regions, in association with human Ig constant regions for both chains
  • Homologous framework regions of a heavy chain variable region from a human antibody are identified using computerized databases, e g , KABAT®, and a human antibody characterized by a homology to the V region frameworks of the donor antibody or V region subfamily consensus sequences (on an amino acid basis) to one of the antibodies of the invention is selected as the acceptor antibody
  • the sequences of synthetic heavy chain variable regions containing the CDR-encoding regions within the human antibody frameworks are designed with optional nucleotide replacements in the framework regions to incorporate restriction sites
  • the invention provides a humanized antibody derived from the chimenc antibody, or preferably, made synthetically by inserting the donor mAb CDR-encoding regions from the heavy and light chains appropriately within the selected heavy and light chain framework Alternatively, a humanized antibody of the invention may be prepared using standard mutagenesis techniques Thus, the resulting humanized antibody contains human framework regions and donor mAb CDR-encoding regions There may be subsequent manipulation of framework residues The resulting humanized antibody can be expressed in recombinant host cells, e g , COS, CHO or myeloma cells
  • a conventional expression vector or recombinant plasmid is produced by placing these coding sequences for the altered antibody in operative association with conventional regulatory control sequences capable of controlling the replication and expression m, and/or secretion from, a host cell Regulatory sequences include promoter sequences, e g , CMV or Rous Sarcoma virus promoter, and signal sequences, that can be derived from other known antibodies Similarly, a second expression vector can be produced having a DNA sequence encoding a complementary antibody light or heavy chain Preferably, this second expression vector is identical to the first except with respect to the coding sequences and selectable markers, in order to ensure, as much as possible, that each polypeptide chain is functionally expressed Alternatively, the heavy and light chain coding sequences for the altered antibody may reside on a single vector 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
  • Suitable vectors for the cloning and subclomng steps employed in the methods and construction of the compositions of this invention may be selected by one of skill in the art
  • the pUC series of cloning vectors such as pUC19, which is commercially available from supply houses, such as Amersham or Pharmacia
  • any vector that is capable of replicating readily, has an abundance of cloning sites and selectable genes (e g , antibiotic resistance) 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 (such as CMV or Rous Sarcoma virus promoters) that direct the replication and expression of heterologous DNA sequences in selected host cells
  • These vectors contain the above-described DNA sequences that code for the engineered antibody or altered immunoglobuhn coding region
  • the expression vectors may also be charactenzed by genes suitable for amplifying expression of the heterologous DNA sequences, e g , the mammalian dihydrofolate reductase gene (DHFR)
  • Other preferable vector sequences include a poly A signal sequence, such as from bovine growth hormone (BGH) and the betaglobm promoter sequence (betaglopro)
  • BGH bovine growth hormone
  • betaglopro betaglobm promoter sequence
  • the present invention also provides a cell line transfected with a recombinant plasmid containing the coding sequences of the engineered antibodies or altered immunoglobuhn molecules thereof
  • 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 colt are used for replication of the cloning vectors and other steps in the construction of altered antibodies of this invention
  • Suitable host cells or cell lines for the expression of the engineered antibody or altered antibody of the invention are preferably mammalian cells such as CHO, COS.
  • a fibroblast cell e g , 3T3
  • myeloid cells e g , 3T3
  • a CHO or a myeloid cell Human cells are suitable, thus enabling the molecule to be modified with human glycosylation patterns
  • other eukaryotic cell lines are suitable.
  • 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 , supra
  • bacterial cells are useful as host cells suitable for the expression of the recombinant Fabs of the present invention (see, e g Pluckthun, A , Immunol Re ⁇ , 130, 151 - 188 ( 1992))
  • any recombinant Fab produced in a bacterial cell would have to be screened for retention of antigen binding ability If the molecule expressed by the bactenal cell was produced in a properly folded form, that bacterial cell would be a desirable host
  • various strains of E. co used for expression are well-known as host cells in the field of biotechnology Vanous strains of B subtdis, Streptomyces, other bacilli and the like may also be employed
  • strains of yeast cells known to those skilled in the art are also available as host cells, as well as insect cells, e g , Drosophda and Lepidoptera, and viral expression systems See, e g , Miller, et al , Genetic Engineering, 8, 277-298, Plenum Press ( 1986) and references cited therein
  • the transfection methods required to produce the host cells of the invention, and culture methods necessary to produce the altered antibody of the invention from such host cell are all conventional techniques
  • the altered 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
  • the humanized antibodies are expressed in a transgenic animal, such as described in U S Patent No 4,873,316
  • 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 ⁇ itro activity by use of an appropriate assay
  • in vitro assays such as the human blood chemiluminescence assay
  • in vitro assays may also be used to determine agonist or antagonist activity prior to subsequent human clinical studies performed to evaluate the persistence of the engineered antibody in the body despite the usual clearance mechanisms
  • one of skill in the art may also construct humanized antibodies from other donor antibodies, variable region sequences and CDR peptides described herein
  • Engineered antibodies 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 increases m antigen binding, agonist, or antagonist activity without appreciable increased immunogenicity for the recipient
  • This invention also relates to a method for treating or preventing infectious disease states in a mammal comprising administenng to a subject in need thereof an effective dose of a SAF-1 agonist or antagonist mAb of the invention The m
  • the altered antibodies, antibodies and fragments thereof of this invention are used in conjunction with other antibodies, particularly human mAbs reactive with other markers (epitopes) responsible for the condition against which the engineered antibody of the invention is directed
  • compositions can be formulated into pharmaceutical compositions and administered in the same manner as desc ⁇ bed for mature proteins See, e g , International Patent Application, Publication No WO 90/02762, published March 22, 1990 Generally, these compositions contain a therapeutically effective amount of an agonist or antagonist antibody of this invention and an acceptable pharmaceutical earner Suitable earners are well known to those of skill in the art and include, for example, saline Alternatively, such compositions may include conventional delivery systems into which protein of the invention is incorporated Optionally, these compositions may contain other active ingredients
  • the SAF-1 agonist and antagonist antibodies of the present invention may be administered to a patient in need thereof in conjunction with an antibiotic and a suitable carrier
  • the therapeutic agents of this invention are suitable for administration by any appropriate internal route, and may be repeated as needed, . g as frequently as one to three times daily for between 1 day to about three weeks to once per week or once biweekly
  • the agonist or antagonist antibody is administered less frequently than is the hgand, when it is used therapeutically
  • the dose and duration of treatment relates to the relative duration of the molecules of the present invention in the human circulation, and can be adjusted by one of skill in the art depending upon the condition being treated and the general health of the patient
  • the mode of administration of the therapeutic agent of the invention can be any suitable route that delivers the agent to the host.
  • the altered antibodies, antibodies, engineered antibodies, and fragments thereof, and pharmaceutical compositions of the invention are particularly useful for parenteral administration, i e subcutaneously. intramuscularly, intravenously or intranasally
  • therapeutic agents of the invention are prepared as pharmaceutical compositions containing an effective amount of the engineered (e g , humanized) antibody of the invention as an active ingredient in a pharmaceutically acceptable carrier
  • an 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 pharmaceutically acceptable carrier, preferably an aqueous earner
  • aqueous carriers may be employed, e g , 0 4% saline, 0 3% glycine and the like These solutions are stenle and generally free of particulate matter
  • These solutions may be sterilized by conventional, well known sterilization techniques (e g , filtration)
  • the compositions ma ⁇ contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, etc
  • a pharmaceutical composition of the invention for intramuscular injection could be prepared to contain 1 mL sterile buffered water, and between about 1 ng to about 100 mg, e g about 50 ng to about 30 mg or more preferably, about 5 mg to about 25 mg, of an engineered antibodv of the invention Similarly a pharmaceutical composition of the invention for intravenous infusion could oe made up to contain about 250 ml of sterile
  • Ringer s solution and about 1 mg to about 30 mg and preferabh 5 mg to about 25 mg of an engineered antibody of the invention
  • 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 bv those of skill in the art
  • one dose of approximately 0 01 mg to approximateh 20 mg per kg body weight of a protein or an antibodv of this ention should be administered parentally , preferably i v or i m
  • Such dose may, if necessary, be repeated at appropriate time intervals selected as appropriate by a physician during the response period
  • mice were immunized with 25 ⁇ g recombinant SAF- 1 in Freund's complete adjuvant and 2 and 4 weeks later with 25 ⁇ g SAF- 1 in Freund's incomplete adjuvant On the basis of a good serum antibody titre to SAF-1, the mice received a further immunization of 20 ⁇ g SAF1 (.
  • mice v in saline
  • a splenectomy was performed on the mice
  • Mouse spleen cells were used to prepare hybndomas by standard procedures, and positive hyb ⁇ domas were cloned by the limiting dilution method See Zola, H Ed , MONOCLONAL ANTIBODIES, CRC Press Inc , 1987)
  • 96- well plates were coated with SAF- l-Fc (recombinant SAF- 1 with human IgGl Fc, 2 ⁇ g/ml, lOO ⁇ l/well in PBS) by incubation overnight at 4°C The solution was then aspirated and non-specific binding sites were blocked with 250 ⁇ l/well of 1 % bovine serum albumin (BSA) in TBS buffer (50 mM Tns, 150 mM NaCl, 0 02% Kathon, pH 7 4) for 5-60 minutes at RT Following this and each of the following steps, the plate was washed 4 times in wash buffer (10 mM Tns, 150 mM NaCl, 0 05% Tween 20.
  • BSA bovine serum albumin
  • Mabs were punfied by ProsepA (Bio Processing, Consett, UK) chromatography, respectively, using the manufacturer's instructions Mabs were >95% pure by SDS-PAGE
  • the epitope analysis of the purified mAbs was measured in the BIAcore Using a flow rate of 10 ⁇ l/min, the first mAb (diluted in HBS buffer) was injected over a rabbit anti-mouse IgG Fc surface, followed by an injection of SAF- 1 for 240s, an injection of blocking mAbs for 48s, and an injection of the second mAb for 240s The surface was regenerated by an injection of 0 1M phosphoric acid, and the RU was recorded after each injection Mabs 13G3 clone 19 and 1 1G4 clone D were discovered to have different epitopes
  • Incubation of human blood with supernatants containing SAF- 1 mAbs (clone 1 1G4) for 30 minutes enhanced the respiratory burst activity, as measured by cellular chemiluminescence, in response to stimulation with PMA or FMLP
  • the 13G3 ant ⁇ -SAF- 1 Mab, that binds to a epitope distinct from 1 1 G4 on SAF- 1 resulted in a significantly lower p ⁇ ming effect
  • Addition of antibodies without addition of PMA or FMLP had no significant effect on chemiluminescence, indicating that anti-S AF- 1 antibodies are not direct activators of respiratory burst activity in human blood cells
  • RNA was purified reverse transcribed and PCR amplified
  • the RNA DNA hybrid was PCR amplified using a mouse IgG CH 1 - specific primer and a degenerate primer based on the N-terminal protein sequence
  • the RNA/DNA hybrid was PCR amplified using a mouse C kappa primer and a degenerate primer based on the N-terminal protein sequence PCR products of the appropnate size, i e , -350 were cloned into a plasmid vector, and sequenced by a modification of the Sanger method See Sanger, et al , PNAS 74 5463 ( 1977) In each case, the sequences of multiple V H clones and the sequences of multiple V ⁇ clones were compared to generate a consensus heavy chain variable region sequence and consensus light chain variable region sequence, respectively

Abstract

La présente invention concerne des anticorps d'agonistes et d'antagonistes monoclonaux du SAF-1 humain (sialoadhesine facteur-1) et concerne par ailleurs l'utilisation des ces anticorps monoclonaux comme agents thérapeutiques pour le traitement des états dus à des maladies infectieuses.
PCT/US2000/022663 1999-08-19 2000-08-18 Anticorps d'agonistes et d'antagonistes de sialoadhesine facteur-1 WO2001012646A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0800536A2 (fr) * 1994-12-23 1997-10-15 Smithkline Beecham Corporation Antagonistes de recombinaison de l'il-5 utiles dans le traitement des troubles induits par l'il-5
US6946130B2 (en) 1994-12-23 2005-09-20 Smithkline Beecham Corporation Recombinant IL-5 antagonists useful in treatment of IL-5 mediated disorders
US7998485B2 (en) 2006-05-11 2011-08-16 Universiteit Gent Sialoadhesin-related compositions and methods
WO2015150326A1 (fr) * 2014-03-31 2015-10-08 Universiteit Gent Méthode de traitement d'une maladie osseuse
US9834600B2 (en) 2008-03-28 2017-12-05 Glaxosmithkline Llc Methods of treatment of eosinophilic bronchitis with an anti-IL-5 antibody
CN113388032A (zh) * 2014-02-06 2021-09-14 X4制药(奥地利)有限责任公司 大肠杆菌特异性抗体序列

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998006733A1 (fr) * 1996-08-09 1998-02-19 Human Genome Sciences, Inc. Sequence nucleotidique codant la proteine de type cd33

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998006733A1 (fr) * 1996-08-09 1998-02-19 Human Genome Sciences, Inc. Sequence nucleotidique codant la proteine de type cd33

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0800536A2 (fr) * 1994-12-23 1997-10-15 Smithkline Beecham Corporation Antagonistes de recombinaison de l'il-5 utiles dans le traitement des troubles induits par l'il-5
EP0800536A4 (fr) * 1994-12-23 2004-12-01 Smithkline Beecham Corp Antagonistes de recombinaison de l'il-5 utiles dans le traitement des troubles induits par l'il-5
US6946130B2 (en) 1994-12-23 2005-09-20 Smithkline Beecham Corporation Recombinant IL-5 antagonists useful in treatment of IL-5 mediated disorders
US7998485B2 (en) 2006-05-11 2011-08-16 Universiteit Gent Sialoadhesin-related compositions and methods
US9834600B2 (en) 2008-03-28 2017-12-05 Glaxosmithkline Llc Methods of treatment of eosinophilic bronchitis with an anti-IL-5 antibody
US11325972B2 (en) 2008-03-28 2022-05-10 Glaxosmithkline Llc Methods of treatment of eosinophilic bronchitis with an anti-IL-5 antibody
CN113388032A (zh) * 2014-02-06 2021-09-14 X4制药(奥地利)有限责任公司 大肠杆菌特异性抗体序列
WO2015150326A1 (fr) * 2014-03-31 2015-10-08 Universiteit Gent Méthode de traitement d'une maladie osseuse
US10479832B2 (en) 2014-03-31 2019-11-19 Universiteit Gent Method of treating bone disease

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