WO2016079276A1 - Utilisation d'un antagoniste anti-gm-csf et d'un antagoniste anti-ccr2 pour traiter une maladie infectieuse - Google Patents

Utilisation d'un antagoniste anti-gm-csf et d'un antagoniste anti-ccr2 pour traiter une maladie infectieuse Download PDF

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WO2016079276A1
WO2016079276A1 PCT/EP2015/077174 EP2015077174W WO2016079276A1 WO 2016079276 A1 WO2016079276 A1 WO 2016079276A1 EP 2015077174 W EP2015077174 W EP 2015077174W WO 2016079276 A1 WO2016079276 A1 WO 2016079276A1
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antibody
exposed
seq
ccr2
fragment
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PCT/EP2015/077174
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English (en)
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Jonathan Zalevsky
Bernard SOUBERBIELLE
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Takeda Gmbh
Millennium Pharmaceuticals Inc.
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Publication of WO2016079276A1 publication Critical patent/WO2016079276A1/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
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/243—Colony Stimulating Factors
    • 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
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention relates to the treatment and/or prevention of infections induced by agents inducing hemorrhagic fever and/or the modulation of immune and/or cellular response in individuals, e.g. humans, infected with, exposed to or suspected to be exposed to agents inducing hemorrhagic fever using agents antagonizing GM-CSF in combination with agents antagonizing CCR2.
  • the antagonists are neutralizing antibodies against primate GM-CSF and antagonists specifically binding mammalian CCR2.
  • These antagonists may be used in combination with other drugs in the treatment and/or prevention and/or modulation of the cellular or immune response of an individual infected with or exposed or suspected to be exposed to, for example, Ebola virus or any other agent causing hemorrhagic fever, flaviviruses such as dengue viruses.
  • Ebola virus disease is caused by a negative single-stranded RNA filovirus.
  • Ebola virus disease has been self-contained and relatively few previous epidemics due to this virus [Feldmann H, 2014 and 2011] but the current epidemic, which began almost a year ago in the Republic of Guinea [Gatherer D, 2014], has taken the world by surprise because of its rapid spread over other Western African countries (Liberia, Sierra Leone, Nigeria, Senegal and Mali), reaching even global proportion when few exported cases were reported and treated on other continents.
  • the fatality rate in EVD is very high, up to 90% in some outbreaks, and the current epidemic has around a 70% mortality rate [Schieffelin JS et al, 2014], though the rate may be slightly lower (-60%) if the patients are hospitalized and have access to standard medical supportive and critical care facilities [WHO Ebola Response Team, 2014].
  • the CDC has classified the filoviruses as category A potential bioterrorism agents [CDC, website]. Indeed, in addition to the immediate need for new medicines and vaccines to alleviate the suffering of the affected populations, this current epidemic has also made even clearer the necessity for stock-piling new drugs in preparation for future epidemics.
  • the virus is not air-borne, and this is determined
  • the virus is transmitted through body fluid (blood, sweat, vomit, diarrhea etc), and likely spread originally from consumption of wild animals such as bats, which are known carriers of the virus [Feldmann H et al, 2011].
  • body fluid blood, sweat, vomit, diarrhea etc
  • bats which are known carriers of the virus [Feldmann H et al, 2011].
  • the incubation of EVD is between 3- 21 days, though the median is only 11 days [WHO Ebola response team, 2014], and therefore the majority of patients develop symptoms around 11 days after contact with an infected patient.
  • RT-PCR and viral antigen detection by ELISA are the primary assays and can be positive as soon as the onset of symptoms, but usually 2-3 days after onset [Feldmann H et al,
  • IV crystalloid intravenous fluids replacement
  • electrolyte balance and nutrition as well as anti-microbial, anti-malarial and symptomatic treatment for pain, GI and neurological symptoms
  • the present invention relates to what is sometimes called "indirect anti-viral" therapy, which targets the virus host cells.
  • This approach is based on the unique biology of the virus, which uses monocyte/macrophage/dendritic cells (DC) as key mediators of its infection. Infection of these cells is the main cellular tropism of the virus in approximately the first 3 symptomatic days.
  • monocyte/macrophage/DC activation triggers an unregulated cytokine cascade, critical for endothelial cells dysfunction, a hallmark of the disease.
  • Viral proteins re- program the host's anti-viral response at the level of JAK/STAT, IRF, and dsRNA sensors and dysregulate monocyte/macrophage/DC homeostasis, and hijack the infected monocytes as a vehicle to disseminate the virus throughout the body [Sullivan N et al. 2003] [Martinez O et al, 2013][Wong G et al, 2014][Ansari AA, 2014].
  • the herein disclosed GM-CSF antagonists target different aspects of the monocytes/macrophage maturation, and by doing so slow down and minimize the virus dissemination and related symptoms.
  • the present invention relates to new combinatory treatments and combinations of antagonists binding GM-CSF and CCR2, which address the above objectives.
  • antagonists of GM-CSF comprise antibodies or fragments thereof, preferably antibodies or fragments having the capability of neutralizing GM-CSF, peptidomimetics, polypeptides, small molecules or nucleic acids.
  • antagonists of CCR2 comprise antibodies or fragments thereof, preferably antibodies or fragments having the capability of neutralizing CCR2, peptidomimetics, polypeptides, small molecules or nucleic acids.
  • the invention relates to a combination of an anti-GM-CSF antagonist and an anti- CCR2 antagonist for use in the treatment and/or prevention and/or modulation of the immune response or the cellular response in an individual to an infection with or the exposure to an agent causing hemorrhagic fever.
  • the individual treated by the method of the present invention includes a primate, wherein the primate is a human, or a non-human primate.
  • Non-human primates include chimpanzee, gorilla, orangutan, monkey, Rhesus Macaque. Preferred is the treatment of humans.
  • the invention relates to a combination of an anti-GM-CSF antagonist and an anti- CCR2 antagonist for use in the treatment and/or prevention and/or modulation of the immune response or the cellular response in an individual to an infection with or the exposure to an agent causing hemorrhagic fever, wherein said combination
  • c) provides time for the individual's immune system to raise a response.
  • the invention relates to a combination of an anti-GM-CSF antagonist and an anti- CCR2 antagonist for use in the treatment and/or prevention and/or modulation of the immune response or the cellular response in an individual to an infection with or the exposure to an agent causing hemorrhagic fever, comprising at least one of the following features:
  • agent-induced cytokine cascade comprising release of monocyte - derived, macrophage-derived, dendritic cell-derived, immune cell-derived, endothelial cell-derived cytokines;
  • monocytes macrophages, dendritic cells and neutrophils
  • JAK/STAT JAK/STAT, IRF, and/or dsRNA sensors
  • symptoms selected from hypotonia, loss of body fluids, fever, blood loss, diarrhea, sore throat, muscle pain and headaches.
  • the invention relates to a combination of an anti-GM-CSF antagonist and an anti-CCR2 antagonist for use in the treatment and/or prevention and/or modulation of the immune response or the cellular response in an individual to an infection with or the exposure to an agent causing hemorrhagic fever, wherein the agent causing hemorrhagic fever is selected from the group comprising filoviridae, flaviviridae, arenaviridae, togaviridae and bunyaviridae (hantavirus).
  • Arenaviridae comprise, e.g., Lassa virus, Junin virus and Machupo virus as respresentative agents inducing hemorrhagic fever.
  • Togaviridae comprises e.g., Chikungunya virus as respresentative agent causing hemorrhagic fever.
  • Flaviviridae comprises, e.g., Dengue viruses (all four known serotypes) as respresentative agent inducing hemorrhagic fever.
  • the family of bunyaviridae comprises the genus Hantavirus as representative agent inducing hemorrhagic fever comprising as representative viruses, e.g. Hantaan river virus, Seoul virus, Puumula virus, Dobrava-Belgrade virus.
  • the herein described combination of GM-CSF antagonists and CCR-2 antagonists can be used also in the treatment of infections and diseases caused by the above viruses.
  • the invention relates to a combination of an anti-GM-CSF antagonist and an anti- CCR2 antagonist for use in the treatment and/or prevention and/or modulation of the immune response or the cellular response in an individual to an infection with or the exposure to an agent causing hemorrhagic fever, wherein the agent causing hemorrhagic fever selected from filoviridae comprises the genera Ebola virus and Marburg virus.
  • the invention relates to a combination of an anti-GM-CSF antagonist and an anti-CCR2 antagonist for use in the treatment and/or prevention and/or modulation of the immune response or the cellular response in an individual to an infection with or the exposure to Ebola virus
  • the Ebola virus is selected from the group comprising the strains Zaire Ebola virus (ZEBOV), Tai Forrest Ebola virus (TEBOV), Sudan Ebola virus (SEBOV), Reston Ebola virus (REBOV), Bundibugyo Ebola virus (BEBOV).
  • the present invention also provides an anti-GM-CSF antagonist and an anti-CCR2 antagonist for use in the treatment and/or prevention of an infection with an agent causing hemorrhagic fever, wherein the treatment increases the survival rate of the individual by at least by 5%, at least by 10 %, at least by 15% , at least by 20%, at least by 25% or at least by 30%, or at least up to 50% as compared to no treatment, based on a current survival rate of 40%.
  • the survival rate is determined at day 7, or at day 14, or at day 21 from onset of symptoms.
  • the use or method of treatment reduces the severity of the following symptoms comprising fever, headache, respiratory and heart rates, diarrhoea, vomiting, compared to time of randomisation, reduces the survival rate at day 21 or day 28 from onset of symptoms, inhibits the progression to a later phase of the disease, decreases the incidence of organ failure, decreases the time to hospital discharge, the time to return to pre-morbid state, decreases the virus load and available laboratory assessments such as immune response at 2 weeks, and 4 weeks for survivors (IgG and IgM specific Ebola).
  • the invention relates to a combination of an anti-GM-CSF antagonist and an anti-CCR2 antagonist for use in the treatment and/or prevention and/or modulation of the immune response or the cellular response in an individual to an infection with or the exposure to an agent causing hemorrhagic fever as defined in any one of the foregoing paragraphs relating to the invention, wherein said anti-CCR2 antagonist or a fragment thereof has binding specificity for CCR2.
  • Embodiments of combinations referred to in the previous paragraph comprise yet further embodiments, wherein said anti-CCR2 antagonist or fragment thereof is a humanized antibody or fragment thereof.
  • said anti-CCR2 antibody or fragment thereof comprises at least one amino acid sequence having at least 70%, at least 80%, at least 90% or at least 95% identity to the amino acid sequence of any one of SEQ ID NO: 57-60.
  • the anti-CCR2 antibody or a fragment thereof comprises an immunoglobulin heavy chain or fragment thereof comprising the amino acid sequence of SEQ ID NO: 58 and/or the amino acid sequence of SEQ ID NO: 59.
  • the said anti-CCR2 antibody or a fragment thereof comprises an immunoglobulin heavy chain or fragment thereof comprising the amino acid sequence of SEQ ID NO: 58 and/or the amino acid sequence of SEQ ID NO: 59.
  • the said anti-CCR2 antibody or a fragment thereof comprises an
  • immunoglobulin light chain or fragment thereof comprising the amino acid sequence of SEQ ID NO: 57 and/or the amino acid sequence of SEQ ID NO: 60.
  • said antibody or a fragment thereof comprises an immunoglobulin heavy chain or fragment thereof comprising the amino acid sequence of SEQ ID NO: 58 and/or the amino acid sequence of SEQ ID NO: 59 in combination with an immunoglobulin light chain or fragment thereof comprising the amino acid sequence of SEQ ID NO: 57 and/or the amino acid sequence of SEQ ID NO: 60.
  • said anti-CCR2 antagonist specifically binds mammalian CCR2, in particular human CCR2.
  • said anti-GM-CSF antagonist is a neutralizing antibody or a fragment thereof.
  • said anti-GM-CSF antibody or fragment thereof comprises in its heavy chain variable region a CDR 3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-13 or 56.
  • said anti-GM-CSF antibody or a fragment thereof may comprise a heavy chain variable region CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-13 or 56 in combination with a heavy chain variable region CDR1 having an amino acid sequence set out in SEQ ID NO: 14 and a heavy chain variable region CDR2 having an amino acid sequence set out in SEQ ID NO: 15.
  • said anti-GM-CSF antibody or a fragment thereof may comprise a light chain variable region CDR1 having an amino acid sequence set out in SEQ ID NO: 16, a CDR2 having an amino acid sequence set out in SEQ ID NO: 17 and a CDR3 having an amino acid sequence set out in SEQ ID NO: 18.
  • said anti-GM-CSF antibody or fragment thereof comprises a light chain variable region CDR1 having an amino acid sequence as set out in SEQ ID NO: 16, a CDR2 having an amino acid sequence set out in SEQ ID NO: 17 and a CDR3 having an amino acid sequence set out in SEQ ID NO: 18 and comprising a heavy chain variable region CDR1 having an amino acid sequence set out in SEQ ID NO: 14, a CDR2 having an amino acid sequence set out in SEQ ID NO: 15 and a CDR3 having an amino acid sequence set out in SEQ ID NO: 2.
  • said anti-GM-CSF antibody or fragment thereof comprises a light chain variable region sequence set out in SEQ ID NO: 19 and/or a heavy chain variable region sequence set out in SEQ ID NO: 21.
  • said anti-GM-CSF antibody or a fragment thereof comprises a light chain sequence set out in SEQ ID NO: 34 and/or a heavy chain sequence set out in SEQ ID NO: 35.
  • said neutralizing antibody or fragment thereof comprises at least one amino acid sequence having at least 70%, at least 80%, at least 90% or at least 95% identity to the amino acid sequence of any of SEQ ID NO: lb-48 and/or 52-56.
  • said anti-GM-CSF antagonist specifically binds primate GM-CSF.
  • said fragment may be an scFv, a single domain antibody, an Fv, a VHH antibody, a diabody, a tandem antibody, a Fab, a Fab' or a F(ab) 2 .
  • the anti-GM-CSF antagonist for use according to the present invention preferably neutralizes GM-CSF.
  • E10 and G9 are IgG class antibodies.
  • E10 has an 870 pM binding affinity for GM-CSF and G9 has a 14 pM affinity for GM-CSF. Both antibodies are specific for binding to human GM-CSF and show strong neutralizing activity as assessed with a TF-1 cell proliferation assay.
  • human anti-GM-CSF antibodies as disclosed in WO2006/122797.
  • GM-CSF antagonists or neutralizers that are anti-GM-CSF receptor antibodies can also be employed in the present invention.
  • Such GM-CSF antagonists include antibodies to the GM-CSF receptor alpha chain or beta chain.
  • An anti-GM-CSF receptor antibody employed in the invention can be in any antibody form as explained above, e.g., intact, chimeric, monoclonal, polyclonal, antibody fragment or derivative, single-chain, humanized, humaneered, and the like.
  • anti-GM-CSF receptor antibodies, e.g., neutralizing high-affinity antibodies, suitable for use in the invention are known in the art (see e.g., US Patent 5,747,032 and Nicola et al, Blood 82: 15 1724, 1993).
  • sequences for suitable antibodies are provided in applications and are
  • Anti-GM-CSF antibodies are provided in WO2006/122797, WO2007/049472, WO2007/092939, WO2009/134805,
  • the present invention also relates to a combination for use in the treatment and/or prevention of an infection with an agent causing hemorrhagic fever according to any one of the preceding claims, wherein said combination is used in combination with further drugs for use in the treatment and/or prevention of an infection with an agent causing hemorrhagic fever, optionally comprising a filovirus, further optionally comprising Ebola virus, wherein said drug is selected from a group comprising antibodies, comprising the antibody ZMAPP, vaccines against agents causing hemorrhagic fever, medicaments for the treatment of fever, inflammation, infectious diseases, diarrhea, pain, vomiting, bleeding, hypotonia, virus infections, or any other symptoms associated with hemorrhagic fever.
  • GM-CSF antagonists and anti-CCR-2 antagonists may be used in combination with vaccines and/or therapeutics against agents causing hemorrhagic fever, e.g. Ebola virus.
  • Antiviral agents may comprise (i) compounds directly targeting the virus, e.g. the viral polymerase, and/or (ii) compounds that target the host- viral life-cycle interaction (e.g. the budding, vesicle fusion, trafficking, sorting, packaging, etc.).
  • Vaccines and/or therapeutics against Ebola virus comprise Vesicular Stomatitis Virus (VSV) comprising and expressing Ebola-derived antigens (e.g.
  • VSV-EBOV manufactured by NewLink Genetics Corp., BioProtection Corp., or VesiculoVax Ebola and Marburg virus developed by Profectus Biosciences Inc.
  • modified adenoviruses comprising and expressing Ebola antigens (e.g. cAd3-EBO developed by Glaxo-Smith- Kline), Vaccinia virus (e.g. MVA)-derived vaccines comprising and expressing Ebola antigens (e.g. MVA-BN Filo manufactured by Bavarian Nordic A/S), synthetic vaccines (e.g. SynCon Ebola and Marburg virus developed by Inovio Pharmaceuticals Inc.), siRNA molecules blocking virus replication (e.g. TKM-Ebola manufactured by Tekmira
  • RNA-antisense molecules inhibiting VP24 gene expression e.g. AVI-7537 manufactured by Sarepta Therapeutics Inc.
  • inhibitors of viral RNA e.g. AVI-7537 manufactured by Sarepta Therapeutics Inc.
  • antivial agents comprise retrovirus protease inhibitor, optionally selected from the group comprising darunavir, atazanavir, indinavir, lopinavir, ritonavir, and saquinavir.
  • semicarbazone proteasome inhibitors for use, structural and/or functional analogue or a derivative thereof, dipeptidyl-boronic acid derivatives, or a pharmaceutically acceptable salt of either, optionally selected from the group comprising the semicarbazone S-2209 ([l-[l- ⁇ l-[(2,4- Dioxo- imidazo lidin-l-ylimino)-methyl] -2-phenyl-ethylcarbamoyl ⁇ -2-(lH-indo 1-3-yl)- ethylcarbamoyl]-2-(lH-indol)]).
  • This list of antiviral agents is not considered limiting. Any additional presently existing antiviral agents that alleviate, ameliorate, prevent and/or cure infections with the herein disclosed viruses are explicitly contemplated for combined treatments with the disclosed antagonists of GM-CSF and CCR-2.
  • the herein disclosed antagonists of GM- CSF e.g. neutralizing antibodies or fragments thereof, and CCR-2 antagonists are administered without additional presently existing antiviral agents.
  • the present invention also relates to a method of treatment and/or prevention and/or modulation of the immune response or cellular response in an individual infected with Ebola virus or any other agent causing hemorrhagic fever, said method comprising administering a combination as defined in any one of the foregoing paragraphs.
  • the method comprises the administration of said combination to
  • c. provide time for the individual's immune system to raise a response.
  • the present invention also relates to a method of treatment and/or prevention and/or modulation of the immune response or cellular response in an individual infected with Ebola virus or any other agent causing hemorrhagic fever comprising:
  • neutrophils vi. Inhibition of agent-induced cytokine cascade comprising release of monocyte - derived, macrophage-derived, dendritic cell-derived, immune cell-derived, endothelial cell-derived cytokines;
  • monocytes macrophages, dendritic cells and neutrophils
  • JAK/STAT JAK/STAT, IRF, and/or dsRNA sensors
  • symptoms selected from hypotonia, loss of body fluids, fever, blood loss, diarrhea, sore throat, muscle pain and headaches.
  • the active ingredients of the combination i.e. antagonists of GM-CSF and CCR2, respectively, may be administered together or individually in a single composition or in two separate compositions. Further, it is possible to administer different dosages of the respective antagonists, or to administer the same interrupted by an interval, e.g. 30 minutes, 1 hour, 6 hours or one day or more.
  • the present invention relates to methods of treatment and/or prevention of an infection with an agent causing hemorrhagic fever as defined in the preceding sections, wherein the individual treated is a human or a non-human primate.
  • anti-GM-CSF antagonist and “anti-CCR2 antagonist” is occasionally used, which means that compounds, e.g. antibodies, are meant that antagonize GM-CSF and/or CCR-2. It is clear that the term does not relate to antagonists of compounds antagonizing GM-CSF and/or CCR-2.
  • the term "antibody” or its grammatically related variations relate to full length antibodies, human antibodies, humanized antibodies, fully human antibodies, genetically engineered antibodies (e.g. monoclonal antibodies, polyclonal antibodies, chimeric antibodies, recombinant antibodies) and multispecific antibodies, as well as to fragments of such antibodies retaining the characteristic binding properties of the full length antibody.
  • the antibody used in the present invention is a human antibody, in particular a human monoclonal antibody.
  • the human monoclonal antibody of the invention is the result of overcoming significant technical hurdles generally acknowledged to exist in the field of antibody technology.
  • the monoclonal nature of the antibody makes it particularly well suited for use as a therapeutic agent, since such antibody will exist as a single, homogeneous molecular species which can be well-characterized and reproducibly made and purified. These factors result in a product whose biological activity can be predicted with a high level of precision. This is very important if such a molecule is going to gain regulatory approval for therapeutic administration in humans.
  • the monoclonal antibody (or the corresponding fragment) according to the invention be a human antibody (or the corresponding fragment).
  • this antibody is of human origin. Following administration to a human patient, a human antibody or fragment thereof will most probably not elicit a strong immunogenic response by the patient's immune system, i.e. will not be recognized as being a "foreign", that is a non-human protein. This means that no patient antibodies will be generated against the therapeutic antibody which would otherwise block the therapeutic antibody's activity and/or accelerate the therapeutic antibody's elimination from the body of the patient, thus preventing it from exerting its desired therapeutic effect.
  • human antibody as used herein is to be understood as meaning that the antibody of the invention, or its fragment, comprises (an) amino acid sequence(s) contained in the human germ line antibody repertoire.
  • an antibody, or its fragment may therefore be considered human if it consists of such (a) human germ line amino acid sequence(s), i.e. if the amino acid sequence(s) of the antibody in question or fragment thereof is (are) identical to (an) expressed human germ line amino acid sequence(s).
  • An antibody or fragment thereof may also be regarded as human if it consists of (a) sequence(s) that deviate(s) from its (their) closest human germ line sequence(s) by no more than would be expected due to the imprint of somatic hyper mutation.
  • the antibodies of many non-human mammals for example rodents such as mice and rats, comprise VH CDR3 amino acid sequences which one may expect to exist in the expressed human antibody repertoire as well. Any such sequence(s) of human or non-human origin which may be expected to exist in the expressed human repertoire would also be considered "human" for the purposes of the present invention.
  • the human monoclonal antibody may be an IgG antibody.
  • IgG antibody relates to a therapeutically useful antibody or fragment thereof falling within the IgG class (isotype) of antibodies and having a gamma-type ( ⁇ ) heavy chain.
  • an IgG antibody comprises not only the variable antibody regions responsible for the highly discriminative antigen recognition and binding, but also the constant regions of the heavy and light antibody polypeptide chains normally present in endogenously produced antibodies and, in some cases, even decoration at one or more sites with carbohydrates.
  • Such glycosylation is generally a hallmark of the IgG format, and portions of these constant regions make up the so-called Fc region of a full antibody which is known to elicit various effector functions in vivo.
  • the Fc region mediates binding of IgG to Fc receptor, hence prolonging half-life in vivo as well as facilitating homing of the IgG to locations with increased Fc receptor presence.
  • the IgG antibody is an IgGl antibody or an IgG4 antibody, formats which are particularly desirable in the context of the present invention since their mechanism of action in vivo is particularly well understood and characterized. This is especially the case for IgGl antibodies.
  • the antibody according to the present invention is an IgGl antibody.
  • antibody fragment or “fragment thereof or its grammatically related variations relate to a part of a full length antibody specifically binding with the same antigen, i.e. primate GM-CSF as the full length antibody or mammalian CCR2.
  • a pharmaceutically active fragment of an antibody i.e. having the same pharmaceutical effects as the full length anti-GM-CSF antibody or mammalian CCR2.
  • This part of a full length antibody may be at least the antigen binding portion or at least the variable region thereof.
  • Genetically engineered proteins acting like an antibody are also included within the meaning of antibody fragment as used herein.
  • Such genetically engineered antibodies may be scFv, i.e.
  • antibody fragments according to the present invention are Fab, Fab', F(ab')2, VHH antibodies, diabodies, tandem antibodies, single domain antibodies and Fv.
  • These formats may generally be divided into two subclasses, namely those which consist of a single polypeptide chain, and those which comprise at least two polypeptide chains.
  • Members of the former subclass include a scFv (comprising one VH region and one VL region joined into a single polypeptide chain via a polypeptide linker); a single domain antibody (comprising a single antibody variable region) such as a VHH antibody
  • a diabody comprising two non-covalently associated polypeptide chains, each of which comprises two antibody variable regions - normally one VH and one VL per polypeptide chain - the two polypeptide chains being arranged in a head-to-tail conformation so that a bivalent antibody molecule results); a tandem diabody (bispecific single-chain Fv antibodies comprising four covalently linked immunoglobulin variable - VH and VL -regions of two different specificities, forming a homodimer that is twice as large as the diabody described above); a Fab (comprising as one polypeptide chain an entire antibody light chain, itself comprising a VL region and the entire light chain constant region and, as another polypeptide chain, a part of an antibody heavy chain comprising a complete VH region and part of the heavy chain constant region, said two polypeptide chains being intermole
  • antibody fragments of the type described herein allow great flexibility in tailoring, for example, the pharmacokinetic properties of an antibody desired for therapeutic administration to the particular exigencies at hand. For example, it may be desirable to reduce the size of the antibody administered in order to increase the degree of tissue penetration when treating tissues known to be poorly vascularized (for example, joints). Under some circumstances, it may also be desirable to increase the rate at which the therapeutic antibody is eliminated from the body, said rate generally being accelerable by decreasing the size of the antibody administered.
  • the antibody according to the present invention or the fragment thereof neutralizes the activity of primate GM-CSF or specifically binds mammalian CCR2.
  • "neutralization,” “neutralizer,” “neutralizing”, “specifically binds”, and grammatically related variants thereof refer to partial or complete attenuation of the biological effect(s) of GM-CSF or CCR2.
  • Such partial or complete attenuation of the biological effect(s) of GM-CSF or mammalian CCR2 results from modification, interruption and/or abrogation of GM-CSF-mediated or mammalian CCR2 -mediated signal transduction, as manifested, for example, in altering activation of cells, e.g.
  • the anti-CCR2 antibody has been engineered for its Fc region to contain two point mutations that abolish ADCC and CDC effector function. Said antibody does not promote cytokine release. It binds to CCR2 and blocks MCP-1 -binding and MCP-1 -mediated cellular functions, which can be measured using appropriate tests.
  • an agent for example an antibody in question or a fragment thereof is to be classified as a neutralizer.
  • this may be accomplished by a standard in vitro test performed generally as follows: In a first proliferation experiment, a cell line, the degree of proliferation of which is known to depend on the activity of GM-CSF, is incubated in a series of samples with varying concentrations of GM-CSF, following which incubation the degree of proliferation of the cell line is measured. From this measurement, the concentration of GM-CSF allowing half-maximal proliferation of the cells is determined.
  • a second proliferation experiment is then performed employing in each of a series of samples the same number of cells as used in the first proliferation experiment, the above- determined concentration of GM-CSF and, this time, varying concentrations of an antibody or fragment thereof suspected of being a neutralizer of GM-CSF.
  • Cell proliferation is again measured to determine the concentration of antibody or fragment thereof sufficient to effect half-maximal growth inhibition. If the resulting graph of growth inhibition vs. concentration of antibody (or fragment thereof) is sigmoid in shape, resulting in decreased cell proliferation with increasing concentration of antibody (or fragment thereof), then some degree of antibody-dependent growth inhibition has been effected, i.e. the activity of GM-CSF has been neutralized to some extent.
  • the antibody or fragment thereof may be considered a "neutralizer" in the sense of the present invention.
  • a cell line the degree of proliferation of which is known to depend on the activity of GM-CSF, is the TF-1 cell line, as described in
  • the degree of cellular proliferation is not the only parameter by which neutralizing capacity may be established.
  • measurement of the level of signaling molecules e.g. cytokines
  • the level of secretion of which depends on GM-CSF may be used to identify a suspected GM-CSF neutralizer.
  • Other examples of cell lines which can be used to determine whether an antibody in question or fragment thereof is a neutralizer of primate GM-CSF activity include AML-193 (Lange, B. et al. (1987). Blood 70, 192-9); GF-D8 (Rambaldi, A. et al. (1993).
  • binds specifically or “specifically binding” or grammatically related variations thereof relate to an antibody having a binding affinity to primate GM- CSF or CCR2 as defined herein of ⁇ 10 ⁇ 9 mol/1.
  • the antibody or a fragment thereof bind to primate GM-CSF with extremely high affinity. KD values of from about 4 x 10 ⁇ 9 M down to as low as about 0.04 x 10 ⁇ 9 M, the latter corresponding to about 40 pM, have been observed for molecules of this class.
  • the high binding affinity of human monoclonal antibodies or fragments thereof to primate GM-CSF has an additional advantage. Normally, antibodies or fragments thereof will be eliminated from the bloodstream of a patient in a size-dependent fashion, with smaller molecules being excreted and eliminated before larger ones. Since the complex of the two polypeptides - antibody or antibody fragment and bound GM-CSF - is obviously larger than the antibody alone, the low k 0ff mentioned above has the effect that therapeutic neutralizer is excreted and eliminated from the patient's body more slowly than would be the case, were it not bound to GM-CSF. Thus, not only the magnitude of the neutralizing activity but also its duration in vivo is increased.
  • the primate GM-CSF to which the antibody or fragment thereof specifically binds is human GM-CSF (Homo sapiens, SEQ ID NO: 49) or non-human primate GM-CSF.
  • non-human primate GM-CSF include gibbon monkey GM-CSF (Nomascus concolor, also known as the western black crested gibbon, SEQ ID NO: 51) and GM-CSF of monkeys of the macaca family (SEQ ID NO: 50), for example rhesus monkey (Macaca mulatta) GM-CSF and cynomolgous monkey GM-CSF (Macaca fascicularis).
  • the human monoclonal antibody or fragment thereof exhibits cross reactivity between both human and at least one of the monkey species mentioned above.
  • This is especially desirable for an antibody molecule which is intended for therapeutic administration in human subjects, since such an antibody will normally have to proceed through a multitude of tests prior to regulatory approval, of which certain early tests involve non-human animal species.
  • it is generally desirable to use as a non-human species a species bearing a high degree of genetic similarity to humans, since the results so obtained will generally be highly predictive of corresponding results which may be expected when administering the same molecule to humans.
  • the human monoclonal antibody or fragment thereof specifically binds to an epitope, in particular to a discontinuous epitope, of human or non-human primate GM-CSF comprising amino acids 23-27 (R LLN) and/or amino acids 65-77 (GLR/QGSLTKLKGPL).
  • the variability at position 67 within the amino acid sequence stretch 65-77 depicted above reflects the heterogeneity in this portion of primate GM-CSF between, on the one hand, human and gibbon GM-CSF (in which position 67 is R) and, on the other hand, monkeys of the macaca family, for example cynomolgous and rhesus monkeys (in which position 67 is Q).
  • human and non-human primate GM-CSF refers to that of mature GM-CSF, i.e. GM-CSF without its 17 amino acid signal sequence (the total length of mature GM-CSF in both human and non-human primate species described above is 127 amino acids).
  • the sequence of human GM-CSF and gibbon GM-CSF is as follows: APARSPSPST QPWEHVNAIQ EARRLLNLSR DTAAEMNETV EVISEMFDLQ EPTCLQTRLE LYKQGLRGSL TKLKGPLTMM ASHYKQHCPP TPETSCATQI ITFESFKENL KDFLLVIPFD CWEPVQE. (SEQ ID NO: 49)
  • GM-CSF The sequence of GM-CSF in certain members of the macaca monkey family such as for example rhesus monkey and cynomolgous monkey is as follows:
  • the minimum epitope, advantageously a discontinuous epitope, bound by the human monoclonal antibody of the invention (or fragment thereof) as described herein is indicated in the above GM-CSF sequence in boldface.
  • discontinuous epitope is to be understood as at least two non-adjacent amino acid sequence stretches within a given polypeptide chain, here mature human and non-human primate GM-CSF, which are simultaneously and specifically (as defined above) bound by an antibody. According to this definition, such simultaneous specific binding may be of the GM-CSF polypeptide in linear form.
  • the mature GM-CSF polypeptide forming an extended loop, in one region of which the two sequences indicated in boldface above line up, for example more or less in parallel and in proximity of one another. In this state they are specifically and simultaneously bound by the antibody fragment of the invention.
  • simultaneous specific binding of the two sequence stretches of mature GM-CSF indicated above may also take the form of antibody binding to a conformational epitope.
  • mature GM-CSF has already formed its tertiary conformation as it normally exists in vivo (Sun, H. W., J. Bernhagen, et al. (1996). Proc Natl Acad Sci USA 93, 5191-6).
  • the polypeptide chain of mature GM-CSF is folded in such a manner as to bring the two sequence stretches indicated above into spatial proximity, for example on the outer surface of a particular region of mature, folded GM-CSF, where they are then recognized by virtue of their three-dimensional conformation in the context of the surrounding polypeptide sequences.
  • the above (discontinuous) epitope to which the antibody or the fragment thereof specifically binds further comprises amino acids 28-31 (LSRD), italicized in the above sequences of human and non-human primate GM-CSF.
  • either of the above (discontinuous) epitopes further comprises amino acids 32-33 (TA) and/or amino acids 21-22 (EA), each of which stretch is underlined in the above sequences of human and non-human primate GM-CSF.
  • the human monoclonal antibody or fragment thereof, or compositions or medicaments according to the invention comprising such antibodies or fragments comprise in its heavy chain variable region a CDR3 comprising an amino acid sequence chosen from the group consisting of those set out in any of the SEQ ID NOs: 1-13 or 56, optionally SEQ ID NO: 2.
  • One embodiment relates to a human monoclonal antibody or fragment thereof comprising a heavy chain variable region CDRl sequence as set out in SEQ ID NO: 14, a heavy chain variable region CDR2 sequence as set out in SEQ ID NO: 15 and a heavy chain variable region CDR3 sequence as set out in SEQ ID NO: 1; or comprising a heavy chain variable region CDRl sequence as set out in SEQ ID NO: 14, a heavy chain variable region CDR2 sequence as set out in SEQ ID NO: 15 and a heavy chain variable region CDR3 sequence as set out in SEQ ID NO: 2; or comprising a heavy chain variable region CDRl sequence as set out in SEQ ID NO: 14, a heavy chain variable region CDR2 sequence as set out in SEQ ID NO: 15 and a heavy chain variable region CDR3 sequence as set out in SEQ ID NO: 3; or comprising a heavy chain variable region CDRl sequence as set out in SEQ ID NO: 14, a heavy chain variable region CDR2 sequence as set out in SEQ ID NO: 15 and
  • any of the above 14 combinations of CDR1, CDR2 and CDR3 sequences exists in a human monoclonal antibody or fragment thereof further comprising in its light chain variable region a CDR1 comprising the amino acid sequence set out in SEQ ID NO: 16, a CDR2 comprising the amino acid sequence set out in SEQ ID NO: 17, and a CDR3 comprising the amino acid sequence set out in SEQ ID NO: 18.
  • the antibodies or fragments thereof may be derivatized, for example with an organic polymer, for example with one or more molecules of polyethylene glycol (“PEG”) and/or polyvinyl pyrrolidone (“PVP").
  • PEG polyethylene glycol
  • PVP polyvinyl pyrrolidone
  • PEG molecules derivatized as PEG-maleimide, enabling conjugation with the antibody or fragment thereof in a site-specific manner via the sulfhydryl group of a cysteine amino acid.
  • PEG-maleimide especially preferred are 20 kD and/or 40 kD PEG-maleimide, in either branched or straight-chain form. It may be especially desirable to increase the effective molecular weight of smaller human anti-primate GM-CSF antibody fragments or anti- mammalian CCR2 fragments such as scFv fragments by coupling the latter to one or more molecules of PEG, especially PEG-maleimide.
  • anti-GM-CSF antibodies and fragments may be conducted through any method known in the art and is disclosed in detail in WO2006/111353, the contents of which are incorporated herein in their entirety.
  • Another aspect of the present invention relates to a pharmaceutical composition for use in the treatment and/or prevention of any of the conditions according to the appending claims, comprising the antibody or a fragment thereof specifically binding (primate) GM- CSF and specifically binding mammalian CCR2.
  • the pharmaceutical composition for use according to the present invention may further comprise at least one pharmaceutically acceptable carrier.
  • pharmaceutically acceptable relates to any compound which may be used in a pharmaceutical composition without causing any undesired effects (such as negative side effects) in a patient to which the composition is administered.
  • Pharmaceutically acceptable carriers may be those well known in the art such as phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions, liposomes, etc.. It is to be understood that the pharmaceutical composition for use according to the present invention may further include any compound considered suitable by the person skilled in the art, selected e.g. depending from the mode of administration for which the pharmaceutical composition is prepared. Preparations for parenteral administration include e.g. sterile aqueous or nonaqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents examples include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.
  • compositions of the present invention may also be present in the composition of the present invention such as, for example, antimicrobials, anti-oxidants, chelating agents, inert gases and the like.
  • pharmaceutical composition of the present invention might comprise proteinaceous carriers, like, e.g., serum albumin or
  • immunoglobulin preferably of human origin.
  • the antagonists, neutralizing antibodies, specifically binding antibodies and/or functional fragments thereof or the pharmaceutical composition comprising the same should provide sufficient stability upon storage. It is possible to produce a wide variety of proteins for therapeutic applications. After their production, protein pharmaceuticals are usually stored prior to their use. Due to the fact that proteins are generally larger and more complex than "traditional" pharmaceuticals, formulation and processing of protein pharmaceuticals that are suitable for storage can be particularly challenging. For reviews of protein pharmaceutical formulation and process design, see Carpenter et al. (1997), Pharm. Res. 14: 969-975; Wang (2000), Int. J. Pharmaceutics 203: 1 -60; and Tang and Pikal (2004), Pharm. Res. 21 : 191-200. Several factors can be considered in designing formulations and processes for protein pharmaceutical production.
  • Stable formulation comprising the anti-GM-CSF antagonist, a neutralizing anti-GM-CSF antibody or fragments thereof according to the present invention may be an aqueous solution, wherein the antibody or fragments thereof are directly dissolved and/or dispersed therein.
  • One embodiment of the present invention is a liquid formulation containing the antagonist, antibody or fragments thereof which is stable and does not undergo the formation of conjugates/aggregates or fragments/degradation products when stored for a long period, and which formulation is suitable for subcutaneous administration.
  • the antagonist, the neutralizing anti-GM-CSF antibody or fragments thereof could be stabilized if a tonicity modifier is added to the solution which is to be stored.
  • tonicity modifiers include, but are not limited to, sugars and sugar alcohols.
  • Simple sugars are called monosaccharides and include glucose, fructose, galactose, xylose, ribose, mannose, lactulose, allose, altrose, gulose, idose, talose, arabinose and lyxose.
  • disaccharides which include for example sucrose, maltose, lactose, isomaltose, trehalose and cellubiose may be used.
  • Sugar alcohols include sorbitol, mannitol, glycerin, erythritol, maltitol, xylitol, polyglycitol.
  • the sugar is a non-reducing sugar such as sucrose or trehalose.
  • Non- reducing sugars are characterized by the absence of an open chain structure, so they are not susceptible to oxidation-reduction reactions. Therefore one or more of non-reducing sugars, such as sucrose or trehalose, or one or more of sugar alcohols, such as mannitol or sorbitol could be added to the formulation comprising a neutralizing antibody or a fragment thereof as described herein.
  • non-reducing sugars and sugar alcohols could be added to the solution, such as sucrose and mannitol, sucrose and sorbitol, trehalose and mannitol, or trehalose and sorbitol.
  • the sugar alcohols mannitol and/or sorbitol are added, optionally in their D-form, more specifically sorbitol is added to the solution.
  • the concentration of the tonicity modifier, optionally sorbitol is between about 1% and about 15% (w/v), optionally between about 2% and about 10% (w/v), specifically between about 3% and about 7% (w/v), more specifically between about 4% and about 6%> (w/v) and most preferably about 5% (w/v).
  • a buffer system with a pH of between about 4 and about 10, optionally between about 4 and about 7, specifically between about 4 and about 6 or between about 5 and about 7, more specifically between about 5.5 and about 6.5, or with a pH of about 5.8.
  • the buffer may be selected from a histidine buffer, an acetate buffer and a citrate buffer.
  • an amino acid is meant to be an L-amino acid or D-amino acid, wherein L-amino is preferred.
  • histidine or a salt thereof is used for the buffer system.
  • the salt is a chloride, phosphate, acetate or sulphate, optionally the salt is a chloride.
  • the pH of the histidine buffer system is between about 5 and about 7, optionally between about 5.5 and about 6.5, specifically the pH is about or exactly 5.8. The pH may be adjusted by the use of conventionally used bases and acids, optionally NaOH.
  • the concentration of the buffer system, optionally the histidine buffer system is between about 10 mM and about 50 mM, optionally between about 20 mM and about 40 mM, specifically about 30mM.
  • a combination of the buffer system, optionally the histidine buffer, and the tonicity modifier, optionally the sugar alcohol, specifically mannitol or sorbitol is used to stabilize the antagonist, the neutralizing anti-GM-CSF antibody or fragments thereof in the solution, in order to prevent aggregation and to render the formulation sufficiently stable for long-term storage and/or for one or more freeze/thaw cycles. It was shown that it is preferable in terms of stability to have about 6% (w/v) and higher of sugar alcohol, optionally sorbitol, in the formulation. However, the upper limit for osmolality of the formulation is set to be about 470mOsm/kg which is still hyperosmotic.
  • the concentration of sugar alcohol, optionally sorbitol is therefore between about 3% and about 7% (w/v), optionally between about 4% and about 6% (w/v) and specifically about 5% (w/v).
  • the formulations or compositions of the invention comprising the neutralizing anti-GM-CSF antibody or fragments thereof do not require further excipients in addition to those disclosed above (i.e., a buffer and a tonicity modifier), such as, for example, surfactants and amino acids, which are used in traditional formulations to stabilize proteins in solution.
  • a buffer and a tonicity modifier such as, for example, surfactants and amino acids, which are used in traditional formulations to stabilize proteins in solution.
  • the formulations described herein are preferred over standard formulations because they have decreased immunogenicity due to the lack of additional agents commonly needed for protein stabilization.
  • amino acids are useful to stabilize proteins at a high concentration by, inter alia, mediating protein solubility and/or inhibiting protein aggregation.
  • threonine e.g. at 250mM
  • the liquid formulation comprising the antagonist, the neutralizing anti-GM-CSF antibody or fragments thereof is optionally free from further amino acids.
  • the present formulation is free or essentially free of sodium chloride.
  • essentially free is meant that the concentration of sodium chloride is at or very near to 0 (zero) mM, e.g. less than about 50 mM, optionally less than about 20 mM, less than about 10 mM, less than about 5 mM, less than about 2b mM or even less than about 1 mM.
  • the addition of surfactants can be useful to reduce protein degradation during storage.
  • the polysorbates 20 and 80 (Tween 20 and Tween 80) are well established excipients for this purpose.
  • the polysorbate 20 to protein ratio is between about 0.01 : 1 to about 3: 1, preferably between about 0.05: 1 to about 2:1, more preferably between about 0.1 : 1 and about 1.5: 1, even more preferably between about 0.1 :1 to about 0.8: 1, and most preferably between about 0.1 : 1 to about 0.2: 1.
  • the polysorbate 20 concentration is between about 0.001% (w/v) and about 0.2% (w/v), preferably between about 0.005%) (w/v) and about 0.15% (w/v), more preferably between about 0.007% (w/v) and about 0.1% (w/v), even more preferably between about 0.007%> (w/v) and about 0.06%> (w/v) and most preferably about 0.01% (w/v).
  • the polysorbate 20 concentration is between about 0.001% (w/v) and about 0.4%> (w/v), preferably between about 0.006%> (w/v) and about 0.25%> (w/v), more preferably between about 0.01% (w/v) and about 0.18%) (w/v), even more preferably between about 0.01% (w/v) and about 0.1 % (w/v) and most preferably about 0.02%> (w/v).
  • the polysorbate 80 to protein ratio is between about 0.01 : 1 to about 3:1, preferably between about 0.05: 1 to about 2: 1, more preferably between about 0.1 : 1 and about 1.5: 1, even more preferably between about 0.1 :1 to about 0.6: 1 , and most preferably from about 0.3 : 1 to about 0.6: 1.
  • the polysorbate 80 concentration is between about 0.001% (w/v) and about 0.2%) (w/v), preferably between about 0.004%> (w/v) and about 0.14% (w/v), more preferably between about 0.007%> (w/v) and about 0.1% (w/v), even more preferably between about 0.007%> (w/v) and about 0.05% (w/v), and most preferably about 0.04% (w/v).
  • the polysorbate 80 concentration is between about 0.001% (w/v) and about 0.4% (w/v), preferably between about 0.007%) (w/v) and about 0.26% (w/v), more preferably between about 0.01% (w/v) and about 0.2% (w/v), even more preferably between about 0.01% (w/v) and about 0.08% (w/v), most preferably about 0.04% (w/v).
  • the concentration of the antagonist, the neutralizing anti-GM-CSF antibody or fragments thereof used is at least about 20 mg/ml, at least about 50 mg/ml or at least about 100 mg/ml in the liquid formulation which is to be stored, freeze/thawed and/or ready to use.
  • 200 mg/ml about 100 mg/ml to about 180 mg/ml, about 130 mg/ml to about 170 mg/ml, about 135 mg/ml to about 165 mg/ml or about 150 mg/ml of the (neutralizing) antibody or a fragment thereof may be used in the present invention.
  • the present formulation of the neutralizing anti-GM-CSF antibody or functional fragments thereof comprises from about 80 mg/ml to about 150 mg/ml of the neutralizing antibody, about 5% (w/v) sorbitol, about 30 mM L-histidine, and from about 0.01% to about 0.08% (w/v) polysorbate 80 and has a pH of about 5.8.
  • the present formulation of the antagonist, the anti-GM- CSF antibody or a fragment thereof comprises from about 135 mg/ml to about 165 mg/ml of the neutralizing antibody, about 5% (w/v) sorbitol, about 30 mM L-histidine and has a pH of about 5.8.
  • the present formulation of the antagonist, the anti-GM- CSF antibody or a fragment thereof comprises 150 mg/ml of the neutralizing antibody, about 5% (w/v) sorbitol, about 30mM L-histidine and has a pH of about 5.8.
  • the present formulation of the neutralizing anti-GM- CSF antibody or functional fragments thereof in the combinations of the present invention comprises about 80 mg/ml to of the neutralizing antibody, about 5% (w/v) sorbitol, about 30 mM L-histidine, about 0.04%> (w/v) polysorbate 80 and has a pH of about 5.8.
  • the present formulation of the neutralizing anti-GM- CSF antibody or functional fragments thereof in the combinations of the present invention comprises about 150 mg/ml to of the neutralizing antibody, about 5% (w/v) sorbitol, about 30 mM L-histidine, about 0.04%> (w/v) polysorbate 80 and has a pH of about 5.8.
  • the shelf life of the produced formulation may have a minimum requirement of 24 months at 2 to 8°C, 36 months at 2 to 8°C, 48 months at 2 to 8°C or at least 28 days at ambient temperature (25°C ⁇ 2°C).
  • said anti-CCR2 antagonist is an antibody or a fragment thereof having binding specificity for CCR2.
  • said antibody or fragment thereof is a humanized antibody or fragment thereof.
  • the anti-CCR2 antibody is monoclonal antibody LS 132.1D9 (1D9) or monoclonal antibody LS 132.8G2 (8G2).
  • the anti-CCR2 antibody is a humanized monoclonal 1D9 or 8G2 antibody.
  • the anti-CCR2 antibody is MLN1202.
  • the anti-CCR2 antibody comprises a heavy chain variable region comprising the amino acid sequence depicted in SEQ ID NO:58, which is encoded by the nucleic acid sequence depicted in SEQ ID NO: 62.
  • the anti-CCR2 antibody comprises the heavy chain constant region comprising the amino acid sequence depicted in SEQ ID NO:59, which is encoded by the nucleic acid sequence depicted in SEQ ID NO: 63.
  • the anti-CCR2 antibody comprises a light chain variable region comprising the amino acid sequence depicted in SEQ ID NO:57, which is encoded by the nucleic acid sequence depicted in SEQ ID NO:61.
  • the anti-CCR2 antibody comprises the light chain constant region comprising the amino acid sequence depicted in SEQ ID NO:60, which is encoded by the nucleic acid sequence depicted in SEQ ID NO: 64.
  • said antibody or fragment thereof comprises at least one amino acid sequence having at least 70%, at least 80%, at least 90% or at least 95% identity to the amino acid sequence of any one of SEQ ID NO: 57-60.
  • said antibody or a fragment thereof comprises an immunoglobulin heavy chain or fragment thereof comprising the amino acid sequence of SEQ ID NO: 58 and/or the amino acid sequence of SEQ ID NO: 62.
  • said antibody or a fragment thereof comprises an immunoglobulin light chain or fragment thereof comprising the amino acid sequence of SEQ ID NO: 57 and/or the amino acid sequence of SEQ ID NO: 61.
  • said antibody or a fragment thereof comprises an immunoglobulin heavy chain or fragment thereof comprising the amino acid sequence of SEQ ID NO: 58 and/or the amino acid sequence of SEQ ID NO: 59 in combination with an immunoglobulin light chain or fragment thereof comprising the amino acid sequence of SEQ ID NO: 57 and/or the amino acid sequence of SEQ ID NO: 60.
  • the anti-CCR2 antibody is an anti-CCR2 antibody described in US 2004/0151721, the disclosure of which is incorporated herein by reference.
  • the combinations of the invention thus comprise ananti-CCR2 antibody as described herein, or an anti-CCR2 antibody which can compete for binding to human CCR2 or a portion of human CCR2 with anti-CCR2 antibodies described herein for use as described herein.
  • the fragment is an scFv, a single domain antibody, an Fv, a VHH antibody, a diabody, a tandem antibody, a Fab, a Fab' or a F(ab)2.
  • said antagonist specifically binds mammalian CCR-2, in particular human CCR-2.
  • the present invention also relates to combinations as defined above comprising an antibody (immunoglobulin) or functional fragment thereof (e.g., an antigen-binding fragment) which binds to a mammalian CC-chemokine receptor 2 (also referred to as CCR2, CKR-2, MCP-1RA or MCP-1 RB) or portion of the receptor (anti-CCR2) for the use as described herein.
  • an antibody immunoglobulin
  • functional fragment thereof e.g., an antigen-binding fragment
  • the antibody or fragment thereof for the use as described herein has specificity for human or rhesus CCR2 or a portion thereof.
  • the present invention also relates to combinations as defined above comprising an antibody or fragment blocking, binding of a ligand (e.g., MCP-1, MCP-2, MCP-3, MCP- 4) to the receptor and inhibiting a function associated with binding of the ligand to the receptor (e.g., leukocyte trafficking).
  • a ligand e.g., MCP-1, MCP-2, MCP-3, MCP- 4
  • a function associated with binding of the ligand to the receptor e.g., leukocyte trafficking
  • a chemokine e.g., MCP-1, MCP-2, MCP-3, MCP-4
  • the present invention also relates to combinations as defined above comprising the antibody or functional fragment of the present invention binds human CCR2 or a portion thereof.
  • the antibody is monoclonal antibody 1D9 or an antibody which can compete with 1D9 for binding to human CCR2 or a portion of human CCR2.
  • the present invention also relates to combinations as defined above comprising antibodies which bind to a mammalian CCR2 or portion of the receptor and provides increased fluorescent staining intensity of CCR2 or compositions comprising CCR2 relative to other anti-CCR2 antibodies.
  • the antibody is monoclonal antibody 1D9 or 8G2 or an antibody which can compete with 1D9 or 8G2 for binding to human CCR2 or a portion of human CCR2.
  • the CCR2-antagonist as used herein can be a humanized immunoglobulin or antigen- binding fragment thereof for use as described herein having binding specificity for CCR2, said immunoglobulin comprising an antigen binding region of nonhuman origin (e.g., rodent) and at least a portion of an immunoglobulin of human origin (e.g., a human framework region, a human constant region of the gamma type).
  • the humanized immunoglobulin or fragment thereof described herein can compete with 1D9 for binding to CCR2.
  • the antigen binding region of the humanized immunoglobulin is derived from monoclonal antibody 1D9 (e.g., an immunoglobulin comprising the variable regions of the light and heavy chains as shown in SEQ ID NO: 57 and SEQ ID NO: 58, respectively).
  • the humanized immunoglobulin or antigen-binding fragment thereof can comprise an antigen binding region comprising at least one complementarity determining region (CDR) of nonhuman origin, and a framework region (FR) derived from a human framework region.
  • the humanized immunoglobulin having binding specificity for CCR2 comprises a light chain comprising at least one CDR derived from an antibody of nonhuman origin which binds CCR2 and a FR derived from a light chain of human origin (e.g., from HF- 21/28), and a heavy chain comprising a CDR derived from an antibody of nonhuman origin which binds CCR2 and a FR derived from a heavy chain of human origin (e.g., from 4B4'CL).
  • the light chain comprises three CDRs derived from the light chain of the 1D9 antibody
  • the heavy chain comprises three CDRs derived from the heavy chain of the 1D9 antibody.
  • the CCR2-antagonist as used herein may comprise humanized immunoglobulin light chains and antigen-binding fragments thereof (e.g., comprising CDR1, CDR2 and CDR3 of the light chain of the 1D9 antibody, and a human light chain FR), and humanized immunoglobulin heavy chains and antigen-binding fragments thereof (e.g., comprising CDR1, CDR2 and CDR3 of the heavy chain of the 1D9 antibody, and a human heavy chain FR) for use as described herein.
  • humanized immunoglobulin light chains and antigen-binding fragments thereof e.g., comprising CDR1, CDR2 and CDR3 of the light chain of the 1D9 antibody, and a human light chain FR
  • humanized immunoglobulin heavy chains and antigen-binding fragments thereof e.g., comprising CDR1, CDR2 and CDR3 of the heavy chain of the 1D9 antibody, and a human heavy chain FR
  • the CCR2-antagonist as used herein may also comprise humanized heavy and light chains described herein (e.g., a humanized light chain comprising the variable region of the light chain shown in SEQ ID NO: 57, a humanized heavy chain comprising the variable region of the heavy chain shown in SEQ ID NO: 58 for use as described herein. Also
  • humanized immunoglobulins comprising one or more humanized light and/or heavy chains or use as described herein.
  • the CCR2-antagonist as used herein also relates to a humanized immunoglobulin or antigen-binding fragment thereof having binding specificity for CCR2 comprising a heavy chain and a light chain, wherein said light chain comprises at least one complementarity determining region derived from murine monoclonal antibody 1D9 and a framework region derived from the light chain of human antibody HF-21/28, and wherein said heavy chain comprises at least one complementarity determining region derived from murine monoclonal antibody 1D9 and a framework region derived from the heavy chain of human antibody 4B4'CL for use as described herein.
  • the light chain comprises three complementarity determining regions derived from the light chain of the 1D9 antibody
  • the heavy chain comprises three complementarity determining regions derived from the heavy chain of the 1D9 antibody.
  • the complementarity determining regions derived from the light chain of 1D9 are amino acids 24-39 of SEQ ID NO: 57, amino acids 55-61 of SEQ ID NO: 57 and amino acids 94-102 of SEQ ID NO: 57
  • the complementarity determining regions derived from the heavy chain of 1D9 are amino acids 31-35 of SEQ ID NO: 58, amino acids 50-68 of SEQ ID NO: 58 and amino acids 101-106 of SEQ ID NO: 58.
  • the CCR2-antagonist as used herein also concerns a humanized immunoglobulin or antigen-binding fragment thereof having binding specificity for CCR2 comprising a light chain and a complementary heavy chain, wherein said light chain comprises a variable region comprising SEQ ID NO: 57 for use as described herein.
  • the invention also relates to a humanized immunoglobulin or antigen-binding fragment thereof having binding specificity for CCR2 comprising a heavy chain and a complementary light chain, wherein said heavy chain comprises a variable region comprising SEQ ID NO: 58 for use as described herein.
  • the invention relates to a humanized immunoglobulin or antigen-binding fragment thereof having binding specificity for CCR2 comprising a heavy chain and a light chain, wherein said light chain comprises a variable region comprising SEQ ID NO: 57, and wherein said heavy chain comprises a variable region comprising SEQ ID NO: 58 for use as described herein.
  • the humanized immunoglobulin or antigen-binding fragment can compete with murine antibody 1D9 for binding to CCR2.
  • the humanized immunoglobulin or antigen-binding fragment can compete with murine antibody 1D9 for binding to CCR2.
  • the humanized immunoglobulin or antigen-binding fragment can compete with murine antibody 1D9 for binding to CCR2.
  • the humanized immunoglobulin or antigen-binding fragment can compete with murine antibody 1D9 for binding to CCR2.
  • the humanized immunoglobulin or antigen-binding fragment can compete with murine antibody 1D9 for binding to CCR2.
  • immunoglobulin or antigen-binding fragment inhibits binding of a ligand to CCR2.
  • the CCR2-antagonist as used herein preferably also inhibits the interaction of a cell bearing mammalian (e.g., human, non-human primate or murine) CCR2 with a ligand thereof, comprising contacting the cell with an effective amount of an antibody or functional fragment thereof which binds to a mammalian CCR2 or a portion of CCR2.
  • a cell bearing mammalian (e.g., human, non-human primate or murine) CCR2 with a ligand thereof, comprising contacting the cell with an effective amount of an antibody or functional fragment thereof which binds to a mammalian CCR2 or a portion of CCR2.
  • Suitable cells include granulocytes, leukocytes, such as monocytes, macrophages, basophils and eosinophils, mast cells, and lymphocytes including T cells (e.g., CD8+ cells, CD4+ cells, CD25+ cells, CD45RO+ cells), and other cells expressing CCR2 such as a recombinant cell expressing CCR2 (e.g., transfected cells).
  • the antibody is 1D9 or an antibody which can compete with 1D9 for binding to human CCR2 or a portion of human CCR2.
  • the CCR2-antagonist as used herein preferably also inhibits the interaction of a cell bearing mammalian CCR2 with a chemokine, comprising contacting said cell with an effective amount of an antibody or functional fragment thereof which binds to CCR2 or a portion of said receptor.
  • the antibody or functional fragment thereof is any one or more of 1D9, an antigen-binding fragment of 1D9 or an antibody or fragment thereof having an epitopic specificity which is the same as or similar to that of 1D9.
  • the invention relates to a method of inhibiting a function associated with binding of a chemokine to CCR2, comprising administering an effective amount of an antibody or functional fragment thereof which binds to a mammalian CCR2 protein or a portion of said receptor.
  • the antibody or functional fragment thereof is any one or more of 1D9, an antigen-binding fragment of 1D9 or an antibody or fragment thereof having an epitopic specificity which is the same as or similar to that of 1D9.
  • the CCR2-antagonist as used herein can be an antibody or functional fragment thereof selected from 1D9, an antibody having an epitopic specificity which is the same as or similar to that of 1D9, an antibody which can compete with 1D9 for binding to human CCR2, and antigen-binding fragments thereof.
  • the CCR2-antagonist as used herein can inhibit leukocyte trafficking in a patient, comprising administering to the patient an effective amount of an antibody or functional fragment thereof which binds to a mammalian CCR2 or portion of said receptor and inhibits function associated with binding of a ligand to the receptor.
  • the present invention relates to a combination comprising an antibody (anti-CCR2) or functional fragment thereof which binds mammalian CC-chemokine receptor 2 (CCR2, CKR-2, MCP-1RA or MCP-1RB) or a portion of CCR2 for use as described herein.
  • the antibody has specificity for human or rhesus CCR2 or portion thereof.
  • the antibodies (immunoglobulins) are raised against an isolated and/or recombinant mammalian CCR2 or portion thereof (e.g., peptide) or against a host cell which expresses mammalian CCR2.
  • the antibodies specifically bind human CCR2 receptor(s) (e.g., CCR2a and/or CCR2b) or a portion thereof, and in another embodiment the antibodies have specificity for a naturally occurring or endogenous human CCR2.
  • CCR2 CC-chemokine receptor 2
  • CCR2 refers to CC-chemokine receptor 2a and/or CC-chemokine receptor 2b.
  • Antibodies or functional fragments used herein which can inhibit one or more functions characteristic of a mammalian CCR2, such as a binding activity (e.g., ligand, inhibitor and/or promoter binding), a signaling activity (e.g., activation of a mammalian G protein, induction of a rapid and transient increase in the concentration of cytosolic free calcium [Ca2+]i), and/or stimulation of a cellular response (e.g., stimulation of chemotaxis, exocytosis or inflammatory mediator release by leukocytes, integrin activation) are also encompassed by the present invention, such as an antibody which can inhibit binding of a ligand (i.e., one or more ligands) to CCR2 and/or one or more functions mediated by CCR2 in response to a ligand.
  • a binding activity e.g., ligand, inhibitor and/or promoter binding
  • a signaling activity e.g., activation of a
  • the antibodies or functional fragments thereof can inhibit (reduce or prevent) the interaction of receptor with a natural ligand, such as MCP-1, MCP-2, MCP-3 and/or MCP-4.
  • a natural ligand such as MCP-1, MCP-2, MCP-3 and/or MCP-4.
  • an antibody or functional fragment thereof that binds to CCR2 can inhibit binding of MCP-1, MCP-2, MCP-3 and/or MCP-4 and/or HIV to mammalian CCR2 (e.g., human CCR2, non-human primate CCR2, murine CCR2).
  • the antibodies or functional fragments thereof of the present invention can inhibit functions mediated by human CCR2, including leukocyte trafficking, HIV entry into a cell, T cell activation, inflammatory mediator release and/or leukocyte
  • the antibodies or fragments can bind CCR2 with an affinity of at least about 0.1 x 10 ⁇ 9 M, preferably at least about 1 x 10 ⁇ 9 M, and more preferably at least about 3 x 10 "9 M.
  • antibodies or functional fragments thereof demonstrate inhibition of chemokine -induced (e.g., MCP-1 -induced) chemotaxis of cells (e.g., PBMC) at less than about 150 ⁇ g/ml, preferably less than about 100 ⁇ g/ml, more preferably less than about 50 ⁇ g/ml, and even more preferably less than about 20 ⁇ g/ml.
  • the antibodies or functional fragments thereof can inhibit binding of a CCR2 ligand (e.g., a chemokine) to CCR2 with an IC 50 of less than about 1.0 ⁇ g/ml, preferably less than about 0.05 ⁇ g/ml, and more preferably less than about 0.005 ⁇ g/ml.
  • a CCR2 ligand e.g., a chemokine
  • the antibodies bind human CCR2, and have an epitopic specificity which is the same as or similar to that of murine 1D9 or 8G2 antibody described herein.
  • Antibodies with an epitopic specificity which is the same as or similar to that of murine 1D9 monoclonal antibody can be identified by their ability to compete with murine 1D9 monoclonal antibody for binding to human CCR2 (e.g., to cells bearing human CCR2, such as transfectants bearing CCR2, CD8+ cells, CD4+ cells, CDR45RO+ cells, CD25+ cells, monocytes, dendritic cells, macrophages and basophils).
  • antibodies with an epitopic specificity which is the same as or similar to that of murine 8G2 monoclonal antibody can be identified by their ability to compete with murine 8G2 monoclonal antibody for binding to human CCR2.
  • receptor chimeras (Rucker et al, Cell 87:437-446 (1996))
  • the binding site of mAbs 1D9 and 8G2 has been mapped to the amino-terminal domain of human CC-chemokine receptor 2, specifically to an epitope comprising from about amino acid 1 to about amino acid 30 of the protein.
  • antibodies having an epitopic specificity which is the same as or similar to that of an antibody of the present invention can be identified.
  • mAbs 1D9 and 8G2 have epitopic specificity for the amino-terminal domain of the CCR2 receptor, e.g., from about amino acid number 1 to about amino acid number 30 of the receptor protein.
  • the invention also relates to a bispecific antibody, or functional fragment thereof (e.g., F(ab') 2 ), which has the same or similar epitopic specificity as at least two of the antibodies described herein (see, e.g., U.S. Patent No. 5,141,736 (Iwasa et al), U.S. Patent Nos. 4,444,878, 5,292,668, 5,523,210 (all to Paulus et al) and U.S. Patent No. 5,496,549 (Yamazaki et al).
  • a bispecific antibody can have the same or similar epitopic specificity as mAb 1D9 and 8G2, e.g., binds the amino terminal domain, or portion thereof, of mammalian CCR2 protein.
  • Hybridoma cell lines producing antibodies were deposited on July 17, 1998, on behalf of LeukoSite, Inc., 215 First Street, Cambridge, MA 02142, U.S.A. (now Millennium
  • the present invention also pertains to the hybridoma cell lines deposited under ATCC
  • the antibodies can be polyclonal or monoclonal, and the term "antibody” is intended to encompass both polyclonal and monoclonal antibodies.
  • methods described herein which utilize 8G2 can also utilize functional fragments (e.g., antigen-binding fragments) of 8G2, antibodies which have the same or similar epitopic specificity as 8G2, and combinations thereof, optionally in combination with antibodies or fragments having an epitopic specificity which is not the same as or similar to 8G2;
  • methods described as utilizing 1D9 can also utilize functional fragments of 1D9, antibodies which have the same or similar epitopic specificity as 1D9, and combinations thereof, optionally in combination with antibodies or fragments having an epitopic specificity which is not the same as or similar to 1D9.
  • Antibodies can be raised against an appropriate immunogen, such as isolated and/or recombinant mammalian CCR2 protein or portion thereof, or synthetic molecules, such as synthetic peptides.
  • cells which express receptor such as transfected cells, can be used as immunogens or in a screen for antibody which binds receptor.
  • the antibodies of the present invention are useful in therapeutic, applications as described herein.
  • Preparation of immunizing antigen, and polyclonal and monoclonal antibody production can be performed as described herein, or using other suitable techniques.
  • a variety of methods have been described (see e.g., Kohler et al, Nature, 256: 495-497 (1975) and Eur. J. Immunol. 6: 511-519 (1976); Milstein et al, Nature 266: 550-552 (1977);
  • a hybridoma can be produced by fusing a suitable immortal cell line (e.g., a myeloma cell line such as SP2/0) with antibody producing cells.
  • a suitable immortal cell line e.g., a myeloma cell line such as SP2/0
  • the antibody producing cell preferably those of the spleen or lymph nodes, are obtained from animals immunized with the antigen of interest.
  • the fused cells (hybridomas) can be isolated using selective culture conditions, and cloned by limiting dilution. Cells which produce antibodies with the desired binding properties can be selected by a suitable assay (e.g., ELISA).
  • Suitable methods of producing or isolating antibodies which bind CCR2, including human or artificial antibodies can be used, including, for example, methods which select recombinant antibody (e.g., single chain Fv or Fab) from a library, or which rely upon immunization of transgenic animals (e.g., mice) capable of producing a repertoire of human antibodies (see e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551-2555 (1993); Jakobovits et al, Nature, 362: 255-258 (1993); Lonberg et al, U.S. Patent No. 5,545,806; Surani et al, U.S. Patent No. 5,545,807).
  • recombinant antibody e.g., single chain Fv or Fab
  • Single chain antibodies, and chimeric, humanized or primatized (CDR-grafted) antibodies, as well as chimeric or CDR-grafted single chain antibodies, and the like, comprising portions derived from different species, are also encompassed by the present invention and the term "antibody".
  • the various portions of these antibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques. For example, nucleic acids encoding a chimeric or humanized chain can be expressed to produce a contiguous protein. See, e.g., Cabilly et al , U.S. Patent No. 4,816,567; Cabilly et al, European Patent No.
  • functional fragments of antibodies including fragments of chimeric, humanized, primatized or single chain antibodies, can also be produced.
  • Functional fragments of the foregoing antibodies retain at least one binding function and/or modulation function of the full-length antibody from which they are derived.
  • Preferred functional fragments retain an antigen-binding function of a corresponding full-length antibody (e.g., the ability to bind a mammalian CCR2).
  • Particularly preferred functional fragments retain the ability to inhibit one or more functions characteristic of a mammalian CCR2, such as a binding activity, a signaling activity, and/or stimulation of a cellular response.
  • a functional fragment can inhibit the interaction of CCR2 with one or more of its ligands (e.g., MCP-1 , MCP-2, MCP-3 and/or MCP-4) and/or can inhibit one or more receptor-mediated functions, such as leukocyte trafficking, HIV entry into cells, T cell activation, inflammatory mediator release and/or leukocyte degranulation.
  • ligands e.g., MCP-1 , MCP-2, MCP-3 and/or MCP-4
  • receptor-mediated functions such as leukocyte trafficking, HIV entry into cells, T cell activation, inflammatory mediator release and/or leukocyte degranulation.
  • antibody fragments capable of binding to a mammalian CCR2 receptor or portion thereof including, but not limited to, Fv, Fab, Fab' and F(ab')2 fragments are encompassed by combinations of the invention.
  • Such fragments can be produced by enzymatic cleavage or by recombinant techniques, for example. For instance, papain or pepsin cleavage can generate Fab or F(ab') 2 fragments, respectively.
  • Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons has been introduced upstream of the natural stop site. For example, a chimeric gene encoding a F(ab')2 heavy chain portion can be designed to include DNA sequences encoding the CHi domain and hinge region of the heavy chain.
  • the combinations of the invention relate also to a humanized immunoglobulin or antigen- binding fragment thereof having binding specificity for CCR2 for use as described herein, comprising an antigen binding region of nonhuman origin (e.g., rodent) and at least a portion of an immunoglobulin of human origin (e.g., a human framework region, a human constant region or portion thereof).
  • the humanized immunoglobulin includes an antigen binding region of nonhuman origin which binds CCR2 and a constant region derived from a human constant region.
  • the humanized immunoglobulin which binds CCR2 comprises a complementarity determining region of nonhuman origin and a variable framework region of human origin, and optionally, a constant region of human origin.
  • the humanized immunoglobulin can comprise a heavy chain and a light chain, wherein the light chain comprises a
  • the heavy chain comprises a complementarity determining region derived from an antibody of nonhuman origin which binds CCR2 and a framework region derived from a heavy chain of human origin.
  • the humanized immunoglobulin can compete with murine 1D9 or 8G2 monoclonal antibody for binding to human CCR2.
  • the antigen-binding region of the humanized immunoglobulin (a) is derived from 1D9 monoclonal antibody (e.g., as in a humanized immunoglobulin comprising CDRl, CDR2 and CDR3 of the 1D9 light chain and/or CDRl, CDR2 and CDR3 of the 1D9 heavy chain) or (b) is derived from 8G2 monoclonal antibody (e.g., as in a humanized immunoglobulin comprising CDRl, CDR2 and CDR3 of the 8G2 light chain and/or CDRl, CDR2 and CDR3 of the 8G2 heavy chain).
  • Chimeric or CDR-grafted single chain antibodies are also encompassed by the term humanized immunoglobulin.
  • the disclosure also relates to a humanized immunoglobulin light chain or antigen-binding fragment thereof or a humanized immunoglobulin heavy chain or antigen-binding fragment thereof.
  • the invention relates to a humanized light chain comprising a light chain CDR (i.e., one or more CDRs) of nonhuman origin and a human light chain framework region.
  • the present invention relates to a humanized immunoglobulin heavy chain comprising a heavy chain CDR (i.e., one or more CDRs) of nonhuman origin and a human heavy chain framework region.
  • the CDRs can be derived from a nonhuman immunoglobulin.
  • Naturally occurring immunoglobulins have a common core structure in which two identical light chains (about 24 kD) and two identical heavy chains (about 55 or 70 kD) form a tetramer.
  • the amino-terminal portion of each chain is known as the variable (V) region and can be distinguished from the more conserved constant (C) regions of the remainder of each chain.
  • V variable
  • C constant
  • Within the variable region of the light chain is a C-terminal portion known as the J region.
  • Within the variable region of the heavy chain there is a D region in addition to the J region.
  • Most of the amino acid sequence variation in immunoglobulins is confined to three separate locations in the V regions known as hypervariable regions or complementarity determining regions (CDRs) which are directly involved in antigen binding.
  • CDRs complementarity determining regions
  • the CDRs are held in place by more conserved framework regions (FRs). Proceeding from the amino-terminus, these regions are designated FR1, FR2, FR3, and FR4, respectively.
  • FR1, FR2, FR3, and FR4 Proceeding from the amino-terminus, these regions are designated FR1, FR2, FR3, and FR4, respectively.
  • the locations of CDR and FR regions and a numbering system have been defined by Kabat et al. (Kabat et ah,
  • Human immunoglobulins can be divided into classes and subclasses, depending on the isotype of the heavy chain.
  • the classes include IgG, IgM, IgA, IgD and IgE, in which the heavy chains are of the gamma ( ⁇ ), mu ( ⁇ ), alpha (a), delta ( ⁇ ) or epsilon ( ⁇ ) type, respectively.
  • Subclasses include IgGl, IgG2, IgG3, IgG4, IgAl and IgA2, in which the heavy chains are of the ⁇ , ⁇ 2, ⁇ 3, ⁇ 4, ⁇ and ⁇ 2 type, respectively.
  • immunoglobulin molecules of a selected class or subclass may contain either a kappa (6) or lambda (8) light chain. See e.g., Cellular and Molecular Immunology, Wonsiewicz, M.J., Ed., Chapter 45, pp. 41-50, W. B. Saunders Co, Philadelphia, PA (1991); Nisonoff, A., Introduction to Molecular Immunology, 2nd Ed., Chapter 4, pp. 45-65, Sinauer Associates, Inc., Sunderland, MA (1984).
  • the term "immunoglobulin” as used herein includes whole antibodies and biologically functional fragments thereof.
  • Such biologically functional fragments retain at least one antigen-binding function of a corresponding full-length antibody (e.g., specificity for CCR2 of antibody 1D9), and preferably, retain the ability to inhibit the interaction of CCR2 with one or more of its ligands (e.g., HIV, MCP-1, MCP-2, MCP-3, MCP-4).
  • a corresponding full-length antibody e.g., specificity for CCR2 of antibody 1D9
  • Examples of biologically functional antibody fragments which can be used include fragments capable of binding to CCR2, such as single chain antibodies, Fv, Fab, Fab' and F(abN) 2 fragments. Such fragments can be produced by enzymatic cleavage or by recombinant techniques. For instance, papain or pepsin cleavage can be used to generate Fab or F(ab') 2 fragments, respectively. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site.
  • a chimeric gene encoding the heavy chain of an F(ab') 2 fragment can be designed to include DNA sequences encoding the CHi domain and hinge region of the heavy chain.
  • an antigen-binding fragment of a humanized immunoglobulin heavy or light chain is intended to mean a fragment which binds to an antigen when paired with a complementary chain.
  • an antigen-binding fragment of a humanized light chain will bind to an antigen when paired with a heavy chain (e.g., murine, chimeric, humanized) comprising a variable region
  • an antigen-binding fragment of a humanized heavy chain will bind to an antigen when paired with a light chain (e.g., murine, chimeric, humanized) comprising a variable region.
  • humanized immunoglobulin refers to an immunoglobulin comprising portions of immunoglobulins of different origin, wherein at least one portion is of human origin.
  • the humanized antibody can comprise portions derived from an immunoglobulin of nonhuman origin with the requisite specificity, such as a mouse, and from immunoglobulin sequences of human origin (e.g., chimeric
  • immunoglobulin joined together chemically by conventional techniques (e.g., synthetic) or prepared as a contiguous polypeptide using genetic engineering techniques (e.g., DNA encoding the protein portions of the chimeric antibody can be expressed to produce a contiguous polypeptide chain).
  • a humanized immunoglobulin of the present invention is an immunoglobulin containing one or more immunoglobulin chains comprising a CDR derived from an antibody of nonhuman origin and a framework region derived from a light and/or heavy chain of human origin (e.g., CDR-grafted antibodies with or without framework changes). Chimeric or CDR-grafted single chain antibodies are also encompassed by the term humanized immunoglobulin.
  • humanized immunoglobulins can be produced using synthetic and/or recombinant nucleic acids to prepare genes (e.g., cDNA) encoding the desired humanized chain.
  • genes e.g., cDNA
  • nucleic acid (e.g., DNA) sequences coding for humanized variable regions can be constructed using PCR mutagenesis methods to alter DNA sequences encoding a human or humanized chain, such as a DNA template from a previously humanized variable region (see e.g., Kamman, M., et al, Nucl Acids Res., 17: 5404 (1989)); Sato, K., et al, Cancer Research, 53: 851-856 (1993); Daugherty, B.L.
  • variants can also be readily produced.
  • cloned variable regions can be mutagenized, and sequences encoding variants with the desired specificity can be selected (e.g., from a phage library; see e.g., Krebber et al, U.S. 5,514,548; Hoogenboom et al, WO 93/06213, published April 1, 1993)).
  • the antigen binding region of the humanized immunoglobulin can be derived from an immunoglobulin of nonhuman origin (referred to as a donor immunoglobulin) having binding specificity for CCR2.
  • a suitable antigen binding region can be derived from the murine monoclonal antibody 1D9.
  • Other sources include CCR2-specific antibodies obtained from nonhuman sources, such as rodent (e.g., mouse, rat), rabbit, pig goat or non-human primate (e.g., monkey).
  • antibodies which bind to the same or similar epitope as the 1D9 antibody
  • antibodies can be made (e.g., Kohler et al, Nature, 25(5:495-497 (1975); Harlow et al, 1988, Antibodies: A Laboratory Manual, (Cold Spring Harbor, NY); and Current Protocols in Molecular Biology, Vol. 2 (Supplement 27, Summer '94), Ausubel et al, Eds. (John Wiley & Sons: New York, NY), Chapter 11 (1991)).
  • antibodies can be raised against an appropriate immunogen in a suitable mammal (e.g., a mouse, rat, rabbit or sheep).
  • Immunoglobulins of nonhuman origin having binding specificity for CCR2 can also be obtained from antibody libraries (e.g., a phage library comprising nonhuman Fab molecules).
  • the antigen binding region of the humanized immunoglobulin comprises a CDR of nonhuman origin.
  • the humanized immunoglobulin comprises a CDR of nonhuman origin.
  • immunoglobulin having binding specificity for CCR2 comprises at least one CDR of nonhuman origin.
  • CDRs can be derived from the light and heavy chain variable regions of immunoglobulins of nonhuman origin, such that a humanized immunoglobulin includes substantially heavy chain CDRl, CDR2 and/or CDR3, and/or light chain CDRl, CDR2 and/or CDR3, from one or more immunoglobulins of nonhuman origin, and the resulting humanized immunoglobulin has binding specificity for CCR2.
  • all three CDRs of a selected chain are substantially the same as the CDRs of the corresponding chain of a donor, and more preferably, all three CDRs of the light and heavy chains are substantially the same as the CDRs of the corresponding donor chain.
  • the invention relates to an immunoglobulin having binding specificity for CCR2 comprising a humanized light chain or antigen-binding fragment thereof comprising CDRl, CDR2 and CDR3 of the light chain of the 1D9 antibody and a heavy chain, e.g., a human heavy chain.
  • the invention also includes an immunoglobulin having binding specificity for CCR2 comprising a humanized heavy chain or antigen-binding fragment thereof comprising CDRl, CDR2 and CDR3 of the heavy chain of the 1D9 antibody and a light chain, e.g., a human light chain.
  • the invention also relates to an immunoglobulin having binding specificity for CCR2 comprising a light chain and a heavy chain, wherein the light chain comprises at least 1 CDR of an antibody of non-human origin (e.g., 1D9) and framework and constant regions of human origin (e.g., SEQ ID NO: 57 and SEQ ID NO: 60), and wherein the heavy chain comprises a variable region of non-human origin (e.g., from 1D9) and a constant region of human origin.
  • the invention also provides antigen-binding fragments of these
  • the invention also relates to an immunoglobulin having binding specificity for CCR2 comprising a light chain and a heavy chain, wherein the light chain comprises a variable chain of non-human origin (e.g., from 1D9) and a constant region of human origin, and wherein the heavy chain comprises at least 1 CDR of an antibody of non-human origin (e.g., 1D9) and framework and constant regions of human origin (e.g., SEQ ID NO: 58 and SEQ ID NO: 59).
  • the invention also provides antigen-binding fragments of these immunoglobulins.
  • human framework regions are preferably derived from a human antibody variable region having sequence similarity to the analogous or equivalent region (e.g., light chain variable region) of the antigen binding region donor.
  • Other sources of framework regions for portions of human origin of a humanized immunoglobulin include human variable consensus sequences (see, e.g., Kettleborough, C.A. et al, Protein Engineering 4:773-783 (1991); Carter et al, WO 94/04679, published March 3, 1994)).
  • the sequence of the antibody or variable region used to obtain the nonhuman portion can be compared to human sequences as described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, U.S. Government Printing Office (1991).
  • the framework regions of a humanized immunoglobulin chain are derived from a human variable region having at least about 60% overall sequence identity, preferably at least about 70% overall sequence identity and more preferably at least about 85% overall sequence identity, with the variable region of the nonhuman donor (e.g., murine antibody 1D9).
  • a human portion can also be derived from a human antibody having at least about 65% sequence identity, and preferably at least about 70% sequence identity, within the particular portion (e.g., FR) being used, when compared to the equivalent portion (e.g., FR) of the nonhuman donor.
  • the humanized immunoglobulin comprises at least one of the framework regions (FR) derived from one or more chains of an antibody of human origin.
  • the FR can include a FR1 and/or FR2 and/or FR3 and/or FR4 derived from one or more antibodies of human origin.
  • the human portion of a selected humanized chain includes FR1, FR2, FR3 and FR4 derived from a variable region of human origin (e.g., from a human immunoglobulin chain, from a human consensus sequence).
  • the immunoglobulin portions of nonhuman and human origin for use in the present invention have sequences identical to immunoglobulins or immunoglobulin portions from which they are derived or to variants thereof. Such variants include mutants differing by the addition, deletion, or substitution of one or more residues.
  • the CDRs which are of nonhuman origin are substantially the same as in the nonhuman donor, and preferably are identical to the CDRs of the nonhuman donor. Changes in the framework region, such as those which substitute a residue of the framework region of human origin with a residue from the corresponding position of the donor, can be made.
  • One or more mutations in the framework region can be made, including deletions, insertions and substitutions of one or more.
  • framework mutations can be designed as described herein.
  • the humanized immunoglobulins can bind CCR2 with an affinity similar to or better than that of the nonhuman donor.
  • Variants can be produced by a variety of suitable methods, including mutagenesis of nonhuman donor or acceptor human chains.
  • the humanized immunoglobulins have binding specificity for human CCR2.
  • the humanized immunoglobulin of the present invention has at least one functional characteristic of murine antibody 1D9, such as binding function (e.g., having specificity for CCR2, having the same or similar epitopic specificity), and/or inhibitory function (e.g., the ability to inhibit CCR2-dependent function in vitro and/or in vivo, such as the ability to inhibit the binding of a cell bearing CCR2 to a ligand thereof (e.g., a chemokine)).
  • binding function e.g., having specificity for CCR2, having the same or similar epitopic specificity
  • inhibitory function e.g., the ability to inhibit CCR2-dependent function in vitro and/or in vivo, such as the ability to inhibit the binding of a cell bearing CCR2 to a ligand thereof (e.g., a chemokine)
  • preferred humanized immunoglobulins can have the binding specificity of the murine antibody 1D9, the epitopic specificity of murine antibody 1D9 (e.g., can compete with murine 1D9, a chimeric 1D9 antibody, or humanized 1D9 for binding to CCR2 (e.g., on a cell bearing CCR2)), and/or inhibitory function of murine antibody 1D9.
  • the binding function of a humanized immunoglobulin having binding specificity for CCR2 can be detected by standard immunological methods, for example using assays which monitor formation of a complex between humanized immunoglobulin and CCR2 (e.g., a membrane fraction comprising CCR2, on a cell bearing CCR2, human cell line or recombinant host cell comprising nucleic acid encoding CCR2 which expresses CCR2).
  • assays which monitor formation of a complex between humanized immunoglobulin and CCR2 (e.g., a membrane fraction comprising CCR2, on a cell bearing CCR2, human cell line or recombinant host cell comprising nucleic acid encoding CCR2 which expresses CCR2).
  • Binding and/or adhesion assays or other suitable methods can also be used in procedures for the identification and/or isolation of humanized immunoglobulins (e.g., from a library) with the requisite specificity (e.g., an assay which monitors adhesion between a cell bearing CCR2 and a ligand thereof (e.g., HIV, MCP-1, MCP-2, MCP-3, MCP-4), or other suitable methods.
  • a ligand thereof e.g., HIV, MCP-1, MCP-2, MCP-3, MCP-4
  • the immunoglobulin portions of nonhuman and human origin for use in the present invention include light chains, heavy chains and portions of light and heavy chains. These immunoglobulin portions can be obtained or derived from immunoglobulins (e.g., by de novo synthesis of a portion), or nucleic acid molecules encoding an immunoglobulin or chain thereof having the desired property (e.g., binding CCR2, sequence similarity) can be produced and expressed.
  • Humanized immunoglobulins comprising the desired portions (e.g., antigen binding region, CDR, FR, C region) of human and nonhuman origin can be produced using synthetic and/or recombinant nucleic acids to prepare genes (e.g., cDNA) encoding the desired humanized chain.
  • nucleic acid (e.g., DNA) sequences coding for newly designed humanized variable regions can be constructed using PCR mutagenesis methods to alter existing DNA sequences (see e.g., Kamman, M., et ah, Nucl. Acids Res. 17:5404 (1989)).
  • PCR primers coding for the new CDRs can be hybridized to a DNA template of a previously humanized variable region which is based on the same, or a very similar, human variable region (Sato, K., et al., Cancer Research 55:851-856 (1993)).
  • a nucleic acid comprising a sequence encoding a variable region sequence can be constructed from synthetic oligonucleotides (see e.g., Kolbinger, F., Protein Engineering 8:971-980 (1993)).
  • a sequence encoding a signal peptide can also be incorporated into the nucleic acid (e.g., on synthesis, upon insertion into a vector). If the natural signal peptide sequence is unavailable, a signal peptide sequence from another antibody can be used (see, e.g., Kettleborough, C.A., Protein Engineering 4:773-783 (1991)). Using these methods, methods described herein or other suitable methods, variants can be readily produced.
  • cloned variable regions can be mutagenized, and sequences encoding variants with the desired specificity can be selected (e.g., from a phage library; see e.g., Krebber et ah, U.S. 5,514,548; Hoogenboom et ah, WO 93/06213, published April 1, 1993)).
  • sequences encoding variants with the desired specificity can be selected (e.g., from a phage library; see e.g., Krebber et ah, U.S. 5,514,548; Hoogenboom et ah, WO 93/06213, published April 1, 1993)).
  • the disclosure relates to a humanized immunoglobulin light chain or antigen-binding fragment thereof for use as described herein, said light chain or antigen-binding fragment thereof having an amino acid sequence comprising at least a functional portion of the light chain variable region amino acid sequence of SEQ ID NO: 57.
  • said light chain or antigen-binding fragment thereof having an amino acid sequence comprising at least a functional portion of the light chain variable region amino acid sequence of SEQ ID NO: 57.
  • the amino acid sequence comprises at least one, preferably two, and more preferably three of the CDRs of SEQ ID NO: 57.
  • the invention also relates to a humanized immunoglobulin heavy chain or antigen-binding fragment thereof, said heavy chain or antigen-binding fragment thereof having an amino acid sequence comprising at least a functional portion of the heavy chain variable region amino acid sequence shown in SEQ ID NO: 58.
  • the amino acid sequence comprises at least one, preferably two, and more preferably three of the CDRs of SEQ ID NO: 58. It is noted that all murine sequences described herein are derived from Mus musculus.
  • a humanized immunoglobulin light chain or antigen-binding fragment thereof having binding specificity for CCR2 can comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 57 and SEQ ID NO: 60.
  • a humanized immunoglobulin heavy chain or antigen-binding fragment thereof having binding specificity for CCR2 can comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 58, and SEQ ID NO: 59.
  • a humanized immunoglobulin of the invention can comprise both a light chain or antigen-binding fragment thereof having binding specificity for CCR2, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 57 and SEQ ID NO: 60, and a heavy chain or antigen-binding fragment thereof having binding specificity for CCR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 58, and SEQ ID NO: 59.
  • the disclosure relates to a humanized immunoglobulin having binding specificity for CCR2 comprising a light chain comprising the amino acid sequence of SEQ ID NO: 57 and a complementary heavy chain, or an antigen-binding fragment of said humanized immunoglobulin having binding specificity for CCR2.
  • the disclosure relates to a humanized immunoglobulin having binding specificity for CCR2 comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 58 and a complementary light chain, or an antigen-binding fragment of said humanized immunoglobulin having binding specificity for CCR2.
  • a complementary light or heavy chain is one which is capable of associating with a selected heavy or light, respectively, chain, resulting in the ability of an immunoglobulin comprising said complementary heavy and light chains to have binding specificity for CCR2.
  • the invention relates to a humanized immunoglobulin having binding specificity for CCR2 comprising a light chain comprising the amino acid sequence of SEQ ID NO: 57 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 58, or an antigen- binding fragment of said humanized immunoglobulin having binding specificity for CCR2.
  • the humanized immunoglobulin light chain or antigen-binding fragment thereof having binding specificity for CCR2 can be encoded by a nucleic acid molecule comprising SEQ ID NO: 61.
  • the humanized immunoglobulin heavy chain or antigen-binding fragment thereof having binding specificity for CCR2 can be encoded by a nucleic acid molecule comprising SEQ ID NO: 62.
  • the disclosure relates also to a chimeric immunoglobulin or antigen-binding fragment thereof having binding specificity for CCR2 comprising a light chain variable region of nonhuman origin and a human constant region (e.g., a light chain constant region).
  • the invention further relates to a chimeric immunoglobulin or antigen-binding fragment thereof having binding specificity for CCR2 comprising a heavy chain variable region of nonhuman origin and a human constant region (e.g., a heavy chain constant region).
  • the chimeric immunoglobulin or antigen-binding fragment thereof having binding specificity for CCR2 comprises a light chain variable chain region of nonhuman origin and a heavy chain variable region of nonhuman origin and further comprises a human constant region (e.g., a human light chain constant region and/or a human heavy chain constant region).
  • a human constant region e.g., a human light chain constant region and/or a human heavy chain constant region
  • the disclosure also relates to isolated and/or recombinant (including, e.g., essentially pure) nucleic acid molecules comprising nucleic acid sequences which encode a humanized immunoglobulin or humanized immunoglobulin light or heavy chain as described herein.
  • Nucleic acid molecules referred to herein as "isolated” are nucleic acid molecules which have been separated away from the nucleic acids of the genomic DNA or cellular RNA of their source of origin (e.g., as it exists in cells or in a mixture of nucleic acids such as a library), and include nucleic acid molecules obtained by methods described herein or other suitable methods, including essentially pure nucleic acid molecules, nucleic acid molecules produced by chemical synthesis, by combinations of biological and chemical methods, and recombinant nucleic acid molecules which are isolated (see e.g., Daugherty, B.L. et al, Nucleic Acids Res., 19(9): 2471-2476 (1991); Lewis, A.P. and J.S. Crowe, Gene, 101 : 297-302 (1991)).
  • Nucleic acid molecules referred to herein as "recombinant” are nucleic acid molecules which have been produced by recombinant DNA methodology, including those nucleic acid molecules that are generated by procedures which rely upon a method of artificial recombination, such as the polymerase chain reaction (PCR) and/or cloning into a vector using restriction enzymes.
  • “Recombinant” nucleic acid molecules are also those that result from recombination events that occur through the natural mechanisms of cells, but are selected for after the introduction to the cells of nucleic acids designed to allow and make probable a desired recombination event.
  • the disclosure also relates more specifically to isolated and/or recombinant nucleic acid molecules comprising a nucleotide sequence which encodes a humanized 1D9
  • the light chain comprises three complementarity determining regions derived from the light chain of the 1D9 antibody
  • the heavy chain comprises three complementarity determining regions derived from the heavy chain of the 1D9 antibody.
  • nucleic acid molecules include, for example, (a) a nucleic acid molecule comprising a sequence which encodes a polypeptide comprising the amino acid sequence of the heavy chain variable region of a humanized 1D9 immunoglobulin (e.g., heavy chain variable region of SEQ ID NO:58, which is encoded by the nucleic acid sequence of SEQ ID NO:62); (b) a nucleic acid molecule comprising a sequence which encodes a polypeptide comprising the amino acid sequence of the light chain variable region of a humanized 1D9 immunoglobulin (e.g., light chain variable region of SEQ ID NO: 57, which is encoded by the nucleic acid sequence of SEQ ID NO:61); (c) a nucleic acid molecule comprising a sequence which encodes at least a functional portion of the light or heavy chain variable region of a humanized 1D9 immunoglobulin (e.g., a portion sufficient for antigen binding of a humanized immunoglobulin which comprises said chain).
  • nucleic acids can be made which encode a selected polypeptide.
  • the nucleic acid comprises the nucleotide sequence of the variable region as set forth or substantially as set forth in SEQ ID NO:62or as set forth or substantially as set forth in SEQ ID NO:61, including double or single-stranded polynucleotides.
  • polypeptides which are larger than the variable region (i.e., include a signal peptide coding sequence or a portion of a constant region coding sequence)
  • reference to the variable region of a particular figure is meant to include the variable region portion of the sequence shown.
  • Isolated and/or recombinant nucleic acid molecules meeting these criteria can comprise nucleic acid molecules encoding sequences identical to sequences of humanized 1D9 antibody or variants thereof as discussed above.
  • Nucleic acid molecules of the present disclosure can be used in the production of humanized immunoglobulins having binding specificity for CCR2.
  • a nucleic acid molecule e.g., DNA
  • a suitable construct e.g., a vector
  • the disclosure also relates to targeting molecules for use as described herein which can effectuate the interaction of a CCR2-expressing cell with a target cell.
  • the targeting molecule includes a first binding moiety which can bind mammalian CCR2, and a second binding moiety which can bind a molecule expressed on the surface of a target cell.
  • Preferred target cells include virus infected cells.
  • tumor antigens such as Lewis Y, HER-2/neu, disialoganglioside G3, carcinoembrionic antigen, CD30
  • virus infected cells e.g., viral antigens, such as influenza virus hemagglutinin, Epstein-Barr virus LMP-1, hepatitis C virus E2 glycoprotein, HIV gpl60, HIV gp 120 and agents causing hemorrhagic fever
  • viral antigens such as influenza virus hemagglutinin, Epstein-Barr virus LMP-1, hepatitis C virus E2 glycoprotein, HIV gpl60, HIV gp 120 and agents causing hemorrhagic fever
  • the targeting molecule can contain any suitable binding second moiety which binds to a molecule expressed on a desired target cell (see, for example Ring, U.S. Patent No. 5,948,647, the entire teachings of which are incorporated herein by reference).
  • Suitable binding moieties include, for example, proteins and peptides (including post-translationally modified forms e.g., glycosylated, phosphorylated, lipidated), sugars, lipids, peptidomimetics, small organic molecules, nucleic acids and other agents which bind mammalian CCR2 or a molecule expressed on the surface of a target cell.
  • Suitable binding moieties can be identified using any suitable method, such as the binding assays described herein.
  • the first binding moiety can be, for example, a humanized immunoglobulin of the invention which binds mammalian CCR2 or antigen-binding fragment thereof (e.g., Fab, Fv, Fab', F(ab)' 2 ).
  • the second binding moiety can be, for example, an antibody (e.g., a second humanized immunoglobulin) or antigen-binding fragment thereof which binds to a molecule expressed on the target cell or antigen binding fragment thereof.
  • the targeting molecule comprises a first binding moiety which is a humanized anti-CCR2 immunoglobulin or antigen-binding fragment thereof, it is preferred that the humanized anti-CCR2 immunoglobulin does not inhibit binding of ligand to CCR2.
  • the first binding moiety can be directly or indirectly bonded to the second binding moiety through a variety of suitable linkages.
  • the moieties can be part of a contiguous polypeptide (i.e., a fusion protein).
  • the targeting molecule is a fusion protein
  • the first and second binding moieties can be arranged on the polypeptide in any suitable configuration.
  • the first and second binding moieties can be indirectly bonded through a (i.e., one or more) peptide linker, or bonded directly to each other through a peptide bond.
  • binding moieties are not part of a contiguous polypeptide they can be directly bonded by a chemical bond formed by reaction of a functional group (or activated derivative thereof) on the first moiety with a second functional group (or activated derivative thereof) on the second moiety.
  • a functional group or activated derivative thereof
  • two thiols can react to form a disulfide bond and an amine can react with a carboxylic acid or acyl halide to form an amide.
  • a variety of other suitable reactions which can be used are known in the art (see, for example, Hermanson, G. T., Bioconjugate Techniques, Academic Press: San Diego, CA (1996)).
  • the binding moieties can be indirectly bonded through a suitable linker (e.g., a peptide linker).
  • a linker contains two reactive groups which can react to form bonds with the first binding moiety and/or the second binding moiety.
  • Linkers which contain two different reactive groups can be used to selectively conjugate the first binding moiety to the second binding moiety.
  • Many linkers which are suitable for forming conjugates between proteins, nucleic acids, peptides, vitamins, sugars, lipids, small organic molecules and other suitable agents are known (see, for example, U.S. Patent Nos. 5,856,571, 5,880,270; Hermanson, G. T., Bioconjugate Techniques, Academic Press: San Diego, CA (1996)).
  • the independent activities of the binding moieties (e.g., binding activities, chemoattractant activity) of the targeting molecule are not significantly different from the activities of the binding moieties as separate molecular entities.
  • the targeting molecule can bind to CCR2 with an affinity which is within a factor of about 1000, preferably within a factor of 100, more preferably within a factor of 10 or substantially the same as the affinity of the free antibody or antigen-binding fragment.
  • Target molecules with these preferred characteristics can be prepared using any suitable method. The resulting targeting molecule can then be assayed for binding (e.g., by ELISA) and for chemoattractant activity.
  • the targeting molecule may be a bispecific humanized antibody or bispecific antigen- binding fragment thereof (e.g., F(ab') 2 ) which has specificity for mammalian CCR2 and a molecule expressed on a target cell (e.g., tumor antigen, viral antigen).
  • Bispecific antibodies can be secreted by triomas and hybrid hybridomas. The supernatants of triomas and hybrid hybridomas can be assayed for bispecific antibody using a suitable assay (e.g., ELISA), and bispecific antibodies can be purified using conventional methods. These antibodies can then be humanized according to methods described herein.
  • the invention provides a targeting molecule which is a humanized bispecific antibody having binding specificity for CCR2 and an antigen expressed on a target cell, or a bivalent antigen-binding fragment of the bispecific antibody.
  • the invention also relates to a method of effectuating the interaction of a CCR2 -bearing cell with a target cell in a patient, comprising administering to the patient an effective amount of a targeting molecule which is a humanized bispecific antibody having binding specificity for CCR2 and an antigen expressed on a target cell, or a bivalent antigen-binding fragment of the bispecific antibody.
  • Another aspect of the disclosure relates to a method of preparing a humanized
  • immunoglobulin which has binding specificity for CCR2.
  • the humanized immunoglobulin which has binding specificity for CCR2.
  • immunoglobulin can be obtained, for example, by the expression of one or more recombinant nucleic acids encoding a humanized immunoglobulin having binding specificity for CCR2 in a suitable host cell, for example.
  • immunoglobulin having binding specificity for CCR2 are also provided.
  • the constructs can be introduced into a suitable host cell, and cells which express a humanized immunoglobulin of the present invention can be produced and maintained in culture.
  • Suitable host cells can be prokaryotic, including bacterial cells such as E. coli, B.
  • subtilis and or other suitable bacteria or eucaryotic, such as fungal or yeast cells (e.g., Pichia pastoris, Aspergillus species, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Neurospora crassa), or other lower eucaryotic cells, and cells of higher eucaryotes such as those from insects (e.g., Sf9 insect cells (WO 94/26087, O'Connor, published November 24, 1994)) or mammals (e.g., COS cells, such as COS-1 (ATCC Accession No. CRL- 1650) and COS-7 (ATCC Accession No. CRL-1651), CHO (e.g., ATCC Accession No.
  • Host cells which produce a humanized immunoglobulin having binding specificity for CCR2 can be produced as follows.
  • a nucleic acid encoding all or part of the coding sequence for the desired humanized immunoglobulin can be inserted into a nucleic acid vector, e.g., a DNA vector, such as a plasmid, virus or other suitable replicon for expression.
  • a variety of vectors are available, including vectors which are maintained in single copy or multiple copy, or which become integrated into the host cell chromosome.
  • Suitable expression vectors can contain a number of components, including, but not limited to one or more of the following: an origin of replication; a selectable marker gene; one or more expression control elements, such as a transcriptional control element (e.g., a promoter, an enhancer, terminator), and/or one or more translation signals; a signal sequence or leader sequence for membrane targeting or secretion.
  • a signal sequence can be provided by the vector or other source.
  • the transcriptional and/or translational signals of an immunoglobulin can be used to direct expression.
  • a promoter can be provided for expression in a suitable host cell. Promoters can be constitutive or inducible.
  • a promoter can be operably linked to a nucleic acid encoding a humanized immunoglobulin or immunoglobulin chain, such that it directs expression of the encoded polypeptide.
  • suitable promoters for procaryotic e.g., lac, tac, T3, T7 promoters for E. coli
  • eucaryotic e.g., yeast alcohol dehydrogenase (ADH1), SV40, CMV
  • the expression vectors typically comprise a selectable marker for selection of host cells carrying the vector, and, in the case of replicable expression vector, an origin or replication.
  • Genes encoding products which confer antibiotic or drug resistance are common selectable markers and may be used in procaryotic (e.g., ⁇ -lactamase gene (ampicillin resistance), Tet gene for tetracycline resistance) and eucaryotic cells (e.g., neomycin (G418 or geneticin), gpt (mycophenolic acid), ampicillin, or hygromycin resistance genes).
  • procaryotic e.g., ⁇ -lactamase gene (ampicillin resistance), Tet gene for tetracycline resistance
  • eucaryotic cells e.g., neomycin (G418 or geneticin), gpt (mycophenolic acid), ampicillin, or hygromycin resistance genes.
  • Dihydrofolate reductase marker genes permit selection with methotrexate in a variety of hosts. Genes encoding the gene product of auxotrophic markers of the host (e.g., LEU2, URA3, HISS) are often used as selectable markers in yeast. Use of viral (e.g., baculovirus) or phage vectors, and vectors which are capable of integrating into the genome of the host cell, such as retroviral vectors, are also contemplated. In one embodiment, the vector is pLKTOK38. The present invention also relates to cells carrying these expression vectors.
  • An expression vector comprising a fused gene encoding a humanized immunoglobulin light chain, said gene comprising a nucleotide sequence encoding a CDR derived from a light chain of a nonhuman antibody having binding specificity for CCR2 and a framework region derived from a light chain of human origin.
  • the disclosure relates to an expression vector comprising a gene encoding a humanized immunoglobulin light chain, said gene comprising a nucleotide sequence encoding a CDR derived from a light chain of a nonhuman antibody having binding specificity for CCR2 and a framework region derived from a light chain of human origin.
  • the disclosure also relates to an expression vector comprising a gene encoding a humanized immunoglobulin heavy chain, said gene comprising a nucleotide sequence encoding a CDR derived from a heavy chain of a nonhuman antibody having binding specificity for CCR2 and a framework region derived from a heavy chain of human origin.
  • the nonhuman antibody is murine antibody 1D9.
  • the disclosure also includes host cells comprising the expression vectors of the invention.
  • the disclosure also relates to an isolated or recombinant gene encoding a humanized immunoglobulin light or heavy chain comprising a first nucleic acid sequence encoding an antigen binding region derived from murine monoclonal antibody 1D9; and a second nucleic acid sequence encoding at least a portion of a constant region of an immunoglobulin of human origin.
  • the disclosure also relates to a host cell (e.g., which expresses a humanized
  • immunoglobulin or an antigen binding fragment thereof having specificity for CCR2 comprising a first recombinant nucleic acid molecule encoding a humanized
  • immunoglobulin light chain or fragment thereof and a second recombinant nucleic acid molecule encoding a humanized immunoglobulin heavy chain or fragment thereof
  • said first nucleic acid molecule comprises a nucleotide sequence encoding a CDR derived from the light chain of murine antibody 1D9 and a framework region derived from a light chain of human origin
  • said second nucleic acid molecule comprises a nucleotide sequence encoding a CDR derived from the heavy chain of murine antibody 1D9 and a framework region derived from a heavy chain of human origin.
  • the invention also includes a method of preparing a humanized immunoglobulin or antigen- binding fragment thereof comprising maintaining a host cell of the invention under conditions appropriate for expression of a humanized immunoglobulin, whereby humanized immunoglobulin chains are expressed and a humanized immunoglobulin or antigen-binding fragment thereof having specificity for CCR2 is produced.
  • the method can further comprise the step of isolating the humanized immunoglobulin or fragment thereof.
  • nucleic acid molecule i.e., one or more nucleic acid molecules
  • a construct i.e., one or more constructs comprising such nucleic acid molecule(s)
  • a suitable host cell by a method appropriate to the host cell selected (e.g., transformation, transfection, electroporation, infection), such that the nucleic acid molecule(s) are operably linked to one or more expression control elements (e.g., in a vector, in a construct created by processes in the cell, integrated into the host cell genome).
  • Host cells can be maintained under conditions suitable for expression (e.g., in the presence of inducer, suitable media supplemented with appropriate salts, growth factors, antibiotic, nutritional supplements, etc.), whereby the encoded polypeptide(s) are produced.
  • the encoded protein e.g., humanized 1D9 antibody
  • the encoded protein can be isolated from, e.g., the host cells, medium, milk. This process encompasses expression in a host cell of a transgenic animal (see e.g., WO 92/03918, GenPharm International, published March 19, 1992).
  • Fusion proteins can be produced in which a humanized immunoglobulin or
  • immunoglobulin chain is linked to a non-immunoglobulin moiety (i.e., a moiety which does not occur in immunoglobulins as found in nature) in an N-terminal location, C- terminal location or internal to the fusion protein.
  • a suitable expression vector such as a pET vector (e.g., pET-15b, Novagen), a phage vector (e.g., pCANTAB 5 E, Pharmacia), or other vector (e.g., pRIT2T Protein A fusion vector, Pharmacia).
  • a suitable expression vector such as a pET vector (e.g., pET-15b, Novagen), a phage vector (e.g., pCANTAB 5 E, Pharmacia), or other vector (e.g., pRIT2T Protein A fusion vector, Pharmacia).
  • the resulting construct can be introduced into a suitable host cell for expression.
  • fusion proteins can be isolated or purified from a cell lysate by means of a suitable affinity matrix (see e.g., Current Protocols in Molecular Biology (Ausubel, F.M. et al, eds., Vol. 2, Suppl. 26, pp. 16.4.1-16.7.8 (1991)).
  • the humanized immunoglobulin is administered in an effective amount which inhibits binding of CCR2 to a ligand thereof.
  • an effective amount will be sufficient to achieve the desired therapeutic (including prophylactic) effect (such as an amount sufficient to reduce or prevent CCR2 -mediated binding and/or signaling).
  • the humanized immunoglobulin can be administered in a single dose or multiple doses.
  • the dosage can be determined by methods known in the art and can be dependent, for example, upon the individual's age, sensitivity, tolerance and overall well-being. Suitable dosages for antibodies can be from about 0.1 mg/kg body weight to about 10.0 mg/kg body weight per treatment.
  • the humanized immunoglobulin can be administered to an individual (e.g., a human) alone or in conjunction with another agent.
  • a humanized immunoglobulin can be administered before, along with or subsequent to administration of the additional agent.
  • the disclosure includes pharmaceutical compositions comprising a humanized immunoglobulin or antigen-binding fragment thereof of the invention and a suitable carrier. In one embodiment, more than one humanized
  • an additional monoclonal antibody is administered in addition to a humanized immunoglobulin of the present invention.
  • an additional pharmacologically active ingredient e.g., a further agent suitable in the treatment of an infection with an agent causing hemorrhagic fever
  • parenteral e.g., intravenous, intraarterial, intramuscular, subcutaneous injection
  • oral e.g., dietary
  • topical inhalation
  • intranasal or oral inhalation intranasal drops
  • rectal depending on the disease or condition to be treated.
  • Parenteral administration is a preferred mode of administration.
  • Formulation will vary according to the route of administration selected (e.g., solution, emulsion).
  • An appropriate composition comprising the humanized antibody to be administered can be prepared in a physiologically acceptable vehicle or carrier.
  • suitable carriers include, for example, aqueous or
  • Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.
  • Intravenous vehicles can include various additives, preservatives, or fluid, nutrient or electrolyte replenishers (See, generally, Remington's Pharmaceutical Sciences, 17th Edition, Mack Publishing Co., PA, 1985).
  • a suitable dispenser for administration e.g., an atomizer, nebulizer or pressurized aerosol dispenser.
  • mammalian CCR2 protein refers to naturally occurring or endogenous mammalian CCR2 proteins and to proteins having an amino acid sequence which is the same as that of a naturally occurring or endogenous corresponding mammalian CCR2 protein (e.g., recombinant proteins). Accordingly, as defined herein, the term includes mature receptor protein, polymorphic or allelic variants, and other isoforms of a mammalian CCR2 (e.g., produced by alternative splicing or other cellular processes), and modified or unmodified forms of the foregoing (e.g., glycosylated, unglycosylated).
  • Mammalian CCR2 proteins can be isolated and/or recombinant proteins (including synthetically produced proteins).
  • Naturally occurring or endogenous mammalian CCR2 proteins include wild type proteins such as mature CCR2, polymorphic or allelic variants and other isoforms which occur naturally in mammals (e.g., humans, non-human primates), such as the CCR2a and CCR2b forms of the receptor protein which are produced by alternative splicing of the carboxy-terminus of the protein.
  • Such proteins can be recovered or isolated from a source which naturally produces mammalian CCR2, for example.
  • proteins and mammalian CCR2 proteins having the same amino acid sequence as a naturally occurring or endogenous corresponding mammalian CCR2 are referred to by the name of the corresponding mammal.
  • the protein is designated as a human CCR2 protein (e.g., a recombinant human CCR2 produced in a suitable host cell).
  • One or more antibodies or fragments can be administered to an individual by an appropriate route, either alone or in combination with (before, simultaneous with, or after) another drug or agent, or before, simultaneous with or after surgical, mechanical or therapeutic intervention.
  • the antibodies of the present invention can also be used in combination with other monoclonal or polyclonal antibodies (e.g., in combination with any agent suitable for the treatment of an infection with an agent causing
  • hemorrhagic fever or with existing blood plasma products, such as commercially available gamma globulin and immune globulin products used in prophylactic or therapeutic treatments.
  • an effective amount of an antibody or fragment i.e., one or more antibodies or fragments
  • An effective amount is an amount sufficient to achieve the desired therapeutic (including prophylactic) effect, under the conditions of administration, such as an amount sufficient for inhibition of a CCR2 function, and thereby, inhibition of an inflammatory response or treating or preventing an infection with an agent causing hemorrhagic fever, or an amount sufficient for promotion of a CCR2 function, as indicated.
  • the antibody or fragment can be administered in a single dose or multiple doses. The dosage can be determined by methods known in the art and is dependent, for example, upon the antibody or fragment chosen, the subject's age, sensitivity and tolerance to drugs, and overall well-being.
  • Antibodies and antigen-binding fragments thereof can often be administered with less frequency than other types of therapeutics.
  • an effective amount of an antibody can range from about 0.01 mg/kg to about 5 or 10 mg/kg administered daily, weekly, biweekly or monthly.
  • routes of administration are possible including, but not necessarily limited to, oral, dietary, topical, parenteral (e.g., intravenous, intraarterial, intramuscular,
  • inhalation e.g., intrabronchial, intraocular, intranasal or oral inhalation, intranasal drops
  • Other suitable methods of administration can also include rechargeable or biodegradable devices and slow release polymeric devices.
  • the pharmaceutical compositions of this invention can also be administered as part of a combinatorial therapy with other agents.
  • Formulation of an antibody or fragment to be administered will vary according to the route of administration and formulation (e.g., solution, emulsion, capsule) selected.
  • An appropriate pharmaceutical composition comprising an antibody or functional fragment thereof to be administered can be prepared in a physiologically acceptable vehicle or carrier.
  • suitable carriers include, for example, aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.
  • aqueous carriers include water, buffered water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), dextrose solution and glycine.
  • Intravenous vehicles can include various additives, preservatives, or fluid, nutrient or electrolyte replenishers (See, generally, Remington's Pharmaceutical Science, 16th Edition, Mack, Ed. 1980).
  • the compositions can optionally contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents and toxicity adjusting agents, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride and sodium lactate.
  • the antibodies and fragments of this invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use according to art-known lyophilization and reconstitution techniques.
  • the optimum concentration of the active ingredient(s) in the chosen medium can be determined empirically, according to procedures well known to the skilled artisan, and will depend on the ultimate pharmaceutical formulation desired.
  • the antibody or fragment can be solubilized and loaded into a suitable dispenser for administration (e.g., an atomizer, nebulizer or pressurized aerosol dispenser).
  • individuals to whom the herein described combinations are administered are selected from the following group of individuals:
  • qPCR-based test e.g. the WHO approved test manufactured by Altona Diagnositcs, Hamburg, Germany (RealStar® Filovirus Screen RT-PCR Kit 1.0).
  • inventive anti-GM-CSF-agonists or compositions comprising the same.
  • Respective qPCR assays are available from various diagnostic companies (e.g. form Altona Diagnostics, Vela Diagnostics, etc. for Dengue virus, Chikungunya virus) and for Lassa virus and Hantavirus infections specialized laboratories exist, e.g. in clinical institutions in various countries;
  • the antibody is administered subcutaneously (sc), in other embodiments, the antibody is administered intravenously (iv)
  • sc subcutaneously
  • iv intravenously
  • the administration route may be subcutaneously.
  • the antibody may be administered intravenously.
  • the antibody dose is selected from a range of 0.5 mg/kg up to about 10 mg/kg, e.g. the dose may be 0.5 mg/kg or 2 mg/kg, or it may be between 3-5 mg/kg, or it may be between 5-8 mg/kg.
  • a high dose e.g. a dose of about 250-750 mg/day, e.g. about 300 mg/day or about 560 mg/day (corresponding to 8 mg/kg for an individual with a weight of 70 kg).
  • a loading dose e.g. a dose of about 250-750 mg/day, e.g. about 300 mg/day or about 560 mg/day (corresponding to 8 mg/kg for an individual with a weight of 70 kg).
  • a dose e.g. a dose of about 100 mg/day to about 200 mg/day, preferably about 150 mg/day after about 7 days, optionally after about 14 days and further optionally (depending on the severity of the clinical symptoms) after about 28 days.
  • a loading dose when a loading dose is not administered, it is possible to initially administer from 0.5 mg/kg to about 8.0 mg/kg of the inventive antibody (i.e. between 35 mg/body weight to about 560 mg/body weight for an individual with a body weight of 70 kg).
  • a third or further administration may be contemplated depending on the clinical symptoms. For example, it is possible to administer from 0.5 mg/kg to 3.0 mg/kg on days 0 and 14 (optionally also on days 7 and 28).
  • a loading dose when a loading dose is not administered, it is possible to initially administer from 3.0 mg/kg to about 5.0 mg/kg of the inventive antibody formulation on day 0 and from 3.0 mg/kg to about 5.0 mg/kg on day 14 (optionally also from 3.0 mg/kg to about 5.0 mg/kg on day 7). It is also possible to administer from 5.0 mg/kg to 8.0 mg/kg on days 0 and 14 (optionally also on days 7 and 28).
  • the antibody is administered subcutaneously (sc), in other embodiments, the antibody is administered intravenously (iv)
  • sc subcutaneously
  • iv intravenously
  • the administration route may be subcutaneously.
  • the administration route may be subcutaneously.
  • individuals with early onset clinical symptoms e.g. those with detectable clinical symptoms correlating with a positive qPCR test for viral load, as well as individuals suffering from hemorrhagic and diarrhea complications, fever, etc. that are in later stages of infections with the herein described agents it may be preferred to administer the antibody intravenously.
  • the antibody dose is selected from a range of 0.5 mg/kg up to about 10 mg/kg, e.g. the dose may be 0.5 mg/kg or 2 mg/kg, or it may be between 3-5 mg/kg, or it may be between 5-10 mg/kg.
  • a high dose e.g. a dose of about 250-750 mg/day, e.g. about 450 mg/day.
  • the loading dose may be a single intravenous dose of 10 mg/kg (about 750 mg) on day 0, or it may be a single subcutaneous dose of 450 mg on day 0.
  • a dose of about 100 mg/day to about 200 mg/day preferably about 150 mg/day after about 7 days, optionally after about 14 days and further optionally (depending on the severity of the clinical symptoms) after about 28 days.
  • a loading dose when a loading dose is not administered, it is possible to initially administer from 0.5 mg/kg to about 10.0 mg/kg of the inventive antibody (i.e. between 35 mg/body weight to about 700 mg/body weight for an individual with a body weight of 70 kg).
  • administering In some embodiments is contemplated to administer about 2.0 mg/kg to about 3.0 mg/kg, on day 0 and on day 14 (optionally also on day 7). Administration of at least two doses is preferred, but a third or further administration may be contemplated depending on the clinical symptoms. For example, it is possible to administer from 2.0 mg/kg to 5.0 mg/kg on days 0 and 14 (optionally also on days 7 and 28).
  • a loading dose when a loading dose is not administered, it is possible to initially administer from 5.0 mg/kg to about 10.0 mg/kg of the inventive antibody formulation on day 0 and from 3.0 mg/kg to about 5.0 mg/kg on day 14 (optionally about 5.0 mg/kg on day 7).
  • the formulations of antibodies referred to above may comprise a concentration of 150 mg/ml in vials for distribution.
  • a Individuals, e.g. humans, receiving pre-/post-exposure prophylaxis. These individuals receive an early administration/treatment before onset of symptoms. Administrations occurs from about two weeks, from about 1 week, form about 1 day prior to exposure and continues to about 1 week, to about 2 weeks, or to about 3 weeks after exposure.
  • b Individuals with early onset clinical symptoms and with oral rehydration only (usually day 0-3 from onset of symptoms), e.g. those with detectable clinical symptoms correlating with a positive qPCR test for viral load for increasing the survival rate and other symptoms as described above.
  • Administration occurs from day 0, from day 1, from day 2, from day 3 diagnosis of symptoms and continues with the above described dosing regimen.
  • a single non-human primate study is performed to assess the efficacy of a combination treatment of an anti-CCR2 antibody and an anti-GM-CSF antibody as described herein, to treat Ebola.
  • the anti-CCR2 antibody used is the anti-CCR2 antibody MLN1202, as described herein, the amino acid sequence of which is depicted in SEQ ID NOs: 57-60, the antibody against GM-CSF used in the example is a human monoclonal IgGl antibody that binds to and neutralizes with high affinity and specificity human GM-CSF and that is described in WO2006/111353. Its generation is described in Example 2 of
  • the antibody comprises the light chain and heavy chain CDR sequences as depicted in SEQ ID NOs: 16, 17, 18, 14, 15 and 2. These CDR sequences are comprised in the heavy and light chain variable domain, respectively, that are shown in SEQ ID NOs: 34 and 35, respectively.
  • Animals are to be dosed according to established dosing regimens in humans and observed for changes in disease severity relative to control animals (dosed with formulation buffer only), including, but not limited to, a decrease in morbidity and/or mortality, delay in time to death, or decreased viral burden.
  • Non-Human Primates are randomized into 2 groups of 3 animals each.
  • the groups consist of 1) Formulation buffer only, 2) anti-CCR2 antibody and anti-GM-CSF antibody. Animals are infected intramuscularly with 100 PFU of Ebola Zaire on study day 0.
  • the first treatment occurs on day -1, with a dose of either 30 mg/kg, or 10 mg/kg or 3 mg/kg of antibody in a concentration of e.g. 150 mg/ml.
  • the antibody may be formulated according to one of the formulations provided in the description.
  • a second dose is provided on study day 4.
  • Samples are collected every other day (if the animal weights and blood volume allow) for hematology, chemistries, viral load (by PCR), flow cytometry, and Bio-Plex analysis. Animals are monitored daily for signs of clinical disease and evaluated for euthanasia. The endpoints of the study are death/euthanasia or survival past study day 28. All animals have a full necropsy after death/euthanasia. Histology is performed on inguinal, tracheobronchial, mediastinal, and mandibular lymph nodes as well as liver, kidney, pancreas, lung, and spleen.
  • Non-Human Primates (Rhesus Macaques) are randomized into 2 groups of 3 animals each.
  • the groups consist of 1) Formulation buffer only, 2) anti-CCR2 antibody and anti-GM-CSF antibody.
  • Animals are infected intramuscularly with 100 PFU of Ebola Zaire on study day 0. The animals are tested daily with qPCR for viral antigen load. As soon as the viral antigen is detectable, the first treatment occurs with doses of either 30 mg/kg, or 10 mg/kg or 3 mg/kg of each antibody in a concentration of e.g.150 mg/ml.
  • the antibodies may be formulated according to one of the formulations provided in the description.
  • a second dose is administered on day 4. Samples are being collected every other day (if the animal weights and blood volume allow) for hematology, chemistries, viral load (by PCR), flow cytometry, and Bio-Plex analysis. Animals are monitored daily for signs of clinical disease and evaluated for euthanasia. The endpoints of the study are death/euthanasia or survival past study day 28. All animals have a full necropsy after death/euthanasia.
  • Histology is performed on inguinal, tracheobronchial, mediastinal, and mandibular lymph nodes as well as liver, kidney, pancreas, lung, and spleen. Immunohistochemistry for viral antigen and appropriate macrophage cell markers is performed on the same tissues.
  • phase 2 human clinical study to evaluate the efficacy and safety of 1 to 3 doses of intravenous or subcutaneous administration of an anti-CCR2 antibody and an anti-GM- CSF antibody in combination for the treatment of confirmed Ebola virus disease (EVD).
  • the anti-CCR2 antibody used is the anti-CCR2 antibody MLN1202, as described herein, the amino acid sequence of which is depicted in SEQ ID NOs: 57-60, the antibody against GM-CSF used in the example is a human monoclonal IgGl antibody that binds to and neutralizes with high affinity and specificity human GM-CSF and that is described in WO2006/111353.
  • the antibody comprises the light chain and heavy chain CDR sequences as depicted in SEQ ID NOs: 16, 17, 18, 14, 15 and 2. These CDR sequences are comprised in the heavy and light chain variable domain, respectively, that are shown in SEQ ID NOs: 34 and 35, respectively.
  • the antibodies are used at doses of 150 mg, 300mg or 450 mg or higher as described below, and administered subcutaneously or intravenously to subjects with EBV either in the first phase of disease (i.e. suspected or confirmed Ebola case patients with early- defined symptoms and with oral rehydration only (usually day 0-3 from onset of symptoms), or who are in the second phase of disease (confirmed cases with second phase- symptoms (mainly gastro-intestinal 3-10 days) requiring oral or IV rehydration).
  • first phase of disease i.e. suspected or confirmed Ebola case patients with early- defined symptoms and with oral rehydration only (usually day 0-3 from onset of symptoms)
  • second phase of disease confirmed cases with second phase- symptoms (mainly gastro-intestinal 3-10 days) requiring oral or IV rehydration).
  • the dose for the anti-GM-CSF antibody is 350 mg on day zero and then 150 mg on day 7, though the dose can be modulated up to 8 mg/kg if thought necessary, or decrease down to 1 mg/kg if necessary.
  • the optimum dose is 10 mg/ml ( ⁇ 750 mg IV) intravenously, but if not possible to inject intravenously, the subcutaneous dose is 450 mg on day 0 and then 150 mg weekly as needed, but for no more than 3 additional administrations.
  • the study population is following a Simon-2 design with step-wise enrolment supervised by a data safety monitoring committee provided with live available data. A total of 42 patients are necessary for each cohorts to prove a decrease of 20% in mortality (increase in survival by 20%), this is on the basis of a current survival rate of 40% and an alpha error of 0.05 and a 80%> power.
  • the study comprises three different periods, i.e.
  • the primary end-point is survival at day 14 from onset of symptoms. Secondary end- points are over the time frame of 4 weeks from onset of symptoms reduction in symptoms severity (fever, headache, respiratory & heart rates, diarrhea and vomiting, chemistry) compared to time of randomisation, survival at other time points, progression to later phase of the disease, incidence of organ failure, time to hospital discharge, time to return to pre -morbid state, virus load and available laboratory assessments such as immune response at 2weeks, and 4 weeks for survivors (IgG and IgM specific Ebola), in addition to assessing safety and tolerability
  • eligibility of the subjects for the study is assessed and the eligibility criteria include
  • Eligible subjects are hospitalised in isolation in the Ebola response care emergency facilities or in appropriate isolated treatment rooms in hospital.
  • the subject is given the antibodies intravenously or if not possible subcutaneously.

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Abstract

L'invention concerne des combinaisons comprenant des antagonistes anti-GM-CSF et des antagonistes anti-CCR2 pour traiter et prévenir des infections provoquées par des agents induisant une fièvre hémorragique ou pour moduler la réponse cellulaire ou immunitaire d'un individu qui a été exposé, infecté ou susceptible d'être infecté par de tels agents, par exemple, le virus Ebola.
PCT/EP2015/077174 2014-11-21 2015-11-20 Utilisation d'un antagoniste anti-gm-csf et d'un antagoniste anti-ccr2 pour traiter une maladie infectieuse WO2016079276A1 (fr)

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CN106191291A (zh) * 2016-08-15 2016-12-07 中国人民解放军军事医学科学院微生物流行病研究所 rs1042658在预测新布尼亚病毒引起的发热伴血小板减少综合征死亡风险中的应用
WO2023072916A1 (fr) 2021-10-27 2023-05-04 Granite Bio Ag Anticorps ciblant ccr2

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WO2001057226A1 (fr) * 2000-02-03 2001-08-09 Millennium Pharmaceuticals, Inc. Anticorps humanises anti-ccr2 et procedes d'utilisation de ces anticorps
WO2006111353A2 (fr) * 2005-04-18 2006-10-26 Micromet Ag Neutralisants d'anticorps du facteur de stimulation des colonies de granulocytes/macrophages (gm-csf) humain
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WO2001057226A1 (fr) * 2000-02-03 2001-08-09 Millennium Pharmaceuticals, Inc. Anticorps humanises anti-ccr2 et procedes d'utilisation de ces anticorps
WO2006111353A2 (fr) * 2005-04-18 2006-10-26 Micromet Ag Neutralisants d'anticorps du facteur de stimulation des colonies de granulocytes/macrophages (gm-csf) humain
AU2013201228A1 (en) * 2006-02-08 2013-03-21 Eisai, Inc. Antigenic gm-csf peptides and antibodies to gm-csf
WO2009038760A2 (fr) * 2007-09-18 2009-03-26 Amgen Inc. Protéines de liaison à un antigène gm-csf humain
WO2010124136A1 (fr) * 2009-04-24 2010-10-28 Delta Vidyo, Inc. Écumoire vidéo efficace
WO2013004806A1 (fr) * 2011-07-06 2013-01-10 Morphosys Ag Associations thérapeutiques d'anticorps anti-cd20 et anti-gm-csf, et leurs utilisations

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106191291A (zh) * 2016-08-15 2016-12-07 中国人民解放军军事医学科学院微生物流行病研究所 rs1042658在预测新布尼亚病毒引起的发热伴血小板减少综合征死亡风险中的应用
WO2023072916A1 (fr) 2021-10-27 2023-05-04 Granite Bio Ag Anticorps ciblant ccr2

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