WO2016081801A1 - Utilisation d'un antagoniste anti-ccr2 dans le traitement d'une maladie infectieuse - Google Patents

Utilisation d'un antagoniste anti-ccr2 dans le traitement d'une maladie infectieuse Download PDF

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Publication number
WO2016081801A1
WO2016081801A1 PCT/US2015/061765 US2015061765W WO2016081801A1 WO 2016081801 A1 WO2016081801 A1 WO 2016081801A1 US 2015061765 W US2015061765 W US 2015061765W WO 2016081801 A1 WO2016081801 A1 WO 2016081801A1
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ccr2
antibody
hemorrhagic fever
treatment
infection
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PCT/US2015/061765
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English (en)
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Jonathan ZALVESKY
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Millennium Pharmaceuticals, Inc.
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Publication of WO2016081801A1 publication Critical patent/WO2016081801A1/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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—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 infectious diseases in individuals e.g. humans, for example, those caused by agents inducing hemorrhagic fever using agents antagonizing CCR2.
  • the antagonists are neutralizing antibodies against mammalian CCR2. These antagonists can be used alone or in combination with other drugs in the treatment and/or prevention of infections caused, e.g., by Ebola virus.
  • 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 proportions 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. Currently, 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
  • 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 cell dysfunction, a hallmark of the disease.
  • Viral proteins re- program the host's anti-viral response at the level of JAK/STAT, IRF, and dsR A sensors and deregulate monocyte/macrophage/DC homeostasis, and hijack the infected monocytes as a vehicle to disseminate the virus throughout the body [Sullivan N et al.
  • CCR2 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 provides anti-CCR2 antagonist for use in the treatment and/or prevention of an infection with an agent causing hemorrhagic fever.
  • anti-CCR2 antagonists comprise antibodies or fragments thereof
  • peptidomimetics amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids.
  • the present invention relates to an anti-CCR2 antagonist or fragment thereof for use in the treatment and/or prevention of an infection and/or modulation of the immune response or cellular response in an individual exposed to, infected with or suspected to be exposed 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 treatment and/or prevention comprises modulating the cellular and/or immune response of an individual. In another embodiment the treatment and/or prevention
  • c) provides time for the individual's immune system to raise a response.
  • the treatment comprises modulating the cellular and/or immune response of an individual, wherein the modulation comprises at least one of the following features:
  • the present invention also provides anti-CCR2 antagonists for use in the treatment and/or prevention of an infection with 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
  • 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 anti-CCR2 antagonists can be used also in the treatment of infections and diseases caused by the above viruses.
  • the agent causing hemorrhagic fever selected from filoviridae comprises the genera Ebola virus and Marburg 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).
  • ZEBOV strains Zaire Ebola virus
  • TEBOV Tai Forrest Ebola virus
  • SEBOV Sudan Ebola virus
  • REBOV Reston Ebola virus
  • BEBOV Bundibugyo Ebola virus
  • the present invention also provides 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 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).
  • 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 LS132.1D9 (1D9) or monoclonal antibody LS132.8G2 (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:2, which is encoded by the nucleic acid sequence depicted in SEQ ID NO:6.
  • the anti-CCR2 antibody comprises the heavy chain constant region comprising the amino acid sequence depicted in SEQ ID NO:3, which is encoded by the nucleic acid sequence depicted in SEQ ID NO: 7.
  • the anti-CCR2 antibody comprises a light chain variable region comprising the amino acid sequence depicted in SEQ ID NO: 1, which is encoded by the nucleic acid sequence depicted in SEQ ID NO: 5.
  • the anti-CCR2 antibody comprises the light chain constant region comprising the amino acid sequence depicted in SEQ ID NO:4, which is encoded by the nucleic acid sequence depicted in SEQ ID NO:8.
  • 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: 1-4.
  • said antibody or a fragment thereof comprises an immunoglobulin heavy chain or fragment thereof comprising the amino acid sequence of SEQ ID NO: 2 and/or the amino acid sequence of SEQ ID NO: 3.
  • said antibody or a fragment thereof comprises an immunoglobulin light chain or fragment thereof comprising the amino acid sequence of SEQ ID NO: 1 and/or the amino acid sequence of SEQ ID NO: 4.
  • said antibody or a fragment thereof comprises an immunoglobulin heavy chain or fragment thereof comprising the amino acid sequence of SEQ ID NO: 2 and/or the amino acid sequence of SEQ ID NO: 3 in combination with an immunoglobulin light chain or fragment thereof comprising the amino acid sequence of SEQ ID NO: 1 and/or the amino acid sequence of SEQ ID NO: 4.
  • the anti-CCR2 antibody is an anti-CCR2 antibody described in US 2004/0151721, the disclosure of which is incorporated herein by reference.
  • the invention thus relates to an anti-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 CCR2, in particular human CCR2.
  • said antagonist is used in combination with 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.
  • a further aspect of the invention relates to a method of treatment and/or prevention of an individual infected with Ebola virus or any other agent causing hemorrhagic fever, said method comprising administering an anti-CCR2 antagonist as defined herein, optionally in combination with 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 as described herein.
  • the herein disclosed CCR2 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 comprise Vesicular Stomatitis Virus (VSV) comprising and expressing Ebola-derived antigens (e.g.
  • VSV Vesicular Stomatitis Virus
  • 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 polymerase e.g. Favipiravir T-705 manufactured by Fujifilm Holdings Corp. MediVector Inc.
  • nucleosides as inhibitors of the viral RNA polymerase e.g. BCX4430 developed by Biocryst Pharmaceuticals Inc.
  • Further 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 CCR2.
  • the herein disclosed antagonists of CCR2 e.g. antibodies or fragments thereof, are administered alone, i.e. without additional presently existing antiviral agents.
  • Another embodiment relates to the method described herein wherein the administration of said anti-CCR2 antagonist
  • c) provides time for the individual's immune system to raise a response.
  • the administration of said anti-CCR2 antagonist modulates the cellular and/or immune response of an individual, wherein the modulation comprises at least of the following features:
  • m) Inhibition, slowing down or minimization of clinical symptoms in said individual comprising symptoms selected from hypotonia, loss of body fluids, fever, blood loss, diarrhea, sore throat, muscle pain and headaches.
  • 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-CCR2 antagonist is frequently used, which means that compounds, e.g. antibodies, are meant that antagonize CCR2. It is clear that the term does not relate to antagonists of compounds antagonizing CCR2.
  • 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 humanized antibody, in particular a humanized monoclonal 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.
  • mammalian CCR2 as the full length antibody.
  • it relates to a pharmaceutically active fragment of an antibody, i.e. having the same pharmaceutical effects as the full length anti-CCR2 antibody.
  • 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. fusion proteins of a heavy and a light chain variable region connected by a peptide linker.
  • Further exemplary 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
  • a single antibody variable region such as a VHH antibody (comprising a single VH region).
  • Members of the latter subclass include an Fv (comprising one VH region and one VL region as separate polypeptide chains which are non-covalently associated with one another); 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
  • 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 present invention also encompasses pharmaceutical compositions comprising the anti- CCR2 antagonist or a fragment thereof described herein.
  • 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
  • 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 non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are 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.
  • Preservatives and other additives 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.
  • the pharmaceutical composition of the present invention might comprise proteinaceous carriers, like, e.g., serum albumin or immunoglobulin, preferably of human origin.
  • the present invention also relates to 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-1RB) or portion of the receptor (anti-CCR2) for the use as described herein.
  • CC-chemokine receptor 2 also referred to as CCR2, CKR-2, MCP-1RA or MCP-1RB
  • anti-CCR2 mammalian CC-chemokine receptor 2
  • the antibody of the present invention or fragment thereof for the use as described herein has specificity for human or rhesus CCR2 or a portion thereof.
  • the antibody or fragment of the invention blocks binding of a ligand (e.g., MCP-1, MCP-2, MCP-3, MCP-4) to the receptor and inhibits 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 chemokine e.g., MCP-1, MCP-2, MCP-3, MCP-4
  • the present invention also relates to an antibody or functional fragment thereof (e.g., an antigen-binding fragment) which binds 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 or8G2 or an antibody which can compete with 1D9 or 8G2 for binding to human CCR2 or a portion of human CCR2.
  • the present invention also relates to a humanized immunoglobulin or antigen-binding fragments 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).
  • nonhuman origin e.g., rodent
  • an immunoglobulin of human origin e.g., a human framework region, a human constant region of the gamma type.
  • immunoglobulin or fragment thereof described herein can compete with 1D9 for binding to CCR2.
  • the antigen binding region of the humanized immunoglobulin or fragment thereof described herein can compete with 1D9 for binding to CCR2.
  • the antigen binding region of the humanized immunoglobulin or fragment thereof described herein can compete with 1D9 for binding to CCR2.
  • the antigen binding region of the humanized immunoglobulin or fragment thereof described herein can compete with 1D9 for binding to CCR2.
  • 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: 1 and SEQ E) NO: 2, 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 present invention also relates to humanized immunoglobulin light chains and antigen- binding fragments thereof (e.g., comprising CDRl, CDR2 and CDR3 of the light chain of the 1D9 antibody, and a human light chain FR), and to humanized immunoglobulin heavy chains and antigen-binding fragments thereof (e.g., comprising CDRl, 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 CDRl, 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 CDRl, CDR2 and CDR3 of the heavy chain of the 1D9 antibody, and a human heavy chain FR
  • the invention relates to 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: 1, a humanized heavy chain comprising the variable region of the heavy chain shown in SEQ ID NO: 2 for use as described herein. Also encompassed are humanized immunoglobulins comprising one or more humanized light and/or heavy chains or 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 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 FIF-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: 1, amino acids 55-61 of SEQ ID NO: 1 and amino acids 94-102 of SEQ ID NO: 1
  • the complementarity determining regions derived from the heavy chain of 1D9 are amino acids 31-35 of SEQ ID NO: 2, amino acids 50-68 of SEQ ID NO: 2 and amino acids 101-106 of SEQ ID NO: 2.
  • the invention further relates to 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: 1 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: 2 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: 1, and wherein said heavy chain comprises a variable region comprising SEQ ID NO: 2 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 inhibits binding of a ligand to CCR2.
  • the present invention further relates to a construct comprising a nucleic acid molecule encoding a humanized immunoglobulin having binding specificity for CCR2 or a chain of such an immunoglobulin.
  • a construct comprising a nucleic acid molecule encoding a humanized immunoglobulin having binding specificity for CCR2 or a chain of such an immunoglobulin.
  • an expression vector comprising a gene (e.g., a fused gene) encoding a humanized immunoglobulin light chain, 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, is provided.
  • An expression vector comprising a gene encoding a humanized immunoglobulin heavy chain, 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 is another example of such a construct.
  • the present description also relates to a host cell comprising a nucleic acid molecule of the present invention, including one or more constructs comprising a nucleic acid molecule of the present invention.
  • the invention relates to a host cell comprising a first recombinant nucleic acid encoding a humanized immunoglobulin light chain, and a second recombinant nucleic acid encoding a humanized immunoglobulin heavy chain, said first nucleic acid comprising a nucleotide sequence encoding a CDR derived from the light chain of murine 1D9 antibody and a framework region derived from a light chain of human origin; and said second nucleic acid comprising a nucleotide sequence encoding a CDR derived from the heavy chain of murine 1D9 antibody and a framework region derived from a heavy chain of human origin.
  • the disclosure also provides a method of preparing a humanized immunoglobulin comprising maintaining a host cell of the present description under conditions appropriate for expression of a humanized immunoglobulin, whereby a humanized immunoglobulin chain(s) is expressed and a humanized immunoglobulin is produced.
  • the method can further comprise the step of isolating the humanized immunoglobulin.
  • the humanized immunoglobulins of the present invention can be less immunogenic than their murine or other nonhuman counterparts.
  • the humanized immunoglobulins described herein can be used as therapeutic agents in humans, for example in the treatment of Ebola or treatment of any other infection causing hemorrhagic fever.
  • the present invention further relates to a method of inhibiting 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.
  • Another embodiment of the invention relates to a method of inhibiting 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 antibody or functional fragment thereof is any of 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 present invention also encompasses a method of inhibiting 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 an antibody (anti-CCR2) or functional fragment thereof which binds mammalian CC-chemokine receptor 2 (CCR2, CKR-2, MCP-IRA or MCP-IRB) 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 a particularly preferred 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.
  • a binding activity e.g., ligand, inhibitor and/or promoter binding
  • a signaling activity e.g., activation of a mamma
  • 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 2antibodies 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 degranulation.
  • 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 of the invention can inhibit binding of a CCR2 ligand (e.g., a chemokine) to CCR2 with an IC50 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 of the present invention 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.
  • a bispecific antibody of the present invention 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 according to the present invention were deposited on July 17, 1998, on behalf of LeukoSite, Inc., 215 First Street, Cambridge, MA 02142, U.S.A. (now Millennium Pharmaceuticals, Inc., 40 Landsdowne Street, Cambridge, MA 02139, U.S.A.), at the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110, U.S.A., under Accession Nos. HB-12549 (1D9) and HB-12550 (8G2).
  • the present invention also pertains to the hybridoma cell lines deposited under ATCC Accession No. HB-12549 and ATCC Accession No. HB-12550, as well as to the monoclonal antibodies produced by the hybridoma cell lines deposited under ATCC Accession Nos. HB- 12549 and HB-12550.
  • the antibodies of the present invention 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; similarly, 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.
  • functional fragments e.g., antigen-binding fragments
  • Antibodies of the present invention 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.
  • 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, and fragments thereof, are useful in therapeutic, applications as described herein.
  • the present invention encompasses an antibody or functional portion thereof of the present invention (e.g., mAb 1D9, 8G2 or MLN1202, or antigen-binding fragments thereof) for use in therapy (including prophylaxis) or diagnosis (e.g., of particular diseases or conditions as described herein), and use of such antibodies or functional portions thereof for the manufacture of a medicament for use in treatment of diseases or conditions 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.
  • 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.
  • 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).
  • 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.
  • 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 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 present invention relates 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 complementarity determining region derived from an antibody of nonhuman origin which binds CCR2 and a framework region derived from a light chain of human origin, and 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 present invention 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 ah (Kabat et ah, Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.
  • 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 (1, (2, (3, (4, ⁇ and ⁇ 2 type, respectively.
  • Human immunoglobulin molecules of a selected class or subclass may contain either a kappa (6) or lambda (8) light chain.
  • immunoglobulin as used herein includes whole antibodies and biologically functional fragments thereof.
  • 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).
  • ligands e.g., HIV, MCP-1, MCP-2, MCP-3, MCP-4.
  • 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.
  • Fab or F(ab') 2 fragments can 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).
  • 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. See, e.g., Cabilly et al., U.S. Patent No. 4,816,567; Cabilly et al, European Patent No. 0,125,023 Bl; Boss et al, U.S. Patent No. 4,816,397; Boss et al, European Patent No.
  • 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 (the nonhuman portion) can be derived from an immunoglobulin of nonhuman origin (referred to as a donor
  • 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).
  • rodent e.g., mouse, rat
  • rabbit pig goat
  • non-human primate e.g., monkey
  • other polyclonal or monoclonal antibodies such as antibodies which bind to the same or similar epitope as the 1D9 antibody, can be made (e.g., Kohler et al., Nature, 256:495-497 (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).
  • a suitable mammal e.g., a mouse, rat, rabbit or sheep.
  • Cells bearing CCR2, membrane fractions containing CCR2, and immunogenic fragments of CCR2 are examples of suitable mammal.
  • Antibody-producing cells e.g., a lymphocyte
  • a suitable immortalized cell e.g., a myeloma cell line
  • Fused cells can be isolated employing selective culturing techniques.
  • Cells which produce antibodies with the desired specificity can be selected by a suitable assay (e.g., ELISA).
  • 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 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
  • 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 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 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 light chain
  • 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:l and SEQ ID NO: 4), 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 immunoglobulins.
  • 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:2 and SEQ ID NO: 3).
  • the invention also provides antigen-binding fragments of these immunoglobulins.
  • the portion of the humanized immunoglobulin or immunoglobulin chain which is of human origin can be derived from any suitable human immunoglobulin or immunoglobulin chain.
  • a human constant region or portion thereof if present, can be derived from the 6 or 8 light chains, and/or the ( (e.g., ⁇ , j2, j3, ⁇ 4), ⁇ , a (e.g., ⁇ , ⁇ 2), ⁇ or ⁇ heavy chains of human antibodies, including allelic variants.
  • a particular constant region (e.g., IgGl), variant or portions thereof can be selected in order to tailor effector function.
  • a mutated constant region can be incorporated into a fusion protein to minimize binding to Fc receptors and/or ability to fix complement (see e.g., Winter et al, GB 2,209,757 B; Morrison et al, WO 89/07142; Morgan et al, WO 94/29351, December 22, 1994).
  • 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.
  • 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)).
  • 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 FRl 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 FRl, 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 of the present invention 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
  • 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.
  • 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. To prepare a portion of a chain, one or more stop codons can be introduced at the desired position.
  • 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. 77:5404 (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 53:851-856 (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 S: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 al, U.S. 5,514,548; Hoogenboom et al, WO 93/06213, published April 1, 1993)).
  • the invention 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: 1.
  • the amino acid sequence comprises at least one, preferably two, and more preferably three of the CDRs of SEQ ID NO: 1.
  • 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: 2.
  • the amino acid sequence comprises at least one, preferably two, and more preferably three of the CDRs of SEQ ID NO: 2. 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: 1 and SEQ ID NO: 4.
  • 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: 2, and SEQ ID NO: 3.
  • 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: 1 and SEQ ID NO: 4, 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: 2, and SEQ ID NO: 3.
  • 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: 1 and a complementary heavy chain, or an antigen-binding fragment of said humanized immunoglobulin having binding specificity for CCR2.
  • the invention relates to a humanized immunoglobulin having binding specificity for CCR2 comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 2 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: 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 2, 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: 5.
  • 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: 6.
  • the invention also relates 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 present invention 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 of the present invention for use 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 present invention also relates more specifically to isolated and/or recombinant nucleic acid molecules comprising a nucleotide sequence which encodes a humanized 1D9 immunoglobulin (i.e., a humanized immunoglobulin of the present invention in which the nonhuman portion is derived from the murine monoclonal antibody 1D9) or chain thereof.
  • 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:2, which is encoded by the nucleic acid sequence of SEQ ID NO:6; (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: 1, which is encoded by the nucleic acid sequence of SEQ ID NO:5; (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
  • the nucleic acid comprises the nucleotide sequence of the variable region as set forth or substantially as set forth in SEQ ID NO:6 or as set forth or substantially as set forth in SEQ ID NO:5, including double or single-stranded polynucleotides.
  • SEQ ID NO:6 the nucleotide sequence of the variable region as set forth or substantially as set forth in SEQ ID NO:6 or as set forth or substantially as set forth in SEQ ID NO:5, including double or single-stranded polynucleotides.
  • 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 invention 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 invention 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 tumor cells and 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, HTV 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, HTV 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.
  • 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 e.g., a heterobifunctional linker
  • 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;
  • 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 is 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 invention relates to a method of preparing a humanized
  • the humanized immunoglobulin which has binding specificity for CCR2.
  • the humanized 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.
  • Constructs or expression vectors suitable for the expression of a humanized 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,
  • 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 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 transcriptional control element e.g., a promoter, an enhancer, terminator
  • translation signals e.g., a signal 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. For example, 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).
  • 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, HIS3) 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 invention includes 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 invention 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 invention also includes host cells comprising the expression vectors of the invention.
  • the invention 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 invention 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, wherein 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, and wherein 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.
  • a host cell e.g., which expresses a humanized immunoglobulin or an antigen binding fragment thereof having specificity for CCR
  • 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., pCA TAB 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.
  • some 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 invention includes pharmaceutical compositions comprising a humanized immunoglobulin or antigen-binding fragment thereof of the invention and a suitable carrier.
  • more than one humanized immunoglobulin which inhibits the binding of CCR2 to its ligands is administered.
  • 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 (e.g., intrabronchial, intranasal or oral inhalation, intranasal drops), or rectal, depending on the disease or condition to be treated.
  • 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 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.
  • 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).
  • the compound can be solubilized and loaded into 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. These 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. For example, where the corresponding mammal is a human, the protein is designated as a human CCR2 protein (e.g., a recombinant human CCR2 produced in a suitable host cell).
  • a human CCR2 protein e.g., a recombinant human CCR2 produced in a suitable host cell
  • One or more antibodies or fragments of the present invention 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 is administered.
  • 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, subcutaneous injection or infusion), inhalation (e.g., intrabronchial, intraocular, intranasal or oral inhalation, intranasal drops), depending on the disease or condition to be treated.
  • parenteral e.g., intravenous, intraarterial, intramuscular, subcutaneous injection or infusion
  • 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 replenishes (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
  • the antibody or fragment can be solubilized and loaded into a suitable dispenser for administration (e.g., an atomizer, nebulizer or pressurized aerosol dispenser).
  • a suitable dispenser for administration e.g., an atomizer, nebulizer or pressurized aerosol dispenser.
  • individuals to whom the herein described antagonists of CCR2 are administered are selected from the following group of individuals:
  • Individuals e.g. humans, receiving pre-/post-exposure prophylaxis. These individuals receive an early administration/treatment before onset of symptoms. Such individuals may be selected from the group comprising relief workers, family members exposed to infected individuals, etc. Such individuals are usually still negative in qPCR
  • 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; Individuals with early onset clinical symptoms, e.g. those with detectable clinical symptoms correlating with a positive qPCR test for viral load; Individuals suffering from hemorrhagic and diarrhea complications, fever, etc. that are in later stages of infections with the herein described agents.
  • diagnostic companies e.g. form Altona Diagnostics, Vela Diagnostics, etc. for Dengue virus, Chikungunya virus
  • Lassa virus and Hantavirus infections specialized laboratories exist, e.g. in clinical institutions in various countries; Individuals with early onset clinical symptoms, e.g. those with detectable clinical symptoms correlating with a positive qPCR test for viral load; Individuals suffering from hemorrhagic and diarrhea complications, fever
  • anti-CCR2 antibodies or fragments or derivatives thereof are particularly preferred.
  • the antibody is administered subcutaneously (sc), in other embodiments, the antibody is administered intravenously (iv)
  • the administration route may be subcutaneously.
  • individuals with early onset clinical symptoms e.g.
  • 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). 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, form day 2, from day 3 of onset of symptoms and continues with the above described dosing regimen.
  • 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.
  • Non-Human Primates are randomized into 2 groups of 3 animals each.
  • the groups consist of 1) Formulation buffer only and 2) anti-CCR2 antibody.
  • the anti- CCR2 antibody used in the example is MLN1202, as described herein, the amino acid sequence of which is depicted in SEQ ID NOs: 1-4.
  • 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 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.
  • 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.
  • the anti- CCR2 antibody used in the example is MLN1202, as described herein, the amino acid sequence of which is depicted in SEQ ID NOs: 1-4.
  • 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 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 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 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: 1 - 4.
  • the antibody is 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 rehydratation 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 gastrointestinal 3-10 days) requiring oral or IV rehydratation).
  • first phase of disease i.e. suspected or confirmed Ebola case patients with early-defined symptoms and with oral rehydratation only (usually day 0-3 from onset of symptoms)
  • second phase of disease confirmed cases with second phase-symptoms (mainly gastrointestinal 3-10 days) requiring oral or IV rehydratation).
  • the optimum dose for the anti-CCR2 antibody 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, diarrhoea 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 antibody intravenously or if not possible subcutaneously.

Abstract

L'invention concerne l'utilisation d'agents antagonistes anti-CCR2, notamment des anticorps neutralisants dirigés contre CCR2 de mammifère, pour le traitement et/ou la prévention de maladies infectieuses chez des individus provoquées par des agents induisant une fièvre hémorragique. Ces antagonistes peuvent être utilisés seuls ou en association avec d'autres médicaments dans le traitement et/ou la prévention d'infections provoquées, par exemple, par le virus Ebola.
PCT/US2015/061765 2014-11-21 2015-11-20 Utilisation d'un antagoniste anti-ccr2 dans le traitement d'une maladie infectieuse WO2016081801A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6084075A (en) * 1996-03-01 2000-07-04 Pharmacia & Upjohn Ab Agonist and antagonist antibodies to the chemokine receptor-2 (CCR2)
WO2005060368A2 (fr) * 2003-12-10 2005-07-07 Millennium Pharmaceuticals, Inc. Anticorps humanises anti-ccr2 et leurs procedes d'utilisation
US20110274696A1 (en) * 2008-08-18 2011-11-10 Amgen Fremont Inc. Antibodies to ccr2
US20120004252A1 (en) * 2009-12-17 2012-01-05 Boehringer Ingelheim International Gmbh New ccr2 receptor antagonists and uses thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6084075A (en) * 1996-03-01 2000-07-04 Pharmacia & Upjohn Ab Agonist and antagonist antibodies to the chemokine receptor-2 (CCR2)
WO2005060368A2 (fr) * 2003-12-10 2005-07-07 Millennium Pharmaceuticals, Inc. Anticorps humanises anti-ccr2 et leurs procedes d'utilisation
US20110274696A1 (en) * 2008-08-18 2011-11-10 Amgen Fremont Inc. Antibodies to ccr2
US20120004252A1 (en) * 2009-12-17 2012-01-05 Boehringer Ingelheim International Gmbh New ccr2 receptor antagonists and uses thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LIN ET AL.: "CCR2-antagonist prophylaxis reduces pulmonary immune pathology and markedly improves survival during influenza infection.", J IMMUNOL., vol. 186, no. Iss. 1, 1 January 2011 (2011-01-01), pages 508 - 515 *

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