WO2006125201A2 - Anticorps anti-muteines mcp-1 biotine-pegylees, compositions, procedes et utilisations correspondants - Google Patents

Anticorps anti-muteines mcp-1 biotine-pegylees, compositions, procedes et utilisations correspondants Download PDF

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WO2006125201A2
WO2006125201A2 PCT/US2006/019625 US2006019625W WO2006125201A2 WO 2006125201 A2 WO2006125201 A2 WO 2006125201A2 US 2006019625 W US2006019625 W US 2006019625W WO 2006125201 A2 WO2006125201 A2 WO 2006125201A2
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mcp
antibody
human
binding
cells
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WO2006125201A3 (fr
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Marian F. Kruszynski
Raymond Sweet
Ping Tsui
Nicole Stowell
Jinguan Luo
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Centocor, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • 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
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to chemically synthesized ligand variants which can be site specifically modified, wherein the ligand can be used for the selection of antibodies in a variety of solid phase assays and assays relying on ligand capture and antibody-ligand complex capture methods. More specifically the invention relates to MCP-I muteins, including specified portions or variants thereof, which may, optionally, be PEGylated or biotin-PEGylated for use in selecting human antibodies from a library.
  • Monocyte chemoattractant protein 1 belongs to the family of chemotactic cytokines known as chemokines. MCP-I is expressed by a variety of cell types including monocytes, vascular endothelial cells, smooth muscle cells, glomerular mesangial cell, osteoblastic cells, and human pulmonary type-2-like epithelial cells. It is believed that MCP-I may play an active role in the initiation and progression of inflammatory diseases, by promoting monocyte influx and subsequent activation in tissues. MCP-I is chemotactic for monocytes but not neutrophils.
  • CHAK CC- chemokine activated killer
  • IL-2 cell surface antigens
  • IL-6 cytokines
  • MCP-I is a potent activator of human basophils, inducing the degranulation and the release of histamines.
  • MCP-I is synthesized in human articular chondrocytes in response to a variety of inflammatory cytokines, and thus may play an active role in the initiation and progression of degenerative and inflammatory arthropathies by promoting monocyte influx and activation in synovial joints. Moreover, elevated levels of MCP-I are observed in macrophage-rich atherosclerotic plaques. The factor activates the tumoricidal activity of monocytes and macrophages in vivo.
  • MCP-I is known to bind and signal through the chemokine receptor, CCR2.
  • CCR2 is a seven trans- membrane-spanning G-protein-coupled receptor expressed on many cells including monocytes, T- cells, B-cells, and basophils.
  • Two MCP-I specific receptors, CCR2A and CCR2B, have been cloned which signal in response to nanomolar (nM) concentrations of MCP-I
  • MCP-I is clearly involved in inflammatory and oxidative stress responses in the vasculature. MCP-I appears to play a role in angiogenesis, the formation of new blood vessels. Tumor cell-secreted MCP-I levels correlate with blood vessel density in a number of tumors, including breast cancer, squamous cell carcinoma of head and neck and esophagus, gastric carcinoma, and hemangioma. Furthermore, high levels of tumor MCP-I were also found to serve as a prognostic biomarker indicating poor prognosis and early relapse.
  • MCP-I in tumor tissues may stimulate angiogenesis by recruiting tumor infiltrating macrophages and subsequent production of angiogenenic growth factors such as vascular endothelial growth factor, tumor necrosis factor oc, and interleukins 6 and 8.
  • angiogenenic growth factors such as vascular endothelial growth factor, tumor necrosis factor oc, and interleukins 6 and 8.
  • MCP-2 proteins with certain functional and sequence homology to human MCP-I are known. Especially similar to MCP-I are MCP-2 and eotaxin, MCP-2 having 68.7 percent sequence identity, respectively, to MCP-I. The range of activities and spectrum of involvement of these proteins in human homeostatic mechanisms and pathology is not as well understood or how it is different than that of MCP-I.
  • MCP-2 is related closely to MCP-I and MCP-3 and uses both CCRl as well as CCR2B as its functional receptors. MCP-3 binds to a receptor designated D6. MCP-3 also binds to CCRlO.
  • the MCP-3 protein (97 amino acids) sequence shows 74 percent identity with MCP-I and 58 percent homology with MCP-2 .
  • MCP-3 differs from MCP-I in being N-glycosylated.
  • MCP-4 shares 56-61 percent sequence identity with the three known monocyte chemotactic proteins and is 60 percent identical with Eotaxin.
  • the functions of MCP-4 appear to be highly similar to those of MCP-3 and Eotaxin .
  • MCP-4 is a potent chemoattractant for monocytes and T-lymphocytes. It is inactive on neutrophils.
  • MCP-4 binds to receptors that recognize MCP-I, MCP-3, and RANTES, such as CCR2.
  • MCP-4 On eosinophils MCP-4 has similar efficacy and potency as MCP-3, RANTES, and Eotaxin.
  • MCP-4 shares receptors with eotaxin (CCRl and CCR3) and shows full cross-desensitization with eotaxin.
  • Other antibodies capable of binding MCP-I or a plurality of beta-chemokines have been reported.
  • Many assays involving protein/protein interactions such as ELISA and antibody/peptide selection from phage display libraries, require immobilization of the target protein.
  • a commonly used method of protein immobilization is to coat the protein directly to a solid support such as ELISA wells.
  • the direct contact between the protein and the solid support via non-specific adsorption can destroy or mask important epitopes, thus negatively impacting the assay results.
  • biotinylated proteins are used to isolate antibodies to these proteins using the technique of antibody phage display.
  • the biotinylated antigen is mixed in solution with a phage antibody library followed by capture via an avidin or streptavidin conjugated solid support such as coated microwell plates or magnetic microbeads.
  • This technique has the advantages of solution based kinetics, controlled antigen concentration to bias affinity selection, and facile separation of bound phage from the library of non-binders. These features are particularly applicable to the selection of high affinity antibodies.
  • the present invention provides isolated biologically active, MCP-I muteins.
  • the invention further provides a synthetic method for producing the biologically active MCP-I muteins of the invention and, optionally, methods of modifying the synthesized proteins.
  • the MCP-I muteins have specific utility in identifying, selecting, or purifying antibodies capable of binding native MCP-I and blocking at least one of the biological activities of native MCP- 1 protein.
  • the modified MCP-I mutein of the invention is a biotin-PEG-MCP-1 mutein.
  • the MCP-I mutein is selected from is selected from human MCP-I Ser 40 , human MCP-I lie 41 , and human MCP-I Tyr 43 .
  • the modified MCP-I mutein is selected from human MCP-I lie 41 , Lys(Biotin-PEG 4 ) 69 ), and (lie 41 , Lys(Biotin-PEG 4 ) 75 .
  • the modified proteins described herein as biotin-PEG-MCP-1 muteins can be used to generate an antibody according to known methods.
  • An antibody of the invention can include or be derived from any mammal, such as but not limited to a human, a mouse, a rabbit, a rat, a rodent, a primate, or any combination thereof, and the like, or the antibody can be derived from a synthetic source, such as a synthetic phage display library or any other suitable library as known in the art.
  • the antibodies of the invention include human monoclonal antibodies which bind to an epitope contained or derived from the disclosed or described human MCP-I muteins, biotin-PEG- MCP-1 muteins, or antibodies that compete with human MCP-I muteins or biotin-PEG-MCP-1 muteins for binding to native human MCP-I.
  • the present invention also provides at least one isolated anti-MCP-1 mutein antibody as described herein, wherein the antibody has at least one activity, such as, but not limited to inhibition of MCP-I binding to chemokine receptors of the C-X-C family, in particular, the CCR2 receptor or, binding to CCR2 receptor expressing cells.
  • An anti-human MCP-I antibody can thus be screened for a corresponding activity according to known methods, such as but not limited to, competition with the a known or made MCP- 1 antibody for at least one biological activity towards an MCP-I protein and inhibition of CCR2 receptor downstream signalling. DESCRIPTION OF THEFIGURES
  • Figure 1 shows the amino acid sequence of human MCP-I (SEQ ID NO: 9) and a ribbons representation of the molecular model.
  • Figure 2 shows a graph representing IC50 binding data showing that the synthetic human MCP-I (He 41 ), (lie 41 , Lys(Biotin-PEG 4 ) 69 ), and (He 41 , Lys(Biotin-PEG 4 ) 75 ) and the wild type human MCP-I competed for the CCR2 receptor with similar potencies
  • Figure 3 shows a graphical representation of data where the synthetic human MCP-I (He 41 ), (He 41 , Lys(Biotin-PEG 4 ) 69 ), and (He 41 , Lys(Biotin-PEG 4 ) 75 ) and/or the wild type human MCP-I was added to 2xlO 5 Thp-1 cells stained with a Ca 2+ flurophore. Binding of MCP-Is to CCR2 of the Thp-1 cells induced change in fluorescence, which was measured in real time before and after the addition of the chemokines.
  • VHlA heavy chain variable sequence FRl, CDRl, FR2, CDR2 variants, FR3, CDR3, FR4
  • VH3 Heavy chain variable sequence FRl, CDRl, FR2, CDR2 variants, FR3, CDR3, FR4
  • Mab - monoclonal antibody PEG - polyethylene glycol
  • PSA penicillin, streptomycin, amphotericin
  • RT room temperature
  • TBS Tris buffered saline
  • v/v - volume per volume w/v - weight per volume
  • an “antibody” includes whole antibodies and any antigen binding fragment or a single chain thereof.
  • the antibody includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule, such as but not limited to at least one complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework (FR) region, or any portion thereof, or at least one portion of a binding protein, which can be incorporated into an antibody of the present invention.
  • CDR complementarity determining region
  • Anti-biotin-PEG-MCP-1- mutein antibody such antibody genereated or derived from antibodies generated using at least one biotinylated and PEGylated MCP- 1 mutein is an antibody that affects the MCP-I ligand, such as but not limited to where such antibody modulates, decreases, increases, antagonizes, angonizes, mitigates, aleviates, blocks, inhibits, abrogates and/or interferes with at least one MCP-I activity or binding, or with MCP-I activity or binding, in vitro, in situ and/or in vivo.
  • a suitable anti-human MCP-I antibody, specified portion or variant of the present invention can bind at least one MCP-I, or specified portions, variants or domains thereof.
  • a suitable anti-human MCP-I antibody, specified portion, or variant can also optionally affect at least one of MCP-I activity or function, such as but not limited to, RNA, DNA or protein synthesis, protein release, MCP-I receptor signaling, membrane MCP-I cleavage, MCP-I activity, MCP-I production and/or synthesis.
  • Biotin refers to biotin (hexahydro-2-oxo-lH-thieno[3,4-d]imidazoline-4-valeric acid); molecular weight 244 g/mol, also known as a B-complex vitamin, and includes avidin-binding analogs thereof.
  • biotinylation is meant a method of attaching the biotin residue to another chemical or biological structure. Biotinylation of a protein is conveniently achieved by chemical conjugation of the biotin to certain functional groups such as amine moiety of lysine side chains or the free amino terminus of a protein, e.g. NHS chemistry. Other methods or sites or conjugation, such as to free sulfhydryls, is also contemplated.
  • MCP-I is used herein to mean the 76 amino acid sequence referenced in NCBI record accession No. NP_002973 and variously known as MCP (monocyte chemotactic protein), SMC-CF (smooth muscle cell chemotactic factor), LDCF (lymphocyte-derived chemotactic factor), GDCF (glioma-derived monocyte chemotactic factor), TDCF (tumor-derived chemotactic factors), HCIl (human cytokine 11), MCAF (monocyte chemotactic and activating factor).
  • MCP monoocyte chemotactic protein
  • SMC-CF smooth muscle cell chemotactic factor
  • LDCF lymphocyte-derived chemotactic factor
  • GDCF glioma-derived monocyte chemotactic factor
  • TDCF tumor-derived chemotactic factors
  • HCIl human cytokine 11
  • MCAF monocyte chemotactic and activating factor
  • the gene symbol is
  • JE is the mouse homolog of human MCP- 1/CCL2.
  • human antibodies of the invention generated from at least one bioinylated and PEGylated MCP-I mutein as described herein may, in certain cases, cross-react with MCP-I from species other than human, or other proteins which are structurally related to human MCP-I (e.g., human MCP-I homologs). In other cases, the antibodies may be completely specific for human MCP-I and not exhibit species or other types of cross-reactivity.
  • monoclonal antibody or “monoclonal antibody composition” as used herein refer to a preparation of antibody molecules of single molecular composition.
  • a monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
  • human monoclonal antibody refers to antibodies displaying a single binding specificity which have variable and constant regions derived from human germline immunoglobulin sequences.
  • binding refers to the affinity of binding to a predetermined ligand or antigen.
  • an antibody binds with a dissociation constant (KD) of 10 "7 M or less, and binds to the predetermined antigen with a K D that is at least twofold less than its K D for binding to a non-specific antigen (e.g., BSA, casein, or any other specified polypeptide) other than the predetermined antigen.
  • KD dissociation constant
  • an antibody recognizing an antigen and “an antibody specific for an antigen” are used interchangeably herein with the term “an antibody which binds specifically to an antigen”.
  • highly specific binding means that the relative K D of the affinity for the specific target ligand, antigen, or epitope of and antigen is at least 10-fold less than the KD for binding that antibody to other ligands.
  • the term "high affinity” with respect to the binding of one protein, for example an antibody, to a ligand, antigen, or epitope on an antigen refers to an interaction having a K D of 10 "8 M or less, more preferably 10 "9 M or less and even more preferably 10 "10 M or less.
  • Kdis or " K 0 ,” or “Kd' as used herein, is intended to refer to the dissociation rate of a particular protein-ligand interaction.
  • the “K D ", is the ratio of the rate of dissociation (k 2 ), also called the “off-rate (k Off )", to the rate of association rate (Ic 1 ) or "on-rate (k on )".
  • K D k2/kl or k off / k on and is expressed as a molar concentration (M). It follows that the smaller KD, the stronger the binding. So a KD of 10 "6 M (or 1 ⁇ M) indicates weak binding compared to 10 "9 M (or InM).
  • MCP-I human monocyte chemoattractant protein 1
  • Figure 1 shows the amino acid sequence of human MCP-I and a ribbons representation of the molecular model and the residues targeted substitution. MCP-I and three analogs were synthesized by step-wise Fmoc solid phase synthesis.
  • biotinylation procedure using this amine chemistry modifies lysine residues on a protein or peptide.
  • the number of biotin per protein or peptide molecule can vary or be consistent, depending on the type of methods and reagents used, as known in the art and as described herein.
  • site-specific biotinylation is used to generated biotinylated MCP-I muteins for use in the invention and such biotinylated targets only selected residues and yield homogenous preparations of modified protein.
  • conjugation on resin was performed by deprotecting the carboxy-terminal octa- or decapeptide, thereby allowing regeneration of the free epsilon amino group of lys 69 or lys 75 .
  • the protected octapeptide-resin Fmoc- Lys(Aloc)-Gln(Trt)-Thr(Bu t )-Gln(Trt)-Thr(Bu t )-Pro-Lys(Boc)-Thr(Bu t )-resin, was first prepared on an automatic peptide synthesizer.
  • the Aloe protecting group at the lys which was to become residue 69 in the final product was removed selectively using palladium tetrakis(tri ⁇ henylphospine), acetic acid, and N-methylmorpholine.
  • the coupling reagent, NHS-dPEG4-biotin was then reacted with the free amine and the final (1-68) amino acids were added.
  • the protected decapeptide-resin Fmoc-Leu-Asp(OBu t )-Lys(Boc)-Gln(Trt)-Thr(Bu t )-Gln(Trt)-Thr(Bu')-Pro-
  • Lys(Mtt)-Thr(Bu')-resin was prepared on an automatic peptide synthesizer.
  • the Mtt group was selectively removed using 1% TFA in dicolormethane with 5% TIS.
  • the coupling reagent, NHS- dPEG4-biotin was then reacted with the free amine and final (1-66) amino acids were added.
  • Human antibodies that are specific for human MCP-I protein or fragments thereof, such as isolated and/or MCP-I protein or a portion thereof (including synthetic molecules, such as synthetic peptides) can be performed using any suitable technique known in the art.
  • Human antibodies can be produced using various techniques known in the art.
  • the human antibody is selected from a phage library, where that phage library expresses human antibodies (Vaughan et Io al. Nature Biotechnology 14:309-314 (1996): Sheets et al. PITAS (USA) 95:6157-6162 (1998)); Hoogenboom and Winter, J. MoI. Biol., 227:381 (1991); Marks et al.' J. MoI.
  • Human antibodies can also be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated.
  • transgenic animals e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated.
  • a transgenic mouse comprising a functionally rearranged human immunoglobulin heavy chain transgene and a transgene comprising DNA from a human immunoglobulin light chain locus that can undergo functional rearrangement, can be immunized with human MCP-I or a fragment thereof to elicit the production of antibodies.
  • the antibody producing cells can be isolated and hybridomas or other immortalized antibody-producing cells can be prepared as described herein and/or as known in the art.
  • the antibody, specified portion or variant can be expressed using the encoding nucleic acid or portion thereof in a suitable host cell.
  • human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos.
  • the human antibody may be prepared via immortalization of human B lymphocytes producing an antibody directed against a target antigen (such B lymphocytes may be recovered from an individual or may have been immunized in vitro). See, e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147 (l):86-95 (1991); and US Pat No. 5,750,373.
  • Antibody producing cells can also be obtained from the peripheral blood or, preferably the spleen or lymph nodes, of humans or other suitable animals that have been immunized with the antigen of interest.
  • Any other suitable host cell can also be used for expressing heterologous or endogenous nucleic acid encoding an antibody, specified fragment or variant thereof, of the present invention.
  • the fused cells (hybridomas) or recombinant cells can be isolated using selective culture conditions or other suitable known methods, and cloned by limiting dilution or cell sorting, or other known methods. Cells which produce antibodies with the desired specificity can be selected by a suitable assay (e.g., ELISA).
  • a hybridoma is produced by fusing a suitable immortal cell line (e.g., a myeloma cell line such as, but not limited to, Sp2/0, Sp2/0-AG14, NSO, NSl, NS2, AE-I, L.5, >243, P3X63Ag8.653, Sp2 SA3, Sp2 MAI, Sp2 SSl, Sp2 SA5, U937, MLA 144, ACT IV, M0LT4, DA-I, JURKAT, WEHI, K-562, COS, RAH, NIH 3T3, HL-60, MLA 144, NAMAIWA, NEURO 2A, or the like, or heteromylomas, fusion products thereof, or any cell or fusion cell derived therefrom, or any other suitable cell line as known in the art.
  • a suitable immortal cell line e.g., a myeloma cell line such as, but not limited to, Sp2/0, Sp2/0-AG14, NSO, NS
  • antibody producing cells such as, but not limited to, isolated or cloned spleen, peripheral blood, lymph, tonsil, or other immune or B cell containing cells, or any other cells expressing heavy or light chain constant or variable or framework or CDR sequences, either as endogenous or heterologous nucleic acid, as recombinant or endogenous, viral, bacterial, algal, prokaryotic, amphibian, insect, reptilian, fish, mammalian, rodent, equine, ovine, goat, sheep, primate, eukaryotic, genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA, chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single, double or triple stranded, hybridized, and the like or any combination thereof. See, e.g., Ausubel, supra, and Colligan, Immunology, supra
  • Human antibodies that bind to human MCP-I and that comprise a defined heavy or light chain variable region can be prepared using suitable methods, such as phage display (Katsube, Y., et al, Int J MoI. Med, l(5):863-868 (1998)).
  • Suitable methods of producing or isolating antibodies of the requisite specificity can be used, including, but not limited to, methods that select recombinant antibody from a peptide or protein library (e.g., but not limited to, a bacteriophage, ribosome, oligonucleotide, RNA, cDNA, or the like, display library; e.g., as available from Cambridge antibody Technologies, Cambridgeshire, UK; MorphoSys, Martinsreid/Planegg, DE;
  • a peptide or protein library e.g., but not limited to, a bacteriophage, ribosome, oligonucleotide, RNA, cDNA, or the like, display library; e.g., as available from Cambridge antibody Technologies, Cambridgeshire, UK; MorphoSys, Martinsreid/Planegg, DE;
  • Applicants exemplified a method of selecting and making human antibodies with the desired affinity, specifity and bioactivity towards human MCP-I starting from a phage display human Fab library.
  • Applicants exemplified a method of selecting and making human antibodies with the desired affinity, specifity and bioactivity towards human MCP-I starting from a phage display human Fab library.
  • human MCP-I and its analogs or "muteins” were chemically synthesized and modified for specific uses in selection, affinity, and biological assays.
  • Human MCP-I He 41 , and human MCP-I Tyr 43 were used in the initial solid phase panning as well as other aspects of antibody selection and affintity maturation assays and described herein as were the biotinylated versions of MCP-I mutein: ⁇ e41, Lys(Biotin-PEG 4 ) 69 ) and ( ⁇ e41, Lys(Biotin-PEG 4 ) 75 (SEQ ID NO: 1).
  • Biacore affinity measurements in the Fab capture mode with MCP-I in solution worked well for ranking of the maturation candidates and the affinities were in the range of 49 to 406 nM.
  • the best parental Fab showed an affinity of 50 nM, indicating that the affinity had to be optimized at least 100 fold to reach the affinity success criterion.
  • the binding to cynomolgus monkey and native human MCP-I could be detected in the Fab capture mode, which was an additional pre-requisite for maturation.
  • the affinities to cynomolgus monkey MCP-I were in the same range as for the human MCP-I.
  • MCP-I Specificity to MCP-I was measured in the antibody capture mode in Biacore, adding 100 nM of each chemokine and detecting the binding signal. Most of the candidate Fabs for maturation were specific, while a couple showed some cross-reactivity to homologue chemokines.
  • a very important feature of the Fab was the neutralizing activity and several different assays were set up to analyze this activity. 125 I MCP-I THP-I cell binding assay was the most sensitive assay, which was especially important after the optimization.
  • the parental Fabs showed IC 50 values from 10 - 650 nM. Beside the radio ligand binding assay other secondary bioassays were planned to prove the neutralizing activity at different levels of the downstream signaling pathway of MCP-I.
  • Attraction of monocytes is one of the major functions of MCP-I but most probably due to missing activity, co-purified factors or endotoxin the parental Fabs did not work in the chemotaxis assay and therefore it was agreed to test the respective IgGl only, instead of trying to get the Fabs working in this assay.
  • Another downstream signaling event is the calcium release into the cytoplasm. Indeed all Fabs, that showed neutralizing activity in the radio ligand binding assay, inhibited the MCP-I induced calcium mobilization in THP-I cells with an a IC 50 range from 0.1 to 3 ⁇ M. It had to be shown that the biological activity of the parental Fabs was completely retained after conversion into the IgG format.
  • Fabs with K 0 in the range of 10 - 400 nM and IC 50 values in the range of 10 - 650 nM in the radio ligand binding assay were selected according to their characteristics. Subsequently they were grouped into 3 groups for the library cloning and the subsequent selection. L-CDR3 and H-CDR2 optimization were performed in parallel. High quality libraries were generated. Solution panning was used for the selection process and the stringency of selection was increased by reduction of antigen, off-rate selection and very long washing steps. For the following screening process a BioVeris screening was used allowing high throughput ranking of the optimized binders. The screening worked very efficiently for identification of improved binders.
  • MOR03471 two were optimized in L-CDR3 only and two came from cross-cloning.
  • the affinity matured candidate analyses and sequences are detailed in Examples 3 and 4, Tables 4 - 6, and SEQ ID Nos: 2-28.
  • Biacore mainly as the detection limits were reached.
  • MorphoSys a very sensitive K D determination method was used, being solution equilibrium titration (SET) combined with BioVeris technology. Monovalent dissociation constants could be calculated by means of appropriate fit models for Fab and IgG. In addition to affinity measurement, this method was used for cross- reactivity studies. The affinities of the final candidates were in the range of 10 to 320 pM to human and cynomolgus MCP-I measured in BioVeris and confirmed by KinexA at Centocor. Specificity testing using BioVeris showed no cross-reactivity to human MCP-2 for all tested 16 Fabs and IgGs.
  • the radio ligand binding assay was the most sensitive assay in this project with an assay IC 50 limit of about 100 pM for Fab and even 20 pM for IgG. Beside affinity, the activity in this assay was used for ranking and selection of optimized binders for detailed characterization. The overall improvement in activity during optimization was up to a factor of 100Ox and finally one MOR03471 derived Fab, MOR03878, showed the highest affinity at
  • MOR03781 and MOR03878 showed IC 50 values in the range of 3 to 5 nM.
  • Native MCP-I was needed to confirm the activities of the MCP-I antibodies isolated against the synthetic or the recombinant MCP-I.
  • Native MCP-I was purified from PANCl supernatant and used for the induction of calcium release. Optimized Fabs showed inhibition of native MCP-I induced
  • MOR03850 and MOR03878 fulfilled all success criteria including specificity criterion and the
  • a humanized or engineered antibody has one or more amino acid residues from a source which is non-human, e.g., but not limited to, mouse, rat, rabbit, non-human primate or other mammal.
  • Antibodies can also optionally be humanized with retention of high affinity for the antigen and other favorable biological properties.
  • humanized antibodies can be optionally prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences.
  • Humanization or engineering of antibodies of the present invention can be performed using any known method, such as but not limited to those described in, Winter (Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen et al., Science 239:1534 (1988)), Sims et al., J. Immunol. 151: 2296 (1993); Chothia and Lesk, J. MoI. Biol. 196:901 (1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285 (1992); Presta et al., J. Immunol.
  • Transgenic mice that can produce a repertoire of human antibodies that bind to human antigens can be produced by known methods (e.g., but not limited to, U.S. Pat. Nos: 5,770,428, 5,569,825, 5,545,806, 5,625,126, 5,625,825, 5,633,425, 5,661,016 and 5,789,650 issued to Lonberg et al; Jakobovits et al. WO 98/50433, Jakobovits et al. WO 98/24893, Lonberg et al. WO 98/24884, Lonberg et al.
  • mice comprise at least one transgene comprising DNA from at least one human immunoglobulin locus that is functionally rearranged, or which can undergo functional rearrangement.
  • the endogenous immunoglobulin loci in such mice can be disrupted or deleted to eliminate the capacity of the animal to produce antibodies encoded by endogenous genes.
  • peptide display libraries Screening antibodies for specific binding to similar proteins or fragments can be conveniently achieved using peptide display libraries.
  • This method involves the screening of large collections of peptides for individual members having the desired function or structure, antibody screening of peptide display libraries is well known in the art.
  • the displayed peptide sequences can be from 3 to 5000 or more amino acids in length, frequently from 5-100 amino acids long, and often from about 8 to 25 amino acids long.
  • several recombinant DNA methods have been described.
  • One type involves the display of a peptide sequence on the surface of a bacteriophage or cell. Each bacteriophage or cell contains the nucleotide sequence encoding the particular displayed peptide sequence.
  • nucleic acid molecule of the present invention encoding at least one anti-MCP-1 antibody can be obtained using methods described herein or as known in the art.
  • Isolated nucleic acid molecules of the present invention can include nucleic acid molecules comprising an open reading frame (ORF), optionally with one or more introns, e.g., but not limited to, at least one specified portion of at least one CDR, as CDRl, CDR2 and/or CDR3 of at least one heavy chain (e.g., SEQ ID NOS: 6-12, 22 and 23) or light chain (e.g., SEQ ID NOS: 13-21 and 24-26); nucleic acid molecules comprising the coding sequence for an anti-MCP-1 antibody or variable region (e.g., SEQ ID NOS:2-5, 27 and 28); and nucleic acid molecules which comprise a nucleotide sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode at least one anti-MCP-1 antibody as described herein and/or as known in the art.
  • ORF open reading frame
  • nucleic acid variants that code for specific anti-MCP-1 antibodies of the present invention. See, e.g., Ausubel, et al., supra, and such nucleic acid variants are included in the present invention.
  • nucleic acid molecules of the present invention which comprise a nucleic acid encoding an anti-MCP-1 antibody can include, but are not limited to, those encoding the amino acid sequence of an antibody fragment, by itself; the coding sequence for the entire antibody or a portion thereof; the coding sequence for an antibody, fragment or portion, as well as additional sequences, such as the coding sequence of at least one signal leader or fusion peptide, with or without the aforementioned additional coding sequences, such as at least one intron, together with additional, non-coding sequences, including but not limited to, non- coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals (for example - ribosome binding and stability of mRNA); an additional coding sequence that codes for additional amino acids, such as those that provide additional functionalities.
  • the sequence encoding an antibody can be fused to a marker sequence, such as
  • the present invention provides isolated nucleic acids that hybridize under selective hybridization conditions to a polynucleotide disclosed herein.
  • the polynucleotides of this embodiment can be used for isolating, 10 detecting, and/or quantifying nucleic acids comprising such polynucleotides.
  • polynucleotides of the present invention can be used to identify, isolate, or amplify partial or full-length clones in a deposited library.
  • the polynucleotides are genomic or cDNA sequences isolated, or otherwise complementary to, a cDNA from a human or mammalian nucleic acid library.
  • the cDNA library comprises at least 80% full-length sequences, preferably at least 85% or
  • the cDNA libraries can be normalized to increase the representation of rare sequences.
  • Low or moderate stringency hybridization conditions are typically, but not exclusively, employed with sequences having a reduced sequence identity relative to complementary sequences.
  • Moderate and high stringency conditions can optionally be employed for sequences of greater identity.
  • Low. stringency conditions allow selective 0 hybridization of sequences having about 70% sequence identity and can be employed to identify orthologous or paralogous sequences.
  • polynucleotides of this invention will encode at least a portion of an antibody encoded by the polynucleotides described herein.
  • the polynucleotides of this invention embrace nucleic acid sequences that can be employed for selective hybridization to a polynucleotide encoding an antibody of the present 5 invention. See, e.g., Ausubel, supra; Colligan, supra, each entirely incorporated herein by reference.
  • the isolated nucleic acids of the present invention can be made using (a) recombinant methods, (b) synthetic techniques, (c) purification techniques, or combinations thereof, as well-known in the art.
  • RNA, cDNA, genomic DNA, or any combination thereof can be obtained from biological sources using any number of cloning methodologies known to those of skill in the art.
  • oligonucleotide probes that selectively hybridize, under stringent conditions, to the polynucleotides of the present invention are used to identify the desired sequence in a cDNA or genomic DNA library.
  • the isolation of RNA, and construction of cDNA and genomic libraries, is well known to those of ordinary skill in the art. (See, e.g., Ausubel, supra; or Sambrook, supra)
  • a cDNA or genomic library can be screened using a probe based upon the sequence of a polynucleotide of the present invention, such as those disclosed herein. Probes can be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different organisms. Those of skill in the art will appreciate that various degrees of stringency of hybridization can be employed in the assay; and either the hybridization or the wash medium can be stringent. As the conditions for hybridization become more stringent, there must be a greater degree of complementarity between the probe and the target for duplex formation to occur.
  • the degree of stringency can be controlled by one or more of temperature, ionic strength, pH and the presence of a partially denaturing solvent such as formamide.
  • the stringency of hybridization is conveniently varied by changing the polarity of the reactant solution through, for example, manipulation of the concentration of formamide within the range of 0% to 50%.
  • the degree of complementarity (sequence identity) required for detectable binding will vary in accordance with the stringency of the hybridization medium and/or wash medium.
  • the degree of complementarity will optimally be 100%, or 70-100%, or any range or value therein. However, it should be understood that minor sequence variations in the probes and primers can be compensated for by reducing the stringency of the hybridization and/or wash medium.
  • RNA or DNA Methods of amplification of RNA or DNA are well known in the art and can be used according to the present invention without undue experimentation, based on the teaching and guidance presented herein.
  • Known methods of DNA or RNA amplification include, but are not limited to, polymerase chain reaction (PCR) and related amplification processes (Mullis, et al., U.S. Patent No. 4,683,202 (1987); and Innis, et al., PCR Protocols A Guide to Methods and Applications, Eds., Academic Press Inc., San Diego, CA (1990).
  • PCR polymerase chain reaction
  • the isolated nucleic acids of the present invention can also be prepared by direct chemical synthesis by known methods (see, e.g., Ausubel, et al., supra). Chemical synthesis generally produces a single-stranded oligonucleotide, which can be converted into double-stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template.
  • a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template.
  • One of skill in the art will recognize that while chemical synthesis of DNA can be limited to sequences of about 100 or more bases, longer sequences can be obtained by the ligation of shorter sequences. A particularly preferred method for chemical synthesis of coding sequences is taught in US Patent Nos. 6521427 and 6670127. 3. Vectors and Expression Systems
  • the invention provides vectors, preferably, expression vectors, containing a nucleic acid encoding the anti-MCP-1 antibody, or may be used to obtain plasmids containing various antibody HC or LC genes or portions thereof.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector wherein additional DNA segments can be ligated into the viral genome.
  • the present invention also relates to vectors that include isolated nucleic acid molecules of the present invention, host cells that are genetically engineered with the recombinant vectors, and the production of at least one anti-MCP-1 antibody by recombinant techniques, as is well known in the art. See, e.g., Sambrook, et al., supra; Ausubel, et al., supra, each entirely incorporated herein by reference.
  • DNAs encoding partial or full-length light and heavy chains can be inserted into expression cassettes or vectors such that the genes are operatively linked to transcriptional and translational control sequences.
  • a cassette which encodes an antibody can be assembled as a construct.
  • a construct can be prepared using methods known in the art. The construct can be prepared as part of a larger plasmid. Such preparation allows the cloning and selection of the correct constructions in an efficient manner. The construct can be located between convenient restriction sites on the plasmid or other vector so that they can be easily isolated from the remaining plasmid sequences.
  • a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid of DEAE-dextran. If the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line and then transduced into host cells. Introduction of a vector construct into a host cell can also be effected by electroporation or other known methods. Such methods are described in the art, such as Sambrook, supra, Chapters 1-4 and 16-18; Ausubel, supra, Chapters 1, 9, 13, 15, 16.
  • the term "operatively linked" is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene.
  • the expression vector and expression control sequences are chosen to be compatible with the expression host cell used.
  • the antibody light chain gene and the antibody heavy chain gene can be inserted into separate vector or, more typically, both genes are inserted into the same expression vector.
  • the antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present).
  • the light and heavy chain variable regions of the antibodies described herein can be used to create full-length antibody genes of any antibody isotype by inserting them into expression vectors already encoding heavy chain constant and light chain constant regions of the desired isotype such that the VH segment is operatively linked to the CH segment(s) within the vector and the VI, segment is operatively linked to the CL segment within the vector.
  • the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell.
  • the antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene.
  • the signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).
  • a mammalian expression vector will contain (1) regulatory elements, usually in the form of viral promoter or enhancer sequences and characterized by a broad host and tissue range; (2) a "polylinker" sequence, facilitating the insertion of a DNA fragment which comprises the antibody coding sequence within the plasmid vector; and (3) the sequences responsible for intron splicing and polyadenylation of mRNA transcripts. This contiguous region of the promoter-polylinker- polyadenylation site is commonly referred to as the transcription unit.
  • the vector will likely also contain (4) a selectable marker gene(s) (e.g., the beta-lactamase gene), often conferring resistance to an antibiotic (such as ampicillin), allowing selection of initial positive transformants in E.
  • a plasmid origin of replication are included for propagation of the expression construct in E. coli and for transient expression in Cos cells, the SV40 origin of replication is included in the expression plasmid.
  • a promoter may be selected from a SV40 promoter, (e.g., late or early SV40 promoters, the CMV promoter (US PatNos. 5,168,062; 5,385,839), an HSV tk promoter, a pgk (phosphoglycerate kinase) promoter, an EF-I alpha promoter (US Pat.No. 5,266,491), at least one human immunoglobulin promoter.
  • a SV40 promoter e.g., late or early SV40 promoters, the CMV promoter (US PatNos. 5,168,062; 5,385,839), an HSV tk promoter, a pgk (phosphoglycerate kinase) promoter, an EF-I alpha promoter
  • Expression vectors will preferably but optionally include at least one selectable marker.
  • markers include, e.g., but not limited to, methotrexate (MTX), dihydrofolate reductase (DHFR, US PatNos. 4,399,216; 4,634,665; 4,656,134; 4,956,288; 5,149,636; 5,179,017, ampicillin, neomycin (G418), mycophenolic acid, or glutamine synthetase (GS, US Pat.Nos. 5,122,464; 5,770,359; 5,827,739) resistance for eukaryotic cell culture, and tetracycline or ampicillin resistance genes for culturing in E.
  • MTX methotrexate
  • DHFR dihydrofolate reductase
  • DHFR dihydrofolate reductase
  • DHFR dihydrofolate reductase
  • DHFR dihydrofolate reductase
  • coli and other bacteria or prokaryotics are entirely incorporated hereby by reference.
  • Appropriate culture mediums and conditions for the above- described host cells are known in the art. Suitable vectors will be readily apparent to the skilled artisan.
  • polyadenlyation or transcription terminator sequences are typically incorporated into the vector.
  • An example of a terminator sequence is the polyadenlyation sequence from the bovine growth hormone gene. Sequences for accurate splicing of the transcript can also be included.
  • An example of a splicing sequence is the VPl intron from SV40 (Sprague, et al., J. Virol. 45:773-781 (1983)).
  • gene sequences to control replication in the host cell can be incorporated into the vector, as known in the art. Also, to avoid high surface expression of heavy chain molecules, it may be necessary to use an expression vector that eliminates transmembrane domain variant splices.
  • Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription can be achieved with the early and late promoters from SV40, the long terminal repeats (LTRS) from Retroviruses, e.g., RSV, HTLVI, HTVI and the early promoter of the cytomegalovirus (CMV). However, cellular elements can also be used (e.g., the human actin promoter).
  • LTRS long terminal repeats
  • CMV cytomegalovirus
  • cellular elements can also be used (e.g., the human actin promoter).
  • Suitable expression vectors for use in practicing the present invention include, for example, vectors such as pIRESlneo, pRetro-Off, pRetro-On, PLXSN, or pLNCX (Clonetech Labs, Palo Alto, CA), pcDNA3.1 (+/-), pcDNA/Zeo (+/-) or pcDNA3.1/Hygro (+/-) (Invitrogen), PSVL and PMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109).
  • vectors such as pIRESlneo, pRetro-Off, pRetro-On, PLXSN, or pLNCX (Clonetech Labs, Palo Alto, CA), pcDNA3.1 (+/-), pcDNA/Zeo (+/-) or pcDNA3.1/Hy
  • the nucleic acids encoding the antibody sequence can be expressed in stable cell lines that contain the gene integrated into a chromosome.
  • a selectable marker such as dhfr, gpt, neomycin, or hygromycin allows the identification and isolation of the transfected cells which express large amounts of the encoded antibody.
  • the DHFR (dihydrofolate reductase) marker is useful to develop cell lines that carry several hundred or even several thousand copies of the gene of interest.
  • Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy, et al., Biochem. J.
  • the DNA constructs used in the production of the antibodies of the invention can optionally include at least one insulator sequence.
  • the terms "insulator”, “insulator sequence” and “insulator element” are used interchangeably herein.
  • An insulator element is a control element which insulates the transcription of genes placed within its range of action but which does not perturb gene expression, either negatively or positively.
  • an insulator sequence is inserted on either side of the DNA sequence to be transcribed.
  • the insulator can be positioned about 200 bp to about 1 kb, 5' from the promoter, and at least about 1 kb to 5 kb from the promoter, at the 3' end of the gene of interest.
  • the distance of the insulator sequence from the promoter and the 3' end of the gene of interest can be determined by those skilled in the art, depending on the relative sizes of the gene of interest, the promoter and the enhancer used in the construct.
  • more than one insulator sequence can be positioned 5' from the promoter or at the 3' end of the transgene.
  • two or more insulator sequences can be positioned 5' from the promoter.
  • the insulator or insulators at the 3' end of the transgene can be positioned at the 3' end of the gene of interest, or at the 3'end of a 3' regulatory sequence, e.g., a 3' untranslated region (UTR) or a 3' flanking sequence.
  • UTR 3' untranslated region
  • Suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pET Hd (Studier et al., Gene Expression Technology:
  • Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter.
  • Target gene expression from the pET Hd vector relies on transcription from a T7 gnlO- lac fusion promoter mediated by a co-expressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident ⁇ prophage harboring a T7 gnl gene under the transcriptional control of the lacUV 5 promoter.
  • the expression vector is a yeast expression vector.
  • yeast expression vectors examples include pYepSecl (Baldari et al. (1987) EMBO J.
  • the expression vector is a baculovirus expression vector.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al. (1983) MoI. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).
  • a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6: 187- 195).
  • the expression vector's control functions are often provided by viral regulatory elements.
  • commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
  • suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook et al., supra.
  • the recombinant mammalian expression vector is capable of directing expression of the nucleic acid, preferentially in a particular cell type, such as lymphoma cells (e.g., mouse myeloma cells).
  • tissue-specific regulatory elements are used to express the nucleic acid. Tissue-specific regulatory elements are known in the art.
  • suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol.
  • promoters of T cell receptors Winoto and Baltimore (1989) EMBO J. 8:729-733 and immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S.
  • the DNA molecule is operably linked to a regulatory sequence in a manner that allows for expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to the mRNA encoding a polypeptide.
  • Regulatory sequences operably linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types. For instance, viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific, or cell type specific expression of antisense RNA.
  • the antisense expression vector can be in the form of a recombinant plasmid, phagemid, or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced.
  • a high efficiency regulatory region the activity of which can be determined by the cell type into which the vector is introduced.
  • cM-T412 A chimeric mouse/human IgGIk monoclonal antibody against human CD4, known as cM-T412 (EP0511308 entirely incorporated by reference), was observed to be expressed at high levels in transfected mouse myeloma cells (Looney et al. 1992. Hum Antibodies Hybridomas 3(4): 191-200). Without a large effort at optimizing culture conditions, production levels of > 500 mg/L (specific productivity on a pg/cell/day basis not known) were readily obtained at Centocor, Inc. Malvern, PA in 1990.
  • antibody-cloning vectors were developed useful for HC and LC cloning which include the gene promoter/transcription initiation nucleic acid sequence, the 5' untranslated sequences and translation initiation nucleic acid sequences, the nucleic acid sequences encoding the signal sequence, the intron/exon splice donor sequences for the signal intron and the J-C intron, and the J-C intron enhancer nucleic acid sequences.
  • Plasmid ⁇ l39 a pUC19 plasmid, contains a 5.8 kb EcoRI-EcoRI genomic fragment cloned from C123 hybridoma cells secreting the fully mouse M-T412 Ab; the fragment contains the promoter and V region part of the cM-T412 HC gene.
  • the starting material for LC V region vector engineering was plasmid ⁇ 39, a pUC plasmid that contains a 3 kb HindUJ-Hindlll genomic fragment cloned from C123 hybridoma cells; this fragment contains the promoter and V region part of the cM- T412 LC gene.
  • the engineered vectors derived from ⁇ l39 and ⁇ 39 were designed to enable convenient assembly of HC or LC genes suitable for expression in a mammalian host cell in a two- step process that entails 1) cloning DNA encoding a sequence of interest between specially-prepared restriction sites in a V region vector, whereby the V-region coding sequence is positioned immediately downstream of the vector-encoded signal sequence, as well as downstream of part or all of the gene promoter; and 2) transferring a fragment that spans the inserted sequence from the V region vector to the C region vector in the proper orientation whereby the resulting plasmid constitutes the final expression plasmid suitable for expression in cells (Scallon et al. 1995 Cytokine 7(8):759-769).
  • Plasmid pC4 is a derivative of the plasmid pSV2-dhfr (ATCC Accession No. 37146).
  • the plasmid contains the mouse DHFR gene under control of the SV40 early promoter.
  • Chinese hamster ovary- or other cells lacking dihydrofolate activity that are transfected with these plasmids can be selected by growing the cells in a selective medium (e.g., alpha minus MEM, Life Technologies, Gaithersburg, MD) supplemented with the chemotherapeutic agent methotrexate.
  • a selective medium e.g., alpha minus MEM, Life Technologies, Gaithersburg, MD
  • methotrexate methotrexate
  • Plasmid pC4 contains for expressing the gene of interest the strong promoter of the long terminal repeat (LTR) of the Rous Sarcoma Virus (Cullen, et al., Molec. Cell. Biol. 5:438-447 (1985)) plus a fragment isolated from the enhancer of the immediate early gene of human cytomegalovirus (CMV) (Boshart, et al., Cell 41:521-530 (1985)). Downstream of the promoter are BamHI, Xbal, and Asp718 restriction enzyme cleavage sites that allow integration of the genes. Behind these cloning sites the plasmid contains the 3' intron and polyadenylation site of the rat preproinsulin gene.
  • LTR long terminal repeat
  • CMV cytomegalovirus
  • High efficiency promoters can also be used for the expression, e.g., the human b-actin promoter, the SV40 early or late promoters or the long terminal repeats from other retroviruses, e.g., HTV and HTLVI.
  • Clontech's Tet-Off and Tet-On gene expression systems and similar systems can be used to express the MCP-I antibody in a regulated way in mammalian cells (M. Gossen, and H. Bujard, Proc. Natl. Acad. Sci. USA 89: 5547-5551 (1992)).
  • Other signals e.g., from the human growth hormone or globin genes can be used as well.
  • At least one anti-MCP-1 antibody of the present invention can be optionally produced by a cell line, a mixed cell line, an immortalized cell or clonal population of immortalized cells, as well known in the art. See, e.g., Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2004); Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2 nd Edition, Cold Spring Harbor, NY (1989); Harlow and Lane, antibodies, a Laboratory Manual, Cold Spring Harbor, NY (1989); Colligan, et al., eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2004); Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997-2004), each entirely incorporated herein by reference.
  • a production cell line capable of efficient and reproducible expression of a recombinant polypeptide(s) is required.
  • the cell line is stable and bankable.
  • a variety of host cell lines can be employed for this purpose. As the understanding of the complexities of how the cellular machinery impact the final amount and composition of a biotherapeutic product, the selection of a host cell line which will impart the needed attributes to the production and the composition of the product become more evident.
  • antibody genes are assembled from gene segments that may be widely separated in the germ line.
  • heavy chain genes are formed by recombination of three genomic segments encoding the variable (V), diversity (D) and joining (J)/constant (C) regions of the antibody.
  • Functional light chain genes are formed by joining two gene segments; one encodes the V region and the other encodes the J/C region.
  • Both the heavy chain and kappa light chain loci contain many V gene segments (estimates vary between 100s and 1000s) estimated to span well over 1000 kb.
  • the lambda locus is, by contrast, much smaller and has been shown to span approximately 300 kb on chromosome 16 in the mouse. It consists of two variable gene segments and four joining/constant (J/C) region gene segments. Formation of a functional gene requires recombination between a V and a J/C element.
  • control of transcription of both rearranged heavy and kappa light chain genes depends both on the activity of a tissue specific promoter upstream of the V region and a tissue specific enhancer located in the J-C intron. These elements act synergistically. Also, a second B-cell specific enhancer has been identified in the kappa light chain locus. This further enhancer is located 9 kb downstream of C kappa .
  • the hybridoma method of immortalizing antibody expression genes relies on the endogenous promoter and enhancer sequences of the parent B-cell lineage.
  • nucleic acids of the present invention can be expressed in a host cell by turning on (by manipulation) in a host cell that contains endogenous DNA encoding an antibody of the present invention.
  • Such methods are well known in the art, e.g., as described in US patent Nos. 5,580,734, 5,641,670, 5,733,746, and 5,733,761, entirely incorporated herein by reference.
  • Antibodies of the invention can be produced in a host cell transfectoma using, for example, a combination of recombinant DNA techniques and gene transfection methods as is well known in the art (e.g., Morrison, S. (1985) Science 229:1202).
  • Suitable host cells include bacteria, mammalian cells, plant cells,
  • Mammalian cell lines available in the art for expression of a heterologous polypeptide intact glycosylated proteins include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells (BHK), NSO mouse melanoma cells and derived cell lines, e.g. SP2/0, YB2/0 (ATC CRL-1662) rat myeloma cells, human embryonic kidney cells (HEK), human embryonic retina cells PerC.6 cells, hep G2 cells, BSC-I (e.g., ATCC CRL-26)
  • a common, preferred bacterial host is E. coli.
  • Mammalian cells such as CHO cells, myeloma cells, HEK293 cells, BHK cells (BHK21, ATCC CRL- 10), mouse Ltk- cells, and NIH3T3 cells have been frequently used for stable expression of heterologous genes.
  • cell lines such as Cos (COS-I ATCC CRL 1650; COS-7, ATCC CRL- 15 1651) and HEK293 are routinely used for transient expression of recombinant proteins.
  • Preferred mammalian host cells for expressing the recombinant antibodies of the invention include myeloma cells such as Sp2/0, YB2/0 (ATC CRL-1662), NSO, and P3X63.Ag8.653 (e.g. SP2/0-Agl4) because of their high rate of expression.
  • myeloma cells such as Sp2/0, YB2/0 (ATC CRL-1662), NSO, and P3X63.Ag8.653 (e.g. SP2/0-Agl4) because of their high rate of expression.
  • another preferred expression system is the GS gene expression system disclosed in WO 87/04462, WO 20 89/01036 and EP 338,841.
  • the antibodies When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods.
  • mammalian cells are mammalian cells. Mammalian cell systems often will be in the form of monolayers of cells although mammalian cell suspensions or bioreactors can also be used.
  • COS-I e.g., ATCC CRL 1650
  • COS-7 e.g., ATCC CRL-1651
  • 30 HEK293, BHK21 e.g., ATCC CRL-IO
  • CHO e.g., ATCC CRL 1610
  • BSC-I e.g., ATCC CRL-26 cell lines
  • Cos-7 cells CHO cells, hep G2 cells, P3X63Ag8.653, SP2/0-Agl4, 293 cells, HeLa cells and the like, which are readily available from, for example, American Type Culture Collection, Manassas,
  • Preferred host cells include cells of lymphoid origin such as myeloma and lymphoma cells. Particularly preferred host cells are P3X63Ag8.653 cells (ATCC Accession Number CRL-1580) and SP2/0-Agl4 cells (ATCC Accession Number CRL-1851).
  • CHO-Kl and DHFR- CHO cells DG44 and DUK-BI l are used for high-level protein production because the amplification of genes of interest is enabled by the incorporation of a selectable, amplifiable marker, DHFR using e.g. the drug methotrexate (MTX) (RJ. Kaufman, 1990. Methods Enzymol. 185: 537-566).
  • DHFR " CHO cells can be successfully used to produce recombinant mAbs at a high level.
  • DHFR " CHO may produce ant-MCP-1 antibodies at the rate of 80-110 mg 10 6 cells "1 day “1 or more than 200 mg 10 6 cells "1 day “1 .
  • a variety of promoters have been used to obtain expression of H- and L-chains in these CHO cells, for example, the b-actin promoter, the human CMV MIE promoter, the Ad virus major late promoter (MLP), the RSV promoter, and a murine leukemia virus LTR.
  • MLP Ad virus major late promoter
  • RSV RSV promoter
  • a murine leukemia virus LTR murine leukemia virus LTR.
  • vectors for mAb expression are described in the literature in which the two Ig chains are carried by two different plasmids with an independent selectable/amplifiable marker. Vectors containing one antibody chain, e.g.
  • the H-chain, linked to a DHFR marker, and an L-chain expression cassette with the Neo r marker or vice versa to can be used obtain up tol80 mg of a humanized mAb L "1 7 day "1 in spinner flasks.
  • the methods used for initial selection and subsequent amplification can be varied and are well known to those skilled in the art.
  • high-level mAb expression can be obtained using the following steps: initial selection and subsequent amplification of candidate clones, coselection (e.g., in cases where both H-chain and L-chain expression vectors carry DHFR expression unit) and amplification, coamplification using different amplifiable markers, and initial selection and amplification in mass culture, followed by dilution cloning to identify individual high-expressing clones.
  • coselection e.g., in cases where both H-chain and L-chain expression vectors carry DHFR expression unit
  • coamplification using different amplifiable markers e.g., in cases where both H-chain and L-chain expression vectors carry DHFR expression unit
  • initial selection and amplification in mass culture e.g., in cases where both H-chain and L-chain expression vectors carry DHFR expression unit
  • coamplification using different amplifiable markers e.g., in cases where both H-chain and L-chain expression vectors carry DHFR expression unit
  • transgenic tobacco leaves expressing recombinant proteins have been successfully used to provide large amounts of recombinant proteins, e.g., using an inducible promoter. See, e.g., Cramer et al., Curr. Top. Microbol. Immunol. 240:95-118 (1999) and references cited therein.
  • transgenic maize have been used to express mammalian proteins at commercial production levels, with biological activities equivalent to those produced in other recombinant systems or purified from natural sources. See, e.g., Hood et al., Adv. Exp. Med.
  • Antibodies have also been produced in large amounts from transgenic plant seeds including antibody fragments, such as single chain antibodies (scFv's), including tobacco seeds and potato tubers. See, e.g., Conrad et al., Plant MoI. Biol. 38:101-109 (1998) and reference cited therein.
  • scFv's single chain antibodies
  • transgenic plants can also be produced using transgenic plants, according to know methods. See also, e.g., Fischer et al., Biotechnol. Appl. Biochem. 30:99-108 (Oct., 1999), Ma et al., Trends Biotechnol.
  • An anti-MCP-1 antibody can be recovered and purified from recombinant cell cultures by well- known methods including, but not limited to, protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography.
  • High performance liquid chromatography can also be employed for purification. See, e.g., Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997-2001), e.g., Chapters 1, 4, 6, 8, 9, 10, each entirely incorporated herein by reference.
  • Antibodies of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a eukaryotic host, including, for example, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the antibody of the present invention can be glycosylated or can be non-glycosylated, with glycosylated preferred. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Sections 17.37- 17.42; Ausubel, supra, Chapters 10, 12, 13, 16, 18 and 20, Colligan, Protein Science, supra, Chapters 12-14, all entirely incorporated herein by reference.
  • Anti-MCP-1 antibodies (also termed anti-CCL-2 antibodies or MCP-I antibodies) useful in the 5 methods and compositions of the present invention can optionally be characterized by high affinity binding to MCP-I, highly specific binding to MCP-I, ability to inhibit one or more of the biologic activities associated with MCP-I, and optionally and preferably having low toxicity.
  • the antibodies of the invention can bind human MCP-I with a wide range of affinities (K D ).
  • at least one human mAb of the present invention can optionally bind human 10 MCP-I with high affinity.
  • a human mAb can bind human MCP-I with a K D equal to or less than about 10 "7 M, such as but not limited to, 0.1-9.9 (or any range or value therein) X 10 '7 , 10 "8 , lO ⁇ lO 40 , l ⁇ 11 , Kr 12 , 10 "13 or any range or value therein.
  • the affinity or avidity of an antibody for an antigen can be determined experimentally using any suitable method. (See, for example, Berzofsky, et al, "Antibody-Antigen Interactions," In
  • the measured affinity of a particular antibody-antigen interaction can vary if measured under different conditions (e.g., salt concentration, pH).
  • affinity and other antigen-binding parameters e.g., K D , K a , K d
  • K D , K a , K d are preferably made with standardized solutions of antibody and antigen
  • a standardized buffer such as the standard soluctions and buffers described herein.
  • the isolated antibodies of the present invention comprise an antibody amino acid sequences disclosed herein encoded by any suitable polynucleotide, or any isolated or prepared antibody.
  • the human antibody or antigen-binding fragment binds human MCP-I and, thereby partially or substantially neutralizes at least one biological activity of the protein.
  • neutralizing antibody refers to an antibody that can inhibit an MCP-I -dependent activity by about 20-120%, preferably by at least about
  • an anti-MCP-1 antibody to inhibit an MCP-1-dependent activity is preferably assessed by at least one suitable MCP-I protein or receptor assay, as described herein and/or as known in the art.
  • a human antibody of the invention can be of any class (IgG, IgA, IgM, IgE, IgD, etc.) or isotype and can comprise a kappa or lambda light chain.
  • the human antibody comprises an IgG heavy chain or defined fragment, for example, at least one of isotypes, IgGl, IgG2, IgG3 or IgG4.
  • Antibodies of this type can be prepared by employing a transgenic mouse or other trangenic non-human mammal comprising at least one human light chain (e.g., IgG, IgA, and IgM (e.g., ⁇ l., ⁇ 2, ⁇ 3, ⁇ 4) transgenes as described herein and/or as known in the art.
  • the anti-human MCP-I human antibody comprises an IgGl heavy chain and an IgGl light chain.
  • At least one antibody of the invention binds at least one specified epitope specific to at least one MCP-I protein, fragment, portion or any combination thereof.
  • the at least one epitope can comprise at least one antibody binding region that comprises at least one portion of the protein, which epitope is preferably comprised of at least 1-3 amino acids to the entire specified portion of contiguous amino acids of the SEQ ID NO: 1.
  • the human antibody or antigen-binding fragment of the present invention will comprise an antigen-binding region that comprises at least one human complementarity determining region (CDRl, CDR2 and CDR3) or variant of at least one heavy chain variable region and at least one human complementarity determining region (CDRl, CDR2 and CDR3) or variant of at least one light chain variable region.
  • the antibody or antigen-binding portion or variant can comprise at least one of the heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 9 OR 12, and/or a light chain CDR3 having the amino acid sequence of SEQ ID NO: 15 - 17, 20 OR 21.
  • the antibody or antigen-binding fragment can have an antigen-binding region that comprises at least a portion of at least one heavy chain CDR (i.e., CDRl, CDR2 and/or CDR3) having the amino acid sequence of the corresponding CDRs 1, 2, and/or 3 (e.g., SEQ ID NOS: 6-12 and/or 22, 23, and 26).
  • the antibody or antigen-binding portion or variant can have an antigen-binding region that comprises at least a portion of at least one light chain CDR (i.e., CDRl, CDR2 and/or CDR3) having the amino acid sequence of the corresponding CDRs 1, 2 and/or 3 (e.g., SEQ ID NOS: 13-21 and/or 24 and 25).
  • CDRl light chain CDR
  • CDR2 light chain CDR3
  • CDR3 having the amino acid sequence of the corresponding CDRs 1, 2 and/or 3 (e.g., SEQ ID NOS: 13-21 and/or 24 and 25).
  • the three heavy chain CDRs and the three light chain CDRs of the anitbody or antigen-binding fragment an amino acid sequence derived from the corresponding CDR of at least one of Fab MOR0336, MOR03464, MOR03468, MOR03470, MOR03471, MOR03473, MOR03548, as described herein and the heavy chain framework regions derived from a VHB antibody (SEQ ID NO. 2) and the light chain framework regions derived from the a kappa-type antibody (SEQ ID No. 4).
  • Such antibodies can be prepared by chemically joining together the various portions (the CDRs and frameworks) of the antibody using conventional techniques, by preparing and expressing a nucleic acid molecule that encodes the antibody using conventional techniques of recombinant DNA technology or by using any other suitable method.
  • the anti-MCP-1 antibody can comprise at least one of a heavy or light chain variable region having a defined amino acid sequence in the framework regions.
  • the anti-MCP-1 antibody comprises at least one of at least one heavy chain variable region, optionally having the amino acid sequence of SEQ ID NO: 2 or 3 and/or at least one light chain variable region, optionally having the amino acid sequence of SEQ ID NO: 4 or 5.
  • Antibody class or isotype (IgA, IgD, IgE, IgG, or IgM) is conferred by the constant regions that are encoded by heavy chain constant region genes.
  • human IgG class there are four subclasses or subtypes: IgGl, IgG2, IgG3 and IgG4 named in order of their natural abundance in serum starting from highest to lowest.
  • IgA antibodies are found as two subclasses, IgAl and IgA2.
  • isotype switching also refers to a change between IgG subclasses or subtypes.
  • the invention also relates to antibodies, antigen-binding fragments, immunoglobulin chains and CDRs comprising amino acids in a sequence that is substantially the same as an amino acid sequence described herein.
  • such antibodies or antigen-binding fragments and antibodies comprising such chains or CDRs can bind human MCP-I with high affinity (e.g., K D less than or equal to about 10 " 9 M).
  • Amino acid sequences that are substantially the same as the sequences described herein include sequences comprising conservative amino acid substitutions, as well as amino acid deletions and/or insertions.
  • a conservative amino acid substitution refers to the replacement of a first amino acid by a second amino acid that has chemical and/or physical properties (e.g, charge, structure, polarity, hydrophobicity/ hydrophilicity) that are similar to those of the first amino acid.
  • Conservative substitutions include replacement of one amino acid by another within the following groups: lysine (K), arginine (R) and histidine (H); aspartate (D) and glutamate (E); asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), K, R, H, D and E; alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), tryptophan (W), methionine (M), cysteine (C) and glycine (G); F, W and Y; C, S and T.
  • An anti-MCP-1 antibody of the present invention can include one or more amino acid substitutions, deletions or additions, either from natural mutations or human manipulation, as specified herein or as taught in Knappik et al. US6828422 for variable regions derived from human germline gene sequences and categorized by sequence similarities into families designated as VHlA, VHlB, VH2, etc. and by light chains as kappa or lambda subgroups.
  • the number of amino acid substitutions a skilled artisan would make depends on many factors, including those described above. Generally speaking, the number of amino acid substitutions, insertions or deletions for any given anti-MCP-1 antibody, fragment or variant will not be more than 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, such as 1-30 or any range or value therein, as specified herein.
  • Amino acids in an anti-MCP-1 antibody of the present invention that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine- scanning mutagenesis (e.g., Ausubel, supra, Chapters 8, 15; Cunningham and Wells, Science
  • Anti-MCP-1 antibodies of the present invention can include, but are not limited to, at least one portion, sequence or combination selected from 5 to all of the contiguous amino acids of at least one of SEQ ID NOS: 2-5 and 27-28.
  • An anti-MCP-1 antibody can further optionally comprise a polypeptide of at least one of SEQ ID NOS: 27 and 28.
  • the amino acid sequence of an immunoglobulin chain, or portion thereof has about 100% identity to the amino acid sequence of the corresponding chain of at least one of SEQ ID NOS: 27-28 but for conservative substitutions which do not change the binding specificity of the anti-MCP-1 antibody.
  • the amino acid sequence of a light chain variable region can be compared with the sequence of SEQ ID NO: 4 or 5, or the amino acid sequence of a heavy chain can be compared with SEQ ID NO: 2 or 3.
  • the amino acid identity is determined using a suitable computer algorithm, as known in the art.
  • the present invention includes at least one biologically active antibody of the present invention.
  • Biologically active antibodies have a specific activity at least 20%, 30%, or 40%, and preferably at least 50%, 60%, or 70%, and most preferably at least 80%, 90%, or 95%-1000% of that of the native (non-synthetic), endogenous or related and known antibody.
  • Methods of assaying and quantifying measures of enzymatic activity and substrate specificity are well known to those of skill in the art and described herein.
  • the invention relates to human antibodies and antigen-binding fragments, as described herein, which are modified by the covalent attachment of an organic moiety.
  • the organic moiety can be a linear or branched hydrophilic polymeric group, fatty acid group, or fatty acid ester group.
  • the hydrophilic polymeric group can have a molecular weight of about 800 to about 120,000 Daltons and can be a polyalkane glycol (e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)), carbohydrate polymer, amino acid polymer or polyvinyl pyrolidone, and the fatty acid or fatty acid ester group can comprise from about eight to about forty carbon atoms.
  • the modified antibodies and antigen-binding fragments of the invention can comprise one or more organic moieties that are covalently bonded, directly or indirectly, to the antibody.
  • Each organic moiety that is bonded to an antibody or antigen-binding fragment of the invention can independently be a hydrophilic polymeric group, a fatty acid group or a fatty acid ester group.
  • fatty acid encompasses mono-carboxylic acids and di-carboxylic acids.
  • Hydrophilic polymers suitable for modifying antibodies of the invention can be linear or branched and include, for example, polyalkane glycols (e.g., PEG, monomethoxy-polyethylene glycol (mPEG), PPG and the like), carbohydrates (e.g., dextran, cellulose, oligosaccharides, polysaccharides and the like), polymers of hydrophilic amino acids (e.g., polylysine, polyarginine, polyaspartate and the like), polyalkane oxides (e.g., polyethylene oxide, polypropylene oxide and the like) and polyvinyl pyrolidone.
  • polyalkane glycols e.g., PEG, monomethoxy-polyethylene glycol (mPEG), PPG and the like
  • carbohydrates e.g., dextran, cellulose, oligosaccharides, polysaccharides and the like
  • polymers of hydrophilic amino acids e.g., polylysine,
  • the hydrophilic polymer that modifies the antibody of the invention has a molecular weight of about 800 to about 150,000 Daltons as a separate molecular entity.
  • PEG 50 Oo and PEG 2 o,ooo wherein the subscript is the average molecular weight of the polymer in Daltons, can be used.
  • the hydrophilic polymeric group can be substituted with one to about six alkyl, fatty acid or fatty acid ester groups. Hydrophilic polymers that are substituted with a fatty acid or fatty acid ester group can be prepared by employing suitable methods.
  • a polymer comprising an amine group can be coupled to a carboxylate of the fatty acid or fatty acid ester, and an activated carboxylate (e.g., activated with N, N- carbonyl diimidazole) on a fatty acid or fatty acid ester can be coupled to a hydroxyl group on a polymer.
  • an activated carboxylate e.g., activated with N, N- carbonyl diimidazole
  • Fatty acids and fatty acid esters suitable for modifying antibodies of the invention can be saturated or can contain one or more units of unsaturation.
  • Fatty acids that are suitable for modifying antibodies of the invention include, for example, n-dodecanoate (C 12 , laurate), n-tetradecanoate (Ci 4 , myristate), n- octadecanoate (Ci 8 , stearate), n-eicosanoate (C 2 o, arachidate) , n-docosanoate (C 22 , behenate), n- triacontanoate (C 30 ), n-tetracontanoate (C40), cw-A9-octadecanoate (Ci 8 , oleate), all ci ⁇ - ⁇ 5,8,ll,14- eicosatetraenoate (C 20 , arachidonate), octanedioic acid, te
  • modified human antibodies and antigen-binding fragments can be prepared using suitable methods, such as by reaction with one or more modifying agents.
  • An “activating group” is a chemical moiety or functional group that can, under appropriate conditions, react with a second chemical group thereby forming a covalent bond between the modifying agent and the second chemical group.
  • amine-reactive activating groups include electrophilic groups such as tosylate, mesylate, halo (chloro, bromo, fluoro, iodo), N- hydroxysuccinimidyl esters (NHS), and the like.
  • Activating groups that can react with thiols include, for example, maleimide, iodoacetyl, acrylolyl, pyridyl disulfides, 5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like.
  • An aldehyde functional group can be coupled to amine- or hydrazide- containing molecules, and an azide group can react with a trivalent phosphorous group to form phosphoramidate or phosphorimide linkages.
  • Suitable methods to introduce activating groups into molecules are known in the art (see for example, Hermanson, G. T., Bioconjugate Techniques, Academic Press: San Diego, CA (1996)).
  • An activating group can be bonded directly to the organic group (e.g., hydrophilic polymer, fatty acid, fatty acid ester), or through a linker moiety, for example a divalent C 1 -C 12 group wherein one or more carbon atoms can be replaced by a heteroatom such as oxygen, nitrogen or sulfur.
  • Suitable linker moieties include, for example, tetraethylene glycol, -(CH 2 ) 3 - , -NH-(CHDe-NH-, -(CH 2 ) 2 -NH- and -CH 2 -O-CH 2 -CH 2 -O-CH 2 -CH 2 -O-CH-NH-.
  • Modifying agents that comprise a linker moiety can be produced, for example, by reacting a mono-Boc-alkyldiamine (e.g., mono-Boc-ethylenediamine, mono-Boc-diaminohexane) with a fatty acid in the presence of l-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) to form an amide bond between the free amine and the fatty acid carboxylate.
  • a mono-Boc-alkyldiamine e.g., mono-Boc-ethylenediamine, mono-Boc-diaminohexane
  • EDC l-ethyl-3- (3-dimethylaminopropyl) carbodiimide
  • the Boc protecting group can be removed from the product by treatment with trifluoroacetic acid (TFA) to expose a primary amine that can be coupled to another carboxylate as described, or can be reacted with maleic anhydride and the resulting product cyclized to produce an activated maleimido derivative of the fatty acid.
  • TFA trifluoroacetic acid
  • the modified antibodies of the invention can be produced by reacting a human antibody or antigen- binding fragment with a modifying agent.
  • the organic moieties can be bonded to the antibody in a non-site specific manner by employing an amine-reactive modifying agent, for example, an NHS ester of PEG.
  • Modified human antibodies or antigen-binding fragments can also be prepared by reducing disulfide bonds (e.g., intra-chain disulfide bonds) of an antibody or antigen-binding fragment.
  • the reduced antibody or antigen-binding fragment can then be reacted with a thiol-reactive modifying agent to produce the modified antibody of the invention.
  • Modified human antibodies and antigen-binding fragments comprising an organic moiety that is bonded to specific sites of an antibody of the present invention can be prepared using suitable methods, such as reverse proteolysis (Fisch et al., Bioconjugate Chem., 3:147-153 (1992); Werlen et al, Bioconjugate Chem., 5:411-417 (1994); Kumaran et al, Protein ScL 6(10):2233-2241 (1997); Itoh et al, Bioorg. Chem., 24(1): 59-68 (1996); Capellas et al, Biotechnol. Bioeng., 56(4):456-463 (1997)), and the methods described in Hermanson, G. T., Bioconjugate Techniques, Academic Press: San Diego, CA (1996). 7. Anti-Idiotype Antibodies to Anti-MCP-1 Antibodies
  • an anti-idiotypic (anti-Id) antibody specific for such antibodies of the invention is an antibody which recognizes unique determinants generally associated with the antigen-binding region of another antibody.
  • the anti-Id can be prepared by immunizing an animal of the same species and genetic type (e.g. mouse strain) as the source of the Id antibody with the antibody or a CDR containing region thereof. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody and produce an anti-Id antibody.
  • the anti-Id antibody may also be used as an antigen-binding region thereof.
  • immunogen to induce an immune response in yet another animal, producing a so-called anti-anti-Id antibody.
  • Antibody Compositions comprising further therapeutically active ingredients
  • the composition can optionally further comprise an effective amount of at least one compound or protein selected from at least one of a dermatological drug, an anti-inflammatory drug, an analgesic, a renal drug (e.g., an angiotensin receptor blocker (ARB) or antagonist), an anti-infective drug, a cardiovascular (CV) system drug, a central nervous system (CNS) drug, an autonomic nervous system (ANS) drug, a respiratory tract drug, a gastrointestinal (GI) tract drug, a hormonal drug, a drug for fluid or electrolyte balance, a hematologic drug, an antineoplastic, an immunomodulation drug, an ophthalmic, otic or nasal drug, a topical drug, a nutritional drug or the like.
  • a dermatological drug an anti-inflammatory drug
  • an analgesic e.g., an angiotensin receptor blocker (ARB) or antagonist
  • ARB angiotensin receptor blocker
  • ANS autonomic nervous system
  • a respiratory tract drug
  • Such drugs are well known in the art, including formulations, indications, dosing and administration for each presented herein (see., e.g., Nursing 2001 Handbook of Drugs, 21 st edition, Springhouse Corp., Springhouse, PA, 2001; Health Professional's Drug Guide 2001, ed., Shannon, Wilson, Stang, Prentice-Hall, Inc, Upper Saddle River, NJ; Pharmcotherapy Handbook, Wells et al., ed., Appleton & Lange, Stamford, CT, each entirely incorporated herein by reference).
  • Anti-MCP-1 antibody compositions of the present invention can further comprise at least one of any suitable and effective amount of a composition or pharmaceutical composition comprising at least one anti-MCP-1 antibody to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy, optionally further comprising at least one selected from at least one TNF antagonist (e.g., but not limited to a TNF chemical or protein antagonist, TNF monoclonal or polyclonal antibody or fragment, a soluble TNF receptor (e.g., p55, p70 or p85) or fragment, fusion polypeptides thereof, or a small molecule TNF antagonist, e.g., TNF binding protein I or II (TBP-I or
  • TBP-II nerelimonmab, infliximab, enteracept, CDP-571, CDP-870, afelimomab, lenercept, and the like
  • an antirheumatic e.g., methotrexate, auranofin, aurothioglucose, azathioprine, etanercept, gold sodium thiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalzine
  • a muscle relaxant a narcotic, a non-steroid anti-inflammatory drug (NSAE )
  • an analgesic an anesthetic, a sedative, a local anethetic, a neuromuscular blocker
  • an antimicrobial e.g., aminoglycoside, an antifungal, an antiparasitic, an antiviral, a carbapenem, cephalosporin, a flurorquinolone,
  • Non-limiting examples of such cytokines include, but are not limted to, any of IL-I to TL-29.
  • Suitable dosages are well known in the art. See, e.g., Wells et al., eds., Pharmacotherapy Handbook, 2 nd Edition, Appleton and Lange, Stamford, CT (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, CA (2000), each of which references are entirely incorporated herein by reference.
  • Such anti-cancer or anti-infectives can also include toxin molecules that are associated, bound, co- formulated or co-administered with at least one antibody of the present invention.
  • the toxin can optionally act to selectively kill the pathologic cell or tissue.
  • the pathologic cell can be a cancer or other cell.
  • Such toxins can be, but are not limited to, purified or recombinant toxin or toxin fragment comprising at least one functional cytotoxic domain of toxin, e.g., selected from at least one of ricin, diphtheria toxin, a venom toxin, or a bacterial toxin.
  • toxin also includes both endotoxins and exotoxins produced by any naturally occurring, mutant or recombinant bacteria or viruses which may cause any pathological condition in humans and other mammals, including toxin shock, which can result in death.
  • toxins may include, but are not limited to, enterotoxigenic E. coli heat-labile enterotoxin (LT), heat-stable enterotoxin (ST), Shigella cytotoxin, Aeromonas enterotoxins, toxic shock syndrome toxin-1 (TSST-I), Staphylococcal enterotoxin A (SEA), B (SEB), or C (SEC), Streptococcal enterotoxins and the like.
  • Such bacteria include, but are not limited to, strains of a species of enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli (e.g., strains of serotype 0157:H7), Staphylococcus species (e.g., Staphylococcus aureus, Staphylococcus pyogenes), Shigella species (e.g., Shigella dysenteriae, Shigella flexneri, Shigella boydii, and Shigella sonnet), Salmonella species (e.g., Salmonella typhi, Salmonella cholera-suis, Salmonella enteritidis), Clostridium species (e.g., Clostridium perfringens, Clostridium perfringens, Clostridium perfringens, Clostridium pere, Clostridium botulinum), Camphlobacter species (e.g., Camphlobacter jejuni, Camphlobacter
  • Anti-MCP-1 antibody compounds, compositions or combinations of the present invention can further comprise at least one of any suitable auxiliary agent, such as, but not limited to, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like. Pharmaceutically acceptable auxiliaries are preferred.
  • Non-limiting examples of, and methods of preparing such sterile solutions are well known in the art, such as, but limited to, Gennaro, Ed., Remington's Pharmaceutical Sciences, 18 th Edition, Mack Publishing Co. (Easton, PA) 1990.
  • Pharmaceutically acceptable carriers can be routinely selected that are suitable for the mode of administration, solubility and/or stability of the anti-MCP-1 antibody, fragment or variant composition as well known in the art or as described herein.
  • compositions include but are not limited to proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1-99.99% by weight or volume.
  • Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like.
  • amino acid/antibody components which can also function in a buffering capacity, include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like.
  • One preferred amino acid is glycine.
  • Carbohydrate excipients suitable for use in the invention include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffmose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), myoinositol and the like.
  • Preferred carbohydrate excipients for use in the present invention are mannitol, trehalose, and raffinose.
  • Anti-MCP-1 antibody compositions can also include a buffer or a pH-adjusting agent; typically, the buffer is a salt prepared from an organic acid or base.
  • Representative buffers include organic acid salts such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris, tromethamine hydrochloride, or phosphate buffers.
  • Preferred buffers for use in the present compositions are organic acid salts such as citrate.
  • anti-MCP-1 antibody compositions of the invention can include polymeric excipients/additives such as polyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates (e.g., cyclodextrins, such as 2-hydroxypropyl- ⁇ -cyclodextrin), polyethylene glycols, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates such as "TWEEN 20" and "TWEEN 80"), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol), and chelating agents (e.g., EDTA).
  • polymeric excipients/additives such as polyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates (e.g., cyclodextrins, such as 2-hydroxypropyl- ⁇ -cyclodextrin), polyethylene glycols
  • compositions according to the invention are known in the art, e.g., as listed in "Remington: The Science & Practice of Pharmacy", 19 th ed., Williams & Williams, (1995), and in the “Physician's Desk Reference", 52 nd ed., Medical Economics, Montvale, NJ (1998), the disclosures of which are entirely incorporated herein by reference.
  • Preferrred carrier or excipient materials are carbohydrates (e.g., saccharides and alditols) and buffers (e.g., citrate) or polymeric agents.
  • the invention provides for stable formulations suitable for pharmaceutical or veterinary use, comprising at least one anti-MCP-1 antibody in a pharmaceutically acceptable formulation.
  • the invention provides an article of manufacture, comprising packaging material and at least one vial comprising a solution of at least one anti-MCP-1 antibody with the prescribed buffers and/or preservatives, optionally in an aqueous diluent, wherein said packaging material comprises a label that indicates that such solution can be held over a period of 1, 2, 3, 4, 5, 6, 9, 12, 18, 20, 24, 30, 36, 40, 48, 54, 60, 66, 72 hours or greater.
  • the invention further comprises an article of manufacture, comprising packaging material, a first vial comprising lyophilized at least one anti-MCP-1 antibody, and a second vial comprising an aqueous diluent of prescribed buffer or preservative, wherein said packaging material comprises a label that instructs a patient to reconstitute the at least one anti-MCP-1 antibody in the aqueous diluent to form a solution that can be held over a period of twenty-four hours or greater.
  • the range of at least one anti-MCP-1 antibody in the product of the present invention includes amounts yielding upon reconstitution, if in a wet/dry system, concentrations from about 1.0 ⁇ g/ml to about 1000 mg/ml, although lower and higher concentrations are operable and are dependent on the intended delivery vehicle, e.g., solution formulations will differ from transdermal patch, pulmonary, transmucosal, or osmotic or micro pump methods.
  • the aqueous diluent optionally further comprises a pharmaceutically acceptable preservative.
  • Preferred preservatives include those selected from the group consisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben (methyl, ethyl, propyl, butyl and the like), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal, or mixtures thereof.
  • concentration of preservative used in the formulation is a concentration sufficient to yield an anti-microbial effect. Such concentrations are dependent on the preservative selected and are readily determined by the skilled artisan.
  • excipients e.g., isotonicity agents, buffers, antioxidants, preservative enhancers
  • An isotonicity agent such as glycerin, is commonly used at known concentrations.
  • a physiologically tolerated buffer is preferably added to provide improved pH control.
  • the formulations can cover a wide range of pHs, such as from about pH 4 to about pH 10, and preferred ranges from about pH 5 to about pH 9, and a most preferred range of about 6.0 to about 8.0.
  • the formulations of the present invention have pH between about 6.8 and about 7.8.
  • Preferred buffers include phosphate buffers, most preferably sodium phosphate, particularly phosphate buffered saline (PBS).
  • additives such as a pharmaceutically acceptable solubilizers like Tween 20 (polyoxyethylene (20) sorbitan monolaurate), Tween 40 (polyoxyethylene (20) sorbitan monopalmitate), Tween 80 (polyoxyethylene (20) sorbitan monooleate), Pluronic F68 (polyoxyethylene polyoxypropylene block copolymers), and PEG (polyethylene glycol) or non-ionic surfactants such as polysorbate 20 or 80 or poloxamer 184 or 188, Pluronic® polyls, other block co-polymers, and chelators such as EDTA and EGTA can optionally be added to the formulations or compositions to reduce aggregation. These additives are particularly useful if a pump or plastic container is used to administer the formulation. The presence of pharmaceutically acceptable surfactant mitigates the propensity for the protein to aggregate.
  • a pharmaceutically acceptable solubilizers like Tween 20 (polyoxyethylene (20) sorbitan monolau
  • the formulations of the present invention can be prepared by a process which comprises mixing at least one anti-MCP-1 antibody and a buffered solution in quantities sufficient to provide the protein at the desired concentrations. Variations of this process would be recognized by one of ordinary skill in the art. For example, the order the components are added, whether additional additives are used, the temperature and pH at which the formulation is prepared, are all factors that can be optimized for the concentration and means of administration used.
  • the claimed formulations can be provided to patients as solutions or as dual vials comprising a vial of lyophilized at least one anti-MCP-1 antibody that is reconstituted with a second vial containing water, a preservative and/or excipients, preferably a phosphate buffer and/or saline and a chosen salt, in an aqueous diluent.
  • a preservative and/or excipients preferably a phosphate buffer and/or saline and a chosen salt
  • Formulations of the invention can optionally be safely stored at temperatures of from about 2 to about 40 0 C and retain the biologically activity of the protein for extended periods of time, thus, allowing a package label indicating that the solution can be held and/or used over a period of 6, 12, 18, 24, 36, 48, 72, or 96 hours or greater. If preserved diluent is used, such label can include use up to 1-12 months, one-half, one and a half, and/or two years.
  • the claimed products can be provided indirectly to patients by providing to pharmacies, clinics, or other such institutions and facilities, clear solutions or dual vials comprising a vial of lyophilized at least one anti-MCP-1 antibody that is reconstituted with a second vial containing the aqueous diluent.
  • the clear solution in this case can be up to one liter or even larger in size, providing a large reservoir from which smaller portions of the at least one antibody solution can be retrieved one or multiple times for transfer into smaller vials and provided by the pharmacy or clinic to their customers and/or patients.
  • Recognized devices comprising these single vial systems include those pen-injector devices for delivery of a solution such as BD Pens, BD Autojector ® , Humaject ®1 NovoPen ® , B-D ® Pen, AutoPen ® , and OptiPen ® , GenotropinPen ® , Genotronorm Pen ® , Humatro Pen ® , Reco-Pen ® , Roferon Pen ® , Biojector ® , Iject ® , J-tip Needle-Free Injector ® , Intraject ® , Medi-Ject ® , e.g., as made or developed by Becton Dickensen (Franklin Lakes, NJ, www.bectondickenson.com), Disetronic (Burgdorf, Switzerland, www.disetronic.com; Bioject, Portland, Oregon (www.bioject.com); National Medical Products , Weston Medical (Peterborough
  • the products presently claimed include packaging material.
  • the packaging material provides, in addition to the information required by the regulatory agencies, the conditions under which the product can be used.
  • the packaging material of the present invention provides instructions to the patient to reconstitute the at least one anti-MCP-1 antibody in the aqueous diluent to form a solution and to use the solution over a period of 2-24 hours or greater for the two vial, wet/dry, product.
  • the label indicates that such solution can be used over a period of 2-24 hours or greater.
  • the presently claimed products are useful for human pharmaceutical product use.
  • formulations or methods of stablizing the anti-MCP-1 antibody may result in other than a clear solution of lyophilized powder comprising said antibody.
  • non-clear solutions are formulations comprising particulate suspensions, said particulates being a composition containing the anti-MCP-1 antibody in a structure of variable dimension and known variously as a microsphere, microparticle, nanoparticle, nanosphere, or liposome.
  • Such relatively homogenous essentially spherical particulate formulations containing an active agent can be formed by contacting an aqueous phase containing the active and a polymer and a nonaqueous phase followed by evaporation of the nonaqueous phase to cause the coalescence of particles from the aqueous phase as taught in U.S.
  • Porous microparticles can be prepared using a first phase containing active and a polymer dispersed in a continuous solvent and removing said solvent from the suspension by freeze-drying or dilution- extraction-precipitation as taught in U.S. 4,818,542.
  • Preferred polymers for such preparations are natural or synthetic copolymers or polymer selected from the group consisting of gleatin agar, starch, arabinogalactan, albumin, collagen, polyglycolic acid, polylactic aced, glycolide-L(-) lactide poly(episilon-ca ⁇ rolactone, poly(epsilon-ca ⁇ rolactone-CO-lactic acid), poly(epsilon-caprolactone-CO- glycolic acid), poly( ⁇ -hydroxy butyric acid), polyethylene oxide, polyethylene, poly(alkyl-2- cyanoacrylate), poly(hydroxyethyl methacrylate), polyamides, poly(amino acids), poly(2-hydroxyethyl DL-aspartamide), poly(ester urea), poly(L-phenylalanine/ethylene glycol/1 ,6-diisocyanatohexane) and poly(methyl methacrylate).
  • Particularly preferred polymers are polyesters such as polyglycolic acid, polylactic aced, glycolide-L(-) lactide poly(episilon-caprolactone, poly(epsilon-caprolactone-CO-lactic acid), and poly(epsilon-caprolactone-CO-glycolic acid.
  • Solvents useful for dissolving the polymer and/or the active include: water, hexafluoroisopropanol, methylenechloride, tetrahydrofuran, hexane, benzene, or hexafluoroacetone sesquihydrate.
  • the process of dispersing the active containing phase with a second phase may include pressure forcing said first phase through an orifice in a nozzle to affect droplet formation.
  • Dry powder formulations may result from processes other than lyophilization such as by spray drying or solvent extraction by evaporation or by precipitation of a crystalline composition followed by one or more steps to remove aqueous or nonaqueous solvent.
  • Preparation of a spray-dried antibody preparation is taught in U.S. 6,019,968.
  • the antibody-based dry powder compositions may be produced by spray drying solutions or slurries of the antibody and, optionally, excipients, in a solvent under conditions to provide a respirable dry powder.
  • Solvents may include polar compounds such as water and ethanol, which may be readily dried.
  • Antibody stability may be enhanced by performing the spray drying procedures in the absence of oxygen, such as under a nitrogen blanket or by using nitrogen as the drying gas.
  • Another relatively dry formulation is a dispersion of a plurality of perforated microstructures dispersed in a suspension medium that typically comprises a hydrofluoroalkane propellant as taught in WO 9916419.
  • the stabilized dispersions may be administered to the lung of a patient using a metered dose inhaler.
  • Equipment useful in the commercial manufacture of spray dried medicaments are manufactured by Buchi Ltd. or Niro Corp.
  • At least one anti-MCP-1 antibody in either the stable or preserved formulations or solutions described herein can be administered to a patient in accordance with the present invention via a variety of delivery methods including SC or IM injection; transdermal, pulmonary, transmucosal, implant, osmotic pump, cartridge, micro pump, or other means appreciated by the skilled artisan, as well-known in the art.
  • the present invention also provides a method for modulating or treating at least one MCP-I related disease, in a cell, tissue, organ, animal, or patient, as known in the art or as described herein, using at least one MCP-I antibody of the present invention.
  • the present invention also provides a method for modulating or treating at least one MCP-I related disease, in a cell, tissue, organ, animal, or patient including, but not limited to, at least one of malignant disease, metabolic disease, an immune or inflammatory related disease, a cardiovascular disease, an infectious disease, or a neurologic disease.
  • Such conditions are selected from, but not limited to, diseases or conditions mediated by cell adhesion and/or angiogenesis.
  • diseases or conditions include an immune disorder or disease, a cardiovascular disorder or disease, an infectious, malignant, and/or neurologic disorder or disease, or other known or specified MCP-lrelated conditions.
  • the antibodies are useful for the treatment of diseases that involve angiogenesis such as disease of the eye and neoplastic disease, tissue remodeling such as restenosis, and proliferation of certain cells types particularly epithelial and squamous cell carcinomas.
  • diseases that involve angiogenesis such as disease of the eye and neoplastic disease, tissue remodeling such as restenosis, and proliferation of certain cells types particularly epithelial and squamous cell carcinomas.
  • diseases include use in the treatment of atherosclerosis, restenosis, cancer metastasis, rheumatoid arthritis, diabetic retinopathy and macular degeneration.
  • the neutralizing antibodies of the invention are also useful to prevent or treat unwanted bone resorption or degradation, for example as found in osteoporosis or resulting from PTHrP overexpression by some tumors.
  • the antibodies may also be useful in the treatment of various fibrotic diseases such as idiopathic pulmonary fibrosis, diabetic nephropathy, hepatitis, and cirrhosis.
  • the present invention provides a method for modulating or treating at least one MCP-I related disease, in a cell, tissue, organ, animal, or patient, as known in the art or as described herein, using at least one MCP-I antibody of the present invention. Particular indications are discussed below:
  • the present invention also provides a method for modulating or treating at least one malignant disease in a cell, tissue, organ, animal or patient, including, but not limited to, at least one of: pneumonia; lung abscess; occupational lung diseases caused be agents in the form or dusts, gases, or mists; asthma, bronchiolitis fibrosa obliterans, respiratory failure, hypersensitivity diseases of the lungs includeing hypersensitivity pneumonitis (extrinsic allergic alveolitis), allergic bronchopulmonary aspergillosis, and drug reactions; adult respiratory distress syndrome (ARDS), Goodpasture's Syndrome, chronic obstructive airway disorders (COPD), idiopathic interstitial lung diseases such as idiopathic pulmonary fibrosis and sarcoidosis, desquamative interstitial pneumonia, acute interstitial pneumonia, respiratory bronchiolitis-associated interstitial lung disease, idiopathic bronchiolitis obliterans with organizing pneumonia, lymphocytic interstitial pneumonitis
  • the present invention also provides a method for modulating or treating at least one malignant disease in a cell, tissue, organ, animal or patient, including, but not limited to, at least one of: leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), B-cell, T-cell or FAB ALL, acute myeloid leukemia (AML), chromic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, myelodyplastic syndrome (MDS), a lymphoma, Hodgkin's disease, a malignamt lymphoma, non-hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, Kaposi's sarcoma, colorectal carcinoma, pancreatic carcinoma, renal cell carcinoma,!
  • leukemia acute leukemia, acute lymphoblastic leukemia (ALL), B-cell, T-cell or FAB ALL
  • AML acute my
  • breast cancer nasopharyngeal carcinoma, malignant histiocytosis, paraneoplastic syndrome/hypercalcemia of malignancy, solid tumors, adenocarcinomas, squamous cell carcinomas, sarcomas, malignant melanoma, particularly metastatic melanoma, hemangioma, metastatic disease, cancer related bone resorption, cancer related bone pain, and the like.
  • the present invention also provides a method for modulating or treating at least one immune related disease, in a cell, tissue, organ, animal, or patient including, but not limited to, at least one of rheumatoid arthritis, juvenile rheumatoid arthritis, systemic onset juvenile rheumatoid arthritis, psoriatic arthritis, ankylosing spondilitis, gastric ulcer, seronegative arthropathies, osteoarthritis, inflammatory bowel disease, ulcerative colitis, systemic lupus erythematosis, antiphospholipid syndrome, iridocyclitis/uveitis/optic neuritis, idiopathic pulmonary fibrosis, systemic vasculitis/ admireer's granulomatosis, sarcoidosis, orchitis/vasectomy reversal procedures, allergic/atopic diseases, asthma, allergic rhinitis, eczema, allergic contact dermatitis, allergic conjunctivitis, hypersensitivity pneu
  • the present invention also provides a method for modulating or treating at least one cardiovascular disease in a cell, tissue, organ, animal, or patient, including, but not limited to, at least one of cardiac stun syndrome, myocardial infarction, congestive heart failure, stroke, ischemic stroke, hemorrhage, arteriosclerosis, atherosclerosis, restenosis, diabetic aterosclerotic disease, hypertension, arterial hypertension, renovascular hypertension, syncope, shock, syphilis of the cardiovascular system, heart failure, cor pulmonale, primary pulmonary hypertension, cardiac arrhythmias, atrial ectopic beats, atrial flutter, atrial fibrillation (sustained or paroxysmal), post perfusion syndrome, cardiopulmonary bypass inflammation response, chaotic or multifocal atrial tachycardia, regular narrow QRS tachycardia, specific arrythmias, ventricular fibrillation, His bundle arrythmias, atrioventricular block, bundle branch block
  • the present invention also provides a method for modulating or treating at least one neurologic disease in a cell, tissue, organ, animal or patient, including, but not limited to, at least one of: neurodegenerative diseases, multiple sclerosis, migraine headache, AIDS dementia complex, demyelinating diseases, such as multiple sclerosis and acute transverse myelitis; extrapyramidal and cerebellar disorders' such as lesions of the corticospinal system; disorders of the basal ganglia or cerebellar disorders; hyperkinetic movement disorders such as Huntington's Chorea and senile chorea; drug-induced movement disorders, such as those induced by drugs which block CNS dopamine receptors; hypokinetic movement disorders, such as Parkinson's disease; Progressive supra- nucleo Palsy; structural lesions of the cerebellum; spinocerebellar degenerations, such as spinal ataxia, Friedreich's ataxia, cerebellar cortical degenerations, multiple systems degenerations (Mencel, Dejerine-Thomas, Shi-Drager
  • Such a method can optionally comprise administering an effective amount of a composition or pharmaceutical composition comprising at least one TNF antibody or specified portion or variant to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy.
  • a composition or pharmaceutical composition comprising at least one TNF antibody or specified portion or variant to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy. See, e.g., the Merck Manual, 16 th Edition, Merck & Company, Rah way, NJ (1992).
  • the present invention also provides a method for modulating or treating fibrotic conditions of various etiologies such as liver fibrosis (including but not limited to alcohol-induced cirrhosis, viral-induced cirrhosis, autoimmune- induced hepatitis); lung fibrosis (including but not limited to scleroderma, idiopathic pulmonary fibrosis); kidney fibrosis (including but not limited to scleroderma, diabetic nephritis, glomerular pehpritis, lupus nephritis); dermal fibrosis (including but not limited to scleroderma, hypertrophic and keloid scarring, burns); myelofibrosis; Neurofibromatosis; fibroma; intestinal fibrosis; and fibrotic adhesions resulting from surgical procedures.
  • liver fibrosis including but not limited to alcohol-induced cirrhosis, viral-induced cirrhosis, autoimmune- induced
  • the present invention also provides a method for modulating or treating at least one wound, trauma or tissue injury or chronic condition resulting from or related thereto, in a cell, tissue, organ, animal or patient, including, but not limited to, at least one of: bodily injury or a trauma associated with surgery including thoracic, abdominal, cranial, or oral surgery; or wherein the wound is selected from the group consisting of aseptic wounds, contused wounds, incised wounds, lacerated wounds, non-penetrating wounds, open wounds, penetrating wounds, perforating wounds, puncture wounds, septic wounds, infarctions and subcutaneous wounds; or wherein the wound is selected from the group consisting of ischemic ulcers, pressure sores, fistulae, severe bites, thermal burns and donor site wounds; or wherein the wound is an aphthous wound, a traumatic wound or a herpes associated wound.
  • Donor site wounds are wounds which e.g. occur in connection with removal of hard tissue from one part of the body to another part of the body e.g. in connection with transplantation.
  • the wounds resulting from such operations are very painful and an improved healing is therefore most valuable.
  • Wound fibrosis is also amenable to anti-MCP-1 antibody therapy as the first cells to invade the wound area are neutrophils followed by monocytes which are activated by macrophages.
  • Macrophages are believed to be essential for efficient wound healing in that they also are responsible for phagocytosis of pathogenic organisms and a clearing up of tissue debris. Furthermore, they release numerous factors involved in subsequent events of the healing process.
  • the macrophages attract fibroblasts which start the production of collagen.
  • the anti-MCP-1 antibodies of the invention can be used in methods for modulating, treating or preventing such sequelae of wound healing.
  • the present antibodies of the present invention may also be used in methods for modulating or treating at least one symptom of chronic rejection of a transplanted organ, tissue or cell, such as a cardiac transplant.
  • the present invention also provides a method for modulating or treating at least one infectious disease in a cell, tissue, organ, animal or patient, including, but not limited to, at least one of: acute or chronic bacterial infection, acute and chronic parasitic or infectious processes, including bacterial, viral and fungal infections, HTV infection/HTV neuropathy, meningitis, hepatitis (A 1 B or C, or the like), septic arthritis, peritonitis, pneumonia, epiglottitis, e.
  • acute or chronic bacterial infection including acute and chronic parasitic or infectious processes, including bacterial, viral and fungal infections, HTV infection/HTV neuropathy, meningitis, hepatitis (A 1 B or C, or the like)
  • septic arthritis including peritonitis, pneumonia, epiglottitis, e.
  • coli 0157:h7 hemolytic uremic syndrome/thrombolytic thrombocytopenic purpura, malaria, dengue hemorrhagic fever, leishmaniasis, leprosy, toxic shock syndrome, streptococcal myositis, gas gangrene, mycobacterium tuberculosis, mycobacterium avium intracellulare, Pneumocystis carinii pneumonia, pelvic inflammatory disease, orchitis/epidydimitis, legionella, lyme disease, influenza a, epstein-barr virus, vital-associated hemaphagocytic syndrome, vital encephalitis/aseptic meningitis, and the like.
  • Any method of the present invention can comprise administering an effective amount of a composition or pharmaceutical composition comprising at least one anti-MCP-1 antibody to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy.
  • a method can optionally further at least one selected from at least one TNF antagonist (e.g., but not limited to a TNF antibody or fragment, a soluble TNF receptor or fragment, fusion proteins thereof, or a small molecule TNF antagonist), an antirheumatic (e.g., methotrexate, auranofin, aurothioglucose, azathioprine, etanercept, gold sodium thiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalzine), a muscle relaxant, a narcotic, a non-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative, a local anethetic, a
  • Suitable dosages are well known in the art. See, e.g., Wells et al., eds., Pharmacotherapy Handbook, 2 nd Edition, Appleton and Lange, Stamford, CT (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, CA (2000), each of which references are entirely incorporated herein by reference.
  • Particular combinations for treatment of neoplastic diseases comprise co-administration or combination therapy by administering, before concurrently, and/or after, an antineplastic agent such as an alkylating agent, a nitrogen mustard, a nitrosurea, an antibiotic, an anti-metabolite, a hormonal agonist or antagonist, an immunomodulator, and the like.
  • an antineplastic agent such as an alkylating agent, a nitrogen mustard, a nitrosurea, an antibiotic, an anti-metabolite, a hormonal agonist or antagonist, an immunomodulator, and the like.
  • an antineplastic agent such as an alkylating agent, a nitrogen mustard, a nitrosurea, an antibiotic, an anti-metabolite, a hormonal agonist or antagonist, an immunomodulator, and the like.
  • an antineplastic agent such as an alkylating agent, a nitrogen mustard, a nitrosurea, an antibiotic, an anti-metabolite, a hormonal agonist or antagonist, an immunomodulator, and the like.
  • a method of the present invention can comprise a method for treating a MCP-I mediated disorder, comprising administering an effective amount of a composition or pharmaceutical composition comprising at least one anti-MCP-1 antibody to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy.
  • Such a method can optionally further comprise co-administration or combination therapy for treating such diseases or discorders, wherein the administering of said at least one anti-MCP-1 antibody, specified portion or variant thereof, further comprises administering, before concurrently, and/or after, at least one selected from a renal drug, a dermatogical drug, an anti- angiogenic drug, an anti-infective drug, a cardiovascular (CV) system drug, a central nervous system
  • CNS autonomic nervous system
  • ANS autonomic nervous system
  • GI gastrointestinal
  • hormonal drug a drug for fluid or electrolyte balance
  • a hematologic drug an antineoplactic, an immunomodulation drug
  • an ophthalmic, otic or nasal drug a topical drug, a nutritional drug or the like
  • at least one TNF antagonist e.g., but not limited to a TNF antibody or fragment, a soluble TNF receptor or fragment, fusion proteins thereof, or a small molecule TNF antagonist
  • an antirheumatic e.g., methotrexate, auranofin, aurothioglucose, azathioprine, etanercept, gold sodium thiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalzine
  • NSAID non-steroid anti-inflammatory drug
  • Such drugs are well known in the art, including formulations, indications, dosing and administration for each presented herein (see., e.g., Nursing 2001 Handbook of Drugs, 21 st edition, Springhouse Corp., Springhouse, PA, 2001; Health Professional's Drug Guide 2001, ed., Shannon, Wilson, Stang, Prentice-Hall, Inc, Upper Saddle River, NJ; Pharmcotherapy Handbook, Wells et al., ed., Appleton & Lange, Stamford, CT, each entirely incorporated herein by reference).
  • treatment of pathologic conditions is effected by administering an effective amount or dosage of at least one anti-MCP-1 antibody composition that total, on average, a range from at least about 0.01 to 500 milligrams of at least one anti-MCP-1 antibody per kilogram of patient per dose, and preferably from at least about 0.1 to 100 milligrams antibody /kilogram of patient per single or multiple administration, depending upon the specific activity of contained in the composition.
  • the effective serum concentration can comprise 0.1-5000 ⁇ g/ml serum concentration per single or multiple adminstration.
  • Suitable dosages are known to medical practitioners and will, of course, depend upon the particular disease state, specific activity of the composition being administered, and the particular patient undergoing treatment. In some instances, to achieve the desired therapeutic amount, it can be necessary to provide for repeated administration, i.e., repeated individual administrations of a particular monitored or 5 metered dose, where the individual administrations are repeated until the desired daily dose or effect is achieved.
  • Preferred doses can optionally include 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
  • the dosage administered can vary depending upon known factors, such as the pharmacodynamic characteristics of the particular agent, and its mode and route of administration; age, health, and weight of the recipient; nature and extent of symptoms, kind of concurrent treatment, frequency of treatment, and the effect desired.
  • a dosage of active 5 ingredient can be about 0.1 to 100 milligrams per kilogram of body weight.
  • 0.1 to 50, and preferably 0.1 to 10 milligrams per kilogram per administration or in sustained release form is effective to obtain desired results.
  • treatment of humans or animals can be provided as a one-time or periodic dosage of at least one antibody of the present invention 0.1 to 100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively or additionally, at least one of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or alternatively or additionally, at least one of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
  • Dosage forms (composition) suitable for internal administration generally contain from about 0.001 milligram to about 500 milligrams of active ingredient per unit or container.
  • the active ingredient will ordinarily be present in an amount of about 0.5-99.999% by weight based on the total weight of the composition.
  • the antibody can be formulated as a solution, suspension, emulsion, particle, powder, or lyophilized powder in association, or separately provided, with a pharmaceutically acceptable parenteral vehicle.
  • a pharmaceutically acceptable parenteral vehicle examples include water, saline, Ringer's solution, dextrose solution, and 1-10% human serum albumin. Liposomes and nonaqueous vehicles such as fixed oils can also be used.
  • the vehicle or lyophilized powder can contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives).
  • the formulation is sterilized by known or suitable techniques. Suitable pharmaceutical carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, A. Osol, a standard reference text in this field.
  • MCP-I antibodies of the present invention can be delivered in a carrier, as a solution, emulsion, colloid, or suspension, or as a dry powder, using any of a variety of devices and methods suitable for administration by inhalation or other modes described here within or known in the art.
  • Formulations for parenteral administration can contain as common excipients sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes and the like.
  • Aqueous or oily suspensions for injection can be prepared by using an appropriate emulsifier or humidifier and a suspending agent, according to known methods.
  • Agents for injection can be a non-toxic, non-orally administrable diluting agent such as aquous solution or a sterile injectable solution or suspension in a solvent.
  • As the usable vehicle or solvent water, Ringer's solution, isotonic saline, etc.
  • sterile involatile oil can be used as an ordinary solvent, or suspending solvent.
  • any kind of involatile oil and fatty acid can be used, including natural or synthetic or semisynthetic fatty oils or fatty acids; natural or synthetic or semisynthtetic mono- or di- or tri-glycerides.
  • Parental administration is known in the art and includes, but is not limited to, conventional means of injections, a gas pressured needle-less injection device, or laser perforator devise, as well known in the art (e.g., but not limited to, materials and methods disclosed in U.S. Pat. No. 5,851,198, and U.S. Pat. No. 5,839,446, entirely incorporated herein by reference).
  • Alternative Delivery is known in the art and includes, but is not limited to, conventional means of injections, a gas pressured needle-less injection device, or laser perforator devise, as well known in the art (e.g., but not limited to, materials and methods disclosed in U.S. Pat.
  • the invention further relates to the administration of at least one anti-MCP-1 antibody by parenteral, subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracelebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, intralesional, bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal means.
  • At least one anti-MCP-1 antibody composition can be prepared for use for parenteral (subcutaneous, intramuscular or intravenous) or any other administration particularly in the form of liquid solutions or suspensions; for use in vaginal or rectal administration particularly in semisolid forms such as, but not limited to, creams and suppositories; for buccal, or sublingual administration such as, but not limited to, in the form of tablets or capsules; or intranasally such as, but not limited to, the form of powders, nasal drops or aerosols or certain agents; or transdermally such as not limited to a gel, ointment, lotion, suspension or patch delivery system with chemical enhancers such as dimethyl sulfoxide to either modify the skin structure or to increase the drug concentration in the transdermal patch (Junginger, et al.
  • At least one anti-MCP-1 antibody composition is delivered in a particle size effective for reaching the lower airways of the lung or sinuses.
  • at least one anti-MCP-1 antibody can be delivered by any of a variety of inhalation or nasal devices known in the art for administration of a therapeutic agent by inhalation. These devices capable of depositing aerosolized formulations in the sinus cavity or alveoli of a patient include metered dose inhalers, nebulizers, dry powder generators, sprayers, and the like. Other devices suitable for directing the pulmonary or nasal administration of antibodies are also known in the art. All such devices can use of formulations suitable for the administration for the dispensing of antibody in an aerosol.
  • Such aerosols can be comprised of either solutions (both aqueous and non aqueous) or solid particles.
  • Metered dose inhalers like the Ventolin ® metered dose inhaler, typically use a propellent gas and require actuation during inspiration (See, e.g., WO 94/16970, WO 98/35888).
  • Dry powder inhalers like TurbuhalerTM (Astra), Rotahaler ® (Glaxo), Diskus ® (Glaxo), SpirosTM inhaler (Dura), devices marketed by Inhale Therapeutics, and the Spinhaler ® powder inhaler (Fisons), use breath-actuation of a mixed powder (US 4668218 Astra, EP 237507 Astra, WO 97/25086 Glaxo, WO 94/08552 Dura, US 5458135 Inhale, WO 94/06498 Fisons, entirely incorporated herein by reference).
  • Nebulizers like AERxTM Aradigm, the Ultravent ® nebulizer (Mallinckrodt), and the Acorn ⁇ ® nebulizer (Marquest Medical Products) (US 5404871 Aradigm, WO 97/22376), the above references entirely incorporated herein by reference, produce aerosols from solutions, while metered dose inhalers, dry powder inhalers, etc. generate small particle aerosols.
  • These specific examples of commercially available inhalation devices are intended to be a representative of specific devices suitable for the practice of this invention, and are not intended as limiting the scope of the invention.
  • a composition comprising at least one anti-MCP-1 antibody is delivered by a dry powder inhaler or a sprayer.
  • an inhalation device for administering at least one antibody of the present invention.
  • delivery by the inhalation device is advantageously reliable, reproducible, and accurate.
  • the inhalation device can optionally deliver small dry particles, e.g. less than about 10 ⁇ m, preferably about 1-5 ⁇ m, for good respirability.
  • Solid phase peptide synthetic methods were used to prepare human MCP-I variants with specific biotinylation at Lys 69 or Lys 75 .
  • the procedure involves design and complete chemical synthesis of the entire protein with selected residues biotinylated, followed by efficient refolding of the proteins into a biologically active form.
  • the MCP-I variants thus prepared are chemically pure and biologically active. These homogenous biotinylated derivatives enabled efficient selection and analysis of high affinity antibodies to human MCP-I.
  • MCP-I monocyte chemoattracting protein -1
  • MCP-I monocyte chemoattracting protein -1
  • Fig. 1 shows the amino acid sequence of human MCP-I and a ribbons representation of the molecular model.
  • Lysine residues that are suitable for site specific biotinylation need to be (1) exposed on the surface, (2) distant from the active surface and (3) presenting the active surface towards solvent when attached to a solid support.
  • K35, K38 and K49 are excluded for modification because of their biological importance.
  • Kl 9, K44, K56 and K58 are in the vicinity of the active surface and so are also avoided to minimize potential disruption of MCP-I function.
  • K69 (shown in Fig. 1 as balls-and-sticks) appears to fit the criterion by inspection.
  • K75 is disordered in the structure, but it is more distant than K69 from the active surface, and thus a good potential site for modification.
  • hMCP-1 He 41
  • a hydrophilic spacer was placed between the biotin and the ⁇ -amino group of lysine residue.
  • the spacer is a water soluble, non-immunogenic, discrete PEG (poly(ethylene)glycol) with four ethyleneoxy units (PEG 4 ).
  • the linker provides sufficient spacer length for binding streptavidin conjugates - chain length from biotin amide to terminal carbonyl is 19.2 A.
  • human MCP-I (De 41 ) analogues with a biotin-PEG 4 moiety attached to the ⁇ -amino group of lysine residue at position 69 or 75.
  • the protected octapeptide-resin Fmoc-Lys(Aloc)-Gln(Trt)-Thr(Bu')-Gln(Trt)-Thr(Bu t )-Pro- Lys(Boc)-Thr(Bu')-resin, was prepared on an ABI 431A Peptide Synthesizer using Version 1.12 of the 0.1 mM scale Fmoc software.
  • Fmoc-Thr(Bu')-Wang resin (Bachem, King of Prussia, PA) (Lot 0551540, substitution 0.12 mmol/g)
  • Fmoc-Lys(Aloc) AnaSpec, San Jose, CA
  • the peptide-resin was transferred into a manual shaker and the Aloe group was removed using standard procedure (Pd(PPh 3 ) 4 in chloroform in the presence of N-methylmorpholine) (Kunz, H. and Unverzagt, C, Angewandte Chemie International Edition in English (1984) 23, 436-437 ).
  • the NHS-dPEG 4 -biotin (Quanta BioDesign, Powell, OH) was coupled to ⁇ -amino group of Lys 69 in NMP/HOBt solution.
  • the final (1-68) amino acids were added using a Rainin Multiple Peptide Synthesizer (Symphony, Model SMPS-110) using Version 2.01 of the 0.1 mM scale Fmoc software.
  • the Boc-L-Glp (Bachem, King of Prussia, PA) was coupled as the N-terminal residue.
  • the product was cleaved from the resin (1.41 g, 0.06 mM) by stirring with trifluoroacetic acid TFA (40 mL), ethanedithiol EDT (8.0 mL), phenol (3.0 g), thioanisole (2.0 mL), triisopropylsilane TIS (2.0 mL) and water (2.0 mL) over four hours at ambient temperature to give 496 mg of the crude product.
  • TFA trifluoroacetic acid
  • ethanedithiol EDT 8.0 mL
  • phenol 3.0 g
  • thioanisole 2.0 mL
  • triisopropylsilane TIS 2.0 mL
  • water 2.0 mL
  • the crude, linear protein (480 mg) was purified in multiple injections on two Vydac C-18 columns, 10 ⁇ m, 2.5 x 25 cm, eluting with a 0-30% over 5 min and a 30-60% gradient of 80% acetonitrile in 0.1% TFA over 60 min at a flow rate of 6 mL/min. Fractions were collected, analyzed by HPLC and MS and pure fractions pooled and lyophilized to give 101 mg of the linear protein as a white solid.
  • the linear protein (100 mg) was oxidized by dissolving in 1.0 M guanidine x HCl in 0.1 M Tris x HCl at a pH 8.66 containing 1.0 mM EDTA, 3.0 mM reduced glutathione and 0.3 mM oxidized glutathione at concentration of 20 ⁇ g/mL over 70-100 hours.
  • the oxidized protein was purified on two Vydac C-18 columns, 10 ⁇ m, 2.5 x 25 cm, eluting with a 0-5% over 5 min, a 5-25% over 30 min, a 25-55% over 30 min, and a 55-100% gradient of 80% acetonitrile in 0.1% TFA over 60 min at a flow rate of 6 mL/min. Fractions were collected, analyzed by HPLC and MS and pure fractions pooled and lyophilized to give 33 mg of white solid. A portion of the material (8.2 mg) was repurified using an affinity column with immobilized monomelic avidin (Pierce, Number 20227).
  • the biotinylated protein (1.5 mg) was dissolved in PBS (2 mL) and added onto a column (2 mL). The unbound protein was washed out with PBS (6 x 2 mL), then the bound biotinylated protein was eluted using biotin-containing buffer (2 mM D-biotin in PBS) (6 x 2 mL). The collected fractions were analyzed by HPLC and MS, then pooled, concentrated and dialyzed to PBS to give 0.92 mg of the final product (PBS solution, 5.0 mL) (MALDI-MS: 9,151.7; calculated molecular weight: 9,151.7) (CEN 2494A). The protein was analyzed for its primary amino acid sequence and for disulfide bonds mapping using mass spectrometry. All obtained data were as expected. Human MCP-KDe 41 . LvsCbiotin-PEGa') 75' )
  • Fmoc-Leu-Asp(OBu t )-Lys(Boc)-Gln(Trt)-Thr(Bu')- Gln(Trt)-Thr(Bu t )-Pro-Lys(Mtt)-Thr(Bu')-resin was prepared on an ABI 43 IA Peptide Synthesizer using Version 1.12 of the 0.1 mM scale Fmoc software.
  • Fmoc-Thr(Bu t )-Wang resin (834 mg, 0.1 mM) (Bachem, Lot 0551540, substitution 0.12 mmol/g) and Fmoc-Lys(Mtt) (624.8 mg, 1.0 mM) (NovaBiochem, Lot A29537, molecular weight: 624.8) were used in the synthesis.
  • the peptide-resin was transferred into a manual shaker and the Mtt group was removed using DCM-TFA-TIS (94:1:5, v/v/v) (4 x 2 min) (Aletras, A. et al., Int. J. Peptide Protein Res.
  • the product was cleaved from the resin (0.52 g and 0.94 g) by stirring with TFA (40 mL), EDT (8.0 mL), phenol (3.0 g), thioanisole (2.0 mL), TIS (2.0 mL) and water (2.0 mL) over four hours at ambient temperature to give 463 mg of the crude product.
  • the crude, linear protein (460 mg) was purified in multiple injections on two Vydac C- 18 columns, 10 ⁇ m, 2.5 x 25 cm, eluting with a 0-30% over 5 min and a 30-60% gradient of 80% acetonitrile in 0.1% TFA over 60 min at a flow rate of 6 mL/min. Fractions were collected, analyzed by HPLC and MS and pure fractions pooled and lyophilized to give 83 mg of the linear protein as a white solid.
  • the linear protein (58.3 mg) was oxidized by dissolving in 1.0 M guanidine x HCl in 0.1 M Tris x HCl at a pH 8.66 containing 1.0 mM EDTA, 3.0 mM reduced glutathione and 0.3 mM oxidized glutathione at concentration of 20 ⁇ g/mL over 72 hours.
  • the oxidized protein was purified in three injections on two Vydac C-18 columns, 10 ⁇ m, 2.5 x 25 cm, eluting with a 0-5% over 5 min, a 5-30% over 30 min, a 30-60% over 30 min and a 60-100% gradient of 80% acetonitrile in 0.1% TFA over 40 min at a flow rate of 6 mL/min. Fractions were collected, analyzed by HPLC and MS. Semipure fractions were pooled and lyophilized to give 13.6 mg of white solid.
  • the 13.6 mg was repurified using affinity column with immobilized monomeric avidin (Pierce, Number 20227) as described in 2.1. to give: 1.2 mg of the final product (PBS solution, 2.0 mL, cone. 0.58 mg/mL, MALDI-MS: 9,152.7 (calculated mol. weight 9,151.7) (CEN 2508A).
  • the samples were reduced, alkylated with iodoaceetamide, and digested with pepsin.
  • 5 ⁇ L of each protein was mixed with 4 ⁇ L of 45 mM DTT and 45 ⁇ L of PBS, PH 7.0. The solutions were incubated at 60 °C for 20 min. Then, 5 ⁇ L of 100 mM iodoactamide was added and the solutions incubated in the dark at room temperature for 20 min. After alkylation, 1 ⁇ L of pepsin (0.5 ⁇ g/ ⁇ L) was added and the reaction mixture incubated at 37 °C for 1 h.
  • the samples were desalted using Cl 8 ZipTips with the digested peptides eluting into 50:50:0.1 AcCN/H 2 O/TFA.
  • the samples were spotted onto a MALDI plate with ⁇ - cyano-4-hydroxycinnamic acid (CHCA) as a matrix.
  • CHCA ⁇ - cyano-4-hydroxycinnamic acid
  • the digests were analyzed using the 4700 Proteomics Analyzer (Applied Biosystems, Foster City, CA). Each peptide sequence was confirmed using tandem TOF-TOF mass spectrometry.
  • the disulfide-bond structure was studied after digestion of each protein construct with endopeptidase Lys-C.
  • the digests were performed by mixing 50 ⁇ L peptide solution in PBS (pH 7.0) with 1 ⁇ L of endopeptidase Lys-C at a concentration of 1.3 ⁇ g/ ⁇ L, and incubating the mix for 2 h at 37 °C. After incubation, the samples were desalted using C18 ZipTips (Millipore Corp., Bedford, MA). The samples were analyzed using a Voyager DE-STR MALDI TOF MS (Applied Biosystems, Foster City, CA).
  • Two matrices were used to analyze the Lys-C digestions: a saturated solution of CHCA or a 1:10 mix of 2-(4-hydroxyphenylazo)benzoic acid (HPBA)/HCCA. Comparison of the data from the mixed matrix to the data from the straight CHCA matrix showed which disulphide-bonded peptides were occurring naturally, and which ones were potentially formed during MALDI ionization.
  • a BIAcore 3000 (Biacore Inc. Piscataway, NJ) was used to determine the binding constants of the interaction between anti-MCP-1 antibodies and the recombinant and synthetic proteins.
  • a capture sensor surface was prepared by covalently immobilizing Protein A (Pierce Chemicals, Rockville, IL) onto a CM-5 chip using a NHS/EDC amine coupling kit (Biacore Inc. Piscataway, NJ). Approximately 3000 RU of Protein A were immobilized.
  • Anti-MCP-1 antibody C775 was captured onto the Protein A modified sensor surface by passing a 5 ug/mL solution of the monoclonal antibody diluted into phosphate buffered saline (PBS, 10 mM sodium phosphate, 150 mM sodium chloride, pH 7.4). Approximately 160 response units (RU) of C775 were captured by the surface during experiment cycle. Binding studies were performed by equilibrating the instrument and sensor surface using a running buffer of PBS buffered saline containing 3 mM EDTA and 0.005% Tween-20. Samples consisting of 25 nM of protein were then passed over this surface at a flow rate of 30 ⁇ L/min for 4 min using the KINJECT command.
  • PBS phosphate buffered saline
  • rhMCP-1 (279-MC-050/CF; R&D Systems) free of carrier protein were analyzed. Analysis included ES-MS of the intact sample in its native and alkylated forms, and peptide mass fingerprinting of a tryptic digest of the native sample (data not shown). The rhMCP-1 showed correct molecular weight, amino acid sequence and disulfide bonding. However, there was approximately 15% of Met(O) 64 , 15% of GIn 1 and about 5% of a putative des[ ⁇ Glu-Pro] isoforms.
  • the synthetic proteins were sequenced by mass spectroscopy. After intact mass analysis, the samples were reduced, alkylated and digested with pepsin. The digests were analyzed using the 4700 Proteomics Analyzer. Each peptide sequence was confirmed using tandem mass spectrometry. These experiments confirmed all of the amino acid substitutions. When all of the data were compiled, 97% sequence confirmation was achieved across the entire protein. There was one dipeptide (Asn 14 -Phe 15 ) that was unaccounted for due to loss during desalting.
  • the disulphide-bond structure for the synthetic MCP-I and analogs was studied after digestion of each protein construct with Lys-C. Each digest was analyzed using two different matrixes: CHCA and 1:10 HPBA/CHCA. The mixed matrix suppresses disulfide-bond cleavage during MALDI ionization. The samples were analyzed using a DE-STR MALDI TOF MS. The data from this experiment was used to determine if there were alternate disulfide bond linkages present in the samples. A list of the observed peptides from the Lys-C digestions of the MCP-I proteins is not shown.
  • L3-L1-L6 3255.7 Da
  • L3 peptide corresponds to CPK (36-38; observed 345.4 Da); Ll to ⁇ EPDAINAPVTCCYNFTNRK (1- 19; observed 2136.4 Da) and L6 to EICADPK (50-56; observed 773.9 Da).
  • the Cys 11 and Cys 12 in peptide Ll are adjacent to each other, preventing specific linkage assignment; in other words, it was not possible to distinguish which peptides are bound to which cysteines in peptide Ll. Minor levels of either partially or fully reduced cysteines were detected in Ser 40 , He 41 and Tyr 43 analogs of MCP-I .
  • the peptide fragments produced by the enzymatic digest of the synthetic proteins and rhMCP-1 (279- MC-050/CF; R&D Systems) were analyzed, and found to be identical.
  • association and dissociation rate constants of the synthetic proteins were comparable to those observed with rhMCP-1 (Table 2) .
  • six additional mouse anti-human MCP-I mAbs were shown to have comparable binding kinetics to synthetic and recombinant MCP-I (data not shown).
  • EXAMPLE 2 Biological characterization of human MCP-I(IIe 41 , Lys(Biotin-PEG 4 ) 69 ) and human MCP-I(IIe 41 , Lys(Biotin-PEG 4 ) 75 )
  • the synthetic human MCP-I (He 41 ), (lie 41 , Lys(Biotin-PEG 4 ) 69 ), and (He 41 , Lys(Biotin-PEG 4 ) 75 ) and/or the wild type human MCP-I was added to Thp-1 cells concurrently with 125 I labeled MCP-I.
  • Thp-1 cells One million Thp-1 cells were used for binding.
  • Cells and the labeled and the unlabeled MCP-I were incubated in a filtration well for 1 hour at room temperature. Unbound 125 I MCP-I was removed by applying vacuum. Wells were then counted using a gamma counter.
  • Binding inhibition by the testing MCP-I analogs was expressed as percentage of total binding of 125 I MCP-I in the absence of the unlabeled MCP-Is.
  • THP-1 cells (ATCC, Manassas, VA) is a monocytic cell line derived from a patient with acute monocytic leukemia and express CCR2. Cells were maintained in culture in RPMI 1640 medium containing 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES and 1.0 mM sodium pyruvate, 90%; 10% fetal bovine serum (FBS; Vitacell RPMI 20-2001, ATCC, Manassas, VA) at 37 0 C and 5% CO2 at a density of 4-8 x 105 cells/mL.
  • FBS fetal bovine serum
  • the Ca2+ mobilization assay was performed in a 96-well format, using the FLEXstationTM Ca2+ Plus Assay Kit (Molecular Devices, Sunnyvale, CA) following the manufacturer's protocol for non-adherent cells and a FLEXstationTM (Molecular Devices, Sunnyvale, CA).
  • the peak RFU values were imported into Graphpad Prism and plotted.
  • the synthetic human MCP-I (De 41 ), (He 41 , Lys(Biotin-PEG 4 ) 69 ), and (He 41 , Lys(Biotin-PEG 4 ) 75 ) and/or the wild type human MCP-I was added to 2xlO 5 Thp-1 cells stained with a Ca 2+ flurophore. Binding of MCP-Is to CCR2 of the Thp-1 cells induced change in fluorescence which was measured in real time before and after the addition of the chemokines.
  • Chemotaxis Assay was performed using the 96-well disposable chemotaxis apparatus (8 ⁇ m pore size; Neuro Probe Inc., Gaithersburg, MD). For calcein AM incorporation, THP-1 cells were resuspended in 10 mL of growth media with 2.5 ⁇ g/mL Calcien AM (Molecular Probes, Eugene, OR) and incubated for 1 h at 37 oC and 5% CO2. The bottom chamber contained 315 mL of varying concentrations of either recombinant or synthetic protein.
  • Calcein-loaded cells were washed and resuspended in assay buffer compromising of PBS (Invitrogen Corp., Grand Island, NY) and 2% FBS (Invitrogen Corp., Grand) at a density of 2 x 106 cells/mL. Fifty mL of the cell suspension was added to the top of the membrane filter. The chamber was incubated for 1 h at 37 °C and 5% CO2. Unmigrated cells were rinsed off the chamber with water and blotted dry. The chamber was centrifuged for 30 s at 1200 rpm and then read in the Tecan Fluorometer, (535 nm, bottom read). Data were saved as RFU values in the Microsoft Excel program and plotted using XY line plots using GraphPad Prism.
  • MCP-I human monocyte chemoattractant protein 1
  • Non-Limiting Example Applicants have previously shown desirable therapeutic characteristics of a murine anti-human MCP-I antibody designated C775 and described in applicants co-pending patent application U.S. Ser. No. 11/170453 (SEQ ID NO: 7 and 8 of that application for the heavy and light chain variable regions, respectively) and related filings.
  • the objective of the present effort was to identify at least one human antibody from the HuCAL GOLD ® , which neutralizes the biological activity of the human chemokine MCP-I and displays similar attributes.
  • the attributes of the C775 antibody, the thus the desired human anti-MCP-1 antibody were defined by success criteria outlined below.
  • DNA restriction and modification enzymes as well as polymerases were purchased from Invitrogen (Carlsbad, CA, USA), New England Biolabs (Beverly, MA, USA), Roche Diagnostics (Mannheim, Germany) and MBI Fermentas (Vilnius, Lithuania).
  • Goat anti-human IgG F(ab') 2 fragment specific POD conjugated was supplied by Jacksons (West Grove, PN, USA), sheep anti-human IgG, Fd fragment specific, antibody by The Binding Site (Birmingham, UK) and streptavidin conjugated to alkaline phosphatase (ZyMAXTM grade) by Zymed Laboratories (San Francisco, CA, USA).
  • Recombinant human chemokines, hMCP-1, 2, 3, 4 and hEotaxin 1, 2 and 3 Reagents, Ligands and Antibodies: mAb 279, specific for human MCP-I (R&D systems); synthetic hMCP-1 (Bachem); mAbl mouse anti hCCR2 biotin (R&D systems); human gamma globulin (Jackson Immuno Research); mouse gamma globulin (Jackson Immuno Research); mAb mIgG2b isotype control biotin (R&D systems); streptavidin-PE (BD Pharmingen); Versene (Invitrogen; PBS (Invitrogen).
  • Chemical synthesis also provided a method for the site specific biotinylation of human MCP-I using the epsilon-amino group of lysine not involved in receptor binding or surface activity at K69 and K75 is disordered in the structure (U.S. Serial No. 60/682620 and Kruszynski et al. 2006, J Peptide Sci. 12:354-360).
  • a hydrophilic spacer of four ethyleneoxy units (PEG 4 ) was inserted between the biotin and the ⁇ -amino group of lysine residue.
  • the chain length from biotin amide to terminal carbonyl is 19.2 A.
  • the spacer was chosen to increase solubility and provide sufficient spacer length for binding streptavidin conjugates.
  • MCP-I and variants The sequence of MCP-I and variants is given in SEQ ID NO: 1. Variants were determined to retain the ability to induce Ca2+ mobilization in THP-I cells. Biotin-Lys 69 and biotin-Lys 75 MCP-I were compared side by side in screenig, consolidation and affinity determination and no significant differences could be obsereved. Using Biacore, 35 optimized Fabs were analyzed on MCP-I He 41 , Lys(biotin-PEG 4 ) 69 and MCP-I He 41 , Lys(biotin-PEG 4 ) 75 immobilized on streptavidin chips in parallel. In general the measured affinities on MCP-I K69 and K75 were comparable.
  • Phage Fab Libray The phagemid library is based on the HuCAL ® concept (Knappik et al., 2000) and employs the CysDisplayTM technology for displaying the Fab on the phage surface (L ⁇ hmng, 2O ⁇ f).
  • the library encodes approximately 10 10 unique Fabs displayed on M13 bacteriophage as fusions to a minor coat protein, pin.
  • HuCAL GOLD ® antibody-phages were divided into three pools comprising different VH master genes. In addition the whole library was used in one pool (VHl- 6). 2 X 10 13 HuCAL GOLD ® input phages were used for each panning.
  • the pre-adsorbed phages and the biotinylated antigens (3:1 biotin to antigen ratio for biotinylation; 200 nM final cone.) were added to the pre-blocked 1.5 ml Eppendorf tubes and incubated for Ih at RT on a rotating wheel.
  • further Reacti-Bind Neutravidin Coated Polystyrene microtiter plate strips were rinsed with 2 x 300 ⁇ l PBS, blocked with 300 ⁇ l Chemiblocker 1:1 diluted with PBS for Ih and washed Ix 300 ⁇ l PBS.
  • the Fab encoding inserts of the selected HuCAL GOLD ® phages were subcloned via Xbal and Ec ⁇ BI into the expression vector ⁇ MORPH ® X9_FH.
  • Fab fragments carry a C-terminal FLAGTM tag (Prickett Jt aL ⁇ l9SP) and as a second C-terminal tag the 6x His-tag (Chenj ⁇ t al.7 ⁇ 1994j).
  • biotinylated hMCP-1 V41I was performed using Reacti-BindTM NeutrAvidinTM 384 well plates (Pierce, Rockford, IL, USA) coated with 20 ⁇ l 0.5 ⁇ l/ml biotinylated hMCP-1 analog-1 (V41I) or analog-2 (F43Y) diluted in PBS, pH 7.4, for 16 h at 4 0 C. After blocking with 1% BSA in TBS, 0.05% Tween20 (Sigma, St. Louis, MO, USA) for 1 h at RT, periplasmic extracts were added.
  • Fab-fragments were performed by incubation with goat anti- human IgG, F(ab') 2 fragment specific antibody.
  • Solution phase screening with biotinylated hMCP-1 Analog-1 (V41I) was performed by coating Maxisorp (Nunc, Rochester, NY, USA) 384 well plates with 20 ⁇ l sheep anti-human IgG, Fd fragment specific, antibody diluted 1:1000 in PBS, pH 7.4 for 16 h at 4°C. After blocking with 3% BSA in TBS,
  • THP-I cells human acute monocytic leukemia cells
  • RPMI fetal bovine serum
  • Radioligand Binding Assay were performed in Millipore filter plates (Millipore, Bedford, MA). 1 x 10 6 THP-I cells/well were incubated with 125 I-MCP-I (1 ng/mL; Perkin Elmer Life Science, Boston, MA) together with different concentrations of recombinant human (rh) MCP-I (279- MC, R&D Systems, Minneapolis, MN) or synthetic proteins. All reagents were diluted in binding buffer consisting of RPMI Medium 1640 (Invitrogen Corp., Grand Island, NY) and 0.1% BSA.
  • the Ca 2+ mobilization assay was performed in a 96-well format, using the FLEXstationTM Ca 2+ Plus Assay Kit (Molecular Devices, Sunnyvale, CA) following the manufacturer's protocol for non-adherent cells and a FLEXstationTM (Molecular Devices, Sunnyvale, CA).
  • the peak RFU values were imported into Graphpad Prism for analysis.
  • MCP-I Induced CCR2 Receptor Internalization FACS Assay After optimization of ligand concentration (EC50 of synthetic MCP-I ⁇ 100 ng/ml) and incubation time (after 1 h most internalization had occurred) the IC50 was determinated by adding different concentrations of antibodies. Cultured CCR2 expressing cells were washed with PBS and detached with Versene (Invitrogen) for about lOmin at 37°C. AU centrifugation steps of the cells were at about 200Xg. Cells were washed twice with FACS buffer (PBS / 3% FCS), counted and checked for viability (trypan blue).
  • FACS buffer PBS / 3% FCS
  • 96 V- bottom well plates were filled with ⁇ 2.5x10 5 cells in lOO ⁇ l per well and put on ice.
  • the antibodies were diluted in cell culture medium (MEME) to give about 200 ⁇ g/ml down to 0,001 ⁇ g/ml in triplicate samples.
  • the different concentrations of the antibodies were pre-incubate with a final concentration of 100 ng/ml synthetic MCP-I (Bachem) for 10 min at RT.
  • the cells were re-suspended with the pre-incubated 100 ⁇ l MCP-1/antibody mixture and incubated for Ih at 37°C in an incubator for receptor internalization.
  • Cells were re-suspended in 50 ⁇ l anti-CCR2/gamma globulin mix (or control IgG2b/gamma globulin mix) and incubated for 1 h on ice. Cells were washed twice with 180 ⁇ l FACS buffer, re- suspended in 50 ⁇ l 1:400 diluted Streptavidin-PE (BD Pharmingen) and incubated for 1 h at 4 °C on ice in the dark. Cells were washed twice with 180 ⁇ l FACS buffer, re-suspended in 100 ⁇ l 2% PFA / PBS and stored overnight at 4 0 C for fixation (alternatively direct measurement without PFA fixation is possible). For FACS measurement the cells were re-suspended with 200 ⁇ l FACS buffer and at least 5000 cells were counted each. Affinity Assays
  • Biacore K D Determination on Directly Coated Antigen.
  • the kinetic constants Ic 0n and k off were determined with serial dilutions of the respective Fab binding to covalently immobilized MCP-I using the BIAcore 3000 instrument (Biacore, Uppsala, Sweden).
  • EDC-NHS amine coupling chemistry was used for covalent antigen immobilization standard.
  • Kinetic measurements were done in PBS (136 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1,76 mM KH 2 PO 4 pH 7.4) at a flow rate of 20 ⁇ l/min using Fab concentration range from 1.5-500 nM.
  • Injection time for each concentration was 1 min, followed by 3 min dissociation phase. For regeneration 5 ⁇ l 1OmM HCl was used. All sensograms were fitted using BIA evaluation software 3.1 (Biacore).Biacore K D Determination on Biotin-K69 Human MCP-I and Cyno MCP-I. Biotin-K69 human MCP-I and biotinylated cyno MCP-I were coated to streptavidin chip surface and cyno-MCP-1 was directly coated to CM5 chips. Binding of the Fabs was tested using the standard methods.
  • Biacore K D Determination in the Antibody Capture Mode. Fabs were captured at 50OnM with anti- hFab (s.3.15) on a CM5 chip (flow-rate 5 ⁇ l/min), solution of each analog (MCPl, 2, 3, 4 and Eotaxin, Eotaxin-2 and -3) was injected. All cytokines were carrier free and used in a concentration range from 15 to 500 nM (for parental Fabs before optimization) as an analyte for Affinity determination. Sensorgrams were analyzed using the BIAevaluation software. Biacore affinity determination to MCP- 1 in the antibody capture mode was not possible for the optimized binders because the detection limits of Biacore were reached.
  • Fabs surface plasmone resonance was used (Biacore 3000, Uppsala, Sweden) using the capture assay was used. Fabs were captured and the various proteins (MCP-2, -3, -4 and Eotaxin-1, -2 and -3) were used as analytes.
  • CM5 chips (Biacore, Sweden) were coated with 6500- 8000 RU anti-F(ab) 2 (Dianova, Affipure F(ab) 2 fragment goat anti-human IgG, F(ab) 2 fragment specific; 1OmM acetate buffer, pH 4.5) on all 4 flow cells, using standard EDC-NHS amine coupling chemistry.
  • the flow cells 2-4 were captured with specific anti-MCP-1 Fabs (20 ⁇ l of 500 nM Fab at a flow rate of 10 ⁇ l/ml, resulted capture density 300-400RU). After capturing of Fabs, the chemokines were injected (20 ⁇ l, flow rate 20 ⁇ l/min, PBS pH 7.4) at a concentration of 100 nM. Chemokines were stored in small aliquots and only freshly thawed material with maximum 1 freeze thaw cycle was used for the measurements. To avoid a combination of off rates aroused by the off rate of MCP-I, Fab specific interaction and the anti-Fab/ Fab interaction, buffer was injected, to determine the dissociation of anti-Fab/Fab interaction.
  • the achieved buffer sensorgram was subtracted from the specific one.
  • the response units were normalized to the amount of capture antibody onto the surface.
  • Binding to Native MCP-I in the Antibody Capture Mode The method was used as described above. Native MCP-I was purified from the PANCl supernatant and used for binding analysis. Binding to native MCP-I in the Fab capture mode was well above the detection limit, however, a true affinity measurement was not possible owing to the impurities in the extract obscuring the correct concentration of the native MCP-I. Conversion to IgG
  • variable domain fragments of heavy (VH) and light chains (VL) were subcloned from Fab expression vectors into appropriate pMorphJiIg vectors for human IgGl, human IgG4, chimeric human/mouse IgGl and IgG2a.
  • Restriction enzymes Eco ⁇ I, Mfel, BIpI were used for subcloning of the VH domain fragment into pMorph_hIgGl.l, pMorph_hIgG4.1, pMorph_mIgGl.l or pMorph_mIgG2a.l and EcoRY, BsiWl for subcloning of the VL domain fragment into pMorph_hIg ⁇ _l, pMorph_hIg ⁇ _l, pMor ⁇ h_mIg ⁇ _l or pMorph_mIg ⁇ _l vectors respectively. Resulting IgG constructs were expressed at CNTO.
  • Solid phase panning was performed on hMCP-1 (411) and hMCP-1 (43Y) directly coated to Maxisorp plates. Four different panning strategies were applied, containing three selection rounds each. After sub-cloning into the expression vector pMORPHx9_Fab_FH the solid phase screening was performed on directly coated hMCP-1 (411) and on biotinylated hMCP-1. In total 8832 clones were analyzed in primary screening and 983 primary hits were obtained. Finally 5 unique Fabs were identified, but all 5
  • Fabs did not neutralize MCP-I in cellular assays, indicating that direct coating to Maxisorp might impair the conformation or at least the accessibility of neutralizing epitopes.
  • a semi-solution panning was performed incubating the biotinylated human MCP-I analog-1 (V41I) and analog-2 (F43Y) with the HuCAL GOLD ® phages in solution followed by capturing of the phage antigen complexes as described.
  • Two different main panning strategies were applied including 3 rounds of panning on biotinylated human MCP-I protein analog-1 (V41I) and analog-2 (F43Y) respectively (no alternating panning). In total 9024 clones were analyzed in primary screening and 121 primary hits were obtained, finally revealing 18 unique binders.
  • Luminex based re-screening of 192 clones from panning on biotinylated human MCP-I protein analog-1 (V41I) lead to 9 additional primary hits and 3 additional unique binders, showing that Luminex screening is a suitable alternative screening method to the capture screening.
  • Luminex screening is a suitable alternative screening method to the capture screening.
  • 21 unique binders were identified from the semi- solution panning and 14 of these binders showed neutralizing activity. All neutralizing Fabs from HuCAL GOLD ® derived from this panning. Characterization of HuCAL GOLD ® Fabs. Unique Fabs were expressed and purified for further characterization.
  • hMCP-1 binding affinity was determined by BIAcore and Fabs were characterized in the following assays: 1) inhibition of binding of 125 I-CCL-2 to Thp-1 cells and 2) inhibition of hMCP - 2 induced Ca2+ mobilization in Th ⁇ -1 cells.
  • Fabs that demonstrated neutralization activity in the cell based assays were further tested for 1) binding to synthetic cynomolgous hMCP-2; 2) binding to hMCP -2 family related human chemokinesfor binding specificity (i.e. MCP-2, 3, 4 and Eotaxin 1,2, 3); and 3) binding to native human hMCP-2 to ensure the Fabs selected using the synthetic hMCP-2 peptide, recognize native hMCP-2.
  • Fabs with the most optimal properties were chosen for additional affinity maturation. Properties of the Fabs selected for affinity maturation were summarized in Table 3. The seven Fabs were selected for affinity maturation were assigned to 3 groups for the library cloning and the selection. L-CDR3 and H-CDR2 optimization was performed in parallel. The parallel optimization of the light and the heavy variable chains created the potential for combining improved heavy and light chain via cross cloning to generate even further improved antibodies.
  • EXAMPLE 4 Evaluation of high affinity, MCP-I specific antibodies from Fabs.
  • mobilization assay mobilization assay, affinity to human MCP-I measured by Biacore, specificity to human MCP-I, affinity to cyno MCP-I and binding to native MCP-I detected in Biacore. Additional criteria for grouping the parental Fabs were C775 competition in ELISA and were based on the framework family of the variable heavy and light chain. Characterization of maturation candidates as IgG, especially in chemotaxis assay, was performed in parallel with the maturation selection process.
  • Fabs 03336, 03464, 03468 and 03470 had V ⁇ 3 light chain frameworkswith one of two different heavy chain frameworks.
  • Fabs 03336 and 03464 had VH3 heavy chain frameworks and Fabs 03468 and 03470 had VHlB heavy chain frameworks.
  • the second class of Fabs (Group 2, Table 3), 03471 and 03473, had VHlA heavy chain frameworks and V ⁇ 3 light chain frameworks.
  • Fab 03548 had the same the heavy and the light chain frameworks as two of the Fabs in the first class (VH3, V ⁇ 3) but was maintained separately (Group 3, Table 3) because it had exceptionally potent biological activity and binding cross reactivity with Eotaxin.
  • VH3, V ⁇ 3 variable region sequence classification
  • Table 3 Table 3
  • MCP-I response unit
  • X MW of mAb/RU of mAb captured onto the surface
  • molar binding ratio lower than 0.5 was expected to be not significant.
  • Four of the IgGs showed normalized binding ratio to MCP-I >0.5 and to all homologue chemokines ⁇ 0.5 and were therefore termed specific on the level of IgG.
  • One IgG also showed some binding to MCP-2 and Eotaxin, which was already detected on the level of Fab, but this cross-reactivity was reduced on the level of IgG. Data of MOR03468 IgG are not shown.
  • L-CDR3 and H-CDR2 regions were optimized in parallel by cassette mutagenesis using trinucleotide directed mutagenesis (iVirnekas et al, 1994j), while the framework regions were kept constant.
  • cassette mutagenesis using trinucleotide directed mutagenesis (iVirnekas et al, 1994j)
  • the framework regions were kept constant.
  • all parental Fab fragments were transferred from the corresponding expression vector (pMORPH ® X9_FH) into the CysDisplayTM vector pMORPH ® 25_LHC via XbaVEcoW.
  • pMORPH ® 25_LHC was created from the HuCAL GOLD ® display vector pMORPH ® 23_LHC by removal of one Bss ⁇ E site interfering with library cloning for H-CDR2 optimization.
  • L-CDR3 For optimizing L-CDR3 of a pool of parental Fab fragments the L-CDR3, framework 4 and the constant region of the light chains (405 bp) of the binder pool were removed by BpiUSphl and replaced by a repertoire of diversified L-CDR3s together with framework 4 and the constant domain. Design, synthesis and cloning of this L-CDR3 cassette will be described elsewhere (manuscript in preparation).
  • the pre-adsorbed phages and different concentrations of biotin-K69 MCP-I (0.02 - 50 nM) were incubated for 1.5 h at 22 0 C in solution, followed by capturing of the phage-antigen complexes to Reacti-Bind Neutravidin Coated Polystyrene microtiter plate strips (PERBIO). Washing steps at 22 0 C were extended up to 12 h. Elution by 20 mM DTT in 10 mM Tris/HCl, pH 8.0, and phagemid amplification between each panning round were conducted as described above.
  • Amino acid changes in the matured Fabs were located in either the H-CDR2 or the L-CDR3 of the parental clones 03741 and 03548.
  • Cross cloning of the best improved heavy chain CDR2 with the best light chain CDR3 of the Fabs was then carried out to try to generate Fabs with even higher affinity. Approximately 36 cross-clones were generated. All unique Fab sequences were also screened for prediction of N-linked glycosylation sites. A few Fabs were identified with the NIS consensus sequence for glycosylation in heavy chain CDR2. These Fabs were excluded from further characterization. A total of 84 Fabs were expressed and purified at Morphosys and transferred to Centocor for biological characterization.
  • parental MOR03471 was very successfully optimized in this maturation with 23 Fabs optimized in L-CDR3 and one optimized in H-CDR2. Improved Fabs derived from 4 put of 7 parental Fabs, indicating that each parental binder had different potential for being optimized. Finally 4 Fabs fulfilling all success criteria derived from MOR03471, two optimized in L-CDR3 only and two from cross-cloning, optimized in L-CDR3 and H-CDR2.
  • the clones representing the optimized Fabs are represented by the sequences given in Table 5A-C, where clone MOR03471 parental Fab has VH3 x kappa3 frameworks and MOR03548 has on the VHlA x Iamda3 frameworks.
  • Binding to Native MCP-I Measured by Biacore. Binding to native MCP-I was tested in the Biacore Fab capture mode and all selected Fabs showed binding to native MCP-I. Especially as the detection limits for K D determination in Biacore were reached with the optimized Fabs, alternative methods for affinity determination and verification of specificity had to be used.
  • IgG Conversions All optimized Fabs selected for detailed characterization were converted into IgGl format, in addition 4 Fabs were sub-cloned into IgG4 format. The expression data and the activity in different assays of the tested human IgG4 were as good as of the respective IgGl .
  • binders including MOR03757, MOR03781, MOR03790, MOR03850, MOR03878 as Fab and IgG and MOR03899 as IgG fulfilled the affinity success criteria against human MCP-I being ⁇ 0.5 nM and cyno MCP-I being ⁇ 20 nM. Best affinities to human MCP-I were 20 to 40 pM on the level of Fab and 10 to 20 pM on the level of IgG (Table 6). Best affinities to cynomolgus MCP-I were 10 to 40 pM on the level of Fab and 20 pM on the level of IgG (Table 6).
  • Fabs Binding to MCP-2 do not Inhibit 125i MCP-2 Binding to Thp-1 Cells (CNTO).
  • CNTO Thp-1 Cells
  • radio ligand whole cell binding assays were developed at Centocor. I 125 MCP-2 showed nice binding to Thp-1 cells and the binding was inhibited by the addition of unlabeled MCP-2, but not by the addition of the MCP-I specific reference antibody C775. The results provided an important functional assay for testing the binding/neutralization specificity.
  • MCP-I 1 ng/ml MCP-I was used in receptor binding assay, while about 100 ng/ml MCP-2 were necessary in this assay, as MCP-2 labeling might have caused a loss in activity.
  • MOR03754 showed no significant inhibition of 1251 labeled MCP-2 binding to CCR2 receptor on Thp-1 cells (IC 50 > 2 ⁇ M).
  • Parental MOR03471 Fab showed an IC 50 of 180 nM and optimized MOR03471 Fab derivatives (MOR03781 with 180 pM, MOR03790 with 260 pM, MOR03850 with 160 pM,
  • MOR03878 with 110 pM and MOR03899 with 130 pM showed an overall improvement in activity during optimization up to a factor of 100Ox. Although this assay was the most sensitive bioassay available in this project, even in this assay the optimized binders seemed to have reached the assay limits.
  • the receptor internalization assays were performed using cells expressing CCR-2 that showed higher CCR2 expression than THP-I cells, leading to a better signal to noise ratio.
  • the EC 50 value for MCP-I was found to be of 116 ng/ml. Therefore, 100 ng/ml (-11 nM) MCP-I waschosen for further FACS assays.
  • the optimal incubation time to obtain complete internalization was evaluated at 37°C. Most of the internalization occurred within the first 30 min. Therefore, a 1 h incubation time was used in all subsequent assays. The assay was successfully developed to allow an IC 50 determination.
  • MCP-I induces calcium mobilization in THP-I cells which can be detected with the help of a flurophore.
  • the optimized antibodies showed potent inhibition of calcium mobilization the 4 final candidates MOR03781, MOR03790, MOR03850 and MOR03878 Fab showed IC 50 values from 18 to 28 nM.
  • the respective IgGs again retained the activity and showed even slightly better IC 50 values from about 6 to 10 nM due to their ability to neutralize 2 MCP-I molecules per IgG. Again the assay limits seemed to be reached at about 10 nM.
  • N-terminus sequence of mAb 3790 certain variances from the human germline sequences, due to the amino acid changes introduced during cloning.
  • amino acid codons i.e. the DNA sequence
  • MAb DNA was re- synthesized to correct the imperfect N-terminus alignment to germline sequence and to change the codon bias to those favored in highly expressed human proteins.
  • sequence modified 3790 mAb is designated as CNTO 888 comprising heavy and light chain variable region sequences of SEQ ID NO: 27 and 28, respectively, and below (with CDRs underlined), where the N-terminal residues of the heavy chain are QVQ (Gln-Val-Gln) and or the light chain are EIV (Glu-Ee-Val).
  • CNTO 888 is a fully human IgGl kappa antibody. There are no predicted N-linked glycosylation sites in the sequence.
  • the biochemical and biophysical properties of CNTO 888 (transiently expressed in HEK293 cells and purified by protein A affinity chromatography) were characterized in SDS-PAGE, size exclusion chromatography (SEC), mass spectrum (MS) and BIAcore, for binding affinity (Kd) and specificity.
  • SDS-PAGE the native CNTO 888 migrates as a single band at approximately 150 kDa.
  • the reduced/alkalyated IgG migrates as two bands at approximately 60 kDa and 33 kDa.
  • CNTO888 In vitro characterization of CNTO888.
  • the biological activities of CNTO 888 were evaluated in a variety cell based assays.
  • CNTO 888 expressed transiently evaluated in all of the success criteria assays had activities of which were indistinguishable from the parent mAb 3790 (Table 7).
  • the plasmid p2844 contains the optimized heavy chain coding sequence of CNTO888 coding regions under the anti-CD4 heavy chain promoter and the plasmid p2882 contains the optimized light chain of CNTO888 coding regions under the anti-CD4 light chain promoter.
  • Both constructs include the gpt selection gene to confer chemical resistance to MHX (Mycophenolic acid, Hypoxanthine and Xanthine). Each plasmid was purified, characterized, quantified, and sequenced.
  • 1 MHX 0.5 mg/L Mycophenolic acid, 2.5 mg/L Hypoxanthine and 50 mg/L Xanthine
  • the highest parental cell line (1C4) had a 24-well overgrowth titer of 70 mg/L and a titer of 108.5 mg/L in shake flasks (in CD-Hybridoma media).
  • This parental cell line, C1262A was chosen for further evaluation in shake flasks.
  • C1262A was submitted to the Cell Banking Group for generation of a Development Cell Bank (DCB). Cells from the DCB, designated C1262A:DCB;02SEP04, tested negative for mycoplasma and sterility.
  • DCB Development Cell Bank
  • the parental cell line, C1262A was subcloned using the Halo procedure and yielded five high- producing subclone cell lines.
  • the best subclone cell line (4D5) had a 24-well overgrowth titer of 150.5 mg/L and a titer of 167 mg/L in shake flasks (in CD-Hybridoma).
  • This subclone cell line was coded C1262B. It will be clear that the invention can be practiced otherwise than as particularly described in the foregoing description and examples.

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Abstract

Cette invention concerne des mutéines MCP-1, y compris des parties ou des variantes spécifiées de ces mutéines, qui peuvent éventuellement être PEGylées ou biotine-PEGylées. Cette invention concerne en outre des procédés de synthèse chimique pour produire ces mutéines MCP-1 et des conjugués correspondants. Cette invention concerne l'utilisation de ces mutéines et, en particulier, l'utilisation des conjugués biotinylés pour sélectionner des anticorps de neutralisation contre la protéine MCP-1 humaine, ainsi que les anticorps ainsi sélectionnés. Ces anticorps sont spécifiques pour un épitope conservé sur une mutéine MCP-1 native conservée dans la ou les protéines mutéines MCP-1 biotinylées et PEGylées ou un fragment de celle(s)-ci..
PCT/US2006/019625 2005-05-19 2006-05-19 Anticorps anti-muteines mcp-1 biotine-pegylees, compositions, procedes et utilisations correspondants WO2006125201A2 (fr)

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WO2008060783A3 (fr) * 2006-10-05 2008-10-23 Centocor Antagonistes de ccr2 pour le traitement de la fibrose
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