Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
  • C07K16/243—Colony Stimulating Factors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
  • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
  • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/18—Antivirals for RNA viruses for HIV
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• • 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

• This invention relates to methods for preventing and treating atherosclerotic and associated cardiovascular diseases and diseases relating to HIV by administering an M- CSF-specific antibody to a subject.
• Colony stimulating factor also known as macrophage colony stimulating factor (M-CSF) stimulates the production and proliferation of macrophages.
• Macrophages are well known mediators of the atherosclerotic process and contribute to the formation of occlusive plaques by migrating into early lesions and engulfing lipid. The resulting narrowing of blood vessels, including arteries that supply the heart, brain and limbs, causes angina and other symptoms of vascular occlusion. Macrophages also may contribute to the formation of unstable plaques by secreting proteases and other bioactive molecules that cause stable plaques to become unstable.
• Unstable plaques play a causative role in triggering blood clotting that may cause a total blockage of the blood vessel, resulting in a myocardial infarction or stroke.
• macrophages may also play a role in the over-exuberant repair process that leads to restenosis after angioplasty.
• therapies there is still a great medical need for more effective preventative and therapeutic strategies for atherosclerotic vascular disease and its associated symptoms and damaging consequences.
• Macrophage colony-stimulating factor enhances the susceptibility of macrophages to infection with human immunodeficiency virus (HIV), in part by increasing the expression of CD4 and CCR5 (Kutza, J., et al., AIDS Res Hum Retroviruses, 18(9):619- 25 (2002)).
• M-CSF may function in an autocrine/paracrine manner to sustain HIV replication, and that inhibitors of M-CSF activity dramatically reduce HIV-I replication (Kutza, J., et al., J Immunol, 164(9):4955-60 (2000).
• biologic antagonists for M-CSF may represent novel strategies for inhibiting the spread of HIV-I by blocking virus replication in macrophages and preventing the establishment and maintenance of infected macrophages as a reservoir for HIV.
• a method of treating a macrophage-associated disease comprising administering to a subject having a macrophage- associated disease a non-murine antibody that competes with monoclonal antibody RXl for binding to M-CSF by more than 75%, wherein the monoclonal antibody RXl comprises the heavy chain and light chain amino acid sequences set forth in SEQ ID NOs: 2 and 4, respectively, is provided.
• the aforementioned non-murine antibody specifically binds to the same epitope of M-CSF as the monoclonal antibody RXl.
• the macrophage-associated disease to be treated according to the present invention may be, for example, an atherosclerotic disease or a condition associated with HIV infection. However, it is contemplated that that the macrophage-associated disease to be treated according to the present invention will be useful to treat any disease state in which macrophage activity contributes to the pathology.
• the non-murine antibody binds an epitope of M-CSF that comprises at least 4 contiguous residues of SEQ ED NO: 120 or 121.
• the non-murine antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, a human engineered antibody, a human antibody, a single chain antibody, or an IgG antibody.
• a non-murine antibody useful in the treatment methods of the present invention may retain an affinity K d (dissociation equilibrium constant) with respect to M-CSF of SEQ ED NO: 9 of, for example, at least 10 "7 M or higher, at least 10 "8 M or higher, or at least 10 '9 M or 10 "10 M or higher.
• the non-murine antibody comprises an amino acid sequence 90% identical to SEQ ID NO: 24, or comprises SEQ ID NO: 24.
• the non-murine antibody comprises at least 1, at least 2, at least 3, at least 4, at least 5, or all of (a) SEQ ED NOs: 18, 21, 24, 29, 32, and 36; or (b) SEQ ID NOs: 18, 21, 24, 32, 36 and QASQSIGTSIH (SEQ ID NO: ).
• the non-murine antibody of any of the preceding embodiments may further comprise one or more CDRs from another anti-M-CSF antibody, such as SEQ ID NO: 16, 19, 22, 27, 30, or 34 from 5H4; SEQ ID NO: 17, 20, 23, 28, 31, or 35 from MCl; SEQ ID NO: 18, 21, 25, 29, 32, or 37 from MC3; or a consensus CDR as set forth in SEQ ID NOs: 18, 21, 26, 29, 33, or 38.
• the non-murine antibody comprises a CDR in which at least one amino acid within a CDR is substituted by a corresponding residue of a corresponding CDR of another anti-M-CSF antibody.
• the non-murine antibody comprises a variable light chain amino acid sequence which is at least 65% homologous to the amino acid sequence set forth in SEQ ID NO: 4, and/or a variable heavy chain amino acid sequence which is at least 65% homologous to the amino acid sequence set forth in SEQ ID NO: 2.
• the non-murine antibody may comprise a constant region of a human antibody sequence and one or more heavy and light chain variable framework regions of a human antibody sequence.
• exemplary human antibody sequences include an individual human sequence, a human consensus sequence, an individual human germline sequence, or a human consensus germline sequence.
• any of the preceding described antibodies may comprise a fragment of an IgGl constant region, optionally including a mutation within the IgGl constant region that reduces antibody-dependent cellular cytotoxicity or complement dependent cytotoxicity activity.
• any of the preceding described antibodies may comprise a fragment of an IgG4 constant region, optionally including a mutation in the IgG4 constant region that reduces formation of half-antibodies.
• the non-murine antibody comprises a heavy chain variable region that comprises the amino acid sequence
• the non-murine antibody comprises a heavy chain variable region that comprises the amino acid sequence DVXLXEXGPXXVXPXXXLXLXCXVTDYSITSDYAWNWIRQXPXXKLEWMGYISYS GSTSYNPSLKXRIXIXRXTXXNXFXLXLXXVXXXDXATYYCASFDYAHAMDYWGX GTXVXVXX, wherein X is any amino acid.
• the non-murine antibody comprises a heavy chain variable region that comprises the amino acid sequence XVQLQESGPGLVKPSQXLSLTCTVXDYSITSDYAWNWIRQFPGXXLEWMGYISYSGS TSYNPSLKSRIXIXRDTSKNQFXLQLNSVTXXDTAXYYCASFDYAHAMDYWGQGTX VTVSS, wherein X is any amino acid.
• the non-murine antibody comprises a heavy chain variable region that comprises the amino acid sequence DVQLQESGPGLVKPSQXLSLTCTVTDYSITSDYAWNWIRQFPGXKLEWMGYISYSGS TSYNPSLKSRIXIXRDTSKNQFXLQLNSVTXXDTATYYCASFDYAHAMD YWGQGTX VTVSS, wherein X is any amino acid.
• the non-murine antibody comprises a heavy chain variable region that comprises the amino acid sequence DVQLQESGPGLVKPSQTLSLTCTVTDYSITSDYAWNWIRQFPGKKLEWMGYISYSGS TSYNPSLKSR ⁇ TISRDTSKNQFSLQLNSVTAADTATYYCASFDYAHAMD YWGQGTTV TVSS.
• the non-murine antibody comprises a heavy chain variable region that comprises the amino acid sequence
• the non-murine antibody comprises a light chain variable region that comprises the amino acid sequence
• the non-murine antibody comprises a light chain variable region that comprises the amino acid sequence
• the non-murine antibody comprises a light chain variable region that comprises the amino acid sequence
• the non-murine antibody comprises a light chain variable region that comprises the amino acid sequence
• the non-murine antibody comprises a light chain variable region that comprises the amino acid sequence XKLTQSPXXLSVSPGERVXFSCRASQSIGTSIHWYQQXTXXXPRLLIKYASESISGIPX RFSGSGSGTDFTLXKXVESEDXADYYCQQINSWPTTFGXGTKLEIKRX, wherein X is any amino acid
• the non-murine antibody comprises a light chain variable region that comprises the amino acid sequence EIVLTQSPGTLSVSPGERVTFSCRASQSIGTSIHWYQQKTGQAPRLLIKYASESISGIPD RFSGSGSGTDFTLTISRVESEDFADYYCQQINSWPTTFGQGTKLEIKRT.
• the non-murine antibody comprises a light chain variable region that comprises the amino acid sequence
• the non-murine antibody comprises a light chain variable region that comprises the amino acid sequence
• At least one X of the aforementioned antibody is the same as an amino acid at the same corresponding position in SEQ ID NOs: 2 or 4 using Kabat numbering.
• at least one X is a conservative substitution of an amino acid at the same corresponding position in SEQ ID NOs: 2 or 4 using Kabat numbering.
• at least one X is a non-conservative substitution of an amino acid at the same corresponding position in SEQ ID NOs: 2 or 4 using Kabat numbering.
• At least one X is an amino acid at the same corresponding position within a human antibody sequence, using Kabat numbering.
• the aforementioned human antibody sequence may be, for example, a human consensus sequence, human germline sequence, human consensus germline sequence, or any one of the human antibody sequences in Kabat.
• the aforementioned non-murine antibody comprises any one of the heavy chain sequences set forth in SEQ ID NOS: 114, 116, or 119.
• the non-murine antibody comprises any one of the heavy chain variable region sequences set forth in SEQ ID NOS: 41 or 43.
• the non-murine antibody comprises any one of the light chain sequences set forth in SEQ ID NOS: 45, 47, 48, 51, 53 or 136.
• the non-murine antibody comprises the heavy chain sequence set forth in SEQ ID NO: 114 and the light chain sequence set forth in SEQ ID NO: 47.
• the non-murine antibody comprises the heavy chain sequence set forth in SEQ ID NO: 116 and the light chain sequence set forth in SEQ ID NO: 47. In yet another embodiment, the non-murine antibody comprises the heavy chain sequence set forth in SEQ ID NO: 119 and the light chain sequence set forth in SEQ ID NO: 47.
• the non-murine antibody comprises a variable heavy chain amino acid sequence which is at least 65%, or at least 80%, identical to the variable heavy chain amino acid sequence set forth in SEQ ID NOs: 41 or 43, and/or a variable light chain amino acid sequence which is at least 65%, or at least 80%, identical to the variable light chain amino acid sequence set forth in SEQ ID NOs: 45, 47, 48, 51, or 53.
• the antibody of the invention may, for example, be administered at a dose between about 1 ⁇ g/kg to 100 mg/kg body weight, between about 2 ⁇ g/kg to 30 mg/kg body weight, between about 0.1 mg/kg to 30 mg/kg body weight, or between about 0.1 mg/kg to 10 mg/kg body weight, hi other aspects, the aforementioned methods may further comprise administering a second therapeutic agent.
• kits comprising a therapeutically effective amount of the aforementioned antibody, either in lyophilized or solution form, packaged in a container, such as a vial or bottle or prefilled syringe.
• the container further may comprise a label attached to or packaged with the container, the label describing the contents of the container and providing indications and/or instructions regarding use of the contents of the container to treat a macrophage-associated disease.
• the container optionally comprises another vial with suitable solution for reconstituting lyophilized antibody.
• Figure 1 is a topology diagram showing the disulfide bonds in truncated dimeric M-CSF.
• Figure 2 is a stereodiagram of the C-alpha backbone of M-CSF with every tenth residue labeled and with the non-crystallographic symmetry axis indicated by a dotted line.
• Figure 3 A shows the amino acid sequence of M-CSF-specific murine antibody
• RXl (SEQ ID NOs: 2 and 4) (encoded by the cDNA insert of the plasmid deposited with the American Type Culture Collection, Manassas, VA, USA, under ATCC deposit number PTA- 6113) and a corresponding nucleic acid sequence (SEQ ID NOs: 1 and 3).
• the CDR regions are numbered and shown in bold.
• Figures 3 B and 3 C show the amino acid sequences of M-CSF specific murine antibody RXl light (SEQ ID NO: 5) and heavy chains (SEQ ID NO: 6), respectively, with high risk (bold), moderate risk (underline), and low risk residues identified according to Studnicka et al., WO93/11794.
• Figure 4A shows that M-CSF antibodies RXl and 5Al are species specific.
• Figure 4B shows the M-CSF neutralization activity of antibodies MCl and MC3.
• Figure 5 is the amino acid sequence of M-CSFa (SEQ ID NO: 7).
• Figure 6 is the amino acid sequence of M-CSF ⁇ (SEQ ID NO: 8).
• Figure 7 is the amino acid sequence of M-CSFy (SEQ ID NO: 9).
• a number of polymorphisms in the DNA sequence may result in amino acid differences.
• a common polymorphism provides an Ala rather than Pro at position 104.
• Figures 8, 9, and 10 show the amino acid sequences of M-CSF-specific murine antibodies 5H4 (SEQ ID NOs: 10 and 11), MCl (SEQ ID NOs: 12 and 13) (produced by the hybridoma deposited under ATCC deposit number PTA-6263) and MC3 (SEQ ID NOs: 14 and 15) (produced by the hybridoma deposited under ATCC deposit number PTA-6264), respectively.
• Figures 1 IA and 1 IB are an alignment of CDR regions of the heavy and light chain amino acid sequences of human M-CSF specific murine antibodies RXl; 5H4; MCl; and MC3 (SEQ ID NOs: 16-38).
• Figure HC shows the neutralization activities of intact versus Fab fragments for RXl versus 5H4.
• Figure 12 shows the structure of M-CSF with RXl, 5H4, and MC3 epitopes highlighted (SEQ ID NOs: 120, 122, and 123).
• Figure 13B shows the amino acid sequences of the two exemplary heavy chain Human EngineeredTM sequences (SEQ. ID NOs: 41 and 43), designated "low risk” and "low+moderate risk” as well as corresponding nucleic acid sequences (SEQ ID NOs: 40 and 42).
• Figure 14B shows the amino acid sequences of the two exemplary light chain Human EngineeredTM sequences (SEQ ID NOs: 45 and 47), designated "low risk” and "low+moderate risk” as well as corresponding nucleic acid sequences (SEQ ID NOs: 44 and 46).
• Figure 15B shows the amino acid sequences of two exemplary alternate light chain Human EngineeredTM sequences (SEQ ID NOs: 48, 136), as well as corresponding nucleic acid sequences (SEQ DD NOs: 137 and 135).
• Figure 16B shows the amino acid sequences of the two exemplary light chain Human EngineeredTM sequences (SEQ DD NOs: 51 and 53), designated “low risk” and “low+moderate risk” as well as the corresponding nucleic acid sequence (SEQ DD NO: 52).
• Figures 17 A and 17B show the alignment of murine RXl light chain amino acid sequence (SEQ DD NO: 54) with various human consensus and human germline consensus sequences using the Kabat numbering system (amino acid numbering indicated in line designated "POS") (SEQ DD NOs: 55-82).
• Figures 18A and 18B show the alignment of murine RXl heavy chain amino acid sequence (SEQ ID NO: 83) with various human consensus and human germline consensus sequences using the Kabat numbering system (amino acid numbering indicated in line designated "POS") (SEQ JD NOs: 84-112).
• Figures 18C-18E show how the amino acid residues of antibodies 5H4, MCl and MC3 correspond to the Kabat numbering system (SEQ ID NOs: 10 and 11; SEQ ID NOs: 12 and 13; SEQ ID NOs: 14 and 15, respectively).
• Figure 19A shows the amino acid (SEQ ID NO: 114) and nucleotide sequence (SEQ ID NO: 113) for lieRXl-l.IgGl with low risk amino acid changes.
• Figure 19B shows the amino acid (SEQ ID NO: 116) and nucleotide sequence (SEQ ID NO: 115) for heRXl- LIgGl with low + moderate risk amino acid changes.
• Figure 20 shows the amino acid (SEQ DD NO: 119) and nucleotide sequence
• Colony stimulating factor also known as macrophage colony stimulating factor (M-CSF)
• M-CSF macrophage colony stimulating factor
• M-CSF has been shown to stimulate the production and proliferation of macrophages and osteoclasts, among other cells.
• macrophage activity is beneficial in many situations, macrophage activity is deleterious in a number of situations.
• Macrophages have been shown to play a role in formation of atherosclerotic plaques, destabilization of plaques and restenosis after angioplasty.
• Macrophages and production of M-CSF have also been shown to be associated with HIV replication, and infected macrophages may serve as a reservoir of the virus.
• the invention provides methods of using anti-M-CSF-antibodies to prevent or treat macrophage-associated diseases.
• Macrophage-associated diseases as used herein means conditions or disorders associated with or caused by deleterious macrophage activity.
• Exemplary macrophage-associated diseases include atherosclerotic diseases, or HIV infection and conditions associated therewith.
• the anti-M-CSF antibodies contemplated for use in such methods include any antibody described herein, including RXl-derived antibodies, RXl-competing antibodies, 5H4-derived antibodies, 5H4-competing antibodies, MCl- derived antibodies, MCl -competing antibodies, MC3-derived antibodies and MC3- competing antibodies.
• Atherosclerosis in any blood vessel, diseases or conditions resulting from atherosclerosis, and conditions associated with increased risk of vessel occlusion or thrombosis, for example, hypertension, diabetes, and other risk factors for myocardial infarction or stroke.
• Exemplary atherosclerotic diseases include arterial thrombosis, stenosis or ischemia; cardiovascular disease, including occlusive cardiovascular diseases such as angina; arterial thrombosis, such as coronary artery thrombosis or resulting myocardial ischemia or myocardial infarction; restenosis, particularly following angiography or angioplasty; cerebral artery thrombosis or resulting cerebral ischemia or stroke; intracardiac thrombosis (due to, e.g., atrial fibrillation) or resulting stroke; peripheral vascular disease or peripheral arterial thrombosis or occlusion; neointimal hyperplasia, disruption of intercellular junctions in vascular endothelium, or vessel injury.
• cardiovascular disease including occlusive cardiovascular diseases such as angina
• arterial thrombosis such as coronary artery thrombosis or resulting myocardial ischemia or myocardial infarction
• restenosis particularly following ang
• HIV infectious neoplasma capsulatum
• Coccidioides immitis Candida species, Aspergillus species, Mycobacterium avium-complex, Toxoplasma gondii, Strongyloides stercoralis, cytomegalovirus, herpes simplex viruSjlymphoid interstitial pneumonitis, odynophagia, hairy leukoplakia, erosive gingivitis, aphthous ulcers, gastrointestinal/colitis, Salmonella species, Shigella species, Campylobacter jejuni, Cryptosporidium species, Isospora belli, Blastocystis hominis, Entamoeba histolytic
• M-CSF The full-length human M-CSF mRNA encodes a precursor protein of 554 amino acids.
• M-CSF can either be secreted into the circulation as a glycoprotein or chondroitin sulfate containing proteoglycan or be expressed as a membrane spanning glycoprotein on the surface of M-CSF producing cells.
• M-CSF The three-dimensional structure of the bacterially expressed amino terminal 150 amino acids of human M-CSF, the minimal sequence required for full in vitro biological activity, indicates that this protein is a disulfide linked dimer with each monomer consisting of four alpha helical bundles and an anti-parallel beta sheet (Pandit et al., Science 258: 1358-62 (1992)).
• M-CSF species are produced through alternative mRNA splicing.
• the three polypeptide precursors are M-CFSa of 256 amino acids, M-CSF ⁇ of 554 amino acids, and M-CSF ⁇ of 438 amino acids.
• M-CSF ⁇ is a secreted protein that does not occur in a membrane-bound form.
• M-CSFa is expressed as an integral membrane protein that is slowly released by proteolytic cleavage. M-CSFa is cleaved at amino acids 191-197 of the sequence set out in Figure 5. The membrane-bound form of M-CSF can interact with receptors on nearby cells and therefore mediates specific cell-to-cell contacts.
• the term "M-CSF" may also include amino acids 36-438 of Figure 7.
• M-CSFR receptor spanning molecule with five extracellular immunoglobulin-like domains, a transmembrane domain and an intracellular interrupted Src related tyrosine kinase domain. M-CSFR is encoded by the c-fms proto- oncogene. Binding of M-CSF to the extracellular domain of M-CSFR leads to dimerization of the receptor, which activates the cytoplasmic kinase domain, leading to autophosphorylation and phosphorylation of other cellular proteins (Hamilton J. A., J Leukoc Biol.,62(2): 145-55 (1997); Hamilton J, A., Immuno Today., 18(7): 313-7(1997).
• Phosphorylated cellular proteins induce a cascade of biochemical events leading to cellular responses: mitosis, secretion of cytokines, membrane ruffling, and regulation of transcription of its own receptor (Fixe and Praloran, Cytokine 10: 32-37 (1998)).
• Murine RXl An anti-M-CSF antibody designated murine RXl and having the sequence set forth in SEQ ID NO: 1 was discovered to have superior M-CSF-neutralizing properties compared to other antibodies.
• Murine RXl antibody was modified to be less immunogenic in humans based on the Human EngineeringTM method of Studnicka et al.
• 8 to 12 surface exposed amino acid residues of the heavy chain variable region and 16 to 19 surface exposed residues in the light chain region were modified to human residues in positions determined to be unlikely to adversely effect either antigen binding or protein folding, while reducing its immunogenicity with respect to a human environment.
• Synthetic genes containing modified heavy and/or light chain variable regions were constructed and linked to human ⁇ heavy chain and/or kappa light chain constant regions.
• Any human heavy chain and light chain constant regions may be used in combination with the Human EngineeredTM antibody variable regions.
• the human heavy and light chain genes were introduced into mammalian cells and the resultant recombinant immunoglobulin products were obtained and characterized.
• Other exemplary anti-M-CSF antibodies such as 5H4, MCl, or MC3 are similarly Human EngineeredTM.
• RX 1-derived antibody includes any one of the following:
• an amino acid variant of murine antibody RXl having the amino acid sequence set out in Figure 3, including variants comprising a variable heavy chain amino acid sequence which is at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% homologous to the amino acid sequence as set forth in Figure 3, and/or comprising a variable light chain amino acid sequence which is at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% homologous to the amino acid sequence as set forth in Figure 3, taking into account similar amino acids for the homology determination;
• M-CSF-binding polypeptides (excluding murine antibody RX 1) comprising one or more complementary determining regions (CDRs) of murine antibody RX 1 having the amino acid sequence set out in Figure 3, preferably comprising at least CDR3 of the RX 1 heavy chain, and preferably comprising two or more, or three or more, or four or more, or five or more, or all six CDRs;
• Human EngineeredTM antibodies having the heavy and light chain amino acid sequences set out in Figures 13B through 16B or variants thereof comprising a heavy or light chain having at least 60% amino acid sequence identity with the original Human EngineeredTM heavy or the light chain of Figures 13B through 16B, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95%, including for example, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% identical;
• M-CSF-binding polypeptides comprising the high risk residues of one or more CDRs of the Human EngineeredTM antibodies of Figures 13B through 16B, and preferably comprising high risk residues of two or more, or three or more, or four or more, or five or more, or all six CDRs;
• Human EngineeredTM antibodies or variants retaining the high risk amino acid residues set out in Figure 3B, and comprising one or more changes at the low or moderate risk residues set out in Figure 3B; for example, comprising one or more changes at a low risk residue and conservative substitutions at a moderate risk residue set out in Figure 3B, or for example, retaining the moderate and high risk amino acid residues set out in Figure 3B and comprising one or more changes at a low risk residue, where changes include insertions, deletions or substitutions and may be conservative substitutions or may cause the engineered antibody to be closer in sequence to a human light chain or heavy chain sequence, a human germline light chain or heavy
• MC3-derived antibody includes any one of the following:
• an amino acid variant of murine antibody MC3 having the amino acid sequence set out in Figure 10 including variants comprising a variable heavy chain amino acid sequence which is at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% homologous to the amino acid sequence as set forth in Figure 10, and/or comprising a variable light chain amino acid sequence which is at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% homologous to the amino acid sequence as set forth in Figure 10, taking into account similar amino acids for the homology determination; 2) M-CSF-binding polypeptides (optionally including or excluding murine antibody MC3) comprising one or more complementary determining regions (CDRs) of murine antibody MC3 having the amino acid sequence set out in Figure 10, preferably comprising at least CDR3 of the MC3 heavy chain, and preferably comprising two
• variants of the aforementioned antibodies in preceding paragraph (3) comprising a heavy or light chain having at least 60% amino acid sequence identity with the original Human EngineeredTM heavy or the light chain, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95%, including for example, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% identical;
• M-CSF-binding polypeptides (optionally including or excluding murine antibody MC3) comprising the high risk residues of one or more CDRs of the murine MC3 antibody of Figure 10, and preferably comprising high risk residues of two or more, or three or more, or four or more, or five or more, or all six CDRs;
• Human EngineeredTM antibodies or variants retaining the high risk amino acid residues of murine MC3 antibody, and comprising one or more changes at the low or moderate risk residues; for example, comprising one or more changes at a low risk residue and conservative substitutions at a moderate risk residue, or for example, retaining the moderate and high risk amino acid residues and comprising one or more changes at a low risk residue, where changes include insertions, deletions or substitutions and may be conservative substitutions or may cause the engineered antibody to be closer in sequence to a human light chain or heavy chain sequence, a human germline light chain or heavy chain sequence, a consensus human light chain or heavy chain sequence, or a consensus human germline light chain or heavy chain sequence; that retain ability to bind M-CSF.
• Such antibodies preferably bind to M-CSF with an affinity of at least 10 "7 , 10 "8 or 10 "9 or higher and preferably neutralize the desired biological activity of M-CSF.
• the term "5H4-derived antibody” or "MCl-derived antibody” is similarly defined according to the above description.
• Anti-M-CSF antibodies such asRXl, 5H4, MCl or MC3-derived antibodies, including Human Engineered antibodies or variants, may be of different isotypes, such as IgG, IgA, IgM or IgE.
• Antibodies of the IgG class may include a different constant region, e.g. an IgG2 antibody may be modified to display an IgGl or IgG4 constant region.
• the invention provides Human Engineered antibodies or variants comprising a modified or unmodified IgGl or IgG4 constant region.
• modifications to the constant region particularly the hinge or CH2 region, may increase or decrease effector function, including ADCC and/or CDC activity.
• an IgG2 constant region is modified to decrease antibody-antigen aggregate formation.
• modifications to the constant region, particularly the hinge region may reduce the formation of half-antibodies.
• mutating the IgG4 hinge sequence Cys-Pro-Ser-Cys to the IgGl hinge sequence Cys-Pro-Pro-Cys is provided.
• Human Engineered antibodies containing IgGl or IgG4 constant regions have improved properties compared to Human EngineeredTM antibodies containing IgG2 constant regions.
• Choice of the IgGl or IgG4 Fc region improved binding affinity and M- CSF neutralization activity.
• choice of the IgGl or IgG4 Fc region provided antigen-antibody complexes that more closely resembled those formed by the parent murine antibody. The mobility at the hinge region thus appears to markedly affect binding of antibody to the dimeric antigen M-CSF as well as neutralization activity of the antibody.
• the invention contemplates generally that preparation of antibodies containing a heavy chain comprising a modified or unmodified IgGl or IgG4 constant region, particularly the hinge and CH2 domains, or preferably at least the hinge domains, improves binding affinity and/or slows dissociation of antibody from dimeric antigens.
• RXl-competing antibody includes
• Such antibodies preferably bind to M- CSF with an affinity of at least 10 "7 , 10 "8 or 10 "9 or higher and preferably neutralize the desired biological activity of M-CSF.
• MCl -competing antibody or “MC3 -competing antibody” or “5H4- competing antibody” is similarly defined with reference to the murine 5H4, MCl or MC3 antibodies having the complete light and heavy chain sequences set out in Figure 8, 9 or 10, respectively, and with reference to the epitope of M-CSF bound by the antibody, e.g. amino acids 65-73 or 138-144 of Figure 7 (corresponding to M-CSF epitopes recognized by 5H4 or MC3).
• Non-rodent monoclonal antibody is any antibody, as broadly defined herein, that is not a complete intact rodent monoclonal antibody generated by a rodent hybridoma.
• non-rodent antibodies specifically include, but are not limited to, variants of rodent antibodies, rodent antibody fragments, linear antibodies, chimeric antibodies, humanized antibodies, Human EngineeredTM antibodies and human antibodies, including human antibodies produced from transgenic animals or via phage display technology.
• non-murine antibodies include but are not limited to variants of murine antibodies, murine antibody fragments, linear antibodies, chimeric, humanized, Human Engineered and human antibodies.
• Treatment is an intervention performed with the intention of preventing the development or altering the pathology of a disorder. Accordingly, “treatment” refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. Treatment of patients suffering from clinical, biochemical, radiological or subjective symptoms of the disease, such as a macrophage-associated disease, may include alleviating some or all of such symptoms or reducing the predisposition to the disease.
• the "pathology" of the disease includes all phenomena that compromise the well being of the patient.
• mammal for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal is human.
• therapeutically effective amount is meant to refer to an amount of therapeutic or prophylactic M-CSF antibody that would be appropriate for an embodiment of the present invention, that will elicit the desired therapeutic or prophylactic effect or response, including alleviating some or all of such symptoms of disease or reducing the predisposition to the disease, when administered in accordance with the desired treatment regimen.
• M-CSF refers to a human polypeptide having substantially the same amino acid sequence as the mature human M-CSFa, M-CSF ⁇ , or M- CSF ⁇ polypeptides described in Kawasaki et al., Science 230:291 (1985), Cerretti et al., Molecular Immunology, 25:761 (1988), or Ladner et al., EMBO Journal 6:2693 (1987), each of which are incorporated herein by reference.
• M-CSF dimer refers to two M-CSF polypeptide monomers that have dimerized and includes both homodimers (consisting of two of the same type of M-CSF monomer) and heterodimers (consisting of two different monomers). M-CSF monomers may be converted to M-CSF dimers in vitro as described in U.S. Pat. No. 4,929,700, which is incorporated herein by reference.
• the present invention provides methods of treating subjects suffering from macrophage-associated diseases using the M-CSF-specific antibodies described herein, including preparation of a medicament for treating subjects suffering from macrophage- associated diseases.
• antibody is used in the broadest sense and includes fully assembled antibodies, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments that can bind antigen (e.g., Fab', F'(ab)2, Fv, single chain antibodies, diabodies), and recombinant peptides comprising the forgoing as long as they exhibit the desired biological activity.
• antibody also refers to fragments thereof (such as, e.g., scFv, Fv, Fd, Fab, Fab' and F(ab)'2 fragments) or multimers or aggregates of intact molecules and/or fragments that bind to M-CSF (or M-CSFR). These antibody fragments bind antigen and may be derivatized to exhibit structural features that facilitate clearance and uptake, e.g., by incorporation of galactose residues.
• the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that are typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the homogeneous culture, uncontaminated by other immunoglobulins with different specificities and characteristics.
• the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
• the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature, 256:495 [19751, or may be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567).
• the "monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described inClackson et al.,Nature,352:624628[1991] end Marks et al., J. MoI. Biol, 222:581-597 (1991), for example.
• immunoglobulins can be assigned to different classes. There are five major classes, IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses or isotypes, e.g. IgGl, IgG2, IgG3, IgG4, IgAl and IgA2.
• the heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma and mu respectively.
• the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Different isotypes have different effector functions; for example, IgGl and IgG3 isotypes have ADCC activity.
• Antibody fragments comprise a portion of an intact full length antibody, preferably the antigen binding or variable region of the intact antibody.
• antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng.,8(10): 1057-1062 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
• Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab” fragments, each with a single antigen-binding site, and a residual "Fc” fragment, whose name reflects its ability to crystallize 35 readily.
• Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the Fv to form the desired structure for antigen binding.
• hypervariable region refers to the amino acid residues of an antibody which are responsible for antigen-binding.
• the hypervariable region comprises amino acid residues from a complementarity determining region or CDR [i.e., residues 24-34 (Ll), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (Hl), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain as described by Kabat et al., Sequences of Proteins of Immunological Interest, 5 th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.
• Framework or FR residues are those variable domain residues other than the hypervariable region residues.
• diabodies refers to small antibody fragments with two antigen- binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH VL).
• VH heavy-chain variable domain
• VL light-chain variable domain
• VH VL polypeptide chain
• multispecific monoclonal antibody including monoclonal, human, humanized, Human
• bispecific antibodies having binding specificities for at least two different epitopes.
• exemplary bispecific antibodies may bind to two different epitopes of M- CSF.
• an anti-M-CSF arm may be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g., CD2 or CD3), or Fc receptors for IgG (Fc ⁇ R), such as Fc ⁇ RI (CD64), Fc ⁇ RII (CD32) and Fc ⁇ RIII (CD16) so as to focus cellular defense mechanisms to the M-CSF-expressing cell.
• Bispecific antibodies may also be used to localize therapeutic agents to cells which express M-CSF. These antibodies possess an M-CSF-binding arm and an arm which binds the therapeutic agent.
• Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F(ab').sub.2 bispecific antibodies).
• the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture.
• the preferred interface comprises at least a part of the C H 3 domain of an antibody constant domain.
• one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan).
• Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers. See WO96/27011 published Sep. 6, 1996.
• Bispecific antibodies include cross-linked or "heteroconjugate" antibodies.
• one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin.
• Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.
• bispecific antibodies can be prepared using chemical linkage.
• Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab') 2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation.
• the Fab 1 fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
• Fab '-TNB derivatives is then reconverted to the Fab '-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab '-TNB derivative to form the bispecific antibody.
• the bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
• Fab'-SH fragments directly recovered from E. coli can be chemically coupled in vitro to form bispecific antibodies.
• bispecific antibodies have been produced using leucine zippers.
• the leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion.
• the antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers.
• the "diabody” technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments.
• the fragments comprise a heavy chain variable region (VH) connected to a light-chain variable region (V L ) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and V L domains of one fragment are forced to pair with the complementary V L and VH domains of another fragment, thereby forming two antigen-binding sites.
• VH and V L domains of one fragment are forced to pair with the complementary V L and VH domains of another fragment, thereby forming two antigen-binding sites.
• sFv single-chain Fv
• the bispecific antibody may be a "linear antibody” produced as described in Zapata et al. Protein Eng. 8(10): 1057-1062 (1995). Briefly, these antibodies comprise a pair of tandem Fd segments (VH -C H I-V H -CHI) which form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.
• Antibodies with more than two valencies are also contemplated.
• trispecific antibodies can be prepared. (Tutt et al., J. Immunol. 147:60 (1991))
• the monoclonal, human, humanized, Human Engineered or variant anti-M-CSF antibody is an antibody fragment, such as an RXl, 5H4, MCl, or MC3 antibody fragment.
• an antibody fragment such as an RXl, 5H4, MCl, or MC3 antibody fragment.
• Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells.
• Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab') 2 fragments (Carter et al., Bio/Technology 10:163-167 (1992)).
• the F(ab') 2 is formed using the leucine zipper GCN4 to promote assembly of the F(ab') 2 molecule.
• Fv, Fab or F(ab') 2 fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner.
• an “isolated” antibody is one that has been identified and separated and recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
• the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain.
• Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
• V, D, J or only V and J in the case of light chain genes
• V, D, J or only V and J in the case of light chain genes
• This gene segment rearrangement process appears to be sequential.
• heavy chain D-to-J joints are made, followed by heavy chain V-to-DJ joints and light chain V-to-J joints.
• the recombination of variable region gene segments to form functional heavy and light chain variable regions is mediated by recombination signal sequences (RSS's) that flank recombinationally competent V, D and J segments.
• RSS's recombination signal sequences
• RSS's necessary and sufficient to direct recombination comprise a dyad-symmetric heptamer, an AT-rich nonamer and an intervening spacer region of either 12 or 23 base pairs. These signals are conserved among the different loci and species that carry out D-J (or V-J) recombination and are functionally interchangeable. See Oettinger, et al. (1990), Science, 248, 1517-1523 and references cited therein.
• the heptamer comprises the sequence CACAGTG or its analogue followed by a spacer of unconserved sequence and then a nonamer having the sequence ACAAAAACC or its analogue. These sequences are found on the J, or downstream side, of each V and D gene segment.
• heptameric and nonameric sequences following a V L , V H or D segment are complementary to those preceding the JL, D or JH segments with which they recombine.
• the spacers between the heptameric and nonameric sequences are either 12 base pairs long or between 22 and 24 base pairs long.
• variable region gene segments and variable recombination which may occur during such joining is the production of a primary antibody repertoire.
• Fv is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy- and one light- chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH VI dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
• the Fab fragment also contains the constant domain of the light chain and the first constant domain (CHl) of the heavy chain.
• Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHl domain including one or more cysteines from the antibody hinge region.
• Fab '-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group.
• F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them.
• neutralizing antibody an antibody molecule that is able to eliminate or significantly reduce an effecter function of a target antigen to which is binds. Accordingly, a “neutralizing" anti-target antibody is capable of eliminating or significantly reducing an effecter function, such as enzyme activity, ligand binding, or intracellular signaling.
• compositions for and methods of treating macrophage- associated diseases may utilize one or more antibody used singularly or in combination with other therapeutics to achieve the desired effects.
• Antibodies according to the present invention may be isolated from an animal producing the antibody as a result of either direct contact with an environmental antigen or immunization with the antigen.
• antibodies may be produced by recombinant DNA methodology using one of the antibody expression systems well known in the art (See, e.g., Harlow and Lane, Antibodies: A
• M-CSF monoclonal antibodies may be prepared essentially as described in Halenbeck et al. U.S. Pat. No. 5,491,065 (1997), incorporated herein by reference.
• Exemplary M-CSF monoclonal antibodies include those that bind to an apparent conformational epitope associated with recombinant or native dimeric M-CSF with concomitant neutralization of biological activity. These antibodies are substantially unreactive with biologically inactive forms of M-CSF including monomeric and chemically derivatized dimeric M-CSF.
• Human EngineeredTM anti-M- CSF monoclonal antibodies are provided.
• the phrase "Human EngineeredTM antibody” refers to an antibody derived from a non-human antibody, typically a mouse monoclonal antibody.
• a Human EngineeredTM antibody may be derived from a chimeric antibody that retains or substantially retains the antigen binding properties of the parental, non-human, antibody but which exhibits diminished immunogenicity as compared to the parental antibody when administered to humans.
• chimeric antibody refers to an antibody containing sequence derived from two different antibodies (see, e.g., U.S. Patent No. 4,816,567) which typically originate from different species. Most typically, chimeric antibodies comprise human and murine antibody fragments, generally human constant and mouse variable regions.
• CDR complementarity determining region
• constant region refers to the portion of the antibody molecule that confers effector functions.
• mouse constant regions are preferably substituted by human constant regions.
• the constant regions of the subject antibodies are derived from human immunoglobulins.
• the heavy chain constant region can be selected from any of the five isotypes: alpha, delta, epsilon, gamma or mu.
• the antibodies of the present invention are said to be immunospecific or specifically binding if they bind to antigen with a K a of greater than or equal to about 10 6 M "1 preferably greater than or equal to about 10 7 M "1 , more preferably greater than or equal to about 10 8 M "1 , and most preferably greater than or equal to about 10 9 M "1 , 10 10 M "1 , 10 11 M '1 or
• the anti-M-CSF antibodies may bind to different naturally occurring forms of M- CSF.
• the monoclonal antibodies disclosed herein, such as RXl, 5H4, MCl, or MC3 antibody, have affinity for M-CSF and are characterized by a dissociation equilibrium constant (Kd) of at least 10 "4 M, preferably at least about lO "7 M to about 10 "8 M, more preferably at least about 10- 9 M, 10- 10 M, 10- 11 M or 10- 12 M.
• Kd dissociation equilibrium constant
• affinities may be readily determined using conventional techniques, such as by equilibrium dialysis; by using the BIAcore 2000 instrument, using general procedures outlined by the manufacturer; by radioimmunoassay using I labeled M-CSF; or by another method known to the skilled artisan.
• the affinity data may be analyzed, for example, by the method of Scatchard et al., Ann N.Y. Acad. ScL, 51:660 (1949).
• Preferred antibodies bind M-CSF with a similar affinity as murine RXl of Figure 3 binds to M-CSF, exhibit low immunogenicity, and inhibit macrophage-associated diseases when tested in animal models.
• Other exemplary antibodies bind M-CSF with a similar affinity as murine 5H4, MCl or MC3 of Figure 8, 9 or 10, respectively, binds to M-CSF.
• the antigen to be used for production of antibodies may be, e.g., intact M-CSF or a fragment of M-CSF that retains the desired epitope, optionally fused to another polypeptide that allows the epitope to be displayed in its native conformation.
• cells expressing M-CSF at their cell surface can be used to generate antibodies. Such cells can be transformed to express M-CSF or may be other naturally occurring cells that express M-CSF. Other forms of M-CSF useful for generating antibodies will be apparent to those skilled in the art.
• Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant.
• An improved antibody response may be obtained by conjugating the relevant antigen to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride or other agents known in the art.
• a protein that is immunogenic in the species to be immunized e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a
• Animals are immunized against the antigen, immunogenic conjugates, or derivatives by combining, e.g., 100 ⁇ g or 5 ⁇ g of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites.
• the animals are boosted with 1/5 to ⁇ fraction (1/10) ⁇ the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites.
• the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus.
• the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking reagent.
• Conjugates also can be made in recombinant cell culture as protein fusions.
• aggregating agents such as alum are suitably used to enhance the immune response.
• Monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods.
• a mouse or other appropriate host animal such as a hamster or macaque monkey
• lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization.
• lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp.59- 103 (Academic Press, 1986)).
• the hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
• a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
• the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
• Preferred myeloma cells are those that fuse efficiently, support stable high- level production of antibody by the selected antibody-producing cells, and are sensitive to a medium.
• Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984) ;Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).
• Exemplary murine myeloma lines include those derived from MOP-21 and M.C.-l 1 mouse tumors available from the SaIk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Md. USA.
• Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen.
• the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
• RIA radioimmunoassay
• ELISA enzyme-linked immunoabsorbent assay
• the binding affinity of the monoclonal antibody can, for example, be determined by Scatchard analysis (Munson et al., Anal. Biochem., 107:220 (1980)).
• the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI- 1640 medium.
• the hybridoma cells may be grown in vivo as ascites tumors in an animal.
• the monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A- Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
• immunoglobulin purification procedures such as, for example, protein A- Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
• DNA encoding the monoclonal antibodies may be isolated and sequenced from the hybridoma cells using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibodies). Sequence determination will generally require isolation of at least a portion of the gene or cDNA of interest. Usually this requires cloning the DNA or, preferably, rnRNA (i.e., cDNA) encoding the monoclonal antibodies. Cloning is carried out using standard techniques (see, e.g., Sambrook et al. (1989) Molecular Cloning: A
• a cDNA library may be constructed by reverse transcription of polyA+ rnRNA, preferably membrane-associated rnRNA, and the library screened using probes specific for human immunoglobulin polypeptide gene sequences.
• the polymerase chain reaction PCR
• PCR polymerase chain reaction
• the amplified sequences can be readily cloned into any suitable vector, e.g., expression vectors, minigene vectors, or phage display vectors.
• an "isolated" nucleic acid molecule or “isolated” nucleic acid sequence is a nucleic acid molecule that is either (1) identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the nucleic acid or (2) cloned, amplified, tagged, or otherwise distinguished from background nucleic acids such that the sequence of the nucleic acid of interest can be determined, is considered isolated.
• An isolated nucleic acid molecule is other than in the form or setting in which it is found in nature.
• Isolated nucleic acid molecules therefore are distinguished from the nucleic acid molecule as it exists in natural cells.
• an isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that ordinarily express the antibody where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.
• RNA used for cloning and sequencing is a hybridoma produced by obtaining a B cell from the transgenic mouse and fusing the B cell to an immortal cell.
• An advantage of using hybridomas is that they can be easily screened, and a hybridoma that produces a human monoclonal antibody of interest selected.
• RNA can be isolated from B cells (or whole spleen) of the immunized animal.
• sources other than hybridomas it may be desirable to screen for sequences encoding immunoglobulins or immunoglobulin polypeptides with specific binding characteristics.
• One method for such screening is the use of phage display technology.
• Phage display is described in e.g., Dower et al, WO 91/17271, McCafferty et al., WO 92/01047, and Caton and Koprowski, Proc. Natl. Acad. Sci. USA, 87:6450-6454 (1990), each of which is incorporated herein by reference.
• cDNA from an immunized transgenic mouse e.g., total spleen cDNA
• the polymerase chain reaction is used to amplify a cDNA sequences that encode a portion of an immunoglobulin polypeptide, e.g., CDR regions, and the amplified sequences are inserted into a phage vector.
• cDNAs encoding peptides of interest e.g., variable region peptides with desired binding characteristics, are identified by standard techniques such as panning. The sequence of the amplified or cloned nucleic acid is then determined.
• sequence encoding an entire variable region of the immunoglobulin polypeptide is determined, however, it will sometimes by adequate to sequence only a portion of a variable region, for example, the CDR-encoding portion.
• portion sequenced will be at least 30 bases in length, more often based coding for at least about one-third or at least about one-half of the length of the variable region will be sequenced.
• Sequencing can be carried out on clones isolated from a cDNA library, or, when PCR is used, after subcloning the amplified sequence or by direct PCR sequencing of the amplified segment. Sequencing is carried out using standard techniques (see, e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Guide, VoIs 1-3, Cold Spring Harbor Press, and Sanger, F. et al. (1977) Proc. Natl. Acad. Sci. USA 74: 5463-5467, which is incorporated herein by reference).
• the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, human embryonic kidney 293 cells (e.g., 293 ⁇ cells), Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Recombinant production of antibodies is well known in the art.
• Expression control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
• the control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site.
• Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
• Nucleic acid is operably linked when it is placed into a functional relationship with another nucleic acid sequence.
• DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
• a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or
• a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
• operably linked means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
• Cell, cell line, and cell culture are often used interchangeably and all such designations herein include progeny.
• Transformants and transformed cells include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.
• amino acid sequence of an immunoglobulin of interest may be determined by direct protein sequencing. Suitable encoding nucleotide sequences can be designed according to a universal codon table.
• Amino acid sequence variants of the desired antibody may be prepared by introducing appropriate nucleotide changes into the encoding DNA, or by peptide synthesis. Such variants include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the antibodies. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics.
• the amino acid changes also may alter post- translational processes of the monoclonal, human, humanized, Human EngineeredTM or variant antibody, such as changing the number or position of glycosylation sites.
• Nucleic acid molecules encoding amino acid sequence variants of the antibody are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non- variant version of the antibody.
• the invention also provides isolated nucleic acid encoding antibodies of the invention, optionally operably linked to control sequences recognized by a host cell, vectors and host cells comprising the nucleic acids, and recombinant techniques for the production of the antibodies, which may comprise culturing the host cell so that the nucleic acid is expressed and, optionally, recovering the antibody from the host cell culture or culture medium.
• recombinant production of the antibody the nucleic acid encoding it is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression.
• DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures ⁇ e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).
• Many vectors are available.
• the vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more selective marker genes, an enhancer element, a promoter, and a transcription termination sequence.
• the antibody of this invention may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which is preferably a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide.
• a heterologous polypeptide which is preferably a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide.
• the signal sequence selected preferably is one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell.
• the signal sequence may be substituted by a signal sequence selected, for example, from the group of the pectate lyase (e.g., pelB) alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders.
• the native signal sequence may be substituted by, e.g., the yeast invertase leader, ⁇ factor leader (including Saccharomyces and Kluyveromyces ⁇ -factor leaders), or acid phosphatase leader, the C. albicans glucoamylase leader, or the signal described in WO90/13646.
• mammalian signal sequences as well as viral secretory leaders for example, the herpes simplex gD signal, are available.
• the DNA for such precursor region is ligated in reading frame to DNA encoding the antibody.
• Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells.
• this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of replication or autonomously replicating sequences.
• origins of replication or autonomously replicating sequences are well known for a variety of bacteria, yeast, and viruses.
• the origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2 ⁇ plasmid origin is suitable for yeast, and various viral origins are useful for cloning vectors in mammalian cells.
• the origin of replication component is not needed for mammalian expression vectors (the SV40 origin may typically be used only because it contains the early promoter).
• Selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, tetracycline, G418, geneticin, histidinol, or mycophenolic acid (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.
• One example of a selection scheme utilizes a drug to arrest growth of a host cell. Those cells that are successfully transformed with a heterologous gene produce a protein conferring drug resistance and thus survive the selection regimen. Examples of such dominant selection use the drugs methotrexate, neomycin, histidinol, puromycin, mycophenolic acid and hygromycin.
• Suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the antibody-encoding nucleic acid, such as DHFR, thymidine kinase, metallothionein-I and -II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc.
• cells transformed with the DHFR selection gene are first identified by culturing all of the transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR.
• Mtx methotrexate
• An appropriate host cell when wild-type DHFR is employed is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity.
• host cells particularly wild-type hosts that contain endogenous DHFR transformed or co-transformed with DNA sequences encoding antibody of the invention, wild-type DHFR protein, and another selectable marker such as aminoglycoside 3' ⁇ phosphotransferase (APH) can be selected by cell growth in medium containing a selection agent for the selectable marker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See U.S. Patent No. 4,965,199.
• APH aminoglycoside 3' ⁇ phosphotransferase
• a suitable selection gene for use in yeast is the trpl gene present in the yeast plasmid YRp7 (Stinchcomb et al., Nature, 282: 39 (1979)).
• the trpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1. Jones, Genetics, 85: 12 (1977).
• the presence of the trpl lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
• Leu2-deficient yeast strains (ATCC 20,622 or 38,626) are complemented by known plasmids bearing the Leu2 gene.
• Urc ⁇ -deficient yeast strains are complemented by plasmids bearing the ura3 gene.
• vectors derived from the 1.6 ⁇ m circular plasmid pKDl can be used for transformation of Kluyveromyces yeasts.
• an expression system for large-scale production of recombinant calf chymosin was reported for K. lactis. Van den Berg, Bio/Technology, 8: 135 (1990).
• Stable multi-copy expression vectors for secretion of mature recombinant human serum albumin by industrial strains of Kluyveromyces have also been disclosed. Fleer et al, Bio/Technology, 9: 968-975 (1991).
• Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the antibody-encoding nucleic acid.
• Promoters suitable for use with prokaryotic hosts include the arabinose (e.g., araB) promoter phoA promoter , ⁇ -lactamase and lactose promoter systems, alkaline phosphatase, a tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter.
• arabinose e.g., araB
• trp tryptophan
• Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S .D.) sequence operably linked to the DNA encoding the antibody of the invention.
• Promoter sequences are known for eukaryotes. Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CNCAAT region where N may be any nucleotide. At the 3' end of most eukaryotic genes is an AATAAA sequence that may be the signal for addition of the poly A tail to the 3' end of the coding sequence. All of these sequences are suitably inserted into eukaryotic expression vectors.
• suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3 -phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
• 3-phosphoglycerate kinase or other glycolytic enzymes such as enolase, glyceraldehyde-3 -phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruv
• yeast promoters which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3 -phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization.
• Suitable vectors and promoters for use in yeast expression are further described in EP 73,657.
• Yeast enhancers also are advantageously used with yeast promoters.
• Antibody transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as Abelson leukemia virus, polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, most preferably cytomegalovirus, a retrovirus, hepatitis-B virus, Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, from heat-shock promoters, provided such promoters are compatible with the host cell systems.
• viruses such as Abelson leukemia virus, polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, most preferably cytomegalovirus, a retrovirus, he
• the early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication.
• the immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment.
• a system for expressing DNA in mammalian hosts using the bovine papilloma virus as a vector is disclosed in U.S. Patent No. 4,419,446. A modification of this system is described in U.S. Patent No. 4,601,978. See also Reyes et al., Nature 297: 598-601 (1982) on expression of human ⁇ -interferon cDNA in mouse cells under the control of a thymidine kinase promoter from herpes simplex virus. Alternatively, the rous sarcoma virus long terminal repeat can be used as the promoter.
• Enhancer sequences are now known from mammalian genes (globin, elastase, albumin, alpha- fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the S V40 enhancer on the late side of the replication origin (bp 100- 270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
• the enhancer may be spliced into the vector at a position 5' or 3' to the antibody-encoding sequence, but is preferably located at a site 5' from the promoter.
• Expression vectors used in eukaryotic host cells will also contain sequences necessary for the termination of transcription and for stabilizing the rnRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the rnRNA encoding antibody.
• One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO94/11026 and the expression vector disclosed therein. Another is the mouse immunoglobulin light chain transcription terminator.
• Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described above.
• Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (e.g., B.
• E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable. These examples are illustrative rather than limiting.
• eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors.
• Saccharomyces cerevisiae or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms.
• Kluyveromyces hosts such as, e.g., K. lactis, Kfragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K.
• drosophilamm ATCC 36,906), K. thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastors (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.
• Suitable host cells for the expression of glycosylated antibody are derived from multicellular organisms.
• invertebrate cells include plant and insect cells.
• Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori have been identified.
• a variety of viral strains for transfection are publicly available, e.g., the L-I variant of Autographa calif ornica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells.
• Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, tobacco, lemna, and other plant cells can also be utilized as hosts.
• hamster ovary cells including CHOKl cells (ATCC CCL61), DXB-Il, DG-44, and Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al, Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); monkey kidney CVl line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, [Graham et al., J. Gen Virol.
• Host cells are transformed or transfected with the above-described expression or cloning vectors for antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences, hi addition, novel vectors and transfected cell lines with multiple copies of transcription units separated by a selective marker are particularly useful and preferred for the expression of antibodies that target M-CSF.
• the host cells used to produce the antibody of this invention may be cultured in a variety of media.
• Commercially available media such as Ham's FlO (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI- 1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells.
• any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GentamycinTM drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.
• the culture conditions such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
• the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium, including from microbial cultures. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Better et al. Science 240: 1041-1043 (1988); ICSU Short Reports 10: 105 (1990); and Proc. Natl. Acad. Sci. USA 90: 457-461 (1993) describe a procedure for isolating antibodies which are secreted to the periplasmic space of E. coli. ⁇ See also, [Carter et al., Bio/Technology 10: 163-167 (1992)].
• the antibody composition prepared from microbial or mammalian cells can be purified using, for example, hydroxylapatite chromatography cation or avian exchange chromatography, and affinity chromatography, with affinity chromatography being the preferred purification technique.
• the suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody.
• Protein A can be used to purify antibodies that are based on human ⁇ l, ⁇ 2, or ⁇ 4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983)). Protein G is recommended for all mouse isotypes and for human ⁇ 3 (Guss et al., ⁇ MBO J.
• the matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose.
• the antibody comprises a C H 3 domain
• the Bakerbond ABXTMresin J. T. Baker, Phillipsburg, NJ. is useful for purification.
• chimeric or humanized antibodies are less immunogenic in humans than the parental mouse monoclonal antibodies, they can be used for the treatment of humans with far less risk of anaphylaxis. Thus, these antibodies may be preferred in therapeutic applications that involve in vivo administration to a human.
• Chimeric monoclonal antibodies in which the variable Ig domains of a mouse monoclonal antibody are fused to human constant Ig domains, can be generated using standard procedures known in the art (See Morrison, S. L., et al. (1984) Chimeric Human Antibody Molecules; Mouse Antigen Binding Domains with Human Constant Region Domains, Proc. Natl. Acad. Sci. USA 81, 6841-6855; and, Boulianne, G. L., et al, Nature 312, 643-646 . (1984)). Although some chimeric monoclonal antibodies have proved less immunogenic in humans, the mouse variable Ig domains can still lead to a significant human anti-mouse response.
• Humanized antibodies may be achieved by a variety of methods including, for example: (1) grafting the non-human complementarity determining regions (CDRs) onto a human framework and constant region (a process referred to in the art as humanizing through “CDR grafting"), or, alternatively, (2) transplanting the entire non-human variable domains, but “cloaking" them with a human-like surface by replacement of surface residues (a process referred to in the art as “veneering”).
• CDRs complementarity determining regions
• a rodent antibody on repeated in vivo administration in man either alone or as a conjugate will bring about an immune response in the recipient against the rodent antibody; the so-called HAMA response (Human Anti Mouse Antibody).
• the HAMA response may limit the effectiveness of the pharmaceutical if repeated dosing is required.
• the immunogenicity of the antibody may be reduced by chemical modification of the antibody with a hydrophilic polymer such as polyethylene glycol or by using the methods of genetic engineering to make the antibody binding structure more human like.
• the gene sequences for the variable domains of the rodent antibody which bind CEA can be substituted for the variable domains of a human myeloma protein, thus producing a recombinant chimaeric antibody.
• CDR grafting involves introducing one or more of the six CDRs from the mouse heavy and light chain variable Ig domains into the appropriate four framework regions of human variable Ig domains is also called CDR grafting.
• This technique (Riechmann, L., et al., Nature 332, 323 (1988)), utilizes the conserved framework regions (FR1-FR4) as a scaffold to support the CDR loops which are the primary contacts with antigen.
• FR1-FR4 conserved framework regions
• a disadvantage of CDR grafting is that it can result in a humanized antibody that has a substantially lower binding affinity than the original mouse antibody, because amino acids of the framework regions can contribute to antigen binding, and because amino acids of the CDR loops can influence the association of the two variable Ig domains.
• the CDR grafting technique can be improved by choosing human framework regions that most closely resemble the framework regions of the original mouse antibody, and by site-directed mutagenesis of single amino acids within the framework or CDRs aided by computer modeling of the antigen binding site (e.g., Co, M. S., et al. (1994), J. Immunol. 152, 2968-2976).
• One method of humanizing antibodies comprises aligning the non-human heavy and light chain sequences to human heavy and light chain sequences, selecting and replacing the non-human framework with a human framework based on such alignment, molecular modeling to predict the conformation of the humanized sequence and comparing to the conformation of the parent antibody. This process is followed by repeated back mutation of residues in the CDR region which disturb the structure of the CDRs until the predicted conformation of the humanized sequence model closely approximates the conformation of the non-human CDRs of the parent non-human antibody.
• Such humanized antibodies may be further derivatized to facilitate uptake and clearance, e.g., via Ashwell receptors (See, e.g., U.S. Patent Nos.
• Amino acid sequence variants A useful method for identification of certain residues or regions of the antibody that are preferred locations for mutagenesis is called “alanine scanning mutagenesis," as described by Cunningham and Wells Science, 244:1081-1085 (1989).
• a residue or group of target residues are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acids with antigen.
• Those amino acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at, or for, the sites of substitution.
• the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the performance of a mutation at a given site, ala scanning or random mutagenesis is conducted at the target codon or region and the expressed antibody variants are screened for the desired activity.
• Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intra-sequence insertions of single or multiple amino acid residues.
• terminal insertions include an antibody with an N-terminal methionyl residue or the antibody (including antibody fragment) fused to an epitope tag or a salvage receptor epitope.
• Other insertional variants of the antibody molecule include the fusion to a polypeptide which increases the serum half-life of the antibody, e.g. at the N-terminus or C- terminus.
• epitope tag tagged refers to the antibody fused to an epitope tag.
• the epitope tag polypeptide has enough residues to provide an epitope against which an antibody there against can be made, yet is short enough such that it does not interfere with activity of the antibody.
• the epitope tag preferably is sufficiently unique so that the antibody there against does not substantially cross-react with other epitopes.
• Suitable tag polypeptides generally have at least 6 amino acid residues and usually between about 8-50 amino acid residues (preferably between about 9-30 residues). Examples include the flu HA tag polypeptide and its antibody 12CA5 [Field et al., MoI. Cell. Biol.
• tags are a poly-histidine sequence, generally around six histidine residues, that permits isolation of a compound so labeled using nickel chelation.
• tags such as the FLAG® tag (Eastman Kodak, Rochester, NY), well known and routinely used in the art, are embraced by the invention.
• the term "salvage receptor binding epitope” refers to an epitope of the Fc region of an IgG molecule (e.g., IgG 1 , IgG 2 , IgG 3 , or IgG 4 ) that is responsible for increasing the in vivo serum half -life of the IgG molecule.
• variants are an amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule removed and a different residue inserted in its place. Substitutional mutagenesis within any of the hypervariable or CDR regions or framework regions is contemplated. Conservative substitutions are shown in Table 1. The most conservative substitution is found under the heading of "preferred substitutions”. If such substitutions result in no change in biological activity, then more substantial changes, denominated "exemplary substitutions" in Table 1, or as further described below in reference to amino acid classes, may be introduced and the products screened.
• substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
• Naturally occurring residues are divided into groups based on common side-chain properties:
• hydrophobic norleucine, met, ala, val, leu, ile
• cysteine residues not involved in maintaining the proper conformation of the monoclonal, human, humanized, Human Engineered or variant antibody also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking.
• cysteine bond(s) may be added to the antibody to improve its stability (particularly where the antibody is an antibody fragment such as an Fv fragment).
• Affinity maturation involves preparing and screening antibody variants that have substitutions within the CDRs of a parent antibody and selecting variants that have improved biological properties such as binding affinity relative to the parent antibody.
• a convenient way for generating such substitutional variants is affinity maturation using phage display. Briefly, several hypervariable region sites (e.g. 6-7 sites) are mutated to generate all possible amino substitutions at each site.
• the antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g. binding affinity).
• Alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively, or in addition, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein. Once such variants are generated, the panel of variants is subjected to screening as described herein and antibodies with superior properties in one or more relevant assays may be selected for further development.
• Antibody variants can also be produced that have a modified glycosylation pattern relative to the parent antibody, for example, deleting one or more carbohydrate moieties found in the antibody, and/or adding one or more glycosylation sites that are not present in the antibody.
• Glycosylation of antibodies is typically either N-lmked or O-linked.
• N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue.
• the tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain.
• N-linked glycosylation sites may be added to an antibody by altering the amino acid sequence such that it contains one or more of these tripeptide sequences.
• O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5- hydroxylysine may also be used.
• O-linked glycosylation sites may be added to an antibody by inserting or substituting one or more serine or threonine residues to the sequence of the original antibody.
• the amino acids of RXl at positions 41-43 of Figure 3A may be retained.
• only amino acids 41 and 42 (NG) may be retained.
• amino acid sequence variants of the Human EngineeredTM antibody will have an amino acid sequence having at least 60% amino acid sequence identity with the original Human EngineeredTM antibody amino acid sequences of either the heavy or the light chain (e.g., as in any of Figures 13B through 16B) more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95%, including for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.
• sequence identity is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the Human EngineeredTM residues, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions (as defined in Table 1 above) as part of the sequence identity. None of N-terminal, C-terminal, or internal extensions, deletions, or insertions into the antibody sequence shall be construed as affecting sequence identity or homology. Thus, sequence identity can be determined by standard methods that are commonly used to compare the similarity in position of the amino acids of two polypeptides.
• two polypeptides are aligned for optimal matching of their respective amino acids (either along the full length of one or both sequences, or along a pre-determined portion of one or both sequences).
• the programs provide a default opening penalty and a default gap penalty, and a scoring matrix such as PAM 250 [a standard scoring matrix; see Dayhoff et al., in Atlas of Protein Sequence and Structure, vol. 5, supp. 3 (1978)] can be used in conjunction with the computer program.
• PAM 250 a standard scoring matrix; see Dayhoff et al., in Atlas of Protein Sequence and Structure, vol. 5, supp. 3 (1978)] can be used in conjunction with the computer program.
• the percent identity can then be calculated as: the total number of identical matches multiplied by 100 and then divided by the sum of the length of the longer sequence within the matched span and the number of gaps introduced into the longer sequences in order to align the two sequences.
• the antibody of the invention may be desirable to modify the antibody of the invention with respect to effector function, so as to enhance the effectiveness of the antibody in treating macrophage-associated diseases, for example.
• cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region.
• the homodimeric antibody thus generated may have improved internalization capability and/or increased complement- mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med. 176: 1191-1195 (1992) and Shopes, B. J. Immunol. 148: 2918-2922 (1992).
• Homodimeric antibodies with enhanced activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al., Cancer Research 53: 2560-2565 (1993).
• an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti- Cancer Drug Design 3: 219-230 (1989).
• sequences within the CDR can cause an antibody to bind to MHC Class II and trigger an unwanted helper T- cell response.
• a conservative substitution can allow the antibody to retain binding activity yet lose its ability to trigger an unwanted T-cell response.
• Steplewski et al. Proc Natl Acad Sci U S A. 1988;85(13):4852-6, incorporated herein by reference in its entirety, which described chimeric antibodies wherein a murine variable region was joined with human gamma 1, gamma 2, gamma 3, and gamma 4 constant regions.
• an antibody fragment rather than an intact antibody, to increase tissue penetration, for example.
• This may also be achieved, for example, by incorporation of a salvage receptor binding epitope into the antibody fragment (e.g., by mutation of the appropriate region in the antibody fragment or by incorporating the epitope into a peptide tag that is then fused to the antibody fragment at either end or in the middle, e.g., by DNA or peptide synthesis) (see, e.g., WO96/32478).
• a salvage receptor binding epitope into the antibody fragment (e.g., by mutation of the appropriate region in the antibody fragment or by incorporating the epitope into a peptide tag that is then fused to the antibody fragment at either end or in the middle, e.g., by DNA or peptide synthesis) (see, e.g., WO96/32478).
• the salvage receptor binding epitope preferably constitutes a region wherein any one or more amino acid residues from one or two loops of a Fc domain are transferred to an analogous position of the antibody fragment. Even more preferably, three or more residues from one or two loops of the Fc domain are transferred. Still more preferred, the epitope is taken from the CH2 domain of the Fc region (e.g., of an IgG) and transferred to the CHl, CH3, or VH region, or more than one such region, of the antibody. Alternatively, the epitope is taken from the CH2 domain of the Fc region and transferred to the C.sub.L region or V.sub.L region, or both, of the antibody fragment. See also International applications WO 97/34631 and WO 96/32478 which describe Fc variants and their interaction with the salvage receptor.
• antibodies of the invention may comprise a human Fc portion, a human consensus Fc portion, or a variant thereof that retains the ability to interact with the Fc salvage receptor, including variants in which cysteines involved in disulfide bonding are modified or removed, and/or in which the a met is added at the N-terminus and/or one or more of the N-terminal 20 amino acids are removed, and/or regions that interact with complement, such as the CIq binding site, are removed, and/or the ADCC site is removed [see, e.g., Molec. Immunol. 29 (5): 633-9 (1992)].
• Mutation of residues within Fc receptor binding sites can result in altered effector function, such as altered ADCC or CDC activity, or altered half-life.
• potential mutations include insertion, deletion or substitution of one or more residues, including substitution with alanine, a conservative substitution, a non-conservative substitution, or replacement with a corresponding amino acid residue at the same position from a different IgG subclass (e.g. replacing an IgGl residue with a corresponding IgG2 residue at that position). Shields et al.
• IgGl residues involved in binding to all human Fc receptors are located in the CH2 domain proximal to the hinge and fall into two categories as follows: 1) positions that may interact directly with all FcR include Leu234-Pro238, Ala327, and Pro329 (and possibly Asp265); 2) positions that influence carbohydrate nature or position include Asp265 and Asn297.
• IgGl residues that affected binding to Fc receptor II are as follows: (largest effect) Arg255, Thr256, Glu258, Ser267, As ⁇ 270, Glu272, Asp280, Arg292, Ser298, and (less effect) His268, Asn276, His285, Asn286, Lys290, Gln295, Arg301, Thr307, Leu309, Asn315, Lys322, Lys326, Pro331, Ser337, Ala339, Ala378, and Lys414. A327Q, A327S, P329A, D265A and D270A reduced binding.
• IgGl residues that reduced binding to Fc receptor IIIA by 40% or more are as follows: Ser239, Ser267 (GIy only), His268, Glu293, Gln295, Tyr296, Arg301, Val303, Lys338, and Asp376.
• Variants that improved binding to FcRIIIA include T256A, K290A, S298A, E333A, K334A, and A339T.
• Lys414 showed a 40% reduction in binding for FcRIIA and FcRIIB, Arg416 a 30% reduction for FcRIIA and FcRIILA, Gln419 a 30% reduction to FcRIIA and a 40% reduction to FcRIIB, and Lys360 a 23% improvement to FcRIILA. See also Presta et al., Biochem. Soc. Trans. (2001) 30, 487-490.
• United States Patent No. 6,194,551 incorporated herein by reference in its entirety, describes variants with altered effector function containing mutations in the human IgG Fc region, at amino acid position 329, 331 or 322 (using Kabat numbering), some of which display reduced CIq binding or CDC activity.
• United States Patent No. 6,194,551 incorporated herein by reference in its entirety, describes variants with altered effector function containing mutations in the human IgG Fc region, at amino acid position 329, 331 or 322 (using Kabat numbering), some of which display reduced CIq binding or CDC activity.
• United States Patent No. 6,194,551 incorporated herein by reference in its entirety, describes variants with altered effector function containing mutations in the human IgG Fc region, at amino acid position 329, 331 or 322 (using Kabat numbering), some of which display reduced CIq binding or CDC activity.
• a mutation at amino acid position 238, 265, 269, 270, 327 or 329 are stated to reduce binding to FcRI
• a mutation at amino acid position 238, 265, 269, 270, 292, 294, 295, 298, 303, 324, 327, 329, 333, 335, 338, 373, 376, 414, 416, 419, 435, 438 or 439 are stated to reduce binding to FcRII
• a mutation at amino acid position 238, 239, 248, 249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 293, 294, 295, 296, 301, 303, 322, 327, 329, 338, 340, 373, 376, 382, 388, 389, 416, 434, 435 or 437 is stated to reduce binding to FcRIII.
• Amino acid substitutions are distinguished by one of three risk categories : (1) low risk changes are those that have the greatest potential for reducing immunogenicity with the least chance of disrupting antigen binding; (2) moderate risk changes are those that would further reduce immunogenicity, but have a greater chance of affecting antigen binding or protein folding; (3) high risk residues are those that are important for binding or for maintaining antibody structure and carry the highest risk that antigen binding or protein folding will be affected. Due to the three-dimensional structural role of prolines, modifications at prolines are generally considered to be at least moderate risk changes, even if the position is typically a low risk position. Variable regions of the light and heavy chains of a rodent antibody are Human
• EngineeredTM as follows to substitute human amino acids at positions determined to be unlikely to adversely effect either antigen binding or protein folding, but likely to reduce irnmunogenicity in a human environment.
• Amino acid residues that are at "low risk" positions and that are candidates for modification according to the method are identified by aligning the amino acid sequences of the rodent variable regions with a human variable region sequence. Any human variable region can be used, including an individual VH or VL sequence or a human consensus VH or VL sequence or an individual or consensus human germline sequence.
• the amino acid residues at any number of the low risk positions, or at all of the low risk positions, can be changed.
• an amino acid modification is introduced that replaces the rodent residue with the human residue.
• the amino acid residues at all of the low risk positions and at any number of the moderate risk positions can be changed.
• all of the low and moderate risk positions are changed from rodent to human sequence.
• Synthetic genes containing modified heavy and/or light chain variable regions are constructed and linked to human ⁇ heavy chain and/or kappa light chain constant regions.
• Any human heavy chain and light chain constant regions may be used in combination with the Human EngineeredTM antibody variable regions, including IgA (of any subclass, such as IgAl or IgA2), IgD, IgE, IgG (of any subclass, such as IgGl, IgG2, IgG3, or IgG4), or IgM.
• the human heavy and light chain genes are introduced into host cells, such as mammalian cells, and the resultant recombinant immunoglobulin products are obtained and characterized. Human antibodies from transgenic animals
• Human antibodies to M-CSF can also be produced using transgenic animals that have no endogenous immunoglobulin production and are engineered to contain human immunoglobulin loci.
• WO 98/24893 discloses transgenic animals having a human Ig locus wherein the animals do not produce functional endogenous immunoglobulins due to the inactivation of endogenous heavy and light chain loci.
• WO 91/741 also discloses transgenic non-primate mammalian hosts capable of mounting an immune response to an immunogen, wherein the antibodies have primate constant and/or variable regions, and wherein the endogenous immunoglobulin encoding loci are substituted or inactivated.
• WO 96/30498 discloses the use of the Cre/Lox system to modify the immunoglobulin locus in a mammal, such as to replace all or a portion of the constant or variable region to form a modified antibody molecule.
• WO 94/02602 discloses non-human mammalian hosts having inactivated endogenous Ig loci and functional human Ig loci.
• U.S. Patent No. 5,939,598 discloses methods of making transgenic mice in which the mice lack endogenous heavy chains, and express an exogenous immunoglobulin locus comprising one or more xenogeneic constant regions.
• an immune response can be produced to a selected antigenic molecule, and antibody producing cells can be removed from the animal and used to produce hybridomas that secrete human monoclonal antibodies.
• Immunization protocols, adjuvants, and the like are known in the art, and are used in immunization of, for example, a transgenic mouse as described in WO 96/33735.
• This publication discloses monoclonal antibodies against a variety of antigenic molecules including EL 6, IL 8, TNFa, human CD4, L selectin, gp39, and tetanus toxin.
• the monoclonal antibodies can be tested for the ability to inhibit or neutralize the biological activity or physiological effect of the corresponding protein.
• WO 96/33735 discloses that monoclonal antibodies against IL- 8, derived from immune cells of transgenic mice immunized with EL-8, blocked IL-8 induced functions of neutrophils. Human monoclonal antibodies with specificity for the antigen used to immunize transgenic animals are also disclosed in WO 96/34096 and U.S. patent application no. 20030194404; and U.S. patent application no. 20030031667).
• 20030092125 describes methods for biasing the immune response of an animal to the desired epitope.
• Human antibodies may also be generated by in vitro activated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).
• Human antibodies from phage display technology The development of technologies for making repertoires of recombinant human antibody genes, and the display of the encoded antibody fragments on the surface of filamentous bacteriophage, has provided a means for making human antibodies directly.
• the antibodies produced by phage technology are produced as antigen binding fragments-usually Fv or Fab fragments-in bacteria and thus lack effector functions. Effector functions can be introduced by one of two strategies: The fragments can be engineered either into complete antibodies for expression in mammalian cells, or into bispecific antibody fragments with a second binding site capable of triggering an effector function.
• the Fd fragment (VH-CHI) and light chain (V L -C L ) of antibodies are separately cloned by PCR and recombined randomly in combinatorial phage display libraries, which can then be selected for binding to a particular antigen.
• the Fab fragments are expressed on the phage surface, i.e., physically linked to the genes that encode them.
• selection of Fab by antigen binding co-selects for the Fab encoding sequences which can be amplified subsequently.
• a procedure termed panning Fab specific for the antigen are enriched and finally isolated.
• Guided selection utilizes the power of the phage display technique for the humanization of mouse monoclonal antibody (See Jespers, L. S., et al.,
• the Fd fragment of the mouse monoclonal antibody can be displayed in combination with a human light chain library, and the resulting hybrid Fab library may then be selected with antigen.
• the mouse Fd fragment thereby provides a template to guide the selection.
• the selected human light chains are combined with a human Fd fragment library. Selection of the resulting library yields entirely human Fab.
• the antibody products may be screened for activity and for suitability in the treatment methods of the invention using assays as described in the section entitled "Screening Methods" herein or using any suitable assays known in the art.
• Covalent modifications of the antibody are also included within the scope of this invention. They may be made by chemical synthesis or by enzymatic or chemical cleavage of the antibody, if applicable. Other types of covalent modifications of the antibody are introduced into the molecule by reacting targeted amino acid residues of the antibody with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues. Cysteinyl residues most commonly are reacted with ⁇ -haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives.
• Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, .alpha.-bromo- ⁇ -(5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p- chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-l,3- diazole.
• j Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH
• Lysinyl and amino-terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues.
• Other suitable reagents for derivatizing .arpha.-amino- containing residues include imidoesters such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4- pentanedione, and transaminase-catalyzed reaction with glyoxylate.
• Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pK a of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.
• tyrosyl residues may be made, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosyl residues are iodinated using 12 1 or 1 I to prepare labeled proteins for use in radioimmunoassay.
• Carboxyl side groups are selectively modified by reaction with carbodiimides (R-N.dbd.C.dbd.N-R 1 ), where R and R' are different alkyl groups, such as l-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or l-ethyl-3-(4-azonia- 4,4-dimethylpentyl) carbodiimide.
• R and R' are different alkyl groups, such as l-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or l-ethyl-3-(4-azonia- 4,4-dimethylpentyl) carbodiimide.
• aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
• Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues, respectively. These residues are deamidated under neutral or basic conditions. The deamidated form of these residues falls within the scope of this invention.
• the sugar(s) may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine.
• Enzymatic cleavage of carbohydrate moieties on antibodies can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al. Meth. Enzymol. 138: 350 (1987).
• Another type of covalent modification of the antibody comprises linking the antibody to one of a variety of nonproteinaceous polymers, e.g. , polyethylene glycol, polypropylene glycol, polyoxyethylated polyols, polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol, polyoxyalkylenes, or polysaccharide polymers such as dextran.
• nonproteinaceous polymers e.g. , polyethylene glycol, polypropylene glycol, polyoxyethylated polyols, polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol, polyoxyalkylenes, or polysaccharide polymers such as dextran.
• nonproteinaceous polymers e.g., polyethylene glycol, polypropylene glycol, polyoxyethylated polyols, polyoxyethylated
• Gene Therapy Delivery of a therapeutic antibody to appropriate cells can be effected via gene therapy ex vivo, in situ, or in vivo by use of any suitable approach known in the art, including by use of physical DNA transfer methods (e.g., liposomes or chemical treatments) or by use of viral vectors (e.g., adenovirus, adeno-associated virus, or a retrovirus).
• a nucleic acid encoding the desired antibody either alone or in conjunction with a vector, liposome, or precipitate may be injected directly into the subject, and in some embodiments, may be injected at the site where the expression of the antibody compound is desired.
• the subject's cells are removed, the nucleic acid is introduced into these cells, and the modified cells are returned to the subject either directly or, for example, encapsulated within porous membranes which are implanted into the patient. See, e.g. U.S. Pat. Nos. 4,892,538 and 5,283,187.
• nucleic acids are introduced into viable cells. The techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or in vivo in the cells of the intended host.
• a commonly used vector for ex vivo delivery of a nucleic acid is a retrovirus.
• nucleic acid transfer techniques include transfection with viral vectors (such as adenovirus, Herpes simplex I virus, or adeno-associated virus) and lipid- based systems.
• viral vectors such as adenovirus, Herpes simplex I virus, or adeno-associated virus
• lipid- based systems include lipid- based systems.
• the nucleic acid and transfection agent are optionally associated with a microparticle.
• Exemplary transfection agents include calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, quaternary ammonium amphiphile DOTMA ((dioleoyloxypropyl) trimethylammonium bromide, commercialized as Lipofectin by GIBCO-BRL))(Felgner et al, (1987) Proc. Natl. Acad. Sci.
• CTAB cetyltrimethylammonium bromide
• DOPE 1,3-dimethyl-3-diol
• TMAG lipophilic diester of glutamic acid
• DDAB stearylamine in admixture with phosphatidylethanolamine
• Rose et al., (1991) Biotechnique 10, 520-525 DDAB/DOPE (TransfectACE, GIBCO BRL)
• exemplary transfection enhancer agents that increase the efficiency of transfer include, for example, DEAE-dextran, polybrene, lysosome-disruptive peptide (Ohmori N I et al, Biochem Biophys Res Commun Jun.
• nucleic acid with an agent that directs the nucleic acid-containing vector to target cells.
• targeting molecules include antibodies specific for a cell-surface membrane protein on the target cell, or a ligand for a receptor on the target cell.
• proteins which bind to a cell-surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake. Examples of such proteins include capsid proteins and fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, and proteins that target intracellular localization and enhance intracellular half-life.
• receptor-mediated endocytosis can be used.
• Antibodies may be screened for binding affinity by methods known in the art. For example, gel-shift assays, Western blots, radiolabeled competition assay, co- fractionation by chromatography, co-precipitation, cross linking, ELISA, and the like may be used, which are described in, for example, Current Protocols in Molecular Biology (1999) John Wiley & Sons, NY, which is incorporated herein by reference in its entirety.
• M-CSF Routine competitive binding assays may also be used, in which the unknown antibody is characterized by its ability to inhibit binding of M-CSF to an M-CSF specific antibody of the invention.
• Intact M-CSF, fragments thereof, or linear epitopes such as represented by amino acids 98- 105 of M-CSF of Figure 7, or amino acids 65-73 or 138-144 of Figure 7 (corresponding to M-CSF epitopes recognized by 5H4 or MC3), can be used. Epitope mapping is described in Champe et al, J. Biol. Chem. 270: 1388-1394 (1995).
• the antibodies are next tested for their effect on M-CSF biological activity with respect to inducing production or proliferation of macrophages, followed by administration to animals.
• Compounds potentially useful in macrophage-associated diseases may be screened using various assays. For instance, a candidate antagonist may first be characterized in a cultured cell system to determine its ability to neutralize M-CSF biological activity.
• Such a system may include the co-culture of mouse stromal cell lines (e.g., MC3T3-G2/PA6 and ST2) and mouse spleen cells (Udagawa et al., Endocrinology 125: 1805 13, 1989), and the co-culture of ST2 cells and bone marrow cells, peripheral blood mononuclear cells or alveolar macrophages (Udagawa et al., Proc. Natl. Acad. Sci. USA 87: 7260 4, 1990; Sasaki et al., Cancer Res. 58: 462 7, 1998; Mancino et al., J. Surg. Res.100: 18-24, 2001).
• mouse stromal cell lines e.g., MC3T3-G2/PA6 and ST2
• mouse spleen cells Udagawa et al., Endocrinology 125: 1805 13, 1989
• ST2 cells and bone marrow cells e.gawa et al.
• Efficacy of a given M-CSF antibody in reducing the effect of M-CSF on production or proliferation of macrophages, or in preventing or treating macrophage-associated diseases such as atherosclerotic diseases, or HIV infection and conditions associated therewith, may also be tested in any of the in vitro assays or animal model systems familiar to those skilled in the art.
• the invention provides a method comprising the steps of (a) contacting an immobilized M-CSF with a candidate antibody and (b) detecting binding of the candidate antibody to the M-CSF.
• the candidate antibody is immobilized and binding of M-CSF is detected. Immobilization is accomplished using any of the methods well known in the art, including covalent bonding to a support, a bead, or a chromatographic resin, as well as non-covalent, high affinity interaction such as antibody binding, or use of streptavidin/biotin binding wherein the immobilized compound includes a biotin moiety.
• Detection of binding can be accomplished (i) using a radioactive label on the compound that is not immobilized, (ii) using a fluorescent label on the non-immobilized compound, (iii) using an antibody immunospecific for the non- immobilized compound, (iv) using a label on the non-immobilized compound that excites a fluorescent support to which the immobilized compound is attached, as well as other techniques well known and routinely practiced in the art.
• Antibodies that modulate (i.e., increase, decrease, or block) the activity or expression of M-CSF may be identified by incubating a putative modulator with a cell expressing a M-CSF and determining the effect of the putative modulator on the activity or expression of the M-CSF.
• the selectivity of an antibody that modulates the activity of a M- CSF polypeptide or polynucleotide can be evaluated by comparing its effects on the M-CSF polypeptide or polynucleotide to its effect on other related compounds.
• Selective modulators may include, for example, antibodies and other proteins, peptides, or organic molecules which specifically bind to M-CSF polypeptides or to a nucleic acid encoding a M-CSF polypeptide. Modulators of M-CSF activity will be therapeutically useful in treatment of diseases and physiological conditions in which normal or aberrant activity of M-CSF polypeptide is involved.
• the invention also comprehends high throughput screening (HTS) assays to identify antibodies that interact with or inhibit biological activity (i.e., inhibit enzymatic activity, binding activity, etc.) of a M-CSF polypeptide.
• HTS assays permit screening of large numbers of compounds in an efficient manner.
• Cell-based HTS systems are contemplated to investigate the interaction between M-CSF polypeptides and their binding partners.
• HTS assays are designed to identify "hits” or "lead compounds” having the desired property, from which modifications can be designed to improve the desired property. Chemical modification of the "hit” or “lead compound” is often based on an identifiable structure/activity relationship between the "hit” and M-CSF polypeptides.
• Another aspect of the present invention is directed to methods of identifying antibodies which modulate (i.e., decrease) activity of a M-CSF comprising contacting a M- CSF with an antibody, and determining whether the antibody modifies activity of the M-CSF.
• the activity in the presence of the test antibody is compared to the activity in the absence of the test antibody. Where the activity of the sample containing the test antibody is lower than the activity in the sample lacking the test antibody, the antibody will have inhibited activity.
• heterologous systems are available for functional expression of recombinant polypeptides that are well known to those skilled in the art.
• Such systems include bacteria (Strosberg, et al., Trends in Pharmacological Sciences (1992) 13:95-98), yeast (Pausch, Trends in Biotechnology (1997) 15:487-494), several kinds of insect cells (Vanden Broeck, Int. Rev. Cytology (1996) 164:189-268), amphibian cells (Jayawickreme et al., Current Opinion in Biotechnology (1997) 8: 629-634) and several mammalian cell lines (CHO, HEK293, COS, etc.; see Gerhardt, et al., Eur. J.
• methods of screening for antibodies which modulate the activity of M-CSF comprise contacting test antibodies with a M-CSF polypeptide and assaying for the presence of a complex between the antibody and the M- CSF.
• the ligand is typically labeled. After suitable incubation, free ligand is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of the particular antibody to bind to the M-CSF or M-CSFR polypeptide.
• high throughput screening for antibody fragments or CDRs having suitable binding affinity to a M-CSF polypeptide is employed. Briefly, large numbers of different small peptide test compounds are synthesized on a solid substrate. The peptide test antibodies are contacted with a M-CSF polypeptide and washed. Bound M-CSF polypeptides are then detected by methods well known in the art. Purified polypeptides of the invention can also be coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies can be used to capture the protein and immobilize it on the solid support. Combination Therapy
• compositions comprising one or more M-CSF antibody may be administered to persons or mammals suffering from, or predisposed to suffer from, macrophage-associated diseases.
• Concurrent administration of two therapeutic agents does not require that the agents be administered at the same time or by the same route, as long as there is an overlap in the time period during which the agents are exerting their therapeutic effect.
• Simultaneous or sequential administration is contemplated, as is administration on different days or weeks.
• the method of the invention contemplate the administration of single anti-M-
• CSF antibodies as well as combinations, or "cocktails", of different antibodies.
• Such antibody cocktails may have certain advantages inasmuch as they contain antibodies which exploit different effector mechanisms.
• Such antibodies in combination may exhibit synergistic or additive therapeutic effects.
• RXl or Human EngineeredTM derivative of RXl antibody with other therapeutics can have an effect on a patient experiencing macrophage-associated diseases.
• anti-M-CSF antibody in the manufacture of a medicament for treating a patient having an atherosclerotic disease or a disease associated with HIV, or treating a patient that has been pre-treated with a second therapeutic agent, or a patient that is not responsive to treatment with a second therapeutic agent.
• Pre-treatment means that a patient had been treated with the second therapeutic agent within 2 years, 1 year, 6 months, 3 months 2 months, 1 month, 2 weeks, 1 week, or at least one day before treatment with M-CSF antibody.
• Such a medicament containing anti-M-CSF antibody may be a medicament that is coordinated with treatment using a second therapeutic agent or a procedure, such as angioplasty.
• a second therapeutic agent in the manufacture of a medicament that is coordinated with treatment using the anti-M-CSF antibody.
• the combination might also have a synergistic effect in a treated patient.
• the two therapeutics need not be administered simultaneously; for example, they can be administered within 1 day, 1 week, 2 weeks, 4 weeks, 2 months, 3 months, 6 months, 1 year or two years of each other.
• Exemplary second therapeutic agents for treating diseases associated with atherosclerosis include a second anti-M-CSF antibody, a drug which beneficially alters the serum lipid profile (e.g., statins such as lovastatin, simvastatin and pravastatin, fluvastatin, atorvastatin, cerivastatin and rosuvastatin, drugs that lower intestinal absorption of cholesterol such as ezetimibe, fibrates, cholestyramine or colestipol resins, or nicotinic acid, or drugs containing highly polyunsaturated or omega-3 fatty acids, e.g.
• statins such as lovastatin, simvastatin and pravastatin, fluvastatin, atorvastatin, cerivastatin and rosuvastatin
• drugs that lower intestinal absorption of cholesterol such as ezetimibe, fibrates, cholestyramine or colestipol resins, or nicotinic acid
• anti-anginal agents such as nitrates, beta-blockers, angiotensin converting enzyme inhibitors, angiotensin receptor blockers, calcium channel antagonists, anti-platelet agents, or anticoagulants.
• Exemplary second therapeutic agents for treating diseases associated with HIV infection include a second anti-M-CSF antibody, or agents used in highly active antiretroviral therapy (HAART) as described in Barbara G, et al., Curr Pharm Des.;ll(14):1805-43 (2005), herein incorporated by reference in its entirety.
• HAART highly active antiretroviral therapy
• any reverse transcriptase inhibitor or protease inhibitor known in the art may be used.
• Prodrug refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic or non-cytotoxic to cells compared to the parent drug and is capable of being enzymatically activated or converted into an active or the more active parent form.
• Prodrugs include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, ⁇ -lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs which can be converted into the more active cytotoxic free drag.
• the anti-M-CSF antibodies used in the practice of a method of the invention may be formulated into pharmaceutical compositions comprising a carrier suitable for the desired delivery method.
• Suitable carriers include any material which, when combined with the anti-M-CSF antibodies, retains the desired activity of the antibody and is nonreactive with the subject's immune systems. Examples include, but are not limited to, any of a number of standard pharmaceutical carriers such as sterile phosphate buffered saline solutions, bacteriostatic water, and the like.
• aqueous carriers may be used, e.g., water, buffered water, 0.4% saline, 0.3% glycine and the like, and may include other proteins for enhanced stability, such as albumin, lipoprotein, globulin, etc., subjected to mild chemical modifications or the like.
• Therapeutic formulations of the antibody are prepared for storage by mixing the antibody having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.
• Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine,
• the formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
• Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
• the active ingredients may also be entrapped in microcapsule prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
• colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
• macroemulsions for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
• the formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
• the antibody is administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration.
• Parenteral infusions include intravenous, intraarterial, intraperitoneal, intramuscular, intradermal or subcutaneous administration.
• the antibody is suitably administered by pulse infusion, particularly with declining doses of the antibody.
• the dosing is given by injections, most preferably intravenous or subcutaneous injections.
• Other administration methods are contemplated, including topical, particularly transdermal, transmucosal, rectal, oral or local administration e.g. through a catheter placed close to the desired site.
• the pharmaceutical formulations and medicaments may be a spray or aerosol containing an appropriate solvent(s) and optionally other compounds such as, but not limited to, stabilizers, antimicrobial agents, antioxidants, pH modifiers, surfactants, bioavailability modifiers and combinations of these.
• a propellant for an aerosol formulation may include compressed air, nitrogen, carbon dioxide, or a hydrocarbon based low boiling solvent.
• Injectable dosage forms generally include aqueous suspensions or oil suspensions which may be prepared using a suitable dispersant or wetting agent and a suspending agent. Injectable forms may be in solution phase or in the form of a suspension, which is prepared with a solvent or diluent.
• Acceptable solvents or vehicles include sterilized water, Ringer's solution, or an isotonic aqueous saline solution.
• sterile oils may be employed as solvents or suspending agents.
• the oil or fatty acid is nonvolatile, including natural or synthetic oils, fatty acids, mono-, di- or tri-glycerides.
• the pharmaceutical formulation and/or medicament may be a powder suitable for reconstitution with an appropriate solution as described above.
• these include, but are not limited to, freeze dried, rotary dried or spray dried powders, amorphous powders, granules, precipitates, or particulates.
• the formulations may optionally contain stabilizers, pH modifiers, surfactants, bioavailability modifiers and combinations of these.
• Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsule. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Patent No.
• copolymers of L-glutamic acid and y ethyl-L- glutamate non-degradable ethylene- vinyl acetate
• degradable lactic acid-glycolic acid copolymers such as the Lupron DepotTM (injectable microspheres composed of lactic acid- glycolic acid copolymer and leuprolide acetate)
• poly-D-(-)-3-hydroxybutyric acid While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
• encapsulated antibodies When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S-S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions. Other strategies known in the art may be used.
• the formulations of the invention may be designed to be short-acting, fast- releasing, long-acting, or sustained-releasing as described herein.
• the pharmaceutical formulations may also be formulated for controlled release or for slow release.
• compositions may also comprise, for example, micelles or liposomes, or some other encapsulated form, or may be administered in an extended release form to provide a prolonged storage and/or delivery effect. Therefore, the pharmaceutical formulations and medicaments may be compressed into pellets or cylinders and implanted intramuscularly or subcutaneously as depot injections or as implants such as stents. Such implants may employ known inert materials such as silicones and biodegradable polymers.
• pharmaceutically acceptable excipients and carries are generally known to those skilled in the art and are thus included in the instant invention. Such excipients and carriers are described, for example, in "Remingtons Pharmaceutical Sciences” Mack Pub. Co., New Jersey (1991), which is incorporated herein by reference.
• Specific dosages may be adjusted depending on conditions of disease, the age, body weight, general health conditions, genotype, sex, and diet of the subject, dose intervals, administration routes, excretion rate, and combinations of drugs. Any of the above dosage forms containing effective amounts are well within the bounds of routine experimentation and therefore, well within the scope of the instant invention.
• M-CSF antibodies useful as therapeutics for macrophage-associated diseases will often be prepared substantially free of other naturally occurring immunoglobulins or other biological molecules. Preferred M-CSF antibodies will also exhibit minimal toxicity when administered to a mammal afflicted with, or predisposed to suffer from macrophage- associated diseases.
• compositions of the invention may be sterilized by conventional, well known sterilization techniques.
• the resulting solutions may be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.
• the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride and stabilizers (e.g., 1 20% maltose, etc.).
• the M-CSF antibodies of the present invention may also be administered via liposomes, which are small vesicles composed of various types of lipids and/or phospholipids and/or surfactant which are useful for delivery of a drug (such as the antibodies disclosed herein and, optionally, a chemotherapeutic agent).
• liposomes include emulsions, foams, micelles, insoluble monolayers, phospholipid dispersions, lamellar layers and the like, and can serve as vehicles to target the M-CSF antibodies to a particular tissue as well as to increase the half life of the composition.
• a variety of methods are available for preparing liposomes, as described in, e.g., U.S. Patent Nos. 4,837,028 and 5,019,369, which patents are incorporated herein by reference.
• Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA 77: 4030 (1980); and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556. Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE).
• PEG-PE PEG-derivatized phosphatidylethanolamine
• Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
• Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al., J. Biol. Chem. 257: 286-288 (1982) via a disulfide interchange reaction.
• a chemotherapeutic agent such as Doxorubicin is optionally contained within the liposome [see, e.g., Gabizon et al., J. National Cancer Inst. 81(19): 1484 (1989)].
• the concentration of the M-CSF antibody in these compositions can vary widely, i.e., from less than about 10%, usually at least about 25% to as much as 75% or 90% by weight and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
• Actual methods for preparing orally, topically and parenterally administrable compositions will be known or apparent to those skilled in the art and are described in detail in, for example, Remington's Pharmaceutical Science, 19th ed., Mack Publishing Co., Easton, PA (1995), which is incorporated herein by reference. Determination of an effective amount of a composition of the invention to treat macrophage-associated diseases in a patient can be accomplished through standard empirical methods which are well known in the art.
• the in vivo neutralizing activity of sera from a subject treated with a given dosage of M-CSF antibody may be evaluated using an assay that determines the ability of the sera to block M-CSF induced proliferation and survival of murine monocytes (CDlIb+ cell, a subset of CDIl cells, which expresses high levels of receptor to M-CSF) in vitro as described in Cenci et al., J Clin. Invest. 1055: 1279-87, 2000.
• CDlIb+ cell murine monocytes
• compositions of the invention are administered to a mammal already suffering from, or predisposed to or at risk of a macrophage-associated disease in an amount sufficient to prevent or at least partially arrest the development of disease.
• An amount adequate to accomplish this is defined as a "therapeutically effective dose.”
• Effective amounts of a M- CSF antibody will vary and depend on the severity of the disease and the weight and general state of the patient being treated, but generally range from about 1.0 ⁇ g/kg to about 100 mg/kg body weight. Exemplary doses may range from about 10 ⁇ g/kg to about 30 mg/kg, or from about 0.1 mg/kg to about 20 mg/kg or from about 1 mg/kg to about 10 mg/kg per application.
• Antibody may also be dosed by body surface area (e.g.
• antibody up to 4.5 g/square meter).
• Other exemplary doses of antibody include up to 8g total in a single administration (assuming a body weight of 80 kg or body surface area of 1.8 square meters).
• Administration may be by any means known in the art.
• antibody may be administered by one or more separate bolus administrations, or by short or long term infusion over a period of, e.g., 5, 10, 15, 30, 60, 90 or 120 minutes.
• maintenance doses may be administered, e.g., weekly, biweekly, every 3 weeks, every 4 weeks, monthly, bimonthly, every 3 months, or every 6 months, as needed to maintain patient response.
• More frequent dosages may be needed until a desired suppression of disease symptoms occurs, and dosages may be adjusted as necessary.
• the progress of this therapy is easily monitored by conventional techniques and assays.
• the therapy may be for a defined period or may be chronic and continue over a period of years until disease progression or death.
• Single or multiple administrations of the compositions can be carried out with the dose levels and pattern being selected by the treating physician.
• the appropriate dosage of antibody will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician.
• the antibody is suitably administered to the patient at one time or over a series of treatments.
• the formulations should provide a quantity of M-CSF antibody over time that is sufficient to effectively prevent or minimize the severity of macrophage- associated disease.
• the compositions of the present invention may be administered alone or as an adjunct therapy in conjunction with other therapeutics known in the art for the treatment of macrophage-associated disease.
• the antibody composition will be formulated, dosed, and administered in a fashion consistent with good medical practice.
• Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.
• the therapeutically effective amount of the antibody to be administered will be governed by such considerations, and is the minimum amount necessary to prevent, ameliorate, or treat the M-CSF mediated disease, condition or disorder. Such amount is preferably below the amount that is toxic to the host or renders the host significantly more susceptible to infections.
• the antibody need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question.
• the effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disease, condition or disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as used hereinbefore or about from 1 to 99% of the heretofore employed dosages.
• an article of manufacture containing materials useful for the treatment of a macrophage-associated disease comprises a container and a label.
• Suitable containers include, for example, bottles, vials, syringes, and test tubes.
• the containers may be formed from a variety of materials such as glass or plastic.
• the container holds a composition which is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
• the active agent in the composition is the antibody of the invention.
• the label on, or associated with, the container indicates that the composition is used for treating the condition of choice.
• the article of manufacture may further comprise a second container containing a second therapeutic agent (including any of the second therapeutic agents for macrophage-associated diseases discussed herein or known in the art).
• the article of manufacture may further comprise another container containing a pharmaceutically- acceptable buffer, such as phosphate-buffered saline, Ringer's solution or dextrose solution for reconstituting a lyophilized antibody formulation. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
• Anti-M-CSF antibodies may be administered in their "naked” or unconjugated form, or may be conjugated directly to other therapeutic or diagnostic agents, or may be conjugated indirectly to carrier polymers comprising such other therapeutic or diagnostic agents.
• Antibodies can be detectably labeled through the use of radioisotopes, affinity labels (such as biotin, avidin, etc.), enzymatic labels (such as horseradish peroxidase, alkaline phosphatase, etc.) fluorescent or luminescent or bioluminescent labels (such as FITC or rhodamine, etc.), paramagnetic atoms, and the like. Procedures for accomplishing such labeling are well known in the art; for example, see (Sternberger, L. A. et al., J. Histochem. Cytochem. 18:315 (1970); Bayer, E.A. et al., Meth. Enzym. 62:308 (1979); Engval, E. et al., Immunol. 109:129 (1972); Goding, J.W. J. Immunol. Meth. 13:215 (1976)).
• radioisotopes such as biotin, avidin, etc.
• enzymatic labels
• the carrier polymer may be, for example, an aminodextran or polypeptide of at least 50 amino acid residues.
• a polypeptide carrier can be used instead of aminodextran, but the polypeptide carrier should have at least 50 amino acid residues in the chain, preferably 100-5000 amino acid residues. At least some of the amino acids should be lysine residues or glutamate or aspartate residues.
• the pendant amines of lysine residues and pendant carboxylates of glutamine and aspartate are convenient for attaching a drug, toxin, immunomodulator, chelator, boron addend or other therapeutic agent.
• conjugated antibodies can be prepared by directly conjugating an antibody component with a therapeutic agent.
• the general procedure is analogous to the indirect method of conjugation except that a therapeutic agent is directly attached to an oxidized antibody component.
• a carbohydrate moiety of an antibody can be attached to polyethyleneglycol to extend half-life.
• a therapeutic agent can be attached at the hinge region of a reduced antibody component via disulfide bond formation, or using a heterobifunctional cross-linker, such as N-succinyl 3-(2-pyridyldithio)proprionate (SPDP). Yu et al, Int. J. Cancer56:244 (1994). General techniques for such conjugation are well-known in the art. See, for example, Wong, Chemistry Of Protein Conjugation and Cross-Linking (CRC Press 1991); Upeslacis et al., "Modification of Antibodies by Chemical Methods," in Monoclonal Antibodies: Principles and Applications, Birch et al.
• a heterobifunctional cross-linker such as N-succinyl 3-(2-pyridyldithio)proprionate (SPDP).
• bifunctional protein coupling agents such as N-succinirnidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4- dinitrobenzene).
• SPDP N-succinirnidyl-3-(2-pyridyldithiol)
• fusion proteins can be constructed that comprise one or more anti-M- CSF antibody moieties and another polypeptide.
• Methods of making antibody fusion proteins are well known in the art. See, e.g., U.S. Patent No. 6,306,393.
• Antibody fusion proteins comprising an interleukin-2 moiety are described by Boleti et al., Ann. Oncol. 6:945 (1995), Nicolet et al., Cancer Gene Ther. 2:161 (1995), Becker et al., Proc. Natl Acad. Sci. USA 93:7826 (1996), Hank et al., Clin. Cancer Res. 2:1951 (1996), and Hu et al., Cancer Res. 56:4998 (1996).
• M-CSF antibodies RXl and 5Al are species specific and that antibodies RXl, MCl, and MC3 neutralize human M-CSF activity.
• RXl is a commercially sold antibody that was available more than a year prior to the filing date of this application.
• Exemplary commercial sources include, but are not limited to, mouse anti- human M-CSF monoclonal antibody clones 116, 692, and 21 (Anogen); anti-human M-CSF antibody clones 21113.131, 26730, and 26786 (R & D Systems, Inc.); and anti-human M- CSF antibodyclone M16 (Antigenix America, Inc.).
• M- NFS-60 cell line was used (American Type Culture Collection Accession No. CRL- 1838, available from ATCC in Rockville, MD, USA, derived from a myelogenous leukemia induced with the Cas-Br-MuLV wild mouse ecotropic retrovirous, responsive to both interleukin 3 and M-CSF and which contain a truncated c-myb proto-oncogene caused by the integration of a retrovirus).
• Proliferation of M-NFS -60 requires active M-CSF in a dose- dependent fashion.
• M-NFS-60 cells were washed and plated in RPMI 1640 medium with 10% FBS and 3000 U/ml of M-CSF and 1% Pen/Strep.
• Recombinant human M-CSF (at 10 ng/ml final concentration), human or murine-specific, was incubated with various concentrations of antibodies for 1 hour at 37 0 C in 5% CO 2 in an incubator. Following the incubation, the mixture was added to the M-NFS-60 culture in 96 well microtiter plates. The total assay volume per well was lOO ⁇ l, with 10 ng/ml M-CSF, and the antibody concentration indicated in Figure 4.
• M-CSF antibodies RXl and 5Al are species specific. Cell proliferation is presented as the fluorescent reading from CellTiter GIo assay, which is linear with cell number. Species specific neutralizing activity of RXl and 5Al is shown by its ability to inhibit M-NFS-60 in the presence of either human or murine M-CSF. Finally, as shown in Figure 4B, antibodies MC3 and MCl are also effective inhibitors of M-CSF activity.
• This example sets out a procedure for humanization of the RXl antibody. 5H4, MCl and MC3 are humanized using similar procedures.
• the nucleotide and amino acid sequence for murine RXl are set forth in Figure 3B.
• the sequence of a human antibody identified using the National Biomedical Foundation Protein Identification Resource or similar database is used to provide the framework of the humanized antibody.
• the murine RXl heavy chain sequence is aligned with the sequence of the human antibody heavy chain.
• the human antibody amino acid is selected for the humanized sequence, unless that position falls in any one of four categories defined below, in which case the murine RXl amino acid is selected: (1) The position falls within a complementarity determining region (CDR), as defined by Kabat, J. Immunol., 125, 961-969 (1980);
• the human antibody amino acid is rare for human heavy chains at that position, whereas the murine RXl amino acid is common for human heavy chains at that position; (3) The position is immediately adjacent to a CDR in the amino acid sequence of the murine RXl heavy chain; or
• the murine RXl light chain sequence is aligned with the sequence of the human antibody light chain.
• the human antibody amino acid is selected at each position for the humanized sequence, unless the position again falls into one of the categories described above and repeated below:
• the actual nucleotide sequence of the heavy and light chain genes is selected as follows:
• nucleotide sequences code for the amino acid sequences chosen as described above; (2) 5' of these coding sequences, the nucleotide sequences code for a leader
• nucleotide sequences are the sequences that follow the mouse light chain J5 segment and the mouse heavy chain J2 segment, which are part of the murine RXl sequence. These sequences are included because they contain splice donor signals;
• oligonucleotides are synthesized using an Applied Biosystems 380B DNA synthesizer. Two of the oligonucleotides are part of each strand of the heavy chain, and each oligonucleotide overlaps the next one by about 20 nucleotides to allow annealing. Together, the oligonucleotides cover the entire humanized heavy chain variable region with a few extra nucleotides at each end to allow cutting at the Xba I sites. The oligonucleotides are purified from polyacrylamide gels.
• Each oligonucleotide is phosphorylated using ATP and T4 polynucleotide kinase by standard procedures (Maniatis et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989)).
• TA 33 mM Tris acetate, pH 7.9, 66 mM potassium acetate, 10 mM magnesium acetate
• Construction of plasmids to express humanized light and heavy chains is accomplished by isolating the light and heavy chain Xba I fragments from the pUC19 plasmid in which it had been inserted and then inserting it into the Xba I site of an appropriate expression vector which will express high levels of a complete heavy chain when transfected into an appropriate host cell.
• the expression vectors are transfected into mouse Sp2/0 cells, and cells that integrate the plasmids are selected on the basis of the selectable marker(s) conferred by the expression vectors by standard methods. To verify that these cells secreted antibody that binds to M-CSF, supernatant from the cells are incubated with cells that are known to express M-CSF. After washing, the cells are incubated with fluorescein-conjugated goat anti-human antibody, washed, and analyzed for fluorescence on a FACSCAN cytofluorometer. .
• mice For the next experiments, cells producing the humanized antibody are injected into mice, and the resultant ascites is collected.
• Humanized antibody is purified to substantial homogeneity from the ascites by passage through an affinity column of goat anti-human immunoglobulin antibody, prepared on an Affigel-10 support (Bio-Rad Laboratories, Inc., Richmond, Calif.) according to standard techniques.
• Affigel-10 support Bio-Rad Laboratories, Inc., Richmond, Calif.
• a competitive binding experiment is performed according to techniques known in the art.
• Amino acid substitutions are distinguished by one of three risk categories : (1) low risk changes are those that have the greatest potential for reducing immunogenicity with the least chance of disrupting antigen binding; (2) moderate risk changes are those that would further reduce immunogenicity, but have a greater chance of affecting antigen binding or protein folding; (3) high risk residues are those that are important for binding or for maintaining antibody structure and carry the highest risk that antigen binding or protein folding will be affected. Due to the three-dimensional structural role of prolines, modifications at prolines are generally considered to be at least moderate risk changes, even if the position is typically a low risk position. Subtitutional changes are preferred but insertions and deletions are also possible. Figures 3B and 3C show the risk assignment for each amino acid residue of murine RXl light and heavy chains, respectively, categorized as a high, moderate or low risk change.
• Variable regions of the light and heavy chains of the murine RXl antibody were Human EngineeredTM using this method.
• Amino acid residues that are candidates for modification according to the method at low risk positions were identified by aligning the amino acid sequences of the murine variable regions with a human variable region sequence. Any human variable region can be used, including an individual VH or VL sequence or a human consensus VH or VL sequence.
• the amino acid residues at any number of the low risk positions, or at all of the low risk positions, can be changed.
• EngineeredTM "low risk" light chain sequence in Figures 14A-B human consensus kappa 3 (based on Kabat) was used as the template, and for each position where the murine and human amino acid residues differed at low risk positions, an amino acid modification was introduced that replaced the murine residue with the human residue. A total of 16 amino acid low risk modifications were made to the light chain and 8 low risk modifications were made to the heavy chain.
• amino acid residues that are candidates for modification according to the method at all of the low and moderate risk positions were identified by aligning the amino acid sequences of the murine variable regions with a human variable region sequence.
• the amino acid residues at any number of the low or moderate risk positions, or at all of the low and moderate risk positions, can be changed.
• human consensus Vh2 (based on Kabat) was used as the template, and for each position where the murine and human amino acid residues differed at low or moderate risk positions, an amino acid modification was introduced that replaced the murine residue with the human residue.
• human consensus kappa 3 (based on Kabat) was used as the template, and for each position where the murine and human amino acid residues differed at low or moderate risk positions, an amino acid modification was introduced that replaced the murine residue with the human residue. A total of 19 low and moderate risk amino acid modifications were made to the light chain and 12 low and moderate modifications were made to the heavy chain.
• DNA fragments encoding each of the above-described heavy and light chain V region sequences along with antibody-derived signal sequences were constructed using synthetic nucleotide synthesis.
• DNA encoding each of the light chain V region amino acid sequences described above were inserted into vector pMXPIO containing the human Kappa light chain constant region.
• DNA encoding each of the heavy chain V region amino acid sequences described above were inserted into vector pMXP6 containing the human Gamma-2 heavy chain constant region.
• Additional vectors were constructed containing the the heavy chain V region amino acid sequences fused to the human Gamma- 1 (cDNA)and Gamma-4 (genomic and cDNA) constant regions having sequences displayed in figures 19A, 19b, and 20.
• All of these vectors contain a hCMV promoter and a mouse kappa light chain 3' untranslated region as well as selectable marker genes such as neo or or his for selection of G418 - or histidinol - resistant transfectants, respectively.
• selectable marker genes such as neo or or his for selection of G418 - or histidinol - resistant transfectants, respectively.
• the light and heavy chain vectors are described in Tables 2 and 3, respectively.
• Vectors comprising the desired Human EngineeredTM light plus heavy chain genes (Gamma- 1, Gamma-2 and Gamma-4) were then constructed.
• These "2-Gene” vectors contain genes encoding each antibody chain, heavy and light, under control of the hCMV promoter, CMV splice donor, SV40 16S splice acceptor and the mouse kappa light chain 3' untranslated DNA including the polyA site. They also contain a selectable marker gene such as neo or his and the ampicillin resistance gene.
• Vectors containing both heavy and light chain genes are described in Table 4. Vectors comprising two copies of each light and heavy chain genes (four gene vectors) also can be constructed.
• Vectors containing either the light or heavy chain genes described above also were constructed for transient transfection. These vectors are similar to those described above for permanent transfections except that instead of the neo or his genes, they contain the Epstein- Barr virus oriP for replication in HEK293 cells that express the Epstein-Barr virus nuclear antigen.
• the vectors for transient transfection are described in Tables 5 and 6.
• the vectors described above (Table 4) containing one copy each of the light and heavy genes together are transfected into Ex-Cell 302-adapted CHO-Kl cells.
• CHO-Kl cells adapted to suspension growth in Ex-Cell 302 medium are typically electroporated with 40 ug of linearized vector.
• linearized DNA can be complexed with linear polyethyleneimine (PEI) and used for transfection.
• the cells are plated in 96 well plates containing Ex-Cell 302 medium supplemented with 1% FBS and G418. Clones are screened in 96 well plates and the top -10% of clones from each transfection are transferred to 24 well plates containing Ex-Cell 302 medium.
• a productivity test is performed in 24 well plates in Ex-Cell 302 medium for cultures grown for 7 and 14 days at which time culture supernatants are tested for levels of secreted antibody by an immunoglobulin ELISA assay for IgG.
• the top clones are transferred to shake flasks containing Ex-Cell 302 medium. As soon as the cells are adapted to suspension growth, a shake flask test is performed with these clones in Ex-Cell 302 medium. The cells are grown for up to 10 days in 125 ml Erlenmeyer flasks containing 25 ml media. The flasks are opened at least every other day of the incubation period to allow for gas exchange and the levels of immunoglobulin polypeptide in the culture medium are determined by IgG ELISA at the end of the incubation period. Multiple sequential transfections of the same cell line with two or three multi-unit transcription vectors results in clones and cell lines that exhibit further increases in levels of immunoglobulin production, preferably to 300 ⁇ g/ml or more. Purification
• a process for the purification of immunoglobulin polypeptides from vectors and all lines according to the invention may be designed. According to methods well known in the art, cells are removed by filtration after termination. The filtrate is loaded onto a Protein A column (in multiple passes, if needed). The column is washed and then the expressed and secreted immunoglobulin polypeptides are eluted from the column. For preparation of antibody product, the Protein A pool is held at a low pH (pH 3 for a minimum of 30 minutes and a maximum of one hour) as a viral inactivation step. An adsorptive cation exchange step is next used to further purify the product.
• the eluate from the adsorptive separation column is passed through a virus retaining filter to provide further clearance of potential viral particles.
• the filtrate is further purified by passing through an anion exchange column in which the product does not bind. Finally, the purification process is concluded by transferring the product into the formulation buffer through diafiltration.
• the retentate is adjusted to a protein concentration of at least 1 mg/mL and a stabilizer is added.
• Binding activity The M-CSF binding activity of the recombinant Human EngineeredTM antibodies is evaluated. Protein is purified from shake flask culture supernatants by passage over a protein A column followed by concentration determination by A 2S o- Binding assays are performed as described in Example 1 above or 7 below. Immulon II plates are precoated with the sM-CSF antigen pre-diluted in a PBS coating solution to immobilize it to the microplate. Various test concentrations of M-CSF ranging from 0.25 to 20 ug/ml are added at 50 ul/well and incubated at 4°C overnight. The plates are then washed 3 times with PBS- 0.05% Tween.
• Blocking is performed by adding in PBS-0.05% Tween 1% BSA followed by a 30 minute incubation at 37 0 C. Dilutions of immunoglobulin polypeptides are prepared in PBS-0.05% Tween 1% BSA solution. 2- or 3-fold serial dilutions are prepared and added (100 ul/well) in duplicate or triplicate. After a 90 minute incubation at 37 0 C, the microplate is washed 3 times with PBS-0.05% Tween.
• goat anti-human IgG (gamma- or Fc-specific) secondary antibody conjugated to peroxidase is added to each well and incubated for 60 minutes at 37°C followed by addition of OPD at 0.4 mg/ml in citrate buffer plus 0.012% H 2 O 2 . After 5 - 10 minutes at room temperature the assay is stopped by the addition of 100 ul IM H 2 SO 4 and the plates are read at 490nm. Both goat anti-human IgG (gamma-specific) and goat anti-human IgG (Fc-specific) antibodies have been employed.
• the following example sets out a procedure for the treatment of humans using M-CSF-specific antibody, such as an RXl-derived or RXl-competing antibody, including an RXl Human EngineeredTM antibody with a modified or unmodified IgGl or IgG4 constant region.
• M-CSF-specific antibody such as an RXl-derived or RXl-competing antibody, including an RXl Human EngineeredTM antibody with a modified or unmodified IgGl or IgG4 constant region.
• the procedure can also be followed for an MCl- or MC3-derived or MCl- or MC3- competing antibody.
• the measured M-CSF level in human plasma is about 1 ng/ml.
• M-CSF neutralizing antibody RXl has a measured EC 50 of 2 ng/ml against 1 ng/ml human M-CSF. Accordingly, the effective antibody concentration in human plasma is expected to be 10 to 50,000 fold over its EC 50 , i.e. 20 ng/ml to 100 ug/ml antibody in human plasma.
• Subjects suffering from a macrophage-associated disease are administered anti-M-CSF antibody at an initial dose of 10 mg/kg on a weekly basis and observed for signs of adverse effects or improvement in symptoms of clinical disease.
• Subjects that show no signs of adverse effects are administered gradually escalating doses of 15, 20, 25 or 30 mg/kg and observed for signs of improvement in symptoms of clinical disease.
• EXAMPLE 5 The following example shows the procedure for producing antibodies MCl and MC3.
• MCl and MC3 are two monoclonal murine antibodies that neutralize human M-CSF antibody and bind to human M-CSF.
• the amino acid sequences of these antibodies are shown in Figures 9 and 10, respectively.
• Figures HA and HB show the alignment of the CDRs of the heavy and light chains, respectively, of antibodies RXl, 5H4, MCl and MC3.
• Humanized and Human EngineeredTM versions are generated as described in the examples above.
• Binding properties of the aforementioned antibodies were analyzed using Biacore analyses.
• rabbit anti-mouse Fc was immobilized onto a CM5 biosensor chip via amine coupling.
• the aforementioned antibodies were then captured on the anti-mouse Fc/CM5 biosensor chip at 1.5 ⁇ g/ml for 3 min at 2 ⁇ l/min.
• M-CSF was flowed over the modified biosensor surface at varying concentrations (Rmax -15).
• Test antibodies and antigen were diluted in 0.01 M HEPES pH 7.4, 0.15 M NaCL, 3 niM EDTA, 0.005% Surfactant P20 (HBS-EP).
• the following example reveals the linear epitope (i.e., amino acid sequence) on M-CSF recognized by murine antibodies RXl, 5H4, and MC3.
• the epitope mapping strategy was designed to determine whether antibodies RXl, 5H4, and MC3 recognized linear epitopes or conformational epitopes within M-CSF. Accordingly, the anti-M-CSF antibodies were tested against 0.1 ⁇ g M-CSF under reducing as well as non-reducing conditions. Only the the non-reduced form of M-CSF was recognized by each of the antibodies, suggesting the epitopes recognized are discontinuous in nature.
• the linear epitope of M-CSF was determined for each antibody. Specifically, SPOTs membranes (Sigma Genosys) were prepared where the M-CSF fragment sequence of interest, overlapping lOmer peptides synthesized with one amino acid offset, were loaded onto the cellulose membrane support. These membranes were then probed with the aforementioned antibodies and reactive SPOTs were identified. The peptide sequence was then identified by its corresponding location on the membrane, and overlapping amino acids within the positive reacting peptides were identified as the epitope. As shown in Figure 12, RXl binds to a different linear epitope than 5H4 and MC3, which map to a different location on M-CSF.
• RXl binds to a linear epitope represented by RFRDNTPN (SEQ ID NO: 120) or RFRDNTAN (SEQ ID NO: 121), amino acids 98-105 of M-CSF of Figure 7.
• 5H4 binds to a linear epitope represented by ITFEFVDQE (SEQ ID NO: 122), amino acids 65-73 of M-CSF of Figure 7.
• MC3 binds to two linear epitopes represented by (1) ITFEFVDQE (SEQ ID NO: 122), amino acids 65-73 of M-CSF of Figure 7 and (2) FYETPLQ (SEQ ID NO: 123), amino acids 138-144 of M-CSF of Figure 7.
• Anti-M-CSF neutralizing antibody for example an RXl-derived or RXl- competing antibody
• rhesus monkeys Macaca mulatto
• Macaques with induced lesions are treated with anti-M-CSF antibody according to an escalating dosing regimen (0.2 mg/kg to 20 mg/kg) weekly for up to six months.
• a control group of macaques with induced lesions are injected with expedient solutions.
• the extent of lesions in three major coronary arteries and the right carotid artery is evaluated morphometrically by light microscopy in all groups of animals. Lesion regression is evaluated in the RXl treated group versus the control group. It is expected that treatment with RXl will significantly induce the regression of atherosclerosis lesions.
• the experiments may also include dosing with a second therapeutic agent for treating atherosclerotic disease, such as a drag which beneficially alters the serum lipid profile (e.g., statins such as lovastatin, simvastatin and pravastatin, fluvastatin, atorvastatin, cerivastatin and rosuvastatin, drags that lower intestinal absorption of cholesterol such as ezetimibe, fibrates, cholestyramine or colestipol resins, or nicotinic acid, or drags containing highly polyunsaturated or omega-3 fatty acids, e.g.
• a drag which beneficially alters the serum lipid profile e.g., statins such as lovastatin, simvastatin and pravastatin, fluvastatin, atorvastatin, cerivastatin and rosuvastatin, drags that lower intestinal absorption of cholesterol such as ezetimibe, fibrates, cholestyramine or colesti
• anti-anginal agents such as nitrates, beta-blockers, angiotensin converting enzyme inhibitors, angiotensin receptor blockers, calcium channel antagonists, anti-platelet agents, and anticoagulants.
• the following example sets forth a procedure for an in vivo study on the therapeutic efficacy of anti-M-CSF neutralizing antibody of the invention in treating AIDS.
• Anti-M-CSF neutralizing antibody is tested in an AIDS model in rhesus macaques infected with a chimera (RT-SHIV) of simian immunodeficiency virus containing reverse transcriptase from human immunodeficiency virus type-1 (HIV-I).
• RT-SHIV- infected macaques are treated with RXl with escalating dosing regimen (0.2 mg/kg to 20 mg/kg) weekly up to six months.
• a control group of RT-SHIV- infected macaques is injected with expedient solutions.
• Plasma viral RNA levels in all animals are tracked for reduction after 4 weeks and followed up to 10 weeks. Virus loads are followed throughout the treatment. Both plasma viral RNA levels and viral loads are followed after the stop of treatment.
• Post-treatment RT-SHIV isolates are examined for mutations associated with resistance to the treatment. It is expected that the treatment with anti-M-CSF antibody will block HIV infection through reduction of its plasma viral RNA level and virus loads.
• the experiments may include dosing with a second therapeutic agent for HIV, including, for example, a second anti-M-CSF antibody, or agents used in highly active antiretroviral therapy (HAART) as described in Barbara G, et al., Curr Pharm Des.;l 1(14): 1805-43 (2005), herein incorporated by reference in its entirety.
• a second therapeutic agent for HIV including, for example, a second anti-M-CSF antibody, or agents used in highly active antiretroviral therapy (HAART) as described in Barbara G, et al., Curr Pharm Des.;l 1(14): 1805-43 (2005), herein incorporated by reference in its entirety.
• HAART highly active antiretroviral therapy
• the following example sets forth the procedure for measuring the ability of anti-M-CSF antibody of the invention to inhibit the spread of HIV-I.
• PBMC peripheral blood cells
• monocytes are purified by countercurrent centrifugal cell elutriation (Gruber, M. F., et al., J. Immunol. 154:5528 (1995); Gerrard, T. L., et al., Cell. Immunol. 82:394 (1983)).
• Elutriated monocyte viability is determined by trypan blue exclusion, and presence of CD 14 is determined by flow cytometry (FACS) analysis of representative samples.
• Monocytes are differentiated in culture for 8 days at 37 0 C in 5% CO2 at a concentration of 4 x 10 6 /2 ml in six-well tissue culture plates (Costar, Cambridge, MA) using DMEM (Life Technologies, Gaithersburg, MD) complete medium containing 10% pooled human serum, 2 mM L-glutamine (Life Technologies), 1 niM sodium pyruvate (Life Technologies), and penicillin (50 U/ml)/streptomycin (50 ⁇ g/ml) (Life Technologies) to generate MDM. AU reagents used in the isolation and culture of MDM are tested for endotoxin.
• MDM are harvested by scraping and plated into 24-well tissue culture plates (Nunc, Naperville, IL), at a concentration of 500,000 cells/ml, 1.5 ml/well. After 24-48 h, MDM are infected with HIV-I, (Gruber, M. F., et al, J. Immunol. 154:5528 (1995)). Every 3 days thereafter, 80% of the culture medium is collected, stored at -80°, then replaced. In some experiments, AZT (Sigma, St. Louis, MO) is added at a concentration of 1 ⁇ M following virus adsorption and replenished every 3 days. In other experiments, anti-M-CSF antibody, is added after virus adsorption and replenished every 3 days.
• the concentration of anti-M-CSF added should be sufficient to neutralize 100 ng/ml of M-CSF bioactivity.
• MDM cultures infected with HIV-I are generally maintained in DMEM complete medium, as described above.
• the experiments may include addition of a second therapeutic anti-HIV agent, such as a reverse transcriptase inhibitor or protease inhibitor.
• RT assay A reverse-transcriptase (RT) assay is used to measure the progression of infection in MDM infected with HIV-I.
• the RT assay used is a 3 H-based modification of the methods described by Hoffman (Hoffman, A. D., Virology 147:326 (1985)). Briefly, 60 ⁇ l of harvested culture supernatants are diluted with 60 ⁇ lpl of Tris buffer (pH 7.8)/0.05% Triton X-100.
• Replicate 50- ⁇ l samples are then added to 100 ⁇ l of a solution containing poly (rA) (Pharmacia LKB, Piscataway, NJ), oligo (dT) (Pharmacia LKB), MgCl, and 3 H-labeled dTTP (NEN, Boston, MA) in a 96-well U-bottom microtiter plate (Falcon 3910) and incubated for 2 h at 37 0 C. After incubation, 100 ⁇ l of a solution containing 10% TCA is added to each well.
• the individual wells are transferred to glass-fiber filters (Wallac) by using a cell harvester (Skatron) connected to two fluid reservoirs containing 5% TCA/5% sodium pyrophosphate and 70% ethanol, which are run in sequence. Finally, the filters are counted on a beta scintillation counter (beta platereader, Pharmacia LKB).
• M-CSF antagonists such as antibody RXl, which bind to M-CSF and prevent it from interacting with its receptor, is analyzed for its ability to inhibit HIV-I replication

Landscapes

• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Medicinal Chemistry (AREA)
• Organic Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Animal Behavior & Ethology (AREA)
• Pharmacology & Pharmacy (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Engineering & Computer Science (AREA)
• General Chemical & Material Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Immunology (AREA)
• Molecular Biology (AREA)
• Biophysics (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Genetics & Genomics (AREA)
• Biochemistry (AREA)
• Physical Education & Sports Medicine (AREA)
• Virology (AREA)
• Epidemiology (AREA)
• Mycology (AREA)
• Microbiology (AREA)
• Endocrinology (AREA)
• Vascular Medicine (AREA)
• Urology & Nephrology (AREA)
• Orthopedic Medicine & Surgery (AREA)
• AIDS & HIV (AREA)
• Heart & Thoracic Surgery (AREA)
• Cardiology (AREA)
• Tropical Medicine & Parasitology (AREA)
• Rheumatology (AREA)
• Oncology (AREA)
• Communicable Diseases (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Peptides Or Proteins (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Priority Applications (5)

Applications Claiming Priority (2)

Related Child Applications (2)

Publications (2)

Family

ID=37517276

Family Applications (1)

Country Status (5)

Cited By (4)

Families Citing this family (6)

Family Cites Families (63)

• 2006
  • 2006-07-27 WO PCT/US2006/029186 patent/WO2007016240A2/en not_active Ceased
  • 2006-07-27 EP EP10177804A patent/EP2277916A3/en not_active Withdrawn
  • 2006-07-27 ES ES06800398.7T patent/ES2538036T3/es active Active
  • 2006-07-27 US US11/996,909 patent/US20080233118A1/en not_active Abandoned
  • 2006-07-27 JP JP2008524146A patent/JP5657862B2/ja not_active Expired - Fee Related
  • 2006-07-27 EP EP06800398.7A patent/EP1913028B1/en active Active
• 2012
  • 2012-06-01 JP JP2012126356A patent/JP2012162572A/ja not_active Withdrawn
• 2014
  • 2014-06-20 US US14/310,149 patent/US20150050280A1/en not_active Abandoned
  • 2014-08-01 JP JP2014157391A patent/JP2014208703A/ja not_active Withdrawn

Also Published As

Similar Documents

Legal Events