EP1706137A2 - Muteines de facteur de stimulation de colonies de macrophages (m-csf) et leurs utilisations - Google Patents

Muteines de facteur de stimulation de colonies de macrophages (m-csf) et leurs utilisations

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Publication number
EP1706137A2
EP1706137A2 EP05726267A EP05726267A EP1706137A2 EP 1706137 A2 EP1706137 A2 EP 1706137A2 EP 05726267 A EP05726267 A EP 05726267A EP 05726267 A EP05726267 A EP 05726267A EP 1706137 A2 EP1706137 A2 EP 1706137A2
Authority
EP
European Patent Office
Prior art keywords
mutein
csf
cancer
product
bone
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP05726267A
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German (de)
English (en)
Inventor
Deborah Lee Zimmerman
Gregory Martin Harrowe
Cheng Liu
Kirston Koths
William Michael Kavanaugh
Li Chiron Corporation LONG
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novartis Vaccines and Diagnostics Inc
Original Assignee
Chiron Corp
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Filing date
Publication date
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Publication of EP1706137A2 publication Critical patent/EP1706137A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/193—Colony stimulating factors [CSF]
    • 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
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • 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

Definitions

  • Osteoclasts mediate bone readsorption. Osteoclasts are multinucleated cells differentiating from haemopoietic cells. It is generally accepted that osteoclasts are formed by the fusion of mononuclear precursors derived from haemopoietic stem cells in the bone manow, rather than incomplete cell divisions (Chambers, Bone and Mineral Research, 6: 1- 25, 1989; G ⁇ thling et al., Clin Orthop Relat R.
  • Osteoblastic or bone manow stromal cells are also required for osteoclast differentiation.
  • M-CSF macrophage- colony stimulating factor
  • Receptor activator for NF- ⁇ B ligand is another signal (Suda et al., Endocr Rev 13: 66-80, 1992) through which osteoblastic/stromal cells stimulate osteoclast formation and resorption via a receptor, RANK (TRANCER), located on osteoclasts and osteoclast precursors (Lacey et al., Cell 93: 165-176, 1998; Tsuda et al, Biochem Biophys Res Co 234: 137-142, 1997; Wong et al., J Exp Med 186: 2075-2080, 1997; Wong et al., J Biol.
  • TRANCER RANK
  • Osteoblasts also secrete a protein that strongly inhibits osteoclast formation called osteoprotegerin (OPG, also known as OCIF), which acts as a decoy receptor for the RANKL thus inhibiting the positive signal between osteoclasts and osteoblasts via RANK and RANKL.
  • OPG osteoprotegerin
  • Osteoclasts are responsible for dissolving both the mineral and organic bone matrix (Blair et al., J Cell Biol 102: 1164-1172, 1986).
  • Osteoclasts represent terminally differentiated cells expressing a unique polarized morphology with specialized membrane areas and several membrane and cytoplasmic markers, such as tartrate resistant acid phosphatase (TRAP) (Anderson et al. 1979), carbonic anhydrase II (Vaananen et al., Histochemistry 78: 481- 485, 1983), calcitonin receptor (Warshafsky et al., Bone 6: 179-185, 1985) and vitronectin receptor (Davies et al., J Cell Biol 109: 1817-1826, 1989).
  • TRIP tartrate resistant acid phosphatase
  • Multinucleated osteoclasts usually contain less than 10 nuclei, but they may contain up to 100 nuclei being between 10 and 100 ⁇ m in diameter (Gothling et al., Clin Orthop Relat R 120: 201-228, 1976). This makes them relatively easy to identify by light microscopy. They are highly vacuolated when in the active state, and also contain many mitochondria, indicative of a high metabolic rate (Mundy, in Primer on the metabolic bone diseases and disorders of mineral metabolism, pages 18-22, 1990). Since osteoclasts play a major role in osteolytic bone metastases, there is a need in the art for new agents and methods for preventing osteoclast stimulation. Thus, ' there remains a need in. the art to identify new agents and methods for preventing or treating cancer metastasis, including osteolytic bone metastases.
  • a method of preventing bone metastases comprising administering to a subject afflicted with metastatic cancer a therapeutically effective amount of a M-CSF mutein or mutein product thereby preventing bone loss associated with the metastatic cancer.
  • a method of treating a subject afflicted with a metastatic cancer to bone comprising administering to the subject a therapeutically effective amount of a M-CSF mutein or mutein product thereby reducing the severity of bone loss associated with the metastatic cancer is provided.
  • the aforementioned methods are provided wherein the subject is a mammal or a human.
  • a method is provided wherein the mutein or mutein product inhibits the interaction between M-CSF and its receptor (M-CSFR).
  • M-CSFR M-CSF and its receptor
  • a method wherein the M-CSF mutein or mutein product inhibits osteoclast proliferation and/or differentiation induced by tumor cells is provided.
  • a method wherein the metastatic cancer is breast, lung, renal, multiple myeloma, thyroid, prostate, adenocarcinoma, blood cell malignancies, including leukemia and lymphoma; head and neck cancers; gastrointestinal cancers, including stomach cancer, colon cancer, colorectal cancer, pancreatic cancer, liver cancer; malignancies of the female genital tract, including ovarian carcinoma, uterine endometrial cancers and cervical cancer; bladder cancer; brain cancer, including neuroblastoma; sarcoma, osteosarcoma; and skin cancer, including malignant melanoma or squamous cell cancer.
  • a method of screening for a M-CSF mutein or mutein product comprising the steps of contacting metastatic tumor cell medium, osteoclasts and a candidate M-CSF mutein or mutein product; detecting osteoclast formation, proliferation and/or differentiation; and identifying the candidate as an M-CSF mutein or mutein product if a decrease in osteoclast formation, proliferation and/or differentiation is detected.
  • the metastatic tumor cell medium includes tumor cells.
  • a method wherein the contacting step (a) occurs in vivo, the detecting step (b) comprises detecting size and/or number of bone metastases, and the candidate is identified as a M-CSF mutein or mutein product if a decrease in size and/or number of bone metastases is detected is provided.
  • a method is provided further comprising the step of determining if the candidate M-CSF mutein or mutein product inhibits interaction between M-CSF and its receptor M-CSFR.
  • a method of identifying a M-CSF mutein or mutein product that can prevent or treat metastatic cancer to bone comprising the " steps of detecting binding of a candidate M-CSF mutein or mutein product to M-CSFR; and assaying the ability of the candidate M-CSF mutein or mutein product to prevent or treat metastatic cancer to bone in vitro ox in vivo,
  • a method of identifying a M-CSF mutein or mutein product that can prevent or treat metastatic cancer to bone comprising the steps of identifying a candidate M-CSF mutein or mutein product that inhibits the interaction between M-CSF and M-CSFR; and assaying the ability of the candidate M-CSF mutein or mutein product to prevent or treat metastatic cancer to bone in vitro ox in vivo is provided.
  • a method of preventing bone metastases and tumor growth comprising administering to a subject afflicted with metastatic cancer therapeutically effective amounts of M-CSF mutein or mutein product and a therapeutic agent, thereby preventing bone loss associated with the metastatic cancer and preventing tumor growth.
  • a method of treating a subject afflicted with a metastatic cancer comprising administering to the subject therapeutically effective amounts of M-CSF mutein or mutein product and a therapeutic agent, thereby reducing the severity of bone loss associated with the metastatic cancer and inhibiting tumor growth is provided.
  • a method is provided wherein the subject is a mammal or human.
  • a method is provided wherein the M-CSF mutein or mutein product inhibits the interaction between M-CSF and its receptor M-CSFR. In yet another related aspect, a method is provided wherein the M- CSF mutein or mutein product inhibits osteoclast proliferation and/or differentiation induced by tumor cells. In still another related aspect, a method is provided wherein the therapeutic agent is a bisphosphonate. In a related aspect, the bisphonate is zeledronate, pamidronate, clodronate, etidronate, tilundronate, alendronate, or ibandronate. In still another related aspect, the aforementioned methods are provided wherin the therapeutic agent is a chemotherapeutic agent.
  • the aforementioned methods are provided wherein the subject is precluded from receiving bisphophonate treatment, h a related aspects of the invention, the aforementioned methods are provided wherein the M-CSF mutein or mutein product is effective to reduce the dosage of therapeutic agent required to achieve a therapeutic effect.
  • a method is provided further comprising the step of administering a non-M-CSF colony stimulating factor, for example G-CSF.
  • a pharmaceutical composition comprising a M- .
  • a package, vial or container comprising a medicament comprising an M-CSF mutein or mutein product and instructions that the medicament should be used in combination with surgery or radiation therapy.
  • a method of preventing or treating metastatic cancer to bone comprising the steps of administering a M-CSF mutein or mutein product to a subject and treating the subject with surgery or radiation therapy.
  • a method of treating a subject suffering from a cancer, wherein the cells comprising the cancer do not secrete M-CSF is provided, comprising the step of administering a M-CSF mutein or mutein product.
  • a M-CSF mutein or mutein product in the manufacture of a medicament for preventing bone metastases in a subject afflicted with metastatic cancer is provided by the present invention.
  • a use of a M-CSF mutein or mutein product in the manufacture of a medicament for preventing, in a subject afflicted with metastatic cancer, bone loss associated with the cancer is provided.
  • a use of a M-CSF mutein or mutein product in the manufacture of a medicament for treating a subject afflicted with a metastatic cancer to bone is provided by the instant invention.
  • a use of a M-CSF mutein or mutein product for the manufacture of a medicament for reducing, in a subject afflicted with a metastatic cancer to bone, the severity of bone loss associated with the cancer.
  • the subject is a mammal.
  • the mammal is human.
  • an aforementioned use is provided wherein' the mutein or mutein product inhibits the interaction between M-CSF and its receptor (M-CSFR).
  • M-CSFR M-CSFR
  • the mutein or mutein product inhibits osteoclast proliferation and/or differentiation induced by tumor cells.
  • the metastatic cancer is breast, lung, renal, multiple myeloma, thyroid, prostate, adenocarcinoma, blood cell malignancies, including leukemia or lymphoma; head or neck cancers; gastrointestinal cancers, including stomach cancer, colon cancer, colorectal cancer, pancreatic cancer,, liver cancer; malignancies of the female genital tract, including ovarian carcinoma, uterine endometrial cancers or cervical cancer; bladder cancer; brain cancer, including neuroblastoma; sarcoma, osteosarcoma; or skin cancer, including malignant melanoma or squamous cell cancer.
  • a use of a M-CSF mutein or mutein product and a second therapeutic agent in the manufacture of a medicament for preventing, in a subject afflicted with metastatic cancer, bone metastases and tumor growth is provided.
  • a use of a M-CSF mutein or mutein product and a second therapeutic agent in the manufacture of a medicament for preventing, in a subject afflicted with metastatic cancer, bone loss associated with the cancer is provided.
  • a use of a M-CSF mutein or mutein product and a second therapeutic agent is provided for the manufacture of a medicament for treating a metastatic cancer to bone.
  • a use of a M- CSF mutein or mutein product and a second therapeutic agent in the manufacture of a medicament for reducing the severity of bone loss associated with the cancer and inhibiting tumor growth in a subject afflicted with metstatic cancer is provided.
  • a product comprising an M-CSF mutein or mutein product and a second therapeutic agent as a combined preparation is provided for use in treating cancer.
  • a use of a M-CSF mutein or mutein product in preparation of a medicament for preventing or treating metastatic cancer to bone, wherein the medicament is simultaneously separately or sequentially administered with a second cancer therapeutic agent is provided.
  • a use of a M-CSF mutein or mutein product in preparation of a medicament for preventing or treating metastatic cancer to bone, wherein the medicament is coordinated With treatment using a second therapeutic agent is provided.
  • a use of a M-CSF mutein or mutein.product is provided for preparation of a medicament for treating a subject afflicted with a metastatic cancer to bone, wherein the subject has been pre-treated with the second therapeutic agent.
  • a use of a synergistic combination of a MCSF mutein or mutein product in the manufacture of a medicament for treating a patient having an osteolytic disease wherein said medicament is coordinated with treatment using an anti-MCSF antibody, anti-RANKL antibody, soluble RANKL receptor or bisphosphonate is provided.
  • a use of a second therapeutic agent in preparation of a medicament for preventing or treating metastatic cancer to bone, wherein the medicament is simultaneously separately or sequentially administered with an M-CSF mutein or mutein product is provided.
  • a package, vial or container comprising a medicament comprising an M-CSF mutein or mutein product and instructions that the medicament should be used in combination with surgery or radiation therapy.
  • the subject is a mammal.
  • the mammal is human.
  • an aformetioned use is provided wherein said mutein or mutein product inhibits the interaction between M-CSF and its receptor M-CSFR.
  • the mutein or mutein product inhibits osteoclast proliferation and/or differentiation induced by tumor cells.
  • the second therapeutic agent is a bisphosphonate.
  • the bisphonate is zeledronate, pamidronate, clodronate, etidronate, tilundronate, alendronate, or ibandronate.
  • the second therapeutic agent is a chemotherapeutic agent.
  • the subject is precluded from receiving bisphophonate treatment.
  • an aforementioned use is provided wherein the second , therapeutic agent is a non-M-CSF colony stimulating factor.
  • an aforementioned use is provided wherein the non-M-CSF colony stimulating factor is G-CSF.
  • the present invention also contemplates a use of a M-CSf mutein or mutein product in the manufacture of a medicament for reducing the dose of a therapeutic agent administered to a subject to treat or prevent bone metastases and tumor growth.
  • a use of a M-CSF mutein or mutein product, in an amount that is larger than the amount effective to neutralize M-CSF produced by cancer cells, in the manufacture of a medicament for preventing bone metastases is provided.
  • a use of a M-CSF mutein or mutein product in an amount that is larger than the amount effective to neutralize M-CSF produced by cancer cells, is provided for the manufacture of a medicament for neutralizing M-CSF produced by a subject's cells.
  • the invention also contemplates a use of a M-CSF mutein or mutein product, in an amount that is larger than the amount effective to neutralize M-CSF produced by cancer cells, in the manufacture of a medicament for treating a subject afflicted with a metastatic cancer to bone.
  • a use of a M-CSF mutein or mutein product in an amount that is larger than the amount effective to neutralize M-CSF produced by cancer cells, in the manufacture of a medicament for treating cancer is provided.
  • 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 with every tenth residue labeled and with the non-crystallographic symmetry axis indicated by a dotted line.
  • Figure 3 is a comparison of osteoclast inducing activity between purified M-CSF and conditioned medium (CM) from MDA 231 cells and MCF7 cells.
  • Figure 4 is the amino acid sequence of M-CSF ⁇ (SEQ ID NO: 2).
  • Figure 5 is is the amino acid sequence of M-CSF ⁇ (SEQ ID NO: 4).
  • Figure 6 is is the amino acid sequence of M-CSF ⁇ (SEQ ID NO: 6).
  • Metastasis refers to the spread of cancer cells to other parts of the body or to the condition produced by this spread. Metastasis is a complex multi-step process that includes changes in the genetic material of a cell, uncontrolled proliferation of the altered cell to form a primary tumor, development of a new blood supply for the primary tumor, invasion of the circulatory system by cells from the primary tumor, dispersal of small clumps of primary tumor cells to other parts of the body, and the growth of secondary tumors in those sites.
  • Bone is one of the most common sites of metastasis in human breast, lung, prostate and thyroid cancers, as well as other cancers, and in autopsies as many as 60% of cancer patients are found to have bone metastasis.
  • Osteolytic bone metastasis shows a unique step of osteoclastic bone reso ⁇ tion that is not seen in metastasis to other organs.
  • Bone loss associated with cancer metastasis is mediated by osteoclasts (multinucleated giant cells with the capacity to resorb mineralized tissues), which seem to be activated by tumor products.
  • Colony stimulating factor (CSF 1) also known as macrophage colony stimulating factor (M-CSF) has been found crucial for osteoclast formation.
  • M-CSF has been shown to modulate the osteoclastic functions of mature osteoclasts, their migration and their survival in cooperation with other soluble factors and cell to cell interactions provided by osteoblasts and fibroblasts (Fixe and Praloran, Cytokine 10: 3-7, 1998; Martin et al., Critical Rev. in Eukaryotic Gene Expression 8: 107-23 (1998)).
  • the full-length human M-CSF mRNA encodes a precursor protein of 554 amino ' acids (SEQ ID NO: 4).
  • 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.
  • Three distinct M-CSF species are produced through alternative mRNA splicing.
  • the three polypeptide precursors are M-CFS ⁇ of 256 amino acids (DNA and amino acid sequences set forth in SEQ ID NOS: 1 and 2), M-CSF ⁇ of 554 amino acids (DNA and amino acid sequences set forth in SEQ ID NOS: 3 and 4), and M- CSF ⁇ of 438 amino acids (DNA and amino acid sequences set forth in SEQ ID NOS: 5 and 6).
  • M-CSF ⁇ is a secreted protein that does not occur in a membrane-bound form.
  • M-CSF ⁇ is expressed as an integral membrane protein that is slowly released by proteolytic cleavage.
  • M- CSF ⁇ is cleaved at amino acids 191-197 of SEQ ID NO: 2.
  • M-CSFR DNA and amino acid sequences set forth in SEQ ID NOS: 7 and 8) is a membrane 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.
  • 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)).
  • M-CSF is expressed in stromal cells, osteoblasts, and other cells. It is also expressed in breast, uterine, and ovarian tumor cells. The extent of expression in these tumors conelates with high grade and poor prognosis (Kacinski Ann. Med. 27: 79-85 (1995); Smith et al., Clin. Cancer Res. 1: 313-25 (1995)). In breast carcinomas, M-CSF expression is prevalent in invasive tumor cells as opposed to the intraductal (pre-invasive) cancer (Sch ⁇ ll et al., J. Natl. Cancer hist. 86: 120-6 (1994)). In addition, M-CSF is shown to promote progression of mammary tumors to malignancy (Lin et al., J. Exp.
  • M-CSF For breast and ovarian cancer, the production of M-CSF seems to be responsible for the recruitment of macrophages to the tumor. It has been discoverd that M-CSF neutralizing antibody inhibits osteoclast induction by factors produced by metastatic type cancer cells (See the disclosure of, United States Serial No. 10/713, 895; inco ⁇ orated by reference in its entirety). It is expected that M- CSF muteins, via neutralizing active M-CSF activity through means such as blocking ligand- receptor interaction, will block osteoclast induction by metastatic cancer eels. Thus, the present invention provides compositions and methods for treating or preventing cancer metastasis and bone loss associated with cancer metastasis.
  • Tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include but are not limited to, carcinoma,; lymphoma, blastoma, sarcoma, and leukemia.
  • cancers include breast cancer, prostate cancer, colon cancer, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer.
  • 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.
  • a therapeutic agent may directly decrease the pathology of tumor cells, or render the tumor cells more susceptible to treatment by other therapeutic agents, e.g., radiation and/or chemotherapy.
  • the "pathology" of cancer includes all phenomena that compromise the well being of the patient. This includes, without limitation, abnormal or uncontrollable cell growth, metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels, suppression or aggravation of inflammatory or immunological response, etc.
  • the phrase "metastatic cancer” is defined as cancers that have potential to spread to other areas of the body, particularly bone.
  • cancers can metastasize to the bone, but the most common metastasizing cancers are breast, lung, renal, multiple myeloma, thyroid and prostate.
  • other cancers that have the potential to metastasize to bone include but are not limited to adenocarcinoma, blood cell malignancies, including leukemia and lymphoma; head and neck cancers; gastrointestinal cancers, including stomach cancer, colon cancer, colorectal cancer, pancreatic cancer, liver cancer; malignancies of the female genital tract, including ovarian carcinoma, uterine endometrial cancers and cervical cancer; bladder cancer; brain cancer, including neuroblastoma; sarcoma, osteosarcoma; and skin cancer, including malignant melanoma and squamous cell cancer.
  • M-CSF Muteins The invention provides M-CSF muteins that may be used as MCSF antagonists according to the methods of the invention.
  • Mutein as used herein with respect to polypeptides means a variant of the intact native molecule or a variant of a fragment of the native molecule, in which one or more amino acids have been substituted, inserted or deleted and that exhibit ability to inhibit M-CSF osteoclast-stimulating activity. Such substitutions, insertions or deletions can be at the N- terminus, C-terminus or internal to the molecule.
  • muteins includes within its scope fragments of the native molecule.
  • Insertional muteins include fusions at the N- or C- terminus, e.g. fusion to the Fc portion of an immunoglobulin to increase half-life.
  • “Mutein product” as used herein means a mutein or modification thereof that retains the desired activity.
  • Frament as used herein means a portion of the intact native molecule; for example, a fragment polypeptide is a fragment of the native polypeptide in which one or more amino acids from either the N-terminal or C-terminal have been deleted.
  • Prefened muteins according to the invention exhibit at least about 65%, 70%.
  • sequence identity 75%, 80%, 85%, 90%, 95%, 97% or more sequence identity (homology) to the native polypeptide, as determined by the Smith- Waterman homology search algorithm (Meth. Mol. Biol. 70:173-187 (1997)) as implemented in the MSPRCH program (Oxford Molecular) using an affine gap search with the following search parameters: gap open penalty of 12,-and gap extension penalty of 1.
  • Other well-known and routinely used homology/identity scanning algorithm programs include Pearson and Lipman, PNAS USA, 85:2444-2448 (1988); Lipman and Pearson, Science, 222:1435 (1985); Devereaux et al., Nuc.
  • Modification as used herein means any modification of the native polypeptide, fragment or mutein, such as glycosylation, phosphorylation, polymer conjugation (such as with polyethylene glycol), or other addition of foreign moieties, so long as the desired activity (agonist or antagonist) is retained.
  • FIG. 1 is a topology diagram showing the disulfide bonds in truncated dimeric M-CSF;
  • FIG. 2 is a stereodiagram of the C-alpha backbone with every tenth residue labelled and with the non-crystallographic symmetry axis indicated by a dotted line.
  • the overall topology of this form of M-CSF as shown in FIG. 1 is that of an antiparallel four alpha-helical bundle, in which the helices run up-up-down-down, unlike the more commonly observed up- down-up-down connectivity of most four helical bundles.
  • a long crossover connection links helix A to helix B and a similar connection is found between helices C and D.
  • the disulfide- linked dimeric form the bundles are linked end-to-end, forming an extremely flat, elongated structure (approximate dimensions 85 x 35 x 25 ).
  • There are three intramolecular disulfide bonds in each monomer (Cys7-Cys90, Cys48-Cysl39, Cysl02-Cysl46) all of which are at the distal end of the molecule.
  • One interchain disulfide bond (Cys31— Cys31) is located at the dimer interface with the noncrystallographic two-fold symmetry axis passing through it as shown in FIG. 2.
  • M-CSF ⁇ has intrachain disulfide bonds involving cysteines 157 and/or 159
  • the C-terminal region of M-CSF likely extends from the "rear" of the structure, providing a variable-length "tether" for membrane-bound forms of M-CSF.
  • the "front" or receptor-binding region of M-CSF is on the opposite side of the molecules, consisting of solvent-accessible residues in or near helices A, C, and D, including residues from about 6 to 26, 71 to 90, and 110 to 130, respectively, of native M-CSF. Altering solvent accessible residues in these regions by site directed mutagenesis to increase or decrease side-chain interactions with the receptor may generate M-CSF agonists or antagonists.
  • Residues having a solvent accessible surface area of greater than about 0.25 and preferably greater than about 0.4 are prefened based on normalization of the surface area of the amino acid accessible when in the trypeptide gly-x-gly (Kabsch, W. et al., Biopolymers 22:2577 (1983)).
  • residues are chosen which do not interact with other parts of the protein such as the dimer interface in order to maintain the relative orientation of monomers and to avoid disturbing the process of protein folding.
  • An optional additional consideration is selecting residues not conserved between human and mouse M-CSF, which does not recognize the human M-CSF receptor.
  • Candidate amino acids are preferably selected for substitution with non-conservative amino acids, so as to disrupt hydrogen bonding and/or hydrophobic interactions with MCSF-R residues. For example, changing one or more histidines to non-hydrogen-donor amino acids of similar size may create an M-CSF with altered receptor binding ability.
  • Prefened amino acids for substitution include but are not limited to: H15; Q79; R86; El 15; E41; K93; D99; L55; S18; Q20; 175; V78; L85; D69; N70; H9; N63; and T34.
  • M-CSF residues important in receptor signaling are believed to be composed of discontinuous regions of M-CSF.
  • a double mutant of M-CSF (Q20A, V78K) was constructed to test the importance of solvent accessible residues in the central portion of helices A and C.
  • This double mutein had slightly lower (8-10 fold) biological activity and conespondingly lower receptor-binding activity.
  • Mutagenesis of residues Q17, R21, E115 and El 19 changed side chain properties of solvent-accessible amino acids in the areas of interest but did not affect biological specific activity, suggesting that these residues need not be altered in muteins designed to have antagonist activity.
  • the invention contemplates M-CSF muteins in which residues of helices A and/or C and/or D involved in receptor-binding (for example, amino acids 6 to 26, 71 to 90 and/or 110 to 130) have been mutated non-conservatively.
  • Such muteins preferably retain at least 65%, 70%, 75%, 80%, 85% or 90% similarity (i.e. amino acids that are identical or have similar properties) to the native sequence within helices A, C or D, but have higher similarity to the native sequence in the remainder of the polypeptide, e.g. , at least 95%, 98% or 99% similarity.
  • the M-CSF mutein is a monomeric form of M-CSF.
  • the dimeric form of M-CSF is the biologically active form, and monomeric forms of M-CSF are generally not active. Disulfide bonding of the monomers appears to occur through the Cys31- Cys31 interchain linkage.
  • monomeric forms of M-CSF may be suitable for use as antagonists.
  • Such forms include muteins comprising cysteine deletions and/or cysteine replacements (e.g., cysteine to alanine substitutions) of Cys31 and/or other cysteines, or muteins in which the cysteine(s), particularly Cys31, have been chemically modified so that they are not available for disulfide bonding.
  • the M-CSF mutein comprises one or more of helices
  • Muteins containing any desired conservative and/or non-conservative muteins are readily prepared using techniques well known in the art, including recombinant production or chemical synthesis. Conservative substitutions, particularly substitutions outside of regions directly involved in ligand-receptor binding, are not expected to significantly change the binding properties of the M-CSF muteins (or M-CSFR muteins).
  • Amino acids can be classified according to physical properties and contribution to secondary and tertiary protein structure. A conservative substitution is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties.
  • M-CSF Modifications to the primary sequence of M-CSF can be made by deletion, addition, or alteration of the amino acids encoded by the DNA sequence without destroying the desired structure (e.g., the receptor binding ability of M-CSF) in accordance with well-known recombinant DNA techniques. Further, a skilled artisan will appreciate that individual amino acids may be substituted or modified by oxidation, reduction or other modification, and the polypeptide may be cleaved to obtain fragments that retain the active binding site and structural information.
  • polypeptides having an amino acid sequence which falls within the definition of polypeptide "having substantially the same amino acid sequence as the mature M-CSF ⁇ (SEQ ID NO: 2), M-CSF ⁇ (SEQ ID NO: 4), and M-CSF ⁇ (SEQ ID NO: 4)polypeptides."
  • Polypeptides may be synthesized using standard solution phase or solid phase peptide synthesis techniques known in the art. In solution phase synthesis, a wide variety of coupling methods and protecting groups may be used (see Gross and Meienhofer, eds., "The Peptides: Analysis, Synthesis, Biology," Vol.
  • the Boc strategy uses a 1% cross- linked polystyrene resin.
  • the standard protecting group for ⁇ -amino functions is the tert- butyloxycarbonyl (Boc) group. This group can be removed with dilute solutions of strong acids such as 25% trifluoroacetic acid (TFA).
  • the next Boc-amino acid is typically coupled to the amino acyl resin using dicyclohexylcarbodiimide (DCC).
  • DCC dicyclohexylcarbodiimide
  • the peptide-resin is treated with anhydrous HF to cleave the benzyl ester link and liberate the free peptide.
  • Side-chain functional groups are usually blocked during synthesis by benzyl-derived blocking groups, which are also cleaved by HF.
  • the free peptide is then extracted from the resin with a suitable solvent, purified and characterized.
  • Newly synthesized peptides can be purified, for example, by gel filtration, HPLC, partition chromatography and/or ion-exchange chromatography, and may be characterized by, for example, mass spectrometry or amino acid sequence analysis.
  • Boc strategy C-terminal amidated peptides can be obtained using benzhydrylamine or methylbenzhydrylamme resins, which yield peptide amides directly upon cleavage with HF.
  • C-amidation is accomplished using an appropriate resin such as methylbenzhydrylamme resin using the Boc technology.
  • modifications of the genes encoding the M-CSF polypeptide are readily accomplished by a variety of well-known techniques, such as site-directed mutagenesis (see, Gillman and Smith, Gene 8:81-97 (1979) and Roberts, S. et al., Nature 328:731-734 (1987) and U.S. Pat. No. 5,032,676, all of which are inco ⁇ orated herein by reference). Most modifications are evaluated by screening in a suitable assay for the desired characteristic.
  • a change in the M-CSF receptor-binding character of the polypeptide can be detected by competitive assays with an appropriate reference polypeptides or by the bioassays described in U.S. Pat. No. 4,847,201, which is inco ⁇ orated herein by reference.
  • Insertional variants of the present invention are those in which one or more amino acid residues are introduced into a predetermined site in the M-CSF.
  • insertional variants can be fusions of heterologous proteins or polypeptides to the amino or carboxyl terminus of the subunits.
  • substitutional variants are those in which at least one residue has been removed and a different residue inserted in its place.
  • Non-natural amino acids i.e., amino acids not normally found in native proteins
  • isosteric analogs amino acid or otherwise
  • suitable substitutions are well known in the art, such as the Glu->Asp, Ser->Cys, and Cys->Ser, His->alanine for example.
  • Another class of variants are deletional variants, which are characterized by the removal of one or more amino acid residues from the M-CSF.
  • Other variants of the present invention may be produced by chemically modifying amino acids of the native protein (e.g., diethylpyrocarbonate treatment which modifies histidine residues). Prefened or chemical modifications which are specific for certain amino acid side chains.
  • Chemical modification includes such reactions as oxidation, reduction, amidation, deamidation, or substitution of bulky groups such as polysaccharides or polyethylene glycol (see e.g., U.S. Pat. No. 4,179,337 and WO91/21029 both of which are inco ⁇ orated herein by reference).
  • Exemplary modifications include the modification of lysinyl and amino terminal residues by reaction with succinic or other carboxylic acid anhydrides. Modification with these agents has the effect of reversing the charge of the lysinyl residues.
  • Suitable reagents for modifying 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, and N- hydroxysuccinamide esters of polyethylenene glycol or other bulky substitutions.
  • Arginyl residues may be modified by reaction with a number of reagents, including phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin.
  • arginine residues require 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 also be modified with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosyl residues may also be iodmated using I or I to prepare labeled proteins for use in radioimmunoassay.
  • Carboxyl side groups may be selectively modified by reaction with carbodiimides (R— N.dbd.C.dbd.N— R.sup.l), where R and R] are different alkyl groups, such as l-cyclohexyl-3-(2-mo ⁇ holinyl-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-mo ⁇ holinyl-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 may be deamidated to the conesponding glutamyl and aspartyl residues, respectively, under mildly acidic conditions. Either form of these residues falls within the scope of this invention.
  • Other modifications include hydroxylation of pro line and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the ⁇ -amino groups of lysine, arginine, and histidine side chains (T. E. Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman & Co., San Francisco, pp.
  • Cystein caping may alos be employed to modify the M-CSF polypeptide.
  • sulfhydryl groups may be selectively modified by reaction with Iodoacetamides (alkyl halide or haloacetamide) or Maleimides (N-ethyulmaeimide (NEM) or 7- diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin CPM).
  • Iodoacetamides alkyl halide or haloacetamide
  • Maleimides N-ethyulmaeimide (NEM) or 7- diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin CPM.
  • NEM N-ethyulmaeimide
  • 7- diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin CPM Different alkyl groups are attached to the protein, resulting in a stable
  • a number of methods can be used to determine the similarity of M-CSF muteins to the native M-CSF protein. For example, percent homology is calculated as the percentage of amino acid residues in the smaller of two sequences which align with identical amino acid residue in the sequence being compared, when four gaps in a length of 100 amino acids may be introduced to maximize alignment (Dayhoff, in Atlas of Protein Sequence and Structure, Vol. 5, p. 124, National Biochemical Research Foundation, Washington, D.C. (1972), inco ⁇ orated herein by reference).
  • Sequence alignment of polypeptides for pu ⁇ oses of sequence comparison also can be done using a variety of multiple alignment servers, most of which are presently available on the Internet, e.g., Clustal W, MAP, PLMA, Block Maker, MSA, MEME, and Match-Box.
  • Clustal W Higgins et al., Gene (1988) 73:237-244; Higgins et al.,
  • Meth. Enzymol.(1996) 266:383-402) is employed for sequence alignment of polypeptides (and also, polynu ' cleotides).
  • the program BLASTP compares an amino acid query sequence against a protein database
  • TBLASTN compares a protein query sequence against a nucleotide sequence database dynamically translated in all six reading frames (both strands), and can be employed in the invention. Determinations of whether two amino acid sequences are substantially homologous (i.e., similar or identical) can also be based on FASTA searches in accordance with Pearson et al, Proc. Natl. Acad. Sci. USA 85:2444-2448 (1988).
  • prefened methods to determine identity and/or similarity are designed to give the largest match between the sequences tested.
  • Methods to determine identity and similarity are codified in publicly available computer programs (e.g., such as those previously described).
  • Prefened computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package, including GAP (Devereux et al, Nucleic Acids Research (1984) 12(1):387; Genetics Computer Group, University of Wisconsin, Madison, WI), BLASTP, BLASTN, and FASTA (Altschul et al, J. Molec. Biol. (1990) 215:403-410).
  • the BLAST X program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (Altschul et al., BLAST Manual, NCB NLM NIH Bethesda, MD 20894; Altschul et al, J. Mol. Biol. (1990) 215 :403 -410).
  • NCBI National Center for Biotechnology Information
  • the well known Smith Waterman algorithm may also be used to determine identity.
  • the relatedness of proteins can also be characterized through the relatedness of their encoding nucleic acids. Methods to determine identity and/or similarity of polynucleotide sequences are described above. In addition, methods to determine similarity of polynucleotide sequences through testing their ability to hybridize under moderately or highly stringent ⁇ conditions may be determined as follows. Exemplary moderately stringent hybridization conditions are as follows: hybridization at 42°C in a hybridization solution comprising 50% formamide, 1% SDS, 1 M NaCl, 10% Dextran sulfate, and washing twice for 30 minutes at
  • hybridization conditions can be calculated as described in Sambrook et al., (Eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York (1989), pp. 9.47 to 9.51.
  • Gene Therapy Delivery of a therapeutic protein 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., Hposomes or chemical treatments) or by use of viral vectors (e.g., adenovims, adeno-associated vims, or a retrovirus).
  • physical DNA transfer methods e.g., Hposomes or chemical treatments
  • viral vectors e.g., adenovims, adeno-associated vims, or a retrovirus.
  • a nucleic acid encoding the desired protein may be injected directly into the subject, and in some embodiments, may be injected at the site where the expression of the protein 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.
  • the techniques vary depending upon whether the nucleic acid is transfened into: cultured cells in vitro, or in vivo in the cells of the intended host.
  • Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of hposomes, electroporation, microinjection, cell fusion, DEAE-dextran, and calcium phosphate precipitation.
  • a commonly used vector for ex vivo delivery of a nucleic acid is a retrovirus.
  • Other in vivo nucleic acid transfer techniques include transfection with viral vectors (such as adenovirus, He ⁇ es simplex I vims, or adeno-associated vims) and 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. USA 84, 7413-7417; Malone et al. (1989) Proc. Natl Acad. Sci.
  • metabolizable quaternary ammonium salts (DOTB, N-(l-[2,3-dioleoyloxy]propyl)- N,N,N-trimethylammonium methylsulfate (DOTAP)(Boehringer Mannheim), polyethyleneimine (PEI), dioleoyl esters, ChoTB, ChoSC, DOSC)(Leventis et al. (1990) Biochim. Inter.
  • DOSC metabolizable quaternary ammonium salts
  • CTAB cetyltrimethylammonium bromide
  • DOPE dimethylammonium bromide
  • DEBDA didodecylammonium bromide
  • DDAB didodecylammonium bromide
  • stearylamine in admixture with phosphatidylethanolamine
  • Rose et al., (1991) Biotechnique 10, 520-525 DDAB/DOPE (TransfectACE, GIBCO BRL), and oligogalactose bearing lipids.
  • 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.
  • a candidate mutein may first be characterized in a cultured cell system to determine its ability to neutralize M-CSF in inducing osteoclastogenesis.
  • a cultured cell system may include the co-culture of mouse calvarial osteoblasts and spleen cells (Suda et al., Modulation of osteoclast differentiation. Endocr. Rev. 13: 66 80, 1992; Martin and Udagawa, Trends Endocrinol. Metab.
  • mice 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 manow cells peripheral blood mononuclear cells or alveolar macrophages
  • multinucleated cells fonned in such co-cultures satisfy the major criteria of osteoclasts such as tartrate resistant acid phosphatase (TRAP, a marker enzyme of osteoclasts) activity, calcitonin receptors, p60C-STC, vitronectin receptors, and the ability to form reso ⁇ tion pits on bone and dentine slices.
  • TRIP tartrate resistant acid phosphatase
  • calcitonin receptors calcitonin receptors
  • p60C-STC calcitonin receptors
  • p60C-STC vitronectin receptors
  • the ability of a candidate M-CSF mutein in inhibiting osteoclastogenesis may be assayed in a stromal cell-free or osteoblast-free system.
  • the M-CSF required for osteoclastogenesis may be provided by co-cultured metastatic cancer cells (e.g., MDA 231) or conditioned medium from these cancer cells (Mancino et al., J. Surg. Res. 0: 18-24, 2001) or by addition of purified M-CSF. Efficacy of a given M-CSF mutein in preventing or treating bone loss associated with cancer metastasis may also be tested in any of the animal bone metastasis model systems familiar to those skilled in the art. Such model systems include those involving direct injection of tumor cells into the medullary cavity of bones (Ingall, Proc. Soc. Exp. Biol. Med., 117: 819- 22, 1964; Falasko, Clin. Orthop.
  • osteolytic bone metastases formed from injected tumor cells may be determined by radiographs (areas of osteolytic bone lesions) or histochemistry (bone and soft tissues). Sasaki et al., Cancer Res. 55: 3551 7, 1995; Yoneda et al, J. Clin. Invest. 99: 2509 17, 1997. Clohisy and Ramnaraine, Orthop Res.
  • M-CSF muteins of the present invention may also be useful in preventing or treating cancer metastasis.
  • the effectiveness of a candidate M-CSF mutein in preventing or treating cancer metastasis may be screened using a human amnionic basement membrane invasion model as described in Filderman et al., Cancer Res 52: 36616, 1992.
  • any of the animal model systems for metastasis of various types of cancers may also be used.
  • Such model systems include, but are not limited to, those described in Wenger et al., Clin. Exp. Metastasis 19: 169 73, 2002; Yi et al., Cancer Res. 62: 917 23, 2002; Tsutsumi et al., Cancer Lett 169: 77-85, 2001; Tsingotjidou et al, Anticancer Res. 21: 971 8, 2001; Wakabayashi et al., Oncology 59: 75 80, 2000; Culp and Kogerman, Front Biosci. 3:D672 83, 1998; Runge et al, Invest Radiol. 32: 212 7; Shioda et al., J. Surg. Oncol.
  • cancer metastases may be prevented, or inhibited to result in fewer and/or smaller metastases.
  • the anti-tumor activity of a particular M-CSF mutein, or combination of M-CSF antagonists may be evaluated in vivo using a suitable animal model. For example, xenogenic lymphoma cancer models wherein human lymphoma cells are introduced into immune compromised animals, such as nude or SCLD mice.
  • the invention provides a method comprising the steps of (a) contacting an immobilized M-CSFR polypeptide with a candidate M-CSF mutein and (b) detecting binding of the candidate M-CSF mutein to the M-CSFR polypeptide.
  • the candidate M-CSF mutein is immobilized and binding of M-CSFR . polypeptide 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.
  • Methods of the invention to identify M-CSF muteins include variations on any of the methods described above, the variations including techniques wherein a M-CSF mutein is identified where binding between M-CSF and M-CSFR polypeptides changes in the presence of the candidate M-CSF mutein compared to binding in the absence of the candidate M-CSF mutein.
  • a M-CSF mutein that increases binding between a M-CSF and M-CSFR polypeptide is described as an enhancer or activator
  • a M-CSF mutein that decreases binding between a M- CSF and M-CSFR polypeptide is described as an inhibitor.
  • the invention also comprehends high throughput screening (HTS) assays to identify compounds that interact with or inhibit biological activity (i.e., inhibit enzymatic activity, binding activity, etc.) of a M-CSF polypeptide to a M-CSFR 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 and M-CSFR polypeptides.
  • 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 the M-CSF-M-CSFR polypeptides.
  • Another aspect of the present invention is directed to methods of identifying M- CSF muteins that bind to a M-CSFR polypeptide, comprising contacting a M-CSFR polypeptide with a M-CSF mutein, and determimng whether the M-CSF mutein binds the M-CSFR polypeptide.
  • Binding can be determined by binding assays which are well known to the skilled artisan, including, but not limited to, gel-shift assays, Western blots, radiolabeled competition assay, phage-based expression cloning, co-fractionation by chromatography, co-precipitation, cross linking, interaction trap/two-hybrid analysis, southwestern analysis, ELISA, and the like, which are described in, for example, Cunent Protocols in Molecular Biology (1999) John Wiley & Sons, NY, which is inco ⁇ orated herein by reference in its entirety.
  • the M-CSFR polypeptide employed in such a test may either be free in solution, attached to a solid support, borne on a cell surface or located intracellularly or associated with a portion of a cell.
  • One skilled in the art can, for example, measure the formation of complexes between a M-CSFR polypeptide and the M- CSF mutein being tested.
  • one skilled in the art can examine the diminution in complex formation between a M-CSF and M-CSFR polypeptide caused by the M-CSF mutein being tested.
  • Another aspect of the present invention is directed to methods of identifying compounds which modulate (i.e., decrease) activity of a M-CSFR polypeptide comprising contacting a M-CSFR polypeptide with a M-CSF mutein, and determining whether the M-CSF mutein modifies activity of the M-CSFR polypeptide.
  • the activity in the presence of the test M- CSF mutein is compared to the activity in the absence of the test M-CSF mutein. Where the activity of the sample containing the test M-CSF muteinis higher than the activity in the sample lacking the test M-CSF mutein, the compound will have increased activity.
  • the compound will have inhibited activity.
  • the present invention is particularly useful for screening M-CSF muteins by using the M-CSF and/or M-CSFR polypeptides in any of a variety of drag screening techniques.
  • the M-CSFR polypeptide employed in such a test may either be free in solution, attached to a solid support, borne on a cell surface or located intracellularly or associated with a portion of a cell.
  • One skilled in the art can, for example, measure the formation of complexes between a M- CSFR polypeptide and the M-CSF mutein being tested.
  • M-CSF M-CSFR polypeptide caused by the M-CSF mutein being tested.
  • a variety of heterologous systems is 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.
  • methods of screening for compounds which modulate the activity M-CSFR polypeptides comprise contacting test M-CSF muteins with a M-CSFR polypeptide and assaying for the presence of a complex between the M-CSF mutein and the M-CSFR polypeptide.
  • the M-CSF mutein is typically labeled. After suitable incubation, free M-CSF mutein 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 M-CSF mutein to bind to the M-CSFR polypeptide.
  • high throughput screening for M-CSF muteins having suitable binding affinity to a M-CSFR polypeptide is employed. Briefly, large numbers of different test M-CSF muteins are immobilzed on a solid substrate. The test M-CSF muteins are contacted with a M-CSFR polypeptide and washed. Bound M-CSFR polypeptides are then detected by methods well known in the art. Purified M-CSF muteins of the invention can also be coated directly onto plates for use in the aforementioned drag screening techniques. In addition, non-neutralizing antibodies can be used to capture the M-CSF mutein and immobilize it on the solid support.
  • an expressed M-CSF mutein can be used for HTS binding assays in conjunction with a substrate, ligand, adaptor or receptor molecule that is labeled with a suitable radioisotope, including, but not limited to, I, H, S or P, by methods that are well known t ⁇ those skilled in the art.
  • a suitable radioisotope including, but not limited to, I, H, S or P
  • the substrate, ligand, adaptor or receptor molecule may b ⁇ labeled by well-known methods with a suitable fluorescent derivative (Baindur, et al.', Drag De Res., (1994) 33:373-398; Rogers, Drag Discovery Today (1997) 2:156-160).
  • Radioactive ligan specifically bound to immobilized M-CSF mutein can be detected in HTS assays in one of several standard ways, including filtration of the M-CSF mutein-ligand complex to separate bound ligand from unbound ligand (Williams, Med. Res. Rev. (1991) 11, 147-184; Sweetnam, e al., J. Natural Products (1993) 56, 441-455).
  • Alternative methods include a scintillation proximity assay (SPA) or a FlashPlate foraiat in which such separation is unnecessary (Nakayama, Cur. Opinion Drag Disc. Dev. (1998) 1:85-91 Bosse, et al., J. Biomolecular Screening (1998) 3: 285-292.).
  • Binding of fluorescent ligands can be detected in various ways, including fluorescence energy transfer (FRET), direct spectrophotofluorometric analysis of bound ligand, or fluorescence polarization (Rogers, Drug Discovery Today (1997) 2, 156-160; Hill, Cur. Opinion Drug Disc. Dev. (1998) 1, 92-97).
  • FRET fluorescence energy transfer
  • the invention contemplates a multitude of assays to screen and identify inhibitor of substrate, ligand, adaptor or receptor binding to M-CSFR.
  • M-CSF or M- CSFR is immobilized and interaction with a binding partner is assessed in the presence and absence of a candidate M-CSF mutein.
  • M-CSF and M- CSFR interaction between M-CSF and M- CSFR is assessed in a solution assay, both in the presence and absence of a candidate M-CSF mutein.
  • an inhibitor is identified as a M-CSF mutein that decreases binding between the M-CSF and M-CSFR.
  • Another contemplated assay involves a variation of the di- hybrid assay wherein an inhibitor of protein/protein interactions is identified by detection of a positive signal in a transformed or transfected host cell, as described in PCT publication numbei WO 95/20652, published August 3, 1995.
  • assays may be used to identify specific ligands of M-CSFR, including assays that identify ligands of the M-CSFR through measuring direct binding of test M-CSF muteins to the target protein, as well as assays that identify ligands of target proteins through affinity ultrafiltration with ion spray mass spectroscopy HPLC methods or other physical and analytical methods.
  • binding interactions are evaluated indirectly using the yeast two-hybrid system described in Fields et al., Nature, 340:245-246 (1989), and Fields et al., Trends in Genetics, 10:286-292 (1994), both of which are inco ⁇ orated herein by reference.
  • the two-hybrid system is a genetic assay for detecting interactions between two proteins or polypeptides. It can be used to identify proteins that bind to a known protein of interest, or to delineate domains or residues critical for an interaction. Variations on this methodology have been developed to clone genes that encode DNA binding proteins, to identify peptides that bind to a protein, and to screen for drags.
  • the two-hybrid system exploits the ability of a pair of interacting proteins to bring a transcription activation domain into close proximity with a DNA binding domain that binds to an upstream activation sequence (UAS) of a reporter gene, and is generally performed in yeast.
  • UAS upstream activation sequence
  • the assay requires the construction of two hybrid genes encoding (1) a DNA-binding domain that is fused to a first protein and (2) an activation domain fused to a second protein.
  • the DNA-binding domain targets the first hybrid protein to the UAS of the reporter gene; however, because most proteins lack an activation domain, this DNA-binding hybrid protein does not activate transcription of the reporter gene.
  • the second hybrid protein which contains the activation domain, cannot by itself activate expression of the reporter gene because it does not bind the UAS. However, when both hybrid proteins are present, the noncovalent interaction of the first and second proteins tethers the activation domain to the UAS, activating transcription of the reporter gene.
  • this assay can be used to detect agents that interfere with the binding interaction.
  • Expression of the reporter gene is monitored as different test agents are added to the system. The presence of an inhibitory agent results in lack of a reporter signal.
  • the yeast two-hybrid assay can also be used to identify proteins that bind to M- CSFR.
  • a fusion polynucleotide encoding a M-CSFR (or subunit or fragment) and a UAS binding domain i.e., a first protein
  • a large number of hybrid genes each encoding a different second protein (i.e., M-CSF mutein) fused to an activation domain are produced and screened in the assay.
  • the second protein is encoded by one or more members of a total cDNA or genomic DNA fusion library, with each second protein-coding region being fused to the activation domain.
  • This system is applicable to a wide variety of proteins, and it is not even necessary to know the identity or function of the second binding protein.
  • the system is highly sensitive and can detect interactions not revealed by other methods; even transient interactions may trigger transcription to produce a stable mRNA that can be repeatedly translated to yield the reporter protein.
  • Other assays may be used to search for agents that bind to the target protein.
  • One such screening method to identify direct binding of test ligands to a target protein is described in U.S. Patent No. 5,585,277, inco ⁇ orated herein by reference. This method relies on the principle that proteins generally exist as a mixture of folded and unfolded states, and continually alternate between the two states.
  • a test ligand binds to the folded form of a target protein (i.e., when the test ligand is a ligand of the target protein), the target protein molecule bound by the ligand remains in its folded state.
  • the folded target protein is present to a greater extent in the presence of a test ligand which binds the target protein, than in the absence of a ligand. Binding of the ligand to the target protein can be determined by any method which distinguishes between the folded and unfolded states of the target protein. The function of the target protein need not be known in order for this assay to be performed.
  • Virtually any agent can be assessed by this method as a test ligand, including, but not limited to, metals, polypeptides, proteins, lipids, polysaccharides, polynucleotides and small organic molecules.
  • Other embodiments of the invention comprise using competitive screening assays in which neutralizing antibodies capable of binding a M-CSFR polypeptide of the invention specifically compete with a test M-CSF mutein for binding to the M-CSFR polypeptide. In this manner, the antibodies can be used to detect the presence of any peptide that shares one or more antigenic determinants with M-CSFR. Radiolabeled competitive binding studies are described in A.H. Lin et al. Antimicrobial Agents and Chemotherapy (1997) vol. 41, no. 10. pp.
  • the M-CSF muteins of the invention are employed as a research tool for identification, characterization and purification of interacting, regulatory proteins.
  • Appropriate labels are inco ⁇ orated into the M-CSF muteins of the invention by various methods known in the art and the polypeptides are used to capture interacting molecules. For example, molecules are incubated with the labeled M-CSF muteins, washed to removed unbound M-CSF muteins, and the M-CSF mutein complex is quantified.
  • M-CSFR polypeptides are also useful as reagents for the purification of molecules with which the polypeptide interacts including, but not limited to, M-CSF muteins.
  • affinity purification a polypeptide is covalently coupled to a chromatography column. Cells and their membranes are extracted, and various cellular subcomponents are passed over the column. Molecules bind to the column by virtue of their affinity to the polypeptide. The polypeptide-complex is recovered from the column, dissociated and the recovered molecule is subjected to protein sequencing.
  • This amino acid sequence is then used to identify the captured molecule or to design degenerate oligonucleotides for cloning the conesponding gene from an appropriate cDNA library.
  • Computer modeling can be used to develop a putative tertiary structure of the M- CSF muteins of the invention based on the available information of M-CSF or M-CSFR.
  • novel ligands based on the predicted structure of M-CSF or M-CSFR can be designed.
  • Another aspect of the present invention is the use of the M-CSF or M-CSFR nucleotide sequences disclosed herein for identifying homologs in other animals.
  • nucleotide sequences disclosed herein, or any portion thereof can be used, for example, as probes to screen databases or nucleic acid libraries, such as, for example, genomic or cDNA libraries, to identify homologs, using screening procedures well known to those skilled in the art. Accordingly, homologs having at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, and most preferably at least 100% homology with M-CSF or M-CSFR sequences can be identified.
  • Combination Therapy Having identified more than one M-CSF mutein that is effective in an animal model, it may be further advantageous to mix two or more such M-CSF antagonists together to provide still improved efficacy against cancer metastasis and/or bone loss associated with cancer metastasis.
  • Compositions comprising one or more M-CSF antagonist may be administered to persons or mammals suffering from, or predisposed to suffer from, cancer metastasis and/or bone loss associated with cancer metastasis.
  • M-CSF antagonist therapy may be useful for all stages of cancers, M- CSF mutein therapy may be particularly appropriate in advanced or metastatic cancers.
  • M-CSF mutein therapy a chemotherapeutic or radiation regimen may be prefened in patients that have not received chemotherapeutic treatment, whereas treatment with the M-CSF mutein therapy may be indicated for patients who have received one or more chemotherapies. Additionally, M-CSF mutein therapy can also enable the use of reduced dosages of concomitant chemotherapy, particularly in patients that do not tolerate the toxicity of the chemotherapeutic agent very well.
  • the method of the invention contemplate the administration of single M-CSF muteins, as well as combinations, or "cocktails", of different M-CSF muteins. Such M-CSF muteins in combination may exhibit synergistic therapeutic effects.
  • M-CSF muteins may be combined with other therapeutic agents and/or procedures, including but not limited to various chemotherapeutic agents, androgen-blockers, and immune modulators (e.g., IL-2, GM-CSF), Bisphosphonate(s) (e.g., Acedia; Zometa; Clodronate), surgery, radiation, chemotherapy, hormone therapy (e.g., Tamoxifen; anti- Androgen therapy), antibody therapy (e.g., RANKL/RANK neutralizing antibodies; PTHrP neutralizing antibody, anti-Her2 antibody, VEGF neutralizing antibody), therapeutic protein therapy (e.g., soluble RANKL receptor; OPG, and PDGF and MMP inhibitors), small molecule drug therapy (e.g., Src-kinase inhibitor), kinase inhibitors of growth factor receptors; oligonucleotides therapy (e.g., RANKL or RANK or PTHrP Anti-sense), gene therapy (
  • Cancer chemotherapeutic agents include, without limitation, alkylating agents, such as carboplatin and cisplatin; nitrogen mustard alkylating agents; nitrosourea alkylating agents, such as carmustine (BCNU); antimetabolites, such as methotrexate; purine analog antimetabolites, mercaptopurine; pyrimidine analog antimetabolites, such as fluorouracil (5-FU) and gemcitabine; hormonal antmeoplastics, such as goserelin, leuprolide, and tamoxifen; natural antineoplastics, such as aldesleukin, interleukin-2, docetaxel, etoposide (NP-16), interferon alfa, paclitaxel, and tretinoin (ATRA); antibiotic natural antmeoplastics, such as bleomycin, dactinomycin, daunora
  • antitumor platelet factors cyclophosphamide, Schizophyllan, cytarabine, dacarbazine, thioinosine, thiotepa, tegafur, , neocafzinostatin, OK-432, bleomycin, furtulon, broxuridine, busulfan, honvan, peplomycin, , Bestatin (ubenimex), interferon- ⁇ , mepitiostane, mitobronitol, me ⁇ halan, laminin peptides, lentinan, Coriolus versicolor extract, tegafur/uracil, estramustine (estrogen/mechlorethamine) .
  • additional agents used as therapy for cancer patients include EPO, G- CSF, ganciclovir; antibiotics, leuprolide; meperidine; zidovudine (AZT); interleukins 1 through 18, including mutants and analogues; interferons or cytokines, such as interferons ⁇ , ⁇ , and ⁇ hormones, such as luteinizing hormone releasing hormone (LHRH) and analogues and, gonadotropin releasing hormone (GnRH); growth factors, such as transforming growth factor- ⁇ (TGF- ⁇ ), fibroblast growth factor (FGF), nerve growth factor (NGF), growth hormone releasing factor (GHRF), epidermal growth factor (EGF), fibroblast growth factor homologous factor (FGFHF), hepatocyte growth factor (HGF), and insulin growth factor (IGF); tumor necrosis factor- ⁇ & ⁇ (TNF- ⁇ & ⁇ ); invasion inhibiting factor-2 (IIF-2); bone mo ⁇ hogenetic proteins 1- 7 (BMP
  • compositions can be in the form of, for example, granules, powders, tablets, capsules, syrup, suppositories, injections, emulsions, elixirs, suspensions or solutions.
  • the instant compositions can be formulated for various routes of administration, for example, by oral administration, by nasal administration, by rectal administration, subcutaneous injection, intravenous injection, intramuscular injections, or intraperitoneal injection.
  • the following dosage forms are given by way of example and should not be constraed as limiting the instant invention.
  • powders, suspensions, granules, tablets, pills, capsules, gelcaps, and caplets are acceptable as solid dosage forms. These can be prepared, for example, by mixing one or more compounds of the instant invention, or pharmaceutically acceptable salts or tautomers thereof, with at least one additive such as a starch' or other additive.
  • Suitable additives are sucrose, lactose, cellulose sugar, mannitol, maltitol, dextran, starch, agar, alginates, chitins, chitosans, pectins, tragacanth gum, gum arabic, gelatins, ' collagens, casein, albumin, synthetic or semi-synthetic polymers or glycerides.
  • oral dosage forms can contain other ingredients to aid in administration, such as an inactive diluent, or lubricants such as magnesium stearate, or preservatives such as paraben or sorbic acid, or anti-oxidants such as ascorbic acid, tocopherol or cysteine, a disintegrating agent, binders, thickeners, buffers, sweeteners, flavoring agents or perfuming agents. Tablets and pills may be further treated with suitable coating materials known in the art.
  • Liquid dosage forms for oral administration may be in the form of pharmaceutically acceptable emulsions, syrups, elixirs, suspensions, and solutions, which may contain an inactive diluent, such as water.
  • compositions and medicaments may be prepared as liquid suspensions or solutions using a sterile liquid, such as, but not limited to, an oil, water, an alcohol, and combinations of these.
  • a sterile liquid such as, but not limited to, an oil, water, an alcohol, and combinations of these.
  • Pharmaceutically suitable surfactants, suspending agents, emulsifying agents may be added for oral or parenteral administration.
  • suspensions may include oils.
  • oils include, but are not limited to, peanut oil, sesame oil, cottonseed oil, corn oil and olive oil.
  • Suspension preparation may also contain esters of fatty acids such as ethyl oleate, isopropyl myristate, fatty acid glycerides and acetylated fatty acid glycerides.
  • Suspension formulations may include alcohols, such as, but not limited to, ethanol, isopropyl alcohol, hexadecyl alcohol, glycerol and propylene glycol.
  • Ethers such as but not limited to, poly(ethyleneglycol), petroleum hydrocarbons such as mineral oil and petrolatum; and water may also be used in suspension formulations.
  • 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. Alternatively, sterile oils may be employed as solvents or suspending agents.
  • the oil or fatty acid is non-volatile, 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. Examples of these include, but are not limited to, freeze dried, rotary dried or spray dried powders, amo ⁇ hous powders, granules, precipitates, or particulates.
  • the formulations may optionally contain stabilizers, pH modifiers, surfactants, bioavailability modifiers and combinations of these.
  • the pharmaceutical formulations and medicaments may be in the form of a suppository, an ointment, an enema, a tablet or a cream for release of compound in the intestines, sigmoid flexure and/or rectum.
  • Rectal suppositories are prepared by mixing one or more compounds of the instant invention, or pharmaceutically acceptable salts or tautomers of the compound, with acceptable vehicles, for example, cocoa butter or polyethylene glycol, which is present in a solid phase at normal storing temperatures, and present in a liquid phase at those temperatures suitable to release a drug inside the body, such as in the rectum. Oils may also be employed in the preparation of formulations of the soft gelatin type and suppositories.
  • acceptable vehicles for example, cocoa butter or polyethylene glycol
  • suspension formulations which may also contain suspending agents such as pectins, carbomers, methyl cellulose, hydroxypropyl cellulose or carboxymethyl cellulose, as well as buffers and preservatives.
  • suspending agents such as pectins, carbomers, methyl cellulose, hydroxypropyl cellulose or carboxymethyl cellulose, as well as buffers and preservatives.
  • pharmaceutically acceptable excipients and canies 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 inco ⁇ orated herein by reference.
  • the formulations of the invention may be designed to be short-acting, fast- , releasing, long-acting, and sustained-releasing as described below.
  • the pharmaceutical formulations may also be formulated for controlled release or for slow release.
  • the instant compositions may also comprise, for example, micelles or hposomes, 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.
  • compositions comprising M-CSF muteins may be administered parenterally, topically, orally or locally for therapeutic treatment.
  • the compositions are administered orally or parenterally, i.e., intravenously, intraperitoneally, intradermally or intramuscularly.
  • compositions for administration which comprise one or more M-CSF antagonists in a pharmaceutically acceptable carrier, preferably an aqueous carrier.
  • a pharmaceutically acceptable carrier preferably an aqueous carrier.
  • 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.
  • M-CSF muteins useful as therapeutics for cancer metastasis or bone loss associated with cancer metastasis will often be prepared substantially free of other naturally occurring immunoglobulins or other biological molecules.
  • Prefened M-CSF muteins will also exhibit minimal toxicity when administered to a mammal afflicted with, or predisposed to suffer from, cancer metastasis and/or bone loss associated with cancer metastasis.
  • the 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.
  • 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 muteins of the present invention may also be administered via hposomes. Liposomes, which include emulsions, foams, micelles, insoluble monolayers, phospholipid dispersions, lamellar layers and the like, can serve as vehicles to target the M-CSF muteins to a particular tissue as well as to increase the half life of the composition.
  • 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.
  • the in vivo neutralizing activity of sera from a subject treated with a given dosage of M-CSF mutein may be evaluated using an assay that determines the ability of the sera to block M-CSF induced proliferation and survival of murine monocytes (CDl lb+ cell, a subset of CDl 1 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.
  • CDl lb+ cell murine monocytes
  • compositions of the invention are administered to a mammal already suffering from, or predisposed to, cancer metastasis and/or bone loss associated with cancer metastasis in an amount sufficient to prevent or at least partially anest the development of cancer metastasis and/or bone loss associated with cancer metastasis.
  • An amount adequate to accomplish this is defined as a "therapeutically effective dose.”
  • Effective amounts of a M-CSF mutein 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, with dosages of from about 10 ⁇ g/kg to about 10 mg kg per application being more commonly used.
  • compositions of the present invention are administered alone or as an adjunct therapy in conjunction with other therapeutics known in the art for the treatment of cancer metastasis and/or bone loss associated with cancer metastasis.
  • EXAMPLE 1 This example shows that highly metastatic breast cancer cell lines express high levels of M-CSF.
  • MDA 231 the M-CSF gene expression by the highly metastatic cell line, MDA 231 was compared with that of the cell lines MCF7 and ZR751.
  • MCF7 the highly metastatic cell line
  • CM conditioned media
  • MDA 231 or MCF7 cells were plated at a density of 1 x 10 6 cells/10 cm dish in 8 mis of 50% DMEM/ 50% HAMs F12 containing 1 x ITS, (BD Biosciences, Lexington, Ky), a culture supplement containing insulin, human transferrin, and selenous acid. After 48 hours of incubation at 37°C in 5% CO 2 , the media were collected and centrifuged for 10 minutes at 1500 RPM to remove any suspended cells.
  • Osteoclast assay Bone manow CD34 + cells were plated at a density of 15,000 cells/ 96 well in 100 ⁇ l of Alpha MEM containing 10% FCS, 1 X Pen/Strep and 1 x fungizone. The next day, 50 ⁇ l of media was removed from each well and replaced with 25 ⁇ l of Alpha MEM media and 75 ⁇ l of CM or 50% DMEM/ 50% HAMs F12 containing 1 x ITS. RANKL was added to each well at a final concentration of 100 ng/ml and 30 ng/ml M-CSF was added to the appropriate wells.
  • CM conditioned media
  • EXAMPLE 3 The following example provides a method for the design and production of M-
  • X-ray crystallographic data described in U.S. Patent No. 6,025,146 provides sufficient structural inforaiation regarding M-CSF to be able to identify a limited subset of the amino acids in the protein that are likely to be cracial for M-CSF receptor binding and biological activity and thus which represented likely candidates for mutagenesis with the ultimate goal of providing M-CSF muteins having altered biological activity (i.e., agonists or antagonists). Based on this information, several criteria are used to generate a list of possible target amino acids for substitution. The first criterion is solvent exposure or solvent accessibility, which refers to amino acids residues at the surface of the protein.
  • Residues having a solvent accessible surface area of greater than about 0.25 and preferably greater than about 0.4 are prefened based on normalization of the surface area of the amino acid accessible when in the trypeptide gly-x-gly (Kabsch, W. et al., Biopolymers 22:2577 (1983)). Residues are chosen which do not interact with other parts of the protein such as the dimer interface in order to maintain the relative orientation of monomers and to avoid disturbing the process of protein folding. Still another criterion used in certain instances in selecting candidate amino acid substances is the relationship of the residues to conesponding residues in mouse M-CSF.
  • M-CSF muteins can be carried essentially as described in U.S. Patent No. 6,025,146. Briefly, a variety of M-CSF muteins with altered solvent-accessible residues from regions of the M-CSF mature N terminus and helices A, C, and D are constructed using techniques known in the art. For example, two histidines in the N-terminal/A helix region are changed to alanine through site-directed mutagenesis of a truncated form of M-CSF ⁇ (encoded by pLCSFl 58A).
  • Plasmid DNA pLCSF158A is prepared from the E. coli strain HW22 carrying the plasmid pLCSF158A (U.S. Pat. No. 4,929,700, Example 6, "E. coli strain HW22 transformed with pJN653 containing the asp.sub.59 SCSF/N.DELTA.3C.DELTA.158 gene").
  • the strain is grown in 350 ml R2 media (2x Luria Broth containing 1% sodium chloride and no glucose, J. Bact, 74:461 (1957)) containing 50 micrograms/ml ampicillin at 30 °C with shaking overnight.
  • Plasmid DNA is prepared from the cells using a Qiagen-tip 100 column according to the manufacturer's directions. Twenty micrograms of pLCSF158A DNA is digested with 66 units of Hindlll and 66 units of Stul at 37 °C for 3 hr. 20 min. in 200 microliters lx New England Biolabs NEBuffer 2 (New England Biolabs, Beverly, Mass.). The DNA is extracted with phenol and chloroform, then ethanol precipitated.
  • the DNA is treated with one unit of Calf Intestinal Alkaline Phosphatase in 100 microliters of lx Dephosphorylation Buffer, supplied by Boebringer Mannheim (Indianapolis, Ind.), at 37 °C for 30 min. An additional unit of Calf Intestinal Alkaline Phosphatase is added to the reaction and incubation was continued at 50 °C for 1 hr. The resulting DNA is then ran on a 1% FMC Bioproducts (Rockland, Me.) Sea KEM.RTM. GTG.RTM. agarose gel.
  • telomere sequence The 5.7 kb pLCSF158A fragment is cut from the gel and purified on Qiagen (Chatsworth, Calif.) Qiaex beads according to the manufacturer's directions. Polymerase chain reaction (PCR) is then performed and a PCR product is produced that contained a mutagenized M-CSF sequence in which, for example, histidines 9 and
  • Positive reactions are pooled, extracted with phenol and chloroform, precipitated with ethanol, resuspended and digested with 250 units of Stul in a final volume of 500 microliters lx NEBuffer 2 at 37 °C for 2 hr., 500 units of Hindlll are added to the reaction, the volume increased to 1 ml in lx NEBuffer 2 and digestion was continued at 37 °C for an additional 2.5 hr.
  • the DNA is electrophoresed on a 3% agarose gel.
  • the digested product is cut from the gel and purified on Qiagen Qiaex beads according to the manufacturer's directions.
  • PCR product is then ligated to pLCSF158A vector DNA at an insert-to- vector ratio of approximately 5:1.
  • Ligation is carried out with 1 unit of Boehringer Mannheim T4 DNA ligase in lx ligation buffer, supplied by the manufacturer, in a 20-microliter volume at
  • the M-CSF muteins can be expressed, purified, refolded to form dimeric protein and assayed essentially as described in U.S. Pat. No. 4,929,700, Example 5, using 8M urea as a denaturant and in the DEAE purification step.
  • EXAMPLE 4 This example provides a method for testing M-CSF muteins for their ability to bind M-CSFR.
  • An essential step in the biological function of M-CSF in vivo is the binding to the M-CSF receptor, also refened to as the c-fms gene product.
  • Recombinant human soluble M- CSF receptor (rhsM-CSFR) representing amino acids 20 to 511 of SEQ ID NO: 8 (Coussens, L et al., Nature, 320:277 (1986)) is used as an in vitro assay reagent to test the receptor-binding ability of M-CSF proteins.
  • tags may be added to the C-terminus of the recombinant receptor, i.e., KT3 antibody recognition sequence, and purified by an anti-tag antibody, i.e., KT3, column, for use in affinity chromatography.
  • lectin chromatography can be used to enrich for specific glycoproteins.
  • the rhsM-CSFR can be used to study ligand/receptor interactions as well as ligand-induced receptor dimerization.
  • the assay used to detect ligand/receptor binding employs the use of size exclusion-HPLC, essentially as described in European Patent Application WO92/21029, C.
  • the column used is a Superose 6 (Pharmacia LKB Biotechnology, Inc.) and the mobile phase is PBS at 0.5 ml/min and a M-CSF to rhsM-CSFR ratio of 1 :2. At this ratio, the M-CSF/rhsM-CSFR complex chromato graphs with an expected hydrodynamic radius of 190,00 molecular weight.
  • Other assays may be employed to measure ligand/receptor binding or receptor dimerization such as chemical crosslinking and SDS-PAGE or immunoprecipitation and SDS-PAGE. Molecules that inhibit receptor dimerization but not ligand binding provide another method to antagonize M- CSF actions.
  • EXAMPLE 5 The following example provides a method for testing M-CSF muteins for their ability to interfere with activation of M-CSFR by native M-CSF. Competition for binding M-CSFR between native M-CSF and a candidate M- CSF mutein is accomplished with a modification of the binding assays described in Example 4. Briefly, the size-exclusion HPLC method described above is carried out in the presence and absence of a candidate M-CSF mutein. M-CSF muteins that have the ability to interefere with the M-CSFR/M-CSF intereaction are then identified.
  • EXAMPLE 6 This example provides a method for testing M-CSF muteins for in vitro activity. To measure the neutralizing ability of the M-CSF muteins against the activity of human M-CSF on murine M NFS 60 cells (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 ecofropic 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), recombinant human CSF-1 (at 10 ng/ml final concentration) is pre- incubated with various concentrations (1 ng/ml to lmg/ml) of a candidate M-CSF mutein for 1 hour at 37°C in 5% CO 2 in an incubator.
  • the mixture is added to the M NFS 60 culture in 96 well microtiter plates.
  • the total assay volume per well is lOO ⁇ l, with 10 ng/ml rhM-CSF, and cell density at 5,000 cells/well.
  • cell proliferation is assayed by CellTiter Glo Kit (Promega). It is expected that mixtures containing M-CSF mutein will inhibit M-CSF- induced M NFS 60 cell proliferation.
  • EXAMPLE 7 The following example provides a method for testing muteins for in vivo activity.
  • Experimental Design To evaluate the efficacy of M-CSF muteins as a therapeutic agent for the treatment of osteolysis, the highly metastatic human breast cancer cell line MDA-231 (3 x 10 5 ) was injected into the tibia bone manow cavity of female nude mice. The mice were at the age of 4-7 weeks old, and had an average weight of ⁇ 20g. The mice were chipped for identification and underwent an acclimation period of at least 7 days prior to the start of the 8-week study. The mice received a total dose of 1.5mpk (0.03mg per mouse) Bupreno ⁇ hine subcutaneously at both flank 30 minutes before intra-tibia injection.
  • mice were anesthetized by isoflurane inhalation and the right hind leg was cleaned with 70% ethanol.
  • Tumor cells (MDA- MB-231-luc, 3xl0 5 ) suspended in 10 ⁇ l of saline was injected into the right tibia bone manow cavity using 50 or 100 ⁇ l micro-syringe.
  • Dosing Treatment with M-CSF mutein begins the day following the injection of the tumor cells. The dosing will be a range from 0.1 mg/kg to 50 mg/kg. An example of the highest dosing value, 50 mg/kg, is as follows.
  • the volume injected is increased or decreased by 5 ⁇ l per gram of weight difference.
  • a 23 . gram mouse receives 115 ⁇ l, while an 18 gram mouse receives 90 ⁇ l.
  • each mouse receives a baseline Faxitron image taken the day following injection of tumor cells. Additionally, a Faxitron image is taken at the end of the study (8 weeks). Tumor growth is simultaneously measured using the Xenogen system since the tumor cells stably express luciferase.
  • the number of animals with a mean osteolysis score of > 2.5 is lowest in the group that received the M-CSF mutein treatment: Osteolytic bone damage is evaluated on a scale from 0-4, with 0 equal to no bone damage; 1-2 equal to some bone damage, such that scores of 2.25 or greater is indicative of severe bone damage.

Abstract

La présente invention a trait à des mutéines M-CSF, ainsi qu'à des compositions pharmaceutiques contenant une mutéine M-CSF, à des trousses contenant une composition pharmaceutique, à des procédés de prévention et de traitement de métastases osseuses chez un sujet atteint de cancer métastatique, et à des procédés de criblage pour de mutéines M-CSF.
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US10119171B2 (en) 2012-10-12 2018-11-06 Inbiomotion S.L. Method for the diagnosis, prognosis and treatment of prostate cancer metastasis
CN106795222A (zh) 2014-06-23 2017-05-31 戊瑞治疗有限公司 用结合集落刺激因子1受体(csf1r)的抗体治疗病状的方法
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