MX2008008570A - Methods for preventing and treating cancer metastasis and bone loss associated with cancer metastasis - Google Patents

Abstract

Methods for preventing and treating osteolysis, cancer metastasis and bone loss associated with cancer metastasis by administering an M-CSF-antagonist in combination with a therapeutic agent to a subject are provided.

Description

METHODS FOR THE PREVENTION AND TREATMENT OF CANCERING METASTASES AND LOSS OF ASSOCIATED BONE WITH METASTASIS CANCEROSA TECHNICAL FIELD This invention relates to methods for the prevention and treatment of osteolytic diseases, including cancer metastasis and bone loss associated with cancer metastasis by administering an M-CSF antagonist in combination with another therapeutic agent. An individual.

BACKGROUND OF THE INVENTION Osteoclasts, which mediate bone resorption, are involved in normal and abnormal bone remodeling processes, including osteolytic diseases. Osteoclasts are multinuclear cells that differ from hematopoietic cells. It is generally accepted that osteoclasts are formed by the fusion of mononuclear precursors derived from hematopoietic stem cells in the bone marrow, instead of incomplete cell divisions (Chamber, Bone and Mineral Research, 6: 1-25, 1989; Góthling et al. al., Clin Orthop Relat R. 120: 201-228, 1976; Kahn et al., Nature 258: 325-327, 1975, Suda et al., Endocr Rev 13: 66-80, 1992; Walter, Science 180: Endocr Rev 13: 66-80, 1992; Walker, Science 180: 875, 1973; Alker, Science 190: 785-787, 1975; Walker, Science 190: 784-785, 1975). They share a common primordial cell with monocyte-macrophage lineage cells (Ash et al., Nature 283: 669-670, 1980, Kerby et al., J. Bone Miner Res 7: 353-62, 1992). The differentiation of osteoclast precursors into mature, multinucleated osteoclasts requires different factors including hormonal and local stimuli (Athanasou et al., Bone Miner 3: 317-333, 1988, Feldman et al., Endocrinology 107: 1137-1143, 1980; Walker, Science 190: 784-785, 1975; Zheng et al., Histochem J 23: 180-188, 1991) and living bone and bone cells have been shown to play an important role in the development of osteoclasts (Hagenaars et al. , Bone Miner 6: 179-189, 1989). Osteoblastic or bone marrow stromal cells are also required for the differentiation of osteoclasts. One of the factors produced by these cells that supports the formation of osteoclasts is the macrophage colony stimulating factor, M-CSF (Wiktor-Jedrze czak et al, Proc Nati Acad Sci USA 87: 4828-4832, 1990; Yoshida et al. al., Nature 345: 442-444, 1990). The receptor activator for the ligand NF-? B (RANKL, also known as TRANCE, ODF and OPGL) is another signal (Suda et al., Endocr Rev 13: 66-80, 1992) through which osteoblastic / stromal cells stimulate the formation of osteoclasts and resorption through a receptor, RANK (TRANCER), located on the osteoclasts and precursors of the osteoclasts ( Lacey et al., Cell 93: 1-65-17-6, 1998; Tsuda et al., Biochem Biophys Res Co 234: 137-142, 1997; Wong et al., J Exp Med 186: 2075-2.080, 1997; Wong et al., J Biol. Chem 272: 25190-25194, 1997; Yasuda et al., Endocrinology 139: 1329-1337, 1998: Yasuda al., Proc Nati Acad Sci US 95: 3597-3602, 1998) . Osteoblasts also secrete a protein that strongly inhibits the formation of osteoclasts, called osteoprotegrin (OPG, also known as OCIF), which acts as a decoy receptor for RANKL thereby inhibiting the positive signal between osteoclasts and osteoblasts through of RANK and RANKL.

Osteoclasts are responsible for the dissolution of the mineral and organic bone matrix. (Blair et al., J Cell Biol 102: 1164-1172, 1986). Osteoclasts represent terminally differentiated cells that express a unique polarized morphology with specialized membrane areas and various membrane and cytoplasmic markers, such as tartrate-resistant phosphatase acid (TRAP) (Anderson et al. 1979), carbonic anhydrase II (Váánánen et al., Histochemistry 78: 481-485, 1983), calcitonin receptor (Warshafsky et al., Bone 6: 179-185, 1985) and the vitronectin receptor (Davies et al., J Cell Biol 109: 1817-1826, 1989). Multinucleated osteoclasts normally contain less than 10 nuclei, but these may contain up to 100 nuclei being between 10 and 100 μm in diameter (Göthling et al., Clin Orthop Relat R 120: 201-228, 1976). This makes them relatively easy to identify by light microscopy. These are highly vacuolated when they are in active state, and also contain many mitochondria, indicative of high metabolic rate (Mundy, in Primer on the metabolic bone diseases and mineral metabolism disorders, pages 18-22, 1990). Since osteoclasts play an important role in osteolytic bone metastases, there is a need in the art for new agents and methods for the prevention of osteoclast stimulation and function.

Cancer metastasis is the primary cause of post-operative or post-therapy recurrence in cancer patients. In spite of the intense efforts to develop treatments, the Cancer metastasis remains considerably refractory to treatment. Bone is one of the most common sites of metastasis of different types of human cancers (for example breast, lung, prostate and thyroid cancers). The incidence of osteolytic bone metastases causes serious morbidity due to pain and difficult healing, high susceptibility to fracture, nerve compression and hypercalcemia. Despite the importance of these clinical problems, there are some treatments available for bone loss associated with cancer metastasis.

Several therapeutic strategies aimed at osteolytic disease are currently being used or in development, where efforts have been focused mainly on the development of drugs to block bone resorption through the inhibition of the formation or activity of osteoclasts. Bisphosphonates (BP), pyrophosphate analogs that are concentrated in bone, are to date the most effective inhibitor of bone resorption. BPs are absorbed by the osteoclasts, inhibiting their activity and causing the cells to suffer apoptosis, thereby inhibiting bone resorption. Alendronate was the first BP inhibitor of bone resorption that showed a significant reduction of fractures of the spine and hip, and is approved for osteoporosis treatment. The most recent generation BP, Zometa, is approved for the treatment of hypercalcemia and bone disease in solid tumors and multiple myeloma and is under investigation for the possible treatment of Pager's disease and bone metastasis resulting from solid tumors and multiple myeloma. Zometa acts in very low doses, and is given as an IV infusion for 15 minutes once a month, but also affects the osteoblasts and may cause side effects such as renal toxicity and osteonecrosis of the jaw (Fromigue and Brody, J, Endocrinol Invest. 25: 39-46, 2002; Ibrahim, A. et al., Clin. Canc. Res. 9: 2394-99, 2003; Body, JJ .. The Breast. S2-.S37-44, 2003; Yaccoby, S. et al., Brit. J. Hemat., 116: 278-80, 2002; Corey, E. et al., Clin. Canc. Res. 9: 295-306, 2003; Coleman, RE, Sem Oncol., 29 (6): 43-49, 2002; Coleman, RE, Eur. Soc. Med. Oncol. 16: 687-95, 2005; Bamias et al., J Clin Oncol 13: 8580-8587, 2005 Thus, in the art it remains a need to identify new agents and methods for the prevention or treatment of osteolytic diseases and / or cancer metastases, including osteolytic bone metastases.

COMPENDIUM OF THE INVENTION The compositions and methods of the present invention meet the aforementioned and other related needs in the art. In one embodiment of the invention, there is provided a method for treating an individual suffering from or at risk of an osteolytic disorder, which consists of administering to the individual a monotherapeutic amount of an M-CSF antagonist and an effective monotherapeutic amount. of a second anti-osteoclast agent during a transition period of about one day to one year, during which the M-CSF antagonist reduces the number of active osteoclasts to a desirable therapeutic level. Exemplary M-CSF antagonists include antibodies against M-CSF and exemplary second anti-osteoclast agents include bisphosphonates and RANKL inhibitors, including antibodies against RANKL. Methods and / or uses which include anti-M-CSF antibody and the osteoclast inhibitor herein optionally exclude the use of antibodies derived from RX1, 5H4, MCI and MC3 described in International Publication No. WO 2005/068503. The duration of the transition period can be, for example, at least one day to one year, and can be monitored, for example, by relevant markers of growth or activity of osteoclasts. Otherwise, these can be given at the same time.

By way of example, markers of bone formation include, but are not limited to, calcium and total and bone-specific alkaline phosphatase (BAP), osteocalcin (OC, gla protein bone), propeptide C procollagen type I (PICP ), N-procollagen propeptide type I (PINP), and markers of bone resorption including, but not limited to, NTX (N-terminal crosslinker bone collagen telopeptide) and CTX (C-terminal crosslinker bone collagen telopeptide), crosslinked pyridinium (pyridinoline and deoxypyridinoline [DPD]) and associated peptides, degradation products of type I bone collagen glycosides of hydroxyproline and hydroxylysine, tartrate-resistant acid phosphatase (TRACP) and bone sialoprotein (BSP). See Fohr et al., J. Clin. Endocrinol Metab., November 2003, 88 (1 1): 5059 ÷ 5075.

In the related embodiments, the aforementioned methods are provided wherein the second antiosteoclast agent is interrupted after the transition period. In other related modalities the aforementioned methods are provided where the The amount of the second antiosteoclast agent is reduced after the transition period. In other related embodiments, the aforementioned methods are provided wherein the amount of the M-CSF antagonist is reduced after the transition period.

It is considered that the methods of the present invention achieve their therapeutic potential by inhibiting the interaction between M-CSF and its receptor (M-CSFR). It is further considered that the inhibition of the M-CSF / M-CSFR interaction inhibits the proliferation and / or differentiation of the osteoclast. In any of the methods or compositions of the invention, the M-CSF antagonist may be an α-polypeptide containing an antibody against M-CSF; a polypeptide containing an antibody against M-CSFR; a soluble polypeptide consisting of a mutein of M-CSF or derivative thereof; or a soluble polypeptide that consists of a mutein of M-CSFR or derivative thereof; or a nucleic acid molecule that inhibits the expression of M-CSF or M-CSFR. The identification, production and modification of the different M-CSF antagonists is described in International Publication No. WO2005 / 068503, hereby incorporated by reference in its entirety.

The antibody against M-CSF can be a polyclonal antibody; a monoclonal antibody; a humanized antibody; a human antibody; a human manipulated antibody; a chimeric antibody; antibody fragment Fab, F (ab ') 2 or Fv; or a mutein of any of the aforementioned antibodies.

The antibodies against M-CSF of the present invention that inhibit osteolysis are described in International Publication No. O2005 / 068503, which is hereby incorporated by reference in its entirety for teaching with respect to antibodies against M-CSF. .

In one embodiment of the invention, there is provided a non-murine monoclonal antibody that includes the fractionated fragment, which specifically binds to the same epitope of M-CSF as any of the murine monoclonal antibody RX1, MCI or MC3 having the amino acid sequences set forth in Figures 1, 3 and 4, respectively. In a related embodiment, a foregoing antibody is provided wherein the antibody is selected from the group consisting of a polyclonal antibody; a monoclonal antibody that includes a Human Engineered ™ antibody; a humanized antibody; a human antibody; a chimeric antibody; a fragment of Fab antibody, F (ab ') 2; Fv; It is Fv or SCA; a diabody; linear antibody; or a mutein of any of these antibodies, which preferably retains the binding affinity of at least 1CT7, 10"8 or 10 or more.A non-murine monoclonal antibody, including the functional fragment, which competes with the monoclonal antibody RX1 , MCI and / or MC3 having the amino acid sequence that is established in Figure 1 for binding to M-CSF by more than 75%, is also considered.

In another embodiment, a non-murine monoclonal antibody is provided that includes the fractional fragment, wherein the non-murine monoclonal antibody or functional fragment thereof binds to an epitope of M-CSF that includes at least 4, 5, 6, 7 or 8 adjacent residues of amino acids 98-105 of Figure 7.

In another embodiment, the invention proposes a non-murine monoclonal antibody, which includes the functional fragment, wherein the non-murine monoclonal antibody or functional fragment thereof is linked to an M-CSF epitope that includes at least residues 4,5. , 6, 7 or 8 adjacent amino acids 65-73 or 138-144 of Figure 7 (corresponding to epitopes of M-CSF recognized by 5H4 or MC3).

In yet another embodiment, the aforementioned antibody or fragment that binds to an epitope of M-CSF including amino acids 98-105 of Figure 7 is provided. In a related embodiment, the monoclonal antibody containing CDR3 of the Figure 1A. In another embodiment, the antibody containing at least 1, 2, 3, 4, 5 or 6 CDRs of murine RXl antibody set forth in Figure 1A is provided. An antibody such as this comprising at least 1, 2, 3, 4 or 5 CDRs of murine RXl antibody can also comprise 1 or less 1, 2, 3, 4 or 5 CDRs of any of the 6 CDRs of the 5H4 antibody established in Figure 8A-B. Otherwise, the antibody containing at least 1, 2, 3, 4 or 5 CDRs of the murine RX1 antibody may also comprise at least 1, 2, 3, 4 or 5 CDRs of any of the 6 CDRs of the MCI antibody. established in Figure 8A-B. In yet another alternative, the aforementioned antibody can also contain at least 1, 2, 3, 4 or 5 CDRs of any of the 6 CDRs of the MC3 antibody set forth in Figure 8A-B. In a related embodiment, the antibody containing at least 1, 2, 3, 4 or 5 CDRs of the murine antibody RX1 may contain at least 1, 2, 3, 4 or 5 CDRs of the consensus CDRs set forth in Figure 8A-B. In still another related modality, in the aforementioned antibody one or more CDR residues consensus is substituted by the corresponding residue of any of the CDRs, of the murine antibody RXI, 5H4, MCI or MC3. The desired binding affinity may be retained even if one or more of the amino acids of the antibody has been mutated, for example by conservative substitutions in the CDRs and / or conservative or non-conservative changes in the residues of low and moderate risk.

In another embodiment of the invention are provided the variants of the aforementioned antibody containing a variable heavy chain amino acid sequence which is at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% homologous to the amino acid sequence set forth in Figures 1A, 2, 3 or 4. In a related embodiment, the antibody contains a variable light chain amino acid sequence. which is at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% homologous to the amino acid sequence set forth in Figures 1A , 2, 3 or 4.

In yet another embodiment, the antibody contains a constant region and one or more variable framework regions of the heavy and light chain of a sequence of human antibody. In a related embodiment, the antibody contains a modified or unmodified constant region of a human IgG1, IgG2, IgG3 or IgG4. In a preferred embodiment, the constant region is human IgGl or IgG4, which can optionally be modified to improve or decrease certain properties, in the case of IgGl, modifications to the constant region, particularly the hinge or CH2 region, can increase or decrease effector function, including ADCC and / or CDC activity. In other embodiments, a constant region of IgG2 is modified to decrease the formation of anti-antigen-antigen aggregates. In the case of IgG4, modifications to the constant region, particularly to the hinge region, can reduce the formation of semi-antibodies.

In one embodiment of the invention, there is provided a non-murine monoclonal antibody that specifically binds to the same epitope of M-CSF as any of the murine antibodies RX1, 5H4, MCI or MC3 as described in International Publication No. WO 2005 / 068503, or compete with any of the aforementioned murine antibodies for binding to M-CSF by more than 10%, more preferably more than 25%, still more preferably more than 50%, even more preferably more than 75% and more preferably more than 90%. Antibodies derived from the sequences of such murine antibodies, including chimeric, human, humanized, manipulated human antibodies, or fragments or muteins or chemically derived versions thereof, are described in WO 2005/068503.

The term "RX1-derived antibody" includes any of the following: 1) an amino acid variant of the murine RX1 antibody having the amino acid sequence set forth in Figure 1, including variants containing an amino acid sequence of the variable heavy chain which is at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% homologous to the amino acid sequence as set forth in the Figure 1 and / or containing an amino acid sequence of the variable light chain which is at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% homologous to the amino acid sequence as established in Figure 1, taking into account similar amino acids for the determination of homology; 2) M-CSF binding polypeptides (excluding murine antibody RX1) that contains one or more complementarity determining regions (the CDRs) of the murine RX1 antibody having the amino acid sequence set forth in Figure 1, preferably containing at least CDR3 of the heavy chain of RX1, and preferably containing two or more, or three or more, or four or more, or five or more more, or all six CDRs; 3) Human Engineered ™ antibodies having the amino acid sequences of the heavy and light chains set forth in Figures 9B through 12B or variants thereof containing a heavy or light chain having at least 60% sequence identity. amino acids with the original Human Engineered ™ heavy or light chain of Figures 9B through 12B, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% and more preferably at least 95% %, including for example, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100% identical; 4) M-CSF binding polypeptides (excluding murine antibody RX1) containing the high-risk residues of one or more of the CDRs of the Human Engineered ™ antibodies of Figures 9B through 12B, and preferably containing residues- high risk of two or more, or three or more, or four or more, or five or more, or all six CDRs; ) Human Engineered ™ antibodies or variants that conserve the high risk amino acid residues established in Figure IB, and that contain one or more changes in the low or moderate risk residues established in. Figure IB; for example, that contain one or more changes in a low risk residue and conservative substitutions in a moderate risk residue established in Figure IB; or for example, which preserve the moderate and high risk amino acid residues set forth in Figure IB and which contain one or more changes in a low risk residue, where the changes include insertions, deletions or substitutions and can be conservative substitutions or can causing the manipulated antibody to be closer in sequence to a light chain or human heavy chain sequence, or a sequence of the light chain or heavy chain of the germline, human, or a sequence of the light chain or the chain heavy human consensus or a sequence of the light chain or the heavy chain of the human germ line, consensus; which retains the ability to join M-CSF. Such antibodies preferentially bind to M-CSF with a affinity of at least 10 ~ 7, 10 ~ 3 or 10"9 or greater and preferably neutralize osteoclastogenesis by inducing the activity of M-CSF.

Similarly, the term "antibody derived from MC3"includes any of the following: 1) an amino acid variant of murine MC3 antibody having the amino acid sequence set forth in Figure 4, including variants containing an amino acid sequence of the variable heavy chain which is at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% homologous to the amino acid sequence as set forth in Figure 4 and / or containing an amino acid sequence of the variable light chain which is at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% homologous to the amino acid sequence as set forth in Figure 4, taking into account similar amino acids for the determination of homology, 2) M-CSF binding polypeptides (which optionally includes or excludes murine MC3 antibody) containing one or more determining regions of the complementarity (the CDRs) of the MC3 murine antibody that has the amino acid sequence established gone in Figure 4, preferably containing at least CDR3 of the heavy chain of MC3, and preferably containing two or more, or three or more, or four or more, or five or more, or all six CDRs; 3) Human Engineered ™ antibodies generated by altering the murine sequence according to the methods set forth in Studnicka et al., US Patent No. 5,766,886 and Example 4A thereof, using the Rabat numbering set forth in Figures 13C-13E to identify residues of low, moderate and high risk; such antibodies containing at least one of the following heavy chains and at least one of the following light chains: (a) a heavy chain in which all low risk residues have been modified, if necessary, to be the same residue as a human reference immunoglobulin sequence, or (b) a heavy chain in which all low and moderate risk residues have been modified, if necessary, to be the same residues as a human reference immunoglobulin sequence , (c) a light chain in which all low risk residues have been modified, if necessary, to be the same residues as a human reference immunoglobulin sequence, or (b) a light chain in which two waste risk low and moderate have been modified, if necessary, to be the same residues as a human reference immunoglobulin sequence 4) variants of the aforementioned antibodies in the preceding paragraph (3) containing a heavy or light chain having at least 60% identity of the amino acid sequence with the original Human Engineered ™ heavy or light chain, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% and more preferably at least 95%, including for example, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100% identical; 5) M-CSF binding polypeptides (which optionally includes or excludes the antibody murine MC3) containing the high-risk residues of one or more of the CDRs of the murine antibody MC3 of Figure 4, and preferably containing the high-risk residues of two or more, or three or more more, or four or more, or five or more, or all six CDRs; 6) Human Engineered ™ antibodies or variants that retain high-risk amino acid residues of the murine MC3 antibody and that contain one or more changes in residues of low or moderate risk; for example, that contain one or more changes in a low risk residue and conservative substitutions in a moderate risk residue, or for example, that conserve moderate and high risk amino acid residues and that contain one or more changes in a residue low risk, wherein the changes include insertions, deletions or substitutions and may be conservative substitutions or may cause the manipulated antibody to be closer in sequence to a light chain or human heavy chain sequence, a light chain or chain sequence heavy of the germline, human, a sequence of the light chain or the human heavy chain, consensus or a sequence of the light chain or the heavy chain of the human germline, consensus; which retains the ability to join M-CSF. Such antibodies preferentially bind to M-CSF with an affinity of at least 1CT7, 10 ~ 8 or 10"9 or greater and preferably neutralize osteoclastogenesis by inducing M-CSF activity.

The term "antibody derived from 5H4" or "antibody derived from MCI" is also defined according to the above description.

As described in detail herein, antibodies derived from RX1, 5H4, MCI or MC3, including Human Engineered ™ antibodies or variants, can be of different isotypes, such as IgG, IgA, IgM or IgE. The antibodies of the IgG class can include a different constant region, for example, an IgG2 antibody can be modified to present a constant region of IgG1 or IgG. In preferred embodiments, the invention provides Human Engineered ™ antibodies or variants containing a modified or unmodified constant region of IgG1 or IgG. In the case of IgGl, modifications to the constant region, particularly the hinge region or CH2, may increase or decrease effector function, including ADCC and / or CDC activity. In other embodiments, a constant region of IgG2 is modified to decrease the formation of the antibody-antigen aggregate. In the case of IgG4, modifications to the constant region, particularly the hinge region, can reduce the formation of half-antibodies. In specific exemplary embodiments, the mutation of the hinge sequence of IgG4 Cys-Pro-Ser-Cys is provided to the hinge sequence of IgGl Cys-Pro-Pro-Cys.

A pharmaceutical composition containing any of the M-CSF antagonists or antibodies against M-CSF and an accepted carrier, efficient or diluent for pharmaceutical use can be administered according to the present invention.

In addition it may be advantageous to mix two or more M-CSF antagonists together or co-administer an M-CSF antagonist and a second anti-osteoclast agent to provide improved efficacy against osteolytic disorders of the invention, including cancer metastases and / or bone loss associated with cancer metastasis.

In exemplary embodiments of the invention, the aforementioned methods are provided wherein the second anti-osteoclast agent is a bisphosphonate. In another embodiment, the bisphosphonate is zoledronate, pamidronate, clodronate, etidronate, tiludronate, alendronate, ibandronate or risedronate. Other exemplary anti-osteoclast agents include bisphosphonates, PTHrP neutralizing agents (e.g., antibody, antisense, siRNA), cathepsin K inhibitors, γ-1-a antagonists, RANK / RA KL neutralizing agents ( eg, anti-RA K antibody, antibody anti-RANKL or antisense, soluble RANKL receptor or muteins thereof), RANKL vaccines, osteoprotegrin (OPG), platelet-derived growth factors (PDGF), src kinase inhibitors, gallium maltolate and metalloproteinase inhibitors' matrix (MMP).

The therapeutic methods of the present invention can be combined with a third therapeutic agent such as a chemotherapeutic agent against cancer or with radiation treatment or surgery. Chemotherapeutic agents against cancer 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 antineoplastic drugs, such as goserelin, leuprolide, and tamoxifen; natural antineoplastics such as aldesleukin, interleukin-2, docetaxel, etoposide (VP-16), interferon alpha, paclitaxel and tretinoin (ATRA); natural antineoplastic antibiotics, such as bleomycin, dactinomycin, daunorubicin, doxorubicin and mitomycin; and natural anticancer agents of vinca alkaloids as I could be vinblastine, vincristine, vindesine; hydroxyurea; aceglatone, adriamycin, ifosfamide, enocitabine, epitiostanol, aclarubicin, ancitabine, nimustine, procarbazine hydrochloride, carboquone, carboplatin, carmofur, chromomycin A3, antitumor polysaccharides, antitumor platelet factors, cyclophosphamide, schizophyllan, cytarabine, dacarbazine, thioinosine, thiotepa, tegafur, neocarzinostatin, OK-432, bleomycin, furtulon, broxuridine, busulfan, honvan, peplomycin, bestatin, (ubenimex), interferon-ß, mepitiostane, mitobronitol, merphalan, laminin peptides, lentinan, Coriolus versicolor extract, tegafur / uracil, estramustine ( estrogen / mechlorethamine).

In addition, other agents that are used as adjunctive therapy for cancer patients include EPO, G-CSF, ganciclovir; antibiotics, leuprolide; meperidine; zidovudine (AZT); interleukins 1 to 18, including mutants and the like; interferons or cytokines, such as interferons a, β and β; hormones, such as luteinizing hormone-releasing hormone (LHRH) and analogs and gonadotropin-releasing hormone (GnRH); factors of growth such as transforming growth factor ß (TGF-ß), fibroblast growth factor (FGF), nerve growth factor (NGF), growth hormone releasing factor (GHRF), epidermal growth factor (EGF), homologous factor of fibroblast growth factor (FGFHF), hepatocyte growth factor (HGF) and growth factor of insulin (IGF) / tumor necrosis factor a and ß (TNF-a and ß); inhibitor factor of invasion-2 (IIF-2); bone morphogenetic proteins 1-7 (BMP 1-7); somatostatin; thymosin-a-1; ?-globulin; superoxide dismutase (SOD); Complementary factors anti-angiogenesis factors; antigenic materials; prodrugs; inhibitors of the growth factor receptor kinase; anti-Her2 antibody; and the VEGF neutralizing antibody.

After the transition period, the amount of the M-CSF antagonist or the amount of the second anti-osteoclast agent required to achieve a therapeutic effect can be reduced. Thus, after such a period, an M-CSF antagonist can improve the efficacy of the second anti-osteoclast agent, or reduce the side effects associated with the administration of the second anti-osteoclast agent, or improve the safety of the second agent. anti-osteoclast. An M-CSF antagonist can also improve the effectiveness, reduce the side effects of, or improve the safety of a third therapeutic modality such as chemotherapy against cancer, other adjunctive therapy, surgery or radiation therapy. In another embodiment of the invention, a package, vial or package is provided containing a medicament containing an M-CSF antagonist and instructions that the medicament should be used in combination with a second and / or third therapeutic agent and / or with surgery or radiation therapy.

It is considered that numerous osteolytic disorders can be treated according to the invention. When used herein, an "osteolytic disorder" is any condition resulting from increased osteoclastic activity. An individual at risk for an osteolytic disorder may be an individual from a group predisposed to develop an osteolytic disorder, or an individual suffering from a disease that causes or contributes to increased osteoclastic activity. In exemplary embodiments of the invention, the osteolytic disorder may be a metabolic bone disease associated with relatively increased osteoclastic activity, including endocrinopathy (hypercortisolism, hypogonadism, primary or secondary hyperparathyroidism, hyperthyroidism), hypercalcemia, state of deficiency (rickets / osteomalacia, scurvy, malnutrition), chronic disease (malabsorption syndromes, chronic renal failure) (renal osteodystrophy), chronic liver disease (hepatic osteodystrophy), drugs, glucocorticoids (osteoporosis induced by glucocorticoids), heparin, alcohol) or hereditary disease (osteogenesis imperfecta, homocystinuria), cancer, osteoporosis, osteopetrosis, inflammation of bone associated with arthritis and rheumatoid arthritis, periodontal disease, fibrous dysplasia and / or Paget's disease.

In other exemplary embodiments, the osteolytic disorder may be a metastatic cancer to bone, wherein the metastatic cancer is malignancy of breast, lung, renal, multiple myeloma, thyroid, prostate, adenocarcinoma, blood cells, including leukemia and lymphoma; Head and neck cancer; Gastrointestinal cancer, including esophageal cancer, stomach cancer, colon cancer, intestinal cancer, colorectal cancer, rectal cancer, pancreatic cancer, liver cancer, cancer of the bile duct or gall bladder, malignancy of the female genital tract, including ovarian carcinoma, uterine endometrial cancer, vaginal cancer or cervical cancer; cancer of the bladder, cancer of the brain; included neuroblastoma; sarcoma, osteosarcoma; or skin cancer, including malignant melanoma or squamous cell cancer.

In exemplary embodiments of the invention, any of the aforementioned methods can prevent or reduce bone loss or prevent or reduce bone metastasis or the severity of bone loss associated with disease.

The antibody against M-CSF administered according to the present invention can be given in a dose between about 2 g / kg to 30 mg / kg, 0.1 mg / kg to 30 mg / kg or 0.1 mg / kg to 10 mg / kg of body weight.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1A shows the amino acid sequence of the murine antibody specific for M-CSF RXI (SEQ ID Nos .: 2 and 4) (encoded by the cDNA insert of the plasmid deposited with the American Type Culture Collection, Manassas , VA, USA, under the ATCC deposit number PTA-6113) and a corresponding nucleic acid sequence (SEQ ID Nos .: 1 and 3). The CDR regions are numbered and shown in bold.

Figures IB and 1C show the amino acid sequences of the murine antibody specific to 'M-CSF RX1 (SEQ ID NO: 5) and the heavy chains (SEQ ID NO: 6), respectively, with the high-risk residues (FIG. bold), moderate risk (underlined) and low risk identified according to Studnicka et al., W093 / 11794.

Figures 2, 3 and 4 show the amino acid sequences of murine antibodies specific for M-CSF 5H4 (SEQ ID Nos .: 10 and 11), MCI (SEQ ID Nos .: 12 and 13) (produced by the deposited hybridoma) under the deposit number ATCC PTA-6263) and MC3 (SEQ ID Nos .: 14 and 15) (produced by the hybridoma deposited under the deposit number ATCC PTA-6264), respectively.

Figure 5 is the amino acid sequence of M-CSF (SEQ ID NO: 7).

Figure 6 is the amino acid sequence of M-CSFP (SEQ ID No. 8).

Figure 7 is the amino acid sequence of M-CSFy (SEQ ID NO: 9). Some polymorphisms in the DNA sequence can cause differences in the amino acids.

For example, a common polymorphism provides an Ala instead of Pro at position 104.

Figures 8A and B are an alignment of the CDR regions of the heavy and light chain amino acid sequences of the human M-CSF specific murine antibodies RX1; 5H4; MCI; and MC3 (SEQ ID Nos.: 16-38).

Figure 9A shows: (a) the risk line for the heavy chain of RXl (H = high risk, M = moderate risk, L = low risk), (b) the amino acid sequence of the heavy chain of RXl (ID) SEC No .: 6), (c) the amino acid sequence of the nearest human consensus sequence, consensus Kabat Vh2, aligned to RXl (SEQ ID No .: 39) and (d) changes that were made to produce two Human sequences Engineered ™ copies (SEQ ID Nos .: 41 and 43). Figure 9B shows the amino acid sequences of the two exemplary Human Engineered ™ heavy chain sequences (SEQ ID Nos .: 41 and 43), designated "low risk" and "low + moderate risk", as well as acid sequences nucleic acid (SEQ ID Nos .: 40 and 42).

Figure 10A shows: (a) the risk line for the murine RXl light chain (H = high risk, M = moderate risk, L = low risk), (b) the amino acid sequence of the light chain of RX1 (SEQ ID NO: 5), (c) the amino acid sequence of the nearest human consensus sequence, consensus Kabat Vk3, aligned to RX1 (SEQ ID No.:49) and (d) changes that were made to produce two exemplary Human Engineered ™ sequences (SEQ ID Nos .: 45 and 47). Figure 10B shows the amino acid sequences of the two exemplary Human Engineered ™ light chain sequences (SEQ ID Nos .: 45 and 47), termed "low risk" and "low + moderate risk", as well as the acid sequences nucleic acid (SEQ ID Nos .: 44 and 46).

Figure 11A shows: (a) the risk line for the murine RX1 light chain (H = high risk, M = moderate risk, L = low risk), (b) the amino acid sequence of the light chain of RX1 ( SEC ID No .: 5), (c) the amino acid sequence of the nearest human consensus sequence, consensus Kabat Vk3, aligned to RX1 (SEQ ID NO: 49) and (d) an alternate exemplary amino acid sequence in which the positions were not changed. -56 (ie, the murine sequence remained) (SEQ ID No.:48). Figure 11B shows the amino acid sequences of two Human Engineered ™ sequences of the exemplary alternative light chain (SEC ID Nos .: 48, 87), as well as the sequences of the corresponding nucleic acid (SEQ ID Nos .: 88 and 86).

Figure 12A shows: (a) the risk line for the murine RX1 light chain (H = high risk, M = moderate risk, L = low risk), (b) the amino acid sequence of the light chain of RX1 ( SEQ ID NO: 5), (c) the amino acid sequence of the closest human consensus germline sequence, Vk6 subgroup 2-1- (1) A14, aligned to RX1 (SEQ ID No .: 50) and (d) changes that were made to produce two exemplary Human Engineered ™ sequences (SEQ ID Nos .: 51 and 53). Figure 12B shows the amino acid sequences of the two exemplary Human Engineered ™ light chain sequences (SEQ ID Nos .: 51 and 53), designated "low risk" and "low + moderate risk" as well as acid sequences nucleic acid (SEQ ID No.:52).

Figures 13A and 13B show the alignment of the amino acid sequence of the murine RX1 heavy chain (SEQ ID No .: 54), with various human consensus and human germ line consensus sequences using the Kabat numbering system (numbering of the amino acids indicated online designated "POS") (SEQ ID Nos .: 55-83). Figures 13C-13E show how, the amino acid residues of antibodies 5H4, MCI and MC3 correspond to the Kabat numbering system (SEQ ID Nos .: 10 and 11, SEQ ID Nos .: 12 and 13, SEQ ID Nos: 14 and 15), respectively).

Figure 14 shows the anti-resorptive effects of Zometa in an animal model.

Figure 15 shows the percentage of animals in each group with detectable steolysis.

Figure 16 shows the average scores of the osteolysis based on the analysis of X-ray images during the last day of the study.

Figure 17 shows Faxitron X-ray images representative of tibias (site of tumor inoculation) during the final day of the study. The arrows point to the sites of osteolysis.

Figure 18 shows the effect of RX1 on osteoclastic activity.

Figure 19 shows the inhibition of osteoclastic activity by Zometa.

Figure 20 shows the results of a pharmacokinetic study with RXl in primates.

Figure 21 shows the results of a pharmacokinetic study with RXl in primates.

DETAILED DESCRIPTION The colony stimulating factor (CSF-1), also known as macrophage colony stimulating factor (M-CSF), has been found important for the formation of osteoclasts. In addition, M-CSF has been shown to modulate the osteoclastic functions of mature osteoclasts, their migration and their survival in cooperation with other soluble factors cell-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. Through alternative splicing of mRNA and differential post-translational proteolytic processing, M-CSF can be secreted into the circulation as a glycoprotein or chondroitin sulfate containing Proteoglycan can be expressed as a glycoprotein that covers the membrane on the surface of the M-CSF producing cells. The three-dimensional structure of the amino-terminal 150 amino acids expressed in human M-CSF bacteria, the minimum sequence necessary for complete in vitro biological activity, indicates that this protein is a dimer with sulfide bonds with each monomer consisting of four alpha helical bundles and one antiparallel beta sheet (Pandit et al., Science 258: 1358-62 (1992)). Three different M-CSF species are produced by splicing the alternative mR A. The three polypeptide precursors are M-CSFa of 256 amino acids, M-CSFP of 554 amino acids and M-CSEy of 438 amino acids. M-CSFP is a secreted protein that does not occur in a membrane bound form. M-CSFa is expressed as an integrated membrane protein that is released slowly by proteolytic cleavage. The M-CSFa cleaved at amino acids 191-197 of the sequence set forth in Figure 5. The membrane-bound form of M-CSF can interact with receptors on nearby cells and therefore mediates cell-to-cell specific contacts . The term "M-CSF" may also include amino acids 36-438 of Figure 7.

Various forms of M-CSF function by binding to their M-CSFR receptor on the target cells. M-CSFR is a membrane-spanning molecule with five extracellular immunoglobulin-like domains, a transmembrane domain and an interrupted, intracellular Src tyrosine kinase domain. The M-CSFR is encoded by the proto-onocgen c-fms. The binding of M-CSF to the extracellular domain of M-CSF leads to the dimerization of the receptor, which activates the cytoplasmic kinase domain, giving rise to autophosphorylation and phosphorylation of other cellular proteins (Hamilton, JA, Leukoc Biol., 62 ( 2): 145-55, 1997; Hamilton, JA, Immuno Today., 18 (7): 313-7, 1997).

Phosphorylated cellular proteins induce a cascade of biochemical events that give rise to cellular responses: mitosis, cytokine secretion, membrane puckering and regulation of transcription of their own receptor (Fixe and Praloran, Cytokine 10: 32-37, 1998) .

"Tumor", when used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign and all precancerous and cancerous cells and tissues.

The terms "cancer" and "cancerous" refer to or describe the physiological state in mammals that is normally characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma; lymphoma, blastoma, sarcoma and leukemia. The most specific examples of these cancers can be 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, carcinoma of salivary granules; kidney cancer, liver cancer, vulval cancer, thyroid cancer, hepatic carcinoma and different 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 therapeutic treatment and prophylactic or preventive measures. Those in need of treatment include those who already have the disorder as well as those in which the disorder is to be prevented. During tumor treatment (for example, cancer), a therapeutic agent can directly decrease the pathology of the tumor cells, or render the tumor cells more susceptible to treatment by other therapeutic agents, for example, radiation and / or chemotherapy. The "pathology" of cancer includes all phenomena that compromise the well-being of the patient. These include, without limitation, the abnormal or uncontrolled growth of cells, metastasis, interference with normal functioning of adjacent cells, release of cytokines or other products of secretion to abnormal levels, suppression or aggravation of response inflammatory or immunological, et cetera.

"Mammal" for the purpose of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo animals, for sports or pets, such as dogs, horses, cats, cows, etc. . Preferably the mammal is human.

When used herein, the phrase "metastatic cancer" is defined as cancers that have the potential to disperse to other areas of the body, particularly bone. Various cancers can Metastasize to bone, but the most common metastatic cancers are breast, lung, kidney, multiple myeloma, thyroid and prostate. For example, other cancers that have the potential to metastasize to bone include, but are not limited to, adenocarcinoma, blood cell malignancies, including leukemia and lymphoma; cancers of the head and neck; Gastrointestinal cancers, including esophageal cancer, stomach cancer, colon cancer, intestinal cancer, colorectal cancer, rectal cancer, pancreatic cancer, liver cancer, cancer of the bile duct or gall bladder, malignancies of the female genital tract, including Ovarian carcinoma, uterine endometrial cancers, vaginal cancer and cervical cancer; bladder cancer; brain cancer, including neuroblastoma; sarcoma, osteosarcoma; and skin cancer, including malignant melanoma and squamous cell cancer. The present invention especially considers the prevention and treatment of osteolytic lesions induced by tumor in the bone.

When used herein, the phrase "effective therapeutic amount" refers to an amount of the therapeutic or prophylactic antagonist of M-CSF, such as the antibody against M-CSF, which would be appropriate for an embodiment of the present invention, what will trigger the desired therapeutic or prophylactic effect or response when administered according to the desired treatment scheme.

"Human M-CSF" when used herein refers to a human polypeptide having substantially the same amino acid sequence as the human, mature M-CSFa, M-CSF or M-CSFy polypeptides described in Kawasaki et al., Science 230: 291 (1985), Cerretti et al., Molecular Immunology, 25: 761 (1988) or Ladner et al., EMBO Journal 6: 2693 (1987), each of which is incorporated herein by reference. Terminology such as this manifests the understanding that the three mature M-CSFs have different amino acid sequences, as described above, and that the active form of M-CSF is a dimer with disulfide bond; so, when the term < »" M-CSF "refers to the active biological form, the dimeric form is proposed. "M-CSF dimer" refers to two polypeptide monomers of M-CSF that have dimerized and includes homodimers (consisting of two of the same type of monomer M-CSF) and heterodimers (consisting of two different monomers). The M-CSF monomers can be converted to M-CSF dimers in vitro as described in US Patent No. 4,929,700, which is incorporated herein by reference. 1. Antagonists When used herein, the term "" antagonist "generally refers to the property of a molecule, compound or other agent to, for example, interfere with the binding of a molecule to another molecule or the stimulation of a cell by another cell through steric hindrance, conformational alterations, or other biochemical mechanisms In one sense, the term "antagonists" refers to the property of an agent to prevent the binding of a receptor to its ligand, e.g. -CSF with M-CSFR, thereby inhibiting the signal transduction pathway activated by M-CSF The term antagonist is not limited by some specific mechanism of action but, rather, generally refers to the functional property currently defined. Antagonists of the present invention include, but are not limited to: antibodies against M-CSF and fragments and muteins and modifications thereof, soluble M-CSF and fragments and mu proteins and modifications thereof, antibodies against M-CSFR and fragments and muteins and modifications thereof, soluble M-CSFR and fragments and muteins and modifications thereto, and peptides as well as other chemical compounds and molecules that bind to M-CSF or M-CSFR and nucleic acid molecules such as antisense compounds or RNAi that inhibit the expression of M-CSF and M-CSFR. Any of the antagonists of the present invention can be administered in any manner known in the art. For example, M-CSF muteins, M-CSFR muteins or antibody fragments that bind to M-CSF or M-CSFR can be administered by gene therapy.

The M-CSF antagonists of the present invention include, where appropriate, functional equivalents. For example, molecules may differ in length, structure, components, etc., but may still retain one or more of the defined functions. More specifically, the functional equivalents of the antibodies, antibody fragments or peptides of the present invention can include mimetic compounds, ie, constructs designed to mimic the proper configuration and / or orientation for antigen binding.

Preferred M-CSF antagonists can optionally be modified by the addition of side groups, ie, for example, by amino terminal acylation, carboxy terminal amidation or by coupling or coupling of additional groups to the amino acid side chains. Antagonists can also contain one or more conservative amino acid substitutions. By "conservative amino acid substitutions" is meant those changes in the amino acid sequence that preserve the general charge, hydrophobicity / hydrophilicity and / or hindrance or steric bulk of the substituted amino acid. For example, substitutions between the following groups are conservative: Gly / Ala, Val / Ile / Leu, Asp / Glu, Lys / Arg, Asn / Gln, Ser / Cys / Thr and Phe / Trp / Tyr. Such modifications will not significantly decrease the efficacy of the M-CSF antagonists and may provide such desired properties as may be, for example, increased half-life in vivo or decreased toxicity.

The invention is also intended to include polypeptides that carry modifications other than the insertion, deletion or substitution of the amino acid residues. For example, the modifications may be covalent in nature and include, for example, chemical linkage with polymers, lipids or other organic and inorganic moieties. Such derivatives can be prepared to increase the circulating half-life of a polypeptide, or they can be designed to improve the choice of the polypeptide to the desired cells, tissues or organs. In the same way, the invention further comprises polypeptides of M-CSF or M-CSFR that have been covalently modified to include one or more bonds of soluble polymer such as polyethylene glycol, polyoxyethylene glycol or polypropylene glycol.

A. Antibodies to M-CSF The term "antibody" is used in the broadest sense and includes fully assembled antibodies, monoclonal antibodies, polyclonal antibodies, multispecies antibodies (eg, bispecific antibodies), fragments of antibodies that can bind antigen ( e.g., Fab ', F' (ab) 2, Fv, single chain antibodies, diabodies) and recombinant peptides containing the above provided that they exhibit the desired biological activity.

The term "monoclonal antibody" when used herein refers to an antibody that is obtained from a population of substantially homogeneous antibodies, i.e., each of the antibodies comprising the population are identical, except for possible mutations that occur in nature that may be present in smaller quantities. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Further, contrary to conventional (polyclonal) antibody preparations, which usually include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by the homogeneous culture, not contaminated by other immunoglobulins with different specificities and characteristics.

The "monoclonal" modifier indicates the type of antibody that is being obtained from a considerably homogeneous population of antibodies, and does not have to be constructed as required by the production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be prepared by the hybridoma method first described by Kohler et al., Nature, 256: 495 [1975], or they can be prepared by the methods of Recombinant DNA (see, e.g., US Patent No. 4,816,567). The "monoclonal antibodies" can also be separated from the phage antibody libraries using the techniques described in Clackson et al., Nature, 352: 624628 [1991] and Marks et al., J. Mol. Biol., 222: 581-597 (1991), for example.

Depending on the amino acid sequence of the constant domain of its heavy chains, the immunoglobulins. they can be assigned to different classes. There are five main classes, IgA, IgD, IgE, IgG and IgM, and some of these can also be divided into subclasses or isotypes, p. ex. , IgGl, IgG2, IgG3, IgG4, IgAl and IgA2. The constant domains of the heavy chain that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma and mu, respectively. The subunit structures and the three-dimensional configurations of the different classes of immunoglobulins are well known. Different isotypes have different effector functions; for example, the IgGl and IgG3 isotypes have ADCC activity.

"Antibody fragments" comprise a portion of an intact, full-length antibody, preferably the antigen-binding or variable region of the intact antibody. Examples of the antibody fragments include Fab, Fab ', F (ab') 2 and Fv; diabodies; linear antibodies (Zapata et al., Protein Eng., 8 (10): 1057-1062 (1995)); antibody molecules single-chain and multispecific antibodies formed from the antibody fragments. Digestion of the antibodies with papain produces two identical fragments that bind to the antigen, called "Fab" fragments, each with a single antigen binding site, and a residual "Fe" fragment, whose name reflects its ability to crystallize. easily. The treatment with pepsin produces an F (ab ') 2 fragment having two "single chain Fv" or "sFv" antibody fragments comprising the VH and VL domains of the antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide also contains a polypeptide linker between the VH and VL domains which allows the Fv to form the desired structure for antigen binding. For a review of sFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. , Springer-Verlag, New York, pp. 269-315 (1994).

The term "hypervariable" region refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region comprises the amino acid residues from a complementarity determining region or CDR [i.e., residues 24-34 (Ll), 50-56 (L2) and 89-97 '(L3) of the variable domain of the light chain and 31-35 (Hl), 50-65 (H2) and 95-102 (H3) of the variable domain of the heavy chain as described by Kabat et al. , Sequences of Protéins of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)] and / or those residues of a hypervariable loop or loop (ie, residues 26-32 (Ll), 50-52 (L2) and 91-96 (L3) of the variable domain of the light chain and 26-32 (Hl), 53-55 (H2) and 96-101 (H3) of the variable domain of the heavy chain as described by [Chothia et al. ., J. Mol Biol. 196: 901-917 (1987)].

The "frame" or FR residues are those residues of the variable domain different from the residues of the hypervariable region.

The term "diabodies" refers to fragments of small antibodies with two antigen binding sites, whose fragments comprise a variable domain of the heavy chain (VH) connected to a variable domain of the light chain (VL) in the same chain polypeptide (VH VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen binding sites. The diabodies are described in greater detail in, for example, EP 404, 097; WO 93/11161; and 30 Hollinger et al., Proc. Nati Acad. Sci, USA, 90: 6444-6448 (1993).

In some embodiments it may be desired to generate multispecific (eg bispecific) monoclonal antibodies including monoclonal, human, humanized, Human Engineered ™ or anti-M-CSF variant with binding specificities for at least two different epitopes. Exemplary bispecific antibodies can bind to two different epitopes of M-CSF. In another version, an anti-M-CSF arm can be combined with an arm that binds to an activating molecule on a leukocyte, such as a T cell receptor molecule (for example CD2 or CD3) or Fe receptors for IgG (FcyR ), such as FcyRI (CD64), FcyRII (CD32) and FcyRIII (CD16) to focus cellular defense mechanisms for the cell expressing M-CSF. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing M-CSF. These antibodies have an N-CSF binding arm and an arm that binds to the cytotoxic agent (eg, saporin, anti-interferon-60, vinca alkaloid, ricin A chain, methotrexate or hapten) of radioactive isotope). Bispecific antibodies can be prepared as full-length antibodies or fragments of antibodies (eg, bispecific antibodies F (ab ') 2) - According to another approach for preparing bispecific antibodies, the interface between a pair of antibody molecules can be manipulated to maximize the percentage of heterodimers that are recovered from the recombinant cell culture. The preferred interface consists of at least a portion of the CH3 domain of the antibody constant domain. In this method, one or more side chains of small amino acids from the interface of the first antibody molecule are replaced with larger side chains (eg, tyrosine or tryptophan). Compensatory "cavities" of identical or similar size for the large chain or side chains are created at the interface of the second antibody molecule by substituting the side chains of large amino acids with smaller ones (for example alanine or threonine). This provides a mechanism to increase the yield of the heterodimer on other undesired terminal products such as homodimers. See WO 96/27011 published on September 6, 1996.

Bispecific antibodies include crosslinked or "heteroconjugate" antibodies. For example, one of the antibodies of the heteroconjugate can be coupled to avidita, the other to biotin. Heteroconjugate antibodies can be prepared using any of the convenient crosslinking methods. Suitable crosslinking agents are well known in the art and are described in US Patent No. 4,676,980, together with various crosslinking techniques.

The techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical bonds. Brennan et al., Science 229: 81 (1985) describe a procedure in which intact antibodies are fragmented proteolytically to generate F (ab ') 2 fragments. These fragments are reduced in the presence of the complexing agent dithiol sodium arsenite to stabilize vicinal dithiols. and prevent the formation of intermolecular disulfide. The generated Fab 'fragments are then converted into thionitrobenzoate derivatives (TNB). One of the Fab'-TNB derivatives is then reconverted into the Fab '-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of the enzymes. In yet another embodiment, the Fab '-SH fragments directly recovered from E. coli can be chemically coupled in vitro to form bispecific antibodies (Shalaby et al., J. Exp. Med. 175: 217-225 (1992)) .

Shalaby et al., J. Exp. Med. 175: 217-225 (1992) describe the production of a fully humanised F (ab ') 2 molecule of bispecific antibody. Each Fab 'fragment was secreted separately from E. coli and subjected to chemical coupling directed in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells by overexpressing the HER2 receptor and normal human T cells, as well as activating the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

Various techniques for preparing and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers (Kostelny et al., J. Immunol., 148 (5): 1547-1553 (1992)) The leucine zipper peptides from the Fos and Jun proteins were ligated to the Fab 'portions of two different antibodies by gene fusion. The antibody homodimers were reduced to the hinge region to form monomers and then reoxidized to form the antibody heterodimers. This method can also be used for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Nati Acad. Sci. USA 90: 6444-6448 (1993) has provided an alternative mechanism for preparing bispecific antibody fragments.

The fragments comprise a variable region of the heavy chain (VH) connected to a light chain variable region (VL) by a linker that is too short to allow pairing between two domains of the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VH and VL domains of another fragment, thereby forming two antigen binding sites. Another strategy for preparing bispecific antibody fragments by the use of single chain Fv dimers (sFv) has also been reported. See Gruber et al., Immunol. 152: 5368 (1994).

In another version, the bispecific antibody can be a "linear antibody" produced as described in Zapata et al. Protein Eng. 8 (10): 1 057-1062 (1995). In summary, these antibodies contain a pair of cascaded Fd fragments (VH-CH1-VH-CH1) which form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.

Antibodies with more than two valencies are also considered. For example, trispecific antibodies can be prepared. (Tutt et al., J. Immunol. 147: 60 (1991)).

In some embodiments, the monoclonal, human, humanized, Human Engineered ™ or anti-M-CSF variant is an antibody fragment, such as an antibody fragment RX1, 5H4, MCI or MC3. Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived by proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992) and Brennan et al., Science 229: 81 ( 1985)). However, these fragments can now be produced directly by recombinant host cells. Better et al., Science 240: 1041-1043 (1988) describe the secretion of functional antibody fragments from bacteria (see, eg, Better et al., Skerra et al., Science 240: 103B-1041 (1988)). For example, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F (ab ') 2 fragments (Carter et al., Bio / Technology 10: 163-167 (1992 )). In another embodiment, the F (ab ') 2 is formed using the leucine zipper GCN4 to favor the assembly of the F (ab') 2 molecule. According to another approach, the Fv, Fab or F (ab ') fragments 2 can be isolated directly from the recombinant host cell culture. Other techniques for the production of the antibody fragments will be apparent to the person skilled in the art.

An "isolated" antibody is one that has been identified and separated and recovered from a component of its natural environment.The contaminating components of its natural environment are materials that would interfere with the diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones or other proteinaceous solutes, or non-proteinaceous In the preferred embodiments, the antibody will be purified (1) to more than 95% by weight of the antibody, as determined by the Lowry method, and more preferably 99 % in weight, (2) to a sufficient degree to obtain at least 15 residues of the N-terminal or internal amino acid sequence by the use of a rotary rate sequencer, or (3) to homogeneity by SDS-PAGE under reducing conditions or no reduction using Coomassie blue or, preferably, silver staining. The isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. However, commonly the isolated antibody will be prepared by at least one purification step.

For a detailed description of the structure and production of the antibodies see, Roth, DB, and Craig, NL, Cell, 94: 411-414 (1998), and U.S. Patent No. 6,255,458, incorporated herein by reference In its whole. In summary, the process for generating DNA encoding heavy and light chain immunoglobulin genes occurs mainly in developing B cells. Prior to rearrangement and binding of the different segments of the immunoglobulin gene, the V, D, J and constant (C) gene segments are generally in relatively close proximity on a single chromosome. During the differentiation of cells B, one of each of the appropriate family members of the V, D, J gene segments (or only V and J in the case of the light chain genes) recombine to form heavy and light immunoglobulin genes functionally rearranged. This process of rearrangement of the gene segments seems to be sequential. First, the D-a-J bonds of the heavy chain are prepared, followed by the V-a-DJ bonds of the heavy chain and the V-a-J bonds of the light chain.

The recombination of the segments of the genes of the variable region to form regions. Functional heavy and light chain variables are mediated by the recombination signal (RSS) sequences flanking segments V, D and J competent for recombination. The necessary and sufficient RSSs for direct recombination comprise a dyad-symmetric heptamer, an AT-rich nonamer and an intervening spacer region of 12 or 23 base pairs. These signals are conserved between different l'oci and species that carry out recombination D-J (or V-J) and are functionally interchangeable. See Oettinger, et al. (1990), Science, 248, 1517-1523 and the references mentioned therein. The heptamer comprises the sequence CACAGTG or its analog followed by a non-sequence separator. preserved and then a nonamer having the sequence ACAAAAACC or its analogue. These sequences are located on the J, or the downstream side, of each gene segment V and D. Immediately preceding the segments D and J of the germ line are again two sequences of the recombination signal, first the nonamer and then the heptamer again separated by a non-conserved sequence. The heptameric and nonameric sequences that follow a segment VL, VH or D are complementary to those that precede the JL, D or JH segments with which they recombine. The separators between the heptameric and nonameric sequences are 12 base pairs long or between 22 and 24 base pairs long.

In addition to the rearrangement of segments V, D and J, another diversity is generated in the primary repertoire of the immunoglobulin heavy and light chain by means of variable recombination in the locations where the V and J segments of the light chain are joined. and where segments D and J of the heavy chain join. Such variation in the light chain normally occurs within the last codon of the segment of the V gene and the first codon of the segment J. Similar imprecision in the junction is observed in the chromosome of the heavy chain between segments D and JH and may extend over so many. about 10 nucleotides. In addition, various nucleotides can be inserted between the D and JH gene segments and between the VH and D gene segments which are not encoded by the genomic DNA. The addition of these nucleotides is known as the diversity of the N region.

The pure effect of these rearrangements on the segments of the variable region gene and the variable recombination that can occur during such binding is the production of a primary antibody repertoire.

"Fv" is the minimal antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of a variable domain of the heavy chain and one of the light chain in close association, non-covalent. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH VI dimer. Collectively, the six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind the antigen, albeit at a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The fragments Fabs differ from Fab 'fragments by the addition of some residues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab '-SH is the designation herein for Fab' in which the residue or cysteine residues of the constant domains carry a free thiol group. The F (ab ') 2 antibody fragments were originally produced as pairs of Fab' fragments which have cysteine hinge between them.

By "neutralizing antibody" is meant an antibody molecule that can eliminate or significantly reduce an effector function of a target antigen to which it binds. Accordingly, a "neutralizing" anti-target antibody is capable of eliminating or significantly reducing an effector function, such as enzymatic activity, ligand binding or intracellular signaling.

As provided herein, the compositions for and methods of treating cancer metastasis and / or bone loss associated with Cancer metastases may use one or more antibodies used individually or in combination with other therapeutics to obtain the desired effects. The antibodies according to the present invention can be isolated from an animal by producing the antibody as a result of direct contact with an environmental antigen or immunization with the antigen. Otherwise, the antibodies can be produced by the recombinant DNA methodology using one of the antibody expression systems well known in the art (see, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory. 1988)). Such antibodies can include recombinant IgGs, chimeric fusion proteins having sequences derived from immunoglobulins or "Human Engineered ™" antibodies which can all be used for the treatment of cancer metastasis and / or bone loss associated with cancer metastasis according to the present invention. In addition to full-length, intact molecules, the term "antibody" also refers to fragments thereof (such as, for example, fragments scFv, Fv, Fd, Fab, Fab 'and F (ab)' 2 ) or multimers or aggregates of intact molecules and / or fragments that bind to M-CSF (or M-CSFR). These Antibody fragments bind to the antigen and can be obtained to present structural features that facilitate clearance or uptake, for example, by incorporation of galactose residues.

In one embodiment of the present invention, monoclonal antibodies against M-CSF can be prepared practically as described in Halenbeck et al., US Patent No. 5,491,065 (1997), incorporated herein by reference. Exemplary monoclonal antibodies against M-CSF include those that bind to an apparent conformational epitope associated with recombinant or native chimeric M-CSF with concomitant neutralization of biological activity. These antibodies are considerably unreactive with the biologically inactive forms of M-CSF including the monomeric M-CSF and chemically derived dimer.

In other embodiments of the present invention, Human Engineered ™ anti-M-CSF monoclonal antibodies are provided. The phrase "Human Engineered ™ antibody" refers to an antibody obtained from a non-human antibody, usually a mouse monoclonal antibody. Otherwise, a Human Engineered ™ antibody can be derived from a chimeric antibody which retains or substantially retains the antigen binding properties of the non-human precursor antibody but which exhibits decreased immunogenicity compared to the precursor antibody when administered to humans. The phrase "chimeric antibody", when used herein, refers to an antibody that contains the sequence derived from two different antibodies (see, for example, US Patent No. 4,816,567) which normally originate from different species. Most commonly, the chimeric antibodies comprise fragments of human and murine antibodies, generally the human constant and variable mouse regions.

The phrase "complementarity determining region" or the term "CDR" refers to the amino acid sequences which together define the binding affinity and specificity of the native Fv region of a native immunoglobulin binding site (see, eg. eg, Chothia et al., J. Mol. Biol. 196: 901 917 (1987); Kabat et ah, US Dept. of Health and Human Services NIH Publication No. 91 3242 (1991)). The phrase "constant region" refers to the portion of the antibody molecule that confers effector functions. In the present invention, the mouse constant regions they are preferably replaced by human constant regions. The constant regions of the subject antibodies are derived from human immunoglobulins. The constant region of the heavy chain can be selected from any of the five isotypes: alpha, delta, epsilon, gamma or mu.

The antibodies of the present invention are said to be immunospecific or to bind specifically if they bind to the antigen with a Ka greater than or equal to about 10 °? -1, preferably greater than or equal to 107M ~ X, more preferably greater than or equal to about 108M_1, and more preferably greater than or equal to about IO1, 1010M_1, IO ^ M "1 or 1012M_1. Anti-M-CSF antibodies can bind to different forms found in the nature of M-CSF , including those exposed by the host / individual tissues, as well as those expressed by the tumor.The monoclonal antibodies described herein, such as the RX1, 5H4, MCI or MC3 antibody, have affinity for M-CSF and are characterized by a dissociation equilibrium constant (Kd) of at least 10 ~ 4M, preferably at least about 10 ~ 7M to about 10"8M, more preferably at least about 10 ~ 8M, 10" 10M, 10_11M or 10"12M . These Affinities can be easily determined using traditional techniques, such as by equilibrium dialysis; using the BIAcore 2000 instrument, using the general procedures described by the manufacturer; by radioimmunoassay using M-CSF labeled with 125I; or by another method known to the person skilled in the art. The affinity data can be analyzed, for example, by the method of Scatchard et al., Aim N. Y. Acad. Sci. , 51: 660 (1949). Thus, it will be apparent that the preferred antibodies against M-CSF will exhibit a high degree of specificity for M-CSF and will bind with considerably lower affinity than other molecules. Preferred antibodies bind to M-CSF with an affinity similar to that of murine RX1 of Figure 4 binds to M-CSF, exhibit low immunogenicity and inhibit metastasis of cancer cells when tested in animal models with metastatic disease . Other exemplary antibodies bind to M-CSF with an affinity similar to that of murine 5H4, MCI or MC3 of Figure 3 or 4, respectively, bind to M-CSF.

The antigen to be used for the production of the antibodies can be, for example, intact M-CSF or a fragment of M-CSF which retains the epitope desired, as an option fused to another polypeptide that allows the epitope to be presented in its natural conformation. Otherwise, cells expressing M-CSF on their cell surface can be used to generate the antibodies. Such cells can be transformed to express M-CSF or they can be other cells that are found in nature and that express M-CSF. Other forms of M-CSF useful for generating antibodies will be apparent to those skilled in the art. i. Polyclonal antibodies Polyclonal antibodies are preferably increased in animals by multiple subcutaneous (se) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. An improved antibody response can be obtained by conjugating the relevant antigen to a protein that is immunogenic in the species to be immunized, for example, keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin or soybean trypsin inhibitor using a bifunctional agent or derivatizing., for example, the maleimido benzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride or other agents known in the art.

The animals are immunized against the antigen, immunogenic conjugates or derivatives combining, for example, 100 μg or 5 g of the protein or conjugate (for rabbits or mice), respectively) with three volumes of Freund's complete adjuvant and injecting the solution intradermally in multiple places. One month later, the animals are reinforced with 1/5 a. { fraction (1/10)} of the original amount or peptide or conjugate in Freund's complete adjuvant by subcutaneous injection in multiple places. At 7-14 days post-booster injection, the animals are bled and the serum is analyzed for the antibody titer. The animals are reinforced until the title is constant. Preferably, the animals are boosted with the conjugate of the same antigen, but conjugated to a different protein and / or by a different cross-linking reagent. The conjugates can also be prepared in recombinant cell culture as protein fusions. Also, aggregation agents such as alum are suitably used to improve the immune response. ii. Monoclonal Antibodies Monoclonal antibodies can be prepared using the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975), or they can be prepared by recombinant DNA methods.

In the hybridoma method, a mouse or other appropriate host animal, such as a hamster or a macaque monkey, is immunized as described herein to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization. Otherwise, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusion agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in appropriate culture medium which preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the hypoxanthine enzyme guanine fosforribociltransferasa (HGPRT or HPRT), the culture medium for the hybridomas will usually include hypoxanthine, aminopterin and thymidite (HAT medium), whose substances prevent the growth of cells deficient in HGPRT.

Preferred myeloma cells are those that are efficiently fused, support the high-level, stable production of the antibodies by the selected antibody-producing cells, and are sensitive to a medium. The lines of human myeloma and mouse-human heteromyeloma cells have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)). Exemplary murine myeloma lines include those obtained from mouse tumors MOP-21 and M.C.-ll available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Md. USA.

The culture medium in which the hybridoma cells are growing is analyzed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of the monoclonal antibodies that are produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as a radioimmunoassay (RIA) or the enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis (Munson et al., Anal. Biochem., 107: 220 (1980)).

After it is identified that the hybridoma cells produce the antibodies of the desired specificity, affinity and / or activity, the clones can be subcloned by limiting the dilution procedures and can be grown by the normal methods (Goding, Monoclonal Antibodies: Principies and Practice, pp.59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells can be grown in vivo as tumor ascites in an animal. The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascitic fluid or serum by the traditional immunoglobulin purification procedures such as, for example, example, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

The antibodies of the present invention are said to be immunospecific or to bind specifically if they bind to the antigen with a Ka greater than or equal to about 10 6, preferably greater than or equal to about 10 7 M, more preferably greater than or equal to approximately 108M_1, and more preferably greater than or equal to approximately ??? t1, ? _1 or 1012M_1. Anti-M-CSF antibodies can bind to different natural forms of M-CSF, including those expressed by the host / individual tissues as well as those expressed by the tumor. The monoclonal antibodies described herein, such as the antibody RX1, 5H4, MCI or MC3, have affinity for M-CSF and are characterized by an equilibrium dissociation constant (Kd) of at least 10"4M, preferably at least about 10"7M to about 10" 8M, more preferably at least about 10-8M, 10"10M, 10_11M or 10" 12 M. Such affinities can be easily determined using traditional techniques, such as by dialysis in equilibrium; using the BIAcore 2000, using the general procedures outlined by the manufacturer; by radioimmunoassay using M-CSF labeled with 125I, or by another method known to the skilled worker. The affinity data can be analyzed, for example, by the method of Scatchard et al., Ann N. Y. Acad. Sci. , 51: 660 (1949). Thus, it will be apparent that preferred anti-M-CSF antibodies will exhibit a high degree of specificity for M-CSF and will bind with considerably lower affinity than other molecules. Preferred antibodies bind to M-CSF with an affinity similar to that of the murine RXI of Figure 1 that binds to M-CSF, exhibit low immunogenicity, and inhibit cancer cell metastasis when tested with animal models of metastatic disease . Other exemplary antibodies bind to M-CSF with similar affinity as the murine 5H4, MCI or MC3 of Figure 2, 3 or 4, respectively, bind to M-CSF.

Conservative substitutions are shown in the Table 1 under the heading of "preferred substitutions". If such substitutions result in a change in biological activity, then more considerable changes, termed "exemplary substitutions" in Table 1, or as described below with reference to classes of amino acids, can be introduced and the products can be screened.

TABLE 1 Substitutions of preferred residues, exemplified original Ala (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his; asp, lys; Asp (D) glu; asn glu gln arg Cys (C) ser; wing be Gln (Q) asn; glu asn Glu (E) asp; gln asp Gly G) ala His (H) asn; gln; lys; arg lie (1) leu; val; met; to; leu phe; norleucine Leu (L) norleucine; ile; val; ile met; to; phe Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; to; Pro (P) wing 7 tyr Ser (S) thr Thr (T) be Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; be phe Val (V) ile; leu; met; phe; leu wing; norleucine Considerable modifications in the biological properties of the antibody are achieved through the selection of substitutions that differ significantly in their effect on the maintenance of (a) the structure of the polypeptide skeleton in the area of substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the volume of the side chain. The residues found in nature are divided into groups based on the common properties of the side chain: (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic: cys, ser, thr; (3) acid: asp, glu; (4) basic: asn, gln, his, lys, arg; (5) residues that influence the orientation of the chain: gly, pro; and (6) aromatic: trp, tyr, phe.

Non-conservative substitutions involve the substitution of a member of one of these classes with a member of another class.

Any cysteine residue not involved in maintaining the proper conformation of the humanized antibody or variant can also be substituted, generally with serine, to maintain conformation Suitable humanized antibody or variant can also be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely. It is possible to add cysteine bonds to the antibody to improve its stability (particularly where the antibody is an antibody fragment as an Fv fragment).

B. M-CSF Muteins The invention further provides M-CSF muteins that can be used as M-CSF antagonists according to the methods of the invention.

"Fragment" when used herein means a portion of the natural molecule intact; for example, a polypeptide fragment is a fragment of the native polypeptide in which one or more N-terminal or C-terminal amino acids has been deleted.

"Mutein" when 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. Such substitutions, insertions or deletions can be in the N terminal, C terminal or internal for the molecule. Thus, the term "muteins" includes fragments of the native molecule within its scope. Insertional muteins include fusions in the N or C terminal, for example, fusion to the Fe portion of an immunoglobulin to increase half-life.

Preferred muteins according to the invention have at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or more sequence identity (homology) for 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 a related gap search. the following search parameters: penalty of opening the gap of 12, and penalty for the extension of the gap of 1. Other well-known and commonly used algorithms for screening homology / identity include Pearson and Lipman, PNAS USA , 85: 2444-2448 (19138); Lipman and Pearson, Science, 222: 1435 (1985); Devereaux et al., Nuc. Acids Res., 12: 387-395 (1984); or the BLASTTP5 BLASTN or BLASTX algorithms of Altschul, et al., Mol. Biol., 215: 403-410 (1990). Computer programs that use these programs are also available and include, but are not limited to: GAP, BESTFIT, BLAST, FASTA and TFASTA, which are available in the Genetics Computing Group (GCG) package, Version 8, Madison Wis., USA; and CLUSTAL in the PC / Gene program at lntellegenetics, Mountain View California. Preferably, the percentage of the identity of the sequences is determined using the parameters predetermined by the program.

"Modification" when used herein means any modification of the native polypeptide, fragment or mutein, such as glycosylation, phosphorylation, conjugation of polymers (such as with polyethylene glycol) or other addition of foreign portions, provided that the desired activity (agonist or antagonist).

U.S. Patent No. 6,025,146, and Koths, Mol. Reprod. Dev. January 1997; 46 (1): 31-38 both are incorporated herein by reference in their entirety, describe the crystallization of M-CSF alone and M-CSF complexed to MCSF-R, and characterize the three-dimensional structure of M-CSF as well as the residues involved in binding to the receptor. U.S. Patent No. 6,025,146 also describes the Methods for selecting candidate amino acid substitutions in M-CSF, based on structural information. The general topology of this form of M-CSF is that of four antiparallel alpha-helical beams, in which the helices run up-up-down-down, unlike the most commonly observed up-down-up-down connectivity. of most of the four helical beams. A long cross-connection links helix A to helix B, and a similar connection is found between helixes C and D. In the dimeric form with disulfide bond, the bundles are bound end-to-end, forming an extremely flat, elongated structure ( of 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 distant end of the molecule. An interchain disulfide bond (Cys31-Cys31) is located at the dimeric interface with the non-crystallographic double symmetry axis passing through it, as shown in Figure 2. Mutation experiments indicate that all cysteine residues in this M-CSF form may be necessary for full biological activity. The structure described here suggests that its function is mainly structural instead of being related to the recognition of the receiver. U.S. Patent No. 6,025,146 provides the tridimensional structure of the truncated M-CSFa dimer, which is truncated as identified by the alpha carbon positions of the amino acid residues of the sequence.

The specific residues in helices A, C and D appear to be involved in the specificity of the receptor binding interaction. Since M-CSFP has intrachain disulfide bonds including cysteines 157 and / or 159, the C-terminal region of M-CSF probably extends from the "backside" of the structure, providing a variable length "tether" for the forms attached to the M-CSF membrane. Thus, the "front" or receptor binding region of M-CSF is on the opposite side of the molecules, consisting of solvent accessible residues at or near helixes A, C and D, including residues from approximately 6 to 26, 71 to 90, and 110 to 130, respectively, of the native M-CSF. Alterations of residues accessible to the solvent in these regions by site-directed mutagenesis to increase or decrease the side chain interactions with the receptor can generate M-CSF agonists or antagonists. Waste that has an accessible surface area to the solvent greater than about 0.25 and preferably greater than about 0.4 are preferred based on the normalization of the surface area of the accessible amino acid when found in the tripeptide gly-x-gly (Kabsch, W. et al., Biopolymers 22: 2577 ( 1983)). Preferably the residues are chosen which do not interact with other parts of the protein, such as the chimeric interface to maintain the relative orientation of the monomers and avoid disturbance of the protein folding process. An additional optional consideration is to select non-conserved residues between human and mouse M-CSF, which do not recognize the human M-CSF receptor. The candidate amino acids are preferably selected for substitution with non-conservative amino acids to break hydrogen bond and / or hydrophobic interactions with MCSF-R residues. For example, the change from one or more histidines to non-hydrogen-donating amino acids of similar size can create an M-CSF with altered receptor binding ability. Preferred amino acids for substitutions include, but are not limited to: H15; Q79; R86; E115; E41; K93; D99; L55; S18; Q20; 175; V78; L85; D69; N70; H9; N63 and T34. The M-CSF residues important in receptor signaling are considered to be composed of discontinuous regions of M-CSF. To minimize the For the likelihood of antibody formation for potentially administered M-CSF-based proteinaceous drugs, it is desirable to preserve the parental M-CSF residues accessible to the solvent (to resemble the natural molecule) where possible.

Mutagenesis of amino acids H15 and H9 in the N-terminal / helix A region resulted in muteins with significantly lower biological activity and significantly lower binding capacity of M-CSF-R. These results indicated that the reduced biological activity was due to decreased receptor binding affinity; thus, these histidine residues represent contacts that are important for binding affinity to the M-CSF receptor and should be left unchanged if full receptor binding capacity is desired. The residues accessible to the nearby solvent such as Y6 and S13 and others can also represent contact residues with the M-CSF receptor. A double mutant of M-CSF (Q20A, V78K) was constructed to test the importance of residues accessible to the solvent in the central portion of helices A and C. This double mutein had slightly lower (8-13 times) biological activity and correspondingly lower receptor binding activity. The mutagenesis of residues Q17, R21, E115 and E119 changed the properties of the side chain of the amino acids accessible to the solvent. In the areas of interest, but did not affect the specific biological activity, suggesting that these residues do not need to be altered in muteins designed to have antagonistic activity.

In one embodiment, the invention considers the use of M-CSF muteins in whose residues of the helices A and / or. C and / or D involved in receptor binding (eg, amino acids 6 to 26, 71 to 90 and / or 110 to 130) have been mutated in a non-conservative manner. Such muteins preferably retain at least 65%, 70%, 75%, 80%, 85% or 90% similarity (ie, amino acids that are identical or have similar properties) to the native sequence within the helices A, C or D, but they have greater similarity to the native sequence in the rest of the polypeptide, for example, at least 95%, 98% or 99% similarity. In addition, the residues that support three-dimensional [sic] confirmation of the receptor binding site can be mutated in a non-conservative fashion.

In another embodiment, the M-CSF mutein is a monomeric form of M-CSF. The dimeric form of M-CSF is the biologically active form, and the monomeric forms of M-CSF are generally not active. The disulfide bond of the monomers appears to be observed through the intercatenary link Cys31-Cys31. Thus, it is considered that the monomeric forms of M-CSF may be appropriate for use as antagonists. Such forms include muteins containing cis-deletions and / or cysteine substitutions (eg, cysteine-alanine substitutions) of Cys31 and / or other cisterns, or muteins in which cysteine (s), particularly Cys31 , has been chemically modified so that these are not available for disulfide bonding.

In still another embodiment, the M-CSF mutein comprises one or more of the helices A, C or D, or portions thereof involved in receptor binding, alone or fused to other polypeptides that allow the presentation of the fragments in the appropriate three-dimensional conformation.

Muteins containing any of the 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 the regions directly involved in the ligand / receptor binding, are not expected to significantly change the binding properties of the M-CSF muteins (or the M-CSFR muteins). The amino acids can be classified according to the physical properties and the contribution to the secondary and tertiary structure of the protein. A conservative substitution is recognized in the art as a substitution of an amino acid by another amino acid having similar properties. Exemplary conservative substitutions are set forth in Table 2 (from WO 97/09433, page 10, published March 13, 1997 (PCT / GB96 / 02197, filed on 6/9/96), immediately below.

Table 2 Conservative substitutions I SIDE CHAIN CHARACTERISTICS AMINO ACID Aliphatic Non-polar G A P I L V Polar-no load C S T M N Q Polar-charged D E K R Aromatic H F W Y Other N Q D E Otherwise, the conservative amino acids may be grouped as described in Lehninger, (Biochemistry, Second Edition, Worth Publishers, Inc. NY: NY (1975), pp.71-77) as set forth in Table 3 below.

Table 3 Conservative substitutions II SIDE CHAIN CHARACTERISTICS AMINOACIDO Non-polar (hydrophobic) A. Aliphatic: ALIVP B. Aromatic: FW CC Containing sulfur: M DD Limit: Polar G without charge A. Hydroxyl: STY B. Amides: NQ C. Sulfhydryl: C D. Limit: G With positive charge (Basic): KRH With negative charge (acid) .: DE As yet another alternative, exemplary conservative substitutions are set forth in Table 4, below: Table 4 Conservative Substitutions III Original Residue Exemplary Substitution Wing (A) Val, Leu, lie Arg (R) Lys, Gln, Asn (N) Gln, His, Lys, Arg Asp (D) Glu Cys (C) Ser Gln ( Q) Asn Glu (E) Asp His (H) Asn, Gln, Lys, Arg lie (I) Leu, Val, Met, Ala, Leu (L) lie, Val, Met, Ala, Lys (K) Arg, Gln , Asn Met (M) Leu, Phe, lie Phe (F) Leu, Val, lie, Ala Pro (P) Gly Ser (S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y) 'Trp, Phe , Thr, Ser Val (V) lie, Leu, Met, Phe, The availability of a DNA sequence that encodes M-CSF allows the use of diverse systems of expression to produce the desired polypeptides. The construction of the expression vectors and the recoiabinant production from the appropriate DNA sequences is done by methods well known in the art. These techniques and various other techniques are generally made in accordance with Sambrook et al., Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989), and Kriegier, M., Gene Transfer and Expression, A Laboratory Manual, Stockton Press, New York (1990), which are incorporated herein by reference.

Some 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 binding capacity to the M-CSF receptor) in accordance with Well-known recombinant DNA techniques. In addition, a skilled worker will appreciate that individual amino acids can be substituted or modified by oxidation, reduction or other modification, and the polypeptide can be divided to obtain fragments that preserve the active binding site and structural information. Such substitutions and alterations result in polypeptides having an amino acid sequence that it falls within the definition of polypeptide "having substantially the same amino acid sequence as the mature polypeptides M-CSFa (SEQ ID NO: 7), M-CSFp (SEQ ID NO: 8), and M-CSFy (SEC ID No .: 9) ".

The polypeptides may be produced by chemical synthesis or recombinant production techniques known in the art.

The ratio of the proteins can also be determined by the relation of their encoding nucleic acids. Methods for determining the identity and / or similarity of polynucleotide sequences are described above. In addition, methods for determining the similarity of the polynucleotide sequences through analysis of their ability to hybridize under moderate or highly restrictive conditions can be determined as follows. Exemplary moderately stringent hybridization conditions are as follows: Hybridization at 42 ° C in a hybridization solution containing 50% formamide, 1% SDS, 1 M NaCl, 10% dextran sulfate, and washing twice for 30 minutes at 60 ° C in a wash solution containing 0.1 x SSC and 1% SDS. Highly restrictive conditions include washings at 68 ° C in a wash solution containing 0.1 x SSC and 1% SDS. It is understood in the art that conditions of equivalent stringency can be achieved by varying the temperature and buffer, or the salt concentration as described in the art (Ausubel, et al. (Eds.), Protocols in Molecular Biology, John iley &Sons (1994), pp. 6.0.3 to 6.4.10). Modifications in hybridization conditions can be determined empirically or can be accurately calculated based on the length and percentage of pairing of guanosine / cytosine (GC) bases in the probe. 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.

C. Soluble M-CSFR Exemplary M-CSFR fragments according to the invention may contain one or more, or two or more, of the domains involved in the M-CSF / receptor binding (they are considered to be domains 1, 2 and 3). Preferred M-CSFR fragments contain all three of domains 1, 2 and 3 of M-CSFR. Mutations and / or further modifications to such fragments or to the entire extracellular domain of M-CSFR are considered and can be produced as described above in the mutein section of M-CSF.

M-CSFR (SEQ ID Nos .: 84 and 85) is a membrane-spanning molecule with five extracellular immunoglobulin-like domains (of which domains 1-3 are considered to be involved in ligand-receptor binding), a transmembrane domain and a Src related, interrupted, intracellular tyrosine kinase domain. With reference to SEQ ID NO: 85, the aforementioned domains are located as follows: Ig domain 1: amino acids 27-102; Ig domain 2: amino acids 112-196; Ig domain 3: amino acids 215-285; Ig domain 4: amino acids 308-399; Ig domain 5: amino acids 410-492; transmembrane domain: amino acids 515-537; and kinase domain: amino acids 582-910. A "common" immunoglobulin-like domain contains a loop or loop structure normally anchored by a disulfide bond between two cysteines at the end of each loop. In M-CSF-R, these cysteines forming Ig-like loops are in the following amino acid positions: Domain 1: 42, 84; Domain 2: 127, 177: Domain 3: 224, 278: Domain 4: no cysteines involved; Domain 5: 419, 485 The intact extracellular portion of M-CSFR or any fragment thereof that retains antigenicity, eg, one or more of the Ig-like loops or loops, may be used to increase the antibodies that are they would unite the native receiver. Polyclonal, monoclonal, chimeric, grafted, humanized, fully human CDR antibodies and antigen-binding fragments thereof can be prepared as described above for antibodies to M-CSF. The products of the antibodies can be screened for activity as an M-CSF antagonist and for adequacy in the methods of treatment of the invention using the assays as described in the section entitled "Screening Methods" herein or using any of the appropriate assays known in the art.

One or more of the aforementioned Ig-like loops within the extracellular domain of the receptor may be sufficient to inhibit the interaction between M-CSF and M-CSFR. Thus, fragments of the extracellular domain of M-CSFR and mutein thereof can be easily prepared using recombinant or chemical synthetic means well known in the art. The products can be screened for activity as an M-CSF antagonist and for adequacy in the methods of treatment of the invention using assays as described in the section entitled "Screening Methods" herein using any of the appropriate assays known in the art.

D. Gene Therapy The delivery of a therapeutic protein to the appropriate cells can be effected through gene therapy ex vivo, in situ or in vivo through the use of any suitable approach known in the art, including the use of physical DNA transfer methods ( for example, liposomes or chemical treatments) or by the use of viral vectors (e.g., adenovirus, adeno-associated virus or retrovirus). Antisense compounds and methods of using them are also provided by the present invention. The level of activity of M-CSF or M-CSFR can be reduced by using antisense, gene knockout, ribozyme, triple helix or RNAi methods well known to decrease the level of gene expression. The techniques for the production and use of such molecules are well known to those skilled in the art.

When used herein, the term "peptidomimetic" is a non-peptidic compound that contains an assembly of amino acid side chains, or pharmacophores, or suitable derivatives thereof, that are supported on scaffolding such that the spatial orientation of pharmacophores significantly mimics the bioactive conformation of a natural peptide. For example, a peptidomimetic may lack amino acids or peptide bonds but retain the particular three-dimensional arrangement of the peptide chain groups of the precursor peptide that is required for binding activity. The scaffolding may consist of a carbon skeleton or bicyclic, tricyclic or polycyclic higher heteroatom or may be based on one or more annular structures (eg pyridine, indazole, etc.) or amide bonds. This scaffold can be linked by separators to an acid group (for example, a carboxylic acid functional group) at one end and a basic group (for example, a portion containing N such as amidine or guanidine) at the other end of the nucleus . Exemplary techniques for synthesizing peptidomimetics are described in U.S. Patent Application No. 200030199531 published October 23, 2003, U.S. Patent Application No. 20030139348 published July 24, 2003.

In addition to antibodies and other proteins, this invention also considers alternative M-CSF antagonists that include, but are not limited to, peptides or small organic molecules that are also effective to inhibit the interaction between M-CSF and M-CSFR or the activation of M-CSFR.

II. Combination therapy The concomitant administration of two therapeutic agents according to the present invention, such as an M-CSF antagonist and a second anti-osteoclastic agent, does not require that the agents be administered at the same time or in the same way, always that there is an overlap in time during which the agents are exerting their therapeutic effect. The simultaneous or successive administration is considered, as is the administration in different days and weeks.

The discovery of a significant time interval to observe the therapeutic effect after starting treatment with an antibody against M-CSF (an exemplary M-CSF antagonist) makes it desirable to co-administer a second anti-osteoclast agent with faster onset of action during this transition period. During the transition period, the two agents must be administered in an effective monotherapeutic amount. After the transition period, the second anti-osteoclast agent can be interrupted or reduced in dose. If the M-CSF antagonist and the second anti-osteoclast agent exert synergistic effects, the dose of one or both may be reduced after the transition period.

The compositions of the invention are administered to a mammal already suffering from, or predisposed to, osteolytic disorders, including cancer metastasis and / or bone loss associated with cancer metastasis, or other diseases related to bone loss, such as osteoporosis, in an amount sufficient to prevent or at least partially arrest the development of such a disease. An amount of a suitable therapeutic agent to achieve this when the therapeutic agent is given alone (not in combination with a second therapeutic agent) is defined as an "effective monotherapeutic dose".

In the combination therapy methods of the present invention, the M-CSF antagonist, such as the antibody against M-CSF, and the second anti-osteoclastic agent can be administered at the same time or at different times. The two agents can be administered, for example, in the course of 8 hours, one day, 14 days, 30 days, 3 months, 6 months, 9 months or one year with each other.

Exemplary second anti-osteoclastic agents include bisphosphonates, including, but not limited to, zoledronate, pamidronate, clodronate, etidronate, tiludronate, alendronate, ibandronate or risedronate. Other exemplary anti-osteoclastic agents include bisphosphonates, neutralizing agents PTHrP (eg, antibody, antisense, siRNA), cathepsin K inhibitors, γ-1-a antagonists, RANK / RANKL neutralizing agents (e.g. , the antibody against RANK, such as AMG-162, or antisense, the soluble RANKL receptor or its muteins), the RANKL vaccine, osteoprotegrin (OPG), platelet derived growth factors (PDGF), s rc kinase, gallium maltolate, and inhibitors of matrix metalloproteinase (MMP).

Exemplary dosages of bisphosphonates include intravenous administration of 4mg. Lower doses can also be administered that include 3.5 mg, 3.3 mg or 3.0 mg. Other routes of administration are possible and include the subcutaneous and, as described in WO 02/087555. Effective amounts of an antibody against M-CSF will vary and will depend on the severity of the disease and the weight and general condition of the patient being treated, but generally range from about 1.0 mg / kg to about 100 mg / kg of weight body, or about 10 mg / kg to about 30 mg / kg, with doses from about 0.1 mg / kg to about 10 mg / kg or about 1 mg / kg to about 10 mg / kg per application being the most commonly used. For example, about 10 mg / kg to 5 mg / kg or about 30 mg / kg to 1 mg / kg of antibody is an initial candidate dose for administration to the patient, e.g., by one or more separate administrations or by continuous infusion. Administration is daily, on alternate days, weekly or less frequently, as necessary, depending on the response to the disease and the patient's tolerance for treatment. Maintenance doses for a long time, such as 4, 5, 6, 7, 8, 10 or 12 weeks or more may be necessary until a desired suppression of disease symptoms is observed, and doses may be adjusted as necessary. The evolution of this treatment is easily monitored by traditional techniques and trials.

While the methods of the present invention may be useful for all stages of cancers, they may be particularly appropriate in advanced or metastatic cancers. The combination of the treatment method with a chemotherapeutic or radiation scheme may be preferred in patients who have not received chemotherapeutic treatment, while the treatment with the treatment method of the present invention may be indicated for patients who have received one or more chemotherapies. In addition, the therapeutic methods of the present invention may also allow the use of reduced doses of concomitant chemotherapy, particularly in patients who do not tolerate the toxicity of the chemotherapeutic agent very well.

The method of the invention considers the administration of unique anti-M-CSF antibodies, as well as combinations or "cocktails" of different antibodies. Such antibody cocktails may have some advantages since they contain antibodies that take advantage of different effector mechanisms or combine directly cytotoxic antibodies with antibodies that depend on the immune effector functionality. Such antibodies in combination may have synergistic therapeutic effects.

The methods of the invention can be used in combination with yet another therapy, such as cancer therapeutics. Exemplary therapeutic agents and / or cancer procedures include, but are not limited to, the various chemotherapeutic agents, androgen blockers, and immune modulators (e.g., IL-2, GM-CSF, SLC), Bisphosphonate (s) (eg, Aredia. (Ie, pamidronate, pamidronic acid, pamidronate disodium, pamidronate disodium pentahydrate), Zometa (ie, Aclasta, zoledronic acid, zoledronate), Clondronate (ie, Bonefos, Loron, clodronate disodium, sodium clondronate), Fosamax (ie alendronate, alendronate sodium salt trihydrate, alendronic acid), Fosavance (ie, Fosamax formulated with vitamin D), Bondronat or Bonviva or Boniva (ie, ibandronate, ibandronic acid, ibandronate sodium), Actonel (ie, risedronate, risedronate sodium, risendronic acid), Didronel or Didrocal (ie, etidronate, etidronic acid, etidronate disodium), Nerixia (ie, neridronate, neridronic acid); Skelid (ie, tiludronate, tiludronic acid), dimethyl-APD (ie, olpadronate, olpadronic acid), and medronic acid or medronate), surgery, radiation, cytotoxic chemotherapy, hormone therapy (eg, Tamoxifen; anti-androgen), antibody therapy (p. ex. , neutralizing antibodies to KANKL / RANK; Neutralizing PTHrP, anti-Her2, anti-CD20, anti-CD40, CD22, VEGF, 1GFR-1, EphA2, HAAH, TMEFF2, CAIX antibodies), therapeutic protein therapy (eg, the soluble RANKL receptor; OPG , and inhibitors of PDGF and MMP), drug therapy of small molecules (eg, Src-kinase inhibitor), ^ kinase inhibitors of growth factor receptors or RANKL inhibitors, oligonucleotide therapy (eg, RANKL or RANK or PTHrP antisense) gene therapy (eg, RANKL or RANK inhibitors, such as anti-RANKL antibodies) ), peptide therapy (eg, RANKL mutein) as well as those proteins, peptides, compounds and small molecules described herein.

Chemotherapeutic agents against cancer 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; folinic acid; purine analog antimetabolites, mercaptopurine; pyrimidine analog antimetabolites, such as fluorouracil (5-FU) and gemcitabine (Gemzar®): hormonal antineoplastic drugs such as goserelin, leuprolide and tamoxifen; natural antineoplastic agents, such as aldesleukin, interleukin-2, docetaxel, etoposide (VP-16), interferon alpha, paclitaxel (Taxol®), and tretinoin (ATRA); natural antineoplastic antibiotics such as bleomycin, dactinomycin, daunorubicin, doxorubicin, daunomycin and mitomycins that include mitomycin C; and natural antineoplastics vinca alkaloids such as vinblastine, vincristine, vindesine; hydroxyurea; aceglatone, adriamycin, ifosfamide, enocitabine, epitiostanol, aclarubicin, ancitabine, nimustine, procarbazine clorhirate, carboquone, carboplatin, carmofur, chromomycin A3, antitumor polysaccharides, antitumor platelet factors, cyclophosphamide (Cytoxin®), schizophyllan, cytarabine (cytosine arabinoside), dacarbazine, thioinosine, thiotepa, tegafur, dolastatins, dolastatin analogues such as auristatin, CPT-11 (irinotecan), mitozantrone, vinorelbine, teniposide, aminopterin, carminomycin, esperamycin (See, eg, US Patent No. 4,675,187 ), neocarzinostatin, OK-432, bleomycin, furtulon, broxuridine, busulfan, honvan, peplomycin, bestatin (Ubenimex®), interferon-ß, mepitiostane, mitobronitol, melphalan, laminin peptides, lentinan, Coriolus versicolor extract, tegafur / uracil , estramustine (estrogen / mechlorethamine).

In addition, other agents that are used as a treatment for cancer patients include EPO, G-CSF, ganciclovir; antibiotics, leuprolide; meperidine; zidovudine (AZT); Interleukins 1 through 18, including mutants and the like; interferons or cytokines, such as interferons a, β and β, hormones, such as luteinizing hormone-releasing hormone (LHRH) and the like, and gonadotropin-releasing hormone (GnRH); Growth factors such as transforming growth factor ß (TGF-ß), fibroblast growth factor (FGF), nerve growth factor (NGF), growth hormone releasing factor (GHRF), factor of. epidermal growth (EGF), homologous factor of fibroblast growth factor (FGFHF), hepatocyte growth factor (HGF), and insulin growth factor (IGF); tumor necrosis factor - & β (TNF-a &ß); Inhibitory factor-2 (IIF-2); bone morphogenetic proteins 1-7 (BMP 1-7); somatostatin; thymosin-a-1; ?-globulin; superoxide dismutase (SOD); complement factors; anti-angiogenesis factors; antigenic materials and prodrugs.

"Prodrugs" refers to a precursor or derivative form of an active pharmaceutical substance that is less cytotoxic or non-cytotoxic to tumor cells compared to the precursor drug and is capable of being enzymatically activated or converted to an active or more active precursor form. See, for example, Wilman, "Prodrugs in Cancer Chemotherapy" Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and Stella et al., "Prodrugs: A Chemical Approach to Targeted Drug Delivery," Directed Drug Delivery, Borchardt et al. , (ed.), pp. 247-267, Humana Press (1985). Prodrugs include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid modified prodrugs, glycosylated prodrugs, prodrugs containing β-lactam, prodrugs that contain optionally substituted phenoxyacetamide or prodrugs containing optionally substituted phenylacetamide, 5-fluorocytosine and other 5-fluorouridine prodrugs that can be converted to the most active cytotoxic free drug. Examples of the cytotoxic drugs that can be derived in a prodrug form for use herein include, but are not limited to, those chemotherapeutic agents described above.

III. Administration and Preparation The effective amounts of an M-CSF antagonist will vary and will depend on the severity of the disease and the weight and general condition of the patient being treated, but will generally range from about 1.0 g / kg to about 100 mg. / kg of body weight, most commonly used doses from about 10 μg / kg to about 10 mg / kg per application. The determination of an effective amount of a composition of the invention can be achieved by standard empirical methods that are well known in the art. For example, the in vivo neutralizing activity of the sera of an individual treated with a given dose of the M-CSF antagonist can be evaluated using an assay that determines the ability of the sera to block the proliferation induced by M-CSF and the survival of monocytes murine (CDllb + cell, a subset of CD11 cells, which expresses high levels of the receptor for M-CSF) in vitro as described in Cenci et al., J Clin. Invest. 1055: 1279-87, 2000.

Administration is daily, every two days, every 3 days, twice a week, weekly or less frequently, as necessary, depending on the response to the disease and the patient's tolerance to treatment. Maintenance doses for prolonged periods may be necessary, and doses may be adjusted as necessary.

Single or multiple administrations of the compositions can be carried out with the dose levels and standards selected by the attending physician.

M-CSF antagonists, including antibodies to M-CSF used in the practice of a method of the invention can be formulated in pharmaceutical compositions containing a suitable carrier for the desired delivery method. Suitable carriers include any material that, when combined with the M-CSF antagonist, retains the antitumor function of the antagonist and is non-reactive with the individual's immune systems. Examples may be, but are not limited to, any of the different normal pharmaceutical carriers such as phosphate buffered saline solutions, sterile, bacteriostatic water, and the like. It is possible to use a variety of aqueous carriers, for example water, buffered water, 0.4% saline, 0.3% glycine and the like, and may include other proteins to improve stability, such as albumin, lipoprotein, globulin, etc., subjected to moderate or similar chemical modifications.

Therapeutic formulations of the antagonists are prepared for storage by mixing the antagonist having the desired degree of purity with carriers, excipients or stabilizers accepted for physiological use, optional (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Accepted carriers, excipients or stabilizers are non-toxic to recipients at the doses and concentrations employed, and include buffer solutions such as phosphate, citrate and other organic acids; antioxidants that include ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m- cresol); polypeptides of low molecular weight (less than about 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; sodium-forming counterions; metal complexes (eg, Zn-protein complexes); and / or non-ionic surfactants such as TWEE ™, PLURONICS ™ or polyethylene glycol (PEG).

The. The formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may also be desired to provide an immunosuppressive agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

The active ingredients can also be contained in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsule and poly- (methylmethacrylate) microcapsule, respectively, in colloidal drug delivery systems ( for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. These techniques are described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations that are used for in vivo administration must be sterile. This is easily achieved by filtration through sterile filtration membranes.

The antagonist is administered by any appropriate means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary and intranasal administration and, if desired for local, intralesional treatment. Parenteral infusions include intravenous, intraarterial, intraperitoneal, intramuscular, intradermal or subcutaneous administration. In addition, the antagonist is appropriately administered by pulse infusion, particularly with declining doses of the antagonist. Preferably, the dosage * is given by injections, more preferably intravenous or subcutaneous injections, depending in part if the administration is brief or chronic. Other methods of administration are considered to include topical, particularly transdermal, transmucosal, rectal, oral or local administration, for example, by a catheter placed near the desired site.

The compositions of the present invention can be in the form of, for example, granules, powders, tablets, capsules, syrup, suppositories, injections, emulsions, elixirs, suspensions or solutions. The present 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.

Injectable dosage forms generally include aqueous suspensions or oily suspensions that can be prepared using a suitable dispersant or wetting agent and a suspending agent. The injectable forms may be in the solution phase or in the form of a suspension which is prepared with a solvent or diluent. Acceptable solvents or vehicles include sterile water, Ringer's solution or an isotonic aqueous saline solution. Otherwise, it is possible to use sterile oils as solvents or suspending agents. Preferably, the oil or fatty acid is non-volatile, including natural or synthetic oils, fatty acids, mono-, di- or triglycerides.

For injection, the pharmaceutical formulation and / or medicament may be a suitable powder for reconstitution with an appropriate solution as described above. Examples of these include, but are not limited to, freeze-dried, spin-dried or spray-dried powders, amorphous powders, granules, precipitates or particulates. For the injection, The formulations may contain, as an option, stabilizers, pH modifiers, surfactants, bioavailability modifiers and combinations thereof.

It is possible to prepare sustained release preparations. Suitable examples of the sustained or prolonged release preparations include semipermeable matrices of solid hydrophobic polymers containing the antagonist, which matrices are in the form of molded articles, for example films or microcapsules. Examples of the sustained release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl-methacrylate), or poly (vinyl alcohol)), polylactides (US Patent No. 3, 773, 919), L acid copolymers -glutamic and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, copolymers of lactic acid-degradable glycolic acid such as Lupron Depot ™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D- (-) -3-hydroxybutyric acid. Although polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid allow the release of the molecules for approximately 100 days, some hydrogels release proteins for shorter times.

When the encapsulated antagonists remain in the body for a long time, these can be denatured or added as a result of exposure to humidity at 37 ° C, resulting in a loss of biological activity and possible changes in immunogenicity. Appropriate strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is found to be the formation of intermolecular SS bonds by thio-disulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilization from acid solutions, control of moisture content, use of appropriate additives and the development of specific polymer matrix compositions.

The formulations of the invention can be designed to be short-acting, fast-release, sustained-release or sustained release as described herein. Thus, pharmaceutical formulations can also be formulated for controlled release or for slow release.

The present compositions may also contain, for example, micelles or liposomes, or some other forms encapsulated or may be administered in the form of prolonged releases to provide prolonged storage and / or delivery effect. Therefore, pharmaceutical formulations and medicaments can be compressed into pellets or cylinders and implanted intramuscularly or subcutaneously as depot injections or as implants as stents. Such implants may employ inert materials known as silicones and biodegradable polymers.

In addition to those representative dosage forms described above, excipients and carriers accepted for pharmaceutical use are generally known to the person skilled in the art and thus are included in the present invention. Such excipients and carriers are described, for example, in "Remingtons Pharmaceutical Sciences" Mack Pub. Co. , New Jersey (1991), which is incorporated herein by reference.

Specific doses can be adjusted depending on the disease states, age, body weight, general health status, sex and diet of the individual, dose intervals, routes of administration, rate of excretion and combinations of drugs. Any of the above dosage forms containing effective amounts is well within the limits of routine experimentation and therefore, well within the scope of the present invention.

M-CSF antagonists or antibodies useful as therapeutics according to the invention will often be prepared virtually free of other natural immunoglobulins or other biological molecules. Preferred M-CSF antagonists will also exhibit minimal toxicity when administered to a mammal affected with, or predisposed to suffering from, osteolytic disorders, including cancer metastasis and / or bone loss associated with cancer metastasis.

The compositions of the invention can be sterilized by traditional, well-known sterilization techniques. The resulting solutions can be packaged for use or filtered under aseptic and lyophilized conditions, the freeze-dried preparation being combined with a sterile solution before administration. The compositions may contain excipients accepted for pharmaceutical use as necessary to approximate the conditions physiological, such as pH adjustment and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride and stabilizers (for example 1 % maltose, etc. [sic]).

The M-CSF antagonists of the present invention can also be administered through liposomes, which are small vesicles composed of different types of lipids and / or phospholipids and / or surfactants that are useful for the delivery of a drug (as can be be the antagonists described herein and, as an option, a chemotherapeutic agent). Liposomes include emulsions, foams, micelles, insoluble monolayers, phospholipid dispersions, lamellar layers and the like, and can serve as vehicles to direct M-CSF antagonists to a particular tissue as well as to increase the half-life of the composition. A variety of methods are available for preparing liposomes, as described in, for example, US Patent Nos. 4,837,028 and 5,019,369, the patents of which are incorporated herein by reference.

Liposomes containing the antagonist are prepared by methods known in the art, as described in Epstein et al., Proc. Nati Acad. Sci. USA 82: 3688 (1985); Hwang et al., Proc. Nati Acad. Sci. USA 77: 4030 (1980); and U.S. Patents Nos. 4, 485, 045 and 4,544,545. Liposomes with improved circulation time are described in U.S. Patent No. 5,013,556. Particularly useful liposomes can be produced by the reverse phase evaporation method with a lipid composition containing phosphatidylcholine, cholesterol and phosphatidylethanolamine derived from PEG (PEG-PE). The liposomes are extruded through filters of defined pore size to produce the liposomes with the desired diameter. The Fab 'fragments of the antibody against M-CSF of the present invention can be conjugated to the liposomes as described in Martin et al., J. Biol. Chem.257: 286-288 (1982) through an exchange reaction disulfide. A chemotherapeutic agent (such as Doxorubicin) is optionally contained within the liposome [see, p. ex. , Gabizon et al., J. National Cancer Inst. 81 (19): 1484 (1989)].

The concentration of the M-CSF antagonist in these compositions can vary widely, that is, 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 the volumes of liquid, viscosities, etc., according to the particular mode of administration that is choose The actual methods for preparing compositions that can be administered orally, topically and parenterally will be known or apparent to those skilled in the art and are described in detail in, for example, Remington's Pharmaceutical Science, 19th ed., Mack Publishing Co., Easton, PA (1995), which is incorporated herein by reference.

The determination of an effective amount of a composition of the invention can be achieved by standard empirical methods which are well known in the art. For example, the in vivo neutralizing activity of the sera of an individual treated with a given dose of the M-CSF antagonist can be assessed using an assay that determines the ability of the sera to block proliferation induced by M-CSF and the survival of murine monocytes (CDllb + cell, a subset of CD11 cells, which express high levels of the receptor for M-CSF) in vitro as described in Cenci et al., J Clin. Invest. 1055: 1279-87, 2000.

The compositions of the invention are administered to a mammal already suffering from, or predisposed to, an osteolytic disorder, including cancer metastasis and / or bone loss associated with cancer metastasis in an amount sufficient to prevent or at least partially arrest the development of such, disease. Effective amounts of an M-CSF antagonist will vary and will depend on the severity of the disease and the weight and general condition of the patient being treated, but generally range from about 1.0 mg / kg to about 100 mg / kg of weight body weight, or about 10 mg / kg to about 90 mg / kg, with doses from about 20 mg / kg to about 80 mg / kg or about 30 mg / kg to about 70 mg / kg or about 40 mg / kg kg at approximately 60mg / kg per application. For example, about 10 mg / kg to 50 mg / kg or about 20 mg / kg to 60 mg / kg of anti-MCSF antibody is an initial candidate dose for administration to the patient, eg, by one or more separate administrations or continuous infusion. Administration is daily, on alternate days, weekly or less frequently, as necessary, depending on the response to the disease and the patient's tolerance for treatment. Maintenance dose for longer periods, such as it may be 4, 5, 6, 7, 8, 10 or 12 weeks or longer may be necessary until a desired suppression of the symptoms of the disease is observed, and the doses can be adjusted as necessary. The progress of this treatment is easily monitored by traditional techniques and trials.

Single or multiple administrations of the compositions can be carried out with dose levels and standards selected by the attending physician. For the prevention or treatment of the disease, the appropriate dose of the M-CSF antagonist, including the antibody against M-CSF, will depend on the type of the disease to be treated, as defined above, the severity and course of the disease , if the antagonist is administered for preventive or therapeutic purposes, previous therapy, patient's clinical history and response to the antagonist, and the criteria of the attending physician. The antagonist is conveniently administered to the patient at one time or in a series of treatments.

The composition of the antagonist will be formulated, dosed and administered in a manner congruent with good medical practice. The factors to be taken into account in this sense includes the specific disorder that is being treated, the mammal being treated, the clinical state of the patient, the origin of the disorder, the place of supply of the agent, the method of administration, the administration program and other factors known to the practically doctor. The effective therapeutic amount of the antagonist to be administered will be governed by such considerations, and is the minimum amount necessary to prevent, ameliorate or treat the disease, condition or disorder mediated by M-CSF, particularly to treat cancer cells, and more particularly to treat metastasis of tumor cells. Such amount is preferably below the amount that is toxic to the host or makes the host significantly more susceptible to infections.

In another embodiment of the invention, an article of manufacture containing the materials useful for the treatment of the diseases, disorders or conditions described above is provided. The article of manufacture consists of a container and a label. Suitable containers include, for example, bottles, vials, syringes and test tubes. The containers can be formed from a variety of materials like glass or plastic. The containers contain a composition that is effective in treating the condition and can have a sterile access port (for example, the container can be an intravenous solution bag or a vial having a stopper that can be punctured by a hypodermic injection needle). ). The active agent of the composition is the M-CSF antagonist or antibody of the invention. The label on, or associated with, the container indicates that the composition is used for the treatment of the state of choice. The article of manufacture may also contain a second container containing a buffer solution accepted for pharmaceutical use, such as phosphate buffered saline, Ringer's solution and dextrose solution. In addition, other materials may be desirable from a commercial and user standpoint, including other buffer solutions, diluents, filters, needles, syringes and package inserts with instructions for use.

The invention is demonstrated by the following examples, which are not intended to be limiting in any way.

EXAMPLES EXAMPLE 1 This example establishes the dose-dependent, anti-resorptive effects of Zometa (zoledronate) in an animal model (Figure 14). The treatment with > = 0.03 mg / kg of Zometa inhibited osteolytic damage caused by tumor growth at the bone site. In addition, a dose-response effect was observed when the mice were treated with increasing concentrations of Zometa. The combined anti-mouse and anti-human M-CSF 5A1 and 5H4 mAbs also protected against bone damage. The antibody against M-CSF alone was more effective than 0.03 mg / kg of Zometa in the treatment of severe osteolytic damage. Osteolysis score based on x-ray image on the last day of the study (Figure 14): 0 = normal; 1 = wrong or minimal injury with normal architecture of the cortex; 2 = definite lytic lesion with minimal disruption of the cortex / architecture 3 = Large lesion (s) with disruption of the cortex / architecture 4 = gross destruction with non-preserved architecture From this initial study a combined study was made using a sub-effective dose of 0.03 mg / kg of Zometa. Two weeks after intratibial inoculation of 6 x 10 ° MDA-MB-231Luc cells, Nu / Nu mice were treated with 5A1 / 5H4 (10 mg / kg once a week), Zometa (0.03 mg / kg twice per week). week) or both antibody and bisphosphonate. The bone lesions were reviewed weekly by Faxitron analysis (x-ray technology), and at the end of the study, all the animals were subjected to final x-rays and the images collected and distributed for the severity score of the lesions. The analysis of the results showed that the anti-MCSF and Zometa mAbs were effective for the treatment of osteolysis, but that the combined treatment of Zometa and the mAb M-CSF inhibited the incidence (Figure 15) and the degree (Figure 16) of bone lysis to a greater degree than any treatment alone.

Figure 16 shows that treatment with 0.03 mg / kg Zometa or 10 mg / kg anti-M-CSF antibody (5A1 + 5H4) inhibited osteolytic damage caused by tumor growth at the bone site. A combined scheme of Zometa plus anti-MCSF antibody also inhibited bone lysis. The average scores of the osteolysis were calculated from: 1) an average of scores from 3 volunteers, and 2) a group average (originally 10 animals / group).

Osteolysis score based on x-ray image on the last day of the study: 0 = normal; 1 = equivocal or minimal lesion with normal architecture of the cortex: 2 = defined lytic lesion with minimal disruption of the cortex / architecture; 3 = large lesion (s) with disruption of the cortex / architecture; 4 = gross destruction with non-preserved architecture The examination of the representative faxitron images further demonstrated the severity of the lesions found in the untreated animals compared to the relatively minor lesions in the animals treated with Zometa and the anti-MCSF antibody (Figure 17). No adverse interactions were observed in the combination treatment group.

In conclusion, the anti-MCSF and Zometa antibody effectively inhibit osteolysis, and the combination of the two treatments result in an increased anti-sweating effect compared to any treatment alone. This suggests that the combination may be a safe and effective option for patients with bone disease who are intolerant of bisphosphonate, or who are already being treated with bisphosphonates.

EXAMPLE 2 This example shows that the inhibition of M-CSF activity had no effect on the differentiated activity of the osteoclasts (Figure 18). The effect of neutralizing antibodies aga M-CSF and bisphosphonate on the differentiated activity of osteoclasts was analyzed with humanized Chir-RXl and Zometa.

Human bone marrow CD34 + cells (Bio hittaker catalog number 2M-101A, 3x 10 cells / vial) were induced to 'differentiate into osteoclasts under experimental conditions described herein. During day 1, CD34 + cells were thawed from a vial frozen in 10 mL of medium (Alpha MEM with 10% FCS, 1 x Pen Strep and 1 x fungizone). The cells were washed once and resuspended in 2 mL of medium and plated on the OsteoLyse plate.

(OsteoLyse ™ Assay Kit (Human Collagen), Cambrex) at 100 uL per well. During day 2, without removing the original media, 50 μL of 4x CSF-1 is added to each well at 30 ng / mL final concentration and 50 μL of 4x RA KL (sRANKL, Chemicon catalog # GF091, 10 ug / package) for final concentration of 100 ng / mL. During day 7, 50 μL of 5x RANKL is added to each well for a final concentration of 100 ng / mL.

During day 15, the antibodies (Chir-RXl or control antibody) or Zometa were added at the concentrations indicated. On day 17, 10 uL of cell culture supernatant was sampled and mixed with 200 μL of Fluorophore Releasing Reagent in each well of the 96-well, black well plate (included in the OsteoLyse Assay Kit).

EXAMPLE 3 This example shows that Zometa inhibits the differentiated activity of osteoclasts in a dose-dependent manner (Figure 19). The effect of neutralizing antibodies · for M-CSF and bisphosphonate on the differentiated activity of osteoclasts was analyzed with humanized Chir-RXl and Zometa.

Human bone marrow CD34 + cells (Biowhittaker catalog number 23M-101A, 3 x 10 5 cells / vial) were induced to differentiate into osteoclasts under experimental conditions described herein. During day 1, CD34 + cells were thawed from a vial frozen in 10 mL of medium (Alfa MEM with 10% FCS, 1 x Pen Strep and lx fungizone). The cells were washed once and resuspended in 2 mL of medium and plated on OsteoLyse plates (OsteoLyse ™ Assay Kit (Human Collagen), Cambrex) at 100 uL per well. During day 2, without removing the original media, 50 μL of 4x CSF-1 is added to each well at 30 ng / mL of final concentration and 50 μL of 4x RA KL (sRANKL, Chemicon cátlogo # GF091, 10 ug / pack ) for the final concentration of 100 ng / mL. During day 7, 50 μL of 5x RANKL is added to each well for final concentration of 100 ng / mL.

During day 15, the antibodies (Chir-RXl or the control antibody) or Zometa were added at the indicated concentrations. On day 17, 10 uL of the cell culture supernatant was sampled and mixed with 200 μL of the Fluorofore Releasing Reagent reagent in each well of the black 96-well assay plate (included in the OsteoLyse Assay Kit).

EXAMPLE 4 This example shows the results of a pharmacokinetic and pharmacodynamic study using RX1 in primates (Figure 20 and Figure 21).

The aim of this study was to investigate the pharmacodynamics and pharmacokinetics of heRXl-lO.Gl, a humanized anti-human M-CSF antibody, when administered to cynomolgus monkeys as an intravenous injection of a single slow bolus (Groups 2 and 3 in the day 1) followed by an observation period of 13 weeks or as repeated doses (Group 1 on days 1, 43, 50 and 57) followed by an observation period of 10 weeks. The humanized anti-M-CSF IgGl monoclonal antibody was administered by intravenous (IV) slow bolus injection through a brachial or saphenous vein.

The use of the animal is required by the global regulatory agencies to evaluate the safety of new drugs. The antibody does not cross-react in rodent species but has been shown to be active in cynomolgus monkeys. Therefore, the cynomolgus monkey was selected since it is a non-rodent species accepted for use in studies with intravenous injection with biological substances.

The animals were randomly assigned in the following groups: a On day 1, group 1 will receive a dose of 0.2 mg / kg and on day 43, 50 and 57, the same animals will receive a dose of 10 mg / kg.

Groups 2 and 3 were administered with the formulation of the experimental article (2 and 20 mg / kg / dose, respectively) during day 1 by slow bolus intravenous injection for a period of approximately 10 minutes. The formulations were administered by a saphenous vein using a catheter and an abbocath. The volume of the dose was 4 mL / kg and the actual dose was based on the most recent practical body weight of each animal. Group 1 was administered a dose of 0.2 mg / kg on day 1, and subsequent doses of 10 mg / kg / dose on days 43, 50 and 57. The formulation was administered by slow bolus intravenous injection. (for an approximate period of 10 minutes) by a saphenous vein using a catheter and an abbocath. The volume of the dose was 4 mL / kg and the actual dose was based on the most recent practical body weight of each animal.

Blood was collected from all the animals for hematology and / or clinical biochemistry, as follows: The following parameters were examined: Hematology: morphology of blood cells; erythrocyte indices (MCV, MCH, MCHC and RD); hema'tocrito; hemoglobin; mean volume of platelets; Platelet count; account of red blood cells; reticulocytes (absolute and percent); and counts white blood cells (total, absolute and differential percent).

Clinical biochemistry: A / G ratio (calculated); alanine aminotransferase; albumin; alkaline phosphatase; aspartate aminotransferase; blood urea nitrogen; calcium; chloride; cholesterol; creatinine; globulin (calculated); glucose; inorganic phosphorus; potassium; sodium; total bilirubin, direct and indirect; total protein; triglycerides and C-reactive protein.

The biochemical markers of bone turnover were analyzed as follows (approximately 2 inL of blood was collected from all animals for the determination of bone biomarkers): (NTX: telopeptide N-terminal cross-linking of bone collagen) (CTX: telopeptide C-terminal cross-linking of bone collagen) Pharmacokinetic evaluation and activity of -CSF in serum: For Groups 2 and 3, blood was collected (1.5 mL each) by venipuncture in SST tubes before the dose, day 1 (immediately after the end of the infusion and 4 hours after the end of the infusion), and days 3, 8, 15, 22, 29, 43, 57, 71 and 85. For the animals of Group 1, collected blood (1.5 mL each) by venipuncture in SST tubes before the dose, on day 1 (immediately after the end of the infusion and 4 hours after the end of the infusion), and on days 3, 8, 15, 22 , 29, 43 (before the dose and 4 hours after the end of the infusion), 50 (before the dose and 4 hours after the end of the infusion), 57 (before the dose and 4 hours after the end of the infusion). infusion), 59, 64, 71, 78, 92, 106 and 120. Samples were analyzed for pharmacokinetic evaluation and for serum M-CSF activity and the remaining heRXl-10GG activity.

The blood samples were allowed to coagulate at room temperature for approximately 30 minutes before centrifugation. The serum was obtained by centrifugation at approximately 2700 rpm for 10 minutes at approximately 4 ° C and the serum obtained was divided into 4 aliquots.

All US Patents, US Patent Application Publications, US Patent Applications, Foreign Patents, Foreign Patent Applications and Non-patent publications referenced in this specification and / or listed in the Application Data Sheet are incorporated herein for reference in their entirety.

From the foregoing it will be noted that, although the specific embodiments of the invention have been described herein for demonstration purposes, it is possible to make various modifications without departing from the spirit and scope of the invention.

Claims (30)

1. A method of treating a patient suffering from an osteolytic disorder consisting of the steps of: administering to the patient an anti-M-CSF antibody in an effective monotherapeutic dose and an osteoclast inhibitor in an effective monotherapeutic dose.

2. A method of treating a patient suffering from an osteolytic disorder consisting of the steps of: administering to the patient an anti-M-CSF antibody in an effective monotherapeutic dose and a bisphosphonate in an effective monotherapeutic dose.

3. The method of claim 1, characterized in that the osteoclast inhibitor is a RANKL inhibitor.

4. The method of claim 2, characterized in that the anti-MCSF antibody is the antibody derived from RX1, antibody derived MCI, an antibody derived from MC3 or an antibody derived from 5H4.

5. The method of claim 2, characterized in that the anti-MCSF antibody is heRXl.

6. The method of any of the preceding claims, characterized in that the osteolytic disorder is osteoporosis, bone loss associated with cancer metastasis, Pager's disease or periprosthetic bone loss.

7. The method of any of the preceding claims, characterized in that the osteoclast inhibitor and the anti-MCSF antibody are administered at the same time.

8. The method of claim, characterized in that the RANKL inhibitor is selected from the group consisting of an anti-RANKL antibody, a soluble RA KL receptor and a RANKL vaccine.

9. The method of claims 3, 4, 6 and 7, characterized in that the anti-RANKL antibody is AMG-162.

10. The method of claims 2, 4, 6 and 7, characterized in that the bisphosphonate is selected from the group consisting of zoledronate, pamidronate, clodronate, etidronate, tiludronate, alendronate, ibandronate and risedronate.

11. The method of claim 10, characterized in that the bisphosphonate is zoledronate.

12. A method of treating a patient suffering from an osteolytic disorder consisting of the steps of administering to the patient an anti-M-CSF antibody in an effective monotherapeutic dose and a bisphosphonate in an effective monotherapeutic dose during a transition period.

13. The method of claim 12, characterized in that the osteoclast inhibitor is a bisphosphonate or a RANKL inhibitor.

14. The method of claim 13, characterized in that the transition period is approximately 1-7 days.

15. The method of claim 13, characterized in that the transition period is one week to one month.

16. The method of claim 13, characterized in that the transition period is 1 month to 3 months.

17. The method of claim 13, characterized in that the transition period is 3 to 6 months.

18. The method of claim 13, characterized in that the transition period is 6 to 12 months.

19. The method of claim 13, further comprises the step of stopping the bisphosphonate treatment after the. transition period.

20. The method of claim 13 further comprises the step of reducing the dose of the bisphosphonate after the transition period.

21. The method of claim 13, characterized in that the dose of bisphosphonate is reduced immediately after the transition period.

22. The method of claim 13 further comprises the step of reducing the dose of the anti-MCSF antibody after the transition period.

23. The method of claim 13, characterized in that the dose of the anti-MCSF antibody is reduced immediately after the transition period.

24. The method of claim 13, characterized in that the bisphosphonate is administered at least once after the transition period.

25. The method of any of the preceding claims, characterized in that the anti-MCSF antibody comprises at least two of the CDRs of the murine RXl antibody of SEQ ID NO: 5 and SEQ ID NO: 6.

26. The method of any of the preceding claims, characterized in that the anti-MCSF antibody comprises at least three of the CDRs of the murine RXl antibody of SEQ ID NO: 5 and SEQ ID NO: 6.

27. The method of any of the preceding claims, characterized in that the anti-MCSF antibody competes with the murine RXI antibody for binding to M-CSF of SEQ ID NO: 5 and SEQ ID NO: 6 in at least 75%.

28. The method of claims 6, 13 and 20, characterized in that the osteoclast inhibitor is zoledronate and the anti-M-CSF antibody is an antibody derived from RXl.

29. The method of claim 29, characterized in that the zoledronate is administered in a dose of between 0.5 mg and 4 mg.

30. The method according to claims 4, 6, 8, 10, 11 or 12, characterized in that the anti-MCSF antibody binds to the same epitope as the RX1, MCI, MC3 or 5H4 antibodies.