FIELD OF THE INVENTION

The present invention relates to methods and pharmaceutical compositions for the treatment of tissue lesions.

BACKGROUND OF THE INVENTION

Tissue lesions are a major concern since it may target a wide diversity of affected tissues, lesion anatomies and etiologies. Despite existing treatments, there remains a need for improved therapies and more creative approaches for treating tissue lesions.

In particular, wound healing is a process involved in many conditions, such as traumas or diseases, including vascular disorders, diabetes, steroid treatments induced changes and genetic abnormalities such as sickle-cell anaemia for example. Wound healing is an intricate and interactive biological process that includes three phases which overlap in time: inflammation, proliferation and tissue remodeling (Gurtner G C, Werner S, Barrandon Y, and Longaker M T. Wound repair and regeneration. Nature. 2008; 453 (7193):314-21). The well-coordinated interplay between tissue structures, mass number of residential cells (keratinocytes, fibroblasts and endothelial cells) and infiltrating immunocytes (neutrophils, macrophages and lymphocytes) are essential for the healing process (Singer A J, and Clark R A. Cutaneous wound healing. N Engl J Med. 1999; 341 (10):738-46). Fetal microchimeric cells (FMCs) have been reported to participate in maternal wound healing when adult mothers have been pregnant (Nassar D, Droitcourt C, Mathieu-d'Argent E, Kim M J, Khosrotehrani K, and Aractingi S. Fetal progenitor cells naturally transferred through pregnancy participate in inflammation and angiogenesis during wound healing. FASEB J. 2012; 26(1):149-57) (Seppanen E, Roy E, Ellis R, Bou-Gharios G, Fisk N M, and Khosrotehrani K. Distant mesenchymal progenitors contribute to skin wound healing and produce collagen: evidence from a murine fetal microchimerism model. PLoS One. 2013; 8 (5):e62662).

Indeed, FMCs, enter maternal circulation during all pregnancies in humans and rodents, and persist in maternal marrow for the remainder of the lifespan after delivery (O'Donoghue K, Chan J, de la Fuente J, Kennea N, Sandison A, Anderson J R, Roberts I A, and Fisk N M. Microchimerism in female bone marrow and bone decades after fetal mesenchymal stem-cell trafficking in pregnancy. Lancet. 2004; 364 (9429):179-82). These cells have been reported to include a heterogeneous population of progenitor cells namely lymphoid, hematopoietic and mesenchymal progenitors: these may differentiate in various maternal tissues in cardiomyocytes, neurons, hepatocytes, fibroblasts, endothelial cells, leukocytes, thyroid, cervix and gut epithelial cells etc. These cells are able to seed in variable proportion different maternal organs (Fujiki Y, Johnson K L, Peter I, Tighiouart H, and Bianchi D W. Fetal cells in the pregnant mouse are diverse and express a variety of progenitor and differentiated cell markers. Biol Reprod. 2009; 81 (1):26-32). Indeed, fetal cells are discovered in high frequency in injured tissues (Khosrotehrani K, Johnson K L, Cha D H, Salomon R N, and Bianchi D W. Transfer of fetal cells with multilineage potential to maternal tissue. JAMA. 2004; 292 (1):75-80). FMCs have therefore been identified in acute and chronic wounds, inflamed skin, tumors such as melanoma and carcinoma, participating in blood vessel endothelium in maternal affected tissues. These data indicate the transfer of fetal endothelial progenitor cells during pregnancy and their involvement in later maternal neovascularization and wound healing process.

Chemokines are small signal proteins secreted by cells, which can direct chemotaxis movement for nearby responsive cells and play a crucial role for tissue homeostasis. CCL2, also named monocyte chemotactic protein 1 (MCP-1) is a member of CC chemokine family, secreted by monocytes and macrophages, exerts its potent chemotaxis ability for monocytes, T lymphocytes, macrophages, natural killer (NK) cells and endothelial cells. The major receptor for CCL2 is CCR2 and it is highly expressed on the surface of various cells, including monocytes, macrophages, endothelial cells. CCL2 attracts target cells through binding strongly to CCR2 on their surface and mediates chemotactic function (Boring L, Gosling J, Chensue S W, Kunkel S L, Farese R V, Jr., Broxmeyer H E, and Charo I F. Impaired monocyte migration and reduced type 1 (Th1) cytokine responses in C-C chemokine receptor 2 knockout mice. J Clin Invest. 1997; 100(10):2552-61).

Because of the presence of their receptors on various immunocytes and residential cells, chemokines play crucial roles in all three phases of wound healing: not only to direct the recruitment of leukocytes during early inflammatory phase but also to contribute to angiogenesis, re-epithelialization and tissue remodeling during the later steps (Gillitzer R, and Goebeler M. Chemokines in cutaneous wound healing. J Leukoc Biol. 2001; 69(4):513-21). CCL2 participates to wound healing process, especially during the early stage (Engelhardt E, Toksoy A, Goebeler M, Debus S, Brocker E B, and Gillitzer R. Chemokines IL-8, GROalpha, MCP-1, IP-10, and Mig are sequentially and differentially expressed during phase-specific infiltration of leukocyte subsets in human wound healing. Am J Pathol. 1998; 153(6):1849-60). Overexpression of CCL2 has been reported in human burn wound (Gibran N S, Ferguson M, Heimbach D M, and Isik F F. Monocyte chemoattractant protein-1 mRNA expression in the human burn wound. J Surg Res. 1997; 70(1):1-6). Deficiency of CCL2, together with other chemokines, resulted in impaired recruitment of myeloid cells and skin healing (Lin Q, Fang D, Fang J, Ren X, Yang X, Wen F, and Su S B. Impaired wound healing with defective expression of chemokines and recruitment of myeloid cells in TLR3-deficient mice. J Immunol. 2011; 186(6):3710-7), while in the same way full CCL2 deficiency causes delayed myocardial infarcts healing (Dewald O, Zymek P, Winkelmann K, Koerting A, Ren G, Abou-Khamis T, Michael L H, Rollins B J, Entman M L, and Frangogiannis N G. CCL2/Monocyte Chemoattractant Protein-1 regulates inflammatory responses critical to healing myocardial infarcts. Circ Res. 2005; 96(8):881-9). These data indicate that the presence of CCL2 is mandatory for normal healing. However, injection of CCL2 at various doses in normal recipients do not modify wound healing (Di Pietro, 2001). In contrast, CCL2 administration improves wound healing when there is a defect in CCL2 secretion such as in diabetic wound (Wood S, Jayaraman V, Huelsmann E J, Bonish B, Burgad D, Sivaramakrishnan G, Qin S, DiPietro L A, Zloza A, Zhang C, et al. Pro-inflammatory chemokine CCL2 (MCP-1) promotes healing in diabetic wounds by restoring the macrophage response. PLoS One. 2014; 9(3):e91574). CCR2, the CCL2 receptor, mediates monocytes, macrophages Ly6C+ and hematopoietic progenitor cells migrating from bone marrow to blood and then to injured or inflammed tissues. Therefore, the CCL2/CCR2 pathway mediates recruitment of immunocytes and marrow cells that have well been established.

SUMMARY OF THE INVENTION

The present invention relates to methods and pharmaceutical compositions for the treatment of tissue lesions. In particular, the invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION

Here, the inventors report that CCL2 is expressed mainly by leukocytes during early stages of skin wound healing. They show that CCR2 is expressed on FMCs, especially on a subpopulation of progenitor cells, that they call “fetal myeloid progenitor cells” (FMPCs), and mediates the recruitment of these cells to maternal wound tissue. Moreover, the inventors report that recruited FMCs/FMPCs improve maternal skin wound healing by organizing blood vessel endothelium and secreting pro-angiogenesis peptides, particularly chemokine CXCL1, to enhance angiogenesis in wound. Their findings suggest the pivotal role of CCL2/CCR2 signaling in recruitment of FMCs/FMPCs and their participant in maternal tissue repair.

A first aspect of the present invention relates to a method of treating a tissue lesion in a subject in need thereof comprising administering the subject with a therapeutically effective amount of a CCR2 agonist.

As used herein, the term “subject” denotes a female mammal, such as a rodent, a feline, a canine, and a primate who is pregnant or who has been pregnant at least one time. Thus, it is understood that the subject according to the present invention is not a male. Preferably the subject according to the invention is a woman. Preferably, the subject according to the present invention is a woman who is pregnant or who has been pregnant at least one time. In the context of the present invention, a “subject in need thereof” denotes a subject, preferably a woman suffering from any tissue lesion.

As used herein, the term “CCR2” for “chemokine (C-C motif) receptor 2” has its general meaning in the art and refers to a chemokine receptor. CCR2 is a member of G-protein coupled receptors superfamily and binds to the CCL2, CCL7, CCL8, CCL12, CCL13 and CCL16 chemokines. CCR2 transduces a signal by increasing intracellular calcium ion levels.

Chemokines receptor CCR2, through either of any isoform A (SEQ ID N:1), or isoform B (SEQ ID N:2):

SEQ ID NO: 1 (isoform A):10         20         30         40MLSTSRSRFI RNTNESGEEV TTFFDYDYGA PCHKFDVKQI50         60         70         80GAQLLPPLYS LVFIFGFVGN MLVVLILINC KKLKCLTDIY90        100        110        120LLNLAISDLL FLITLPLWAH SAANEWVFGN AMCKLFTGLY130        140        150        160HIGYFGGIFF IILLTIDRYL AIVHAVFALK ARTVTFGVVT170        180        190        200SVITWLVAVF ASVPGIIFTK CQKEDSVYVC GPYFPRGWNN210        220        230        240FHTIMRNILG LVLPLLIMVI CYSGILKTLL RCRNEKKRHR250        260        270        280AVRVIFTIMI VYFLFWTPYN IVILLNTFQE FFGLSNCEST290        300        310        320SQLDQATQVT ETLGMTHCCI NPIIYAFVGE KFRSLFHIAL330        340        350        360GCRIAPLQKP VCGGPGVRPG KNVKVTTQGL LDGRGKGKSI370GRAPEASLQD KEGASEQ ID NO: 2 (isoform B):10         20         30         40MLSTSRSRFI RNTNESGEEV TTFFDYDYGA PCHKFDVKQI50         60         70         80GAQLLPPLYS LVFIFGFVGN MLVVLILINC KKLKCLTDIY90        100        110        120LLNLAISDLL FLITIPLWAH SAANEWVFGN AMCKLFTGLY130        140        150        160HIGYFGGIFF IILLTIDRYL AIVHAVFALK ARTVTFGVVT170        180        190        200SVITWLVAVF ASVPGIIFTK CQKEDSVYVC GPYFPRGWNN210        220        230        240FHTIMRNILG LVLPLLIMVI CYSGILKTLL RCRNEKKRHR250        260        270        280AVRVIFTIMI VYFLFWTPYN IVILLNTFQE FFGLSNCEST290        300        310        320SQLDQATQVT ETLGMTHCCI NPIIYAFVGE KFRRYLSVFF330        340        350        360RKHITKRFCK QCPVFYRETV DGVTSTNTPS TGEQEVSAGL

As used herein, the term “CCR2 agonist” refers to any compound, natural or not, that is able to bind to CCR2 and promotes CCR2 activity.

As used herein, the term “tissue lesion” relates to abnormal biological tissue change or damage in the tissue of an organism, usually caused by disease or trauma. It may be a cut, a burn, a wound, or also resulting from the action of a pathogen such as parasite or infection as well as metabolic, physiological, vascular, genetic or immune disorder.

In some embodiment, the tissue lesion is selected from the group consisting of skin lesion, hepatic lesion, cardiac lesion, lung lesion, neurologic lesion, ocular lesion, stomach lesion, pancreas lesion, spleen lesion, bowel lesion, thyroid lesion, thymus lesion, kidney lesion, artery lesion, vein lesion, bone lesion, bone marrow lesion, muscle lesion, tendon lesion, ligament lesion, reproductive organs lesion or endocrine glands lesion.

In some embodiment, the tissue lesion is selected from the group consisting of skin lesion, hepatic lesion, cardiac lesion, lung lesion, neurologic lesion, ocular lesion, stomach lesion, pancreas lesion, spleen lesion, bowel lesion, thyroid lesion, thymus lesion, kidney lesion, artery lesion except carotid aneurysm, vein lesion, bone lesion, bone marrow lesion, muscle lesion, tendon lesion, ligament lesion, reproductive organs lesion or endocrine glands lesion.

In some embodiments, the tissue lesion is a diabetic foot ulcer resulting from diabetes.

In some embodiment, the tissue lesion is leg ulcer resulting from sickle-cell anemia.

In some embodiment, the tissue lesion is leg ulcer resulting from micro or macro vascular disease.

In some embodiments, the tissue lesion results from sickle-cell disease.

It is to be understood that the term “wound” as used herein includes surgical incisions as well as wounds caused by accidental trauma or pathologies.

The term also includes venous stasis ulcer, burns, delayed wound healing observed during corticoid treatments, delayed wound healing observed in elderly (aging defect), stress, delayed wound healing observed in diabetic patients, epithelialization defects of surgical scars or following skin grafts, finger cracks occurring after cold exposure, nail pathologies associated with delayed healing, foot blisters occurring during prolonged walk or run.

Corneal wounds may result from observed various diseases such as corneal ulcer, corneal erosion or trauma, keratitis and dry eye. Wound may also result from topical administration of drugs and surgery situations, such as keratoplasty and during the time-course of recovery from corneal graft.

In particular, the term “wound” as used herein refers to any wound resulting from diabetes, such as diabetic foot ulcer or to any wound resulting from sickle-cell anaemia, such as leg ulcer.

As used herein, the terms “treating” or “treatment” refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subject who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By “therapeutic regimen” is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase “induction regimen” or “induction period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a “loading regimen”, which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase “maintenance regimen” or “maintenance period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

In some embodiments, the CCR2 agonist is a small organic molecule. The term “small organic molecule” refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e. g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, more in particular up to 2000 Da, and most in particular up to about 1000 Da.

In some embodiments, the CCR2 agonist is a CCR2 antibody or a portion thereof. In some embodiments, the CCR2 agonist is selected from the group consisting of chimeric antibodies, humanized antibodies or full human monoclonal antibodies.

As used herein, “antibody” includes both naturally occurring and non-naturally occurring antibodies. Specifically, “antibody” includes polyclonal and monoclonal antibodies, and monovalent and divalent fragments thereof. Furthermore, “antibody” includes chimeric antibodies, wholly synthetic antibodies, single chain antibodies, and fragments thereof. The antibody may be a human or nonhuman antibody. A nonhuman antibody may be humanized by recombinant methods to reduce its immunogenicity in man.

In one embodiment of the antibodies or portions thereof described herein, the antibody is a monoclonal antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a polyclonal antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a humanized antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a chimeric antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a light chain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a heavy chain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fab portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a F(ab′)2 portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fc portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fv portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a variable domain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises one or more CDR domains of the antibody.

Antibodies are prepared according to conventional methodology. Monoclonal antibodies may be generated using the method of Kohler and Milstein (Nature, 256:495, 1975). To prepare monoclonal antibodies useful in the invention, a mouse or other appropriate host animal is immunized at suitable intervals (e.g., twice-weekly, weekly, twice-monthly or monthly) with antigenic forms of CCR2. The animal may be administered a final “boost” of antigen within one week of sacrifice. It is often desirable to use an immunologic adjuvant during immunization. Suitable immunologic adjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, alum, Ribi adjuvant, Hunter's Titermax, saponin adjuvants such as QS21 or Quil A, or CpG-containing immunostimulatory oligonucleotides. Other suitable adjuvants are well-known in the field. The animals may be immunized by subcutaneous, intraperitoneal, intramuscular, intravenous, intranasal or other routes. A given animal may be immunized with multiple forms of the antigen by multiple routes. Briefly, the recombinant CCR2 may be provided by expression with recombinant cell lines. In particular, CCR2 may be provided in the form of human cells expressing CCR2 at their surface. Following the immunization regimen, lymphocytes are isolated from the spleen, lymph node or other organ of the animal and fused with a suitable myeloma cell line using an agent such as polyethylene glycol to form a hydridoma. Following fusion, cells are placed in media permissive for growth of hybridomas but not the fusion partners using standard methods, as described (Coding, Monoclonal Antibodies: Principles and Practice: Production and Application of Monoclonal Antibodies in Cell Biology, Biochemistry and Immunology, 3rd edition, Academic Press, New York, 1996). Following culture of the hybridomas, cell supernatants are analyzed for the presence of antibodies of the desired specificity, i.e., that selectively bind the antigen. Suitable analytical techniques include ELISA, flow cytometry, immunoprecipitation, and western blotting. Other screening techniques are well-known in the field. Preferred techniques are those that confirm binding of antibodies to conformationally intact, natively folded antigen, such as non-denaturing ELISA, flow cytometry, and immunoprecipitation.

Significantly, as is well-known in the art, only a small portion of an antibody molecule, the paratope, is involved in the binding of the antibody to its epitope (see, in general, Clark, W. R. (1986) The Experimental Foundations of Modern Immunology Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed., Blackwell Scientific Publications, Oxford). The Fc′ and Fc regions, for example, are effectors of the complement cascade but are not involved in antigen binding. An antibody from which the pFc′ region has been enzymatically cleaved, or which has been produced without the pFc′ region, designated an F(ab′)2 fragment, retains both of the antigen binding sites of an intact antibody. Similarly, an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, designated an Fab fragment, retains one of the antigen binding sites of an intact antibody molecule. Proceeding further, Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd. The Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation.

Within the antigen-binding portion of an antibody, as is well-known in the art, there are complementarity determining regions (CDRs), which directly interact with the epitope of the antigen, and framework regions (FRs), which maintain the tertiary structure of the paratope (see, in general, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragment and the light chain of IgG immunoglobulins, there are four framework regions (FR1 through FR4) separated respectively by three complementarity determining regions (CDR1 through CDRS). The CDRs, and in particular the CDRS regions, and more particularly the heavy chain CDRS, are largely responsible for antibody specificity.

It is now well-established in the art that the non CDR regions of a mammalian antibody may be replaced with similar regions of conspecific or heterospecific antibodies while retaining the epitopic specificity of the original antibody. This is most clearly manifested in the development and use of “humanized” antibodies in which non-human CDRs are covalently joined to human FR and/or Fc/pFc′ regions to produce a functional antibody.

This invention provides in certain embodiments compositions and methods that include humanized forms of antibodies. As used herein, “humanized” describes antibodies wherein some, most or all of the amino acids outside the CDR regions are replaced with corresponding amino acids derived from human immunoglobulin molecules. Methods of humanization include, but are not limited to, those described in U.S. Pat. Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,761, 5,693,762 and 5,859,205, which are hereby incorporated by reference. The above U.S. Pat. Nos. 5,585,089 and 5,693,761, and WO 90/07861 also propose four possible criteria which may used in designing the humanized antibodies. The first proposal was that for an acceptor, use a framework from a particular human immunoglobulin that is unusually homologous to the donor immunoglobulin to be humanized, or use a consensus framework from many human antibodies. The second proposal was that if an amino acid in the framework of the human immunoglobulin is unusual and the donor amino acid at that position is typical for human sequences, then the donor amino acid rather than the acceptor may be selected. The third proposal was that in the positions immediately adjacent to the 3 CDRs in the humanized immunoglobulin chain, the donor amino acid rather than the acceptor amino acid may be selected. The fourth proposal was to use the donor amino acid reside at the framework positions at which the amino acid is predicted to have a side chain atom within 3A of the CDRs in a three dimensional model of the antibody and is predicted to be capable of interacting with the CDRs. The above methods are merely illustrative of some of the methods that one skilled in the art could employ to make humanized antibodies. One of ordinary skill in the art will be familiar with other methods for antibody humanization.

In one embodiment of the humanized forms of the antibodies, some, most or all of the amino acids outside the CDR regions have been replaced with amino acids from human immunoglobulin molecules but where some, most or all amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they would not abrogate the ability of the antibody to bind a given antigen. Suitable human immunoglobulin molecules would include IgG1, IgG2, IgG3, IgG4, IgA and IgM molecules. A “humanized” antibody retains a similar antigenic specificity as the original antibody. However, using certain methods of humanization, the affinity and/or specificity of binding of the antibody may be increased using methods of “directed evolution”, as described by Wu et al., /. Mol. Biol. 294:151, 1999, the contents of which are incorporated herein by reference.

Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. See, e.g., U.S. Pat. Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and references cited therein, the contents of which are incorporated herein by reference. These animals have been genetically modified such that there is a functional deletion in the production of endogenous (e.g., murine) antibodies. The animals are further modified to contain all or a portion of the human germ-line immunoglobulin gene locus such that immunization of these animals will result in the production of fully human antibodies to the antigen of interest. Following immunization of these mice (e.g., XenoMouse (Abgenix), HuMAb mice (Medarex/GenPharm)), monoclonal antibodies can be prepared according to standard hybridoma technology. These monoclonal antibodies will have human immunoglobulin amino acid sequences and therefore will not provoke human anti-mouse antibody (KAMA) responses when administered to humans.

In vitro methods also exist for producing human antibodies. These include phage display technology (U.S. Pat. Nos. 5,565,332 and 5,573,905) and in vitro stimulation of human B cells (U.S. Pat. Nos. 5,229,275 and 5,567,610). The contents of these patents are incorporated herein by reference.

Thus, as will be apparent to one of ordinary skill in the art, the present invention also provides for F(ab′) 2 Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab′)2 fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and/or CDR1 and/or CDR2 regions have been replaced by homologous human or non-human sequences. The present invention also includes so-called single chain antibodies.

The various antibody molecules and fragments may derive from any of the commonly known immunoglobulin classes, including but not limited to IgA, secretory IgA, IgE, IgG and IgM. IgG subclasses are also well known to those in the art and include but are not limited to human IgG1, IgG2, IgG3 and IgG4.

In another embodiment, the antibody according to the invention is a single domain antibody. The term “single domain antibody” (sdAb) or “VHH” refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called “Nanobody®”.

In some embodiments, the CCR2 agonist is a polypeptide. In a particular embodiment the polypeptide is a functional equivalent of CCL2.

As used herein, the term “CCL2” refers to the chemokine (C-C motif) ligand 2, also referred as monocyte chemotactic protein 1 (MCP1) and small inducible cytokine A2. CCL2 is a small cytokine that belongs to the CC chemokine family.

CCL2 (SEQ ID NO: 3):1MKVSAALLCL LLIAATFIPQ GLAQPDAINA PVTCCYNFTN RKISVQRLAS51YRRITSSKCP KEAVIFKTIV AKEICADPKQ KWVQDSMDHL DKQTQTPKT

As used herein, a “functional equivalent of CCL2” is a polypeptide which is capable of binding to CCR2, thereby promoting a CCR2 activity according to the invention. The term “functional equivalent” includes fragments, mutants, and muteins of CCL2. The term “functionally equivalent” thus includes any equivalent of CCL2 obtained by altering the amino acid sequence, for example by one or more amino acid deletions, substitutions or additions such that the protein analogue retains the ability to bind to CCR2 and promote an CCR2 activity according to the invention. Amino acid substitutions may be made, for example, by point mutation of the DNA encoding the amino acid sequence.

In some embodiments, the functional equivalent of a polypeptide is at least 80% homologous to the corresponding protein.

In some embodiments, the functional equivalent of a polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the corresponding protein.

In some embodiments, the CCR2 agonist is a polypeptide having at least 80% of identity with SEQ ID NO:3.

In a preferred embodiment, the functional equivalent of a polypeptide is at least 90% homologous as assessed by any conventional analysis algorithm such as for example, the Pileup sequence analysis software (Program Manual for the Wisconsin Package, 1996).

In some embodiments, the present invention provides a polypeptide which comprises consecutive amino acids having a sequence which corresponds to the sequence of at least a portion of CCL2, which portion binds to CCR2 and promotes the CCR2 activity according to the invention.

The polypeptides of the invention may be produced by any suitable means, as will be apparent to those of skill in the art. In order to produce sufficient amounts of polypeptides or functional equivalents thereof for use in accordance with the present invention, expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the polypeptide of the invention. In particular, the polypeptide is produced by recombinant means, by expression from an encoding nucleic acid molecule. Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. When expressed in recombinant form, the polypeptide is in particular generated by expression from an encoding nucleic acid in a host cell. Any host cell may be used, depending upon the individual requirements of a particular system. Suitable host cells include bacteria mammalian cells, plant cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells. HeLa cells, baby hamster kidney cells and many others. Bacteria are also preferred hosts for the production of recombinant protein, due to the ease with which bacteria may be manipulated and grown. A common, preferred bacterial host isE coli.

In some embodiments, the polypeptide of the invention is an immunoadhesin.

As used herein, the term “immunoadhesin” designates antibody-like molecules which combine the binding specificity of a heterologous protein (an “adhesin” which is able to bind to CCR2) with the effector functions of immunoglobulin constant domains. Structurally, the immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity to CCR2 (i.e., is “heterologous”), and an immunoglobulin constant domain sequence. The adhesin part of an immunoadhesin molecule typically is a contiguous amino acid sequence comprising at least the binding site for CCR2. In one embodiment, the adhesin comprises the polypeptides characterized by SEQ ID NO:3. The immunoglobulin constant domain sequence in the immunoadhesin may be obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM.

The immunoglobulin sequence typically, but not necessarily, is an immunoglobulin constant domain (Fc region). Immunoadhesins can possess many of the valuable chemical and biological properties of human antibodies. Since immunoadhesins can be constructed from a human protein sequence with a desired specificity linked to an appropriate human immunoglobulin hinge and constant domain (Fc) sequence, the binding specificity of interest can be achieved using entirely human components. Such immunoadhesins are minimally immunogenic to the patient, and are safe for chronic or repeated use.

The polypeptides of the invention, fragments thereof and fusion proteins (e.g. immunoadhesin) according to the invention can exhibit post-translational modifications, including, but not limited to glycosylations, (e.g., N-linked or O-linked glycosylations), myristylations, palmitylations, acetylations and phosphorylations (e.g., serine/threonine or tyrosine).

In specific embodiments, it is contemplated that polypeptides used in the therapeutic methods of the present invention may be modified in order to improve their therapeutic efficacy. Such modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify biodistribution. For example, the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution. In example adding dipeptides can improve the penetration of a circulating agent in the eye through the blood retinal barrier by using endogenous transporters.

In one embodiment, the CCR2 agonist is an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by EXponential enrichment (SELEX) of a random sequence library. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such asE. coliThioredoxin A that are selected from combinatorial libraries by two hybrid methods.

In a particular embodiment, the CCR2 agonist is CCL2.

In some embodiments, the CCR2 agonist of the invention is administered to the subject with a therapeutically effective amount.

By a “therapeutically effective amount” is meant a sufficient amount of CCR2 agonist to treat tissue lesions at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. In particular, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, in particular from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

The compositions according to the invention are formulated for parenteral, transdermal, oral, rectal, subcutaneous, sublingual, intrapulmonary, topical or intranasal administration. Local administration can include simple solutions, gels, emulsions, liposomes; but also devices with patches or dressings or intra dermal or subcutaneous injections.

Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.

In a particular embodiment, the pharmaceutical compositions are formulated for parenteral administration. The pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.

In a particular embodiment, the pharmaceutical compositions are formulated for topical administration.

In one embodiment, the pharmaceutical compositions are formulated for being directly administrated into the lesion site.

The CCR2 agonist of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.

“Pharmaceutically” or “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

In some embodiments, the CCR2 agonist of the invention may be administered in combination with conventional treatment usually used for treating tissue lesions. For example, debridement, offloading, and infection control are conventional treatment for the treatment of ulcers in diabetes.

A further object of the invention relates to a method for screening a drug for the treatment of tissue lesions comprising the steps of i) providing a plurality of test substances ii) determining whether the test substances are CCR2 agonists and iii) positively selecting the test substances that are CCR2 agonists.

Typically, the screening method of the invention involves providing appropriate cells which express CCR2 on their surface. Such cells include cells from mammals, yeast,DrosophilaorE. coli. In particular, a polynucleotide encoding CCR2 is used to transfect cells to express the receptor. The expressed receptor is then contacted with a test substance and a CCR2 ligand (e.g. CCL2), as appropriate, to observe activation of a functional response. In particular comparison steps may involve to compare the activity induced by the test substance and the activity induce by a well known CCR2 agonist such as CCL2. In particular substances capable of having an activity similar or even better than a well known CCR2 agonist are positively selected.

Typically, the screening method of the invention may also involve screening for test substances capable of binding of to CCR2 present at cell surface. Typically the test substance is labelled (e.g. with a radioactive label) and the binding is compared to a well known CCR2 agonist such as CCL2.

Typically, the candidate compound is selected from the group consisting of small organic molecules, peptides, polypeptides or oligonucleotides.

The test substances that have been positively selected may be subjected to further selection steps in view of further assaying its properties for the treatment of tissue lesions. For example, the candidate compounds that have been positively selected may be subjected to further selection steps in view of further assaying its properties on animal models for tissue lesions.

The above assays may be performed using high throughput screening techniques for identifying test substances for developing drugs that may be useful to the treatment of tissue lesions. High throughput screening techniques may be carried out using multi-well plates (e.g., 96-, 389-, or 1536-well plates), in order to carry out multiple assays using an automated robotic system. Thus, large libraries of test substances may be assayed in a highly efficient manner. More particularly, stably-transfected cells growing in wells of micro-titer plates (96 well or 384 well) can be adapted to high through-put screening of libraries of compounds. Compounds in the library will be applied one at a time in an automated fashion to the wells of the microtitre dishes containing the transgenic cells described above. Once the test substances which induce the activity of CCR2 are identified, they can be positively selected for further characterization. These assays offer several advantages. The exposure of the test substance to a whole cell allows for the evaluation of its activity in the natural context in which the test substance may act. Because this assay can readily be performed in a microtitre plate format, the assays described can be performed by an automated robotic system, allowing for testing of large numbers of test samples within a reasonably short time frame. The assays of the invention can be used as a screen to assess the activity of a previously untested compound or extract, in which case a single concentration is tested and compared to controls. These assays can also be used to assess the relative potency of a compound by testing a range of concentrations, in a range of 100 μM to 1 μM, for example, and computing the more efficient concentration.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

