ANTIBODIES AGAINST THE INTEGRINA ALFA 2 AND ITS USESCROSS REFERENCEThis application claims the benefit, under 35 U.S.C. § 119 (e), of the provisional application in the USA. with serial number 60 / 738,303 filed on November 18, 2005, which is incorporated here in its entirety for your reference.
TECHNICAL FIELDThe present invention is generally related to antibodies directed to the a2ß1 integrin and its uses, including humanized antibodies against the alpha 2 integrin (a2) and methods of treatment with antibodies against integrin a.2.
BACKGROUND OF THE INVENTIONThe a2ß1 integrin (very late activation antigen 2, VLA-2) is expressed in a wide variety of cell types including platelets, vascular endothelial cells, epithelial cells, activated monocytes / macrophages, fibroblasts, leukocytes, lymphocytes, neutrophils and activated mast cells . (Hemler, Annu Rev Immunol 8: 365: 365-400 (1999); Wu and Santoro, Dev. Dyn. 206: 169-171 (1994); Edelson e.,., Blood. 103 (6): 2214-20 (2004), Dickeson et al, Cell Adhesion and Communication 5: 273-281 (1998)). The most typical ligands for a2ß1 include collagen and laminin, which are found in the extracellular matrix. In general, domain I of integrin a2 binds to collagen in a divalent cation-dependent manner while the same domain binds to laminin via both independent and divalent cation-dependent mechanisms. (Dickeson et al, Cell Adhesion and Communication 5: 273-281 (1998)). The specificity of the a2ß1 integrin varies with the cell type and serves as a collagen and / or laminin receptor for specific cell types, for example, the integrin a2β1 is known as a collagen receptor for platelets and as a laminin receptor for cells endothelial (Dickeson et al, J Biol. Chem. 272: 7661-7668 (1997)). Echovirus-1, decorin, E-cadherin, matrix metalloproteinase 1 (MMP-I), endorepepin and multiple collectins, and C1q protein of complement are also ligands for the a2ß1 integrin. (Edelson et al., Blood 107 (1): 143-50 (2006)). The a2ß1 integrin has been implicated in several biological and pathological processes including collagen-induced platelet aggregation, cellular migration to collagen, cell-dependent reorganization of collagen fibers, as well as cellular collagen-dependent responses that result in increase in the expression and proliferation of cytokines, (Gendron, J. Biol. Chem. 278: 48633-48643 (2003), Andreasen et al., J. Immunol., 171: 2804-2811 (2003); Rao et al. , J. Immunol., 165 (9): 4935-40 (2000)), aspects of the function of T cells, mast cells and neutrophils (Chan et al., J. Immunol., 147: 398-404 (1991); and de Fougerolles, Curr Opin Immunol 13: 286-290 (2001), Edelson et al., Blood 103 (6): 2214-20 (2004), Werr et al., Blood 95: 1804-1809 (2000) ), aspects of delayed type hypersensitivity, contact hypersensitivity and collagen-induced arthritis (de Fougerolles et al., J. Clin Invest. 105: 721-720 (2000); Kriegelstein et al. al., J. Clin. Invest. 110 (12): 1773-82 (2002)), ductal morphogenesis of the mammary gland (Keely et al., J. Cell Sci. 108: 595-607 (1995); Zutter et al., Am. J. Pathol. 155 (3): 927-940 (1995)), healing of epidermal wounds (Pilcher et al., J. Biol. Chem. 272: 181457-54 (1997)), and processes associated with VEGF-induced angiogenesis ( Senger et al., Am. J. Pathol. 160 (1): 195-204 (2002)). Integrins are heterodimers composed of a subunit a and a β, and make up a large family of proteins on the surface of cells that mediate cell adhesion to the extracellular matrix (ECM) as well as plasma proteins and they are central to some types of cell-cell interactions. Integrins interact with ECM components through their extracellular domains. (Pozzi &Zent, Exp Nephrol 94: 77-84 (2003)). By binding to ligands, integrins transduce intracellular signals to the cytoskeleton that modify cellular activity in response to these cell adhesion events, which is known as outward signaling (see, for example, Hemler, Annu Rev Immunol 8: 365: 365-400 (1999); Hynes, Cell. 110 (6): 673-87 (2002)). That signaling can also activate other subtypes of integrins expressed in the same cell, which is known as signaling from the inside out. Inward-out signaling occurs through regulatory signals that originate within the cytoplasm of the cell as an interruption of the binding between a subunit a and a β, which are then transmitted to the external ligand binding domain of the receptor. Integrins can play important roles in cell adhesion events that control the development, morphogenesis, physiology and pathology of an organ, as well as normal tissue homeostasis and immune and thrombotic responses, as well as serving as environmental sensors for the cell. These proteins are characterized by having a closed conformation under normal conditions that when activated undergo a rapid change in the conformation that exposes the site of ligand binding. X-ray crystallography is a recent tool that has been used in the study of the structure and mechanisms of integrin activation. The understanding of the structural characteristics of the integrin facilitates a better understanding of the binding sites, the differentiated states and their active and inactive formations. In general, the binding site for the ligand / counter-receptor for all integrins is within the a domain and is composed of a metal ion-dependent binding site, known as the MIDAS domain (Dembo ei al. , J Biol. Chem. 274, 32108-32111 (1988); Feuston et al., J. Med. Chem. 46: 5316-5325 (2003); Gadek et al., Science 295 (5557): 1086-9 ( 2002)); Gurrath et al., Eur. J. Biochem. 210: 911-921 (1992)). In the α subunits of the collagen-binding integrins, which include the a1, a2, a10, and a11 integrins, the MIDAS site is located within an additional domain inserted into the N-terminus known as domain I, A ol / A, a characteristic that it shares with subunits a of the family of ß2 leukocyte integrins (Randi and Hogg, J Biol Chem. 269: 12395-8 (1994), Larson et al. J Cell Biol. 108 (2): 703-12 (1989), Lee et al., J Biol Chem. 269: 12395-8 (1995), Emsley et al, J. Biol. Chem. 272: 28512-28517 (1997) and Cell 100: 47-56 (2000) ). The I domains are structurally homologous to the A1 domain of von Willebrandt factor, with a Rossman-type folding topology of six strands of β-strands surrounded by seven a-helices (Colombatti and Bonaldo, Blood 77 (11): 2305-15 (1991); Larson et al, J Cell Biol. 108 (2): 703-712 (1989); Emsley et al, J. Biol. Chem. 272: 28512-28517 (1997); Nolte et al; FEBS Letters, 452 (3) : 379-385 (1999)). The integrins that bind to the collagen have an additional α-helix known as the aC helix (Emsley et al, J. Biol. Chem. 272: 28512-28517 (1997) and Cell 100: 47-56 (2000); Nolte et al; FEBS Letters, 452 (3): 379-385 (1999)). The interactions between integrins and ligands can facilitate extravasation of leukocytes in inflamed tissues (Jackson et al., J. Med. Chem. 40: 3359-3368 (1997); Gadek et al., Science 295 (5557). : 1086-9 (2002), Sircar et al., Bioorg, Med. Chem. 10: 2051-2066 (2002)) and play an important role in subsequent events after the initial extravasation of leukocytes from the circulation to the tissues in response to inflammatory stimuli, including migration, recruitment and activation of proinflammatory cells at the site of inflammation (Eble JA, Curr. Phar. Des 11 (7): 867-880 (2005)). It was reported that some antibodies that block integrin a2ß1 showed an impact on delayed hypersensitivity responses and efficacy in a murine model of rheumatoid arthritis and in a model of inflammatory bowel disease (Kriegelstein et al., J. Clin. Invest. 110 (12): 1773-82 (2002), de Fougerolles et al., J. Clin Invest. 105: 721-720 (2000)), besides that it was reported that they attenuated endothelial cell proliferation and in vitro migration (Senger et al., Am. J. Pathol. 160 (1): 195-204 (2002), which suggests that the blocking of the Ntegpna a2ß1 could prevent / inhibit abnormal or higher than normal angiogenesis, as observed in different cancers Normally, platelets circulate in the blood in an inactive resting state, however, they are primed to respond quickly in the sites of injuries to a wide variety of antagonists: when stimulated, they undergo changes in their form and become highly reactive with plasma proteins, such as fibrinogens and von Willebrand factor (vWf), other platelets and the endothelial lining of the wall All these interactions cooperate to facilitate the rapid formation of a plug of hemostatic fibrin platelets (Cramer, 2002 in Hemostasis and Thrombosis, 4th edition). platelet epitors transduce signal paths from the outside inward, which in turn trigger signaling from the inside out resulting in activation of secondary receptors such as the fibrinogen platelet receptor, integrin allbß3, which results in to platelet aggregation. Antibodies or peptide mimetic ligands that bind to or interact with platelet receptors are anticipated to induce a similar signaling cascade that results in platelet activation. Even a minor platelet activation can result in thrombotic responses in platelets, thrombocytopenia and bleeding complications. Integrin a2ß1 is the only integrin that binds to collagen that is expressed in platelets and is thought to play some important role in the adhesion of platelets to collagen and in hemostasis (Gruner et al., Blood 102: 4021-4027 (2003); Nieswandt and Watson, Blood 102 (2): 449-461 (2003); Santoro eí al., Thromb. Haemost. 74: 813-821 (1995); Siljander went to., Blood 15: 1333-1341 (2004); Vanhoorelbeke eí al., Curr. Drug Targets Cardiovasc. Haematol Disord. 3 (2): 125-40 (2003)). In addition, the a2ß1 platelet could play some important role in regulating the size of the platelet aggregate (Siljander et al., Blood 103 (4): 1333-1341 (2004)). The α2β1 integrin has also been shown to be an integrin that binds laminin expressed on endothelial cells (Languino et al., J Cell Bio 109: 2455-2462 (1989)). It is believed that endothelial cells bind to laminin through an integrin-mediated mechanism; however, it has been suggested that domain I of a2 could function as a ligand-specific sequence involved in the mediation of endothelial cell interactions (Bahou et al., Blood 84 (11): 3734-3741 (1994)). ).
It has been anticipated that a therapeutic antibody binding integrin a2ß1, including integrin a2ß1 in platelets, could result in hemorrhagic complications. For example, antibodies directed towards other platelet receptors such as GPIb (Vanhoorelbeke et al., Curr. Drug Targets Cardiovasc.Hematol.Disord.3 (2): 125-40 (2003) or GP llb / llla (Schell et al., Ann. Hematoi 81: 76-79 (2002), Nieswandt and Watson, Blood 102 (2): 449-461 (2003), Merlini et al., Circulation 109: 2203-2206 (2004)) have been associated with thrombocytopenia although the mechanisms behind this have not been well understood, it is hypothesized that binding an antibody to a platelet receptor can alter its three-dimensional structure and expose epitopes that are not normally exposed, which would result in the elimination of platelets (Merlini et al., Circulation 109: 2203-2206 (2004)). In fact, hemorrhagic complications associated with oral doses of GP antagonists lla / lllb have been described as the "dark side" of this class of compounds ( Bhatt and Topol, Nat. Rev. Drug Discov. 2 (1): 15-28 (2003).) If integrin a2ß1 plays a pap the important in the movement of leukocytes through the inflammatory tissue, it would be desirable to develop therapeutic agents that could target a2ß1 for disorders associated with integrin a2ß1 and / or cellular processes associated with the disorders, including cancer, inflammatory diseases and autoimmune diseases, if said agents will not activate the platelets. Therefore, there is a need in the art to develop compounds capable of targeting the a2ß1 integrin, such as the a2ß1 integrin in leukocytes, which are not associated with adverse bleeding complications. The BHA2.1 human blocker against integrin a2ß1 was first described by Hangan et al., (Cancer Res. 56: 3142-3149 (1996)). Other antibodies against integrin a2ß1 are known and have been used in vitro, such as antibodies AK7 (Mazurov et al., Thromb.Haemost.66 (4): 494-9 (1991), P1 E6 (Wayner et al., J Cell Biol. 107 (5): 1881-91 (1988)), 10G11 (Giltay et al., Blood 73 (5): 1235-41 (1989)) and A2-11 E10 (Bergelson et al., Cell Adheses. Commun. 2 (5): 455-64 (1994)) which are commercially available Hangan et al., (Cancer Res. 56: 3142-3149 (1996)) used the BHA2.1 antibody in vivo to study the effects of blocking the function of the a2ß1 integrin in the extravasation of human tumor cells in the liver, and the ability of these tumor cells to develop metastatic foci under an antibody treatment.The Ha1 / 29 antibody (Mendrick and Kelly, Lab Invest. (6): 690-702 (1993)), specific for rat and murine a2ß1 integrin, has been used in vivo to study the upregulation of integrin a2ß1 in T cells after viral activation of LCMV (by its acronyms in English) (Andreasen et al., J. Immunol. 171: 2804-2811 (2003)), to study the responses of delayed hypersensitivity induced by SRBC (for its acronym in English), contact hypersensitivity induced by FITC (for its acronym in English) and collagen-induced arthritis (from Fougerolles et al., J. Clin Invest. 105: 721-720 (2000)), to study the role of integrin a2β1 in angiogenesis regulated by VEGF (Senger et al., Am. J. Pathol, 160 (1): 195-204 (2002), Senger et al., PNAS 94 (25): 13612-7 (1997)), and to study the role of integrin a2ß1 in the locomotion of PMNs (for its acronym in English) in response to the platelet activation factor (PAF-for its acronym in English) (Werr eí al., Blood 95: 1804-1809 (2000)). The use of murine monoclonal antibodies, such as those described above, as human therapeutic agents in non-immunocompromised patients has been limited by the robust immune responses directed against murine antibodies administered, particularly on repeated administration. Not only can this response shorten the effective half-life of the circulating murine antibody, but it can also lead to profound responses at the site of injection and / or anaphylaxis (Shawler et al., J. Immunol. 135 (2): 1530 (1985)). In addition, effector functions of rodents associated with constant regions (Fc) are much less effective than their human counterparts when administered in humans, which results in a loss of potentially desirable complement activation and cytotoxicity activity antibody dependent cell (ADCC). Therefore, there is a need to develop antibodies directed against the a2ß1 integrin, including those for the treatment of disorders, mechanisms and cellular processes associated with the a2ß1 integrin, among which are inflammatory diseases and autoimmune diseases. Moreover, it would be desirable to develop antibodies against the a2ß1 integrin that were not associated with the development of a response against murine antibodies in a patient.
BRIEF DESCRIPTION OF THE INVENTIONThe present invention provides antibodies against alpha 2 integrin (a2) and methods for its use, notably humanized antibodies against alpha 2 integrin (a2) and methods for its use. In certain variants, the a2 integrin antibody includes one or more human constant regions (e.g., C and / or CH) and a light chain variable region comprised of the amino acid sequenceSEQ ID NO: 19 and / or a heavy chain variable region composed of the amino acid sequence SEQ ID NO: 21 or variants of the same amino acid sequence. Various forms of the antibody are contemplated in this document. For example, the a2 integrin antibody can be a full-length antibody (i.e., composed of constant regions of human immunoglobulin) or an antibody fragment (e.g., Fab, F (ab ') 2, Fab' fragments, Fv or scFv). In addition, the antibody can be labeled with a detectable label, immobilized on a solid phase and / or conjugated with a heterologous compound (such as a cytotoxic agent). Diagnostic and therapeutic uses for antibodies against integrin a2 are contemplated for both prophylactic and preventive uses. For diagnostic uses, a method for determining the presence of the α2β1 integrin protein including the exposure of a sample suspected of containing the integrin α2β1 protein to an antibody against integrin α2, and determining the binding of the antibody to the sample is provided . For this use, a kit including an antibody to integrin a2 and instructions to detect integrin protein a2ß1 is provided. Therapeutic uses include, but are not limited to, the treatment of cellular disorders, mechanisms and processes associated with integrin a2ß1 such as inflammatory diseases and autoimmune diseases, particularly multiple sclerosis. Applications of gene therapy for antibodies to integrin a2 are contemplated. Several vectors (e.g., retroviral vectors, chromosomes) that encode the heavy and light chain genetic sequences against the a2ß1 integrin, can be transferred to cells (e.g., fibroblasts, stem cells) to generate populations of cells that secrete MAb. against integrin a2ß1. These cells may possess specific properties "to house" different types of cells, tissues and / or organs. In turn, these cells that produce antibodies can be introduced into a patient for localized delivery of the MAbs against the a2ß1 integrin. As an example, mesenchymal stem cells modified with a MAb vector against integrin a2ß1 could be injected into the brain of a patient suffering from multiple sclerosis. The stem cells differentiate into neural cells and secrete the MAb against the a2ß1 integrin to treat the inflammation associated with multiple sclerosis. In addition, antibodies against integrin a2ß1 can be conjugated to viruses encoding therapeutic genes (eg, ricin). The modified viruses would bind specifically to cells expressing a2ß1 on the cell surface, allowing increased efficiency of the transgene transfer. In addition, immunoconjugates composed of antibody-liposome complexes against the a2ß1 integrin that encapsulate nucleic acids encoding therapeutic genes can be introduced intravenously into a patient. The antibody against the immunoconjugated integrin a2ß1 would bind to cells expressing the a2ß1 integrin and facilitate the efficient absorption of therapeutic genes. An isolated nucleic acid encoding an antibody against integrin a2 is also provided; a vector that includes that nucleic acid, optionally operably linked to control sequences recognized by a host cell transformed with the vector; a host cell that includes that vector; a method for producing the a2 integrin antibody that includes the culture of the host cell to express the nucleic acid and, optionally, recovering the antibody from the culture of the host cell (e.g., from the culture medium of the host cell). Also provided is a composition that includes a humanized antibody against integrin a2 and a pharmaceutically acceptable carrier or diluent. The compositions for therapeutic uses can be sterile and can be lyophilized. A method is also provided for treating a disorder associated with integrin a2ß1, which includes administering to a subject a pharmaceutically effective amount of an antibody against integrin a2 as a humanized antibody against integrin a2 to a mammal. For those therapeutic uses, other agents (eg, another antagonist of integrin 2β1) can be co-administered to the mammal either before, after, or simultaneously with the antibody against integrin a2. A humanized antibody against integrin a2 including a heavy chain variable region composed of the amino acid sequence (a) HCDR2 (VIWARGFTNYNSALMS, SEQ ID NO: 2), (b) HCDR1 (GFSLTNYGIH, SEQ ID NO: 1) is also provided. ), HCDR2 (VIWARGFTNYNSALMS, SEQ ID NO: 2) and HCDR3 (ANDGVYYAMDY, SEQ ID NO: 3), or (c) SEQ ID NO: 40. In one embodiment, the heavy chain variable region mentioned above comprises the amino acid sequence SEQ ID NO: 185. In a further embodiment, the heavy chain variable region mentioned above comprises by the amino acid sequence SEQ ID NO: 185 where (a) position 71 is Lys, (b) position 73 is Asn, (c) position 78 is Val, or (d) any combination of (a) - (c). In a further embodiment, the heavy chain variable region mentioned above comprises an amino acid sequence selected from SEQ ID NOs: 70-79 and SEQ ID NOs: 109-111.
In one embodiment, the a2 integrin antibody mentioned above also includes a FW4 region composed of the amino acid sequence WGQGTLVTVSS (SEQ ID NO: 13J.) In one embodiment, the a2 integrin antibody mentioned above is composed of the amino acid sequence. HCDR1 (SEQ ID NO: 1), HCDR2 (SEQ ID NO: 2) and HCDR3 (SEQ ID NO: 3) In one embodiment, the antibody against a2 integrin mentioned above also includes a light chain. humanized antibody against integrin a2 that includes a variable region of light chain composed of the amino acid sequence of (a) an LCDR1 selected fromSANSSVNYIH (SEQ ID NO: 4) or SAQSSWNYIH (SEQ ID NO: 112), (b) LCDR2(DTSKLAS, SEQ ID NO: 5) and (c) LCDR3 (QQWTTNPLT, SEQ ID NO: 6). In one embodiment, the aforementioned light chain variable region comprises the amino acid sequence SEQ ID NO: 186. In one embodiment, the aforementioned light chain variable region comprises the amino acid sequence SEQ ID NO: 186 wherein ( a) position 2 is Phe, (b) position 45 is Lys, (c) position 48 is Tyr, or (d) any combination of (a) - (c). In one embodiment, the aforementioned light chain variable region comprises an amino acid sequence selected from SEQ ID NO: 41, SEQ ID NOs: 80-92 and SEQ ID NO: 108.
In one embodiment, the humanized anti-α2 integrin antibody mentioned above also includes a Region FW4 comprising the amino acid sequence FGQGTKVEIK of SEQ ID NO: 38. In one embodiment, the aforementioned humanized anti-integrin 2 antibody is composed of the amino acid sequence LCDR1 (SEQ ID NO: 4), LCDR2 (SEQ ID NO: 5) and LCDR3 (SEQ ID NO: 6). In one embodiment, the aforementioned humanized anti-a.2 integrin antibody also includes a heavy chain. The invention also provides a humanized antibody against integrin a2 which includes: (i) a heavy chain variable region composed of the amino acid sequence (a) HCDR2 (VIWARGFTNYNSALMS, SEQ IDNO: 2), (b) HCDR1 (GFSLTNYGIH, SEQ ID N0: 1), HCDR2 (VIWARGFTNYNSALMS, SEQ ID N0: 2) and HCDR3 (ANDGVYYAMDY, SEQ IDNO: 3), or (c) SEQ ID NO: 40; and (ii) a light chain variable region composed of the amino acid sequence (a) an LCDR1 selected from SANSSVNYIH (SEQ ID NO: 4) or SAQSSWNYIH (SEQ ID N0.H2), (b) LCDR2 (DTSKLAS; SEQ ID; NO: 5) and (c) LCDR3 (QQWTTNPLT, SEQ ID NO: 6). The humanized antibody against the a2 integrin mentioned above is also provided, wherein (a) the heavy chain variable region comprises by the amino acid sequence SEQ ID NO: 185, (b) the light chain variable region comprises the sequence of amino acids SEQ ID NO: 186, or (c) both (a) and (b). The humanized antibody against the a2 integrin mentioned above is also provided, wherein (i) the heavy chain variable region comprises the amino acid sequence SEQ ID NO: 185 wherein (a) position 71 is Lys, (b) the position 73 is Asn, (c) position 78 is Val, or (d) any combination of (a) - (c); (ii) the light chain variable region comprises the amino acid sequence SEQ ID NO: 186 wherein (a) position 2 is Phe, (b) position 45 is Lys, (c) position 48 is Tyr, or (d) any combination of (a) - (c); or (iii) both (i) and (ii). The humanized antibody against the a2 integrin mentioned above is also provided, wherein (a) the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NOs: 70-79 and SEQ ID NOs: 109-111; (b) the light chain variable region comprises an amino acid sequence selected from SEQ ID NO: 41, SEQ ID NOs: 80-92 and SEQ ID NO: 108; or (c) both (a) and (b). In one embodiment, the antibody against integrin a.2 mentioned above recognizes domain I of human a2 integrin. In one embodiment, the antibody against the a2 integrin mentioned above binds the a2ß1 integrin. In one embodiment, the antibody against the a2 integrin mentioned above binds to an α2 integrin epitope, the epitope is composed of: (a) a Lys residue corresponding to position 192 of the amino acid sequence of integrin a2 as expressed in SEQ ID NO: 8 or to position 40 of the amino acid sequence of domain I of integrin a2 as expressed in SEQ ID NO: 11; (b) a residue Asn corresponding to position 225 of the amino acid sequence of integrin a2 as expressed in SEQ ID NO: 8 or to position 73 of the amino acid sequence of domain I of integrin a.2 as expressed in SEQ ID NO: 11; (c) a Gln residue corresponding to position 241 of the amino acid sequence of integrin a2 as expressed in SEQ IDNO: 8 or to position 89 of the amino acid sequence of domain I of integrin a2 as expressed in SEQ ID NO: 11; (d) a Tyr residue corresponding to position 245 of the amino acid sequence of integrin a2 as expressed in SEQ ID NO: 8 or to position 93 of the amino acid sequence of domain I of integrin a2 as expressed in SEQ ID NO: 11; (e) an Arg residue corresponding to position 317 of the amino acid sequence of integrin a2 as expressed in SEQ ID NO: 8 or to position 165 of the amino acid sequence of domain I of integrin a2 as expressed in SEQ ID NO: 11; (f) an Asn residue corresponding to position 318 of the amino acid sequence of integrin a2 as expressed in SEQ ID NO.8 or position 166 of the amino acid sequence of domain I of integrin a2 as expressed in SEQ ID NO: 11; or (g) any combination of (a) to (f). An antibody to integrin a2 is also provided, wherein the antibody binds to an α2 integrin epitope, the epitope being composed of: (a) a Lys residue corresponding to position 192 of the amino acid sequence of integrin a2 as is expressed in SEQ ID NO: 8 or position 40 of the amino acid sequence of domain I of integrin a2 as expressed in SEQ ID NO: 11; (b) a residue Asn corresponding to position 225 of the amino acid sequence of integrin a2 as expressed in SEQ ID NO: 8 or to position 73 of the amino acid sequence of domain I of integrin a2 as expressed in SEQ ID NO: 11; (c) a Gln residue corresponding to position 241 of the amino acid sequence of integrin a2 as expressed in SEQ ID NO: 8 or to position 89 of the amino acid sequence of domain I of integrin a2 as expressed in SEQ ID NO: 11; (d) a Tyr residue corresponding to position 245 of the amino acid sequence of integrin a2 as expressed in SEQ ID NO: 8 or to position 93 of the amino acid sequence of domain I of integrin a2 as expressed in SEQ ID NO: 11;(e) an Arg residue corresponding to position 317 of the amino acid sequence of the ategrin a.2 as expressed in SEQ ID NO: 8 or to position 165 of the amino acid sequence of domain I of integrin a2 as is expressed in SEQ ID NO: 11; (f) an Asn residue corresponding to position 318 of the amino acid sequence of integrin a2 as expressed in SEQ ID NO: 8 or to position 166 of the amino acid sequence of domain I of integrin a2 as expressed in SEQ ID NO: 11; or (g) any combination of (a) to (f). In one embodiment, the humanized antibody against the a2 integrin mentioned above is a full-length antibody. In one embodiment, the humanized antibody against a2 integrin mentioned above is an antibody fragment. In one embodiment, the humanized anti-a.2 integrin antibody mentioned above is linked to a detectable label. In one embodiment, the humanized anti-ategrin antibody a2 mentioned above is immobilized on a solid phase. In one embodiment, the humanized antibody against the a2-integrin a2 mentioned above inhibits the binding of the a2 or a2β1 integrin to a ligand of the a2β1 integrin. In one embodiment, the ligand of the a2β1 integrin mentioned above is selected from collagen, laminin, Echovirus-1, decorin, E-cadherin, matrix metalloproteinase 1 (MMP-I), endorepepin, collectin and complement C1q protein. The invention also provides a method for determining whether a sample contains α2 integrin, α2β1 integrin, or both, and requires contacting the sample with the humanized antibody against the a2-integrin mentioned above and determining whether the antibody binds to the shows, said binding being an indication that the sample contains integrin a2, integrin a2β1, or both. The invention also provides a kit that includes the humanized antibody against a2 ategrin aforementioned, optionally also includes instructions for its use in the detection of the protein ategrina a2 or a2ß1. The invention also provides an isolated nucleic acid encoding the humanized antibody against the α2β1 integrin mentioned above. The invention also provides a vector including the aforementioned nucleic acid. The invention also provides a host cell that includes the nucleic acid or the aforementioned vector. The invention also provides a method for producing a humanized antibody against a2 integrin and requires culturing the aforementioned host cell under conditions that allow the expression of the antibody. In one embodiment, the method also requires recovering the humanized antibody against integrin a2 of the host cell. In another embodiment, the method also requires recovering the humanized antibody against integrin a2 from the culture medium of the host cell. The invention also provides a screening method comprising: detecting the binding of integrin a2 or a2ß1 to an antibody composed of the VL region of SEQ ID NO: 19 and the VH region of SEQ ID NO: 21 in the presence against the absence of a test antibody; and the selection of the test antibody if its presence is related to the decreased binding of the a2 or a2ß1 integrin to the antibody composed of the VL region of SEQ ID NO: 19 and the VH region of SEQ ID NO: 21. In one embodiment, integrin a2 or a2β1 is immobilized on a solid support. The invention also provides a method of screening which comprises: detecting the binding of integrin a2ß1 to collagen in the presence of a test antibody, wherein test antibody refers to an antibody that binds to an I domain of a.2; detecting the binding of the test antibody to domain I of a2 in the presence of Mg ++ ions; the detection of the binding of the test antibody to domain I of a2 in the presence of Ca ++ ions; detecting the binding of the test antibody to domain I of a2 in the presence of a cation-free medium; and the selection of the test antibody if it inhibits the binding of the a2ß1 integrin to the collagen and binds to domain I of a.2 in the presence of Mg ++ ions, Ca ++ atoms and a cation free medium.
The invention also provides a composition that includes the humanized antibody against the a2-integrin a2 mentioned above and a pharmaceutically acceptable carrier. The invention also provides a method for treating a disorder associated with a2β1 integrin in a subject; the method requires administering to the subject a therapeutically effective amount of the antibody or composition against the a2 integrin mentioned above. The invention also provides a method for inhibiting the binding of leukocytes to collagen which requires administering to a subject an effective amount of the antibody against the a2β1 integrin mentioned above to inhibit the binding of leukocytes to collagen. The invention also provides a use of the humanized antibody against the a2 integrin mentioned above as a medicament. The invention also provides a use of the aforementioned humanized anti-a2 integrin or composition for the treatment of a disorder associated with the a2ß1 integrin. The invention also provides a use of the aforementioned humanized anti-a2 integrin or composition to prepare a medicament for the treatment of a disorder associated with integrin a2ß1. The invention also provides a composition for the treatment of a disorder associated with integrin a2ß1, the composition includes the humanized antibody against the a2 integrin mentioned above and a pharmaceutically acceptable carrier or diluent. The invention also provides a package including the aforementioned humanized anti-a2 integrin or composition together with instructions for the treatment of a disorder associated with integrin a2ß1. In some embodiments, the disorder associated with integrin a2β1 is selected from an inflammatory disease, an autoimmune disease and a disease characterized by abnormal or increased angiogenesis. In some modalities, the disorder associated with a2β1 integrin is selected from inflammatory bowel disease, Crohn's disease, ulcerative colitis, reactions to a transplant, optic neuritis, spinal cord trauma, rheumatoid arthritis, systemic lupus erythematosus (SLE-by its acronym in English), diabetes mellitus, multiple sclerosis, Reynaud's syndrome, experimental autoimmune encephalomyelitis, Sjorgen syndrome, scleroderma, juvenile diabetes, diabetic retinopathy, age-related macular degeneration, cardiovascular disease, psoriasis, cancer, as well as infections that induce an inflammatory response. In some variants, the disorder associated with integrin a2ß1 is selected from multiple sclerosis (eg, characterized by relapse, acute treatment, delayed treatment), rheumatoid arthritis, optic neuritis, and spinal cord trauma. In some embodiments, the aforementioned method is not associated with (a) platelet activation, (b) platelet aggregation, (c) a decrease in the number of circulating platelets, (d) hemorrhagic complications, or (e) any combination of ( a) to (d). In one embodiment, the aforementioned integrin antibody a.2 includes a heavy chain composed of SEQ ID NO: 174 or SEQ ID NO: 176 and a light chain composed of SEQ ID NO: 178. In one embodiment, the antibody against the aforementioned integrin a.2 competitively inhibits the binding of an antibody composed of the UL region of SEQ ID NO: 19 and the VH region of SEQ ID NO: 21 to a human a2β1 integrin or to the I domain thereof. In one embodiment, the aforementioned method is associated with a palliation of an exacerbation or neurological sequelae associated with multiple sclerosis. In one embodiment, the aforementioned integrin a.2 antibody inhibits the binding of the a2ß1 integrin to collagen and is not a mimetic ligand. A method for directing a portion, which may be a molecule, protein, nucleic acid, is also provided., vector, composition, complex, etc., to a site characterized by the presence of an integrin ligand a2ß1, the method requires annexing or binding the portion to the humanized antibody against the a2 integrin mentioned above. Also provided is an α2 integrin epitope that binds to an antibody against integrin a2, wherein the epitope does not include the binding site of a2 integrin ligands. In some variants, epitope binding is not associated with (a) platelet activation, (b) platelet aggregation, (c) a decrease in the number of circulating platelets, (d) hemorrhagic complications, (e) activation of integrin a2, or (f) any combination of (a) to (e). Preferred antibodies bind to domain I of human a2β1 integrin. In particular, preferred antibodies are capable of blocking a2-dependent cellular adhesion to the extracellular matrix (ECM), particularly to collagen, laminin or both. Humanized antibodies are provided, including antibodies based on an antibody referred to herein as TMC-2206. Antibodies against integrin a2 are provided which are very specific for human a2β1 integrin, and whose administration is not associated with undesirable effects, such as bleeding complications or complications due to cellular activation. The binding specificity (e.g., the epitope specificity) of these antibodies is associated with their unexpected non-hemorrhagic profiles. The humanized antibody against the a2ß1 integrin may have a heavy chain variable region composed of the amino acid sequence HCDR1 (GFSLTNYGIH; SEQ ID NO: 1) and / or HCDR2 (VIWARGFTNYNSALMS; SEQ ID NO: 2) and / or HCDR3 (ANDGVYYAMDY; SEQ ID NO: 3). The humanized antibody to integrin a2ß1 may have a light chain variable region composed of the amino acid sequence LCDR1 (SANSSVNYIH; SEQ ID NO: 4 or SAQSSWNYIH; SEQ ID NO: 112) and / or LCDR2 (DTSKLAS; SEQ ID NO: 5) and / or LCDR3 (QQWTTNPLT; SEQ ID NO: 6). In certain embodiments, the humanized antibodies against the α2β1 integrin have a heavy chain composed of HCDR1 (GFSLTNYGIH; SEQ ID NO: 1) and / or HCDR2 (VIWARGFTNYNSALMS; SEQ ID NO: 2) and / or HCDR3 (ANDGVYYAMDY; SEQ ID NO; : 3) and a light chain variable region composed of the amino acid sequence LCDR1 (SANSSVNYIH; SEQ ID NO: 4) and / or LCDR2 (DTSKLAS; SEQ ID NO: 5) and / or LCDR3 (QQWTTNPLT; SEQ ID NO: 6). In other embodiments, the antibody is comprised of a variant of the amino acid sequence of one or more of those CDRs, the variant of which comprises one or more amino acid insertions within or adjacent to a CDR residue and / or deletions within or adjacent to a CDR residue and / or CDR residue substitutions (with substitution) being the preferred type of amino acid alteration to generate those variants).
BRIEF DESCRIPTION OF THE DRAWINGSFigure 1: Graphical results of studies of the effects of the antibody against integrin a2 in paralytic diseases in a mouse model EAE (for its acronym in English) when administered at the first sign of the disease (see Example 7). Figure 2: Graphic results of studies of the effects of the antibody against integrin a2 in paralytic diseases when administered during the induction phase (see Example 7).
DETAILED DESCRIPTION OF THE INVENTIONThe present invention provides antibodies specifically reactive with human alpha 2 (a2) integrin, including humanized antibodies and methods for their use. Humanized antibodies can have framework regions (FW) and complementarity determining regions (CDRs) of a non-human antibody, usually mouse, specifically reactive with integrin a2 human The encoding of the nucleotide sequences and amino acid sequences composed of heavy and light chain antibodies are provided. In the preferred variants, one or more of the CDR regions are derived from or are based on the murine antibody secreted by the hybridoma clone, BHA2.1 [referred to herein as the TMC-2206 antibody]. Antibodies having similar binding properties and antibodies (or other antagonists) having similar functionality to the antibodies detailed herein are also provided. Preferred a2 integrin antibodies include those that (a) bind to domain I of integrin a2, (b) inhibit the function of integrin a2 (eg, binding to collagen or laminin), (c) they bind to integrin s.2 in platelets at rest without inducing platelet activation and (d) they recognize the binding epitope of TMC-2206 (for example, they compete with TMC-2206 for binding to integrin a2). These antibodies can preferentially bind to the inactive or closed conformation of the target a2 integrin molecule without competing for the ligand binding site. The unexpected advantages of antibodies against integrin a.2 as those described herein, which bind preferentially to the closed conformation of integrin a2ß1 and / or bind to integrin a2ß1 without competing for the ligand binding site (ie, they are not mimetic ligands), they include the potential prevention of platelet activation, platelet aggregation, the decrease in the number of circulating platelets and / or hemorrhagic complications in a treated subject. "Haemorrhagic complications" as used herein refers to any adverse effect on blood levels and physiology, including platelet thrombotic responses, thrombocytopenia, increased clotting time, increased bleeding time and blood loss limiting use. of the antibody against integrin a2. The a2ß1 integrin is a molecule composed of an a2 integrin subunit (see, for example, SEQ ID NO: 7, for DNA sequence and SEQ ID NO: 8 for human a2 protein sequence) of the alpha integrin family , and by a β1 integrin subunit (see, for example, SEQ ID NO: 9 for DNA sequence and SEQ ID NO: 10 for human β1 protein sequence) of the beta integrin family, and can be of any subject including a mammal, but preferably a human. The a2ß1 integrin can be purified from any natural source, or it can be produced synthetically (for example, by using recombinant DNA technology). The coding sequences of the nucleic acid for integrin a2 and for integrin β1 are described in Takada and Hemler J. Cell Biol. 109 (1): 397-407 (1989, the GenBank shipment X17033, subsequently updated to entry NM 002203) and in Argraves, WS, J. Cell. Biol. Sep. 105 (3): 1183-90 (1987; Genbank shipment X07979.1 and related sequences represent alternately assembled variants), respectively. The "I" domain of the a2β1 integrin molecule refers to a region of this α2β1 integrin molecule within the a2 subunit, and is described, for example, in Kamata et al., J Biol. Chem. 269: 9659- 9663 (1994); Emsley et al., J. Biol. Chem. 272: 28512 (1997) and Cell 101: 47 (2000). The amino acid sequence of an I domain of human a2 integrin is shown as SEQ ID NO: 11 (see also, for example, SEQ ID NO: 107). Domain I of integrin a.2 contains a ligand binding site of the MIDAS type (Metal Ion Dependent Site Accession or metal ion dependent adhesion site) that has a requirement and a specificity for a divalent cation to support the binding of ligands . The amino acid sequences for an I domain of the a2 integrin in rat shown as SEQ ID NO: 93 (see also, for example, SEQ ID NO: 113) and in mouse shown as SEQ ID NO: 94 (see also, for example , SEQ ID NO: 114) are shown in Table 28. Sequences of domain I of Cynomolgus monkey and rhesus monkey were cloned from the fraction of leukocytes derived from whole blood and are provided in SEQ ID NO.103 (DNA), SEQ. ID NO: 171 (amino acid) for cynomolgus and SEQ ID NO: 104 (DNA), SEQ ID NO: 172 (amino acid) for rhesus, respectively. An epitope of TMC-2206 (BHA2.1) refers to a region of domain I of human a2 integrin to which the TMC-2206 antibody binds. This epitope encompasses a region surrounded by amino acid residues, K40, N73, Q89, Y93, R165 and N166 and, optionally, other amino acid residues of domain I of integrin a2. A disorder associated with integrin a2 refers to a disorder, disease or condition involving processes / functions dependent on integrin a2 (eg, binding, activity) that mediate aberrant cellular reactions within the target tissue. Examples of a2-integrin-dependent processes involved in diseases include collagen-dependent cellular responses such as those involved in increases in cytokine expression and proliferation, aspects of T cell function, mast cells and neutrophils, inflammatory disorders, ductal morphogenesis of the gland mammary, healing of epidermal wounds, and angiogenesis. Examples of disorders associated with integrin a2 include, but are not limited to, inflammatory diseases or disorders, for example, inflammatory bowel disease (such as Crohn's disease and ulcerative colitis), reactions to a transplant (including transplant rejection), optic neuritis, spinal cord trauma, rheumatoid arthritis, multiple sclerosis (including the treatment of associated neurological sequelae, as well as multiple sclerosis characterized by relapses), autoimmune diseases or disorders (including systemic lupus erythematosus or SLE, diabetes mellitus, Reynaud's syndrome, encephalomyelitis experimental autoimmune, Sjorgen syndrome, scleroderma), juvenile diabetes, and disorders associated with abnormal or greater than normal angiogenesis (such as diabetic retinopathy, age-related macular degeneration, cardiovascular disease, psoriasis, rheumatoid arthritis, and cancer), as well as infections that induce a response inflammatory The treatment of a disorder associated with integrin a2ß1 refers both to the therapeutic use and to the prophylactic or preventive use of the a2 integrin antibodies described herein. Those in need of treatment include those who have already been diagnosed with the disorder, as well as those for whom the onset of the disorder will be prevented or delayed. A mammal, including for treatment purposes, refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports or pet animals such as dogs, horses, cats, cows, etc. Preferably, the mammal is human.
Intermittent or periodic dosing is a dosage that is continuous for a certain period of time and at regular intervals that are preferably separated by more than one day. The term "antibody" or "immunoglobulin" is used in the broadest sense, and embraces monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies, and antibody fragments as long as they exhibit the desired biological activity. Antibody fragments comprise a portion of a full-length antibody, usually a binding of an antigen or a variable region thereof. Examples of antibody fragments include fragments of Fab, Fab ', F (ab') 2, and Fv, diabodies (diabodies), linear antibodies, single-chain antibody molecules, single-domain antibodies (e.g., from camelids). ), single-domain NAR shark antibodies, and multispecific antibodies formed from antibody fragments. Antibody fragments may also refer to binding portions composed of CDRs or antigen-binding domains including, but not limited to, VH (VH, VH-V) regions, anticalins, PepBodies ™, antibody-epitope cell fusions T (Troybodies) or Peptibodies. A monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, for example, the individual antibodies constituting the population are identical except for mutations that possibly occurred naturally and could be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In addition, in contrast to conventional (e.g., polyclonal) antibody preparations that typically include different antibodies directed against different determinants (e.g., epitopes) on an antigen, each monoclonal antibody is directed against at least one determinant on the antigen. The "monoclonal" modifier indicates the character of the antibody as an antibody obtained from a substantially homogenous population of antibodies, and should not be construed as requiring the production of the antibody by any particular method, for example, monoclonal antibodies may be produced by the antibody. hybridoma method first described by Kohler et al., Nature 256: 495 (1975), or can be produced by recombinant DNA methods (see, for example, U.S. Patent No. 4,816,567.) Monoclonal antibodies also they can be isolated from phage antibody libraries, for example, using the techniques described in Clackson et al., Nature 352: 624-628 (1991) and in Marks et al., J. Mol. Biol. 222: 581-597 ( 1991) Monoclonal antibodies can also be isolated using the techniques described in U.S. Patent Nos. 6,025,155 and 6,077,677 as well as in U.S. Patent Publication Nos. 2002/0160970 and 2003/0083293 (see also, for example, Lindenbaum, et al., Nucleic Acids Research 32 (21): 0177 (2004)).
The monoclonal antibodies may include chimeric antibodies in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibody, while the The remaining part of the chain is identical or homologous to the corresponding sequences in antibodies derived from other species or belonging to another class or subclass of antibody, as well as fragments of those antibodies, as long as they exhibit the desired biological activity (see, for example , U.S. Patent No. 4,816,567; and Morrison e al., Proc. Nati, Acad Sci. USA 81: 6851-6855 (1984) for chimeric mouse-human antibodies). A hypervariable region refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region comprises amino acid residues of a complementarity determining region or CDRs (eg, residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain, and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the variable domain of heavy chain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and / or those residues of a hypervariable cycle (eg residues 26-32 ( L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the variable chain domain heavy, Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987)). The framework or FR residues are those variable domain residues different from the residues of the hypervariable region. For the antibodies described herein, the CDR and framework regions are identified based on the Kabat numbering system, except that the CDR1 of the heavy chain receives the Oxford Molecular AbM definition as expandable residues 26 to 35. The modeling software AbM antibody from Molecular Oxford (http://people.cryst.cck.ac.uk/~ubc07s/) (Martin et al., Proc. Nati Acad. Sci. USA, 86, 9268-9272 (1989); Martin et al., Methods Enzymol., 203, 121-153 (1991), Pedersen et al., Immunomethods, 1, 126 (1992), and Rees et al., In Sternberg MJE (ed.), Protein Structure Prediction. University Press, Oxford, 141-172 (1996)) combines the numbering systems of the hypervariable region Kabat CDR and Chothia to define the CDRs. Humanized forms of non-human antibodies (eg, murine) can be chimeric antibodies that contain a minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (receptor or acceptor antibody) in which the residues of the hypervariable region of the receptor are replaced by residues from the hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate, which have the desired specificity, affinity and capacity. In addition, the individual residues or groups of the framework region (FR) Fv of the human immunoglobulin can be replaced by corresponding non-human residues. Likewise, humanized antibodies could include residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine the performance of the antibody. In general, the humanized antibody will contain substantially all of at least one, and generally two, variable regions or domains, in which all or substantially all hypervariable cycles correspond to those of a non-human immunoglobulin and all or substantially all regions FR are those of the human immunoglobulin sequence. Optionally, the humanized antibody will also contain at least a portion of an immunoglobulin constant region (e.g., Fc), which is usually a human immunoglobulin (see, e.g., Queen et al., Proc. Nati. Acad. Sci. USA 86: 10029 (1989), and Foote and Winter, J. Mol. Biol. 224: 487 (1992)). Single-chain Fv or scFv antibody fragments may be composed of the VH and VL regions or domains of the antibody, wherein these domains are present in a single polypeptide chain. In general, the Fv polypeptide also includes a polypeptide linker between the V and VL domains that allows the scFv to form the desired structure for antigen binding (for a review, see, for example, Pluckthun in The Pharmacology of Monoclonal Antibodies, vol 113, Rosenburg and Moore eds Springer-Verlag, New York, pp. 269-315 (1994)). Diabody (diabody) refers to small fragments of antibodies with two paratopes (antigen binding sites), whose fragments include a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same chain of polypeptide (VH-V). When using a connector that is too short to allow parity between two domains in the same chain, the domains are forced to pair with the complementary domains of another chain and create two paratopes. The diabodies are described more extensively in, for example, EP 404,097; WO 93/11161; and Hollinger went to., Proc. Nati Acad. Sci. USA 90: 6444-6448 (1993). "Linear antibody" refers to antibodies such as those described in Zapata et al., Protein Eng. 8 (10): 1057-1062 (1995). Briefly, these antibodies are composed of a pair of Fd segments in series (VH -CH1-VH-CH1) that form a pair of regions of binding with the antigen. Linear antibodies can be bispecific or monospecific. An isolated antibody refers to one that has been identified and separated and / or recovered from a component of its natural environment. The contaminating components of their natural environment are materials that could interfere with the diagnostic or therapeutic uses for the antibody, and could include enzymes, hormones and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of the antibody as determined in the Lowry method, and preferably to more than 99% by weight, (2) to a sufficient degree to obtain minus 15 residues of the N-terminal or internal amino acid sequence with the use of a rotating cup sequencer, or (3) to homogeneity by SDS-PAGE under reducing or not reducing conditions using Coomassie blue dye or, preferably, silver . The isolated antibody includes the antibody in situ within the recombinant cells since at least one component of the antibody's natural environment will not be present. However, generally, an isolated antibody will be prepared with at least one purification step. An antibody labeled an epitope refers to one in which the antibody of the invention is fused to an epitope tag. The epitope tag polypeptide has enough residues to provide an epitope against which an opposing antibody can be made, although it is short enough so as not to interfere with the activity of the antibody to the a2ß1 integrin. Preferably, the epitope tag is sufficiently unique that the opposing antibody does not substantially cross-react with other epitopes. Polypeptides with a suitable label usually have at least 6 amino acid residues and generally have between 8 and 50 amino acid residues (preferably between 9 and 30 residues). Examples include the polypeptide labeled HA HA and its antibody 12CA5 (Field et al., Mol.Cell. Biol. 8: 2159-2165 (1988)); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereof (Evan et al., Mol.Cell. Biol. 5 (12): 3610-3616 (1985)); and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al., Protein Engineering 3 (6): 547-553 (1990)). In certain variants, the epitope tag is an epitope of binding to the rescue receptor that is an epitope of the Fc region of an IgG molecule (eg, IgG-i, IgG2, IgGβ, or IgG4) which is responsible for increasing the in vivo serum half-life of the IgG molecule. A cytotoxic agent refers to a substance that inhibits or prevents the functioning of the cells and / or causes the destruction of the cells. These may include radioactive isotopes (eg 131l, 125l, 90Y and 186Re), chemotherapeutic agents and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof. A non-cytotoxic agent refers to a substance that does not inhibit or prevent the functioning of the cells and / or cause the destruction of the cells. A non-cytotoxic agent can include an agent that can be activated to become cytotoxic. A non-cytotoxic agent may include a microsphere, a liposome, a matrix or a particle (see, for example, U.S. Patent Publications 2003/0028071 and 2003/0032995 which are incorporated herein by reference). These agents can be conjugated, coupled, linked or associated with an antibody to integrin a2ß1 as described herein. A chemotherapeutic agent refers to a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include, but not limited to, Adriamycin, Doxorubicin, 5-Fluorouracil, Cytosine Arabinoside ("Ara-C"), Cyclophosphamide, Tiotepa, Taxotere (docetaxel), Busulfan, Cytoxin, Taxol, Methotrexate, Cisplatin, Melphalan, Vinblastine, Bleomycin, Etoposide , Ifosfamide, Mithomycin C, Mitoxantrone, Vincristine, Vinorelbine, Carboplatin, Teniposide, Daunomycin, Carminomycin, Aminopteripa, Dactinomycin, Mitomycin, Spiramycin (see US Pat. No. 4,675,187), Melphalan and other related nitrogen mustards. A prodrug refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted to the more active parent 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 ei al., (ed.), pp. 247-267, Humana Press (1985)). Prodrugs include, but are not limited to, prodrugs containing phosphates, prodrugs containing thiophosphates, prodrugs containing sulphates, prodrugs containing peptides, prodrugs modified by D-amino acids, glycosylated prodrugs, prodrugs containing β-lactams, optionally substituted by prodrugs containing phenoxyacetamide or optionally substituted by prodrugs containing phenylacetamide, 5-fluorocytosine prodrugs and other 5-fluorouridine which can be converted into more active cytotoxic free drugs. Examples of cytotoxic drugs that can be derived in a prodrug form can be the chemotherapeutic agents described above.
A label refers to a detectable compound or composition that is conjugated or coupled directly or indirectly with the antibody. The label may be detectable by itself (eg, radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of the compound or composition of a substrate that is detectable. "Solid phase" refers to a non-aqueous matrix to which the antibody of the present invention can adhere. Examples of solid phases encompassed herein include those formed partially or completely of glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol, and silicones. In certain embodiments, depending on the context, the solid phase may comprise the well of a test plate; in others it is a purification column (for example, an affinity chromatography column). This term also includes a discontinuous solid phase of discrete particles, such as those described in U.S. Pat. No. 4,275,149. A "liposome" refers to a small vesicle composed of various types of lipids, phospholipids and / or surfactants that is useful for the delivery of a drug (such as the antibodies of the invention and, optionally, a chemotherapeutic agent) to a mammal. The components of the liposome are commonly arranged in a double-layer formation, similar to the lipid organization of the biological membranes.
An "isolated nucleic acid molecule" refers to a nucleic acid molecule that is identified and separated from at least one contaminating nucleic acid molecule with which it is commonly associated in the natural source of the antibody nucleic acid. An isolated nucleic acid molecule is of a different shape or configuration than that found in nature. Therefore, isolated nucleic acid molecules are distinguished from the nucleic acid molecules found in natural cells. However, an isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that ordinarily express the antibody where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells. A viral vector refers to a vehicle for the transfer of a nucleic acid (eg, DNA or RNA) to cells through viral infection or transduction. Examples of viral vectors include retroviruses, adenoviruses, pox viruses and baculoviruses. A non-viral vector refers to a nucleic acid vehicle such as a CAN, plasmid or chromosome that is delivered to cells by non-viral methods such as electroporation, injections and transfection mediated by cationic reagents. "Expression control sequences" refers to those DNA sequences necessary for the expression of a coding sequence operably linked in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to use promoters, polyadenylation signals and enhancers. A nucleic acid is operably linked when placed in a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to the DNA of a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned in a manner that facilitates translation. In general, DNA sequences operably linked are contiguous and, in the case of a secretory leader, contiguous and in reading phase. However, breeders do not have to be contiguous. Binding is performed by ligation at convenient restriction sites. If these sites do not exist, synthetic oligonucleotide adapters or connectors are used according to conventional practice. Another aspect of the present invention is the treatment of disorders associated with the a2ß1 integrin by administering to a subject a nucleic acid molecule encoding an antibody against the a2 integrin of the invention. Appropriate methods of administration include genetic therapy methods (see below).
A nucleic acid of the invention can be delivered to cells in vivo using methods such as direct DNA injection, receptor-mediated DNA uptake, virus-mediated transfection or non-viral transfection, and lipid-based transfection, all which may involve the use of gene therapy vectors. Direct injection has been used to introduce naked DNA into cells in vivo (see, for example, Acsadi et al. (1991) Nature 332: 815-818; Wolff et al. (1990) Science 247: 1465-1468). A delivery apparatus (eg, a "gene gun") can be used to inject DNA into the cells in vivo. Such an apparatus may be commercially available (for example, from BioRad). The naked DNA can also be introduced into the cells by complexing the DNA to a cation, such as polylysine, which is coupled to a ligand for a cell surface receptor (see, for example, Wu, G. and Wu, CH (1988) J. Biol. Chem. 263: 14621; Wilson et al. (1992) J. Biol. Chem. 267: 963-967; and U.S. Patent No. 5,166,320). The binding of the DNA-ligand complex to the receptor can facilitate the absorption of DNA by endocytosis mediated by a receptor. A DNA-ligand complex bound to the capsids of the adenovirus that interrupts the endosomes, thereby releasing material into the cytoplasm, can be used to prevent degradation of the complex by intracellular lysosomes (see, for example, Curiel et al. (1991). ) Proc. Nati, Acad. Sci. USA 88: 8850, Cristiano et al. (1993) Proc. Nati. Acad. Sci. USA 90: 2122-2126). Defective retroviruses are well characterized by their use as gene therapy vectors (for a review see, Miller, A. D. (1990) Blood 76: 271). Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with those viruses can be found in Current Protocols in Molecular Biology, Ausubel, F. M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and in other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM that are well known to those skilled in the art. Suitable examples of packaged virus lines include .psi.Crip, .psi.Cre, .psi.2 and .psi.Am. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and / or in vivo (see, e.g., Eglitis, et al. (1985) Science 230: 1395-1398, Danos and Mulligan (1988) Proc. Nati, Acad. Sci. USA 85: 6460-6464, Wilson et al. (1988) Proc. Nati. Acad. Sci. USA 85: 3014-3018; Armentano et al. (1990) Proc. Nati, Acad. Sci. USA 87: 6141-6145; Huber et al. (1991) Proc. Nati. Acad. Sci. USA 88: 8039-8043; Ferry et al. (1991) Proc. Nati, Acad. Sci. USA 88: 8377-8381; Chowdhury et al. (1991) Science 254: 1802-1805; van Beusechem et al. (1992) Proc. Nati. Acad. Sci. USA 89: 7640-7644; Kay et al. (1992) Human Gene Therapy 3: 641-647; Dai et al. (1992) Proc. Nati. Acad. Sci. USA 89: 10892-10895; (1993) J. Immunol., 150: 4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCT Application WO 89/07136; PCT ratio WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573).
To be used as a gene therapy vector, the genome of an adenovirus can be engineered to encode and express a nucleic acid compound of the invention, but is inactivated in terms of its ability to replicate in a normal viral life cycle. See, for example, Berkner went to. (1988) BioTechniques 6: 616; Rosenfeld went to. (1991) Science 252: 431-434; and Rosenfeld went to. (1992) Cell 68: 143-155. Adequate adenoviral vectors derived from Ad type 5 dl324 strain of adenovirus or other adenovirus strains (eg, Ad2, Ad3, Ad7, etc.) are well known to those skilled in the art. Recombinant adenoviruses are favorable because they do not require division of cells to function as effective gene delivery vehicles and can be used to infect a wide variety of cell types, including airway epithelials (Rosenfeld et al. (1992) cited above), endothelial cells (Lemarchand et al. (1992) Proc. Nati. Acad. Sci. USA 89: 6482-6486), hepatocytes (Herz et al. Gerard (1993) Proc. Nati, Acad. Sci. USA 90: 2812-2816) and muscle cells (Quantin et al. (1992) Proc. Nati, Acad. Sci. USA 89: 2581-2584). Adeno-associated viruses (AAV) can be used as a gene therapy vector for the delivery of DNA for gene therapy purposes. An AAV is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, to function as an aid virus for efficient replication and a productive life cycle (Muzyczka et al., Curr. , and Immunol. (1992) 158: 97-129). An AAV can be used to integrate DNA into undivided cells (see, for example, Flotte et al. (1992) Am. J. Respir Cell. Mol. Biol. 7: 349-356; Samulski eí al, (1989) J. Virol. 63: 3822-3828; and McLaughlin went to. (1989) J. Virol. 62: 1963-1973). An AAV vector like the one described in Tratschin et al. (1985) Mol. Cell. Biol. 5: 3251-3260 can be used to introduce DNA into cells (see, for example Hermonat et al. (1984) Proc. Nati, Acad. Sci. USA 81: 6466-6470; Tratschin et al. (1985) Mol. Cell Biol. 4: 2072-2081; Wondisford et al. (1988) Mol Endocrinol 2: 32-39; Tratschin et al. (1984) J. Virol. 51: 611-619; and Flotte et al. 1993) J. Biol. Chem. 268: 3781-3790). The lentiviral gene therapy vectors can also be adapted for use in the invention. General methods for gene therapy are known to those skilled in the art. See, for example, Pat. from the USA Do not.,399,346 by Anderson et al. A biocompatible capsule for the delivery of genetic material is described in PCT Publication WO 95/05452 by Baetge et al. Methods of genetic transfer within hematopoietic cells have also been previously reported (see Clapp, DW, et al., Blood 78: 1132-1139 (1991); Anderson, Science 288: 627-9 (2000); and Cavazzana-Calvo et al., Science 288: 669-72 (2000)). Cell, cell line and cell culture are often used interchangeably and all those designations include progeny. Transformaand transformed cells (eg, obtained by transfection, transformation or transduction of nucleic acids, vectors, viruses, etc.) include the cell of the main subject and the cultures derived therefrom regardless of the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or unintentional mutations. Mutant progeny that have the same function or biological activity as when selected in the originally transformed cell are included. When looking for different designations, it will be clear from the context. Humanized antibodies as described herein include antibodies having variable region frameworks derived from a human acceptor antibody molecule, hypervariable sequences or CDRs of a murine donor antibody, and constant regions, if present, derived from human sequences. The antibodies of the present invention have been constructed with both the heavy chain variable region and the light chain variable region CDRs of the BHA2.1 clone of the murine monoclonal antibody (Hangan et al., Cancer Res. 56: 3142-3149 (1996 )). Preferred starting materials for building antibodies are antibodies to integrin a2 such as those secreted by hybridoma BHA2.1 (eg, TMC-2206) which are function blocking antibodies directed against human a2 integrin and are dependent for binding and its activity from the presence of an intact I domain within the target a2 integrin. Antibodies with the epitope specificity of TMC-2206 (or BHA2.1) are preferred, including antibodies that bind to the inactive conformation of the a2 integrin molecule, and / or which do not act as mimetic ligands. Antibodies with the epitope specificity of TMC-2206 (or BHA2.1) are preferred. Although they interact with the a2ß1 integrin present in both leukocytes and platelets, they do not cause platelet activation, they impair the aggregation of activated platelets. in collagen, have a minimal effect or have no effect on bleeding and / or are not associated with hemorrhagic complications at the administered concentrations, including therapeutic doses in vivo. The antibodies can be constructed wherein the human acceptor molecule for the light chain variable region is selected based on the homology considerations between the potential variable regions of the acceptor molecule and the light chain variable region of the murine antibody. The germ lines of candidate human acceptor molecules are preferred to reduce potential antigenicity. The germline databases are formed by antibody sequences that are read at the end of the FW3 region of the heavy chain and partially in the CDR3 sequence. For the selection of a FW4 region, it is preferred to search databases of mature antibody sequences that have been derived from the selected germline molecule, and are also preferred to select a reasonably homologous FW4 region for use in the antibody molecule recombinant. The human acceptor molecules are preferably selected from the same class of light chain as the murine donor molecule, and from the same canonical structural class of the variable region of the murine donor molecule.
Secondary considerations for the selection of the human acceptor molecule for the light chain variable region include CDR length homology between the murine donor molecule and the human acceptor molecule. The human acceptor antibody molecules are preferably selected for homology searches in the V-BASE database, and other databases such as the Kabat and NCBI public databases can also be used. For humanized antibodies against a2 integrin with the same epitope specificity or with a similar specificity and / or functional properties similar to those of TMC-2206, one of the preferred light chain human acceptor molecules is SEQ ID NO: 37 with the germline antibody sequence A14 for the FW region 1-3 and the sequence FGQGTKVEIK for FW4 (SEQ ID NO: 38) representing a common FW-4 of mature kappa 1 light chains (e.g. light chain sequence AAB24132 (NCBI entry gi / 259596 / gb / AAB24132)). Antibodies can be constructed where the human acceptor molecule for the heavy chain variable region is selected based on homology considerations between the variable regions of the potential acceptor molecule and the heavy chain variable region of the murine antibody. The candidate human acceptor molecules of the germ line are preferred to reduce the potential antigenicity. The germline databases are composed of antibody sequences that read to the end of the FW3 region of the heavy chain and partially into the CDR3 sequence. For the selection of an FW4 region, it is preferable to look for databases of mature antibody sequences that have been derived from the selected germline molecule, and it is also preferred to select a reasonably homologous FW4 region for the purpose of using it in the recombinant antibody molecule. The human acceptor molecules are preferably selected from the same heavy chain class as the murine donor molecule, and from the same canonical structural class of the variable region of the murine donor molecule. Secondary considerations for the selection of the human acceptor molecule for the heavy chain variable region include homology in the length of the CDR between the murine donor molecule and the human acceptor molecule. The human acceptor antibody molecules are preferably selected for homology searches in the V-BASE database, and other databases such as the Kabat and NCBI public databases can also be used. For antibodies against integrin a.2 with the same epitope specificity or similar specificity and / or functional properties similar to those of TMC-2206, one of the preferred heavy chain acceptor molecules is SEQ ID NO: 39 with the sequence of germline antibodies 4-59 for the FW region 1-3 (SEQ ID NO: 12) and the antibody CAA48104.1 (NCBI entry, gi / 33583 / emb / CAA48104.1), a mature antibody derived from the sequence 4-59 of the germline for the FW 4 region (SEQ ID NO: 13) (http://www.ncbi.nlm.nih.gov).
Methods for humanizing a non-human a2 integrin antibody are described herein, including the examples below. In order to humanize an antibody against integrin a2 the initial material of the non-human antibody is obtained by preparation of the immunization or by means of the purchase of commercially available antibodies. Exemplary techniques for generating antibodies are described herein. The a2β1 integrin antigen to be used for the production of antibodies may be, for example, a soluble form of a2β1 integrin or another α2β1 integrin fragment, eg, an α2β1 integrin fragment that includes a human α2 integrin domain I (SEQ ID NO: 11) (see also, for example, SEQ ID NO: 107). Other forms of a2 integrin useful for generating antibodies will be obvious to those skilled in the art based on the a2 integrin sequence (e.g., a human a.2 integrin as in SEQ ID NO: 8). Polyclonal antibodies are preferably obtained from animals by means of multiple subcutaneous (sc), intravenous (iv) or intraperitoneal (ip) injections of the relevant antigen with or without an adjuvant. It may be useful to conjugate the relevant antigen with a protein that is immunogenic in the species that will be immunized, for example, keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin or trypsin inhibitor in soybean using a bifunctional or derivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (via lysine residues), glutaraldehyde, succinic anhydride, SOCI2, or R1N = C = NR, wherein R and R1 are different alkyl groups. The animals can be immunized against the antigen, the immunogenic conjugates or the derivatives by combining the antigen or the conjugate (for example, 100 μg for rabbits or 5 μg for mice) with 3 volumes of Freund's complete adjuvant and the solution is injected intradermally in multiple sites. One month later, the animals are supercharged with the antigen or with the conjugate (for example, with 1/5 to 1/10 of the original amount used to immunize) in the complete Freund's adjuvant by means of subcutaneous injections at multiple sites. Seven to 14 days later the animals are bled and the serum is analyzed to titrate the antibody. The animals are overfed until the titer reaches a plateau level. For conjugated immunizations it is preferable that the animal be overfed with the conjugate of the same antigen, but it is conjugated to a different protein and / or through a different cross-linking reagent. The conjugates can also be made in a culture of recombinant cells as protein fusions. In addition, aggregating agents such as alum are suitable for improving the immune response. It is possible to make monoclonal antibodies using the hybridoma method that was first described in Kohler et al., Nature, 256: 495 (1975), or they can be made by recombinant DNA methods (for example, US Pat. No. 6,204,023). Monoclonal antibodies can also be made using the techniques described in U.S. Pat. Nos. 6,025,155 and 6,077,677, as well as in US Patent Applications Nos. 2002/0160970 and 2003/0083293 (see also, for example, Lindenbaum, et al., Nucleic Acids Research 32 (21): 0177 (2004 )). In the hybridoma method a mouse or other suitable host animal such as a rat, a hamster or a monkey is immunized (e.g., as described above in this same document) to obtain lymphocytes that produce or are capable of producing antibodies that are will bind specifically with the antigen used for immunization. Alternatively, lymphocytes can be immunized in vitro. Then, the lymphocytes are fused with the myeloma cells using a suitable fusion agent, such as polyethylene glycol, to form a hybridoma cell (see, for example, Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 ( Academic Press, 1986)). Hybridoma cells prepared in this way are seeded and cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental melanoma cells lack the hypoxanthine guanine phosphoribosyl transferase enzyme (HGPRT or HPRT), the culture medium for the hybridomas will usually include hypoxanthine, aminopterin and thymidine (HAT medium), which substances prevent the growth of the cells deficient in HGPRT. Preferred myeloma cells are those that are efficiently fused, which support a stable and high-level production of antibodies by means of the selected antibody-producing cells, and that are sensitive to a medium like the HAT medium. Among these, the preferred myeloma cell lines are the murine myeloma lines, such as those derived from tumors of MOP-21 and M.C.-11 mice available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA US, and SP-2 or X63-Ag8-653 cells available from American Type Culture Collection, Rockville, Md. USA. UU Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (eg, Kozbor, J. Immunol., 133: 3001 (1984)).; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)). The culture medium in which the hybridoma cells are being cultured is analyzed to determine the production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells will be determined by immunoprecipitation or by an in vitro binding assay, such as a radioimmunoassay (RIA) or an enzyme-linked immunosorbent assay (ELISA).
The binding affinity of the monoclonal antibody can be determined, for example, by the Scatchard analysis of Munson et al., Anal. Biochem., 107: 220 (1980). After hybridoma cells are identified to produce antibodies with the desired specificity, affinity and / or activity, clones can be subcloned by limiting dilution and culture procedures by standard methods (Goding, Monoclonal Antibodies: Principies and Practice, pp.59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, the D-MEM (or its initials in English) or RPMI-1640. In addition, hybridoma cells can be cultured in vivo as ascites tumors in an animal. The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascitic fluid or serum by means of conventional methods of immunoglobulin purification including, for example, protein A chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis and / or affinity chromatography. The DNA encoding the monoclonal antibodies is easily isolated and sequenced using conventional methods (for example, by using oligonucleotide probes that are capable of specifically binding to the coding genes in the heavy and light chains of the monoclonal antibodies). Hybridoma cells serve as a preferred source of said DNA. Once isolated, the DNA can be placed in expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells or myeloma cells, including those that otherwise they do not produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The recombinant production of antibodies is described in more detail below. Examples of this document describe methods for the humanization of an exemplary a2 integrin antibody. In certain embodiments it may be desirable to generate amino acid sequence variants of the humanized antibody, particularly when these improve the binding affinity or other biological properties of the humanized antibody. Variants of the amino acid sequences of the humanized antibody against integrin a2ß1 are prepared by introducing the appropriate changes in the nucleotide within a humanized antibody against the a2β1 DNA integrin or by peptide synthesis. Such variants include, for example, deletions of and / or insertions in and / or substitutions of residues within the amino acid sequences shown for the TMC-2206 antibody against the a2 integrin (eg, derived from or based on variable region sequences). like those shown in SEQ ID NOs: 19 and 21). Any combination of elimination, insertion and substitution of amino acids is done to reach the final construction, as long as the final construction has the desired characteristics. Changes in the amino acid can also alter the post-translational processes of the humanized antibody against integrin a2, such as the change in the number or position of the glycosylation sites. There are various methods used to create human or human type antibodies (eg, "humanization"). Approaches to humanize antibodies have varied over the years. One approach was to generate murine variable regions fused to human constant regions, called chimeras with murine-human Fc regions (see, for example, Morrison et al, Proc. Nati, Acad. Sci. USA 81: 6851-6855 (1984 ), U.S. Patent No. 5,807,715). Another approach exploited the fact that CDRs can be easily identified based on their hypervariable nature (Kabat et al, J. Biol. Chem. 252: 6609-6616 (1977)), Kabat, Adv. Protein Chem. 32: 1-75 (1978)) and its canonical structure (Chothia and Lesk, J. Mol. Biol. 196 (4): 901-17 (1987); Lazakani et al., J. Mol. Biol. 272: 929 (1997) and be humanized by grafting only the non-human CDR regions (known as donor CDRs) into a human framework (known as acceptor lattice) as shown, for example, Jones et al., Nature 321 (6069): 522-5 (1986); (see, for example, U.S. Patent No. 5,225,539; U.S. Patent No. 6,548,640). The six cycles of CDR are presented in a grouping, and based on the crystallographic analysis, the critical residues of the structure within the so-called "Vernier" zone that flank the CDRs or at the heavy-light chain interface can be identified easily (see, for example, Chothia and Lesk, J. Mol. Biol. 196 (4): 901-17 (1987); Chothia et al., J. Mol. Biol. 186 (3): 651-63 (1985). ); Chothia et al., Nature 342 (6252): 877-83 (1989)). These residues can be retromutated to the murine residue to restore the correct relative orientation of the six CDRs (see, for example, Verhoyen et al., Science 239 (4847): 1534-6 (1988); Reichman et al., Nature 332 ( 6162): 323-7 (1988); Tempest et al., Biotechnology (NY) 9 (3): 266-71 (1991)). Because variable regions can be classified into families that have a relatively high homology between mouse and human (reviewed in, for example, Pascual and Capra Adv. Immunol., 49: 1-74 (1991)), these early studies also indicated that the potential for loss in affinity could also be minimized in the grafted antibody by selecting the human germline sequence with the highest homology with the murine antibody of interest for use as the human acceptor molecule (see, for example, Patent No. 5,225,539; Verhoyen et al., Science 239 (4847): 1534-6 (1988)). Homologies of families and structural relationships between frameworks that impact the correct presentation of a given type of canonical CDR structure have been reported (see, for example, Al-Lazakani et al., J. Mol. Biol. 273 (4): 927 -48 (1997) and the references cited there). Preferably, the human or germline sequence that best fits is chosen. The available databases of germ line sequences of antibodies can be used to determine the family subtype of a determined murine heavy or light chain and to identify the sequences that best fit and that are useful as human acceptor frameworks within that human subfamily. Preferably, both the linear amino acid homology of the donor and the acceptor frameworks and the canonical structure of the CDR are taken into account. Exemplary heavy chain residues that can be substituted in a humanized antibody against integrin a.2 include one or more of the following numbers of framework residues: H37, H48, H67, H71, H73, H78, and H91 (system Kabat numbering). Preferably, at least four of these framework residues are replaced. A particularly preferable set of substitutions for the heavy chain in humanized antibodies against integrin a2 as exemplified herein is H37, H71, H73 and H78. Similarly, the residues in the light chain can also be replaced. Exemplary light chain residues for substitution include one or more of the following residue numbers: L1, L2, L4, L6, L46, L47, L49, and L71. Preferably at least three of these framework residues are replaced. A particularly preferable set of substitutions for the light chain in humanized antibodies against integrin a2 as exemplified herein is L2, L46 and L49. A useful method for the identification of certain residues or regions of a humanized antibody against integrin a2 which are preferred locations for mutagenesis is known as "alanine mutagenesis" (see, for example, Cunningham and Wells Science, 244: 1081-1085). (1989)). Here, a waste or a group of target waste is identified (for example, loaded waste such as arg, asp, his, lys, and glu) and is replaced by a neutral or negatively charged amino acid (preferably alanine or polyalanine) to affect the interaction of the amino acids with the a2β1 integrin antigen. Then, those amino acid locations that demonstrate a functional sensitivity to substitutions are refined by introducing more variants or variants of another type into, or for, substitution sites. Therefore, although the site for introducing a variant in the amino acid sequence is predetermined, the nature of the mutation itself need not be predetermined. For example, to analyze the performance of a mutation at a given site, alanine scanning or random mutagenesis is performed at the codon or in the target region and the expressed variants of the humanized antibody against integrin a2 are analyzed in search of the desired activity . Inserts in the amino acid sequence include amino-terminal and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as insertions within the sequence of residues of one or multiple amino acids . Examples of terminal insertions include a humanized antibody against integrin a2 with an N-terminal methionyl residue or the antibody fused to an epitope tag. Other insertional variants of a humanized antibody molecule against a2 integrin include the N-terminal or C-terminal fusion of a humanized antibody against the a2 integrin of an enzyme or a polypeptide, which increases the half-life of the antibody serum (see below). Another type of variant is a variant amino acid substitution. These variants have at least one amino acid residue removed from a humanized antibody molecule against integrin a2 and a different residue inserted in its place. The sites of greatest interest for substitution mutagenesis include the hypervariable cycles, but alterations of the framework are also contemplated. The residues of hypervariable regions or framework residues involved in the binding of antigens are generally substituted relatively conservatively. These conservative substitutions are shown below under the heading of "preferred substitutions". If such substitutions result in a change in biological activity, then more substantial changes are introduced, termed "exemplary substitutions" or as better described below in reference to the amino acid classes, and the products are analyzed.
Substantial modifications in the biological properties of the antibody are achieved by selecting substitutions that differ significantly in their effect to maintain (a) the skeletal structure of the polypeptide in the area of substitution, for example, as a sheet conformation or helical, (b) the load or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. The residues that occur naturally are divided into groups based on the common properties of the chain side: (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 will involve the exchange of a member of one of these classes by another class. Any cysteine residue that is not involved in maintaining the proper confirmation of a humanized antibody against integrin a.2 may also be substituted, usually with serine, to improve the oxidative stability of the molecule and to prevent aberrant crossover. Conversely, the cysteine linkage (s) can be added to the antibody to improve its stability (particularly when the antibody is an antibody fragment as an Fvt fragment). Another type of amino acid variant of the antibody alters the original glycosylation pattern of the antibody. By alteration is meant the removal of one or more carbohydrate moieties that are in the antibody and / or the addition of one or more glycosylation sites that are not present in the antibody. The glycosylation of antibodies is usually N- or O-glycosylation. N-glycosylation refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, wherein X is any amino acid except proline, are the recognition sequences for the enzymatic binding of the carbohydrate moiety in the side chain of asparagine. Therefore, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-glycosylation refers to the binding of one of the sugars N-acetylgalactosamine, galactose or xylose, to a hydroxyamino acid, usually serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used. The addition or removal of glycosylation sites to the antibody is conveniently achieved by altering the amino acid sequence so as to contain or lack one or more of the tripeptide sequences described above (for the N-glycosylation sites) . Alteration can also be performed with the addition or substitution of one or more serine or threonine residues in the original antibody sequence (for the O-glycosylation sites). The nucleic acid molecules encoding the humanized antibody sequence variants against integrin a2 are prepared by a variety of methods known to those skilled in the art. These methods include, but are not limited to, isolation from a natural source (in the case that variants of amino acid sequences occur naturally) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, mutagenesis by PCR or mutagenesis by insertion of a cassette of a variant prepared above or of a non-variant version of the humanized antibody against integrin a2.
Generally, the amino acid sequence variants of a humanized antibody against integrin a2 will have an amino acid sequence that will have at least 75% of the identity of the amino acid sequence with the original amino acid sequences of the humanized antibody of the heavy chain or the light chain (for example, variable region sequences as in SEQ ID NO: 21 or SEQ ID NO: 19, respectively), preferably at least 80%, preferably at least 85%, preferably at least 90% and preferably at minus 95% including, for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% 94%, 95%, 96%, 97%, 98%, 99% and 100%. The identity or homology with respect to this sequence are defined herein as the percentage of amino acid residues in the candidate sequence that are identical to the humanized residues of a2 integrin, after aligning the sequences and introducing the gaps, if necessary, to obtain the sequence identity of the maximum percentage, and without considering conservative substitutions (as described above) as part of the identity of the sequence. It should not be inferred that N-terminals, C-terminals or extensions, deletions or internal insertions in the antibody sequence affect the identity or homology of the sequence. Therefore, the identity of the sequence can be determined by standard methods that are commonly used to compare the similarity in the position of the amino acids of two polypeptides. Using a computer program such as BLAST or FASTA, two polypeptides are aligned to obtain an optimal match of their respective amino acids (either along one or both sequences, or along a predetermined part of one or both sequences) . The programs offer a predetermined punishment in the opening and a predetermined punishment for the gap, and a rating matrix as PAM250 (a standard rating matrix).; see Dayhoff et al., in Atlas of Protein Sequence and Structure, vol. 5, supp. 3 (1978)) can be used in conjunction with the computer program. For example, the identity of the percentage can be calculated later as: the total number of identical matches multiplied by 100 and then divided by the sum of the length of the longest sequence within the match space and the number of gaps entered in the matches. longer sequences in order to align the two sequences. Antibodies having the characteristics identified herein as desirable in a humanized antibody against integrin a.2 are analyzed by means of the methods described herein. For example, methods for analyzing antibodies against the a.2-integrin candidates in search of the preferred features and functionalities are provided and include the analysis of antibodies that bind to the epitope at the a2ß1 integrin binding by means of a antibody of interest (eg, those that compete with, inhibit or block the binding of the TMC-2206 antibody to the a2β1 integrin). The methods and sample materials are described in Example 13. Cross-blocking assays can be performed and described for example in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988). In addition, or alternatively, epitope mapping, as described in Champe et al., J. Biol. Chem. 270: 1388-1394 (1995), can be used to determine whether the antibody binds to an epitope of interest. (see, for example, Example 12 for the epitope mapping studies of TMC-2206). The immobilized α2β1 integrin can be used in a similar manner to determine the binding potencies relative to measuring K K values in competition assays (see, for example, Example 2). For example, Eu-TMC-2206 with fluorescent label is used in the presence of various concentrations of the unlabeled candidate antibody, for example, using a test system similar to that described above. After a specified incubation time, the amount of Eu-TMC-2206 that was bound is determined. The inhibition curves are fitted with the "competition for a site" model using the Prism software (GraphPad, Inc. CA) to obtain IC50 values and to calculate Kj using the Cheng and Prusoff equation (Biochem, Pharmacol. (23): 3099-108 (1973)). It is desirable to prepare, identify and / or select humanized antibodies against integrin a.2 that have beneficial binding properties; for example, under the conditions described in Example 2, wherein candidate antibodies are tested for their ability to block cell adhesion mediated by integrin a2ß1 compared to TMC-2206 and with mouse-human chimeric antibody derivative of TMC-2206 as described in Example 2. For example, CHO cells expressing human a2 integrin and endogenous hamster ßl (Symington et al., J. Cell Biol. 120 (2): 523-35 (1993 )) are prepared and labeled with CFSE (Molecule Probes, OR). The labeled cells are prepared and the cell concentration is adjusted; the cells are kept in the dark until they are used. A plate coated with collagen (rat tail collagen Type I, BD Biosciences) is prepared and each solution of the serially diluted antibody is added to the plate with collagen. The labeled cells are added to the well and the plate is incubated. After washing, the cells are lysed and the intensity of the fluorescence is read (excitation, 485 nm, emission, 535 nm). The inhibitory activity of each antibody is calculated. Additionally, the binding constants of the candidate antibodies for the immobilized integrin ligand a2ß1 can be calculated as described in Example 2. The wells of a 96-well microtiter plate are coated with platelet α2β1 integrin (custom coated with platelets a2β1 made by GTI Inc., Wl) and then they are blocked. For example, to determine the affinity of TMC-2206 for its a2 integrin antigen, TMC-2206 with fluorescent tag or antibody with isotype IgG control is used (see examples below). The fluorescently labeled antibody, including Eu-TMC-2206 or Eu-con isotype IgG control, is applied to microtiter plates blocked with a2ß1 integrin. After the sealed plates are incubated to allow the antibody-antigen interaction to reach equilibrium, the samples are transferred from each well to a nine-well containing an improving solution for the measurement of the free label (without binding). The improving solution is also added to the empty wells for the measurement of the attached label. The Kd values of the antibody against integrin a.2 are calculated by Scatchard analysis. The relative affinity of the TMC-2206 derivatives (including humanized antibodies derived from or based on TMC-2206) can be determined by obtaining the Ki value in a competition assay. For example, for the proficiency test, TMC-2206 with Eu tag is added to wells coated with a2ß1 in the presence of unlabelled anti-integrin a.2 antibodies, including TMC-2206 or chimeric antibodies (including humanized antibodies) derived of or based on TMC-2206, or the antibody with isotype IgG control at various concentrations. After an incubation period to reach equilibrium, the wells are washed and the antibody levels labeled as binding are measured as the Eu tag retained in each well. The Ki value can be derived from the EC50 values using the Kd value obtained for the Eu-TMC-2206 antibody by the direct binding studies as described above. In certain embodiments, the humanized antibody against integrin a2 is an antibody fragment. Several techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived through the proteolytic digestion of intact antibodies (see, for example, 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 be produced directly by recombinant host cells, such as bacteria (see, for example, Better et al., Science 240 (4855): 1041-1043 (1988)).; U.S. Patent No. 6,204,023). For example, Fab'-SH fragments can be directly coated with E. coli and chemically coupled to form F (ab ') 2 fragments (Cárter et al., Bio / Technology 10: 163-167 (1992)). According to another approach, F (ab ') 2 fragments can be isolated directly from the culture of recombinant host cells. Other techniques for the production of antibody fragments will be apparent to the skilled artisan. In some embodiments, it may be desirable to generate humanized antibodies against the a2 multispecific integrin (eg, bispecific) that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies (e.g., with two different binding arms) can bind to two different epitopes of the a2ß1 integrin protein. Alternatively, an arm against integrin a2 may be combined with an arm that binds to a trigger molecule in a leukocyte such as a T cell receptor molecule (e.g., CD2 or CD3), or Fc receptors for IgG (Fc? R), such as Fc? R1 (CD64), Fc? RII (CD32) and Fc? RIII (CD16) so that it approaches cellular defense mechanisms in a cell that has integrin a2ß1 attached to its surface. Bispecific antibodies can be used for localized cytotoxic agents for cells with integrin a2ß1 attached to their surfaces. These antibodies possess an a2ß1 integrin binding arm and an arm that binds the cytotoxic agent (eg, gelonin, saporin, anti-interferon alpha, vinca alkaloid, castor A chain, or radioisotope hapten). Bispecific antibodies can be prepared as full-length antibodies or as antibody fragments (for example, bispecific antibodies F (ab ') 2). According to another approach for the creation of bispecific antibodies, the interface between a pair of antibody molecules can be designed to maximize the percentage of heterodimers that are coated with recombinant cell culture. The preferred interface includes at least a portion of the CH3 domain of a constant domain of the antibody. In this method, one or more small side chains of amino acids are replaced with larger side chains (eg, tyrosine or tryptophan). Compensatory cavities of identical or smaller size for the larger lateral chain (s) are created at the interface of the second antibody by replacing the large side chains of amino acids with smaller ones (eg alanine) or threonine). This provides a mechanism for increasing the performance of heterodimers over other undesired end products such as homodimers (see, for example, WO96 / 27011). Bispecific antibodies include cross-linked or heteroconjugate antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin and the other to biotin. Heteroconjugate antibodies can be made using any convenient method of crosslinking. Suitable crosslinking agents are well known in this area, and are presented, for example, in U.S. Patent No. 4,676,980 together with a number of cross-linking techniques. Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. Bispecific antibodies can be prepared using chemical linkage. For example, Brennan et al., (Science 229: 81 (1985)) describes a procedure wherein intact antibodies are proteolytically cleaved to generate F (ab ') 2 fragments. These fragments are reduced in the presence of sodium arsenite as an agent to complex the dithiol in order to stabilize the vinyl dithiols and prevent the formation of intermolecular disulfide. The Fab 'fragments generated afterwards are converted to thionitrobenzoate derivatives (TNB). One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes. The Fab'-SH fragments, recovered from E. coli, can be chemically coupled to form bispecific antibodies. For example, Shalaby et al., (J. Exp. Med. 175: 217-225 (1992)) describes the production of a fully humanized F (ab ') 2 bispecific antibody molecule. Wherein each Fab 'fragment was secreted separately from E. coli and subjected to chemical coupling directed in vitro to form the bispecific antibody. Therefore, the bispecific antibody formed was able to bind to cells that overexpress the HER2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets. Several techniques for creating and harboring bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine closures (see, for example, Kostgelny et al., J. Immunol., 148 (5): 1547-1553 (1992)). The leucine-closing peptides of the Fos and Jun proteins were linked to the Fab 'portions of two different antibodies by genetic fusion. The antibody homodimers were reduced in the hinge region to form monomers and then reoxidized to form antibody heterodimers. This method can also be used for the production of antibody heterodimers. The diabody technology (see, for example, Hollinger et al., Proc. Nati, Acad. Sci. USA 90: 6444-6448 (1993)) has provided an alternative mechanism for the creation of bispecific antibody fragments. The fragments include a heavy chain variable region (VH) connected to a light chain variable region (VL) by a linker that is too short to allow parity between two domains of the same chain. Consequently, the VH and VL domains of a fragment are forced to establish a parity with the complementary VL and VH domains of another fragment, thus forming two paratopes. Another strategy has also been reported for creating bispecific antibody fragments with the use of single chain Fv (sFv or scFv) dimers (see, for example, Gruber et al., J. Immunol., 152: 5368 (1994)). Alternatively, the bispecific antibody may be a linear antibody, for example, produced as described in Zapata et al., Protein Eng. 8 (10): 1057-1062 (1995). Antibodies produced with more than two valences are contemplated. For example, trispecific antibodies can be prepared (see, for example, Tutt et al., J. Immunol. 147: 60 (1991)). Other modifications of the humanized antibodies against integrin a2 are contemplated. For example, it may be desirable to modify the antibody with respect to effector function, thereby improving or reducing the effectiveness of the antibody, for example, in the treatment against cancer. The cysteine residues can be introduced into the Fc region, thereby allowing interchain chain disulfide bond formation in the region. The homodimeric antibody generated in this way could have an improved internalization capacity and / or an increase in complement-mediated cytolysis (CMC) and / or antibody-dependent cellular cytotoxicity (ADCC-for short). in English) (see for example, Carón eí al., J. Exp. Med. 176: 1191-1195 (1992) and Shopes, BJ Immunol., 148: 2918-2922 (1992)). Homodimeric antibodies with improved antitumor activity could also be prepared using crosslinking heterobifunctionals (see, for example, those described in Wolff et al., Cancer Research 53: 2560-2565 (1993)). Alternatively, an antibody can be designed with dual Fc regions and therefore can have enhanced CMC and / or ADCC capabilities (see, for example, Stevenson et al., Anti-Cancer Drug Design 3: 219-230 (1989)). Immunoconjugates composed of a humanized antibody against a2 integrin conjugated to a portion, for example, a molecule, composition, complex or agent, for example a cytotoxic agent such as a chemotherapeutic agent, toxin (for example, an enzymatically active toxin of bacterial, fungal, plant or animal origin or fragments thereof) or an isotope radioactive (eg, a radioconjugate), to direct the agent to a cell, tissue or organ that is expressed against integrin a.2. Said immunoconjugate can be used in a method for directing the portion or agent to a particular site of action characterized by the presence of integrin a2 or a2ß1. The chemotherapeutic agents useful in the generation of said immunoconjugates have been described above. Enzymatically active toxins and fragments thereof that may be used include the diphtheria A chain, non-binding active fragments of diphtheria toxin, the A chain of an exotoxin (from Pseudomonas aeruginosa), castor A chain, of abrin, A chain of modeccina, alpha-sarcina, proteins Aleurites fordii, diantine proteins, proteins of Phytolaca americana (PAPI, PAPII and PAP-S), inhibitor of momordica, curcin, crotina, inhibitor of sapaonaria officinalis, gelonina , mitogillin, restrictocin, phenomycin, enomycin or trichothecenes. There is a wide variety of radionuclides for the production of radioconjugated alpha 2 integrin antibodies. Some examples include 212 Bi, 131 In, 90Y or 186Re. Antibody and cytotoxic agent conjugates are manufactured using various bifunctional protein coupling agents such as N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), minothiolane (IT), bifunctional imidoester derivatives (such as dimethyl adipimidate HCL) ), active esters (such as disuccinimidyl suberate), aldehydes (such as gluteraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis- (p-diazonium benzoyl) -ethylenediamine), dissociates (eg tolieno 2,6-disocianato) or bisactive fluorine compounds (such as 1,5-difluoride-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). The 1-isothiocyanatobenzyl-3-methyldiethylene triaminopentaacetic acid with carbon label 14 (MX-DTPA) is an exemplary chelating agent for the conjugation of the radionuclide with the antibody (see, for example, W094 / 11026). In another embodiment, the antibody can be conjugated to a receptor (such as streptavidin) to be used in the cell, tissue or pre-target organ that expresses a2 integrin wherein the antibody-receptor conjugation is administered to the patient, followed by removing the circulation of the unbound conjugate using a cleaning agent and then administering a ligand (eg, avidin) which is conjugated with an agent, for example a cytotoxic agent (such as a radionuclide). The α2 integrin antibodies detailed herein may also be formulated as immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art, such as those described in Epstein et al., Proc. Nati Acad. Sci. USA 82: 3688 (1985); Hwang went to., Proc. Nati Acad. Sci. USA 77: 4030 (1980); and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with improved circulation time are disclosed in U.S. Patent No. 5,013,556. Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition including phosphatidylcholine, cholesterol and phosphatidylethanolamine (PEG-PE) PEG derivatives. The liposomes are extruded through filters having a defined pore size to obtain liposomes with the desired diameter. Fab 'fragments of an antibody to integrin a.2 can be conjugated to liposomes as described in Martin et al., J. Biol. Chem. 257: 286-288 (1982) through an exchange reaction disulfide. A chemotherapeutic agent (eg, doxorubicin) is optionally contained within the liposome (see, for example, Gabizon et al., J. National Cancer Inst. 81 (19): 1484 (1989)). Humanized anti-a2 integrin antibodies can also be used in Antibody Directed Enzyme Drug Therapy (ADEPT) by conjugating the antibody with a prodrug activating enzyme that converts a prodrug (e.g., a chemotherapeutic agent) peptidyl, see, for example, WO81 / 01 145) in an active drug, (see, for example, WO88 / 07378 and U.S. Patent No. 4,975,278). The enzyme component of the immunoconjugate that is useful for ADET includes any enzyme capable of acting on a prodrug in such a way as to convert it to its most active form. Enzymes that are useful include, but are not limited to, alkaline phosphatase useful for converting phosphate-containing prodrugs into free drugs; arylsulfatase useful for converting prodrugs containing sulphates into free drugs; cytosine deaminase useful for converting non-toxic 5-fluorocytosine to the anticancer drug 5-fluorouracil; proteases, such as protease serratia, thermolysin, subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L), which are useful for converting prodrugs containing peptides into free drugs; D-alanyl carboxypeptidases, which are useful for converting prodrugs containing amino acid substitutes D; enzymes attached to carbohydrates such as β-galactosidase and neuraminidase useful for converting the glycosylated prodrugs into free drugs; ß-lactamase useful for converting drugs derived with ß-lactams into free drugs; and penicillins amidases, such as penicillin V amidase or penicillin G amidase, useful for converting the derivative drugs into their amino nitrogens with phenoxyacetic or phenylacetic groups, respectively, into free drugs. Alternatively, antibodies with enzymatic activity, also known as abzymes, can be used to convert the prodrugs of the invention into active free drugs (see, for example, Massey, Nature 328: 457-458 (1987)). The antibody-abzyme conjugates can be prepared as described herein, including for the delivery of the abzyme to a cell, tissue or organ expressing a2 integrin. Enzymes can be covalently bound to the a2 integrin antibodies by techniques well known in the art, including the use of the crosslinking heterobifunctional reagents mentioned above. Alternatively, fusion proteins that include at least one antigen binding region of an antibody to integrin a2 linked to at least a portion of active functionality of an enzyme can be constructed using recombinant DNA techniques that are well known in the art (see, for example, Neuberger et al., Nature 312: 604-608 (1984)). In certain embodiments of the invention it may be desirable to use an antibody fragment, instead of an intact antibody, for example, to increase penetration into the tissue or into a tumor. It may also be desirable to modify the antibody fragment in order to increase its serum half-life. This can be achieved by incorporating a rescue receptor epitope that binds to the antibody fragment, for example, by mutation of the appropriate region in the antibody fragment or by incorporating the epitope into a peptide tag which is then fused to the antibody fragment at either end or in the middle, for example, by DNA synthesis or of peptide (see, for example, W096 / 32478).
The covalent modifications of humanized antibodies against integrin a.2 can be carried out, for example, by chemical synthesis or by enzymatic or chemical adhesion of the antibody. Other types of covalent modifications of the antibody are introduced into the molecule by reacting the residues of the antibody target amino acids with an organic derivatizing agent that is capable of reacting with the selected side chains or with the N-terminal or C-terminal residues. Cysteinyl residues, for example, commonly react with α-haloacetates (and their corresponding amines), such as chloroacetic acid or chloracetamide, to generate carboxymethyl or carboxyamidomethyl derivatives. The cysteinyl residues are also derivatized by reaction with bromotrifluoroacetone, α-bromo-β- (5-imidazolium) propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, -chloromercuribenzoate, 2-chloromercury-4-nitrophenol or chloro-7-nitrobenzo-2-oxa-1,3-diazole. Histidyl residues, for example, are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide is also useful; the reaction is preferably carried out in 0.1 M sodium cacodylate at pH 6.0. The lysinyl and amino terminal residues, for example, are reacted with succinic or with other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the linisil residues. Other suitable reagents for derivatizing a-amino-containing residues include imidoesters such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloro borohydride, trinitrobenzene sulphonic acid, O-methylisourea, 2,4-pentanedione and a trans-aminase catalyzed reaction with glyoxylate. The arginyl residues, for example, are modified by reaction with one or more conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione and ninhydrin. Derivatization of the arginine residues requires that the reaction be carried out under alkaline conditions due to the high pKa of the guanidine functional group. In addition, these reagents can react with the lysine groups and with the epsilon-amino group of arginine. Tyrosyl residues, for example, are specifically modified with a particular interest in introducing spectral labels into the tyrosyl residues by reaction with aromatic diazonium or tetranitromethane compounds. Most frequently, N-acetyl imidazole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosyl residues are iodinated using 125 l or 131 l to prepare labeled proteins for use in radioimmunoassays. Groups on the carboxyl side, for example, aspartyl or glutamyl, are selectively modified by reaction with carbodiimides (RN = C = N-R '), where R and R' are different alkyl groups, such as 1-cyclohexyl-3- ( 2-morpholinyl-4-ethyl) carbodiimide or 1-ethyl-3- (4-azonia-4,4-dimethylpentyl) carbodiimide. In addition, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl by reaction with ammonium ions. The glutaminyl and asparaginyl residues are frequently deaminated to the corresponding residues of glutamyl and aspartyl, respectively. These residues are deamidated under neutral or basic conditions. The deaminated form of these residues falls within the scope of this invention. Other modifications include the hydroxylation of proline and lysine, the phosphorylation of the hydroxyl groups of the seryl or threonyl residues, the methylation of the a-amino groups of the side chains of lysine, arginine and histidine (TE Creighton, Proteins : Structure and Molecular Properties, WH Freeman &Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminal amino, and amidation of any C-terminal carboxyl group. Another type of covalent modification involves the chemical or enzymatic coupling of the glycosides with the antibody. These methods are advantageous in that they do not require production of the antibody in a host cell having glycosylation capabilities for O-glycosylation and N-glycosylation. Depending on the coupling mode used, the sugars can bind to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine and hydroxyproline, ( e) aromatic residues such as those of phenylalanine, tyrosine or tryptophan, or (f) the amide group of glutamine (see, for example, WO87 / 05330; Aplin and Wriston, CRC Crit. Rev. Biochem., Pp. 259-306 (1981)). The removal of any portion of carbohydrates present in the antibody can be carried out, for example, chemically or enzymatically. Chemical deglycosylation requires exposure of the antibody to the trifluoromethane sulfonic acid compound, or an equivalent compound. This treatment results in the dissociation of most or all of the sugars except the binding sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the antibody intact (see, for example, Hakimuddin, et al., Arch. Biochem Biophys, 259: 52 (1987), Edge et al., Anal. Biochem., 18: 131 (1981)). Enzymatic cleavage of the carbohydrate moieties in antibodies can be achieved by the use of a wide variety of endo and exoglycosidases, (see, for example, Thotakura et al., Meth. Enzymol, 138: 350 (1987)). Another type of covalent modification of the antibody includes binding of the antibody to a wide variety of non-proteinaceous polymers such as polyethylene glycol, polypropylene glycol or polyoxyalkylenes (see, for example, US Patent Nos. 4,640,835, 4,496,689, 4,301, 144 4,670,417, 4,791, 192 or 4,179,337). The isolated nucleic acids encoding a humanized antibody against the a2 integrin, as well as the vectors and host cells that make up the nucleic acid, and the recombinant techniques for the production of the antibody are described herein. For the recombinant production of the antibody, the nucleic acids encoding the antibody are isolated and inserted into a replicable vector for subsequent cloning (amplification of the DNA) or for expression. The DNA encoding the antibody was already isolated and sequenced using conventional methods (for example, using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the antibody). There are many vectors available. The vector components generally, but not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter and a transcription termination sequence. An antibody to integrin a2 can be produced recombinantly, including by means of a fusion polypeptide with a heterologous polypeptide, which is preferably a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. Preferably the selected heterologous signal sequence must be recognized and processed (eg, dissociated by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process a eukaryotic signal sequence (e.g., an immunoglobulin signal sequence), the signal sequence is replaced by a prokaryotic signal sequence including, for example, pectate lyase leaders ( as pelB), alkaline phosphatase, penicillinase, Ipp or heat-resistant enterotoxins II. For yeast secretion a yeast signal sequence can be used including, for example, the yeast invertase leader, a factor leader (including factor leaders to Saccharomyces and Kluyveromyces), or a leader of acid phosphatase factor , the leader of glucoamylase C. albicans or the signal described in WO90 / 13646. In the expression of mammalian cells, mammalian signal sequences as well as viral secretory leaders such as herpes simplex signal gD, are available and can be used. The DNA for said precursor region (for example, the signal sequence) is linked in the reading frame to the DNA encoding an antibody against integrin a2. Both the expression and the cloning vectors contain a nucleic acid sequence that allows the vector to replicate in one or more of the selected host cells. Usually, in the cloning vectors this sequence allows the vector to replicate independently of the chromosomal DNA of the host and includes origins of the replication or autonomous replication sequences. Said sequences are well known for various bacteria, yeasts and viruses. For example, the origin of replication of plasmid pBR322 is suitable for most gram-negative bacteria, the origin of plasmid 2μ is suitable for yeast and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells. In general, the origin of the replication component is not necessary for the expression vectors of mammalian cells (for example, the SV40 origin could typically be used only because it contains the early promoter). The expression and cloning vectors may contain a selection gene, also referred to as a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, eg, ampicillin, neomycin, methotrexate or tetracycline, (b) auxotrophic complement deficiencies, or (c) provision of critical nutrients not available from the middle of the complex (for example, the D-alanine racemase that codes for Bacilli genes). An example of a selection scheme uses a drug to stop the growth of a host cell. These cells are successfully transformed with a heterologous gene that produces a protein and confers resistance to the drug, thereby surviving the selection regime. Examples of this dominant selection use the drugs methotrexate, neomycin, histidinol, puromycin, mycophenolic acid and hygromycin. Another example of selectable markers suitable for mammalian cells are those that allow the identification of the cells competent to take the nucleic acid of the antibody against integrin a2, such as DHFR (for its acronym in English), thymidine kinase, metallothionein-l and -II, preferably genes of primate metallothionein, adenosine deaminase, ornithine decarboxylase, etc. For example, cells transformed with the DHFR selection gene are first identified by culturing all transformants in a culture medium containing methotrexate (Mtx), a competitive antagonist of DHFR. An appropriate host cell when wild-type DHFR is used is the Chinese Hamster Ovary (CHO) cell line, deficient in DHFR activity.
Alternatively, host cells (in particular wild-type hosts containing endogenous DHFR) transformed or cotransformed with DNA sequences encoding the a2 integrin antibody, wild-type DHFR protein and another selectable marker such as aminoglycoside 3'-phosphotransferase (APH) can be selected by cell growth in a medium containing a selection agent for the selectable marker, including an aminoglycoside antibiotic such as kanamycin, neomycin or G418. { see for example, U.S. Pat. No. 4,965,199). A suitable selection gene for use in yeast is the trp gene present in yeast plasmid YRp7 (Stinchcomb et al., Nature, 282: 39 (1979)). The trpl gene offers a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1 (see, for example, Jones, Genetics, 85:12 (1977 )). The presence of the trpl lesion in the genome of the host cell of the yeast thus provides an effective environment for detecting transformation by growth in the absence of tryptophan. Similarly, yeast strains deficient in Leu2 (ATCC 20,622 or 38,626) are complemented with known plasmids including the Leu2 gene. In addition, vectors derived from the circular plasmid pKD1 1.6 μ can be used for the transformation of Kluyveromyces yeasts. As an alternative, an expression system for large-scale production of recombinant calf chymosin was reported for K. lactis by Van den Berg, Bio / Technology, 8: 135 (1990). Stable multi-copy expression vectors for the secretion of mature recombinant human serum albumin by industrial Kluyveromyces strains have also been disclosed (see, for example, Fleer et al., Bio / Technology, 9: 968-975 (1991)). The expression and cloning vectors generally contain a promoter which is recognized by the host organism and is operably linked to the nucleic acid of the antibody against integrin a2. Promoters suitable for use with prokaryotic hosts include the arabinic promoter systems (eg, araB), phoA, β-lactamase promoters and lactose promoters, alkaline phosphatase, a tryptophan (trp) promoter system and hybrid promoters such as the promoter tac However, other known bacterial promoters are suitable. Promoters for use in bacterial systems will also contain a Shine-Dalgamo (S.D.) sequence operably linked to the DNA encoding the a2 integrin antibody. Promoter sequences are known for eukaryotes. Most eukaryotic genes have an AT-rich region that is located approximately 25 to 30 bases upward from the site where transcription was initiated. Another sequence found from 70 to 80 bases upwards from the start of the transcription of many genes is the CNCAAT region (SEQ ID NO: 115) where N could be any nucleotide. At the 3 'end of most eukaryotic genes is an AATAAA sequence (SEQ ID NO: 116) which could be the signal for the addition of the polyA tail to the 3' end of the coding sequence. Said sequences are suitably inserted into the eukaryotic expression vectors. Examples of promoter sequences suitable for use with yeast hosts include, but are not limited to, the promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexoquinase, pyruvate decarboxylase, phosphofructokinase, glucose. -6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase and glucokinase. Other promoters of yeasts, which are inducible promoters that have the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, cytochrome C iso, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase and enzymes responsible for the use of maltose and galactose. Vectors and promoters suitable for use in the expression of yeast are further described in EP 73,657. Yeast enhancers are also advantageously used with yeast promoters. Transcription of the antibody to integrin a2 from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, avian pox virus, adenovirus (such as Adenovirus 2), papilloma virus bovine, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis B virus or simian virus (SV40), from heterologous promoters of mammals, for example, the actin promoter or an immunoglobulin promoter, from heat shock promoters , as long as said promoters are compatible with the systems of the host cells. The first and last promoters of SV40 viruses are conveniently obtained as an SV40 restriction fragment that also contains the replication SV40 viral origin. The first immediate promoter of human cytomegalovirus is conveniently obtained as a Hinu restriction fragment. A system for expressing DNA in mammalian hosts using bovine papilloma virus as a vector is shown in the US Pat. .US. No. 4,419,446, and a modification of this system is described in U.S. Pat. No. 4,601, 978. { see also Reyes ei ai, Nature 297: 598-601 (1982) for the expression of human β-interferon cDNA in mouse cells under the control of a herpes simplex virus thymidine kinase promoter). Alternatively, the long terminal repeat of rous sarcoma virus can be used as the promoter. Transcription of DNA encoding an antibody to integrin a2 by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein and insulin). However, a eukaryotic cell virus enhancer is often employed. Some examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and the adenovirus enhancers. { see, also, for example, Yaniv, Nature 297: 17-18 (1982) about the enhancer elements for the activation of eukaryotic promoters). The enhancer can be divided in the vector at a position 5 'or 3' to the coding sequence of the antibody against integrin a2, but preferably it is located at the 5 'site from the promoter. Other gene regulation systems well known in the art (eg, inducible systems such as tetracycline and GeneSwitch ™ inducible systems) can be used to control the transcription of DNA encoding an antibody to integrin a2. Expression vectors used in eukaryotic host cells (yeast, fungi, insects, plants, animals, humans or nucleated cells of other multicellular organisms) will also contain the sequences necessary for the termination of transcription and to stabilize the mRNA. Such sequences are commonly available from the 5 ', and occasionally 3', untranslated regions of the eukaryotic or viral DNAs or cDNAs. These regions contain segments of nucleotides transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding an antibody to integrin a2. A useful transcription termination component is the polyadenylation region of bovine growth hormone (see, for example, WO94 / 11026 and the expression vector disclosed therein). Suitable host cells for cloning or expressing the DNA in the vectors herein are prokaryotic cells, yeasts or the highest prokaryotic cells as described above. Suitable prokaryotes for this purpose include eubacteria, including gram positive or gram negative organisms, for example, Enterobacteriaceae such as Escherichia, eg, E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, eg, Salmonella typhimurium, Serratia. , for example, Serratia marcescan and Shigella, as well as Bacilli as ß. subtilis and B. licheniformis, Pseudomonas as P. aeruginosa and Streptomyces. Suitable E. coli cloning hosts include E. coli 294 (ATCC 31,446), E. coli B, E. coli X1776 (ATCC 31, 537) and E. coli W3110 (ATCC 27,325). In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeasts are suitable cloning or expression hosts for the coding vectors of the alpha 2 integrin antibody. Saccharomyces cerevisiae, or common baking yeast, is the most commonly used among the lowest host eukaryotic microorganisms. However, many other genera, species and strains are usually available and useful, such as Schizosaccharomyces pombe; Kluyveromyces hosts including K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans or K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces as Schwanniomyces occidentalis; and filamentous fungi including hosts Neurospora, Penicillium, Tolypocladium or Aspergillus such as A. nidulans or A. niger. Host cells suitable for the expression of the glycosylated integrin α2 antibody are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains and variants have been identified in addition to their corresponding permissive host host insect cells such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly) and Bombyx morí. A wide variety of viral strains for transfection are publicly available, for example, the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx morí NPV, and such viruses can be used, in particular for transfection of Spodoptera cells frugiperda Cell crops of cotton, corn, potato, soybean, petunia, tomato and tobacco plants can also be used as hosts. However, the greatest interest has been in vertebrate cells and the spread of vertebrate cells, including a wide variety of mammalian cells, has become a routine procedure.
Examples of useful mammalian host cells include: a monkey kidney CV1 cell line transformed by SV40 (eg, COS-7, ATCC CRL 1651); an embryonic kidney human line 293 or 293 cells subcloned for growth in suspension culture. { see for example, Graham ei ai, J. Gen Virol. 36: 59 (1977)); baby hamster kidney cells (eg, BHK, ATCC CCL 10); Chinese hamster ovary cells (CHO), including CHO cells lacking DHFR (see, for example, DHFR Uriaub ei ai, Proc Nati Acad Sci USA 77: 4216 (1980)); mouse Sértoli cells (eg, TM4, Mather, Biol. Reprod. 23: 243-251 (1980)); monkey kidney cells (e.g., CV1 ATCC CCL 70); African green monkey kidney cells (e.g., VERO-76, ATCC CRL-1587); human cervical carcinoma cells (e.g., HELA, ATCC CCL 2); canine kidney cells (e.g., MDCK, ATCC CCL 34); buffalo rat liver cells (e.g., BRL 3A, ATCC CRL 1442); human lung cells (e.g., W138, ATCC CCL 75); human liver cells (e.g., Hep G2, HB 8065); human breast tumor (e.g., MMT 060562, ATCC CCL51); TRI cells (see, for example, Mather ei ai, Annals N.Y Acad. Sci. 383: 44-68 (1982)); MRC 5 cells; FS4 cells; or a human hepatoma line (for example, Hep G2). The host cells are transformed with an expression as described above or with cloning vectors for the production of the antibody against integrin a2 and are cultured in a conventional nutrient medium as appropriate to induce the promoters, select the transformants and / or amplify the genes that encode the desired sequences. The host cells used to produce an antibody to integrin a2 could be cultured in a wide variety of media. Commercially available media such as Ham's F10 (Sigma), Essential Minimum Medium ((MEM), (Sigma), RPMI-1640 (Sigma) and Dulbecco's Modified Eagle Medium ((DMEM), Sigma) are suitable for culturing the cells In addition, any of the means described in Ham ei ai, Meth. Enz. 58: 44 (1979), Barnes ei ai, Anal. Biochem. 102: 255 (1980), U.S. Patent Nos. 4,767,704 4,657,866, 4,927,762, 4,560,655, or 5,122,469, WO90103430, WO 87/00195, or US Patent Re. No. 30,985 can be used as culture media for the host cells, either of which can be supplemented according to is necessary with hormones and / or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium and phosphate), buffer solutions (such as HEPES), nucleotides (such as adenosine and thymidine) ), antibiotics (such as the drug GENTAMYCIN ™), trace elements (defined as inor ganoics usually present in the final concentrations on the micromolar scale) and glucose or an equivalent energy source. Any other necessary supplements may also be included at the appropriate concentrations that are known to those skilled in the art. The culture conditions, such as temperature, pH and the like, are selected by those skilled in the art, including those culture conditions previously used with the host cell selected for expression. Antibodies to integrin a2 can be purified from cells, including microbial or mammalian cells using, for example, protein A chromatography, ion exchange chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis and / or chromatography of affinity. The suitability of protein A as an affinity ligand depends on the species and the isotype of any Fc immunoglobulin domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human heavy chains? 1,? 2, or? 4 (see, for example, Lindmark ei ai, J. Immunol. Meth. 62: 1-13 (1983)). The G protein is useful for the mouse and human? 3 isotypes (see, for example, Guss et al, EMBO J. 5: 1516-1517 (1986)). The matrix to which the affinity ligand is connected most frequently is agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly (styrenedivinyl) benzene allow for faster flow rates and shorter processing times than can be obtained with agarose. Where the antibody is composed of a CH3 domain, the Bakerbond ABX ™ (J.T. Baker, Phillipsburg, N.J.) is useful for purification. The purification of the protein may include one or more of the following techniques such as fractionation on an ion exchange column, ethanol precipitation, reverse phase HPLC, silica gel chromatography, SEPHAROSE ™ heparin chromatography, chromatography on an ionic or cation exchange resin (eg, a polyaspartic acid column), chromatofocusing, SDS-PAGE, ammonium sulfate precipitation and / or hydrophobic interaction chromatography. For example, it may be useful to follow any purification step to subject the mixture including the antibody of interest and the contaminants to a low pH hydrophobic interaction chromatography using an elution buffer with a pH between 2.5 and 4.5, made from preference at low salt concentrations (eg, from about 0 to 0.25M salt). Formulations of an antibody to a2-integrin, including those for therapeutic administration, are prepared for storage by mixing the antibody containing the desired degree of purity with optional, physiologically acceptable carriers, diluents, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), as lyophilized formulations or aqueous solutions. Acceptable carriers, diluents, excipients or stabilizers are non-toxic to the receptors in the doses and concentrations used, and include buffer solutions such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride, haxamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, paraben alkyls such as paraben methyl or propyl, catechol, resorcinol, cyclohexanol, 3-pentanol, and m- cresol); low molecular weight polypeptides (less than 10 residues); proteins, such as serum albumin, gelatins or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides or other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zinc protein complexes); and / or non-ionic surfactants such as TWEEN ™, PLURONICS ™ or polyethylene glycol (PEG). The antibody formulation may also contain more than one active compound for the particular indication being treated, preferably those with complementary activities that are not inversely affected with each other. It may be desirable to use an antibody against integrin a2 in addition to one or more agents that are currently employed to prevent or treat the disorder in question. In addition, it may be desirable to also offer an immunosuppressant agent. Said molecules are suitably presented in combination with amounts that are effective for the purpose sought. The active ingredients can also be entrapped in a microcapsule prepared, for example, by coacervation or interfacial polymerization techniques, for example, hydroxymethylcellulose or gelatin microcapsule and poly- (methylmethacrylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles or nanocapsules) or in macroemulsions. Such techniques are disclosed, for example, in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). The formulations that will be used for the in vivo administration are preferably sterile. This is easily achieved, for example, by filtration through sterile filtration membranes. It is possible to generate sustained release preparations. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are found as shaped particles, e.g., films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl methacrylate) or poly (vinylalcohol)), polylactides (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and ethyl -L-glutamate, non-degradable ethylene-vinyl acetate, degradable copolymers of lactic acid-glycolic acid such as Lupron Depot ™ (injectable microspheres composed of copolymer of lactic acid-glycolic acid and leuprolide acetate and poly-D - (-) acid) -3-hydroxybutyric acid While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid allow the release of molecules for more than 100 days, certain hydrogels release proteins for shorter periods, when the encapsulated antibodies remain in the body for a long time. can be denatured or added as a result of exposure to humidity at 37 ° C, which results in the loss of biological activity and possible Changes in immunogenicity Rational strategies for stabilization can be designed, depending on the mechanism involved. For example, if it is discovered that the aggregation mechanism is an intermolecular S-S bond formation through thio-disulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling the moisture content, using appropriate additives and developing specific polymer matrix compositions. The a2 integrin antibodies can be used as affinity purification agents. In this process, the antibodies are immobilized on a solid phase such as a Sephadex resin or filter paper, using methods well known in the art. The immobilized antibody is contacted with a sample containing the a2ß1 integrin protein (or a fragment thereof) to be purified, and subsequently the support is washed with a suitable solvent that will remove substantially all the material in the sample except the integrin protein. a2ß1, which is bound to the immobilized antibody. Finally, the support is washed with another suitable solvent, such as glycine buffer at pH 5.0, which will release the a2ß1 integrin protein from the antibody. Antibodies to integrin a2 may also be useful in diagnostic assays for the a2β1 integrin protein, for example, by detecting its expression in specific cells, tissues or serum. For diagnostic applications, the antibody will usually be labeled with a detectable portion. There are numerous labels available that can usually be grouped into the following categories of radioisotopes, fluorescent labels and enzyme-substrate labels. Radioisotopes such as 35 S, 14 C, 125 I, 3 H and 131 I are useful labels. The antibody can be labeled with the radioisotope, for example, using the techniques described in Current Protocols in Immunology, Volumes 1 and 2, Coligen ei ai, Ed. Wiley-Interscience, New York, N.Y., Pubs. (1991) and radioactivity can be measured, for example, using a scintillation counter. Fluorescent labels such as the rare chelates on earth (chelated europium) or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, lysamine, phycoerythrin and Texas red are also useful. Fluorescent tags can be conjugated to the antibody, for example, using the techniques disclosed in Current Protocols in Immunology, supra. The fluorescence can be quantified, for example, using a fluorimeter. Various enzyme-substrate labels are also useful (see, for example, U.S. Patent No. 4,275,149 for a review). The enzyme generally catalyzes a chemical alteration of the chromogenic substrate that can be measured using various techniques. For example, the enzyme can catalyze a color change in a substrate, which can be measured spectrophotometrically. Alternatively, the enzyme may alter the fluorescence or chemiluminescence of the substrate. Techniques for quantifying a change in fluorescence are described above. The chemiluminescence substrate is excited electronically by means of a chemical reaction and could emit a light that can be measured (for example, using a chemiluminometer) or donate energy to a fluorescent acceptor. Examples of enzymatic labels include luciferases (e.g., firefly luciferase and bacterial luciferase); U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydroftalazinedione, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (eg, glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like. Techniques for conjugating enzymes to antibodies are described, for example, in O'Sullivan et al., Methods for the Preparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, in Methods in Enzym. (ed J. Langone &H. Van Vunakis), Academic press, N.Y., 73: 147-166 (1981). Examples of enzyme-substrate combinations include, for example: (i) horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate, wherein hydrogen peroxidase oxidizes to a dye precursor (eg, orthophenylene diamine (OPD) or 3,3 ', 5,5'-tetramethyl benzidine hydrochloride (TMB)); (ii) alkaline phosphatase (AP) with para-Nitrophenyl phosphate as a chromogenic substrate; and (ii) β-D-galactosidase (β-D-Gal) with a chromogenic substrate (eg, p-nitrophenyl-β-D-galactosidase) or a fluorogenic substrate 4-methylumbelliferyl-β-D-galactosidase. There are many other enzyme-substrate combinations available to those skilled in the art (see, for example, US Patent Nos. 4,275,149 and 4,318,980 for a general review). Sometimes a label is indirectly conjugated with the antibody. Those skilled in the art will know several techniques to achieve this. For example, the antibody can be conjugated to biotin and to any of the three broad categories of the aforementioned labels with avidin or vice versa. Biotin binds selectively to avidin and, therefore, the label can be conjugated to the antibody in this indirect manner. Alternatively, to achieve indirect conjugation of the label with the antibody, the antibody can be conjugated with a small hapten (eg, digoxin) and one of the different types of labels mentioned above can be conjugated with an antibody against the hapten (for example, antibody against digoxin). Therefore, indirect conjugation of the label with the antibody can be achieved. An antibody to integrin a2 does not need to be labeled, and its presence can be detected using a labeled antibody that binds the antibody against integrin a2. Antibodies to integrin a2 can be used with any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays (see, for example, Zola, Monoclonal Antibodies: A Manual of Techniques, pp.147-158 (CRC Press, Inc. 1987)). Competitive binding assays depend on the ability of a labeled standard to compete with the analyte in the test sample to bind with a limited amount of the antibody. For example, the amount of a2β1 integrin protein in the test sample is inversely proportional to the amount of standard that is bound to the antibodies. To facilitate the determination of the amount of standard that remains attached, the antibodies are generally insolubilized before or after competing, so that the standard and the analyte that are bound to the antibodies can be conveniently separated from the standard and the analyte. which are still united. Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein that will be detected. In a sandwich assay, the analyte in the test sample is bound by a first antibody which is immobilized on a solid support, and from there a second antibody binds to the analyte, whereby an insoluble three-part complex is formed ( see, for example, U.S. Patent No. 4, 376, 110). The second antibody can also be labeled with a detectable portion (e.g., direct sandwich assays) or can be measured using an antibody to the immunoglobulin that is labeled with a detectable portion (e.g., indirect sandwich assay). For example, one type of sandwich assay is an ELISA, in which case the detectable portion is an enzyme. For immunohistochemistry a sample of tissue, including a tumor sample, may be fresh, frozen or may be embedded in paraffin and fixed with a preservative such as formalin.
The a2 integrin antibodies can also be used for in vivo diagnostic assays. Generally, the antibody is labeled with a radionuclide (11 ln, "Te, 1 C, 131l, 125l, 3H, 32P or 35S) such that tissue, for example a tumor, can be localized using immunoscintigraphy. convenience, an antibody to integrin a2 can be provided in a kit, such as a packaged combination of reagents in predetermined quantities with instructions, including some to perform a diagnostic assay.When the antibody is labeled with an enzyme, the kit will include the substrates and cofactors required by the enzyme (e.g., a substrate precursor that provides the detectable chromophore or flourophorr.) Other additives such as stabilizers, buffer solutions (e.g., a block buffer solution or a buffer solution) may be included in the kit. of lysis) and others like that The relative amounts of the different reagents provided in the equipment can vary r amply, for example, to provide solution concentrations of the reagents that substantially optimize the sensitivity of the assay. The reagents can be provided as dry powders, generally lyophilized, including excipients, for example, which upon dissolution will provide a reactive solution having the appropriate concentration. An antibody to integrin a2 can be used to treat various disorders associated with integrin a2ß1 as described herein. The a2 integrin antibody is administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary or intranasal. If desired for local immunosuppressive treatment, intralesional administration of the antibody (including pumping or other contact of the graft with the antibody prior to transplantation) is used. Parenteral administration includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. In addition, the a2 integrin antibody is suitably administered by pulse infusion, for example, with decreasing doses of the antibody. Preferably the dose is administered by means of intravenous or subcutaneous injections. This may depend in part on whether the administration will be brief or chronic. For the prevention or treatment of the disease, the appropriate dosage of the antibody will depend on the type of disease to be treated, as defined above, of the severity and course of the disease, whether the antibody against integrin a2 is administered with preventive or therapeutic purposes, previous therapy, the patient's clinical history and the response to the antibody, as well as the criteria of the treating physician. The antibody is suitably administered to the patient once or in a series of treatments. Depending on the type and severity of the disease [from about 1 μg / kg to about 15 mg / kg or from about 0.05 μg / kg to about 20 mg / kg] of antibody is an initial dosing candidate for the administration to the subject in, for example, one or more administrations separately, or by means of continuous infusion. A typical daily dosage may vary [from about 1 μg / kg to about 100 mg / kg] or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, treatment is sustained until the desired suppression of disease symptoms occurs. However, other dosage regimens may also be useful. The progress of this therapy is monitored closely by persons skilled in the art. An antibody composition against integrin a2 will be formulated, dosed and administered in a manner consistent with good medical practice. Factors that should be considered in this context include the particular disorder that is being treated, to the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the schedule of administration, the results of the pharmacological and toxicity studies and other factors known to practitioners of medicine. A therapeutically effective amount of the antibody that will be administered will be determined by considering all factors and will be the minimum amount necessary to prevent, ameliorate or treat a disorder associated with the a2ß1 integrin. Said amount will preferably be below the amount that is toxic to the host or that makes the host significantly more susceptible to infections.
The a2 integrin antibody need not be, although it can be optionally formulated, co-administered or used as an adjunctive therapy with one or more agents currently used to prevent or treat the disorder in question. For example, in rheumatoid arthritis, the antibody can be administered together with a glucocorticosteroid, Remicaid® or with any approved treatment for rheumatoid arthritis. For multiple sclerosis, the antibody can be administered together with an interferonß, Avonex, Copaxon or other approved therapies for the treatment of signs and symptoms of multiple sclerosis. For transplants, the antibody can be administered concurrently together or separately from an immunosuppressive agent as defined above, such as cyclosporin A, to modulate the immunosuppressive effect. Alternatively, or in addition to, α2β1 integrin antagonists can be administered to the mammal suffering from a disorder associated with integrin a2β1. The effective amount of these other agents depends on the amount of the a2 integrin antibody present in the formulation, the type of the disorder or the treatment, and other factors such as those mentioned above. In general, these agents are used in the same dosages and with the administration routes mentioned hereinabove or from 1 to 99% of the dosages used there. A production article containing materials, including an antibody to integrin a2, useful for the treatment of the disorders described above is provided. The production item includes a container and a label. Suitable containers include, for example, bottles, flasks, syringes and test tubes. The containers can be formed by very diverse materials such as glass or plastic. The container stores a composition that is effective in treating the condition and could have a sterile access port (for example, the container can be an intravenous solution bag or a bottle with a lid that can be punctured by means of an injection needle. hypodermic). The active agent in the composition is an antibody against integrin alpha 2. The label of, or associated with, the container indicates that the composition is used to treat the chosen condition. The production article could include a second container that stores a pharmaceutically acceptable buffer, such as a phosphate buffered saline solution, Ringer's solution or dextrose solution. It could also include other desirable materials from the commercial and user's point of view, including other buffer solutions, diluents, filters, needles, syringes and equipment brochures with instructions for use. The principles described above have been applied, for example, to the antibody against integrin a2 secreted by the hybridoma BHA2.1 (Hangan ei ai, Cancer Res., 56 (13): 3142-9 (1996)). This antibody binds to the human and rat a2ß1 integrin, but does not bind to its murine counterpart. The antibody thus produced by the hybridoma is referred to herein as TMC-2206 and is commercially available with Chemicon (now part of Millipore, catalog number MAB1998). Chimeric variants, including humanized, of TMC-2206 were produced and subjected to in vitro analysis. In vivo studies were also performed using the TMC-2206 antibody or a similar antibody, including one capable of recognizing the murine a2ß1 integrin. The following examples are offered by way of illustration and in no way are limiting. The disclosures of all citations of the specification are incorporated herein by reference.
EXAMPLE 1Antibodies with specificity for the a2ß1 integrin were designed and prepared. The previously unknown sequences of the variable regions of a murine antibody designated TMC-2206 secreted by hybridoma BHA2.1 were determined as described herein. The VH and VL cDNAs were cloned from the mRNA of BHA2.1 hybridoma cells by RT-PCR using a set of primers corresponding to the amino acids at the N-terminus of the heavy chain variable region (VH) or light chain (VL) of murine, and a second set of primers corresponding to the respective constant regions of the heavy chain? 1 and light chain K. The sequence that was determined from the cDNA that had been synthesized from the mRNA isolated according to standard methods is described herein. The cytoplasmic mRNA was isolated from approximately 1 million (1 x 106) of BHA2.1 hybridoma cells expressing TMC-2206 using standard molecular techniques for those skilled in the art. To isolate poly-A-mRNA, the cells were lysed in 5M guanidinium thiocyanate, mixed with oligo (dT) cellulose (Ambion, TX) and incubated at room temperature with gentle agitation for 60 minutes. Poly-A-RNA bound to oligo (dT) cellulose was pelleted, washed and then applied to a washing column by means of centrifugation (Ambion, TX). The column was centrifuged at 3000 xg for 1 minute, then the RNA was eluted with 200 μL of 10 mM Tris, 1 mM EDTA (TE) buffer, pH 8.0, and precipitated with 0.1 volume of 5 M ammonium acetate. (NH4Ac) and 2.5 volumes of 100% ethanol at -20 ° C. The RNA was converted into pellets by means of centrifugation, and then it was dried and dissolved in water treated with DEPC. CDNAs were synthesized from mRNA isolated from BHA2.1 through reverse transcription initiated with primers based on the heavy-chain (VH) or light (NL) variable region (VL) of murine, and a second set of corresponding primers to the constant regions of the heavy chain? 1 or light K of murine. The sequence of the BHA2.1 antibody was unknown, therefore, commercial primers were used to degenerate the antibody for the N-terminal variable regions of murine heavy and light chains (Light Primer mixture, # 27- 1583-01 and mixing Heavy Primer, # 27-1586-01, from Amersham Biosciences) as shown in Table 1. It is reported that these primers encompass the heterogeneous amino acid composition at the N-terminus of murine heavy and light chains, respectively. The RT-PCR reactions (Qiagen RT equipment) were configured in the following way: 0.5 μg of mRNA, 10 μL of 5? RT buffer solution, 2 μL of 10 mM dNTP mixture, 5 μL of each 10 mM primer solution and 2 μL of enzyme mixture in 50 μL of total volume. The reaction was initiated with reverse transcription at 50 ° C for 30 minutes followed by a PCR activation step at 95 ° C for 15 minutes and ended with a PCR program suitable for the degenerating priming mixtures used to amplify the variable regions of the light chains and heavy: 94 ° C for 30 seconds, 56 ° C for 30 seconds and 72 ° C for 1 minute for 28 cycles with a final extension run of 10 minutes at 72 ° C. Subsequent PCR reactions used the primer pairs listed in Table 2, which were synthesized by Retrogen (San Diego, CA). All primers are listed as 5 'to 3'.
TABLE 1TABLE 2PCR products of approximately 350 bp in length were obtained both for VH and for VL. These PCR products were recovered from a 1% agarose gel, cloned into the cloning vector pCR2.1-TOPO (Invitrogen, CA) and sequenced. Sequencing was performed on a CEQ DNA sequencer using forward and backward M13 primers (Invitrogen, CA). Plasmid DNA was made from 1.5 mL of bacterial cultures using Qiagen equipment according to the manufacturer's instructions. Approximately 300 ng of DNA were used for each PCR sequencing reaction, generally in a volume of μL. The DNA was denatured at 96 ° C for 2 minutes and then mixed with the sequencing primer at a final concentration of 0.3 μM. Four μL of Quick Start Master Mix DTCS (Beckman Coulter, Fullerton, CA) were added to the mixture and sequencing continued for 30 cycles: 96 ° C for 20 seconds, 50 ° C for 20 seconds and 60 ° C for 2 minutes . The sequencing reactions were precipitated with ethanol in the presence of sodium acetate (NaAc), EDTA and glycogen. The pellet was washed twice with 70% ethanol, dried with air and resuspended in 20 μL of the sample loading solution (provided in the kit). Eight individual VH and VL clones were sequenced by standard techniques and the deduced amino acid sequences of VH (SEQ ID NO: 21) and VL (SEQ ID NO: 19) are shown in Tables 3 and 4, respectively. The sequences obtained from the eight clones were identical except for the first or the first two amino acids. In clones VL, Glu-Asn or Gln-Phe occurred with the same frequency. In clones VH, Gln or Glu occurred with the same frequency. The sequences were checked against the BLAST NCBI protein database (http://www.ncbi.nih.gov/BLAST/, Ye ei al., Nucleic acids Res., Jul 1: 34 (Web Server Issue): W6 -9). All sequences along with the query (for example VH or VL of TMC-2206) were aligned by CLUSTALW (multiple sequence alignment) at http: //clustalw.qenome.ip/ (Aiyar, Methods Mol Biol., 132: 221 -41 (2000)). The cloned inserts showed the best match with the heavy (lgG1) and light (K) murine chains, which was the expected isotype. The sequences of the cloned VH and VL regions suggested leader and flanked sequences of similar constant regions, which were used to design more accurate primers in order to clone complete variable regions of both heavy and light TMC-2206 from the hybridoma mRNA . All primers were synthesized by Retrogen (San Diego, CA). The primer pair, VHL for CCATGGCTGTCTTGGGGCTGCTCTTCT (SEQ ID NO: 14) and HC-rev GGGGCCAGTGGATAGAC (SEQ ID NO: 15; mouse Fc? CH1), was used to re-clone the heavy chain variable region and the primer pair, VLL for CCATGGATTTTCAAGTGCAGATTTTCAG (SEQ ID NO: 16) and LCK-rev GTTGGTGCAGCATCAGC (SEQ ID NO: 17), was used to re-clone the light chain variable region of the hybridoma mRNA using the same PCR conditions delineated above. The sequencing of the products confirmed that the identity of the first two residues in TMC-2206 VL was L1-Q and L2-F and that the identity of the first two residues of the heavy chain was H1-Q and H2-V. The remaining nucleotide sequences were identical to those cloned using degenerate primer mixtures.
TABLE 3CDTABLE 4roThe cloned VL region had a length of 106 amino acids and the VH had 119 amino acids. As shown in Tables 3 and 4, there are three CDRs (CDR1-3) and four frameworks (FW1-4) in the heavy (VH) and light (VL) variable regions. The frameworks and CDRs were identified based on the Kabat numbering system (Kabat eí ai, 1983) except that the CDR1 of the heavy chain was identified by the AbM definition of Oxford Molecular which covers residues from 26 to 35. The modeling software of AbM antibodies from Oxford Molecular(http://people.cryst.bbk.ac.uk/~ubcg07s/, Martin ei ai, Proc. Nati Acad. Sci. USA, 86, 9268-9272 (1989); Martin ei ai, Methods Enzymol., 203 , 121-153 (1991), Pedersen ei ai, Immunomethods, 1, 126 (1992), and Rees ei ai, In Sternberg MJE (ed.), Protein Structure Prediction, Oxford University Press, Oxford, 141-172. )) combines the CDR numbering systems of Kabat and the hypervariable Chothia region to define the CDRs. For the consistency of the numbering, the insertions of the regions of the framework and the CDRs relative to the standards are named according to the position of the residue followed by an alphabetical sequence (for example, residues 82A, 82B, 82C are inserted between residues 82 and 83 in the heavy chain as shown in Table 3). Both the VH and the VL sequence have relatively short CDR3. There is a potential glycosylation site (Asp-Ser-Ser, NSS) within the CDR1 of the cloned light chain. This is consistent with the observation that the light chain of TMC-2206 has a molecular weight of 29 kD by SDS-PAGE that can be changed by treatment of endoglycosidase at 25 kD (molecular weight typical of light chains of antibody). To confirm that the cloned sequences represented the bioactive VH and VL of the TMC-2206 antibody, the antibody purified from the hybridoma medium was subjected to N-terminal peptide sequencing by Edman degradation. The deduced amino acid sequence of the VH and VL clones indicated the possible presence of an N-terminal glutamine in each one, which generated the possibility that a blockade of the N-terminus arose from the cyclization of the N-glutamine residue. -terminal to give rise to pyroglutamate (pGlu). Therefore, to remove any potentially cyclized terminal glutamine, the protein was subjected to the digestion of pyroglutamate aminopeptidase using a thermophilic heat tolerant Pyrococcus furiosus enzyme before subjecting the heavy and light chains to N-terminal peptide sequencing. The purified pyroglutamate aminopeptidase (0.01 U) from Pyrococcus furiosus (Sigma, St. Louis, MO) was reconstituted in 50 μL of digestion buffer (50 mM sodium phosphate, pH 7.0, 1 mM EDTA and 10 mM dithiothreitol (DTT)). A preparation of TMC-2206 was digested using a 1: 100 molar ratio of pyroglutamate aminopeptidase: protein at 95 ° C for 1 hour. The digested proteins were resolved using a standard 10% SDS-PAGE gel (Tris-glycine, BioRad Laboratories, Hercules, CA) with sodium mercaptoacetate (0.1 g in 150 mL of run buffer) in the upper compartment. The gel was then transferred to the PVDF membrane Immobilon P (Millipore, Billerica, MA) in transfer buffer (10 mM CAPS, pH 10.5, 0.5 g / L DTT and 15% methanol) at 250 mAmp for 1 hour. The stain was stained using a fresh solution of 0.1% Ponceau S in 1% acetic acid for 1 minute followed by destaining in 1% acetic acid. The blot was subjected to peptide sequencing where it was discovered that 20 of the first 21 amino acids of the light chain N-termini were successfully sequenced and showed an exact identity with the deduced sequence of the peptide that was obtained by cloning. This confirmed that the identity of the first amino acid in the cloned VL was Glu. The VH digested in pyroglutamate aminopeptidase failed to generate any data of the peptide sequence.
EXAMPLE 2Chimeric antibodies with specificity for integrin a2ß1 were designed and prepared, including mouse-human chimeric antibodies. The VH and VL regions of cloned TMC-2206 as described in Example 1 were used to design and prepare heavy and light chimeric chains, respectively, using standard cloning techniques. { see, for example Molecular Biology Manual by Sambrook and Russell, 2001). The heavy and light chains were cloned with the introduction of restriction sites in the following manner. The primers, TMC-2206-r5 'CCCGAATTCACAGGTGCAGTTGAAGGAGTCA SEQ ID NO: 22) and TMC-2206-r3' CGGGATCCTTAGGATCATTTACCAGGAGAG TGGGA (SEQ ID NO: 23), were used to clone the heavy chain TMC-2206 by RT-PCR from the mRNA of the hybridoma BHA2.1 and the primers TMC-2206-k5 'CCCGAATTCACAATTTGTTCTCACCCAGTCT (SEQ ID NO: 24) and TMC-2206-k3' CGGGATCCTTATCTCTAACACTCATTCCTGTTGAA (SEQ ID NO: 25) were used to clone the light chain of TMC-2206. These primers introduced EcoRI and Bam \? \ Sites at the 5 'and 3' ends, respectively, to allow cloning of the heavy and light chains cloned into the mammalian expression vectors plRES2-GFP and plRES2-Ds Red (Clontech, catalog numbers 632306 and 632420), respectively. Both vectors were designed to transport a leader sequence lg? METDTLLLVWLLLWVPGGSTGD (SEQ ID NO: 26). To isolate the mRNA, approximately 1 million hybridoma cells expressing TMC-2206 were pelleted at low speed (10 minutes at 800 rpm), washed with PBS and lysed with 1 mL of Trizol (Invitrogen, CA). After shaking vigorously at the vortex, the cell suspension was extracted with 0.2 mL of chloroform and after centrifugation (14,000 rpm for 5 minutes at 4 ° C), the supernatant was transferred to a new tube where the RNA was precipitated by mixing it. with 0.5 mL of isopropanol followed by centrifugation (14,000 rpm for 10 minutes at 4 ° C). The RNA pellet was washed with 1 mL of 75% ethanol and dissolved in 50 μL of H20 treated with DEPC.
The RT-PCR reaction (Qiagen RT equipment) was performed as described above using 0.5 μg of RNA, 10 μL of 5x RT buffer, 2 μL of 10 mM dNTP mixture, 5 μL of each 10 mM primer solution and 2 μL of enzymatic mixture in a total volume of 50 μL. The PCR products were digested with restriction enzymes EcoRI and ßamHI and the purified fragments of 1% agarose gel were then ligated into the EcoRI / ßamHI sites of the vectors plRES2-GFP (heavy chain) and plRES2-Ds Red (light chain ). Subsequent sequencing of the variable regions confirmed that no mutations were introduced by means of RT-PCR. pCI-neo (Promega, catalog number E1841) was chosen as the expression vector for cloning chimeric antibody molecules, including humanized, based on, or derived from, TMC-2206 as described below. To reduce the possibility of introducing mutations in the constant regions through PCR, cloning cassettes were prepared for VH and for VL. First, the DNA that encoded a lg leader? (SEQ ID NO: 26) was cloned into the Xho \ and EcoRI cloning sites of pCI-neo using the oligonucleotides lg? -S (SEQ ID NO: 27) and lg? -AS (SEQ ID NO: 28) listed in Table 2, which were annealed together and then ligated directly into digested pCI-neo from Xho \ -EcoR using T4 ligase. This provided the parent vector for all subsequent cloning steps. From this, two expression cassettes were created: one to clone in the VH regions adjacent to a human lgG1 Fc (hFc) and the second to clone in the VL regions upward from the constant region of the human kappa chain (hKc ). No EcoRI, Xba \, Hind \\\ or SalI sites were found in the sequences of human IgG1 Fc (hFc) or in the constant region of the kappa chain (I did), therefore, any of these restriction sites could be introduced at the 5 'end of the constant regions to facilitate cloning. Sa / I was chosen as the cloning site because this would reduce the number of amino acid changes at the junction of the variable region and the constant region. For the heavy chain chimera, the introduction of a Sal site into the binding of human mouse-Fc VH was achieved without causing any change in the amino acid sequence. First, a VH EcoRI-Sall fragment was made by PCR using the primer pairs TMC-2206-r5 '(SEQ ID NO: 22) and TMC2206VH-hlgG1 / 4Fc-Sall (SEQ ID NO: 29) shown in the Table 2 to introduce a restriction site I came out at the 3 'end of the murine VH sequence using the heavy chain cloned in the pIRES-GFP vector as a template. Human IgG1 Fc was obtained from the amplification of the DNA of clone IMAGE 20688 (Invitrogen, catalog number 4764519) using the primers shown in Table 2, hlgG1 / 4Fc-Sall-F (SEQ ID NO: 30) and hlgG1 / 4Fc-Notl-R (SEQ ID NO: 31). The two PCR products were digested with EcoRI / Sa / l and Sa / l /? / Oil, respectively, purified and ligated with the vector pCI-neo-Ig? digested with EcoRI /? / oíl. The resulting vector was named pCI-neo-lg? -TMCVH-hFc.
For the light chain chimera it was not possible to design a Sal site without changing the two amino acids at the VL-C, E105D and L106I junction. This was achieved by generating a PCR product using the primers shown in Table 2, TMC-2206-k5 '(SEQ ID NO: 24) and TMC2206VL-hKc-Sa / l (SEQ ID NO: 32) to amplify the 2206VL region of the plasmid, plRES-DsRed2-TMC-2206LC mentioned above. The PCR product was digested with EcoRI / Sa / l separated on a 1% agarose gel, purified with an extraction gel kit (Qiagen) and ligated with the constant region of the human IgG light chain. amplified from clone IMAGE # 4704496 (ATCC) using the primers hKc-Sa / lF (SEQ ID NO: 33) and hKc-γ / o-l-R (SEQ ID NO: 34) in addition to the vector described above, pCL-neo - lg ?. The resulting plasmid was named pCI-neo-lg? -TMC2206VL-hKc. To evaluate if the change of the two amino acids in the conjunction VL-? C would impact the activity of the antibody, a second light chain chimera was constructed that encoded the parental sequence of the amino acid of the light chain chimeric plasmid. First, the constant VL and human kappa regones were amplified with the primer pair TMC-2206VLwt-hKc-R and TMC-2206-k5 '(SEQ ID NOs: 36 and 24) and the primer pair TMC-2206VLwt-hKc-F and hKc -? / oil-R (SEQ ID NOs: 35 and 34) respectively, using the above vector plRES2-DsRed2-lgk-TMC2206LC as a template. Second, splicing by overlap extension PCR (Horton ei ai, Gene 77 (1): 61-8 (1989)) with primers TMC-2206-k5 '(SEQ ID NO: 24) and hKc -? / OIL-R (SEQ ID NO: 34) was performed to link the two products, and the final PCR product was digested and cloned into pCI-neo-Ig ?. To confirm that the mouse-human cloned chimeric antibody included the same specificity as the monoclonal antibody TMC-2206 secreted by the BHA2.1 hybridoma, the mouse-human chimeric antibody was expressed in 293F cells using a transient transfection methodology with a transfection mixture composed of equal parts of DNA / OptiMEM and 293fectin / OptiMEM (Invitrogen). Each solution was made with OptiMEM preheated at room temperature. The DNA / OptiMEM mixture contained 20 μg of the heavy chain expression (HC) plasmid, 20 μg of the light chain expression (LC) plasmid and OptiMEM for a total volume of 1.3 mL. The effectin / OptiMEM mixture contained 53 μL of 293 effectin and OptiMEM for a total volume of 1.3 mL. The 293fectin mixture was added to the DNA mixture, and were mixed and incubated for 20 minutes at room temperature. The 2.6 mL transfection mixture was added to a flask containing 40 mL of 293F cell culture at 106 cells / mL. The flask was incubated at 37 ° C, 8% C02 with shaking at 120 rpm. After 3 days, the cell suspension was centrifuged and immediately subjected to affinity chromatography of protein A to purify the antibody. The final product was concentrated, analyzed by SDS-PAGE and the protein concentration was determined by Lowry assays.
To confirm that the purified mouse-human chimeric antibody has the same binding activity as the parent TMC-2206 antibody, the purified mouse-human chimeric antibody was evaluated for its ability to block cell adhesion mediated by integrin a2ß1. CHO cells expressing a human a2 integrin (SEQ ID NO: 8) and endogenous hamster β1 (Symington ei ai, J Cell Biol. 120 (2): 523-35. (1993)) were separated from the culture flask on incubation in PBS free Ca ++ / Mg ++ containing 5 mM EDTA. The cells were then centrifuged (1200 rpm for 8 minutes in a Beckman GH 38 rotor) and the pellet was resuspended in 10 mL of RPMI-1640. 30 μL of 17 mM CFSE (Molecular Probes, OR) was added to the cell suspension and the mixture was incubated at 37 ° C for 15 minutes. The labeled cells were pelleted at low speed, resuspended in 10 mL of RPMI-1640 with 0.1% BSA and counted. The cell concentration was adjusted to 8 x 10 5 cells / mL and kept in the dark until it was used. A plate coated with collagen (rat tail collagen Type I, BD Biosciences) was blocked with 100 μL / well of 0.1% BSA in PBS and incubated at room temperature for 30 minutes. Protein samples were serially diluted in a serum-free medium and 50μL of each diluted serial antibody solution were added to the collagen plate. Then, 50 μL / well of labeled cells were added to the well and the plate was incubated for 1.5 hours at 37 ° C. After washing, the cells were lysed with 0.1% Triton X-100 and the intensity of the fluorescence (excitation, 485 nm, emission, 535 nm) was read using a Victor2 1420 Multilabel Counter reader (Perkin-Elmer). Cloned chimeric TMC-2206 was a potent inhibitor of cell adhesion mediated by a2ß1 to Type I collagen and showed a power equivalent to TMC-2206 with an EC50 value of 1.8 nM compared to 1.2 nM, respectively. In these experiments, the use of Ig control did not provide binding inhibition while the use of murine TMC-2206 or the chimeric antibody showed an inhibition in binding when tested over a molar concentration range of 10 ~ 11 to 10" 6. The affinity of the mouse-human chimeric antibody for the immobilized a2β1 integrin was also compared with the parent TMC-2206 antibody for its ability to compete for the binding of TMC-2206 with Eu tag to o2β1 coated plates, for example, when determining Ki values First, the affinity of the parent TMC-2206 antibody for the immobilized α2β1 integrin was determined by an equilibrium binding The wells of a 96-well microtiter plate are coated with platelet α2β1 integrin (personalized platelet coated with a2β1 human products manufactured by GTI Inc., Wl) and then blocked with non-fat milk.For the binding and competition tests, TMC-2206 was used with label fluorescent antibody or an antibody with isotype IgG control. To label antibodies with Eu-N1-ITC reagent, approximately 2 mg of either TMC-2206 or isotype control, MOPC-21 (Invitrogen), were suspended in and dialysed against a phosphate-buffered saline solution (PBS, 1.47 mM KH2P04 , 8.1 mM Na2HP04, pH 7.4, 138 mM NaCl and 2.67 mM KCI). After concentration in prewashed MicroSep concentrators (30-kDa cutoff; Pall Life Sciences at 9500 rpm (7000 xg) in a JA-20 rotor (Beckman Instruments, Inc.) for 20 minutes at 4 ° C), the antibodies were adjusted at 4.0 mg / mL with PBS containing a final concentration of 100 mM NaHCO3, pH 9.3. The MAb / bicarbonate mixture (0.250 mL) was mixed gently in a flask containing 0.2 mg of acid? / 1- (p-benzyl-isothiocyanate) -diethylenetriamine -? / 1,? / 2,? / 3,? / 3 -tetraacetic chelate with Eu3 + (Eu-N1-ITC, Perkin Elmer Life Sciences) and reacting overnight at 4 ° C without shaking. Each labeled antibody mixture was applied to a separate PD-10 column (GE Biosciences, Piscataway, NJ) pre-equilibrated with run buffer (50 mM Tris, pH 7.4 and 138 mM NaCl). The fractions (0.5 mL) were collected and assayed for total protein (Bradford reagent); Bio-Rad Laboratories, Hercules, CA) using an absorbance reader on SpectraMax 384 plates and for europium after a 1: 10,000 dilution in DELFIA enhancing solution (Perkin-Elmer) by time resolved fluorescence (TRF) using a Victor2 Multilabel reader Tweet (Perkin Elmer). Fractions that were positive for protein label and Eu were pooled and applied to new PD-10 columns and samples collected and assayed for total protein and for europium content by TRF calibrated against a standard solution of europium (Perkin-Elmer) for calculate the fluoprotein ratio. The fluorescent labeled antibody, either Eu-TMC-2206 or Eu-con isotype IgG control, was then applied to the blocked microtiter plates of integrin a2ß1 in a volume of 10 μL / well. After incubating the sealed plates for 1 hr at 37 ° C to allow the binding to equilibrate, 2 μL samples were transferred from each well to a fresh well containing DELFIA Enhancer Solution (100 μL / well Perkin-Elmer) for the measurement of the free label (without union). The enhancer solution (100 μL / well) was added to the empty wells for the measurement of the binding label. The plate was shaken (Rate Titler Plate Agitator set at 5 for 5 minutes at room temperature) and the temporal resolution fluorescence intensities (TRF) were read using a Victor2 Multilabel Píate reader (Perkin-Elmer Wallac, Boston, MA). It was calculated that the Kd value by Scatchard analysis was 0.374 nM for TMC-2206. Binding potencies related to the immobilized a2ß1 integrin were analyzed by measuring the K i values in a competition assay using Eu-TMC-2206 with fluorescent label of 100 pM in the presence of variable concentrations of the unlabeled TMC-2206 antibody or antibody chimeric as competitors, using a test system similar to that described above. Then, combinations of antibodies were applied to the wells coated with integrin a2ß1, tested at a concentration index of 10"11 to 10" 7 M, and after the specified time, the binding amount of Eu-TMC-2206 was determined. . The inhibition curves were adjusted with the "competition for a site" model using the Prism software (GraphPad, Inc.) to obtain the IC50 values and to calculate K, using the Cheng and Prusoff equation (1973) and the value for Kd of 0.374 nM above. The parental TMC-2206 antibody exhibited a K of 0.22 ± 0.04 nM (n = 10) compared to a value of 0.27 ± 0.07 nM (n = 5) for the wild-type (wt) chimera. The activity of the wild type chimera was comparable to that of the chimeric form that carried the two LC mutations introduced when designing a Sa / I site (, also 0.27 nM), confirming that these mutations did not affect the activity. In these experiments, control wells coated with BSA tested either with IgG control or with TMC-2206 showed no antibody binding.
EXAMPLE 3Humanized antibodies with specificity for integrin a2ß1 were designed and prepared. Residues of the cloned TMC-2206 antibody which is composed of the CDR regions of heavy and light chains were determined and humanized variants were prepared in the following manner. Three regions of hypervariability within the less variable regions of the framework are found in the variable regions of both heavy and light chains. In most cases, these hypervariable regions correspond to, but may extend beyond, the CDRs. The amino acid sequences of the heavy and light chain variable regions of TMC-2206 are specified above in Tables 3 and 4, respectively. The CDR and framework regions were elucidated in a general manner according to Kabat by alignment with other VH and VL regions using general homology searches using the BLAST database of the NCBI protein (http: //www.ncbi.nih. qov / BLAST /. Ye ei ai, Nucleic acids Res., Jul 1: 34 (Web Server Issue): W6-9)), except for HCDR1. HCDRI was defined by AbM as expandable residues 26 to 35. Oxford Molecular AbM antibody modeling software (http://people.cryst.bbk.ac.uk/~ubcq07s/, Martin ei ai, Proc. Nati Acad. Sci. USA, 86, 9268-9272 (1989), Martin ei ai, Methods Enzymol., 203, 121-153 (1991), Pedersen ei ai, Immunomethods, 1, 126 (1992), and Rees ei ai, In Stemberg MJE (ed.), Protein Structure Prediction, Oxford University Press, Oxford, 141-172 (1996)) combines the Kabat and Chothia numbering systems in the definition of CDRs. Thus, the heavy chain CDR regions were defined as follows: HCDR1 aa26- aa35 HCDR2 aa50- aa65 HCDR3 aa95- aa102 Similarly, the light chain CDR regions were defined as follows:LCDR1 aa24- aa34 LCDR2 aa50- aa56 LCDR3 aa89- aa97 It is desirable to preserve the binding affinity of the murine antibody in the humanized counterpart antibody. It may be desirable to choose a human acceptor molecule that shares homology with the murine antibody. Preferred human acceptors are frameworks of human germ lines because the lack of somatic mutations can reduce the degree of immunogenicity; however, the frameworks of individual mature antibodies can also be used as acceptor molecules. The V-BASE database (http://base.mrc-cpe.cam.ac.uk) provides a very complete list of heavy and light chain human germline sequences and was used as a source of line sequences human germinal for comparison with VH and VL of TMC-2206; the Kabat database was also used (http://kabatdatabase.com/ Johnson, G. and Wu, T.T. (2001), Nucleic Acids Res., 29, 205-206). The VH of TMC-2206 was well aligned with three of 51 human germline sequences in the V-BASE database, 4-59, 4-61 and 4-30.4, and no sequence shows a good fit in the framework 3 The lengths H1 and H2 of CDR in 4-59 were identical to those of the VH of TMC-2206, and 4-59 (SEQ ID NO: 39) was selected as an acceptor framework. It carried the same canonical structure class 1 for CDR H1 and CDR H2 as CDR H1 and CDR H2 in TMC-2206. The VH CDR3 and FW4 regions are not included in the germline sequences of VBASE, because part of the CDR3 and framework regions 4 are derived from a different and non-contiguous gene that varies during the maturation of each antibody. The antibody sequence, CAA48104 (NCBI entry: gi / 33583 / emb / CAA48104; http://www.ncbi.nlm.nih.gov/BLAST) was used to provide the sequences for CDR3 and FW 4 alignment, and a sequence of acceptor molecule FW4. A comparison of the VH of TMC-2206 with 4-59 and with the CDR3 and FW4 region of the CAA48104 antibody sequence is given in Table 5.
TABLE 5cThe germline sequence, A14 (SEQ ID NO: 37), was one of 38 human VL antibody sequences in the V-BASE database and was selected as a lattice VL acceptor. A14 is in the VK VI family and its LCDR1 and LCDR2 fall into canonical classes 2 and 1, respectively. The LCDR2 of TMC-2206 is also class 1, although the LCDR1 of TMC-2206 is similar, though not identical, to a canonical class structure 1. The germline VL sequences extend through CDR- L3, so an additional sequence for FW4 of a human VL was selected. The selected sequence represents a lattice 4 gene that is commonly used for kappa light chains in mature human antibodies (eg, AAB24132, NCBI entry gi / 259596 / gb / AAB24132; http://www.ncbi.nlm.nih.gov/BLAST). Although with the introduction of the Sa / I site, two amino acid changes were made in the sequence during the construction of the light chain chimera (E105D and L106I, which did not impact the antibody binding, see above), the human acceptor of light chain FW-4 already has an isoleucine at position 106 so this change only introduced a conservative amino acid mutation (E105D) in the humanized variants. A comparison of the VL of TMC-2206 with A14 and with the FW4 region of the AAB24132 antibody sequence is given in Table 6.
TABLE 6Humanized variants of TMC-2206 were prepared using CDR sequences of VH and VL sequences of TMC-2206 and of the human frameworks selected as described above. To maintain an adequate CDR presentation, some canonical residues of acceptor framework (see, for example, Chothia et al, 1985, 1992, Queen ei ai, 1989, Foote and Winter, 1992, http://people.cryst.bbk.ac .uk / ~ ubcg07s) can be exchanged for the canonical residues of murine donors from the counterpart, a process called backmowing. Tables 7 and 8 list the residues that can affect the CDR intercalated conformation and packaging, respectively, and show differences between the VH and VL residues of the TMC-2206 donor and the corresponding human acceptor framework residues (highlighted with bold and italics). Residue L46 marked with an asterisk in Table 8 can play a role in both canonical CDR structure and interchain packaging.
As shown in Tables 7 and 8, eleven framework residues that affect the canonical presentation of CDR and two residues that affect the intrachain packaging differ between the germline sequences of the donor TMC-2206 and the human acceptor A14 and 4- 59, with residue L46 falling in both categories. In particular, these differences are positions H37, H48, H67, H71, H73, H78 and H91 for the heavy chain, and L2, L4, L46, L47, L49 and L71 in the light chain variable framework regions. These residues were identified and selected as candidates for backmowing.
TABLE 7TABLE 8The 13 candidate retromutations as identified, with 11 involving the presentation of appropriate canonical structure and two Involving interchain packaging, were included in the first humanized variant of TMC-2206. In addition, an amino-terminal Q was retained in the humanized VL. This position was retained with the murine identity because it is adjacent to the Phe in L2, which is an unusual amino acid for this position. These humanized light chain and heavy chain variants were called TMC-2206VH1.0 and TMC-2206VL1.0. Additional humanized variants were prepared with fewer backmutations by changing murine residues back to human framework residues. In this way, it was identified that framework residues were sensitive to a reversion to the human residue (in terms of maintaining the potency of the antibody). In parallel, a computer modeling was carried out to help in the selection of candidate waste to be changed back to its human counterpart. The pCI-neo expression vectors of light and heavy chain chimera described in Example 1 were used for the expression of all humanized variants. The version 1.0 of the humanized VH of TMC-2206 (hVHLO, SEQ ID NO: 40) and the version 1.0 of the humanized VL (hVLLO, SEQ ID NO: 41) that incorporate the 14 retromutations defined above were translated into a sequence of nucleotides optimized for the expression of mammalian cells using the Vector NTI software. These sequences were synthesized in a customized way in series within a single plasmid elaborated by Retrogen (San Diego, CA) and cloned in the EcoRI and Sal sites of the LC and HC expression vectors of parental TMC-2206, replacing the regions VH and VL of mouse. Specifically, the EcoRI-Sa / l digestion of the plasmid DNA resulted in two fragments of different sizes, I feel the larger fragment hVHLO and the small hVLLO. Then, these two fragments were cloned into the EcoRI and Sa / I sites of the LC and HC chimeric expression vectors of parental TMC-2206, replacing the mouse VH and VL regions, respectively, followed by the EcoRI and Sa digestion. / I and by the gel purification of the larger fragment of pCI-neolgk-TMC2206VG-hFc and pCI-neolgK-TMC-2206VLh? C, respectively. This strategy was used for the preparation of subsequent variants. The resulting plasmids contained an IgG leader, an optimal Kozak translation initiation sequence, a variable region and a human constant region. The version with version 1.0 of VH of TMC-2206 (SEQ IDNO: 40) humanized and with version 1.0 of the humanized VL (SEQ ID NO: 41) as described above was tested for the activity in the cell adhesion assay mediated by integrin a2ß1 and in the competition assay for binding to the a2β1 integrin immobilized together with the antibody TMC-2206 chimera and the original as described in example 2. The K value for the humanized prototype was 0.32 nM, comparable with the measured K¡ value of the parent TMC-2206 antibody ( 0.21 nM) as well as in the chimera (0.27 nM), indicating that this first humanized version retained the binding affinity. Similarly, the first humanized prototype exhibited an inhibitory activity comparable to that of the parent TMC-2206 antibody in blocking cell adhesion to a2ß1-mediated collagen (e.g., EC50 of 1.5 nM for both). Using the data generated by the variant of version 1.0, a series of back-mutations was made to the residues of the human VH or VL framework using the PCR methodology and the minimum numbers of backmutations (murine residues) were determined to avoid compromising the specificity and the affinity of the original TMC-2206 mAb. Desirable human variants include those that retain the biological activity of the parent murine antibody and also contain less murine residues to reduce potential immunogenicity. The individual primer sequences were synthesized by Sigma-Genosys and their sequences are listed in Table 9. The primer pairs and templates used for the generated variants are shown in Tables 10 and 11. PCR reactions were performed using the following conditions: Primer 1 and 2 (0.6 μM final concentration), dNTP (1 mM final concentration), DNA template (1 to 10 ng) and 1 unit of Pfx DNA polymerase (Invítrogen, CA) usually in a final volume of 50 μL. A PCR program consisted of initial denaturation at 95 ° C for 2 minutes, followed by 30 cycles where each cycle was at 95 ° C for 30 seconds, 56 ° C for 45 seconds and 68 ° C for one and a half minutes. The final stage was at 68 ° C for 10 minutes.
TABLE 9Name Nucleotide sequences (5 '- 3') primer AGCGTGGACACCAGCAAGAACCAGTTCAGCCTGAAGCTGAGCAGCGTG hVH3.0-F (SEQ ID NO: 42) GTTCTTGCTGGTGTCCACGCTGATGGTCACGCGGGACATGAGAGCGCTGTT hVH3.0-R (SEQ ID NO: 43) CCTCCAGGCAAGGGCCTGGAGTGGATCGGCGTGATATGGGCTCGCGGC hVH4.0-F (SEQ ID NO: 44) CTCCAGGCCCTTGCCTGGAGGCTGGCGTATCCAGTGGATGCCATAGTTGGT hVH4.0-R (SEQ ID NO: 45) CCCAAGCTCCTGATCTATGACACTTCCAAGCTG hVL3.0-F (SEQ ID NO: 46) hVL3.0-R (SEQ ID NO: 47) AGTGTCATAGATCAGGAGCTTGGGGGCCTGGTCGGGCTTCTG hVL4 OF GACGCGAATTCAGACGTGGTGATGACCCAGTCTCCAGCATTCCTG (SEQ ID NO 48) hVH2 OF GTGACCATCAGCAAGGACAACAGC (SEQ ID NO 49) hVH2 OR GCTGTTGTCCTTGCTGATGGTCACGCGGGACATGAGAGCGCTGTT (SEQ ID NO 50) hVH5 0-F ATCGGCGTGATATGGGCTCGCGGCTTC (SEQ ID NO 51] hVH5 0-R GCCGCGAGCCCATATCACGCCGATCCACTCCAGGCCCTTGCCTGG (SEQ ID NO 52) hVH6 0-F ATATGGGCTCGCGGCTTCACAAAC (SEQ ID NO 53 ) hVH6 0-R GTTTGTGAAGCCGCGAGCCCATAT (SEQ ID NO 54) hVH7 0-F GCCGCGGACACCGCCACTTGTACTACTGCGCCAGAGCCAACGACGGG (SEQ ID NO 55) hVH7 0-R GTAGTACACGGCGGTG TCCGCGGCGGT (SEQ ID NO 56) 0-F hVH8 ATATCCAACTATGGCATCCACTGGGTT (SEQ ID NO 57) hVH8 CCAGTGGATGCCATAGTTGGATATGCTAAATCCAGAGACGGTACAGGT 0-R (SEQ ID NO 58) VH12 0- (K71V) -F GCCTGACCATCAGCGTGGACAACAGCAAGAACCAGGTGAG (SEQ ID NO 97) VH12 0- (K71V) CTCACCTGGTTCTTGCTGTTGTCCACGCTGATGGTCAGGC -R (SEQ ID NO 98) VH13 0- (N73T) -F CTGACCATCAGCAAGGACACCAGCAAGAACCAGGTGAGCC (SEQ ID NO 99) VH13 0- (N73T) -R GGCTCACCTGGTTCTTGCTGGTGTCCTTGCTGATGGTCAG (SEQ ID NO 100) VH14 0- (V78F) -F GCAAGGACAACAGCAAGAACCAGTTTAGCCTGAAGCTGAGC (SEQ ID NO 101) VH14 0- (V78F) -R GCTCAGCTTCAGGCTAAACTGGTTCTTGCTGTTGTCCTTGC (SEQ ID NO 102) hVL2 CAGCTTGGAAGTGTCATAGATCAATTTCTTGGGGGCCTGGTCGGG 0-R (SEQ ID NO 59) 0-F hVL5 GACGCGAATTCAGAC TTCGTGCTGACCCAGTCTCCAGCATTCCTG (SEQ ID NO 60) 0-F VL6 GACGCGAATTCACAG TTCGTGATGACCCAGTCTCCAGCATTCCTG (SEQ ID NO 61) hVL7 0-F GACGCGAATTCAGACTTCGTGATGACCCAGTCTCCAGCATTCCTG (SEQ ID NO 62)TABLE 10TABLE 11Table 12 lists the VH variants and Table 13 lists the VL variants in addition to comparing the chosen human acceptor frameworks with the initial variants (1.0) VH and VL. The VH variants that are listed in Table 12 include: hVHI .O (SEQ ID NO: 21); hVH2.0 (SEQ ID NO: 70); hVH3.0 (SEQ ID No. 71); hVH4.0 (SEQ ID NO: 72); hVH5.0 (SEQ ID NO: 73); hVH6.0 (SEQ ID NO: 74); hVH7 0 (SEQ ID NO: 75); hVH8.0 (SEQ ID NO: 76); hVH9.0 (SEQ ID NO: 77); hVHIO.O (SEQ ID NO: 78); hVH11.0 (SEQ ID NO: 79); hVH12.0 (SEQ ID NO: 109); hVH13.0 (SEQ ID NO: 110); hVH14.0 (SEQ ID NO: 111). The VL variants that are listed in Table 13 include: hVLLO (SEQ ID NO: 41); hVL2.0 (SEQ ID NO: 80); hVL3.0 (SEQ ID NO: 81); hVL4.0 (SEQ ID NO: 82); hVL5.0 (SEQ ID NO: 83); hVL6.0 (SEQ ID NO: 84); hVL7.0 (SEQ ID NO: 85); hVLd.O (SEQ ID NO: 86); hVL9.0 (SEQ ID NO: 87); hVLIO.O (SEQ ID NO: 88); hVL11.0 (SEQ ID NO: 89); hVL12.0 (SEQ ID NO: 108). The retained murine residues are indicated with bold. Each additional built variant (see below) is also shown. Each variant shown in Table 12 below VH1.0 has the same sequence as VH1.0 (indicated by a hyphen [-]) unless a specific substitution of an amino acid is shown, changing the arrested murine residue to its counterpart in the human framework. Similarly, each VL variant shown in Table 13 has the same sequence as the VL1.0 variant except for the specific substitutions of the indicated amino acids.
TABLE 12TABLE 13The alignment of the amino acid sequences of TMC-2206 with the germline sequences showed a clustering of framework residues that had been retromutated to the murine equivalent in the humanized TMC-2206 variant (TMC-2356)]. As shown by the alignment, there were two other groupings that fell within FW2 and FW3 in the heavy chain, and similarly there were two groupings, one located in FW1 and one in FW2, in the light chain. Two hVH and hVL variants that contained back mutations to human residues in sites of interest were variants 3.0 and 4.0, designed to transport clusters of mutations to help define the regions where the residues of interest could be found (Tables 12 and 13). In addition to the differences in the residues highlighted in Tables 7 and 8 for the VL regions, position L1 was changed to human Asp in VL4.0, because it is a common residue for the human K light chains. The heavy chains hVH3.0 and hVH4.0 were cotransfected with light chains hVL3.0 and hVL4.0 in different combinations VH / VL and the resulting antibodies were compared with the antibody hVH1.0 / hVL1.0 described above for affinity of ligands by face-to-face comparisons with the unlabeled monoclonal antibody TMC-2206. In Table 14, remnants of version 1.0 of humanized VH or VL that were converted back to human waste are indicated in italics and bold. The numbers in parentheses in Table 14 represent the change in power compared to the variant hVHLO, hVLI .O.
TABLE 14Starting from the K i values it was evident that the changes in the VH 3.0 variant (human residues inserted in H67, H71, H73 and H78, designated the FW-3 grouping) induced a long decrease in the potency of hVH3.0 that contained antibodies; similarly, a reduction was also observed for the hVL4.0 variants (the human residues inserted in L1, L2 and L4 designated the FW-1 grouping). Except for the combined antibodies hVH1.0 / hVL3.0 and hVH4.0 / hVL1.0 that showed a change of 1.9 and 1.3 times in the potency when compared with the antibody hVH1.0 / hVL1.0 antibody, respectively, all the remaining combinations showed a decrease of more than 4 times in the power (Table 14). These data indicated that the backmutations to H37 (V to I) and H48 (L to I), which were conservative amino acid changes, were well tolerated. Changes L46 (K to L) and L47 (W to L) of murine residues again to human residues were reasonably well tolerated in combination with hVHI .O but had a markedly synergistic adverse effect on antibody affinity when combined with the hVH3.0 variant. The examination of the differences in residues that existed between the murine and human VH and VL frameworks indicated that some conservative changes occurred. Additionally, three-dimensional computer modeling of the VH and VL of murine TMC-2206, the human acceptor molecules and the hVH 1.0 and hVL 1.0 structures was also performed. To guide computer modeling, a BLAST search was performed to identify the database structures with matches close to the VL and VH of TMC-2206. The structure ISY6.pdb (resolution 2.0 A) was chosen for the VL of TMC-2206 and the structure IGIG.pdb (resolution 2.3A) was chosen for the VH of TMC-2206. For the light chain human acceptor module A14, the structure ICELpdb (resolution 1.9A) was chosen, while for the human acceptor molecule of heavy chain VH, 4-59, IDNO (resolution 2.3A) was chosen. The modeling predicted that murine residues retained in the humanized VL 1.0 were likely to contact the antigen, except for two (L1 and L4). The models also indicated that residues of the human germline framework did not contact the CDRs while retained murine framework residues were generally grouped around the CDRs. In the variable regions of the heavy chains, three areas of difference between the murine and human VH modeled regions were identified. The first area was residues H27-H33 of which were predicted to have the probability of contacting the antigen and the CDR H1. These residues can also affect the angle of the VL? / H interface and have additional indirect effects on antigen binding. The second area was the first cycle of CDR H2 that could require a residue FW H71. The third area was the CDR H3. For the regions of the light chain, three regions of difference between the murine and human VL structures modeled were also identified. The first structure was a CDR1 that was a longer residue in the VL of murine TMC-2206. The Y murine in L71 (F in A14) was useful to accommodate this difference. The second area was the murine cycle L40 to L43 that was pushed out in solvent, compared to the human which indicated that the human L40-43 could be problematic, although the activity of the first humanized prototype showed that these retromutations were tolerated in hVLLO . The third area was the residues of the human framework L55 to L59 that were displaced in relation to the murine structure. It was predicted that the L73 residue of the framework (L in murine, F in human) was responsible for this difference, although the backmowing was tolerated in hVLLO. Using in silico analysis, the results were predicted for the specific residues of the heavy chain of interest and are summarized in Table 15. Similar results for the light chain residues of interest are listed in Table 16.
TABLE 15Residue Murino Humano Position Type of changeH37 VI Possibly in the interiaz VH / VL Conservative H48 LI Interior, away from the binding site, near H67 Conservative H67 LV Interior, away from the binding site, cela of H48 Conservative H71 KV Behind CDR H2 residues H53-55 Large H73 NT Behind CDR H2, solvated, near H71 Moderate H78 VF Contact CDR H1 residue H34, buried Large H91 FY At VH / VL Conservative interfaceTABLE 16Kind ofHuman Murine Residual Change positionL1 Q D Behind CDR L3, solvated Preservative Extensive contacts with CDR L3, partially ".
L2 F V,. . Large solvated L4 L M Behind CDR L3, partially solvated ConservativeL46 K L On the VH / VL Large interfaceL47 W L VL interior behind CDR L2 LargeL49 and K Possible direct contacts with the antigen LargeL71 and F Behind CDR L1 ConservativeUsing an in silico analysis, the positions were accessed and sorted in order to reflect the likelihood that a human substitution could cause an effect on antibody performance: H48, H67 < H37, H91 < H73 < H78, H71. In this classification, it was predicted that a human back-mutation at positions H48 or H71 would be the least tolerated. Similarly, the following order was predicted for the positions of interest within the light chain region: L1 < L4 < L71 < L2 < L47 < L46 < L49. Generally these classifications were in agreement with the K values, obtained with the variants hVH3.0, hVH4.0 and hVL4.0. However, a difference between the substitution effects predicted by computer modeling and the observed effects seen with constructed variants was observed in the activity for the hVL3.0 variant. For example, the antibody variant composed of a combination between VH1.0 / VL3.0 offered fair activity, although the computer data mentioned above predicted that the hVL3.0 variant would have reduced activity much more. The impact of retained murine framework residues was further evaluated by constructing additional humanized variants, including eight VH variants and six VL variants, each transporting a single mutation of the murine retained with its human counterpart. The relative contributions of the changes to the activity were measured. Table 12 lists the VH variants and Table 13 lists the VL variants. The K values, which were obtained for these variants indicated that the mouse residues in H71, H78, L2 and L46 were preferably retained to maximize activity, while H37, H48, H67, H91, L1, L4 and L71 could be changed to their human counterparts without resulting in a significant loss in activity. Using computer modeling, when changing the mouse residue, it was predicted that L47 (Tryptophan, a rare residue for this position in human antibodies) and H73 would impact the antigen binding. However, the change to human Val-L47 did not significantly affect antigen binding, and the change to Thr-H73 only caused a minor change (1.6-fold decrease) according to the K i measurement. It was predicted that L49 (tyrosine) would bind antigen by in silico modeling and it was predicted that switching to a human lysine would cause a large change in potency. However, the change to human lysine for this position only caused a 3.3-fold decrease in potency according to the measurement of K ,. A significant change in VH was observed for the change in H78 from murine valine to human phenylalanine, which caused the 70-fold decrease in potency for the variant hVH11.0, hVLLO compared with the variant hVHLO, hVLLO according to the measurement of K¡. The modeling indicates that this residue represents a role in the canonical structure of HCDR1. These results suggest that HCDR1 plays an important role in antigen binding. To maximize activity, H71 is also retained. Changing this Lys residue to one of Val resulted in a 6.4-fold decrease observed with the antibody variant hVH9.0, hVLI .O. Additionally, among the canonical residues in the light chain, the phenylalanine in L2 was sensitive to change as evidenced by the marked loss in binding affinity observed with the variant hVL4.0 compared with the variants hVL5.0, hVLd.O and hVL7.0. Computer modeling indicated that this Phe-L2 can make extensive contact with LCDR3. In addition, searches of human and murine antibodies in the database indicated that this Phe at position L2 is rare, suggesting that it may represent a somatic mutation that has an impact on antigen binding. Those residues selected after the analysis as described above to be tolerant to backmowing, were combined in the hVH variants from 12.0 to 14.0 and in the VL variants from VL10.0 to 12, and the activity of these variants were compared against those of the original monoclonal antibody TMC-2206 and those of the mouse-human chimeric antibody TMC-2206. The results indicated that the number of murine residues in hVL could be reduced to three (eg, L2 [Phe], L46 [Lys] and L49 [Tyr]) without causing any loss in the activity of the variants. Similarly, the number of murine residues in hVH could be reduced from seven to three (eg, H71 [Lys], H73 [Asn] and H78 [Val]) without causing statistically significant changes in affinity or potency. These results are summarized in Table 17.
TABLE 17# of K, (nM) ECso (nM)VH VL Changes back to human mupnos residues Mean ± SD Mean ± SDTMC mAb- N / A 0 22 ± 0 04 1 18 + 0 35 2206 Chimera N / A 0 26 ± 0 07 1 66 ± 0 641 0 1 0 N / A 14 0.27 ± 0.06 2.70 + 1 661 0 1 OQ N / A 14 0.35 + 0.03 3.00 ± 1 2012 0 10 0 H37, H48, H91, L1, L4, L47 8 0 29 ± 0 05 2.20 + 0 5812 0 10 OQ H37, H48, H91. L1. L4, L47 8 0.31 + 0 05 2 36 + 1 0614 0 10 0 H37, H48, H91, H91, L1, L4, L47 7 0.32 ± 0 07 2 90 ± 2 7114 0 10 OQ H37, H48, H91, H91, L1, L4, L47 7 0 29 ± 0.05 2.98 ± 1 98 H37, H48, H67, H91, L1, L4, L47, 14 0 12 0 6 0 38 ± 0.10 2 9311.37 L71 H37, H48, H67, H91, L1, L4, L47, 14 0 12 OQ 6 2 95 ± 0 32 L71 0.33 ± 0 11[ANOVA analysis with Dunnett multiple comparison tests did not show statistically significant differences with TMC-2206 or with the chimera]. In parallel, homologs were constructed to these variants where the consensus of the glycosylation sequence within LCDRI was changed. Removal of glycosylation sites (NSS to QSS) can be useful for the next steps of production and for the development of the process. The change to N26Q in the variants hVLLO, hVLIO.O and hVL12.0 (denoted hVLLOQ [SEQ ID NO: 90], hVL10.0Q [SEQ ID NO: 91] and hVL12.0Q [SEQ ID NO: 92]) was introduced in the relevant VL variant using the primer pairs indicated in Table 18 whose sequences are given in Table 9. The change to N26Q did not have a statistically significant effect on the activity of any of the resulting antibodies, as shown in Table 17. Although this glycosylation site occurs in the wild-type TMC-2206 antibody light chain CDR 1, these data indicate that it does not appear to play a role in the affinity of the TMC antibody function blocking activity. -2206.
TABLE 18EXAMPLE 4Human antibodies of class? 1 transport effector functions associated with functions mediated by the complement and by the Fc receptor. Those skilled in the art consider that to avoid antibody-dependent cellular cytotoxicity (ADCC) and complement responses a chain? lacking this functionality, as a human constant region? 4, is preferable. To generate a? 4 version of the VH12.0, VL10.0Q and VH14.0, VL10.0Q antibodies, a constant region sequence? 1 was replaced by a? 4 constant region sequence in the VH12.0 heavy chains and VH14.0 as described below. The constant region sequence? 4 was obtained from the K01316 sequence of Genbank. Both a 1? Fc sequence derived from the IMAGE clone 20688 used to generate the intact heavy chains of the IgG1 antibodies with the hVH and hVL regions as described herein as the? 4 Fc derived from the sequence K01316 contain an Apa1 restriction site that occurs naturally near the union of the variable and constant regions. This site was used to clone a constant region? 4 to replace a constant region? 1. The restriction sites SamH1 and? / Oi1 were placed at the 3 'end of the sequence to facilitate subcloning into the pCl-neo expression vector. Sequence? 4 (SEQ ID NOs: 105 and 106) was then synthesized as a de novo synthetic gene by Blue Heron Biotechnology (Bothell, WA). The Blue Heron Biotechnology plasmid, which contains the IgG4 constant region synthesized de novo, was digested with Apa \ y? ofl, the 1 kb -? 4 constant region fragment was gel purified and ligated into plasmids pCI-VH12.0 and pCI-VH14.0 digested with Apal / Notl to produce plasmids encoding VH12.0-? 4 and VH14.0-? 4. These were individually combined with the pCI-VL10.0Q plasmid transfected into CHO cells. Four days after transfection, the culture supernatants were harvested and the IgG4 isotypes of the VH12.0, VL10.0Q and VH14.0, VL10.0Q antibodies purified by Protein A affinity chromatography. Transient transfections of the constructs ? 1 of these variants were performed in parallel. After elution and acid neutralization, analytical size exclusion chromatography by HPLC indicated the presence of oligomeric forms of higher order in the purified IgG4 Protein-A preparations. Therefore, a second purification step was performed by size exclusion chromatography Sephacryl S-300 26/60 to obtain the monomer fraction. For this, the Sephacryl S-300 26/60 column was pre-equilibrated in 660 ml of SEC buffer solution (40 mM HEPES, pH 6.5, 20 mM L-histidine, 100 mM NaCl and 0.02% Tween-80. ). The pooled fractions containing protein eluted from the Protein A column were loaded (12.5 ml sample injection) through a Superloop (Amersham Biosciences). The SEC fractions (5 ml each) were collected at a flow rate of 2.0 ml / min. The fractions corresponding to the monomeric form (elution of the peak at 168.4 ml) were pooled and the protein content was determined by Lowry assay. Exemplary IgG1 antibodies have a heavy chain hVH 14.0? (SEQ ID NO: 181) or a heavy chain hVH12.0? 1 (SEQ ID NO: 182) and a light chain hVL10.0Q (SEQ ID NO: 178). Exemplary IgG4 antibodies have a heavy chain hVH14.0? 4 (SEQ ID NO: 174) or a heavy chain hVH12.0? 4 (SEQ ID NO: 176) and a hVL light chain 10.0Q (SEQ ID NO: 178). Purified antibodies were tested in the competition test to compare the potency by K values, as well as in the adhesion testcellular to collagen, where the power is measured as EC50 values. I dont knowobserved a significant difference between the isotypes of the differentvariants did not differ significantly from the original TMC-2206 mAb innone of the trials, as shown in Table 19.
TABLE 19Isotype VH VL K, (nM) EC50 (nM)TMC- 2206 lgG1 /? Murino Murino 0.22 1.03 ± 0.29 hlgG1 /? 14.0 10.0Q 0.24 1.30 ± 0.10 hlgG1 /? 12.0 10.0Q 0.27 2.20 + 012 hlgG4 /? 14.0 10.0Q 0.36 2.82 ± 1.04 hlgG4 /? 12.0 10.0Q 0.27 1.83 ± 0.27EXAMPLE 5The effect of the antibody against integrin a2 in extravasationof neutrophils was studied in a murine peritonitis inflammation modeland in rat. The intraperitoneal administration of certain antigens ascasein, carrageenan or thioglycollate and induces a rapid mast cell response that initiates an acute peritonitis response (Edelson et al, Blood 103 (6): 2214-2220 (2004)). This peritonitis is characterized by a rapid infiltration of neutrophils (in a few hours) followed by a slower infiltration and proliferation of macrophages (3-5 days). Therefore, this model was used to evaluate for the first time the use of antibodies against integrin a2 in the prevention or functional reduction of the neutrophil response. The acute peritonitis model was carried out in rats and mice. The TMC-2206 antibody recognizes the rat a2β1 integrin, but not its murine counterpart. However, many in vivo models of inflammatory models are made in mice and an antibody against integrin a2 substitute, Ha 1/29 (Pharmingen, Becton Dickenson, CA, catalog No. 559987), was used in the acute peritonitis model in murine Animals were injected with an antibody against integrin a2 or isotype control either IV or IP at doses ranging from 0.1 to 10 mg / kg 15 minutes before the neutral test. An injection of 1 mL of either 9% casein (mice) or carrageenan (rats) was administered via IP and the animals returned to their cages for specific periods: 3 hours (mice) or 5 hours (rats) (n) = 4 per group). Then, the animals were euthanized with halothane and the peritoneal cavity was washed with 5 mL (mice) or 10 mL (rats) of PBS containing 5 mM EDTA. The cells were collected by low speed centrifugation, resuspended in 5 mL of PBS / EDTA and a 100μL aliquot was seen by microscope, where it was observed that most of the cells had a polymorphonuclear morphology consistent with neutrophils. The cells in the remaining suspension were subjected to low speed centrifugation to obtain a washed cell pellet. The neutrophil content was quantified by analyzing the activity level of myeloperoxidase (MPO) (for example, Speyer ei ai, Am J Pathol 163 (6): 2319-28 (2003)). The cellular pellet recovered from the washed fluid was resuspended in 500 μL of 50 mM buffer solution of KH2P04 (pH 6.0) containing 0.5% hexadecyltrimethyl ammonium bromide (H , Sigma-Aldrich, Ml). The samples were sonicated for 60-90 seconds and centrifuged at 14,000 rpm for 5 minutes at 4 ° C. A 2: 1 serial dilution of the clarified supernatant was made by transferring 50 μL of the sample to a buffer buffer of 50 μL of HTAB in the well of a microtiter plate. Then, 50 μL of this solution in the well was transferred to another 50 μL buffer, and thus the dilution series continued. 200 μL of substrate buffer solution (50 mM buffer solution of KH2P04 (pH 6.0) containing 0.168 mg / mL of o-dianisidine and 0.0005% H202) was added to each sample to initiate the colorimetric reaction, which was monitored with a Molecular Devices plate reader configured at a wavelength of 460 nm. The number of Enzyme Activity Units in the original cell suspension was then calculated (500 μl washed cell suspension for each individual animal). A calibration curve, configured to adjust neutrophils / mL (assessed by direct neutrophil count) against MPO activity indicated a strong linear correlation between U / mL and the number of neutrophils / mL within the measured scale. As shown in Table 20, murine antibodies against the integrin a2ß1 Ha 1/29 and TMC-2206 had a marked effect on neutrophil infiltration in the peritoneal cavity after a neutral test with 9% casein in mice or carrageenan to 1% in rats as measured by the total MPO activity recovered in the peritoneal lavage fluid. The ED50 value obtained in mice with Ha1 / 29 was -0.07 mg / kg while the ED50 for TMC-2206 in the rat was ~5 mg / kg. This difference correlates in part with the relative affinity of the human anti-integrin a2β1 with the rat α2β1 integrin compared to the affinity of the Ha1 / 29 antibody for the mouse α2β1 integrin, and partly with differences in the antigen used in the mice. rat and mouse models (carrageenan and casein, respectively).
TABLE 20EXAMPLE 6The effect of antibodies against integrin a2 in the mouse model (dextran sulfate-induced colitis) in inflammatory bowel disease was studied. In this model, colitis is induced in mice by administering a solution of 5% sodium dextran sulfate (DSS) in drinking water (Elson et al., Gastroenterology 109 (4): 1344-67 (1995); Egger B). ., et al., Digestion 62 (4): 240-8 (2000)). The effect of the treatment with a murine antibody against the integrin a2ß1 in the development of clinical signs and symptoms of colitis was evaluated as well as the effect on the infiltration of proinflammatory leukocytes in the colon.
BALB / C mice (Harían, IN) weighing 16 to 21 grams were placed in pairs. The animals were given distilled water or water containing 5% sodium dextran sulfate (DSS) (ICN, Irvine, CA) ad libitum for 7 days. In this stage the mice presented diarrhea and a notable weight loss. The design of the study was four groups with six mice each; one that served as a control without altering (without previous treatment), one that served as the DSS control and two assigned to receive intraperitoneal injections with doses of 2 or 5 mg / kg of antibody to integrin a2 PS / 2 on days 0 , 2, 4 and 6. The mice were euthanized on day 7 (168 hours after the start of DSS administration). They were weighted to observe any change from the beginning of the study, the length of the colon was measured and then scored on a scale of 0 to 2, where 2 was the most severe rating, for diarrhea, colon bleeding and rectal bleeding of the following way: rectal bleeding: rating of 0 = blood not visible; 2 = visible blood stool consistency: rating of 0 = normal; 1 = loose; 2 = aqueous bleeding of the colon: rating of 0 = blood not visible; 2 = visible blood. Afterwa the colons were processed for immunohistochemistry. Colons, from the cecum to the rectum, were carefully removed and fixed for 2 hours at 4 ° C in 4% paraformaldehyde (PFA), left overnight in 20% sucrose and then rapidly frozen in freezing compound OCT (Tissue Tek) Serial thin sections (10 μM thick) were cut using a Leica cryostat, air dried, blocked for 2 hours in 3% goat serum in PBS and incubated overnight at room temperature in the primary antibody. The primary antibodies that were used included CD11 b / mac-1 rat anti-murine (a marker for neutrophils macrophages and activated, Clone M1 / 70, BD-Pharmingen), CD3 anti-murine hamster (a T cell marker, BD-Pharmingen) and clone F4 / 80 (a marker of macrophages, Research Diagnostics, Inc.). The slices were then washed, incubated for two hours in the corresponding secondary antibody labeled Alexa 488 or TRITC (Molecular Probes, OR) and washed three times for 5 minutes in PBS and mounted on a Vectashield medium containing DAPI (Vector Labs, CA ). Sections were observed with an epifluorescence microscope connected to a Spot RT (Research Diagnostics) camera. The intensity of the fluorescence and the number of fluorescent cells within a selected region of interest (ROI) that delineated the region between the lamina propria and the tips of the villi but eliminated the serous surface (high fluorescence) and the enteric lumen, from a total of 5 fields of vision in five independent sections, they were quantified for each animal using ImagePro software (Media Cybemectics, MD).
As shown in Table 21, treatment with the murine antibody against integrin a.2 had a statistically significant effect on dose by reversing weight loss and stool consistency associated with DSS feeding. The two treatment groups (2 mg / kg and 5 mg / kg) had significant effects on rectal bleeding and colon bleeding, but did not have a significant effect on the shortening of the colon associated with the development of colitis. The treatment also correlated with a marked reduction in the number of infiltrating leukocytes (data not shown).
TABLE 21* P < 0.05 kp < 0.01 In another study, the effects of the antibody against integrin a4 (PS / 2 clone, Southern Biotech, AL), against integrin a2 (clone Ha1 / 29, BD Pharmingen, CA) and against integrin a1 (clone Ha8 / 31 Invitrogen, CA) were examined in comparison with mice treated only with DSS (n = 8 per group). The treatment doses of the antibody were 5 mg / kg. It has been reported that these function-blocking integrin antibodies modulate experimental colitis (Kriegelstein et al., J Clin Invest. 110 (12): 1773-82 (2002); Watanabe et al., Am J Physiol Gastrointest Liver Physiol. 6): G1379-87 (2002)). As shown in Table 22, the three antibodies against integrin were associated with an inversion in the shortening of the colon, but only the treatment against integrin a2 was associated with a significant improvement in stool consistency (diarrhea). Treatments against integrin a.2 and a4 resulted in a significant improvement in colon bleeding. In this study, none of the treatments with the antibody induced a significant effect on weight loss. When the amounts of T cells, macrophages and neutrophils were evaluated by indirect immunofluorescence using anti CD3, F4 / 80 and anti Mac1 (as previously described), all three groups treated with integrin antibodies showed a significant reduction in comparison with the saline control group as indicated in Table 23. These data support the conclusion that antagonizing the function of a2 has a profound effect on the steady state levels of these immune effector cells that accumulate in the inflamed colon. in response to DSS and that these changes correlate concurrently with an improvement in clinical measurements associated with colitis.
TABLE 22* p < 0.05 ** p < 0.01TABLE 23* p < 0.05 ** p < 0.01 EXAMPLE 7The effects of the antibody against integrin a2 were studied in terms of their clinical signs and symptoms in a model of experimental allergic encephalomyelitis (EAE) of multiple sclerosis. The EAE model of multiple sclerosis, induced by injection of the synthetic encephalogenic peptide PLPi3g_151 together with Freund's adjuvant in SJL mice, is considered a predictive model of relapsing-remitting multiple sclerosis (Encinas eí ai, J Neuroscí Res.45 (6 ): 655-69 (1996)). In a study of four groups of mice (8 / group, see figure 1), two groups were dosed with 5mg / kg on days 10, 11, 12, 14, 15, 18 and 20 with isotype control or with the murine antibody against integrin a2, Ha1 / 29, from the onset of the symptoms of the disease until day 20, which was when the first acute exacerbation occurred. It was an acute dose regimen. Day 0 was defined as at the start of priming with the synthetic peptide PLP139-151 in addition to Freund's adjuvant. The onset of the disease was defined as the second day of consecutive weight loss. The other two groups were assigned to a delayed dose regimen where they received 5 mg / kg of antibody to murine a2 integrin or saline three times a week from day 18 to day 36, which coincided with the second exacerbation / first relapse The mice were rated according to their clinical signs and symptoms as follows: 0.5 rating = 2 consecutive days of weight loss 1 rating = flaccid tail rating 2 = ataxia rating 3 = hindquarter paralysis rating 4 = agony rating of 5 = death As shown in Table 24, treatment with 5 mg / kg of the antibody against integrin a2 from the start of the first exacerbation (eg, acute treatment) bogged down the extent of the first exacerbation and also limited the extent of the second exacerbation. When dosing was started after the end of the first exacerbation on day 18 (for example, delayed treatment); see figure 1), mice treated with 5 mg / kg antibody to murine a2 integrin also showed lower clinical scores during the first relapse until day 32, and at that point the two groups showed similar disease scores, as detailed in Table 24. When the mice were only given the dose during the induction phase, as shown in Figure 2, the a2 integrin mAb had little or no effect on the first attack or subsequent steps of EAE model and mice essentially developed the disease equivalent to that of animals treated with isotype control. These results contrast with those previously obtained using the antibody against integrin a4, PS / 2, in the EAE model (Yednock ei ai, Nature 356 (6364): 63-6 (1992); Theien ei ai, J. Clin Invest. 107 (8): 995-1006 (2001)), used to support the treatment for relapse of multiple sclerosis with the antibody natalizumab against integrin a4 (Miller et al., N. Engl. J. Med. 348 ( 1): 15-23 (2003)). These results indicate that in contrast to the role played by integrin a4 in inflammatory neuro-disorders, where antagonism of this receptor can delay the onset of exacerbated relapses associated with multiple sclerosis, antagonizing integrin a2 is a modality of treatment useful to reduce and treat exacerbations when they occur.
TABLE 24A histological analysis was performed on the brains and spinal cords of mice when they were dying (stage 4) or at the end of the study. The mice were euthanized with halothane when they were dying (clinical score 4), or after the observation period of 55 to 60 days. The animals were perfused with PBS, followed by a 4% paraformaldehyde solution. The brains were divided into five coronal blocks, the spinal cords in ten to twelve transverse blocks and the tissues were embedded in paraffin in sections of 4 μ in thickness stained with Luxol Blue to visualize myelination. The tissues were graded with a blind method according to their degree of myelination, infiltration (meningitis) and perivascular thickening. To qualify the sections of the spinal cord, each section of the spinal cord was divided into quadrants: the anterior funicle, the posterior funicle, and each of the lateral funicles. Any quadrant that presented meningitis, perivascular thickening or demyelination was given a grade of 1 in that pathological class. The total number of positive quadrants for each pathological class was determined, then divided by the total number of quadrants present in the block, and multiplied by 100 to obtain the percentage of turnover for each pathological class. A general pathological score was also determined by giving a positive score if there were injuries present in the quadrant. The notorious demyelinating inflammatory lesions were observed in the spinal cord, often in the gracile fasciculus of the posterior funiculus and in the exit zone of the ventral root in the anterolateral funicle. Only moderate perivascular thickening was observed. Meningitis and demyelination showed a strong correlation with clinical signals (r = 0.84 and 0.79, respectively) and had significantly lower scores in the group treated with antibody to integrin a2 (P <0.01 among the groups against integrin a2 and IgG control for both parameters). These data indicate that treatment with the a2 integrin antibody inhibits meningitis and demyelination associated with an exacerbation and / or facilitates remyelination and repair. In the end you get an improved clinical result. Another study was carried out (n = 17 to 20 per group) to compare the antibody against integrin a2 with the antibody against integrin a4, PS / 2 (obtained from Southern Biotech), treatment during the first acute exacerbation until the beginning of the referral (Days 10 to 20). Two additional groups were treated either with IgG control or with antibody against integrin a.2 from the start of remission (Day 18) to the first relapse (Day 36) and at the beginning of the chronic stage of the disease. Again, the treatment against integrin a2 had a marked effect on the incidence of neurological sequelae (paralysis) and a statistically significant reduction in the maximum mean clinical score during the first EAE exacerbation as well as in the relapse stage (chronic stage). of EAE, Table 24). There was a slight improvement effect with delayed treatment against a2-integrin in the clinical ratings during the chronic stage of the disease. The incidence of the disease during the first attack (against integrin a2, 61%, control IgG 85%) and during the chronic stage (against integrin a2, 77%, control IgG 100%) was lower for the group of mice treated with the antibody against integrin a2 compared to the control. In the case of the group treated with the antibody against integrin a.4, the incidence of the disease during the first attack (treatment against integrin a4, 94%, control 82%) and relapse (treatment against integrin a4, 89%; control, 92%) was similar between the group treated with antibody against integrin a4 against the control group. However, the degree of subsequent relapse was markedly reduced. These data with respect to an antibody to integrin a4 are comparable with previous reports on the effect of treatment with antibody against integrin a4 on the clinical outcome in EAE (Theien ei ai, J. Clin. Invest. 107 (8): 995-1006 (2001)).
EXAMPLE 8The effects of binding to platelet a2ß1 integrin were studied (a2β1 is expressed on the surface of platelets), including the effects on platelet function. Different sets of trials were conducted to study these effects. The first studies evaluated whether the binding of TMC-2206 leads to platelet activation according to the P-selectin up-regulation measurement or to the platelet-3 integrin activation that was measured using an antibody specific for the activation of allbß3 as PAC-1 . Human venous blood was collected from the ulnar vein using a number 21 needle from healthy donors who had stopped taking medication at least 10 days earlier in a volume of 1/10 acidified citrate-dextrose buffer (ACD: 85 mM of sodium citrate, 111 mM dextrose, and 71 mM citric acid, without adjustment in pH) containing 500 ng / mL of prostaglandin 12 (PGI2, Sigma-Aldrich) if it was to be used to create washed platelets. The whole blood was centrifuged at 160 μg for 20 minutes at room temperature and the platelet-rich plasma (PRP) was removed without disturbing the buffy coat. Washed platelets were prepared by diluting the PRP 2.5 times with saline citrate buffer solution with glucose (CGS, 13 mM trisodium citrate, 120 mM sodium chloride and 30 mM dextrose, pH 7.0) and PGI2 (500 ng / ml ), and centrifuged at 160 μg for 20 minutes at room temperature to remove any contaminating leukocyte. The supernatant was collected and centrifuged at 1100 [mu] g for 10 minutes and the resulting platelet pellet was gently resuspended in CGS buffer, washed and resuspended in normal Tyrodes-Hepes buffer (12 mM NaHCO3, 138 mM NaCl, 5.5 mM glucose, 2.9 mM KCl, 10 mM HEPES, 1 mM CaCl2, 1 mM MgCl2, pH 7.4). The platelets were left for recovery up to 30 minutes at 37 ° C. Then, washed platelets were counted before adding CaCl2 and MgCl2 at 1 mM. Washed rat platelets were prepared in a similar manner, although the blood was drawn from the vena cava to minimize platelet activation during blood extraction. 50 μL of freshly prepared PRP were incubated with 5 μg / mL of TMC-2206 or with mouse IgG control antibody for 30 minutes, washed and then incubated with goat anti-mouse labeled Alexa-594 in the presence or absence of P-selectin antibody labeled Alexa 488 (BD Pharmingen, catalog number 555523), PAC-1 antibody labeled Alexa 488 (BD Pharmingen, catalog No. 340507) or 150 ng fibrin geno labeled Alexa 488 (Molecular Probes) for 40 minutes at room temperature. P-selectin and PAC-1 are markers of platelet activation, and activated platelets are capable of binding to fibrinogen. At the end of the incubation period, the platelets were fixed with a volume of 1/10 of 4% paraformaldehyde and analyzed using a flow cytometer using a FACScalibur ™. The results of these experiments unexpectedly demonstrated that although the platelets clearly bind to TMC-2206, as indicated by a change in the record in the increase in fluorescence intensity observed in the treated TMC-2206 but not in the platelets treated with IgG control; there was no concomitant increase in the staining of P-selectin or PAC1, which indicates that the binding of TMC-2206 did not activate the platelets. The following studies evaluated whether the binding of TMC-2206 leads to platelet activation, according to the measurement of collagen-induced effects on platelet aggregation. Soluble collagen is a potent aggregator of platelet aggregation and is used as a routine measurement for platelet reactivity (see, for example, Hemostasis and Thrombosis, (2001), ed. Colman ei ai). Studies in mice with inhibited a2 have suggested that the platelets of these mice exhibit a slightly affected response to collagen (Holtkotter et al, J. Biol. Chem.277 (13): 10789-94 (2002) E. pub Jan, 11, 2002; Chen ei ai, Am. J. Pathol. 161 (1): 337-344 (2002)). To test whether TMC-2206 would have any adverse effect on platelet responses to collagen, platelet aggregation assays of humans and rats were performed by classical light transmission aggregometry using a Bio-data PAR4 aggregometer. The PRP was prepared as described above and the platelet count was adjusted to 3? 108 / mL. 450 μL of PRP or washed platelets were agitated with a magnetic count for 1 minute at 37 ° C in the presence of 5 μg / mL of TMC-2206 before adding Type 1 collagen from calf skin (Biodata Corp) to start the aggregation. The final volume of up to 500 μL was formed with Tyrodes buffer solution for washed platelet aggregation and with platelet poor plasma (PPP) for platelet rich plasma (PRP) assays. 500 μL of Tyrodes or PPP buffer were used as targets for the assays. PPP was prepared by making platelet pellets in PRP by centrifuging at 3000 rpm for 5 minutes in a microfuge. The index and the degree of platelet aggregation after the addition of soluble collagen was comparable in the presence or absence of T C-2206. Contrary to expectations, the results of these experiments showed that the binding of TMC-2206 to platelets did not have an effect on platelet aggregation induced by collagen when in vitro tests were performed at the concentrations evaluated. The following studies evaluated whether the binding of TMC-2206 leads to thrombocytopenia, a potential consequence of the binding of antibodies to platelets in vivo. (Hansen and Balthasar, J Pharmacol Exp Ther 298 (1): 165-71 (2001)). To test if thrombocytopenia would occur when administering TMC-2206, the rats were administered a dose of 10 mg / kg of TMC-2206 or murine IgG control by means of an IP injection. Before the injection, tail bleeding was used to measure the blood cell counts to have the reference value. Blood samples were taken at specific times after administration of the drug via IP (for example, 10, 30, 60 minutes and 4, 24 and 72 hours) of non-anesthetized rats by retro-orbital extraction using the capillary Unopette. Then, approximately 40 μL of blood was transferred to a tube containing 5 μL of ACD and immediately sampled in a Hemavet blood cell counter (Drew Scientific). The results of this study unexpectedly showed that there were no significant changes with respect to the platelet counts of the reference value at doses of 5 mg / kg or 10 mg / kg of TMC-2206. In contrast, the injection of 0.1 mg / kg of an antibody to another platelet receptor (allb antibodies, against CD41 (BD Pharmingen, CA)) induced thrombocytopenia, with the platelet count falling by almost 80% within 15 minutes after the administration of the antibody.
EXAMPLE 9The effects of antibodies against integrin a2 were studied in platelet adhesion to collagen including several subtypes of collagen. The integrin a2ß1 is the only integrin that binds to collagen, although it is not the only receptor for collagen, which is expressed by platelets. However, as discussed above, there are other mechanisms, especially in platelet activation, to facilitate firm adhesion to a collagen matrix. In this example, the ability of the TMC-2206 antibody to block platelet adhesion to Type I, II, III, IV and VI collagens, both for platelets at rest and for platelets activated with the moderate platelet agonist, ADP, was evaluated. Immulon II platelets were coated with collagen types I, II, III, VI (Rockland Immunochemical) and IV (Sigma, St. Louis, MO) that had been solubilized without foaming in 5 mM acetic acid, at a final concentration of 1 mg / mL. The wells were washed twice using modified Tyrode's-HEPES buffer solution without Ca ++ or BSA, but with 2 mM / Mg ++ and blocked with 100 μU of Tyrode's-HEPES buffer with 2 mM / Mg ++ and 0.35% BSA, but without Ca ++ Human venous blood was used for the preparation of platelets, including PRP, as described above, in Example 8. Platelet poor plasma (PPP) was created by centrifuging the PRP at 1100 xg for 10 minutes at room temperature. The resulting platelet pellet was gently resuspended for labeling in 1.0 mL of CGS (13 mM trisodium citrate, 120 mM sodium chloride and 30 mM dextrose, pH 7.0), transferred to a 5 mL tube with rounded bottom and a stock of 3 μL of CFSE (53.7 μM final concentration) was added with a gentle roll for exactly 20 minutes. The labeled platelets were diluted in CGS buffer and washed. The platelet pellet was resuspended in 1 mL of CMFTH buffer (5 mM HEPES, pH 7.3, 12 mM sodium bicarbonate, 137 mM NaCl, 3 mM KCI, 0.3 mM NaH2P0, 5 mM dextrose and BSA 0.35%) and remained in the dark as long as possible. Washed rat platelets were prepared in a similar manner, although blood was drawn from the vena cava in a syringe containing 500 ng / mL PGEi in ACD to minimize platelet activation during blood draw. Platelets with CFSE label were diluted to 2.0x105 / μL using Tyrode's-HEPES buffer containing 0.35% BSA. Labeled platelets (1.0x107 well) were applied to wells containing 20 μM of ADP in Tyrode's-HEPES buffer with 0.35% BSA and variable concentrations of the test inhibitor. Microtiter plates containing platelet mixtures were centrifuged at 550 x g for 10 minutes at room temperature followed by incubation in the dark for an additional 10 minutes. The wells were washed with Tyrode's-HEPES buffer. Fluorescence was read using a Victor2 fluorescence plate reader. To determine the ratio of fluorescence intensity to number of platelets, labeled platelets were diluted to several levels in Tyrode's-HEPES buffer solution containing 0.35% BSA (without Ca ++ or ADP), applied to wells coated with collagen Type I or Type IV, centrifuged, and CFSE fluorescence measurements were taken. As shown in Table 25, TMC-2206 blocked the binding to collagen under these static conditions, with an EC50 of 1.7 nM. Similar studies were performed with rat platelets using TMC-2206. The EC50 values to inhibit the binding of rat platelets to rat Type I collagen were 6.3 nM indicating a change in affinity of about 5 times for the rat compared to human a2β1 in platelets.
TABLE 25As shown in Table 25, TMC-2206 functioned as a potent inhibitor of platelet adhesion for fibrillar collagens, but was less potent for non-fibrilar Type IV collagen (10 nM compared to 1-2 nM). Unexpectedly in the presence of ADP, there was a decrease of approximately 10 to 20 fold in the potency to inhibit the binding to the fibrillar collagens and the antibody was no longer effective in preventing adhesion to Type IV collagen. These unexpected observations suggest that TMC-2206 and antibodies with the epitope binding specificity of TMC-2206 are less active in inhibiting the interactions of activated platelets for fibrillar collagen, and would have little or no effect [on the dosing scale]. Therapy] in the binding to Type IV collagen, the predominant collagen subtype of the wall of the endothelial vessels.
EXAMPLE 10The effects of antibodies against integrin a2 were studied by the time of bleeding. The experts in this subject had expectations that the administration of an antibody against a platelet integrin could cause bleeding disorders and cause an increase in the coagulation time in a subject receiving said antibody after an acute injury. To assess whether antibodies directed against integrin a2 would increase the propensity for bleeding in vivo, the effect of TMC-2206 on bleeding time in a rat was determined. The rats were given an IP or IV injection of TMC-2206 15 minutes before evaluating the bleeding time. The non-anesthetized rats were immobilized in a bra device and cut 0.8 cm from the tip of the tail immediately to initiate bleeding. The glue was quickly inserted into a beaker containing 30 mL of PBS maintained at 37 ° C. The time required for the tail to stop bleeding was recorded as the bleeding time. As shown in Table 26, the data demonstrated that administration of TMC-2206 doses up to 10 mg / kg had no significant effect on bleeding time.
TABLE 26EXAMPLE 11The effects of antibodies against integrin a2 were studied in a model of arterial thrombosis. Another potential manifestation of a bleeding disorder by the administration of antibodies that react to a2ß1 in platelets could be an increase in the time of thrombotic occlusion after arterial injury due to the undesired effects of platelet function. Therefore, antibodies against integrin a2 such as TMC-2206 were evaluated in a model of arterial thrombosis induced by ferric chloride in rats. It is a standard model that has been used for the development of antithrombotic agents and the activity manifests as a delay in the time of occlusion after exposure of the endothelial lining of the blood vessel to a FeCl 3 solution (Kurz eí ai, Thromb Res. 60 (4): 269-80. (1990); Hoekstra ei ai, J Med Chem. 42 (25): 5254-65 (1999)). The TMC-2206 antibody was administered to rats through a tail vein injection approximately 30 minutes before the induction of the arterial lesion in doses ranging from 1 mg / kg to 15 mg / kg. For IV injections, most antibodies were concentrated at 4-5 mg / mL to reduce the injection volumes required for the higher doses. The treatment groups were 1.0, 2.5, 5.0, 10.0 and 15 mg / kg of TMC-2206, 5.0 mg / kg of control IgGI (?) Murine (clone MOPC21), 5.0 mg / kg of anti-vWF (DAKO) polyclonal rabbit or saline solution; Each treatment group included 3 or 4 animals. Sprague-Dawley rats (Harían) weighing 220 to 270 grams were anesthetized with 60 mg / kg of sodium pentobarbital. Once they reached a sufficient level of anesthesia, the carotid artery was exposed and placed on a piece of filter paper (4 mm x 5 mm) folded along the 4 mm side to cradle the carotid artery and provide a surface so that the ferric chloride (35%) bathes the carotid. Twelve μL of 35% FeCl3 were applied for 5 minutes, then the paper filter was removed and the flow probe of a flow system from Transonic Systems Inc. (Ithaca, NY) was placed around the carotid artery. The flow was measured for up to 45 minutes. Mean and SEM values were recorded for the flow scales of several animals per group at specific time points after the administration of ferric acid. There were no significant differences in the time of occlusion observed with any of the doses of TMC-2206 that were evaluated, even as high as 15 mg / kg, compared with the saline control, which indicates that there do not seem to be any effects adverse events in thrombosis due to the administration of TMC-2206. Although the values of initiation of the flow can vary substantially between the animals, the occlusion time occurred consistently between 10 and 16 minutes after the administration of ferric chloride in the groups treated with TMC-2206, which was very similar to the groups treated with saline and with IgG control, which had mean occlusion times of 12 and 14 minutes, respectively. The only treatment evaluated that was associated with prevention of occlusion was the positive control, a polyclonal anti-vWf antibody, which did not result in the reduction of flow parameters for periods as long as 45 minutes after the addition of FeCl 3 .
EXAMPLE 12The binding properties of the a2 integrin antibodies were studied, including epitope mapping studies, to characterize the nature of the TMC-2206 binding site in the a2 integrin subunit. An antibody against the a.2 integrin that binds directly to the target binding site and that serves as a direct competitor for ligand binding, could be the cause of platelet activation upon binding to the a2ß1 integrin. Alternatively, an antibody to integrin a2 that binds to integrin a2ß1 in an inactive state and that does not cause the integrin to become active could have a non-platelet activating profile similar to that unexpectedly found for TMC-2206. Antibodies with the same binding epitope or one similar to that of TMC-2206 will inhibit cell adhesion of leukocytes to collagen, and therefore will have significant therapeutic utility, but will not be associated with the bleeding complications that an antibody might have. that it binds to, and activates, the a2ß1 integrin. Studies were conducted to investigate whether the epitope recognized by TMC-2206 was within the ligand binding of domain I of the a2 integrin subunit, or whether it solely depended on the presence of an intact I domain (Hangan ei ai, Cancer Res. 56: 3142-3149 (1996)). For those studies, a GST-domain I fusion protein from a2 was created using a modified version of the protocol described by Tuckwell ei ai, J. Cell Sci. 108 (Pt 4): 1629-37 (1995). The human a2 domain I was cloned from the mRNA isolated from approximately 10 6 CHO cells expressing human a2 integrin (Symington et al., J Cell Biol. 120 (2): 523-35. (1993) .The cells were lysed in Trizol reagent. (Gibco) and chloroform was added to extract the aqueous stage before adding 0.2 volumes of isopropanol to precipitate the RNA that was collected by centrifugation and resuspended in RNA-free water.The primers flanking human a2 domain I were synthesized by Sigma -Genosys The primers were designed with ßamHI and EcoRI sites at the 5 'and 3' ends respectively to be cloned into the vector pGEX-2TK (GE Biosciences) The halphal F (5'GGGGATCCAGTCCTGATTTTCAGCTCTCAG; SEQ ID NO: 117) primers and halphal R (5'GGGAATTCAACAGTACCTTCAATGCTG; SEQ ID NO: 118) (see Table 27) were used for a single-step RT-PCR reaction employing a standard Qiagen kit to amplify amino acids 123 to 346 of the integrin subunit 2 matures and incorporates a Bam \? \ Site at the amino-terminus (which adds a GS upstream of residue 124 of domain I) and an additional EFIVTD hexapeptide as part of the EcoRI cloning site to the stop codon. A single band was detected by agarose gel electrophoresis. The PCR reaction was cleaned using a Qiagen PCR Quick kit, the product was digested with restriction enzymes and cloned into the vector pGEX-2TK (Amersham, GE) using standard molecular biology techniques. The transformed bacteria were selected for the inserts and several clones were sequenced using a CEQ system from Beckman-Coulter. The deduced amino acid sequence that was cloned was identical to the available sequence of a human a2 domain I (SEQ ID NO: 11, which is shown in Table 28). A single clone containing the correct insert of the DNA was amplified in DH5a cells (Invitrogen) and retransformed in electrocompetent bacteria BL21 (Invitrogen).
TABLE 27GST fusion protein with human a2 domain I was expressed in a logarithmic BL21 bacterium using IPTG as an inducing agent. Approximately 4 hours after the induction, the bacterium was harvested and pelletized at 3000 RPM in 50 mL conical tubes. The pellet was resuspended in PBS containing 1% Triton X-100 and protease inhibitors. The homogenate was sonicated for 1 minute and centrifuged at 3000 RPM to clean the lysate of cell debris. The GST fusion protein was purified from bacterial beads using glutathione sepharose beads (GE-Amersham) according to the manufacturer's instructions and eluted in TBS (pH 8.0) containing 20 mM free of glutathione. Purified a.2 GTS-domain I joined collagen with the same specificity as previously reported (Tuckwell et al., J Cell Sci. 108 (Pt 4) - 1629-37 (1995)), that is, a higher affinity for Type I collagen compared to Type IV collagen. It was bound to immobilized TMC-2206 with an apparent Kd of 0.31 nM by ELISA, which was comparable to the observed affinity of TMC-2206 binding to intact a2β1 integrin of 0.37 nM derived from the direct binding studies described in Example 2. Next, the soluble a2 GST-domain I fusion protein was evaluated for its ability to compete with TMC-2206 labeled Eu for binding to a2β1 coated plates as described in Example 2. The value for soluble GST-domain I of soluble a2 was found to be similar (0.18 nM compared to 0.28 nM) to that obtained for unlabeled TMC-2206, which indicated that the binding site for TMC-2206 is within the domain I of a.2 and did not require the presence of subunit ß1. Studies were carried out to investigate the dependence of cations of the union by means of TMC-2206. The cation dependency indicates that a binding portion is targeting the divalent cation binding site (MIDAS) of an integrin, and therefore acts as a mimetic ligand. The collagen that binds to a2 is dependent on Mg ++ under normal physiological conditions, whereas no binding occurs when Mg ++ is replaced by Ca ++ (Staatz ei ai, Cell Biol. 108 (5): 1917-24 (1989); Emsiey ei ai, Cell 101 (1): 47-56 (2000)). For these studies, the GST-domain I fusion protein from a.2 was immobilized on Reacti-Bind microtiter plates coated with glutathione (Pierce Biotechnology, Inc. Rockford, IL) and the ability of TMC-2206 with Eu tag was determined for join under different conditions of the cations (free of Ca and Mg, Ca ++ or Mg ++ in concentrations ranging from 0.1 μM to 3 mM). Plates were coated by incubating 100 μL / well of GST-domain I a2 fusion protein (2.0 μg / mL in cation-free binding buffer: 50 mM HEPES, pH 7.4, 150 mM NaCl and Tween-20 0.5%) for 1 hour at room temperature, in addition to the wells were washed four times in divalent cation-free washing buffer. Wells were blocked using 100 μL / well of blocking buffer (wash buffer containing 3.0 mg / mL of IgG-free BSA [Jackson ImmunoResearch Laboratories, Inc., West Grove, PA]) for 1 hour at room temperature were washed four times in divalent cation-free wash buffer and soaked in divalent cation-free wash buffer (300 μL / well) for 45 minutes at room temperature. The wells were equilibrated in wash buffer (300 μL / well) containing the desired level of divalent cations for 30 minutes and then incubated for 1 hour at 37 ° C in the presence of 41 pM, 199 pM 345 pM, 1 nM of TMC-2206 with Eu tag or control antibody. The murine TMC-2206 antibody bound in a concentration-dependent manner, with similar potency under all conditions, which indicated that its binding to domain I of a2 was cation-independent, and therefore did not involve the MIDAS site . The IgG control with Eu tag was not bound to the wells coated with integrin a2ß1, which confirmed that the binding was specific. Additional studies were conducted to investigate the binding site of TMC-2206. Integrin ligands usually have a key acid that forms the final chelating link for the divalent metal ion (Haas and Plow, Curr Opin, Cell, Bio, 1994, Lee et al, Structure 1955) a feature shared by many. integrin antagonists, including the anti-ategrin antibody a1 and mAb AQC2 (Karpusas ei ai, J. Mol. Biol. 2003) wherein the acid is provided by residue D101 within CDR-H3. By analogy, the D100 of the CDR-H3 of TMC-2206 could provide a similar interaction with the a.2 MIDAS. Therefore, two variants of antibodies containing murine VH were generated, one carrying a D100A mutation and the other carrying a D100R mutation. Their ability to compete for the Eu-TMC-2206 binding in the K i assay was then evaluated in comparison to the mouse-human chimeric TMC-2206 antibody. Mutant D100A was completely inactive at concentrations up to 0.9 μM which represented a major shift to 1600 fold in the relative potency of the mouse-human chimeric TMC-2206 antibody. In contrast, the reverse charge D100R mutant D100R was almost as potent as the mouse-human chimeric TMC-2206 antibody as evidenced by similar K-values (0.41 nM compared to 0.52 nM). This offers evidence against any paper for the D100 residue of TMC-2206 by coupling to the metal chelation complex that forms the MIDAS ligand site.
Additional studies were conducted to investigate the binding specificity for TMC-2206, including epitope mapping studies, with this murine monoclonal antibody that is directed against domain I of the human a2ß1 integrin. TMC-2206 has a cross reaction with the rat a2ß1 integrin but does not cross-react with the mouse α2β1 integrin. Because integrin a2ß1 proteins share a large homology across species, residues within the a2ß1 that are important for antibody binding were identified by methods that could identify the differences that exist between species that have a reaction crossed with the antibody compared with those that do not (for example, Champe ei ai, J. Biol. Chem. 270 (3): 1388-94 (1995); Karpusas ei ai, J. Mol. Biol. 327 (5) ): 1031-41 (2003), Bonnefoy ei ai, Blood 101 (4): 1375-83). The crystal structure I of the a2 integrin domain alone and when complexed with its target ligand, collagen, has been analyzed (Emsiey et al, J. Biol. Chem. 272 (45): 28512-7 (1997); Emsie et al. ai, Cell 101 (1): 47-56 (2000)). A comparison of the sequence of a2 I of human (SEQ ID NO: 11), a2 I of rat (SEQ ID NO: 93), and a2 I of mouse (SEQ ID NO: 94) are shown in Table 28. domains were obtained from Genbank shipments. This analysis reveals that the mouse domain I contains 14 residues that differ from the a2 domains I of both a rat and a human (shown with bold and underlined in Table 28). These residues were used to further study the binding epitope of TMC-2206.
TABLE 28Both the GST-domain I of mouse a2 and the rat were cloned as GST fusion proteins to confirm that the appropriate cross-reactivity was retained by the respective I domains by PCR methodology as described in Example 3. The domain I of murine a.2 was cloned from mRNA isolated from a Balb / C mouse kidney by RT-PCR using malphal F primers (SEQ ID NO: 121) and malphal R (SEQ ID NO: 122), and domain I of rat a2 was taken from a Sprague Dawley rat kidney by RT-PCR using the primers ralphal F (SEQ ID NO: 123) and ralphal R (SEQ ID NO: 124). In addition, the I domains of primate non-human a2s were cloned from pellets of white blood cells obtained with low speed centrifugation of fresh blood extracted from rhesus and cynomolgus monkeys. Afterwards, the white blood cells were rapidly frozen in liquid nitrogen. A total of 5 * 106 (rhesus) and 2 * 106 (cynomolgus) cells were lysed in 1 mL of Trizol (Invitrogen, Cat # 15596-026) and the total RNA was prepared as described above. The final pellet of RNA was resuspended in 50 μl of H20 treated with DEPC. This served as the template for the first stage of reverse transcriptase (RT). The RT reaction consisted of 8 μl (2.24 μg for Rhesus mRNA, 1.44 μg for cynomolgus mRNA) of cellular RNA, 1 μl (10 mM) of DNTP and 1 μl (2 μM) of human domain I forwarding primer (GGGGATCCAGTCCTGATTT; SEQ ID NO: 119). This mixture was incubated at 65 ° C for 5 minutes, cooled on ice. Then 5 μl of this cDNA was used as the template for the PCR amplification reaction, using the human primers forward and backward (forward: GGGGATCCAGTCCTGATTT, SEQ ID NO: 119; backward: GGAATTCAACAGTACCTT, SEQ ID NO: 120) . Cycle times were 1 cycle at 94 ° C for 30 seconds, 94 ° C for 30 seconds, 55 ° C for 30 seconds, 40 cycles at 68 ° C for 1 minute and 1 cycle at 68 ° C for 5 minutes . The PCR products were separated by electrophoresis in 1% agarose gel and the band (of the expected size) was purified directly from the agarose gel, digested with SamHI and EcoRI and cloned into the same sites in the vector pGEX-2TK and transformed into BL21 bacteria.
Individual colonies were isolated and the inserts were sequenced using a Beckman CEQ 8000 DNA analyzer to verify the identity of murine and rat a.2 domain I., and determine the sequence homology of the two species of monkey with human. The cloned murine sequence showed an exact identity with the region of domain I of the deposited sequence, NM_008396.1. Similarly, the cloned rat sequence was identical to the entry in Genbank for the rat integrin, XM_34156.1, with the exception that the cloned sequence contained 6 additional residues for the deposited sequence, which allowed the region between residues 16 and 21 (residues 139 to 144 of the intact a2 integrin) of the rat domain were accurately translated. This amino acid sequence, ACPSLV, was identical to that of the mouse residues at these positions. At the nucleotide level the two primate sequences showed a very strong homology with the human a2 domain I sequences. The I domain of rhesus a2 in the nucleotide sequence (SEQ ID NO: 104) only showed a difference with the human nucleotide sequence, within codon 50, a change from CTT to CTG, but because both encode a leucine, the deduced protein sequences were identical to the human. The nucleotide sequence of domain I of cynomolgus a2 (SEQ ID NO: 103) was identical to human except for codon 40, where there was a change from human AAG to GAC. This results in a change from a lysine to a residue of aspartic acid in this position. However, subsequent studies revealed that this nucleotide change is due to a polymorphism that is not conserved among animals, since another cynomolgus exhibited a 100% homology to domain I of human a2 (see Example 18). Subsequently, the fusion proteins were expressed and purified as described above for the GST-domain I fusion protein of human a2. Analysis of the material eluted from the glutathione sepharose column indicated that the rodent fusion proteins contained aggregated forms. Therefore, these and the primate fusion proteins were further purified by size exclusion chromatography on a Sephadex 75 10/30 column (GE-Amersham) (primate) by FPLC in an Akta-Basic FPLC system (GE-). Amersham) to produce a monomer fraction. Then, the GST fusion proteins were tested for their binding ability with immobilized TMC-2206, as well as for their ability to compete with TMC-2206 with Eu tag for immobilized human a2β1 integrin binding. The assays were performed as described above in Example 2. To evaluate direct binding to TMC-2206, Immulon 4 plates were coated using 50 μL of a bicarbonate solution (pH 9.0) containing 5 μg / ml of TMC- 2206 The plates were sealed and the coating was left overnight at 4 ° C. The next morning, the plates were washed twice with TBS solution and then blocked using 200 μL of the blocking solution described above for 1 hour at room temperature with shaking. After blocking, the blocking solution was removed but the wells were not washed. Instead, a serial dilution of GST fusion protein was made, added to the wells and then incubated for 2 hours at room temperature with shaking. Then, the wells were aspirated and a TBS wash buffer was applied for 5 minutes at room temperature. The washing step was repeated two more times before the secondary antibody was applied. The secondary antibody stage consisted of rabbit antibody against conjugated GST of Amersham HRP diluted 1: 2000 in blocking buffer. 100 μL of the secondary antibody was added to each well and incubated at room temperature for 1.5 hours with shaking. The wells were again aspirated and washed three times with washing buffer before adding the reaction mixture of the substrate. Then 100 μL of substrate reaction mixture (1: 1 TMB dilution kit) was added to each well for six minutes. The reaction was stopped by adding 100 μL of 0.1 M H2SO4. The reaction within the wells was read and quantified by absorption spectrophotometry using the Molecular Dynamics plate reader and the associated Softmax software, respectively. The Kd values were then estimated from the EC50 values using the Prism software (Graphpad, CA). There was a 3-fold change in Kd for the binding of rat a2 to TMC-2206 compared to human a2, whereas the murine a2 GST-domain I fusion protein showed only a light specific binding at the highest concentrations, representing a change of more than 1500 times in affinity (see Table 29). The rhesus GST-a2 I showed affinity comparable to the human, whereas unexpectedly the GST-a2 I of cynomolgus monkey showed no detectable affinity for TMC-2206 at concentrations up to 1 μMa. These relative classifications were also observed in the Ki test. The lack of cross-reactivity of the GST-domain I cynomolgus fusion protein for TMC-2206 indicates that the K40 residue can be a determinant of the epitope. Comparison of the difference in affinity of GST-a2 I cloned from rat. { Kd of 0.54 nM and K, value 3.8 nM) with the fusion protein GST-domain I of human a2. { Kd of 0.18 and K value of 0.33 nM) is consistent with the change in EC50 values found in TMC-2206 assays for its ability to antagonize the adhesion of fresh rat platelets compared to human platelets for Type I collagen, as described above in Example 9. Similarly, the lack of cross-reactivity of the GST-I domain of a2 fusion for mouse TMC-2206 is consistent with the lack of cross-reactivity of the antibody with intact a2β1 integrin. of mouse.
TABLE 29kND indicates no detectable in concentrations up to ~ 1μMIn further studies, the 14 residues corresponding to the only differences in murine a2 domain I when compared to human and rat a2 domain I were individually mutated in the human a2 domain I fusion protein cloned by PCR using standard methods of molecular biology (the primer sequences are shown in Table 30). Clones of individual bacteria were sequenced to verify that the correct mutation had been incorporated into domain I. One of the attempted variants, the mutant G101R, did not produce a correct clone and was not further studied. The primers designed to create the Y93H mutation resulted in a set of clones that instead carried the Y93D mutation. Both variants of Y93 were evaluated. The rest were correct in their sequence. Variants of the resulting proteins were expressed and purified as described above for the wild type a2 human GST-domain I fusion proteins. They were then evaluated to determine their activity in three ways: first, by their relative ability to bind to the different collagens and ensure that the mutations did not introduce flagrant conformational perturbations that interfered with ligand binding; second, because of its apparent affinity for TMC-2206 (direct binding to immobilized TMC-2206 measured by ELISA) and third, for its ability to act as competitive ligands in the KI assay. The apparent K and Kd data are also summarized in Table 30.
TABLE 30ND = not detectable in concentrations up to ~ 1μM Of the 13 residues evaluated, 12 were changed to the murine counterpart with minor effects on affinity, but changes in Y93 caused a marked loss in affinity, as shown in Table 31. The Y93D mutation suppressed the antigen-binding ability even at 3-log concentrations above the? Value for the wild-type GST-domain I fusion proteins. The change to murine histidine (Y93H) caused a 23-fold decrease in apparent affinity for the TMC-2206 antigen. Both mutations suppressed the ability of GST domain I to antagonize the binding of the Eu-labeled antibody to its antigen. Changing murine H93 to Y conferred the ability of murine a2 domain I to bind to TMC-2206, albeit with a 200-fold decrease in the relative potency of wild-type human a2 domain I, as shown in Table 31 .
TABLE 31Comparison of the crystal structures for the I domain of human a2 in the closed conformation (NCBI PDB entry 1AOX) and the open binding of the ligand (PDB entry 1 DZI) reveals that Y93 is located on one side of the I domain that it is behind helix a7, which was shown to undergo a long downward movement with ligand binding (Emsiey et al, J. Biol. Chem. 272: 28512 (1997) and Cell 100: 47 (2000)). Although it was not previously identified as a conformational change associated with ligand binding, examination of the crystal structures indicates that in the closed conformation, the aromatic ring of Y93 extends out of the surface of the protein, but it flips sideways and downwards to align along the face of domain I in the open conformation of the ligand binding. To investigate whether the binding of TMC-2206 to integrin a2ß1 depends on a given conformational state, mutations were introduced in domain I to favor an open conformation of domain I. It has been reported that the E195W mutation (E318 in integrin a2 intact ) blocks the human a2 domain I in the open conformation (Aquilina ei ai, Eur. J. Biochem. 269 (4): 1136-44 (2002)) so its use allows to make a distinction to know if an antibody recognizes a conformation dependent on activation or not. In addition, crystallography studies have shown that E195 forms a buried salt bridge with the R165 residue located in the aC cycle, which serves to maintain the aC cycle in a conformation that protects the site from ligand binding (Emsiey eí ai, Cell 100: 47 (2000)). The aC cycle assumes an extended conformation in the open position and it has been postulated that both the R165 residue and the adjacent R166 residue contribute to the collagen binding (Emsiey et al, J Biol Chem 272: 28512 (1997) and Cell 100: 47 (2000); Kápylá eí ai, J Biol Chem 275: 3348 (2000)). Therefore, four mutations were constructed, the E195W; an R165D mutation to reverse the charge and therefore break the salt bridge that is formed with E195W in the closed conformation, and a N166D mutation, again to reverse the charge inside the aC helix. The change of E195W caused a 45-fold decrease in K-values, as shown in Table 31 indicating that the TMC-2206 antibody exhibits a higher affinity for closed conformation. Both the change in R165D and in N166D suppressed the ability of domain I to bind to the TMC-2206 epitope even at concentrations as high as 1 μM, suggesting again that the TMC-2206 antibody recognizes a closed conformation. From studies of mutagenesis and conformation, it seems that theY93 in the closed conformation may play a role in the binding of TMC-2206, and may provide a determinant for the binding specificity of the species. The unexpected results obtained with domain I of polymorphic cynomolgus indicated that the K40 residue may also play a role in the antigen-TMC-2206 interaction. A computer modeling of the TMC-2206 antibody indicated that the CDRs form a relatively flat binding site, which suggests that the antibody makes multiple contacts with the antigen. Because many residues within the CDRs are loaded, the charged residues surrounding Y93 in the closed position that also show marked positional changes in the open conformation were identified from the PDB structures of the two open and closed conformators as K40 , R69, N73 and Q89. The loads of three of these residues were inverted when generating the following mutants, K40D, R69D and N73D, and modified in the fourth by generating a variant of Q89H, as shown in Table 31. In addition, a third variant of the residue was made 93, a change from tyrosine to phenylalanine, to determine whether the aromatic character of tyrosine was the important structural feature, or whether the activity was dependent on the aromatic hydroxyl character that is particular to tyrosine. In the case of this set of mutations, all were subjected to HPLC purification to enrich the monomeric fraction of the protein preparations obtained from the glutathione sepharose affinity column. First, the functionality of each variant was tested when evaluating collagen binding. All, except for the R69D variant, were linked to the collagen with an EC50 value similar to that of domain I of wild-type human a2. Consequently, the R69D was not studied further. Of the remaining mutants, the introduction of the K40D variant abolished the ability to compete for binding to the TMC-2206 epitope. This was consistent with the results obtained with domain I of cloned cynomolgus showing polymorphism in this residue (change from lysine to aspartic acid). Likewise, the Y93F mutation also repressed the ability to compete for the union of EU-TMC-2206. N73D and Q89H showed a decrease of 7.8 and 15.9 times in the Ki values respectively (Table 30). Taken together, the mutation information indicates that residues K40, Y93, R165 and N166 may be determinants for the binding of TMC-2206 to its epitope, and that N73 and Q89 also contribute to binding energy. These data indicate that the antibody TMC-2206, its derivatives and antibodies such as TMC-2206 (for example, AK7) that recognize the same epitope as TMC-2206 or a similar one,. { see for example, Example 13) are atypical antagonists, not mimetic ligands of 2ß1-collagen interactions. This conclusion is supported by i) its ability to block adhesion mediated by integrin a2ß1 to collagen independently of divalent cation, ii) this inhibition does not involve the interaction of a critical acid group, such as D100 within H-CDR3, with MIDAS, iii) the antibody binds to the surface of domain I that is distal to the direct site of ligand binding, iv) the binding site of TMC-2206 favors the closed conformation of the receptor and spans amino acid residues K40 , N73, Q89, Y93, R165 and N166. Consequently, TMC-2206 and antibodies such as TMC-2206 (for example, those recognizing the same epitope as TMC-2206 or a similar one) will not support the integrin-mediated out-in signaling that would normally occur upon contact with the ligand of cognate collagen, and it is this mode of binding that could contribute to a non-bleeding profile of this antibody and antibodies such as TMC-2206.
EXAMPLE 13Studies were conducted to compare the binding of other antibodies with blocking of functions against integrin a2 with TMC-2206. The results of the mapping studies described in Example 12 indicated that the TMC-2206 antibody appeared to bind to a closed conformation of the a2 integrin domain I and / or did not act as a mimetic ligand. These unexpected results, together with the unexpected results of the studies related to the platelets described in Examples 8, 9, 10 and 11, demonstrated that the epitope of TMC-2206 is particularly advantageous and that antibodies similar in their functional properties to TMC-2206 They are particularly useful. Selection methods to identify those similar antibodies were developed as described herein, and antibodies were identified according to those methods. To determine which blocking antibodies to functions against integrin a2 bound in a manner similar to TMC-2206, a series of cross-competition studies were performed. For studies of commercially available human a2 integrin antibodies, the GST-domain I fusion protein of a2 was immobilized on microtiter plates as mentioned above. The antibodies evaluated were AK7 (Mazurov ei ai, Thromb Haemost 66 (4): 494-9 (1991)), P1E6 (Wayner ei ai, J. Cell Biol. 107 (5): 1881-91 (1988)) , 10G11 (Giltay ei ai, Blood 73 (5): 1235-41 (1989)) and A2-11 E10 (Bergelson ei ai, Cell Adhes, Commun. 2 (5): 455-64 (1994)) available at the comercial con Chemicon, (Temecula, CA; catalog numbers, CBL477 (AK7); MAB1950 (P1 E6); MAB1988 (10G11) and Upstate, (Waltham, MA; A2-IIE10, catalog number 05-227), respectively. Antibodies were tested along with the same batch of a2ß1 platelet coated microtitre plates used in the epitope mapping studies to determine their ability to antagonize the Eu-tagged TMC-2206 binding. In another set of studies the ability of antibodies to antagonize the binding of platelets at rest newly isolated with type I collagen was determined. Therefore, the ability of different antibodies directed against human a2 integrin to antagonize the binding of the TMC-2206 antibody with Eu tag to microtiter plates coated with a2ß1 platelet was measured as K1 values, and the ability to antagonize the adhesion of platelets at rest with collagen type I under static conditions was measured as EC50 values. The results, presented in Table 32, demonstrate that the AK7 antibody is an effective competitor of TMC-2206. Clone 10G11 showed clear biphasic competence of TMC-2206, suggesting that it did not act as a simple competitive antagonist. A2-IIE10 showed a 10-fold change compared to TMC-2206 in blocking adhesion to the platelet, but a change of approximately 350 times in its ability to compete with TMC-2206 with Eu tag, which again indicates that there was no a direct agreement between the two antibodies. P1 E6 failed to demonstrate any effect in any of the trials, which indicated that it recognizes an activated conformation.
TABLE 32k Not detected under the conditions described for the testThese data demonstrate for the first time that not all antibodies with blocking function bind to integrin a2 in the same way, in addition to demonstrating methods for the identification of a new subgroup of antibodies similar in epitope specificity to TMC-2206 with similar activities blocking functions. These data also demonstrate that this new subgroup of antibodies against integrin a2, which includes TMC-2206 and antibodies similar to TMC-2206 in epitope specificity, is characterized by an unexpected lack of hemorrhagic complications in vivo and / or a lack of activation of integrin a2ß1 in platelets. The specificity of the epitope, the activities of blocking functions and the advantages (for example, the non-activation of platelets) are not characteristic of all antibodies against human a2ß1 integrin with function blockage, but rather a new characteristic of a new subgroup of antibodies including TMC-2206 and similar antibodies, including derivatives and / or variants of TMC-2206 that can be identified and / or selected as described herein. Having shown that not all antibodies that block functions and bind to domain I of a2 bind to the same TMC-2206 epitope or to a similar one (for example, overlaps), studies were conducted to determine whether the surrogate antibody used for studies of effectiveness in murine had properties similar to those of TMC-2206. Because the Ha1 / 29 antibody cross-reacts with the rat and mouse a2 integrin, since the TMC-2206 antibody binds to the a2 integrin of both human and rat, the rat GST fusion protein was used to determine if the two antibodies bind to the overlapping sites (e.g., shared epitope specificity). For this, the rat a2 GST-domain I fusion protein was immobilized in Reacti-Bind microtiter plates coated with glutathione (Pierce Biotechnology, Inc. Rockford, IL). First the Kd of Eu-TMC-2206 binding was determined with GST-domain I of human a2 and of rat at 37 ° C as described in Example 2. The Scatchard analysis of Eu-TMC-2206 bound against free indicated that the values ranged from 0.2 nM for domain I of human a2 and 1.3 nM (a decrease of 6 times) for domain I of rat a2. Then, the ability of TMC-2206 with Eu tag to bind the rat a2 domain I in the presence of different concentrations of the competitive antibody was evaluated as described in Example 2 using the K n value of 1.3 nM to derive the Ki value from the EC50 values observed. The Ha1 / 29 antibody (Mendrick and Kelly, Lab Invest. 69 (6): 690-702 (1993)), but not the HMa2 (Miyake ei ai, Eur. J. Immunol., 24: 2000-2005 (1994)) was an effective antagonist of the Eu-TMC-2206 binding, indicating that the Ha1 / 29 antibody bound to sites that are similar (eg, overlapped) to the binding site of TMC-2206.
EXAMPLE 14Another study was performed on exemplary IgG4 antibodies having a heavy chain hVH14.0? 4 (SEQ ID NO: 174) or a heavy chain hVH12.0? 4 (SEQ ID NO: 176) and a hVL light chain 10.0Q (SEQ. ID NO: 178). This study evaluated whether the binding of these IgG4 antibodies leads to platelet activation, according to the measurement of collagen-induced effects on platelet aggregation. Blood samples were collected through venipuncture of the antecubital vein in vacuum-filled tubes containing 3.8% sodium citrate after discarding the first 3.0 ml of free circulation blood. All antibodies were diluted in saline at final concentrations of 140 μg / ml. Each disposable cuvette (containing a disposable electrode assembly) was proportionally divided into 0.5 ml of citrated whole blood and 0.5 ml of saline or of an antibody solution. Each cuvette was preheated at 37 ° C for 5 minutes in the warming well of the aggregometer (Model 591A, Chrono-Log, Havertown, PA), then placed in the reaction well, the set of the reference value, and then they added 20 μl of saline or collagen solution (1 mg / ml; equine type I, Chrono-Log)) to start the aggregation reaction. During the aggregation an accumulation of platelets formed on the exposed surfaces of the electrodes, which resulted in an increase in impedance. Data acquisition continued for 6 minutes with the impedance change (? O, ohms) recorded by a plotter (Model 707, Chrono-Log). The data (Table 33) were analyzed with the Kruskal-Wailis test, which evaluated the hypothesis that the medians of the population of each of the four groups (saline or collagen) were equivalent, and would reject this hypothesis (95% of confidence) if the P values were less than or equal to 0.05. For the saline group (P value = 0.148) neither the isotype control nor the two humanized antibodies induced human platelet aggregation compared to the negative control of the saline solution. For the collagen group (P value = 0.201), neither the isotype control nor the two humanized antibodies inhibited collagen-induced aggregation compared to the negative control of saline. The results of this study and those of Example 8 show that binding of TMC-2206 and humanized IgG4 variant antibodies had no effect on collagen-induced platelet aggregation when evaluated in vitro at all concentrations tested.
TABLE 33EXAMPLE 15The humanized TMC-2206 (hlgG4 /? VH12.0? /L10.0Q) was evaluated for its ability to block the binding of cell adhesion mediated by integrin a2ß1 with collagen type I using CHO-a2 cells, HT1080 cells (fibrosarcoma human) and human platelets following the procedures delineated in Example 2. Humanized TMC-2206 was a potent inhibitor of collagen cell attachment with EC50 values comparable to those of TMC-2206 (Table 34).
TABLE 34EXAMPLE 16The humanized TMC-2206 was evaluated to determine its ability to bind s1 ß1 human immobilized in an ELISA format. Human a1 ß1 integrin (Chemicon International) was diluted in coating buffer (25 mM Tris, pH 7.5, 150 mM NaCl, 1 mM MgCl2) to a final concentration of 0.5 μg / ml. 96 well immunoplates were coated with a1 ß1 at 50ng / well and incubated overnight at 4 ° C. The plates were washed three times with wash buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 2 mM MgCl2, 0.5% Tween-20) and blocked with 5% w / v / skim milk in Wash buffer solution for one hour at room temperature. Humanized antibodies TMC-2206, IgG4 /? human (isotype control), a1 anti-human mouse (FB-12, Chemicon International) were serially diluted in buffer buffer (0.1 mg / ml BSA, IgG free, in wash buffer). Fifty microliters / well of diluted antibody solutions were added to the plates coated with a1ß1, incubated for one hour at room temperature and then washed three times. The anti-IgG conjugate with alkaline phosphatase prepared from an antihuman goat antibody (secondary antibody, Jackson ImmunoResearch Laboratories, West Grove, PA) was added to the wells containing the isotype control and the humanized TMC-2206; the anti-IgG conjugate with alkaline phosphatase prepared with an anti-mouse antibody (Sigma) was added to the wells containing FB-12. After one hour of incubation at room temperature the plates were washed three times, incubated in substrate solution (1 mg / ml 4-nitrophenyl phosphate, 0.1 M diethanolamine, 5 mM MgCl 2, pH 9.8) for 20 minutes and finished with NaOH. The absorbance (405 nm) was read using a Spectramax Plus plate reader with Softmax Pro software. Similar to TMC-2206, humanized TMC-2206 and IgG4 /? they did not join a1ß1. The antibody against the control a1ß1 integrin (FB-12) was bound to a1ß1 with an EC50 of 0.79 ± 0.15 nM.
EXAMPLE 17The KD and Kj values for humanized TMC-2206 and TMC-2206 MAbs that bound to immobilized a2β1 were determined using the competitive binding assay. The wells of a 96-well microtiter plate were coated with platelet α2β1 integrin (custom coating of human platelet a2β1 by GTI Inc., Wl) and then blocked with skim milk. The humanized antibody TMC-2206 was labeled with the reagent Eu-N1-ITC, approximately 2 mg were suspended and dialysed against a phosphate-buffered saline solution (PBS, 1.47 mM KH2P04, 8.1 mM Na2HP04, pH 7.4, 138 mM NaCl and 2.67 mM KCI). After concentration in prewashed MicroSep concentrators (30-kDa limit, Pall Life Sciences at 9500 rpm (7000 xg) in a JA-20 rotor (Beckman Instruments, Inc.) for 20 minutes at 4 ° C), the antibodies were adjusted at 4.0 mg / mL with PBS containing a final concentration of 100 mM NaHCO3, pH 9.3. The MAb / bicarbonate mixture (0.250 mL) was gently combined in a flask containing 0.2 mg of acid? / P (p-benzyl-isothiocyanato-J-diethylenetriamine - ^? / ^? - ^ - tetraacetic chelated with Eu3 + (Eu-N1- ITC; Perkin Elmer Life Sciences) and incubated overnight at 4 ° C without shaking.The labeled antibody mixture was applied to a PD-10 column (GE Biosciences, Piscataway, NJ) pre-equilibrated with run buffer (50 mM Tris, pH 7.4 and 138 mM NaCl.) Fractions (0.5 mL) were collected and assayed for total protein (Bradford reagent).; Bio-Rad Laboratories, Hercules, CA) using an absorbance reader on SpectraMax 384 plates and for europium after a 1: 10,000 dilution in DELFIA Enhancer Solution (Perkin-Elmer) by time resolved fluorescence (TRF) using a Victor2 Multi reader -label Píate (Perkin Elmer). Fractions that were positive for protein and europium label were pooled and applied to a new PD-10 column, and samples collected and assayed for total protein and for europium content by TRF calibrated against a standard europium solution (Perkin- Elmer) to calculate the fluoprotein ratio. Then, the humanized Eu-TMC-2206 was applied to the blocked a2ß1 integrin microtiter plates in a volume of 10 μL / well. After incubating the sealed plates for 1 hour at 37 ° C to allow the binding to equilibrate, 2 μL samples were transferred from each well to a fresh well containing DELFIA Enhancer Solution (100 μL / well Perkin-Elmer) for the measurement of the free label (without union). The enhancer solution (100 μL / well) was added to the wells emptied for the measurement of the binding label. The plate was shaken (Titration Plate Stirrer, speed set at 5, for 5 minutes at room temperature) and the TRF intensities were read using the Victor2 Multi-label Pita reader. The KD values were calculated by Scatchard analysis. The binding potencies related to immobilized α2β1 integrin were analyzed by measuring the K values in a competition assay using 100 pM humanized Eu-TMC-2206 in the presence of different concentrations of the unlabeled TMC-2206 antibody or TMC-2206 humanized as competitors, using a test system similar to the one already described in this example. Then, combinations of antibodies were applied to the wells coated with integrin a2ß1, tested at a concentration index of 10"11 to 10" 7 M, and after the specified time, the amount of Eu-humanized-TMC binding was determined. - 2206. The inhibition curves were adjusted with the "competition for a site" model using the Prism software (GraphPad, Inc.) to obtain the IC50 values and to calculate K, using the equation of Cheng and Prusoff (1973) and the respective values for KD mentioned above. The KD and K values for the TMC-2206 and the humanized TMC-2206 were located at a distance of two times from each other (Table 35). Therefore, the binding affinities of TMC-2206 and humanized TMC-2206 for immobilized a2β1 were similar.
TABLE 35TMC-2206 and humanized TMC-2206 were subjected to surface plasmon resonance (SPR) analysis to determine kinetic association and dissociation constants, kd and ka (also known as k0ff and kon), respectively, with domain I a2. SPR, a method to characterize macromolecular interactions, is an optical technique that uses the transient wave phenomenon to correctly measure minute changes in a refractive index very close to a sensor surface. The binding between an antigen in solution (eg, fusion protein) and its MAb receptor (immobilized on the surface of a sensor chip) results in a change in the refractive index. The interaction is monitored in real time and the amount of antigen binding and day constant association and dissociation constants can be measured very accurately. The equilibrium dissociation constant can be easily calculated from: KD = kd / ka = k0ft / kon. The cloning of a human I a2 domain and the purification of the expressed human a2 GST-domain I fusion protein was described in Example 12. The analyzes were performed at 20CC using a Biacore 2000 optical sensor with a sensor chip of a CM5 research (Biacore Life Sciences, Uppsala, Sweden) and were equilibrated with run buffer (50 mM HEPES, 150 mM NaCl, 0.25 mM MgCl2, 0.25 mM CaCl2, 0.5% Tween-20, 0.1 mg / ml of BSA, pH 7.4). To capture the TMC-2206 on the sensor chip, two of the chip cell surfaces were coated with anti-mouse IgG; the other two flow cells were coated with Protein A for the capture of humanized TMC-2206. Each cycle of the antigen (GST-domain I a2 fusion protein) bound to anti-mouse IgGs attached to the surface involved three stages. In the first, TMC-2206 was captured on an anti-mouse surface and then the humanized TMC-2206 was captured on a Protein A surface. [The other two surfaces (an anti-mouse and a Protein A) served as references analytical.] In the second stage, the fusion protein GST-domain I of a2 was injected through the four surfaces. The responses obtained from the reference surfaces (due to the inconsistencies of the refractive index between the antigen and the run buffer) were subtracted from the responses obtained from the reaction surfaces. In the third stage, the antigen / antibody complexes were ripped off from the surfaces so that the surfaces could be used for another binding cycle. The highest concentration of the GST-domain I fusion protein of human a2 was 41 nM. The antigen solution flowed on the surface for 2 minutes at 50 μl / min and the dissociation of the antigen from the surface was monitored for six minutes. The scale constants for the binding of the human a2 GST-domain I fusion protein with humanized TMC-2206 and humanized TMC-2206 were determined and found to be similar (Table 37). Competitive binding assays and SPR analyzes confirmed that the humanization process did not affect the binding affinity of humanized TMC-2206 with human a2 domain I.
EXAMPLE 18The cross-reactivity of the species with the humanized TMC-2206 was evaluated by means of analytical biochemical techniques. In the first study, the binding affinities (K, values) of TMC-2206, humanized TMC-2206 and GST-domain I a2 fusion proteins derived from different species were determined (Ki values) by means of competitive binding with TMC-2206 humanized with europium label to plates coated with a2ß1 (Example 17). The cloning of a2 domain of human, rhesus, rat and mouse macaque was described in Example 12. The a2 domains I of the additional cynomolgus monkey and rhesus monkey were cloned from cDNA derived from total RNA extracted from skin tissue (MediCorp, Inc., Montreal, QC). There was a 9-fold decrease in K2 for a2 I rat by binding with humanized TMC-2206 compared to human s.2 domain I, whereas the murine a2 GST-domain I fusion protein showed only a small binding specifies at the highest concentration (4 μM; Table 36). [Neither the negative control of the GST fusion protein nor the negative control of the IgG4 / k isotype showed competitive binding effects at concentrations of 0.4 μM.] The fusion proteins GST-domain I of rhesus a2, cynomolgus and human showed comparable unions. Therefore, all four species demonstrated cross-reactivity to humanized TMC-2206.
TABLE 36In a second study, the range and equilibrium constants of the binding of TMC-2206 and humanized TMC-2206, as well as GST-domain I fusion proteins of a2, were evaluated by SPR analysis (Table 37). All derived kinetic and equilibrium constants for human and parental TMC-2206 for the rat and human a2 domains I were similar. In addition, the humanized TMC-2206 rank constants for the a2 domains of human and cynomolgus were similar. Humanized TMC-2206 did not bind to mouse a2 domain I at concentrations of 4.0 μM of mouse a2 GST-domino I fusion protein. Comparable binding of cynomolgus α2 GST-domain I fusion protein with humanized TMC-2206 was not consistent with the result of Example 12, where no competitive binding was observed at concentrations greater than 1 μM (Table 29) . However, DNA sequence analyzes performed on cDNA populations derived from mRNA extracted from monkeys (Medicorp Inc.) revealed a polymorphism at a single amino acid (position 40) compared to domain I from human a2. This polymorphism was not conserved among the animals, since one cynomolgus monkey and one rhesus monkey exhibited heteromorphism while other animals exhibited 100% homology with the human a2 domain I. The GST-domain I of cynomolgus a2 studied in this example by competitive binding analyzes and SPR encoded the identical sequence of domain I of human a2. These biochemical studies demonstrated that the humanized TMC-2206 reacted cross-reactive with the I domains of a2 derived from human, rhesus, cynomolgus and rat, but not with the I domain of mouse a2. Cell cross-reactivity studies in vitro (Example 20) were performed to verify that the humanized TMC-2206 cross-reacted with blood cells from different species.
TABLE 37EXAMPLE 19The cross-reactivity between the species was further evaluated by linking the humanized TMC-2206 to blood cells of different species by flow cytometry. In this first study, cross-reactivity of humanized TMC-2206 with platelets from different species was evaluated. Blood was obtained through venipuncture of human donors, rats, and rhesus / cynomolgus monkeys. Human blood was collected in 3.8% sodium citrate; the blood of rhesus and cynomolgus was collected in 10 mM EDTA; and the rat blood was collected in heparin. Primate whole blood (human, rhesus, cynomolgus) was incubated with humanized TMC-2206 at a final concentration of 140 μg / ml for 10 minutes at room temperature, followed by a 10-minute incubation with conjugated MAb of anti-human mouse IgG4- FITC (Clone HP6023; Southern Biotech), followed by incubation with platelet-specific marker antibodies of the species, conjugated with fluorescent molecules. Human platelets were identified with conjugated PE of antihuman mouse CD42b (BD Biosciences) and the rhesus / cynomolgus platelets were identified with conjugated PE of anti-human mouse CD41a (BD Biosciences). Whole rat blood was incubated with 500 μg / ml humanized TMC-2206 conjugated to Alexa-488 (Alexa Fluor 488 kit, Protein Labeling, A10235, Molecular Probes) for 10 minutes at room temperature, followed by incubation with PE-conjugated hamster-anti-mouse-CD61 (rat platelet marker, BD Biosciences). All samples were washed once, suspended in phosphate saline, and then subjected to flow cytometric analysis. [The front and side doors of the disseminator were adjusted to logarithmic scales to further discriminate platelets of blood cells and larger leukocytes.] Humanized TMC-2206 bound platelets of the four species (Table 38) . In the second study, cross-reactivity of humanized TMC-2206 with leukocytes from different species was evaluated. The blood was obtained from the same four species, except that human blood was collected in 10 mM EDTA. The humanized TMC-2206 conjugated to Alexa 488 was added to the whole blood (final concentrations of 225-400 μg / mL) for 10 minutes, followed by a 30 minute incubation at room temperature with marker antibodies. Antibodies against CD45 were used to stain all leukocytes [for human leukocytes PE-Cy5-conjugated-mouse-antihuman (clone H130, BD Biosciences); for rhesus and cynomolgus leukocytes PE-Cy5-conjugated-mouse-antihuman (clone T0116, BD Biosciences); and for rat leukocytes PE-Cy5-conjugated-mouse-antirata (BD Biosciences). Marker antibodies were used to stain platelets: for human platelets PE-Cy5-conjugated-mouse-anti-human-CD42b (BD Biosciences); for rhesus and cynomolgus platelets R-PE-conjugated-mouse-antihuman-CD41a (BD Biosciences); and for rat platelets R-PE-conjugate-hamster-anti-mouse-CD61 (BD Biosciences) One milliliter of water was added to the reaction mixture (approximately 250 μl), incubated for 5 minutes at room temperature to lyse the blood cells, and then 2 ml of PBS (to generate levels of toxicity that would prevent leukocyte lysis) were added and centrifuged.The cell pellet was resuspended in 0.5 ml of PBS and subjected to flow cytometry analysis. The disseminator side channel was configured on a linear scale and the CD45 channel was configured on a logarithmic scale to discriminate granulocytes, monocytes and lymphocytes.] Because variable levels of endogenous platelet activation will result in the formation of platelet-leukocyte microaggregates , it was critical to identify the leukocytes that did not bind to platelets (which would essentially express the a2ß1 integrin), therefore, only those cells that were n CD45 + / CD41a ", CD45 + / CD42b" or CD45 + / CD61"were evaluated for binding with humanized TMC-2206. The humanized TMC-2206 bound to the lymphocytes, monocytes and granulocytes of the four species (Table 38). These results are consistent with the results of Example 19 in the sense that TMC-2206 cross-reacts with the GST-domain I fusion proteins of human a2, rhesus, cynomolgus and rat (by analysis K1 and SPR). Relatively low percentages of blood cells from rats that bound to humanized TMC-2206 compared to primate blood cells were presented. In three previous cell studies (Examples 9, 19 and 12), the binding affinities of the parental antibodies and humanized TMC-2206 to the rat a2 integrin subunit were found to be one order of magnitude less than the binding affinities with the human a2 subunit. In the first study, Example 9, the EC50 values of TMC-2206 that inhibited the binding of rat platelets and human platelets to rat type I collagen were 6.3 nM and 1.7 nM, respectively. In the second study, Example 19 (Table 38), the K i values for the inhibition of humanized TMC-2206 by binding to a2β1 immobilized by competitors of human and rat aGST-domain I fusion proteins were 0.57 nM and 5.23. nM, respectively. Similarly, in the third study, Example 12 (Table 29), the K i values for the inhibition of the binding of TMC-2206 to a2ß1 by GST-domain I fusion proteins from human and rat a2 were 0.33 nM and 3.8 nM, respectively. In addition, in both platelet and leukocyte studies, all cell samples were washed before being subjected to flow cytometric analysis, with more humanized TMC-2206 being washed from the rat a2 subunit with less affinity compared to the a2 subunits of primates. Combining this with the above results, we obtain a relatively low percentage of rat blood cells rated "positive" compared to primate blood cells (assuming similar densities of the a2ß1 receptor). In summary, all the platelets, lymphocytes, monocytes and granulocytes of the four species evaluated (human, rhesus monkey, cynomolgus monkey and rat) were bound to the humanized TMC-2206.
TABLE 38EXAMPLE 20Another study evaluated whether the binding of humanized TMC-2206 to a2ß1 resulted in platelet activation, according to the flow cytometry measurement. Platelet activation was measured as the upregulation of P-selectin or the activation of the integrin GPMbllla (allbß3). Blood samples were collected through venipuncture of the antecubital vein in vacuum-filled tubes containing 3.8% sodium citrate after discarding the first 3.0 ml of running blood. The whole blood was diluted 1: 10 in TBS (pH 7.4) and followed by a 10-minute incubation at room temperature with saline, isotype control IgG4 / (final concentration 132 μg / ml) or humanized TMC-2206 ( final concentration of 144 μg / ml). For the platelet activation, the peptide activator of the thrombin-6 receptor (TRAP-6, 10 μM final concentration, AnaSpec Inc., San José, CA) or adenosine diphosphate (ADP, 20 μM final concentration; Sigma), and then incubation for 5 minutes at room temperature. Cells were processed by flow cytometry by incubation with marker antibodies: PE-Cy5-conjugated-mouse-antih? Hand-CD42b (BD Biosciences) to stain platelets; PE-conjugate-mouse-antihuman-CD62P (BD Biosciences) to stain P-selectin; and FITC-conjugate-PAC-1 (BD Biosciences) to stain activated GPlIbllla (PAC-1 binds to the active conformation of integrin GPlIbllla). The sampling error for each sample was less than 5% (95% confidence level). The first experiments evaluated whether the binding of humanized TMC-2206 gives rise to platelet activation according to the measurement of P-selectin up-regulation. The activation was rated as the percentage of platelets (CD42b +) that were stained by the P-selectin marker (CD62P) (Table 39). In ANOVA analyzes (one-way, 95% confidence interval), the expression of P-selectin from platelets incubated with saline, IgG4 /? and humanized TMC-2206 was not statistically different (P = 0.96). Therefore, the binding of humanized TMC-2206 to platelets did not induce platelet activation. In face-to-face experiments, TRAP-6 induced significant increases in the expression of P-selectin. The addition of humanized TMC-2206 did not statistically affect P-selectin expression induced by TRAP-6 compared to saline or isotype control (P-0.96, one-way NOVA, 95% confidence interval). Therefore, binding of humanized TMC-2206 did not inhibit platelet activation induced by TRAP-6.
TABLE 39The following study evaluated the upregulation of P-selectin after incubation with / without the ADP agonist (percentage of platelets expressing P-selectin, Table 40). As before, the expression of P-selectin in platelets incubated with humanized TMC-2206, IgG4 / k and saline was comparable, whereby the humanized TMC-2206 did not induce platelet activation. ADP induced an expression of P-selectin comparable to the induction of TRAP-6. There seemed to be an additional increase in the expression of P-selectin with platelets incubated with ADP and then with IgG4 /? or with humanized TMC-2206. However, the increase in upregulation of P-selectin was similar for both isotype control and humanized TMC-2206, which again indicated that the binding of humanized TMC-2206 to platelets does not induce platelet activation. Concomitantly, the expression of P-selectin of the platelets induced with ADP and incubated with IgG4 /? or with humanized TMC-2206 was not reduced. Therefore, the binding of humanized TMC-2206 did not inhibit platelet activation induced by ADP.
TABLE 40The following study evaluated the activation of GPlIbllla after incubation with / without agonists TRAP-6 or ADP by rating the percentage of platelets bound to the PAC-1 marker antibody (which binds to the active conformation of GPlIbllla; Table 41). Levels of activated GPlIbllla expression in platelets incubated with humanized TMC-2206, IgG4 / k and saline were comparable; humanized TMC-2206 did not induce platelet activation. Neither lgG4 / k nor the humanized TMC-2206 inhibited activation induced by TRAP-6 or by ADP.
TABLE 41In summary, humanized TMC-2206 did not induce platelet activation (there was no increase in upregulation of P-selectin or activation of GPlIbllla), nor did the inhibition agonist (TRAP-6, ADP) induced platelet activation. This information complements the platelet aggregation study (Example 15, Table 34) that showed that humanized TMC-2206 did not induce platelet aggregation or inhibit collagen-induced aggregation.
EXAMPLE 21The humanized TMC-2206 was evaluated to determine its effect on both extrinsic and intrinsic coagulation pathways by measuring prothrombin time (PT) and activated partial thromboplastin time (aPTT-for its acronym in English). ). A qualified lyophilized preparation of human plasma (Citrex I, Bio / Data Corporation, Horsham, PA) was used for the measurement of both PT and aPTT. The humanized TMC-2206 was added to the plasma to achieve final concentrations of 179, 214 and 286 μg / mL (corresponding to the Cmax of a single dose of antibody at 12.5, 15.0 and 20.0 mg / kg, respectively) before the samples were submitted to coagulation tests. Standard procedures for PT and aPTT were followed, and coagulation times were measured with a BBL fibrometer (BD, Franklin Lakes, NJ). Table 42 summarizes the information for a series of six experiments (3 PT and 3 aPTT). A saline control was run for each experiment. Statistical Student's t-tests of each matching pair (humanized TMC-2206 and saline) demonstrated in each experiment that there were no statistically significant differences between the average coagulation times for humanized TMC-2206 compared to saline. (The hypothesis that coagulation times were different would be rejected at 95% confidence levels if the individually calculated P values were less than 0.05.) Therefore humanized TMC-2206 did not have an effect on coagulation in accordance with the measurement of PT and PTT.
TABLE 42EXAMPLE 22The effects of humanized TMC-2206 were evaluated in times of rat bleeding. Sprague-Dawley rats (190-200g) were injected intravenously (tail vein) either saline, heparin (0.6 mg / kg, positive control), or humanized TMC-2206 at doses of 5 and 15 mg / kg one hour before the standardized transection of the tip (0.5 mm) of each tail. The rats were not anesthetized and were conscious during observation of the bleeding time. The tip of the cut tail of each rat was immediately immersed in a 2 cm deep test tube containing saline at 37 ° C. The time required for the beginning of a period of 15 seconds of cessation of bleeding was qualified as the bleeding time. A time limit of 20 minutes was used. The blood loss was scored by the amount of hemoglobin released after hemolysis (spectrophotometrically) of the blood collected in the test tube. The humanized TMC-2206 did not show a statistically significant effect on the bleeding time in both test doses compared to the unaltered and saline controls (Table 43, P = 0.08, one-way ANOVA analysis). The humanized TMC-2206 did not show a statistically significant effect on blood loss in both test doses compared to the unaltered and saline controls (P = 0.22, one-way ANOVA analysis). Therefore, humanized TMC-2206 has no effect on bleeding time or blood loss in vivo.
TABLE 43EXAMPLE 23A study was conducted to determine the effect of a single dose of humanized TMC-2206 on circulating levels of cytokines in rats as a means to determine whether humanized TMC-2206 causes the detectable in vivo activation of leukocytes. Saline solution (negative control), IgG4 /? human, isotype control (15 mg / kg), humanized TMC-2206 (15 mg / kg) or lipopolysaccharide (LPS, positive inflammation control; 0.75 mg / kg) were administered to rats intravenously. Non-injected rats were used as unaltered controls. At 2, 4, 6 and 8 hours after the injection, blood samples were taken through the saphenous vein and processed for plasma. The plasma samples were subjected to an account-based multiplex immunoassay (MIA, Lineo Diagnostics, St. Charles, MO) to determine the levels of IL-1a, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-12, GM-CSF, IFN- ?, and TNF-a (Table 44, pg / mL, mean ± SEM). MIA involves the simultaneous detection of analytes (up to 100) in the same volume of the sample (25 μl) by combining several individual antigen / antibody binding reactions in spectrally distinct sets of microspheres. Depending on the antigen, the sensitivity of MIA is between 1.5 - 50 pg / ml. Each cytokine information was subjected to two-way ANOVA analysis, with a confidence interval of 95%, testing the hypothesis that the individual cytokine levels for the four points and for the four conditions (unchanged, vehicle, IgG4 / ky TMC-2206 humanized) were equivalent. The hypotheses would be rejected if the P values were less than 0.05. There were no statistically significant differences in any of the 10 sets (all P values ranged from 0.18 to 1.0, Table 44) of cytokine levels observed in rats injected with the vehicle, IgG4 /? Humanized TMC-2206, or the non-injected ones (without altering). Therefore, intravenous injection of a single dose (15 mg / kg) of humanized TMC-2206 did not induce an increase in the expression of cytokines involved in inflammation.
TABLE 44Although the present invention has been described with reference to particular embodiments, it should be understood that these embodiments are merely illustrative of some aspects of the invention. Therefore, it should be understood that it is possible to make numerous modifications to the illustrative variants and that other arrangements may be considered without departing from the spirit and scope of the invention.