MXPA06010891A - Receptor coupling agents and therapeutic uses thereof - Google Patents

Abstract

Receptor coupling agents, including multivalent constructs comprising anti-TNF receptor binding moieties, for treating cancer and inhibiting tumor volume in a subject are disclosed.

Description

AGENTS OF COUPLING RECEIVERS AND THERAPEUTIC USES OF THEMSELVES BACKGROUND OF THE INVENTION V The ability to induce cell death by various members of the TNF family has been eagerly sought by oncologists for almost 20 years. Originally, TNF itself was used to treat solid tumors and was eventually found to be applicable for the local treatment of melanoma by perfusion of the entire limb (Lejeune et al., 1998) Curr Opin I munol 10: 573 )). More recently, the activation of T? F receptors or ligands or anti-receptor antibodies has aroused clinical interest. The activation of the Fas receptor, for example, has shown a considerable promise, although this may be limited by liver toxicity. Activation of TRAILR1 or TRAILR2 by the TRAIL ligand, another member of the TNF family, has been reported as a transducer of an apoptotic signal to TRAIL-sensitive cancer cells (Griffith et al., J. Immunol., 162: 2597). , 1999, and Degli-Esposti et al., Immuni ty, 7: 813-820, 1997). Activation of LT-ß-R, another member of the TNF family, by soluble ligands or monoclonal anti-receptor agonists, has also been shown to induce the death of certain carcinomas (La erence ... et al., (2001) Nat Med 7: 383, Ichikawa et al., (2001) Nat Med 'J Ref .: 176011 7: 954). The treatment with TNF agonist activating agents could be in this way useful for the treatment? reduction of the advance, severity or the effects of the neoplasm in subjects (for example human).

BRIEF DESCRIPTION OF THE INVENTION The invention describes a receptor coupling agent, which specifically activates at least two different receptors of the T? F family. In one modality, the. coupling agent to the receptors increases the signaling of the receiver. In yet another embodiment, the coupling agent to the receptors induces the formation of complexes of heteromeric receptors. In one embodiment, the coupling agent to the receptors comprises a first binding specificity for one receptor and a second binding specificity for the other receptor. In one embodiment, the first binding specificity is conferred or effected by an antibody or antigen-binding fragment thereof. In yet another embodiment, the second binding specificity is conferred or effected by an antibody or antigen-binding fragment thereof. The binding specificity can be conferred, for example, by a single chain Fv fragment. In yet another embodiment, the first binding specificity is effected by a natural ligand for the receptor, and the second binding specificity is derived from an antibody or antigen-binding fragment thereof. In yet another embodiment, the first binding specificity is conferred by a natural ligand for the receptor, and the second binding specificity is conferred by a natural ligand for the receptor. The invention describes a receptor coupling agent that effectively activates at least two different receptors of the TNF family, wherein at least one receptor contains a death domain. In one embodiment, the coupling agent to the receptors increases receptor signaling or induces the formation of heteromeric receptor complexes, wherein at least one receptor contains a death domain. In one embodiment, the receptor containing a death domain is selected from the group consisting of TNFR1 (DR1), Fas (DR2), TRAIL-R1 (DR4), TRAIL-R2 (DR5), DR6 and p75NGF-R. The invention includes a receptor coupling agent that activates at least two different receptors of the TNF family, wherein at least one receptor does not contain a death domain. The invention also describes a receptor coupling agent that increases receptor signaling or induces the formation of heteromeric receptor complexes, where at least one receiver does not contain a death domain. In one embodiment, the receptor does not contain a death domain and is involved in tissue differentiation. In yet another embodiment, the receiver that does not contain a death domain is selected from the group consisting of LTBR, RANK, EDARl, XEDAR, Fnl4, Troy / Trade, and TAJ. The invention also describes a receptor coupling agent which specifically activates at least two receptors other than the TNF family, wherein at least one receptor is involved in tissue differentiation. The invention also describes a receptor coupling agent that enhances receptor signaling or induces the formation of heteromeric receptor complexes, wherein at least one receptor is involved in tissue differentiation. In one embodiment, the receiver is selected from the group consisting of LTBR, RANK, EDARl, XEDAR, Fnl4, Troy / Trade / TAJ, and p75NGF-R. In one embodiment, the receptor coupling agent activates a non-killing domain that contains a TNF receptor, and a receptor that contains the death domain, for example LTBR / TRAIL-Rl; LTBR / TRAIL-R2; LTBR / p75? GF-R; Fnl4 / p75NGF-R; and p75NGF-R / TAJ. In still another embodiment, the receptor coupling agent activates at least two TNF receptors that do not contain domains of death, for example LTBR / Fnl4; LTBR / RANK; Fnl4 / AJ; LTBR / WWTP; LTBR / XEDAR; RA? K / WWTP; RANK / XEDAR; and TAJ / EDAR; and TAJ / XEDAR.

In yet another embodiment of the invention, the receptor coupling agent activates at least two receptors that contain the death domain. In addition, the invention describes a receptor coupling agent that specifically activates at least two different receptors of the TNF family, wherein at least one receptor is involved in immune regulation. In one embodiment, the receptor is selected from the group consisting of TNFRII, HVEM, CD27, CD30, CD40, 4-lBB, OX40, GITR, TACI, BAFF-R, BCMA, and RE. The invention provides a receptor coupling agent that specifically activates at least two different receptors of the TNF family, wherein at least one of the receptors is not overexpressed on normal liver or endothelial cells. The invention also discloses a receptor coupling agent that specifically activates at least two receptors other than the TNF family, wherein the receptor coupling agent comprises a first binding specificity for a receptor, and a second binding specificity for a receptor. the other receiver. In one embodiment, the coupling agent to the receptors increases receptor signaling or induces the formation of heteromeric receptor complexes. In one embodiment, the first binding specificity is conferred by or derived from an anti-LTβ receptor (LTßR) antibody or the antigen binding fragment thereof. An example of an anti-LTßR antibody includes a humanized CBEll antibody. In one embodiment, the second binding specificity is conferred by or derived from an anti-TRAIL-R2 antibody, or the antigen-binding fragment thereof. Examples of the anti-TRAIL-R2 antibody are a humanized or chimeric 14A2 antibody. In yet another embodiment, the first binding specificity is conferred by a single chain Fv fragment of a humanized CBEll antibody, and the second binding specificity is conferred by a 14A2 antibody. The invention describes a receptor coupling agent that specifically activates at least two different receptors of the TNF family, wherein the receptor coupling agent comprises a first binding specificity for one receptor and a second binding specificity for the other receptor, wherein the first binding specificity comprises at least two trimeric Fe-ligand constructs that are commonly formed from three dimeric Fc domains and six ligand molecules. In this case, the second binding specificity should comprise three antibody molecules. The invention describes a receptor coupling agent that specifically activates at least two different receptors of the TNF family, wherein at least one of the receptors of the TNF family is not normally found in an environment on the cell surface. In one embodiment, the coupling agent to the receptors increases the signaling of the receptor or induces the formation of hetoromeric complexes of the receptor, wherein at least one of the receptors of the TNF family is not normally found in an environment above the surface. cell phone. The invention includes a receptor coupling agent that specifically activates at least two different receptors of the T? F family, increases receptor signaling or induces heteromeric receptor complex formation, wherein at least one of the family receptors T? F is normally found in an environment on the cell surface. The invention further discloses a receptor coupling agent that specifically activates at least two different receptors of the T? F family or increases signaling of the receptor, wherein the strength of the signal is increased through the receptors. The invention includes a receptor coupling agent comprising at least two antibodies, or antigen binding fragments thereof, wherein each antibody binds to a receptor other than the T? F family., which induces the formation of a heteromeric receptor complex. In one embodiment, the antibody is derived from an anti-LTβR antibody, which includes, for example, a humanized CBEll antibody. In yet another embodiment, the second antibody is derived from an anti-TRAIL-R2 antibody, which includes, for example, a humanized or chimeric 14A2 antibody. In one embodiment, the invention includes a method for locating a receptor of the TNF family to a floating mass of the cell membrane, which comprises administering a receptor coupling agent, which includes a first binding specificity for a receptor of the family TNF transported, and a second binding specificity for a receptor of the non-transported TNF family, wherein the binding of the coupling agent to the receptors locates the non-transported TNF receptor to a transport in the cell membrane. The invention also includes a method for enhancing receptor signaling, which comprises administering a receptor coupling agent that specifically activates at least two different receptors of the TNF family, increases receptor signaling and induces the formation of heteromeric complexes of the receptor. . In still another embodiment, the invention describes a method for decreasing tumor volume, which comprises administering to a subject a coupling agent to receptors that specifically activates at least two different receptors of the TNF family, increases receptor signaling, and induces the formation of heteromeric receptor complexes. In yet another embodiment, the invention includes a method of treating cancer, which comprises administering to a subject a coupling agent to the receptors that specifically activates at least two different receptors of the TNF family, increases the signaling of the receptor, or induces the formation of heteromeric complexes to the receptor. In one embodiment, the coupling agent to the receptors is administered in the presence of INF- ?. In yet another embodiment, the coupling agent to the receptors is administered in the presence of a chemotherapeutic agent. The invention also comprises a receptor coupling agent that activates at least two different receptors of the TNF family, and induces the formation of a heteromeric receptor complex comprising a first binding specificity directed to a first TNF receptor, and a second specificity of binding directed to a second TNF receptor. In one embodiment, the first and second binding specificities are directed to TNF receptors, including a T? F receptor that contains the non-killing domain, and a TNF receptor that contains the death domain; two TNF receptors that contain the non-dying domain; or two TNF receptors that contain the death domain. In yet another embodiment, at least one binding specificity is directed to a TNF receptor that contains the non-killing domain, associated with tissue differentiation. In yet another embodiment, two TNF receptors that contain the non-dying domain are selected from the group consisting of LTBR / Fnl4; LTBR / RA? K; Fnl4 / TAJ; LTBR / WWTP; LTBR / XEDAR; RANK / WWTP; RANK / XEDAR; TAJ / EDAR; and TAJ / XEDAR. In yet another embodiment of the invention, the T? F receptor that contains the non-killing domain and the T? F receptor that contains the death domain are selected from the group consisting of LTBR / TRAIL-RI; LTBR / TRAIL-R2; LTBR / p75? GF-R; Fnl4 / p75NGF-R; and p75? GF-R / TAJ.

BRIEF DESCRIPTION OF THE FIGURES Figures 1A-1B graphically describe the results of a 4-day proliferation assay of WiDr cells.

The results show that anti-TRAIL-R2 antibody 14A2 and CBEll anti-LTßR antibody were able to induce the death of WiDr through agonist activity. Figures 2A-2B graphically describe the results of a 4-day MTT growth assay in WiDr colon carcinoma cells. The results demonstrate that the bispecific antibody LTßR / TRAIL-R2 (LT-BS1) has more cell death activity (figure 2b) than the individual progenitor antibodies CB11 and 14A2 (figure 2a). Figure 3 graphically describes the results of a 4-day MTT growth assay with 80 U / ml of IFN? in WiDr colon carcinoma cells, comparing the bispecific antibody LTßR / R2 (LT-BSl) to the tetravalent LTßR bispecific LL-BS1 antibodies (CBEll antibody and BHA10) and LL-MS1 (CBEll antibody). Figures 4A-4B graphically describe the results of a 4-day MTT growth assay with or without 80 U / ml of IFNα. in LS174T tumor cells. The results show the efficacy of the receptor coupling agent, LT-BS1 and antibodies 14A2 and CBEII in the inhibition of the growth of colon carcinoma cells (LS174T tumor cells). Figures 5A-5B graphically describe the results of a 4-day MTT growth assay with or without 80 U / ml IFβ and in tumor cells ME180. The results demonstrate the efficacy of the receptor coupling agent, LT-BS1, and antibodies 14A2 and CBEll in inhibiting the growth of cervical carcinoma cells (tumor cells ME180). Figures 6A-6B graphically describe the results of a 4-day MET growth assay with or without 80 U / ml IFα and in MDA213 tumor cells. The results show the efficacy of the receptor coupling agent, LT-BS1 and antibodies 14A2 and CBEll in the inhibition of growth of breast carcinoma cells (tumor cells MDA231). Figures 7A-7B graphically describe the results of a 4-day MTT growth assay with or without 80 U / ml IFNα. in Hela tumor cells. The results demonstrate the efficacy of the receptor coupling agent, LT-BSl and the individual antibodies 14A2 and CBEll in inhibiting the growth of Hela cervical carcinoma cells. Figures 8A-8D graphically describe the results of a 4-day MTT growth assay with 80 U / ml of iFN? in a gamma of tumor cell types, including breast, cervical and colon. The results show the effects of the receptor coupling agent, LT-BSl and the pentameric antibody CBEll in the inhibition of various types of growths of carcinoma cells, including breast (A, B), cervical (C) and colon ( D). Figure 9 depicts a schematic drawing of the LT-BSl receptor coupling agent construct, comprising anti-TRAIL-R2 antibody 14A2, and LTßR scFv CBEll antibody (purified).

DETAILED DESCRIPTION OF THE INVENTION I. Definitions For convenience, before the further description of the present invention, certain terms used in the specification, in the examples and in the appended claims are defined herein. The term "administering or administration" includes any method of distributing a compound of the present invention, including but not limited to, a pharmaceutical composition or a therapeutic agent, within the system of a subject or to a particular region in or on a subject. The phrases "systemic administration", "systemically administered", "peripheral administration" and "peripherally administered", as used herein, mean the administration of a compound, drug or other material different from directly to the central nervous system, such that it enters the patient's system and, in this way, it is subjected to metabolism and other similar processes, for example, subcutaneous administration. "Parenteral administration" and "parenterally administered" means modes of administration other than enteral and topical administration, usually by injection, and includes without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal administration , transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subraquinoid, intraspinal and intraexternal, and infusion. As used herein, the term "Antibody" is understood to refer to complete and intact antibodies, as well as to Fab, Fab ', F (ab) 2 / Fv fragments, and other fragments thereof, which impart desired binding specificities to the constructs of the present invention. The antibodies include, for example, monoclonal antibodies such as murine monoclonal antibodies, chimeric antibodies, anti-idiotypic antibodies, anti-anti-idiotypic antibodies and humanized antibodies, as well as the multivalent forms thereof.

The term "immunoglobulin" or "antibody" (used interchangeably herein) refers to an antibody protein to the antigen binding having a basic chain structure of four polypeptides, consisting of two heavy chains and two light chains, said chains being stabilized, for example, by interchain disulfide bonds, which have the ability to bind specifically to the antigen. The heavy and light chains are folded into domains. The term "domain" refers to a globular region of a heavy or light chain polypeptide comprising peptide curls (e.g., comprising 3 to 4 peptide curls) stabilized, e.g., β-pleated sheet and / or disulfide bond intrachain The domains are also referred to herein as "constants" or "variables", based on the relative lack of sequential variation within the domains of various members of the class, in the case of a "constant" domain or significant variation within the domains of various members of the class in the case of a "variable" domain. The "constant" domains on the light chain are interchangeably referred to as "light chain constant regions", "light chain constant domains", ("CL" regions or "CL" domains). The "constant" domains on the heavy chain are interchangeably referred to as "heavy chain constant regions", "heavy chain constant domains", ("CH" regions or "CH" domains). The "variable" domains on the light chain are referred to interchangeably as "light chain variable regions", "light chain variable domains", ("VL" regions or "VL" domains). The "variable" domains on the heavy chain are interchangeably referred to as "heavy chain constant regions" "heavy chain constant regions", ("CH" regions or "CH" domains). The term "region" refers to a part or portion of an antibody chain, and includes constant or variable domains as defined herein, as well as more discrete portions or portions of said domains. For example, the light chain domains or regions include "complementarity determination regions" or "CDR" interspersed between the "structural regions" or "FRs" as defined herein. Immunoglobulins or antibodies can exist in monomeric or polymeric form. The term "antigen binding fragment" refers to a polypeptide fragment of an immunoglobulin or antibody that binds to the antigen, or competes with the intact antibody (for example, with the intact antibody from which they were derived) for the binding to the antigen (for example, specific binding). The term "conformation" refers to the tertiary structure of a protein or polypeptide (e.g., an antibody, an antibody chain, domain or region thereof). For example, the phrase "light (or heavy) chain conformation" refers to the tertiary structure of a light (or heavy) chain variable region, and the phrase "antibody conformation" or "antibody fragment conformation" is refers to the tertiary structure of an antibody or fragment thereof. The binding fragments are produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of the intact immunoglobulins. Link fragments include Fab, Fab ', F (ab') 2, Fabc, Fv, simple chains, and single chain antibodies. Apart from immunoglobulins or "bispecific" antibodies. or "bifunctional" is meant that an immunoglobulin or antibody has each of its identical binding sites. A "bispecific" or "bifunctional" antibody is an artificial hybrid antibody that has two different heavy / light chain pairs and two different binding sites Bispecific antibodies can be produced by a variety of methods, including fusion of hybridomas or binding of Fab 'fragments. See for example, Songsivilai & Lachmann, (1990) Clin. Exp. Immuno 1. 79: 315-321; Kostelny et al. , (1992) J. Immuno 1. 148, 1547-1553. The term "antibody construction" refers to a recombinant molecule comprising two or more antigen binding fragments that come from the variable domains of the heavy chain and the light chain of an antibody. An antibody construct can comprise all or part of the constant regions of an antibody from any of the five Ig classes (eg, IgA, IgD, IgE, IgE and IgM). For example, the antibody construct can be made from an antibody whose heavy chains comprise at its C-terminus a single-chain variable fragment. In yet another example, the antibody construct can be made from all or part of the constant region of the two heavy chains of an antibody comprising at its carboxyl and amino termini a variable fragment of single chain. An example of antibody constructs imparting the desired binding specificities is described schematically in Figure 9. In yet another example, the antibody construct can comprise two heavy chains having two or more variable regions and two light chains having one or more variable regions where the two heavy chains are linked by a disulfide or other covalent bond. In yet another example, the antibody construct can comprise two heavy chains. comprising two or more variable regions, where the two heavy chains are linked by a disulfide or other covalent bond. The term "antigen" as used herein, means a molecule that is reactive with a specific antibody. The term "antigen binding site" or "antigen recognition site" refers to a region of an antibody that specifically binds an epitope on an antigen. The term "apoptosis", "apoptotic cell death" or "programmed cell death" as used herein, refers to any cell death resulting from the cascade of cellular events that occur at specific stages of cell differentiation, and in response to specific stimuli. Apoptotic cell death is often characterized by condensation of the cytoplasm and the nucleus of the dying cells. The term "binding specificity" is a property of the receptor coupling agents, described that is conferred, imparted, effected or derived from a binding portion that is targeted to a specific TNF family receptor. A binding specificity of the invention can be conferred by linking portions including, for example, an antibody, or antigen binding fragment thereof, soluble ligand of the single chain Fv fragment, fc fusions or the like. Those skilled in the art will appreciate that for the purposes of the present invention, the terms "link specificity" and "link portion" may be used interchangeably unless otherwise indicated by context constraints. Thus, a binding specificity (binding portion) can also include a TNF ligand that interacts with a receptor of the TNF family. In one embodiment of the invention, a receptor coupling agent comprises at least one binding specificity (or binding portion) for a T? F receptor and a second binding specificity (or binding portion) for another receptor of TNF. The term "cancer" or "neoplasia" generally refers to any malignant neoplasm or spontaneous growth or cell proliferation. The term as used herein, encompasses fully developed malignant neoplasms, as well as pre-malignant lesions. A subject having "cancer", for example, may have a tumor or a proliferation of white blood cells such as leukemia. In certain embodiments, a subject who has cancer is a subject who has a tumor, such as a solid tumor. Cancers that involve a solid tumor include but are not limited to, cancer of non-small tumor cells (NSCLC), testicular cancer, lung cancer, ovarian cancer, uterine cancer, cervical cancer, pancreatic cancer, colorectal cancer (CRC), breast cancer, as well as cancer of the prostate, gastric, skin, stomach, esophagus, and bladder. The term "chemotherapeutic agent" refers to any small molecule or biological material used to treat the disease caused by a foreign cell or malignant cell, such as a tumor cell. Non-limiting examples of chemotherapeutic agents include agents that disrupt DNA synthesis, are inhibitors of topoisomerase I, are alkylating agents, or are plant alkaloids. Exemplary biological chemotherapeutic agents include rituximab, ibritumomab, bevacizumab and trastuzumab. Those skilled in the art will appreciate that other chemotherapeutic agents, compatible with the teachings of the present invention, are readily discernible. The term "agent that disrupts DNA synthesis" refers to any molecule or compound capable of reducing or inhibiting the process of DNA synthesis. Examples of agents that disrupt DNA synthesis include, but are not limited to, nucleoside analogs such as pyrimidine or purine analogues, including, but not limited to, gemcitabine or alternatively anthracycline compounds, including, for example, but not limited to, adriamycin, daunombicin, doxorubicin, and idambycin and epipodophyllotoxins such as etoposide and teniposide. The term "topoisomerase I inhibitor" refers to a molecule or compound that inhibits or reduces the biological activity of a topoisomerase I enzyme. It is included for example, but is not limited to camptosar. The term "alkylating agent" refers to any molecule or compound capable of reacting with the nucleophilic groups of (eg, amines, alcohols, phenols, organic and inorganic acids) and thereby adding alkyl groups (e.g. methyl) to another molecule, such as a protein or nucleic acid. Examples of alkylating agents used as chemotherapeutic agents include bisulfan, chlorambucil, cyclophosphamide, ifosfamide, mechlorethamine, melphalan, thiotepa, various nitrosourea compounds, and platinum compounds such as cisplatin and carboplatin.

The term "plant alkaloid" refers to a compound that belongs to a family of nitrogen-containing alkaline molecules derived from plants that are biologically active and cytotoxic. Examples of plant alkaloids include, but are not limited to, taxanes, such as taxol, docetaxel and paclitaxel and vinca per vincas such as vinblastine, vincristine and vinorelbine. The term "chimeric antibody" refers to an antibody whose light and heavy chain genes have been constructed, typically by genetic engineering from segments of immunoglobulin genes belonging to different species. For example, the variable elements (V) of the genes derived from a mouse monoclonal antibody can be bound to the human constant segments (C), such as IgG1 and IgG. The human IgGl isotype is preferred. A typical chimeric antibody is thus a hybrid protein consisting of the V or antigen binding domain from a mouse antibody, and the C or effector domain from a human antibody. The term "death domain" refers to a cytoplasmic region of a receptor of the TNF family that is involved in the signaling of cell death mediated by TNF and the induction of cellular cytotoxicity mediated by these receptors. This region couples the receptor to the activation of caspase via the adapter proteins that result in the activation of the extrinsic death pathway. Examples of TNF receptors that contain domains of death include, but are not limited to, TNFRl (DRl), Fas (DR2), TRAIL-Rl (DR4), TRAIL-R2 (DR5), p75NGFR, and DR6. The term "effective amount" refers to that amount of a compound, material or composition comprising a compound of the present invention, which is sufficient to effect a desired result, including, but not limited to, for example, reducing of the tumor volume either in vitro or in vivo. An effective amount of a pharmaceutical composition of the present invention is an amount of the pharmaceutical composition that is sufficient to effect a desired clinical result, including but not limited to, for example, improving, stabilizing, preventing or delaying the development of cancer in a patient. In any case, an effective amount of the compounds of the present invention can be administered in one or more administrations. The detection and measurement of these prior indicators are known to those skilled in the art, including, but not limited to, for example, reduction in tumor burden, inhibition of tumor size, reduction in proliferation of secondary tumors, expression of genes in tumor tissue, presence of biornarcadores, involvement of lymph nodes, histological grade and nuclear grade. The term "epitope •" refers to the region of an antigen to which an antibody or antibody construction is linked, preferably and specifically. A monoclonal antibody is preferably linked to a simple specific epitope of a molecule that can be molecularly defined. In the present invention multiple epitopes can be recognized by a multispecific antibody. The term "Fv fragment" refers to the fragment of an antibody that comprises the variable domains of its heavy chain and light chain. The term "Fc fragment" refers to the fragment of an antibody that comprises the constant domain of its heavy chain. The term "humanized immunoglobulin" or "humanized antibody" refers to an antibody or immunoglobulin that includes at least one immunoglobulin or humanized antibody chain (e.g., at least one humanized light or heavy chain). The term "humanized immunoglobulin chain" or "humanized antibody chain" (e.g., a "humanized immunoglobulin light chain" or "humanized immunoglobulin heavy chain") refers to an immunoglobulin or antibody chain (e.g., a light or heavy chain, respectively) having a variable region that includes a variable structural region substantially derived from an immunoglobulin or human antibody and regions of determination of complementarity (CDR, for its acronym in English) (e.g. minus one CDR, preferably two CDRs, more preferably three CDRs) substantially from an immunoglobulin or non-human antibody, and further includes the constant regions (e.g., at least one constant region or portion thereof, in the case of a chain light, and preferably three constant regions in the case of a heavy chain). The term "humanized variable region" (e.g., "humanized light chain variable region" or "humanized heavy chain variable region") refers to a variable region that includes a variable structural region substantially derived from an immunoglobulin or an antibody human, and regions of complementarity determination (CDR), - substantially from an immunoglobulin or non-human antibody. The term "heteromeric receptor complex" refers to a complex comprising a coupling agent to the receptors and two or more receptors, to which the coupling agent is directed to the receptors. In one embodiment, the heteromeric receptor complex of the invention comprises a coupling agent to the receptors and at least two receptors of the TNF family to which the agent is directed to activate them. Preferably, signaling through the receptors is increased as a result of the formation of the heteromeric receptor complex. In one embodiment of the invention, the heteromeric receptor complex is formed on a lipid transport in the cell membrane. In yet another embodiment, the heteromeric receptor complex of the invention is formed outside of a lipid transport on the cell membrane. The term "tumor volume inhibition" refers to any decrease or reduction in the volume of a tumor. The term "ligand" refers to any molecule that binds to a specific site on a protein or other molecule. A ligand is often a polypeptide or a compound that binds to a receptor protein in a high affinity and specific manner, to promote a functional response. For example, the ligands of the invention include the ligands of the TNF family receptor. The term "natural ligand" refers to a ligand that binds to a receptor under normal physiological conditions. The term "receptor" refers herein, to a structure, usually to a polypeptide, located on or in a cell, which recognizes a binding molecule, eg, a ligand, and thereby induces a cellular response. The receptors of the invention include receptors of the TNF family, including for example, TRAIL-R2, HVEM, and LTßR. The term "TNF family receptor" or "TNF-R" refers to receptors that belong to the TNF receptor superfamily, characterized by disulfide bonds that form "cysteine-rich domains" or CRDs. Members of the TNF receptor family generally consist of an extracellular domain, a transmembrane domain and an intracellular signaling domain (see Locksley et al. (2001) Cell 104: 487 for review). The extracellular domain is constructed from 1 to 6 copies of a domain strongly bound by disulfide bridges and is recognized based on the unique arrangement of cysteine residues (Banner et al. (1993) Cell 73: 431). Each TNF receptor binds to a corresponding ligand, although a ligand can share several receptors. The term "beta lymphotoxin receptor agonist (LTßR)" refers to any agent that can increase the binding of the ligand to LTßR, the accumulation of LTßR on the cell surface and / or the signaling of LTßR. The phrase "multivalent antibody" or "multivalent antibody construct" refers to an antibody or antibody construct that contains more than one antigen recognition site. For example, a "bivalent" antibody construct has two antigen recognition sites, while a "tetravalent" antibody construct has four antigen recognition sites. The terms "monospecific," "bispecific," "trispecific," "tetra-specific," etc., refer to the number of different specificities of the antigen recognition site (as opposed to the number of antigen recognition sites) present in a construct. of multivalent antibodies of the invention. For example, the sites of antigen recognition, of the construction of the "monospecific" antibody are all linked to the same epitope. A "bispecific" antibody construct has at least one antigen recognition site that binds to a first epitope, and at least one antigen recognition site that binds to a second epitope that is different from the first epitope. A "monospecific multivalent" antibody construct has multiple antigen recognition sites that all bind to the same epitope. A multivalent bispecific epitope construct "has multiple antigen recognition sites, some of which bind to a first epitope, and some number of which bind to a second epitope, which is different from the first epitope. of the invention, the antibody is a bispecific, multivalent antibody, as shown in Figure 9. A "patient" or "subject" or "host" refers to either a human or a non-human animal. "Pharmaceutical distribution" refers to any device that can be used to deliver a therapeutic agent or agents to a subject.Non-limiting examples of pharmaceutical delivery devices include hypodermic syringes, multi-chamber syringes, stents, catheters, transcutaneous patches, microneedles , microabrasives and implantable controlled release devices In one modality, the term "blood pressure device "pharmaceutical distribution" refers to a double chamber syringe capable of mixing two components prior to injection. The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals, without toxicity, irritation, excessive allergic response, or other problems or complications, commensurate, with a reasonable benefit / risk ratio. The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a filler, diluent, excipient or solvent, liquid or solid capper material, involved in carrying or transporting the compound target from one organ or portion of the body, to another organ or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation, and not harmful to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and waxes for suppositories; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) damping agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline solution; (18) Ringer's solution; (19) ethyl alcohol; (20) buffer solutions; (21) polyesters, polycarbonates and / or polyanhydrides; and (22) other non-toxic compatible substances employed in pharmaceutical formulations. "Pharmaceutically acceptable salts" refer to the relatively non-toxic inorganic or organic acid addition salts of the compounds. The term "transport" or "lipid transport" refers to a lipid transport or a portion thereof which is a specialized cell membrane domain (see Simons et al. , (2000) Nature Reviews / Molecular Cell Biology 1:31). In particular, the term "lipid transport" describes a microdomain enriched in cholesterol and in glycosphingolipid of any membrane of a eukaryotic cell. Lipid transportes tend to be enriched in signaling molecules, with growth factor receptors and sensor molecules that have been shown to migrate to lipid transport after ligand binding or cross-linking. Lipid transportes are characterized by their resistance to solubilization at low temperature in non-detergents. ionic, and can change size and composition in response to intra- or extra-cellular stimuli. Specific protein-protein interactions can be favored within lipid transport, resulting in modulation of the activities of the signaling cascade in the case, for example, cytokine receptors of the plasma membrane. The potential effects either of "transportation" (defined herein as the incorporation of a membrane component, eg, a receptor, within a lipid transport) or "de-transporting" (defined herein as elimination, departure or sweeping a membrane component, eg, a receptor from a lipid transport) of a given receptor, which includes the modulation of signaling mediated by the cytokine receptor (in some cases the triggering of apoptotic cell death), cell localization of the receiver, and abundance of the receiver. Sometimes lipid transport can be grouped; and it has been reported that such clustering is used artificially and physiologically to trigger signaling cascades. In one embodiment, the receptor coupling agent of the invention carries two receptors of members of the TNF family within a lipid transport. In yet another embodiment, the coupling agent to the receptors of the invention carries a receptor of the TNF family outside a lipid transport. The term "receptor coupling agent" includes any agent or construct that can activate at least two different cell surface receptors. In one embodiment, the coupling agent to the receptors is a protein agent. The coupling agents to the receptors are used to increase the signaling capacity of the cell surface receptors. The coupling agents to the receptors of the invention are directed to the receptors of the TNF family. In some cases, the activation of at least two receptors of the TNF family by a coupling agent to the receptors can induce cell death. In one embodiment of the invention, the receptor coupling agent comprises a bispecific multivalent construct. In yet another embodiment, the receptor coupling agent is a bispecific multivalent construct comprising a binding or specificity of anti-LTβR, and a portion and binding specificity to anti-TRAIL-R2. In yet another embodiment, the receptor coupling agent comprises the binding specificity conferred by an anti-LTβR antibody (e.g., CBEll) and the binding specificity conferred by an anti-TRAIL-R2 antibody (e.g., 14A2) . The term "single-chain variable fragment or scFv" refers to an Fv fragment in which the heavy chain domain and the light chain domain are linked.

One or more scFv fragments can be ligated to other antibody fragments (such as the constant domain of a heavy chain or a light chain) to form antibody constructs having one or more antigen recognition sites. The term "synergistic" refers to a combination that is more effective than the additive effects of any two or more simple agents. In one embodiment of the invention, the synergistic or synergistic term includes a combination type of supra-additive inhibition in which the L-T-β-R agonist and the chemotherapeutic agent individually have the ability to inhibit tumor volume. The term "enhancement" refers to a case in which the simultaneous effect of two or more agents is greater than the sum of the independent effects of the agents. "Treatment" of cancer in a subject or "treatment" of a subject having cancer, refers to subjecting the subject to a pharmaceutical treatment, for example, the administration of a drug, such that the degree of cancer is diminished or prevented. The treatment includes (but is not limited to) the administration of a composition such as a pharmaceutical composition, and may be performed either prophylactically, or subsequently at the onset of a pathological event. The term "tumor volume" refers to the total tumor size, which includes the tumor itself or more affected lymph nodes, if applicable. The volume of the tumor can be determined by a variety of methods known in the art, such as, for example, by measuring tumor dimensions using calipers, computed tomography (CT) or magnetic resonance imaging scans (MRI). ), and calculating the volume using equations based on, for example, the diameter of the z axis, or on the standard forms such as sphere, helipsoid or cube.

II: Objectives of the coupling agent to receptors A limiting factor in the treatment of tumors with the activating agents of the TNF family is that often only a subset of tumors seems to be sensitive to such therapies. Receptor-binding agents can specifically activate receptors of the TNF family, and increase receptor signaling, for example, by putting the TNF family receptors in close proximity (for a review on TNF receptors and the family TNF see Locksley et al (2001) Cell 104: 487). The invention provides coupling agents to receptors that can target more than one receptor of the T? F family and increase signaling, thereby providing an improved method of cancer treatment. As such, receptor coupling agents can deliver stronger or more complex signaling, and therefore are effective over a broader gamma of tumors, as shown in example 3. In one embodiment, the coupling agent the receptors increase the strength of the signal by increasing the number of receptors that are put together (Holler N Fau-Tardivel, et al., (2003) Mol Cell Biol. 23: 1428). In yet another embodiment, the receptor coupling agent activates two different receptors of the TNF family, thereby increasing the strength of the signal and triggering two different cascades of signal transduction. The coupling agent to the receptors of the invention comprises the binding specificities that are directed to at least two different members of receptors of the TNF family. The binding specificities are chosen according to the receptor members of the TNF family of interest to which they are going to be directed. For example, in one embodiment, a receptor coupling agent comprises a first binding specificity for the TNF TRAIL-R2 receptor, and a second binding specificity for the T? F receptor, lymphotoxin ß receptor (LTßR) . Examples of different types of T? F family receptors that can be targeted by a receptor coupling agent are described in more detail below.

A. TNF Receptors Containing a Death Domain Receptor-binding agents can be targeted to receptors of the TNF family that contain domains of death, which may be useful for the treatment of cancer. A "death domain" or "DD" (for its acronym in English) refers to a protein domain of certain TNF receptors comprising six conserved alpha helices. TNF receptors that contain death domain are primary targets of the coupling agents to the receptors of the invention, and an example of such construction is provided in the examples section. An example of a death domain receiver is Fas. The Fas pathway molecules include any molecule involved in or related to a pathway leading to apoptosis or programmed cell death (PCD) induced by Fas. Molecules of the Fas pathway include, but are not limited to, Fas, the Fas ligand (FasL) and members of the TNFR superfamily of receptors. FADD, caspase 8, bid, and caspase 3 are also included with molecules of the Fas pathway. The Fas pathway molecules can also be included in other groups as defined herein.

Some of the cytotoxic effects of lymphocytes are mediated by the interaction of a lymphocyte bound, with Fas-R (also known as DR-2, APO-1 and CD95; GenBank Gl Nos. 4507583, 23510421, 23510423, 23510425, 23510427, 23510429, 23510431, and 23510434), a broadly appearing cell surface receptor, which has the ability to trigger cell death (see Nagata and Golstein, (1995) Science 267: 1449-56). The binding of FasL to the Fas receptor leads to aggregation of the receptor on the cell membrane and the specific recruitment of intracellular signaling molecules known as DISC, or the death-inducing signal complex. The adapter protein, FADD, binds to the intracellular death domain of Fas, which leads to the recruitment of caspase 8, also known as FLICE or MACH. Fas-induced cell death can activate a pathway that alters the mitochondrial permeability transition. Cell death by mononuclear phagocytes involves a ligand-receptor pair, TNF and its receptor, TNFR1, (also known as DR-1, CD120, p55-R, GenBank Gl No. 4507575, see also United States Patent No. 5,395,760), which is structurally related to Fas-R and its ligand (see also Vandenabeele et al., (1995) Trends in Cell Biology 5: 392). Like other effects induced by the receptor, the induction of cell death by TNF and FasR receptors occurs through a series of protein-protein interactions, which lead from the ligand-receptor link to the eventual inactivation of effector functions enzymatic, which in the case of these particular receptors, results in cell death. Under normal circumstances, Fas receptor coupling is achieved by infiltration of inflammatory cells and secondary necrosis, and also causes inflammation, eg, hepatic inflammation, by inducing the expression of cellular chemokines, eg, hepatic chemokines, which they recruit and activate immune cells leading to the death of cells, for example, of hepatocytes, in a pro-inflammatory environment. In contrast, the coupling agents to the receptors of the present invention are designed to induce cell death in specific target or target cells. The targeted therapy of the invention may be more potent due to the increased signaling and, therefore, may allow treatment with lower doses of a drug. Such a strategy can minimize the negative consequences observed when apoptosis is systemically induced by activation of a simple cell surface cytokine receptor. In addition to Fas-R and T? F-Rl, other members of the TNF receptor family that contain domains of death include DR3 (also referred to as TRAMP, TR3, and Apo3, see GenBank Gl Nos. 4507569, 23200021, 23200023, 23200025, 23200027, 23200029, 23200031, 23200033, 23200035, 23200037, and 23200039); TRAIL-Rl (also referred to as DR4 and Apo, see GenBank Gl No. 21361086); TRAIL-R2 (also called as DR5, see GenBank Gl Nos. 22547116 and 22547119); p75NGF-R (also referred to as TNFRSF16;? CBI Reference Seq.

? P_002498; GenBank Gl No. 4505393); and DR6 (TRAIL-R3, GenBAnk Gl No 22547121), each containing domains of death that directly initiate apoptosis. There are four human TRAIL receptors called TRAIL-Rl-4. TRAIL-Rl and R2 also known as death receptors 4 and 5 (DR4-5) contain domains of death in the intracellular region, and are capable of triggering apoptosis (Wang and El-Deiry (2003) Oncogene 22: 8628).

TRAIL-R2 is preferred for tumor therapy in humans, since its activation does not trigger apoptosis of hepatocytes and therefore must have reduced toxicity (Ichikaw et al. (2001) Nat Med 7: 954). In this way, the coupling agents to the receptors that activate various receptors of the T? F family that contain domains of death, alone or in combination with any other T? F, for example, a non-death domain T? F such as LT? R, are encompassed by the invention. In one embodiment, a receptor coupling agent is used to decrease the toxic effects of TNF receptors that contain the death domain. While the activation of some receptors that contain domains of death, for example, T? FRl or Fas, has been shown to be toxic in vivo, it is likely that the binding of these receptors to other T? F receptors can decrease the toxicity and in this way, it makes a toxic antibody less toxic. For example, if the transporter or raft-type association is critical for complete T? FRI signaling, unlocking by hooking to a non-transported receiver may be sufficient to reduce the anti-T? FRI toxicity. In one embodiment, a receptor coupling agent comprises a binding portion comprising an anti-LTβR antibody or the antigen binding fragment thereof, and a binding portion directed to an anti-T? F family receptor, which contains a domain of death.

B. Receptors of the non-dying domain The coupling agents to the receptors of the invention can be directed to receptors of the T? F family that do not contain the death domain. The activation of T? F receptors containing the non-domains, for the treatment of solid tumors, specifically a monoclonal anti-LTßR agonist antibody (mAb), also shows potential as an anti-tumor therapy (Browning, et al. (1996) J Exp Med 183: 867, Wilson and Browning (2003) Cell Death Diff 9: 1321). An example of a member of the TNF receptor family that contains the non-dying domain is LTßR. LTßR is involved in the control of the maturation state of various specialized stromal cells in the immune system and plays a critical role during the development of the stromal elements of the lymph node primordium ((Mebius (2003) Nat Rev Immunol 3: 292). It has been proposed that the activation of a development program in epithelial and fibroblastoid cells, in the context of a transformed cell, is harmful for its survival, and this action may explain some of the antitumor activity of the LTß receptor activation. receptors can also initiate inflammatory programs that involve the release of chemokine or promote immunological antitumor responses (Yu et al. (2004) Nat Immunol 5: 141, Baud (2001) Trends Cell Biol 11: 372). Such release can affect the inflammatory state of the tumor and / or invoking the infiltration of the lymphoid elements that promote an immunological reaction to the tumor. receptor coupling partners that activate various receptors of the TNF family that lack domains of death, alone or in combination with TNF receptors that contain domains of death, are encompassed by the invention. In addition to LTßR, other examples of TNF receptors that lack a death domain include Fnl4 (also referred to as TWEAK-R; see co-pending application of Applicant WO 02/22166); RA? K (see? CBI Nos. Access.

AAB86809, AF018253); TAJ (also referred to as TROY, see ? CBI Nos. Access AAF71828, AAH47321, AAK28395); WWTP (see NCBl Access. Nos. AAD50076, AAD50077, AF130988); XEDAR (see ? CBI Access Nos. AAG28761, AAH34919, AAN73210); and CD40 (also referred to as the CD40L receiver, see NCBl Nos.

Access AAH12419, AAH64518, AAR84238). A subgroup among TNF receptors that lack a death domain includes TNF receptors that are involved in tissue differentiation, including the development and healing of wounds. Several receivers TNFs have well-defined roles in the development, for example, LTßR, RANK, EDAR and XEDAR (Mebius (2003) Nat Rev Immunol 3: 292; Theill et al., (2002) Ann Rev Immunol 20: 795; Larikkala et al. , (2002) Development 129: 2541; Rennert (2000) J "Exp Med 192: 1677) Differentiation is the process by which normal cells undergo physical and structural changes as they develop to form different tissues of the body.Differentiation programs can affect tumors in several ways. , TNF receptors involved in tissue differentiation have the potential to directly delay tumor growth by altering the cell cycle progression.Second, the program in the context of transformation can lead to cell site conflict and apoptosis Thirdly, such an introduction of conflict can make a cell more sensitive to chemotherapy.Examples of TNF receptor molecules that mediate tissue differentiation, which can be directed by a coupling agent to receptors for Increase TNF signaling, include the following: RANK (also known c as T? FRSF11A; GenBank Gl? o. 4507565; ? o? Access AF018523; United States Patent Nos. 6,562,948; 6,537,763; 6,528,482; 6,479,635; 6,271,349; 6,017,729); EDAR1 (also known as Downless; GenBank Gl No. 11641231; Accession No. AF130988; United States Patent No. 6,355,782); and TAJ / Troy / Trade (also known as TNFRSF19; GenBank Gl Nos. 23238202 and 23238204; Accession No. AF167555). In addition, XEDAR (also known as EDA-A2R; GenBank Gl? O. 11140823; Access No. AF130988), signaling is involved in the process of ectodermal differentiation. XEDAR plays a major role in activating the NF-kappaB and J? K pathways. It has been shown that Fnl4 is involved in nerve regeneration (Tanabe et al. (2003) J \ Neurosci.23: 9675). Fnl4 is also known as TWEAKR and TNFRSF12A (see GenBank Gl? O., 7706186; U.S. Patent? Or, 6,727,225; U.S. Patent Application Publication? O. 2004 / 0033225A1). Thus, coupling agents to receptors that activate various T? F family receptors involved in tissue differentiation are encompassed by the invention.

C. Receptors of immune regulation The T? F receptor superfamily also contains several receptors involved in immune regulation, which can be directed by the constructs of the present invention. Such receptors include T? FR2 (also known as T? FRSF1B; GenBank Gl No. 4507577), HVEM (also known as T? FRSF14; GenBank Gl No. 23200041), CD27 (also known as T? FRSF7; GenBank Gl No. 4507587), CD30 (also known as T? FRSF8; GenBank Gl Nos. 4507589 and 23510437), CD40 (also known as TNFRSF5; Gl Nos. 4507581 and 23312371), 4-1BB (also known as TNFRSF9; Gl No. 5730095) , OX40 (also known as TNFRSF4; Gl No. 4507579), GITR (also known as TNFRSF18; GenBank Gl os. 4759246, 23238194 and 23238197), TACI (also known as TNFRSF13B; Gl No. 6912694), BAFF-R (also known as TNFRSF4; known as TNFRSF13C; Gl No. 16445027), BCMA (also known as TNFRSF17; Gl? 23238192), and RELT (also known as T? FRSF19L; Gl Nos. 21361873 and 23238200). Additional TNF family receptors, involved in immune regulation, include TRAIL-R3 and TRAIL-R. Thus, coupling agents to receptors that activate various receptors of the TNF family, involved in immune regulation, are encompassed by the invention.

D. Other TNF receptors Other receptors of the target TNF family can be selected for their role in the formation of tumors, and can be used by identifying existing RNA databases of the expression of the receptor in various cell types that allow to define the receptors of the TNF family that are present or are ideally overexpressed on different tumors. In addition, existing RNA databases provide an additional advantage in that the pair of receptors of the TNF family could be optimized by identifying those pairs of receptors that are more uniquely expressed on a tumor type or subgroup of tumors, but they are not abundant on normal tissues, especially the liver and vasculature. In this way, pairs of receptors (or more) are identified, which could distribute a strong signal to the tumor and effect normal tissues. Methods to test the efficacy of the selected tumors are described in more detail below and in the examples section. The coupling agents to the receptors of the invention are directed to at least two different TNF receptors. Target or target TNF receptors are selected based on the individual characteristics of the receptor. For example, a receptor coupling agent can target two TNF receptors that are involved in differentiation events and, therefore, can be effective in the treatment of solid tumors. Other examples of combinations of TNF receptors to which the coupling agent can be directed to the receptors of the invention are described below.

TNF receptor coupling agent with non-death domain / death domain In one embodiment of the invention, the receptor coupling agent targets and activates a TNF receptor that contains a death domain and a TNF receptor that it does not contain a death domain. The examples of combinations of TNF receptors that contain the non-death domain / death domain, targeted, include: LTBR / TRAIL-Rl; LTBR / TRAIL-R2; LTBR / p75NGF-R; Fnl / p75NGF-R; and p75NGF-R / TAJ. The coupling of a TNF receptor containing the death domain to a receptor containing the death domain, can further decrease the toxicity of the receptor activation that contains the death domain. In yet another embodiment, at least one of the T? F receptors that contain the non-killing domain is involved in cell differentiation, including, but not limited to, LTBR, RA? K, and Fnl4. As described in detail below, LTBR, RANLK and Fnl4 are each involved in cell differentiation. Examples of T? F receptors that contain the non-death domain / death domain, wherein the T? F death domain receptor is involved in cell differentiation include, for example, LTBR / p75? GF-R; Fnl / p75? GF-R; and TAJ / p75? GF-R.

TNF receptor coupling agent of the non-dead domain / no-kill domain In one embodiment of the invention, the receptor coupling agent targets and activates two distinct T? F receptors, none of which contains a death domain. Examples of combinations of T? F receptors that contain the non-domains / non-domains domain, led by a coupling agent to the receptors, include: LTBR / Fnl4; LTBR / RA? K; Fnl4 / TAJ; LTBR / WWTP; LTBR / XEDAR; RANK / WWTP; RANK / XEDAR; TAJ / EDAR; TAJ / XEDAR; and LTBR / CD40. In yet another embodiment, at least one of the TNF receptors that contains the non-death domain / non-death domain is involved in cell differentiation. For example, the coupling agent to the receptors can be directed to LTBR and Fnl. Fnl4 is the receptor for TWEAK, a TNF ligand with the ability to induce cell death in the HT29 adenocarcinoma cell line (see Chicheportiche et al. (1997) J. Biol. Chem. 272: 32401). The apoptotic activity of TWEAK is mediated by Fnl4. Fnl4 is also involved in tissue remodeling after damage. The molecular mechanisms found in tissue remodeling are similar to tissue differentiation, and such programs may not be favorable for tumor growth. In yet another example, a coupling agent to the receptors can be directed to LTBR and RANK RANK signaling triggers the differentiation of the mammary epithelium, and therefore, may have enhanced activity when coupled to another differentiation-inducing agent. Thus, the increased signaling of RANK and LTBR using a coupling agent to the receptors may be useful in the prevention of tumor growth. In addition to LTBR, RA? K, and Fnl4, TAJ / TROY plays a role in tissue differentiation, specifically in the regulation of axonal regeneration (Shao et al. (2005) Neuron 45: 353). TAJ is involved in the repression of neurite growth in response to myelin components, for example, effectively a differentiation event. Since Fnl4 is expressed in post-neuronal damage (Tanabe et al (2003) J. Neurosci 23: 9675), the signal combined with TAJ can be used to treat tumors associated with the central nervous system. Thus, in one embodiment, a coupling agent to the receptors can be directed to TAJ and Fnl4. Other examples of combinations of TNF receptors that contain non-killing domain that are involved in tissue differentiation, and therefore, may be beneficial in the inhibition of tumor growth, include but are not limited to, LTBR / WWTP; LTBR / XEDAR; RANK / WWTP; RANK / XEDAR; TAJ / EDAR; and TAJ / XEDAR.

Death domain TNF receptor coupling agent / death domain In one embodiment of the invention, the receptor coupling agent binds to two different TNF receptors, both of which contain a death domain. TRAIL-R1 / TRAIL-R2 is an example of a combination of TNF receptors comprising the death domain / death domain to which a coupling agent can be directed to the receptors.

TNF / immunological receptor coupling agent In one embodiment of the invention, the receptor coupling agent binds to two different TNF receptors that are involved in an immune response. Examples of combinations of immune response TNF receptors that mediate B cell responses include, CD40 / CD27; CD40 / BAFF-R; CD40 / BCMA; and BAFF-R / CD27. Examples of combinations of TNF receptors that mediate immune responses of T cells include CD27 / CD30; CD27 / OX-40; CD27 / 41BB; and OX-40 / 41BB.

III. Coupling agents to receptors A coupling agent to receptors is capable of inducing the formation of a heteromeric receptor complex comprising a receptor coupling agent, and at least two different receptors of the T? F family. The receptors of the T? F family have common signaling modalities as well as specialized transduction mechanisms, unique to specific receptors. These signal transduction pathways are highly complex with three or more pathways that are activated in many cases. The use of a coupling agent to the receptors to induce the formation of a heteromeric receptor complex can more effectively limit tumor growth. For example, a receptor coupling agent that addresses the relatively unique ability of LTßR to activate the alternative NFKb pathway (Dejardin et al., (2002) Immuni ty 17: 525) coupled with caspase activation that comes from a receptor that contains death domain can (eg, TRAIL-R2) result in reduced tumor growth. In addition, such agents can juxtapose two different receptors of the TNF family in a complex, resulting in the co-assembly of elements of the signal transduction machinery into aggregates that are novel and even potentially non-physiological. The coupling agents to the receptors of the invention can be used to reorient a receptor of the T? F family within a single cellular membrane environment, which affects signaling. The signaling capacity of some receptors depends on their location within specialized membrane environments, such as within transports or lipid rafts. Examples of such TNF family receptors include Fas and possibly the TNFRl receptor (Muppidi and Siegel (2004) Nat Immunol 5: 182, Legler et al., (2003) Immuni ty 18: 655). TRAIL receptors also show complex localization patterns (Zhang et al., (2000) J "Immunol 164: 3961) A receptor coupling agent that is coupled to a receptor that normally resides within a raft or lipid transport for another The receiver, which is not normally transported, can force the second receiver into the transport environment and increase its signaling capacity, likewise a coupling agent to the receivers that is coupled to a receiver that normally resides outside a raft or transport. lipid to another receptor, which is normally within the transport can force the first receptor out of the transport environment and decrease the signaling capacity The coupling agent to the receivers of the invention can increase the strength of the signal, forming new complexes of non-physiological heteromeric receptors that exemplify the new signaling characteristics and / or relocate a receiver within an environment where signaling is more or less effective. In one embodiment, the coupling agent to the receptors of the invention is used to place a recipient of the TNF family in a raft or lipid transport in which the receptor of the TNF family is not normally found. In yet another embodiment, the receptor coupling agent is capable of relocating a receptor of the TNF family out of a raft or lipid transport. The coupling agents to the receptors of the invention include any agent that is capable of forming a heteromeric complex with at least two different TNF receptors. The receptor coupling agent comprises at least two binding specificities that are directed to two different TNF receptors. A binding specificity includes any entity that affects the signaling of the receiver, for example, that increases or decreases the signaling of the receiver. Examples of binding specificity agents that can be used to prepare a coupling agent to the receptors of the invention, include, but are not limited to, antibodies, antigen-binding fragments thereof, ligands for the TNF receptor. or any combination thereof.

A. Antibodies In one embodiment, the coupling agent to the receptors of the invention contains specificities or binding portions that comprise or are derived from at least two antibodies or fragments of antigen binding thereof, directed to family members. of TNF receptors. A receptor coupling agent that is a bifunctional construct can contain sequences obtained from a parent antibody directed to the T? F receptors of interest. Bifunctional constructs that can bind and activate two TNF receptors offer a new procedure, such as constructs that embody the ability to activate two different TNF receptors, and thus avoid the complications of packaging two antibodies within a cocktail of drug, a complex process from a pharmaceutical manufacturing point of view. In one embodiment, the receptor coupling agent is a multivalent construct comprising agonists of the TNF family receptors, wherein the receptor coupling agent comprises at least two domains that are capable of binding to each receptor and inducing an activation signal. The antibody constructs of the invention may include a heavy chain containing two or more variable regions comprising the antigen recognition sites, specific for the binding of a TNF family receptor and a light chain containing one or more variable regions comprising the specific antigen recognition sites for a receptor of the TNF family. Antibody constructs can also be constructed to comprise only heavy chains or light chains containing two or more variable regions comprising specific CDRs for binding to a receptor other than the TNF family. In one embodiment, the multivalent antibody comprises antigen binding sites or linker portions that can bind to TRAIL-R2 and LTßR. In one aspect, the present invention provides multivalent antibody constructs that are TNF receptor agonists, including but not limited to, the LTßR agonists and TRAIL-R2. In one embodiment, a multivalent antibody construct comprises at least one antigen recognition site specific for an LTβR epitope. In yet another embodiment, a multivalent antibody construct comprises at least one antigen-specific recognition site for a TRAIL-R2 epitope. In certain embodiments, at least one of the antigen recognition sites is located within a scFv domain, while in other embodiments, all antigen recognition sites are located within the scFv domains. In certain embodiments, the coupling agent to the receptors is bispecific. In other embodiments, the specific construction for at least two members of the TNF family of receptors, including but not limited to, the LTβR epitopes and TRAIL-R2 epitopes. In any of the multispecific constructs, at least one antigen recognition site can be located on a scFv domain, and in certain embodiments, all antigen recognition sites are located on the scFv domains. In other additional embodiments, the antibody constructs of the invention comprise the polynucleotide sequences described in SEQ ID Nos. : 5 and 7 (LT-BSl construction). The specificities or binding portions comprising antigen recognition sites or entire variable regions can be derived from one or more parent antibodies. Progenitor antibodies may include naturally occurring antibodies or antibody fragments, antibodies or antibody fragments adapted from naturally occurring antibodies, antibodies constructed de novo using antibody sequences or antibody fragments known to be specific for the LT-receptor. beta. Sequences that can be derived from progenitor antibodies include heavy and / or light chain variable regions and / or CDR, structural regions or other portions of the same. In one embodiment of the invention, the progenitor antibodies used to construct a receptor coupling agent are an anti-TRAlL-R2 antibody, for example 14A2, and an anti-LTβR antibody, for example CBEll. Multivalent, multivalent antibodies may contain a heavy chain comprising two or more variable regions and / or a light chain comprising one or more variable portions, wherein at least two of the variable regions recognize different epitopes on the LT-beta receptor. The receptor coupling agents comprising anti-multivalent TNF receptor antibodies can be constructed in a variety of different ways, using a variety of different sequences derived from the anti-LTßR progenitor antibodies including murine or humanized BHA10 (WO 04 / 002431; see also Browning et al. , (1995) J. Immunol. 154: 33; Browning et al. , (1996) J. Exp. Med. 183: 867), murine or humanized CBEll (U.S. Patent No. 6,312,691 and WO 02/30986, respectively), and / or murine or chimeric 14A2 anti-TRAIL-R2 progenitor (see SEQ ID Nos: 1 and 3 ) . Examples of murine anti-LTßR antibodies that can be used for the coupling agent to the receptors of the invention include BKAll, CDH10, BCG6, AGH1, BDA8, CBEll and BHA10. The following hybridoma cell lines that produce anti-LT-βR-monoclonal antibodies can be used to produce anti-LTβR antibodies from which antibody construction sequences are derived, which have been previously deposited with the American Type Culture Collection ( ATCC) in accordance with the provisions of the Budapest Treaty and have been assigned the ATCC accession numbers indicated Cell line Name of the mAb Access number a) AG. Hl .5.1 AGH1 HB 11796 b) BD .A8.AB9 BDA8 HB 11798 c) BC.G6.AF5 BCG6 B 11794 d) BH.A10 BHA10 B 11795 e) BK.A11.AC10 BKA11 B 11799 f) CB.Ell. l CBEll B 11793 g) CD.H10.1 CDH10 B 11797 Examples of humanized anti-LTßR antibodies that can be used in conjunction with the present invention include humanized CBEll and humanized BHA10. The following hybridoma cell lines can be used to produce anti-LTßR antibodies from which to derive the sequences of the antibody construct, which have been previously deposited with the American Type Culture Collection (ATCC) according to the provisions of the Budapest Treaty. and they have been assigned the indicated ATCC accession numbers: PTA-3357 and 3765 (humanized CB? ll, see WO 02/30986) and PTA-4726 (humanized BHA10, see WO 04/002431). Other examples of anti-TNF receptor antibodies that are compatible with the coupling agents to the receptors of the invention can be derived from the antibodies directed to the TNF receptors that contain a death domain. A number of antibodies have been generated for TNF receptors that contain death domain and are well known in the art. Such antibodies include the anti-TNF-Rl anti-TNF-Rl monoclonal antibodies of R & D systems); mAb Tularik # 985, United States Patents Nos. 6,110,690; 6,437,113), anti-Fas receptor CH-11 mAb (U.S. Patent No. 6,312,691; WO 95/10540), anti-DR3 antibodies (U.S. Patent No. 5,985,547; Johnson, et al., (1984 ) ImmunoBiology of HLA, ed. Dupont, BO, Springer, New York, U.S. Patent No. 6,462,176; 6,469,166), and anti-TRAIL-R antibodies (U.S. Patent Nos. 5,763,223; 6,072,047; 6,284,236; 6,521,228) 6,569,642; 6,642,358; and U.S. Patent No. 6,417,328). A number of antibodies have also been produced for T? F receptors involved in tissue differentiation and are known in the art. Examples of anti-T? F receptor antibodies specific for T? F receptors involved in tissue differentiation include: anti-RANG monoclonal antibodies (Immunex - U.S. Patent Nos. 6,562,948; 6,537,763; 6,528,482; 6,479,635; 6,271,349; 6,017,729; Komed-WO 03/080671), anti-EDAR (anti-human) and monoclonal (anti-mouse) monoclonal antibodies (R & D Systems - MAB745, BAF157; Elomaa et al. (2001) Human Molecular Genetics. 10: 953), anti-XEDAR monoclonal and polyclonal antibodies (R & D Systems - MAB1093 and JAF1093), anti-Fnl4 monoclonal antibodies (Nakayama et al. (2003) J. Immunology 170: 341; clones ITEM-1, ITEM- 2, and ITEM-4 available from eBioscience), anti-TROY antibody (T3323 from Sigma-Aldrich), and anti-NGFR (anti-rodent) antibodies (Chemicon EUA). A number of antibodies have also been produced for TNF receptors involved in immune regulation and are known in the art. Examples of anti-TNF receptor antibodies, specific for the T? F receptors involved in immune regulation include: anti-HVEM antibodies (HGSI-WO 03/086301), anti-CD40 antibodies (Biogen - WO 97/20063; Chiron - U.S. Patent Nos. 5,677,165; 5,874,082; 6,004,552; 6,056,959; 6,315,998; U.S. Patent Application Publication No. 2002/0106371; U.S. Patent Application Publication No. 2003/0059427; US20030118588A1; 2003 / 0211100A1; US2002020142358A1; United States Patent Nos.

US6312693; US6051228; Fanslow et al. - US5801227), anti-4-lBB (PCT Publication No. WO 03/084999; EP 0948353; United States Patent No. 6210669; Genecraft - WO 03/083069), anti-BAFF-R (rabbit polyclonal - ProSci catalog # 3097) antibodies, among many other antibodies produced for receptors of immune regulation. Multivalent constructs targeted for TNF receptors can be developed by a person skilled in the art using routine recombinant DNA techniques, for example as described in the PCT International Application No. PCT / US86 / 02269; European Patent Application No. 184,187; European Patent Application? O. 171.496; European Patent Application? O. 173.494; International Publication of the PCT? O. WO 86/01533; United States patent? 4,816,567; European Patent Application? O. 125,023; Better et al. , (1988) Science 240: 1041-1043; Liu et al. , (1987) Proc. Nati Acad. Sci. USA 84: 3439-3443; Liu et al. , (1987) J. Immunol. 139: 3521-3526; Sun et al. (1987) Proc. Nati Acad. Sci. USA 84: 214-218; Nishimura et al. , (1987) Cancer Res. 47: 999-1005; Wood et al. (1985) Nature 314: 446-449; Shaw et al. , (1988) J. Nati. Cancer Inst. 80: 1553-1559); Morrison (1985) Science 229: 1202-1207; Oi et al. , (1986) BioTechniques 4: 214; U.S. Pat. Do not . 5,225,539; Jones et al. , (1986) Nature 321: 552-525; Verhoeyan et al. , (1988) Science 239: 1534; Beidler et al. , (1988) J. Immunol. 141: 4053-4060; and Winter and Milstein, (1991) Nature 349: 293-99). Preferably, the non-human antibodies are "humanized" by binding the non-human antigen binding domain to a human constant domain (eg, Cabilly et al, U.S. Patent No. 4,816,567; Morrison et al. 1984) Proc. Nati, Acad. Sci. USA, 81: 6851-55). Other methods that can be used to prepare multivalent TNF anti-receptor antibody constructs are described in the following publications: Ghetie et al. (2001) Blood 97: 1392-1398; Wolff et al. (1993) Cancer Research 53: 2560-2565; Ghetie et al. (1997) Proc. Nati Acad. Sci. 94: 7509-7514; Kim et al. (2002) Int. J. Cancer 97 (4): 542-547; Todorovska et al. (2001) Journal of Immunological Methods 248: 47-66; Coloma et al. (1997) Nature Biotechnology 15: 159-163; Zuo et al. (2000) Protein Engineering (Supplement) 13 (5): 361-367; Santos et al. (1999) Clinical Cancer Research 5: 3118s-3123s; Presta (2002) Current Pharmaceutical Biotechnology 3: 237-256; van Spriel et al. (2000) Review Immunology Today 21 (8) 391-397.

B. TNF ligands The coupling agent to the receptors of the invention also includes binding specificities comprising at least two ligands of the TNF family, conventional, coupled together. Examples of ligands of the TNF family include, but are not limited to, TNF-alpha (NP_000585.2, Gl No. 25952111) LT-alpha, (NP_000586.2, Gl., 6806893), FasL (? P_000630; GenBank Gl No. 4557329), APO-3L (NP_003800, Gl? 4507597;? P_694557, Gl No. 23510441), TRAIL (APO-2L,? P_003801, Gl? O. 4507593), RA? KL (T? FSFll ,? P_003692, Gl. 4507595;? P_143026, Gl No. 14790152), EDARI ligand and XEDAR (EDl, NP_001390, Gl? 4503449; Monreal et al. (1998) Am J Hum Genet. ), Ligand Fnl4 (APO-3L / TWEAK), ligand of? GF Troy / Trade (? GF-β? P_002497, Gl No. 4505391), NGF family (NGF-2 / NTF3 NP_002518, Gl No. 4505469;? TF5 ,? P_006170, GL? 5453808 BD? F:? P_001700, Gl? 25306267;? P_733927, Gl No. 25306235 NP_733928, Gl? 25306253; NP_733929, Gl No. 25306257 NP_733930, Gl No. 25306261; NP_733931, Gl No. 25306264 IFRD1, NP_001541, Gl No. 4504607), TNFRII ligand (TNF above), HVEM ligand (NP__003798, Gl No. 25952144 NP_742011, Gl No. 25952147), CD27L (formerly gen of CD70 NP_001243, Gl No. 4507605), CD30L (CD153, NP_001235, Gl No 4507607), CD40L (CD154, NP_000065, Gl No. 4557433), 4-lBB-L (ILA ligand,? P_003802, Gl No. 4507609), OX40L (CD134L, NP_003317, Gl No.- 4507603), GITRL (AITRL / TL6,? P_005083, Gl? O. 4827034), and BAFF (TALL1,? P_006564, GI No. 5730097).

C. Antibody / receptor combinations The coupling agents to the receptors of the invention also include any combination of above-mentioned anti-TNF receptor antibodies and TNF ligands. For example, the receptor coupling agent may comprise a combination of a ligand-Fe construct coupled to an antibody to a TNF family receptor in a form that creates a molecule with the two trimeric ligands and three antibodies, or any complexes of higher order. In one embodiment, the first binding specificity comprises at least two Fe-ligand trimeric constructs that are commonly formed from three dimeric Fc domains and six ligand molecules. In this case, the second binding specificity could be comprised of the three antibody molecules. The invention also includes a combination of a conventional ligand (not an Ig fusion protein) to a receptor of the TNF family coupled to an antibody to a receptor of the TNF family.

IV. Methods for manufacturing coupling agents to receptors The efficacy of coupling agents to targeted receptors for a combination of TNF receptors can be evaluated by standard assays, including in vitro standards that assess cytotoxicity or inhibition of growth , soft agar colony formation assays and three-dimensional tumor culture systems, such as those established for breast tumor. Efficacy can also be validated by in vivo xenograft model. The use of the human primary lung, hepatic and endothelial cell lines allow in vitro prediction of notorious toxicity. Typically, induced apoptosis and visualization of surface adhesion molecules such as VCAM or ICAM serve as potential markers. For example, IL-8 and / or IP-10 as good markers for toxicity or the induction of a pro-inflammatory program that could be harmful. Cancer cell lines that can be used to test the coupling agent to the receptors of the invention are known in the art. Examples of cell lines that are frequently used as a standard model of colorectal carcinoma include, for example, the HT29 cell line for the evaluation of TNF receptor activating agents. This cell line exists in two variants, HT29 and WiDr and the HT29 cell line have been used by the National Cancer Institute in their selection panel for new potential chemotherapeutic agents. As such, this is a good tool to evaluate the potential of some anticancer agents. Examples of other colorectal cell lines include KM20L2, LS174T, and CACO-2. Examples of breast cancer cell lines that can be used to test the effectiveness of the coupling agent to the receptors include MCF7 and MDA231. Examples of cervical cancer cell lines that can be used to test the effectiveness of the coupling agent to the receptors include Hela and ME180. In addition, an example of a melanoma cell line includes A375, and the example of a rhabdomyosarcoma includes RD, and an example of a sarcoma cell line is SAOS-2. Candidate antibody constructs can be selected for activity using a variety of known assays. For example, screening assays for binding specificity are well known and routinely practiced in the art. For a comprehensive discussion of such trials, see Harlow et al. (Eds.), ANTIBODY'S: A LABORATORY MANUAL; Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y. , 1988, Chapter 6. The following examples provide the assays for determining the efficacy of the activation of the receptor coupling agent by the candidate anti-TRAIL-R2 and LTßR agonist antibody constructs. The coupling agents to the receptors described above can be purified to a purity suitable for use as a pharmaceutical composition. In general, a purified composition will have one species more than about 85 percent of all species present in the composition, more than about 85%, 90%, 95%, 99% or more of all species present. The target species can be purified to essential homogeneity (contaminating species can not be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single species. One skilled in the art can purify a polypeptide of the invention using standard techniques for protein purification, for example, immunoaffinity chromatography, size exclusion chromatography, etc., in light of the teachings herein. The purity of a polypeptide can be determined by a number of methods known to those skilled in the art, including for example, analysis of the amino-terminal amino acid sequence, gel electrophoresis and mass spectrometric analysis. In one embodiment, the coupling agents to the receptors of the invention can be conjugated to a chemotherapeutic agent to inhibit the tumor volume in a supra-additive manner. Exemplary chemotherapeutic agents that can be conjugated to the antibodies of the present invention include, but are not limited to, radioconjugates (90Y, 1311, 99mTc, IIIn, 186Rh, et al.), Tumor-activated prodrugs (maytansinoids, analogues of CC-1065, cliqueamicin derivatives, anthracyclines, vinca alkaloids per vinca, etc.), ricin, diphtheria toxin, pseudomonas exotocin. In some embodiments, multivalent receptor-binding antibodies and antibody fragments of the invention can be chemically modified to provide a desired effect. For example, the pegylation of the antibodies and the antibody fragments of the invention can be carried out by any of the pegylation reactions known in the art, as described for example in the following references: Focus on Growth Factors 3 : 4-10 (1992); European Patent 0 154 316; and European Patent 0 401 384 (each of which is hereby incorporated by reference in its entirety). Preferably, the pegylation is carried out by means of an acylation reaction or an alkylation reaction, with a reactive polyethylene glycol molecule (or a water soluble, reactive, analogous polymer). A preferred water-soluble polymer for the pegylation of the antibodies and antibody fragments of the invention is polyethylene glycol (PEG). As used herein, "polyethylene glycol" encompasses any of the PEG forms that have been used to derivatize other proteins, such as mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol. The methods for preparing pegylated antibodies and antibody fragments of the invention will generally comprise the steps of (a) reacting the antibody or the antibody fragment with polyethylene glycol, such as a reactive ester or PEG aldehyde derivative, under conditions of wherein the antibody or antibody fragment is linked to one or more PEG groups, and (b) obtaining the reaction products. It will be apparent to a person of ordinary skill in the art to select the optimum reaction conditions or acylation reactions, based on known parameters and based on the desired result. Pegylated antibodies and antibody fragments can generally be used to treat conditions that can be alleviated or modulated by the administration of the antibodies and antibody fragments described herein. In general, pegylated antibodies and antibody fragments have increased half-life, as compared to non-pegylated antibodies and non-pegylated antibody fragments. The antibody fragments and pegylated antibodies can be used alone, together or in combination with other pharmaceutical compositions. In other embodiments of the invention, the antibodies or antigen binding fragments thereof are conjugated to albumin using techniques recognized in the art. In yet another embodiment of the invention, multivalent antibodies, or fragments thereof, are modified to reduce or eliminate potential glycosylation sites. Such modified antibodies are frequently referred to as "agglucosylated" antibodies. In order to improve the binding affinity of an antibody or binding fragment to the antigen thereof, the glycosylation sites of the antibody can be altered, for example, by mutagenesis (eg, site-directed mutagenesis). "Sites of glycosylation" refers to the amino acid residues that are recognized by a eukaryotic cell, such as sites for the binding of sugar residues. The amino acids where the carbohydrate, such as the oligosaccharide, is linked, are typically asparagine (N-linked), serine (O-linked), threonine (0-linked). In order to identify potential glycosylation sites within an antibody or antigen-binding fragment, the antibody sequence is examined, for example, by the use of publicly available databases, such as in the network provided by the Center. for Biological Sequence Analysis (see http://www.cbs.dtu.dk/services/NetNGlyc/ to predict N-linked glycosylation sites) and http://www.cbs.dtu.dk/services/NetOGlyc / to predict the O-linked glycosylation sites). Additional methods for altering the glycosylation sites of the antibodies are described in U.S. Patent Nos. 6,300,861 and 5,714,350. In yet another embodiment of the invention, receptor coupling agents that are multivalent antibodies or fragments thereof can be altered, wherein the constant region of the antibody is modified to reduce at least one biological effector function mediated by the constant region, in relation to an unmodified antibody. To modify an antibody of the invention, such that it shows reduced binding to the Fc receptor (FcR), the immunoglobulin constant region segment of the antibody can be mutated in particular regions necessary for FcR interactions (see, for example, Canfield et al. (1991) "Exp. Med. 173: 1483; and Lund et al., (1991) J. of Immuno 1. 147: 2657.) The reduction in the binding ability to FcR of the antibody can also reduce other effector functions. who rely on FcR interactions, such as opsonization and phagocytosis and antigen-dependent cellular cytotoxicity In a particular embodiment the invention further characterizes multivalent receptor-binding antibodies, which have altered effector function, such as ability to bind the effector molecules, for example, the complement or a receptor on an effector cell In particular, the humanized antibodies of the invention have an altered constant invention, for example, the Fc region, wherein at least one amino acid residue in the Fc region has been replaced with a different residue or chain, which reduces the ability of the antibody to bind to FcR. The reduction in the ability of the FcR binding of the antibody can also reduce other effector functions that rely on FcR interactions, such as opsonization and phagocytosis and antigen-dependent cellular cytotoxicity. In one embodiment, the modified humanized antibody is of the IgG class, comprises at least one amino acid residue replacement in the Fc region such that the humanized antibody has an altered effector function, for example, as compared to an unmodified humanized antibody. In particular embodiments, the humanized antibody of the invention has an altered effector function, such that it is less immunogenic (e.g., does not cause unwanted activity of the effector cell, lysis or complement linkage), and / or has a more desirable half-life while retaining the specificity for LTßR. Alternatively, the invention features multivalent humanized antibodies that are coupled to the receptor, which have altered constant regions to increase the binding to FcR, for example, the link to Fc? R3.

Such antibodies are useful for modulating the function of effector cells, for example, to increase the activity of ADCC, for example, particularly for use in oncology applications of the invention. As used herein, "antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which non-specific cytotoxic cells expressing FcRs (e.g., Natural Killer cells (? K its acronym in English), neutrophils and macrophages) recognize the antibody bound on a target cell or target and subsequently cause the lysis of the target cell. The primary cells to mediate ADCC, the? K cells, express Fc? RIII only, while the monocytes express Fc? RI, Fc? RII and Fc? RIII of the antibody, for example, a conjugate of the antibody and another agent or antibody. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, .AD? recombinant, and immunology, which are within the expertise in the art. Such techniques are described in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., Ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (Glover ed., 1985); Oligonucleotide Synthesis (Gait ed., 1984); Mullis et al. U.S. Patent No.:4,683,195; Nucleic Acid Hybridization (Hames &Higgins eds, 1984); Transcription and Translation (Hames &Higgins eds, 1984); Cul ture Of Animal Cells (Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); Perbal, A Practical Guide to Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors for Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods in Enzymology, Vols. 154 and 155 (Wu et al., Eds.), Immuno chemical Methods In Cell and Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (Weir and Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1986). The production of the muBHAlO and muCBEll variable regions, the murine-human chimeric antibodies BHA10 and CBEll, the reshaped BHA10 and CBEll variable domains, the expression vectors encoding huBHAlO and huCBEll, pentameric chCBEll antibodies, and methods for purifying and To evaluate them, they have been previously described in the Applicant's Co-pending Applications, PCT publication No. WO 96/22788, PCT publication WO 02/30986, PCT application No. WO 04/002431 and WO 04/058191, which are each incorporated by reference herein in their entirety.

V. Pharmaceutical Compositions The invention provides the pharmaceutical compositions comprising the receptor coupling agents described above. In certain embodiments, the pharmaceutical compositions may further comprise a chemotherapeutic agent. In one aspect, the present invention provides the pharmaceutically acceptable compositions comprising a therapeutically effective amount of one or more of the compounds described above, formulated together with one or more carriers (additives) and / or pharmaceutically acceptable diluents. In another aspect, certain embodiments, the compounds of the invention may be administered as such or in mixtures with pharmaceutically acceptable carriers and may also be administered in conjunction with other chemotherapeutic agents. The combination therapy (combination) thus includes sequential, simultaneous and separate, or the co-administration of the active compound in a manner in which the therapeutic effects of the first administered do not completely disappear when the subsequent compound is administered. Notwithstanding the selected administration path, the compounds of the present invention, which may be used in a suitable hydrated form, and / or the pharmaceutical compositions of the present invention, are formulated in pharmaceutically acceptable dosage forms by conventional methods known to a person skilled in the art. While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition). The compounds according to the invention can be formulated for administration in any convenient way for use in human or veterinary medicine, by analogy with other pharmaceutical products. As described in detail below, the pharmaceutical compositions of the present invention can be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, eg, dressings (aqueous solutions or suspensions or non-aqueous), tablets, for example, those directed for buccal, sublingual and systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intravenous or epidural injection, for example, as a sterile solution or sterile suspension, or sustained release formulation; (3) topical application, for example, as a cream, ointment, or a controlled release patch or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8) nasally. In one embodiment, the pharmaceutical compositions are formulated for parenteral administration. In one embodiment, the pharmaceutical composition is formulated for intraarterial injection. In yet another embodiment, the pharmaceutical compositions are formulated for systemic administration. In other cases, the compounds of the present invention may contain one or more acid functional groups and, thus, are capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and flavoring agents, preservatives and antioxidants may also be present in the compositions. Formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and / or parenteral administration. The formulations can conveniently be presented in unit dosage form and can be prepared by any methods well known in the art of pharmacy. The amount of the active ingredient that can be combined with a carrier material to produce a single dose will vary depending on the host being treated, the particular mode of administration. The amount of the active ingredient that can be combined with a carrier material to produce a single dose form will generally be that amount of the compound that produces a therapeutic effect. Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular cottonseed, peanut, corn, germ, olive, castor bean and sesame oils), glycerol, tetrahydrofurfuryl alcohol , polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. In addition to the inert diluents, the oral compositions may also contain adjuvants such as wetting agents, emulsifiers and suspending, sweetening, flavoring, coloring, flavoring and preservative agents. The suspensions, in addition to the active compounds, may contain suspending agents, for example, as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof. . The formulations of the invention, suitable for oral administration may be in the form of capsules, sacks, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia or tragacanth),. powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil emulsion, or as an elixir or syrup, or as a tablet (using an inert base, such as gelatin) and glycerin, or sucrose and acacia) and / or as buccal washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. A compound of the present invention can also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol and / or silicic acid; (2) binders, such as, for example, carboxymethyl cellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and / or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds, - (7) wetting agents, such as, for example, cetyl alcohol, glycerol monostearate, and nonionic surfactants; (8) absorbers, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type can also be used as fillers or in soft and hard gelatin capsules using excipients such as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like. A tablet can be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared using binder (for example, gelatin or hydroxypropylmethylcellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface active agent or dispersant. The molded tablets can be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent. The tablets, and other solid dose forms of the pharmaceutical compositions of the present invention, such as dragees, capsules, pills and granules, can optionally be tested or prepared with coatings and protections, such as enteric coatings and other well-known coatings in the technique of pharmaceutical formulations. These may also be formulated to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions, to provide the desired release profile, other polymer matrices, liposomes and / or microspheres. These can be formulated for rapid release, for example, lyophilized. These can be sterilized by, for example, filtration through a bacteria retaining filter, or by incorporation of sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately prior to use. These compositions may also optionally contain opacifying agents and may be of a composition that releases the active ingredient (s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient may also be in a miero-encapsulated form, if appropriate, with one or more excipients described above. Dosage forms for topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound can be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers or propellants that may be required. The ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, acid silicic acid, talc and zinc oxide, or mixtures thereof. The powders and sprays may contain, in addition to a compound of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. The sprays may also contain customary propellants such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons such as butane and propane. The pharmaceutical compositions of this invention, suitable for parenteral administration, comprise one or more compounds of the invention in combination with one or more solutions, dispersions, suspensions or emulsions aqueous or non-aqueous, isotonic, sterile, pharmaceutically acceptable, or sterile powders that they can be reconstituted in injectable solutions or dispersions just before use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes which are isotonic to the formulation with the blood of the intended recipient or suspending or thickening agents. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.

The prevention of the action of microorganisms on the present compounds can be ensured by the inclusion of the various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like within the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be caused by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin. In some cases, in order to prolong the effect of a drug, it is desirable to delay the absorption of the drug from subcutaneous and intramuscular injection. This can be achieved by the use of a liguid suspension of crystalline or amorphous material that has poor water solubility. The rate of absorption of the drug then depends on its rate of dissolution which, in turn, may depend on the size of the crystal and the crystalline form. Alternatively, the delayed absorption of a parenterally administered dosage form is achieved by dissolving or suspending the drug in an oily vehicle. Injectable depot forms are made by forming microencapsulated matrices of the present compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of the drug to the polymer, and the nature of the particular polymer employed, the rate of release of the drug can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissue SAW. Methods and Distribution Devices The pharmaceutical compositions of this invention may also be administered using a variety of pharmaceutical delivery devices, which may include hypodermic syringes, multi-chamber syringes, catheters, transcutaneous patches, microaguas, microabrazives, and delivery devices. Implantable controlled In one embodiment, a pharmaceutical delivery device contains or is capable of being loaded with at least an effective amount of a coupling agent to the receptors. Such devices may have the ability to reconstitute a lyophilized form of the antibody construct in the device prior to delivery. In some embodiments, the pharmaceutical delivery device contains or is capable of being loaded with at least an effective amount of a coupling agent to the receptors, and an effective amount of a chemotherapeutic agent. The device may, in some embodiments, be capable of distributing or administering the coupling agent to the receptors and chemotherapeutic agent simultaneously. The device may have the ability to mix the antibody construction and the chemotherapeutic agent prior to administration with the device. In other additional embodiments, the device may be capable of administering the construction of agonist antibody and the chemotherapeutic agent consecutively. A pharmaceutical dispensing device is a multi-chamber syringe capable of mixing two compounds before injection, or sequentially distributing them. A typical double-chamber syringe and a process for the automated manufacture of the pre-filling of such syringes is described in Nenue Verpackung, No. 3, 1988, p. 50-52; Drugs Made in Germany, Vol. 30, p. 136-140 (1987); Pharm. Ind. 46, No. 10 (1984) p. 1045-1048 and Pharm. Ind. 46, Nr. 3 (1984) p. 317-318. The syringe-type vial is a dual chamber device with a front bottle-type opening for needle coupling, two pistons and an external type bypass for mixing a lyophilized powder in the front chamber, with a reconstitution liquid in the posterior chamber. The described process includes the main steps of washing and siliconizing the barrels of the syringe, the insertion of multiple barrels into the carrier trays, the sterilization, the introduction of the intermediate piston through the trailing end of the barrel, the turning of the trays face up , the introduction of the powder solution through the front opening, lyophilization until a dry powder is obtained, the closing of the front opening while in the lyophilization chamber, the rotation of the trays, the introduction of the reconstitution liquid to through the rear end of the barrel, the insertion of the rear piston, the removal of the products from the trays and the final control and packaging. The pre-filled ampules with various components can be manufactured for use with syringes. In yet another embodiment, the multi-chamber syringe is a Lyo-ject system (Vetter Pharma Turm, Yardley, PA). The Lyo-Ject system allows the user to lyophilize the drug directly in a syringe, which is packed with the diluent for rapid reconstitution and injection. This is described in the patents 4,874,381 and 5,080,649. In other embodiments, the compounds are administered using two separate syringes, catheters, microneedles, or other device capable of performing the injection. The pharmaceutical compositions of this invention can also be administered using microspheres, liposomes, other microparticle delivery systems or sustained release formulations placed in, near or otherwise in communication with the affected tissues or with the bloodstream. Suitable examples of sustained release carriers include semipermeable polymer matrices in the form of shaped articles such as suppositories or microcapsules. Sustained-release or microcapsular sustained release matrices include polylactides (U.S. Patent No. 3,773,319; European Patentd b.

EP-58,881), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., (1985) Biopolymers, 22: 547-56); poly (2-hydroxyethyl-methacrylate) or ethylene vinyl acetate (Langer et al., (1981), J. Biomed, Mater. Res. 15: 167-277; Langer, (1982) Chem. Tech., 12: 98-105). The compositions of this invention will be administered at an effective dose to treat the particular clinical condition faced. The determination of a preferred pharmaceutical formulation and a therapeutically efficient dose regimen for a given application is within the skill in the art, taking into consideration, for example, the condition and weight of the patient, the degree of treatment desired and the patient's tolerance for treatment. Transdermal patches have the added advantage of providing controlled distribution of a compound of the present invention to the body. Such dosage forms may be elaborated by dissolving or dispersing the compound in the appropriate medium. The absorption enhancers can also be used to increase the flow of the compound through the skin. The velocity of such a flow can be controlled either by the provision of a velocity control membrane or by dispersing the compound in a polymeric matrix or in a gel.

VII. Therapeutic Methods Therefore, the present invention further provides new therapeutic methods of cancer treatment, comprising administering to the subject an effective amount of a pharmaceutical composition, optionally using a delivery device described above. The methods of the present invention can be used to treat any cancer, including but not limited to the treatment of solid tumors. Examples of solid tumors that can be treated by the compounds of the present invention, include but are not limited to breast, testicular, pulmonary, ovarian, uterine, cervical, pancreatic, non-small cell lung (NSCLC), colon, as well as prostate, gastric, skin, stomach, esophagus and bladder. In certain embodiments, the method comprises parenterally administering an effective amount of a pharmaceutical composition of the invention to a subject. In one embodiment, the method comprises intraarterial administration of a composition of the invention to a subject. In other embodiments, the method comprises administering an effective amount of a composition of the invention directly to the arterial blood supply of a tumor in a subject. In one embodiment, the methods comprise administering an effective amount of a composition of the invention directly to the arterial blood supply of the cancerous tumor, using a catheter. In embodiments where a catheter is used to administer a composition of the invention, the insertion of the catheter can be guided or observed by fluoroscopy or other method known in the art, whereby insertion of the catheter can be observed and / or it can be guided. In yet another embodiment, the method comprises chemoembolization. For example, a method of chemoembolization may comprise blocking a vessel that feeds the cancerous tumor, with a composition comprised of a resin-like material mixed with an oily base (eg, polyvinyl alcohol in ethiodol) and one or more chemotherapeutic agents. . In other additional embodiments, the method comprises the systemic administration of a composition of the present invention to a subject. In general, chemoembolization or direct intra-arterial or intravenous injection using pharmaceutical compositions of the present invention is typically performed in a similar manner, notwithstanding the site. In summary, angiography (a road map of blood vessels), or more specifically in certain modalities, arteriography, of the area to be bolized can be done first by injecting a radiopaque contrast through an inserted catheter. within an artery or vein (depending on the site to be embolized or injected) as the X-rays are taken. The catheter can be inserted either percutaneously or by surgery. The blood vessel may then be embolized by refluxing the pharmaceutical compositions of the present invention through the catheter, until it is observed that the flow ceases. The occlusion can be confirmed by repeating the angiogram. In embodiments where direct injection is used, the blood vessel is then infused with a pharmaceutical composition of the invention in the desired dose. Embolization therapy generally results in the distribution of compositions containing inhibitors, all along the interstices of the tumor or vascular mass to be treated. The physical volume of the embolic particles that clog the arterial lumen results in the occlusion of the blood supply. In addition to this effect, the presence of one or several anti-angiogenic factors prevents the formation of new blood vessels to supply the tumor or vascular mass, increasing the effect of devitalization by cutting off the blood supply. Direct intraarterial or intravenous administration in general results in the distribution of compositions containing inhibitors throughout the interstices of the tumor or vascular mass that is to be treated as well. However, the blood supply is generally not expected to become occluded with this method. Within one aspect of the present invention, primary and secondary tumors of the liver or other tissues can be treated using embolization or direct intraarterial or intravenous injection therapy. In summary, a catheter is inserted through the femoral or brachial artery and advanced to the hepatic artery by rotating it through the arterial system under fluoroscopic guidance. The catheter is advanced to the hepatic arterial tree as far as necessary to allow complete blockage of the blood vessels supplying the tumor or tumors, while as many of the arterial branches supplying the normal structures are exempted as possible. . Ideally, this will be a segmental branch of the hepatic artery, but it could be that of the entire hepatic artery distal to the origin of the gastroduodenal artery, or even multiple separate arteries, will need to be blocked depending on the grade of the tumor and its individual blood supply. Once the desired position of the catheter is achieved, the artery is embolized by injection of the compositions (as described above) through the arterial catheter until the flow in the artery to be blocked ceases, preferably even after the artery is closed. observation for 5 minutes. The occlusion of the artery can be confirmed by injection of a radio-opaque contrast through the catheter, and demonstrating by fluoroscopy or by X-ray film that the vessel that was previously filled with the contrast, no longer does so. In embodiments where direct injection is used, the artery is infused by injection of the compositions (as described above) through the arterial catheter at a desired dose. The same procedure can be repeated with each feeding artery that is going to be occluded. In most embodiments, the pharmaceutical compositions of the present invention will incorporate the substance or substances to be distributed, in an amount sufficient to deliver to a patient a therapeutically effective amount of a therapeutic patient incorporated, or other material as part of a prophylactic or therapeutic treatment. The desired concentration of the active compound in the particle will depend on the rates of absorption, inactivation and excretion of the drug, as well as the rate of distribution of the compound. It should be noted that the dose values may also vary with the severity of the condition that is to be alleviated. It should be further understood that for any particular subject, the specific dosage regimens should be adjusted over time according to the individual need and professional judgment of the person administering or overseeing the administration of the compositions.

Typically, the dosage will be determined using techniques known to a person skilled in the art. The level of dose selected will depend on a variety of factors, including the activity of the particular compound of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound that is employed, the duration of the treatment, other drugs, the compounds and / or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and previous medical history of the patient being treated, and similar factors well known in medical techniques. The dose may be based on the amount of the composition per kg of body weight of the patient. Other amounts will be known to those skilled in the art and will be readily determined. Alternatively, the dose of the present invention can be determined by reference to the plasma concentrations of the composition. For example, the maximum plasma concentration (Cmax) and the area under the plasma concentration-time curve from time 0 to infinity (AUC (0-4)) can be used. Doses for the present invention include those that produce the above values for Cmax and AUC (0-4) and other doses that result in larger or smaller values for these parameters. A physician or veterinarian having ordinary skill in the art can easily determine and prescribe the effective amount of the required pharmaceutical composition. For example, the physician or veterinarian could begin the doses of the compounds of the invention used in the pharmaceutical composition, at lower levels than those required, in order to achieve the desired therapeutic effect and gradually increase the dose until the desired effect. Generally, an adequate daily dose of a compound of the invention will be that amount of the compound that is the lowest effective dose to produce a therapeutic effect. Such an effective dose will generally depend on the factors described above. The precise time of administration and the amount of any particular compound that will produce the most effective treatment in a given patient will depend on the activity, pharmacokinetics, and bioavailability of a particular compound, the patient's physiological condition (including age, sex, the type of disease and the stage of it, the general physical condition, the ability to respond to a given dose and a type of medication), the route of administration, and the like. The guidelines presented herein can be used to optimize the treatment, for example, to determine the optimum time and / or the amount of administration, which will only require routine experimentation consisting of monitoring the subject, and adjusting the dose and / or the schedule. While the subject is being treated, the patient's health can be monitored by measuring one or more of the relevant indices at predetermined times during a 24-hour period. The treatment, including the supplement, amounts, administration times and formulation, can be optimized according to the results of such monitoring. The patient can be periodically reevaluated to determine the degree of improvement by measuring the same parameters, the first such re-evaluation typically occurs at the end of four weeks from the start of therapy and subsequent re-evaluations occur every four to eight weeks during therapy, and then every three months after this. The therapy can continue for several months or even years, with a minimum of one month being a typical length of therapy for humans. The adjustments of the quantities of the agent administered and possibly at the time of administration can be made based on these reassessments. Treatment can be initiated with smaller doses that are less than the optimal dose of the compound.

After this, the dose can be increased by small increments until the optimal therapeutic effect is reached. The combined use of various compounds of the present invention, or alternatively other chemotherapeutic agents, can reduce the dosage required for any individual component because the onset and duration of the effect of different components can be complementary. In such combination therapy, the different active agents - can be distributed jointly or separately, and simultaneously or at different times in the day. The toxicity and therapeutic efficacy of the present compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine LD50 and ED50. Compositions that show large therapeutic indices are preferred. Although compounds that show toxic side effects can be used, care must be taken to design a distribution system that sends the compounds to the desired site, in order to reduce side effects. The data obtained from cell culture assays and animal studies can be used in the formulation of a range of doses for human use. The dose of any supplement, or alternatively of any compounds therein, preferably falls within a range of circulating concentrations that include the ED50 with little or no toxicity. The dose may vary within this range, depending on the dosage form employed and the route of administration used. For agents of the present invention, the therapeutically effective dose can be estimated initially from assays in cell culture. A dose can be formulated in animal models to achieve a range of circulating plasma concentration that includes the IC 50 (e.g., the concentration of the test compound that achieves maximum mean inhibition of symptoms) as determined in cell culture. Such information can be used to determine more precisely the useful doses in humans. The plasma levels can be measured, for example, by high performance liquid chromatography.

VIII. Therapeutic Use in Combination of the Agents of Coupling to Receptors In some embodiments, the invention further provides the use of a receptor coupling agent in combination with a chemotherapeutic agent to treat cancer, and / or to inhibit tumor growth. Similarly, any of a variety of chemotherapeutic agents can be used or tested for use in the methods of the invention. Such chemotherapeutic agents can include anti-metabolic agents, alkylating agents, platinum-based agents, anthracyclines, antibiotic agents, topoisomerase inhibitors and others. Various forms of the chemotherapeutic agents and / or other biologically active agents can be used. These include, without limitation, forms such as uncharged molecules, molecular complexes, salts, ethers, esters, amides, and the like, which are biologically activated when implanted, injected or otherwise inserted into the tumor. The chemotherapeutic drugs that can be used in combination with the coupling agent to the receptors of the invention, or in the form of a conjugate (for example immunotoxin) can be divided into several categories, based on how they affect the specific chemicals Within the cancer cells, with which cellular activities or processes the drug interferes, and which specific phases of the cell cycle the drug affects. In certain embodiments, the chemotherapeutic agent is an agent that disrupts DNA synthesis. In one embodiment, the agent that disrupts DNA synthesis is an analogous nucleoside compound. In certain modalities, the nucleoside analogue compound is gemcitabine. In yet another embodiment, the agent that disrupts DNA synthesis is an anthracycline compound, and in certain embodiments, the anthracycline compound is adriamycin. In other embodiments, the chemotherapeutic agent is an inhibitor of topoisomerase-I. In certain embodiments, the topoisomerase I inhibitor is Camptosar. The chemotherapeutic agent in other embodiments may be an alkylating agent. The alkylating agents work directly on the DNA to prevent the cancer cell from reproducing. As a class of drugs, these agents are phase-specific (in other words, they function in all phases of the cell cycle). Alkylating agents are commonly active against chronic leukemia, non-Hodgkin's lymphoma, Hodgkin's disease, multiple myeloma, and certain cancers of the lung, breast, and ovary. Examples of alkylating agents include busulfan, cisplatin, carboplatin, chlorambucil, cyclophosphamide, ifosfamide, dacarbazine (DTIC), mechlorethamine (nitrogen mustard), and melphalan. In one embodiment, the alkylating agent is a platinum compound, and in certain embodiments it can be selected from the group consisting of carboplatin and cisplatin. In certain embodiments, the platinum compound is cisplatin. In other additional embodiments, the chemotherapeutic agent may be a plant alkaloid. In one embodiment, the plant alkaloid is a taxane, and in certain modalities this may be Taxol. Methods for testing candidate coupling agents, candidates in combination with chemotherapeutic agents for the purpose of determining inhibition of a tumor, are shown in the co-pending PCT Application of Applicants No. PCT / US03 / 41243, which is incorporated by reference herein, in its entirety. In yet another aspect, the present invention features modified antibodies and antibody conjugates, or fragments thereof, conjugated to another therapeutic moiety, such as a cytotoxin, a drug or a radioisotope. The term modified antibody is also intended to include antibodies, such as monoclonal antibodies, chimeric antibodies, and humanized antibodies that have been modified, for example, by deletion, addition or substitution of portions of the antibody. For example, an antibody can be modified by deleting the constant region, and replacing it with a constant region to increase the half-life, for example, serum half-life, stability or affinity of the antibody. Exemplary radioisotopes include: 90Y, 125I, 131I, 123I, a? Lln, 105Rh, 153Sm, 67Cu, 67Ga, 166Ho, 177Lu, 186Re and 188Re. Radionuclides act by producing the ionizing radiation that causes multiple strand breaks in the Nuclear DNA, leading to cell death. The isotopes used to produce the therapeutic conjugates typically produce high energy α or β particles having a short path length. Such radionuclides kill the cells with which they are in close proximity, for example the neoplastic cells to which the conjugate has adhered or has entered. These have little or no effect on non-localized cells. The radionuclides are essentially non-immunogenic. With respect to the use of radiolabeled conjugates in conjunction with the present invention, the polypeptides of the invention can be directly labeled (such as through iodination) or can be labeled through the use of a chelating agent. As used herein, the phrases "indirect labeling" and "indirect labeling method" both mean that a chelating agent is covalently linked to an antibody and at least one radionuclide is associated with the chelating agent. Such chelating agents are typically referred to as bifunctional chelating agents since they bind to the polypeptide and the radioisotope. Particularly preferred chelating agents comprise the l-isothiocyanatobenzyl-3-methyldiotelen-triaminopentaacetic acid derivatives ("MX-DTPA") and the cyclohexyl-diethylenetriamine-pentaacetic acid ("CHX-DTPA"). Other chelating agents comprise derivatives of P-DOTA and EDTA. Particularly preferred radionuclides for indirect labeling include 11: LIn and 90Y. When conjugated to a cytotoxin, these antibody conjugates are referred to as "immunotoxins". A cytotoxin or cytotoxic agent includes any agent that is harmful (for example, that kills) the cells or that inhibits their growth. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracendione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propanolol, tumor-activated prodrugs (eg, maytansinoids (e.g., DM-1, as described in U.S. Patent No. 6,441,163), puromycin, and analogs or homologs thereof, dolastatin 10 or analogs thereof (e.g., auristatin E (AE) or monomethylauristatin E (MMAE)) Therapeutic agents also include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil , decarbazine), alkylating agents (eg, mechlorethamine, thioepa chlorambucil, meifalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, and streptozotocin, mitomycin C and cis-dichlorodiamine-platinum (II) (DDP) cisplatin), anthracyclines (eg, daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (eg, dactinomycin (formerly actinomycin), bleomycin, mithramycin and anthramycin ( AMC)), anti-mitotic agents (eg, vincristine and vinblastine), CC-1065 analogues, derivatives of clicheamycin, anthracyclines, and vinca alkaloids per vinca, etc.), ricin, diphtheria toxin, and exotoxin pseudomonas Other examples of therapeutic cytotoxins that can be conjugated to an antibody of the invention include calicheamicins and duocarmicines. In a particular embodiment, a human antibody of the invention is conjugated to a maytansinoid, or a derivative thereof, whereby an immunotoxin is formed. U.S. Patent No. 6,441,163 describes methods for conjugating maytansinoids and maytansinoid derivatives to antibodies using disulfide chemistry. In summary, in a method of making the conjugate, an excess of a maytansinoid compound having a disulfide moiety, is mixed with an antibody in an aqueous solution. The reaction is quenched with an excess of amine and the antibody conjugate is purified by gel purification. The antibody conjugates of the invention can be used to modify a given biological response, and the drug portion should not be considered as limited to classical chemical therapeutic agents. For example, the drug portion can be a protein or polypeptide having a desired biological activity. Such proteins may include, for example, an enzymatically active toxin, or an active fragment thereof, such as abrin, ricin A, pseudomonas exotoxin, tetanus toxoid, or diphtheria toxin.; a protein such as tumor necrosis factor or interferon? or biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 (IL-2"), interleukin 6 ("IL-6"), granulocyte macrophage colony stimulation factor ("GM-CSF"), granulocyte colony stimulation factor ("G-CSF"), or other growth factors. For diagnostic applications, the antibodies may include a portion (eg, biotin, fluorescent portions, radioactive portions, histidine tag or other peptide tags) for easy isolation or detection. The antibodies can also include a portion that can extend its serum half life, for example, a polyethylene glycol (PEG) moiety, and a member of the immunoglobulin super family or a fragment thereof (e.g., a portion of the human IgGl heavy chain constant region such as the hinge, CH2 and CH3 regions). The techniques for conjugating such therapeutic portions to the antibodies are well known, see, for example, Arnon et al. , "Monoclonal Antibodies for Immunotargeting of Drugs in Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al. , "Antibodies for Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al. , "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates" ,. Immunol. Rev., 62: 119-58 (1982).

IX. Equipment The present invention provides the equipment for treating various cancers. For example, a kit may comprise one or more pharmaceutical compositions as described above, and optionally instructions for its use. In other embodiments, the invention provides the equipment comprising one or more pharmaceutical compositions and one or more devices for achieving the administration of such compositions. For example, a user equipment may comprise a pharmaceutical composition and a catheter for carrying out direct intraarterial injection of the composition into a cancerous tumor. In other embodiments, a kit may comprise prefilled bulbs of a receptor coupling agent, optionally formulated as a pharmaceutical, or lyophilized, for use with a dispensing device. This invention is further illustrated by the following examples, which should not be considered as limiting. The contents of all references, patents and published patent applications cited throughout this application, as well as the figures and the Sequence Listing, are incorporated by reference herein.

EXAMPLES Example 1: Efficacy of Multiple Anti-TNF Agonist Agents in the Induction of Tumor Cell Death Multiple anti-TNF receptor antibodies were used simultaneously to induce cell death in colon carcinoma cells to determine whether activation of Two receptors other than the TNF family improved the efficacy of the receptor agonist agents. The results show that multiple antibodies were more effective in killing the tumor cells compared to the administration of a simple type of anti-T? F antibodies. The agonist antibodies 14A2 and CBEll directed to the receptor of the T? F family, TRAIL-R2 and LTßR, respectively, were used in the assay. The anti-LTβR CBEll murine monoclonal antibody has been previously described in PCT publication WO 96/22788 and U.S. Patent No. 6,312,691, and the humanized CBEll antibody has been described in WO 02/30986. Anti-TRAIL-R2 antibodies are well known within the field of TNF, and anti-TRAIL-R2 monoclonal antibodies similar to 14A2 have been described (Ichikawa, K., et al., (2001) Nat Med 7: 954; Chuntharapai, A., et al. , (2001) J Immunol 166: 4891). To obtain antibody 14A2, in short, human anti-TRAIL-R2 monoclonal antibodies generated by standard hybridoma technology were generated by immunizing mice with the human fusion protein TRAIL-R2-Ig. The inhibitors were subsequently selected for binding to the TRAIL-R2 portion. An anti-TRAIL-R2 hybridoma of that type, 14A2, linked to the WiDr colorectal adenocarcinoma cells in a FACS analysis. 14A2 was identified as a monoclonal antibody (mAb) that could induce the death of tumor cells when immobilized on the plastic surface via a murine anti-Ig Fc capture domain antibody (see filled boxes, Figure Ib). To determine the combined effect of activating the T? F TRAIL-R2 and LTßR receptors, the WiDr colon carcinoma cells were exposed to soluble murine antibodies CEBll alone, 14A2 alone, and CBEll and 14A2 in combination. The WiDr cells were used in a four-day MTT assay with 80 U / ml of IFN ?. When WiDr cells were cultured with the monoclonal antibody CBEll anti-LTßR (Browning, JL, et al., (1996) J Exp Med 183: 867) or 14A2 added to the culture medium, the relatively limited inhibition of cell growth was observed. . However, the combination of murine CBEll and 14A2 anti-TNF receptor antibodies was more effective, as shown in Figure 2a. Similarly, anti-CBEII was able to enhance TNF activity in similar assays (Mackay, F., Et al., (1997) J Immunol 159: 3299). Thus, the combination of the murine CBEll and 14A2 antibodies in the activation of two receptors of the TNF family simultaneously were more effective in killing the tumor cells of the antibodies alone.

Example 2: Construction of the T? F, Bispecific Receptor Coupling Agent In order to determine whether a coupling agent that activates at least two different TNF receptors has improved efficacy on individual activation portions, a coupling agent to the receptors in the form of a bispecific multivalent antibody that binds to two different receptors of the TNF family, for example, TRAIL-R2 and LTßR, was created. The bispecific multivalent antibody was constructed containing the epitope binding domains of CBEll and 14A2. The anti-hu TRAILR2 / anti-hu bispecific antibody LTßR (designated LT-BSl) was built as follows. The variable regions of the antibody of the heavy and light immunoglobulin 14A2 chains were determined by PCR using the hybridoma 14A2. The variable regions were then combined with a constant human light chain region to form a complete chimeric mouse-human light chain 14A2. The chimeric light chain 14A2 was constructed using the variable domain of a murine lambda light chain, fused to a human kappa constant domain. The lambda-kappa light chain was then used in the construction of the bispecific antibody. The heavy chain variable region was combined with the non-variable region of the heavy chain of Human IgGl with the C-terminal Fv fragment of CBEll. The construction of the single chain Fv versions of humanized CBEll have been previously described in PCT / US03 / 41393 (WO 04/058191). Thus, the Hercules LT-BS1 heavy chain contains the anti-huTRAILR2 heavy chain 14A2-huIgGl (SEQ ID NO: 1 and 2) with the engineered huCBEll scFv fused to its C-terminus, while the Hercules light chain is the chimeric 14A2-hu kappa light chain (SEQ ID NO: 3 and 4). A schematic representation of the LT-BSl Hercules construct is shown in Figure 9. The nucleotide and amino acid sequence of the LT-BSl antibody construction is shown in SEQ ID NOs: 5-8. Co-expression of the heavy chain and the light chain in CHO cells resulted in the production of the bispecific molecules 14A2 / CBEll called LT-BSl here to simplify (LTßR / TRAIN-R2 bispecific 1). LLT-BS1 was purified from Protein A affinity chromatography of the culture supernatant. There were no detectable aggregates by size exclusion chromatography.

Example 3: Efficacy of the Binding Agent to the TNF Receptor, Biespecific, in the Induction of the Death of Tumor Cells As shown in Figure 1, the original murine monoclonal antibodies 14A2 and CBEll, when immobilized (captured) on plastic, they could inhibit the growth of HT29 cells. However, both antibodies in solution showed only weak efficacy. When combined together as two separate mAbs, the simultaneous activation of LTßR and TRAIL-R2 increased growth capacity, as shown in Figure 2a. Thus, there was an increased potency, achieved by the combination of antibodies to three different receptors of the TNF family. The construction of LT-BSl was as potent as the combination of the two individual mAbs, as shown in Figure -2b. The LT-BSl construct demonstrated that the two mAbs are still active when combined within a molecular entity, and illustrates the principle of combining the anti-receptor mAbs with two different members of the TNF family, for increased benefit. To further examine the potency of the bispecific LT-BSl antibody, purified LT-BSl was used in the proliferation assays of HT29 or WiDr for 3 to 4 days, according to standard protocols. WiDr is a variant line of HT29 with similar behavior. LT-BS1 was tested in WiDr cells in parallel with multivalent antibodies directed to LTßR. The anti-LTβR antibodies used were LL-MSl (a monospecific antibody containing the CBEll antigen recognition sites) and LL-BSl (a bispecific antibody containing the recognition sites of the CBEll and BHA10 antigens). The descriptions and sequences of constructions LL-MS1 and LL-BS1 are described in the co-pending PCT application of Applicant WO 04/058191, incorporated by reference herein. As shown in Figure 3, LT-BSl was able to induce cell death in WiDr colon carcinoma cells, demonstrating the potency in colon carcinoma cells, comparable to that of the LL-MSl and LL-BS1 constructs. . Interestingly, in contrast to the proliferation experiments in the WiDr colon carcinoma cell line, where similar results were obtained compared to LT-BSl and the CBE11 / 14A2 combination, LT-BSl was more effective than the CBEll / combination 14A2 in the induction of cell death in the colon carcinoma cell line LS174T. As shown in Figure 4, the combination of murine CBEll and 14A2, alone, had little effect on cell death, whereas exposure to LT-BSl or LT-BSl in combination with IFN ?, resulted in a marked decrease in cell viability. The enhanced potency of LT-BSl on the combination of two separate murine mAbs was further examined using various tumor cell lines, as shown in Table 1 and in Figures 5-8. Proliferation assays were performed in the presence and absence of 80 U / ml of IFN ?, using the soluble antibody according to standard four-day MTT growth test protocols. A range of tumor types was monitored, including cervical and breast tumor cell lines. The results demonstrate that LT-BSl was effective against a wider range of tumors than the CBEll and 14A2 progenitor mAbs, alone or in combination. The activity of LT-BSl was dependent on the presence of iFN? in some cell lines, such as the HT29 and WiDr tumor lines, as is typical of the activation of the TNF family receptor, with this cell type. However, efficacy against some tumor types was not dependent on the addition of IFN ?. The requirement for IFN? even with WiDr / HT29 is not absolute, for example, good anti-tumor efficacy was observed with the anti-LTBR mAb in HT29, in the tumor models of xenoinjergo in vivo in complete absence of IFN? exogenous (Browning et al (1996) J. Exp. Med. 183: 867). The increased spectrum of anti-tumor activity of LT-BSl demonstrates the principle that various combinations of TNF family receptors may have unique activity. This improved activity could not be predicted by the activity of the individual progenitor mAbs.

The shading indicates the cells that responded to LT-BSl without any substantial response to CB? 11 or other combos the rating ++++, ++, + refers to the degree of death achieved without a greater dependence on the concentration 15 CBEll Anti-LTBR (murine) CBEllp Version Pentameric LL-BSl anti-LTBR bispecific 14A2 Anti-TRAIL-R2 (murine) LT-BSl LTBR / TRAIL-R2 bispecific ME180 and MDA231 represent cervical and breast tumor cell lines that show a different pattern of responsiveness. Both tumor cell lines did not respond to the CBEll or 14A2 mAbs alone, or the CBEll pentameric strong LTßr agonist (CBEllP); however, the LT-BSpecific was more specific in reducing its growth in in vitro crops, as shown in Figure 5 (cervical cell line ME180) and 6 (breast carcinoma cell line MDA231). The activity of LT-BSl was increased with IFN? in the cervical cell line ME180, while the potency in the breast carcinoma cell line MDA231 was not affected by the presence of IFN. The Hela cervical carcinoma cells were also evaluated, as shown in Figure 7. Using Hela cells, the LT-BSl construct proved to be more potent in the induction of cell death than murine CBEll and 14A2, alone or in combination. A comparison of the different types of cell lines and the efficacy of LT-BSl in each, compared to a pentameric version of CBEll (CBEllp, as described in PCT Application No. PCT / US03 / 41393, WO 04 / 058191) is shown in Figure 8. In sum, LT-BSl was much more powerful than CBEllp. Based on the results described above, the bispecific construct LT-BSl was more effective in inducing cell death in tumor cells than introducing each antibody alone or in combination. Since the dimerization of any receptor was inefficient, the bispecific construction can lead to a novel event of signal transduction and / or can alter the location of one of the receptors, to make the signaling more effective. The improved efficacy of the bispecific LT-BSl construct indicates that a new event was induced. The increased potency of LT-BSl on the individual mAbs, 14A2 and CBEII, could result from the mechanisms described above, for example the assembly of the new signal transduction complexes, or the altered location of the receptors. Another potential mechanism could be derived from LT-BSl oligomerization / aggregation and as such resembles the pentameric form of CBEll which has increased efficacy (with sensitive cells) or an oligomeric anti-TRAIL-R2 mAb 14A2. Biochemical analysis of purified LT-BSl showed that it had no higher molecular weight forms, detectable fresh from the freezer or after 5 months at 4 ° C.

Example 4: Cross-linking of TNF Receptors To demonstrate that cross-linking between the two receptors, for example, coupling the receptors using a coupling agent to the receptors, increases the activity, the receptor-immunoglobulin (Ig) fusion proteins were pre-mixed with the coupling agent to LT-BSl receptors to determine if Ig-receptor fusions could block LT-BSl activity. It was predicted that pre-mixing either LTBR-Ig or TRAIL-R2-Ig with LT-BSl to neutralize one side of the construct, should block activity in a similar manner. The experiment was carried out with three cell lines, WiDr, ME180 and MDA231. In all three cases, receptor-Ig fusions (LTßR or TRAIL-R2) were effective blockers of LT-BSl activity. In addition, the simple mAbs in solution were inactive (only soluble CBEll had relatively weak activity against WiDr, since it was the basis for selecting this mAb for clinical work). This experiment indicates that the cross-linking of two TNF receptors with a coupling agent to the receptors provides improved activity.

Example 5: Design and Test of the Additional T? F Family Receptor Pairs Another example of a receptor coupling agent is an agent directed to the human receptor Fnl4, which is the receptor for TWEAK. Antibodies against the human Fnl4 receptor are prepared by immunization of wild-type mice, or mice deficient in the Fnl4 receptor or other species with the recombinant fusion protein Fnl4-Fc, or soluble Fnl4. Hybridomas are prepared by conventional methods, and selected for Fnl binding. The TNF mAbs agonists are identified by analysis of NFKB activation. In the case of Fnl4, the activation of NFKB leads to the release of IL-8 or other chemokines, which forms a selection method based on single cells. To determine if an antibody is an agonist, they are monoclonal aggregates in solution or immobilized on plastic. After the addition of the antibody, the release of a chemokine indicates activation of the receptor. Alternatively, a range of reporter cell lines are now commercially available, or are easily constructed to monitor the activation of ? F B. Another alternative to determine whether an antibody activation includes cell proliferation and cell death and caspase activation assays, commonly used to test the induction of death programs. Once the agonist antibody is identified through standard selection techniques, the AR? The coding for the Ig molecules is sequenced and the recombinant forms of these mAbs can be designed. The CBEll scFv construct is added to the C-terminus of the Fnl4 agonist mAb sequence, and the bispecific forms of such an anti-Fnl4-anti-LTBR combination are expressed and isolated by protein A chromatography. The original murine Fc is either used, or the original anti-Fnl4 mAb is converted to a chimeric mAb with an Fc domain of human IgG. Alternatively, a scFv anti-Fnl4 agonist is designed and added to the chimeric versions of anti-LTBR, for example CBEll. Once the bispecific antibodies which are directed to two different TNF receptors, such as Fnl4 and LTBR, are constructed, the resulting proteins are tested in vi tro for activity on tumor lines expressing both receptors. The additive or kinetic activity is confirmed using in vivo tests of the bispecific with that tumor in a xenograft assay. The databases of human tumors are selected to determine if such matings of the receptor occur with a reasonable frequency, for example, more than 5 to 30% in the population. Active molecules as defined above, targeted to tumors with reasonable frequency, are candidates for testing in humans.

Example 6: Selection of Potential TNF Family Receptor Pairs for Tumor Therapy Existing public databases of gene expression are searched to find those receptors that are expressed in a particular tumor type, for example carcinomas of breast invaders of the pipelines. The specific tumor pairs of the receptors, for example, LTßR and RANk, are chosen, and those that are not abundantly expressed in critical cell types, such as the microvasculature and the hepatocytes, are of lower priority. The in vi tro tests are used to further validate the prediction process. For example, the LT-BSl construct can kill a tumor cell such as MDA231 as defined above. In yet another example, LT-BS1 is added to the primary endothelial cell cultures and the evidence of endothelial activation is then examined in several ways. First, the release of chemokine from these cells is studied because it serves as an indicator of signaling capacity. The pro-inflammatory activation of endothelial cells involves the induced visualization of various adhesion molecules that promote the adhesion and trafficking of leukocytes. The E-selectin, VCAM and ICAM are such molecules, and their induction is followed by FACS analysis (see, for example, Hochmna et al (1995) J. Inflammation 46: 220). It is likely that LT-BS1 does not efficiently activate endothelial cells. In contrast, TNF is a strong pro-inflammatory signal and it upregulates the expression of these three molecules. Gene expression analyzes using chip technology are also exploited to obtain a more detailed picture of whether or not these novel bispecific antibodies have unusual and potentially harmful consequences. Endothelial activation and death of endothelial cells or hepatocytes are negative indicators. By using profiling to find unique combinations of tumor-targeted receptors, coupled with favorable predictions for reduced expression on endothelial cells and hepatocytes, the selection process is optimized. Selections are validated through the use of tumor growth assays in vi tro, as well as endothelial activation and survival trials.

EQUIVALENTS A person skilled in the art will recognize that many variations and changes to the invention can be made as described in the Detailed Description, without departing from the spirit and scope of the invention. The examples provided herein are merely illustrative, and should not be considered as limiting the scope of the invention, which is described in the appended claims. All publications and patents mentioned herein are incorporated by reference herein in their entirety, as if each individual publication or patent was specifically and individually indicated as incorporated by reference.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention.

Claims (42)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A receptor coupling agent, characterized in that it specifically activates at least two different receptors of the TNF family, increases receptor signaling, and induces heteromeric receptor complex formation.

2. The receptor coupling agent according to claim 1, characterized in that it comprises a first binding specificity for a first receptor, and a second binding specificity for a second receptor.

3. The receptor coupling agent according to claim 2, characterized in that the first binding specificity is conferred by an antibody, or the antigen binding fragment thereof.

4. The receptor coupling agent according to claim 3, characterized in that the second binding specificity is conferred by an antibody, or the antigen-binding fragment thereof.

5. The receptor coupling agent according to claim 3, characterized in that the first binding specificity is conferred by a single chain Fv fragment.

6. The receptor coupling agent according to claim 5, characterized in that the second binding specificity is conferred by an antibody, or antigen-binding fragment thereof.

The coupling agent to the receptors according to claim 2, characterized in that the first binding specificity is conferred by a natural ligand for the receptor.

8. The receptor coupling agent according to claim 7, characterized in that the second binding specificity is conferred by an antibody, or antigen-binding fragment thereof.

9. The receptor coupling agent according to claim 7, characterized in that the second binding specificity is conferred by a natural ligand for the receptor.

The coupling agent to the receivers according to claim 1, characterized in that at least one receiver contains a death domain.

11. The receptor coupling agent according to claim 10, characterized in that the receptor is selected from the group consisting of TNFR1 (DR1), Fas (DR2), TRAIL-R1 (DR4), TRAIL-R2 (DR5) , p75NGF-R, and DR6.

The coupling agent to the receptors according to claim 1, characterized in that at least one receptor is involved in tissue differentiation.

The coupling agent to the receptors according to claim 12, characterized in that at least one receptor is selected from the group consisting of LTBR, RANK, EDAR1, XEDAR, Fnl4, Troy / Trade, TAJ, and p75NGF-R.

14. The receptor coupling agent according to claim 1, characterized in that at least one receptor is involved in immune regulation.

15. The receptor coupling agent according to claim 14, characterized in that the receptor is selected from the group consisting of TNFRII, HVEM, CD27, CD30, CD40, 4-lBB, OX40, GITR, TACI, BAFF-R , BCMA, and RELT.

The coupling agent to the receptors according to claim 1, characterized in that at least one of the receptors is overexpressed on tumor cells.

17. The receptor coupling agent according to claim 16, characterized in that at least one of the receptors is not overexpressed on normal hepatic or endothelial cells.

18. The receptor coupling agent according to claim 2, characterized in that the first binding specificity is conferred by an anti-LTβ receptor (LTßR) antibody, or an antigen binding fragment thereof.

19. The coupling agent to the receivers according to claim 18, characterized in that the anti-LTβR antibody is derived from a humanized CBEll antibody.

20. The receptor coupling agent according to claim 18, characterized in that the second binding specificity is conferred by an anti-TRAIL-R2 antibody, or the antigen-binding fragment thereof.

21. The receptor coupling agent according to claim 20, characterized in that the anti-TRAIL-R2 antibody is derived from a humanized 14A2 antibody.

22. The receptor coupling agent according to claim 19, characterized in that the first binding specificity is conferred by a single chain Fv fragment of a humanized CBEll antibody, and the second binding specificity is conferred by a 14A2 antibody. humanized or the antigen binding fragment thereof.

23. The receptor coupling agent according to claim 2, characterized in that the first binding specificity is conferred by at least two trimeric ligand-Fe constructs and the second binding specificity is conferred by three antibodies.

24. The receptor coupling agent according to claim 1, characterized in that at least one of the receptors of the TNF family is not normally found in a transport environment on the cell surface.

25. The receptor coupling agent according to claim 1, characterized in that at least one of the receptors of the TNF family is normally found in a transport environment on the cell surface.

26. The coupling agent to the receivers according to claim 1, characterized in that the strength of the signal is improved through the receivers.

27. A receptor coupling agent comprising at least first and second antibodies, or antigen-binding fragments thereof, characterized in that each antibody binds to a receptor other than the TNF family, thereby inducing formation of a heteromeric receptor complex.

28. The receptor coupling agent according to claim 27, characterized in that the first antibody is derived from an anti-LTßR antibody.

29. The receptor coupling agent according to claim 28, characterized in that the anti-LTßR antibody is derived from a humanized CBEll antibody.

30. The receptor coupling agent according to claim 28, characterized in that the second antibody is derived from an anti-TRAlL-R2 antibody.

31. The receptor coupling agent according to claim 30, characterized in that the anti-TRAIL-R2 antibody is derived from a humanized 14A2 antibody.

32. A method for locating a receptor of the TNF family to a cell membrane transport, characterized in that it comprises the administration of a receptor coupling agent comprising a first binding specificity for a recipient of the TNF family transported, and a second binding specificity for a receptor of the non-transported TNF family, wherein the binding of the coupling agent to the receptor locates the non-transported TNF receptor towards a transport or raft in the cell membrane.

33. A method for increasing receptor signaling, characterized in that it comprises the administration of a receptor coupling agent that specifically activates at least two different receptors of the TNF family, because it increases receptor signaling and induces the formation of heteromeric complexes of the receiver.

34. A method for decreasing tumor volume, characterized in that it comprises administering to a subject a coupling agent to the receptors, which specifically activates at least two different receptors of the TNF family, increases the signaling of the receptor and induces the formation of complexes Heteromeric receptor.

35. A method for treating cancer, characterized in that it comprises administering to a subject a coupling agent to receptors that specifically activates at least two different receptors of the TNF family, increases receptor signaling and induces the formation of heteromeric receptor complexes .

36. The method of compliance with the claim 34 or 35, characterized in that the coupling agent to the receptors is administered in the presence of IFN ?.

37. The method according to claim 34 or 35, characterized in that the coupling agent to the receptors is administered in the presence of a chemotherapeutic agent.

38. A receptor coupling agent, characterized in that it activates at least two different receptors of the TNF family and induces formation of a heteromeric complex of the receptor, comprising a first binding specificity directed to a first TNF receptor, and a second link specificity directed to a second TNF receptor.

39. The receptor coupling agent according to claim 38, characterized in that the first and second binding specificities are directed to TNF receptors selected from the group consisting of: a) a T? F receptor that contains the domain not death and a T? F receptor that contains the death domain; b) two T? F receptors that contain the non-death domain; and c) two T? F receptors that contain the death domain.

40. The receptor coupling agent according to claim 39, characterized in that at least one binding specificity is directed to a TNF receptor that contains the non-dying domain, associated with tissue differentiation.

41. The receptor coupling agent according to claim 39, characterized in that two TNF receptors that contain the non-dying domain are selected from the group consisting of LTBR / Fnl4; LTBR / RA? K; Fnl4 / TAJ; LTBR / WWTP; LTBR / XEDAR; RA? K / WWTP; RANK / XEDAR; TAJ / EDAR; and TAJ / XEDAR.

42. The receptor coupling agent according to claim 39, characterized by the TNF receptor that contains the non-killing domain and the T? F receptor that contains the death domain is selected from the group consisting of LTBR. / TRAIL-Rl; LTBR / TRAIL-R2; LTBR / p75? GF-R; Fnl4 / p75? GF-R; and p75NGF-R / TAJ.