USOO7799902B2

(12) United States Patent (10) Patent No.: US 7,799,902 B2 Browning et a1. (45) Date of Patent: Sep. 21, 2010

(M)IEEDHORCOUHJMLKENBAND 2mmmww4Ai umm @m?an COMPOSITIONS THEREOF 2004/0198635 A1 10/2004 Browning et al. 2005/0037003 A1 2/2005 Browning et al. (75) Inventors: Jeffrey L. Browning, Cambridge, MA 2005/0281811 A1 12/2005 Browning et al. (US); Veronique Bailly, Boxborough, 2006/0104971 A1 5/2006 Garber et a1. MA (US); Ellen Garber, Cambridge, 2006/0134102 A1 6/2006 LePage et a1. MA (US) 2006/0222644 A1 10/2006 Garber et a1. 2006/0280722 A1 12/2006 Browning et al. (73) Assignee: Biogen Idec MA Inc., Cambridge, MA 2007/0154476 A1 7/2007 Browning et al. (Us)

(*) Notice: Subject to any disclaimer, the term of this FOREIGN PATENT DOCUMENTS patent is extended or adjusted under 35 U.S.C. 154(b) by 420 days. EP 0509553 B1 10/1992 EP 0519596 B1 12/1992 (21) App1.No.: 11/524,786 WO WO-91/09967 A1 7/1991 WO WO-92/00329 A1 1/1992 (22) Filed: Sep. 21, 2006 WO WO-94/04679 A1 3/1994 WO WO-94/ 13808 A2 6/ 1994 (65) Prior Publication Data WO WO-94/ 13808 A3 6/ 1994 US 2007/0154476 A1 Jul. 5, 2007 WO WO-94/20625 A1 9/1994 WO WO-96/22788 A1 8/1996 Related US. Application Data WO WO-97/03687 A1 2/1997 (63) Continuation of application No. PCT/US2005/ WO WO-99/38525 A1 8/1999 009967, ?led on Mar. 23, 2005. WO WO-99/58679 A1 11/1999 WO WO-02/066516 A2 8/2002 (60) Provisional application No. 60/555,805, ?led on Mar. WO WO-02/085946 A1 10/2002 23, 2004.

(51) Int. Cl. C07K 1/00 (2006.01) OTHER PUBLICATIONS C07K 14/00 (2006.01) Alderson, Mark R. et al., “Regulation of apoptosis and T cell activa C07K 16/00 (2006.01) tion by Fas-speci?c mAb,” International Immunology, v01. C07K 17/00 (2006.01) 6(11):1799-1806 (1994). C 08H 1/00 (2006.01) AlimZhanov, Marat B. et al., “Abnormal development of secondary A61K 38/00 (2006.01) lymphoid tissues in lyrnphotoxin B-de?cient mice,”Proc. Natl. Acad. C07K 2/00 (2006.01) Sci. USA, vol. 94:9302-9307 (1997). A61K 39/00 (2006.01) Androlewicz, Matthew J. et al., “Lyrnphotoxin Is Expressed as a (52) US. Cl...... 530/402; 424/178.1; 530/300; Heteromeric Complex with a Distinct 33-kDa Glycoprotein 0n the 530/350 surface of an Activated Human T Cell Hybridoma,” The Journal of (58) Field of Classi?cation Search ...... None Biological Chemistry, vol. 267(4):2542-2547 (1992). See application ?le for complete search history. Arulanandam, Antonio R.N. et al., “A Soluble Multimeric Recombi nant CD2 Protein Identi?es CD48 as a Low Af?nity Ligand for (56) References Cited Human CD2: Divergence of CD2 Ligands during the Evolution of Humans and Mice,” J. Exp. Med., v01. 177:1439-1450 (1993). U.S. PATENT DOCUMENTS Baens, Mathus et al., “Construction and Evaluation of a hncDNA Library of Human 12p Transcribed Sequences Derived from a 4,485,045 A 11/1984 Regen Somatic Cell Hybrid,” Genomics, v01. 16:214-218 (1993). 4,544,545 A 10/1985 Ryan et al. 4,816,567 A 3/1989 Cabilly et a1. Banks, Theresa A. et al., “Lymphotoxin-ot-De?cient Mice,” The 5,082,783 A 1/1992 Ernst et al. Journal ofImmunology, v01. 155:1685-1693 (1995). 5,225,538 A 7/1993 Capon et a1. 5,530,101 A 6/1996 Queen et a1. (Continued) 5,618,920 A 4/1997 Robinson et al. Primary ExamineriRobert Landsman 5,661,004 A 8/1997 Browning et al. (74) Attorney, Agent, or FirmiLahive & Cock?eld, LLP; 5,670,149 A 9/1997 Browning et al. Amy E. Mandragouras, Esq.; Megan E. Williams, Esq. 5,795,964 A 8/1998 Browning et al. 5,859,205 A 1/1999 Adair et al. (57) ABSTRACT 5,925,351 A 7/1999 Browning et al. 6,312,691 B1 11/2001 Browning et al. 6,403,087 B1 6/2002 Browning et al. Receptor coupling agents, including multivalent constructs 6,669,941 B1 12/2003 Browning et al. comprising anti-TNF receptor binding moieties, for treating 7,001,598 B2 2/2006 Browning et al. cancer and inhibiting tumor volume in a subject are disclosed. 7,030,080 B2 4/2006 Browning et al. 7,060,667 B1 6/2006 Browning et al. 2002/0197254 A1 12/2002 Browning et al. 41 Claims, 9 Drawing Sheets US 7,799,902 B2 Page 2

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Ware, C.F. et al., “The Ligands and Receptors of the Lymphotoxin Rothe, Joachim et al., “Mice lacking the tumour necrosis factor System,” Pathways for Cytolysis, Current Topics Microbiol. receptor 1 are resistant to TNF-mediated toxicity but highly suscep Immunol., pp. 175-218 (1995). tible to infection by Listeria monocytogenes,” Nature, vol. 364:798 Winter, Greg et al., “Man-made antibodies,” Nature, vol. 349:293 802 (1993). 299 (1991). Ruddle, Nancy H. et al, “An Antibody to Lymphotoxin and Tumor Wu, Qiang et al., “The Requirement of Membrane Lymphotoxin for Necrosis Factor Prevents Transfer of Experimental Allergic the Presence of Dendritic Cells in Lymphoid Tissues,” J. Exp. Med., vol. l90(5):629-638 (1999). Encephalomyelitis,” J. Med. Med., vol. l72:ll93-l200 (1990). Xu, Jianchao et al., “Mice De?cient for the CD40 Ligand,” Immunity, Rudikoff, Stuart et al., “Single amino acid substitution altering anti vol. 1:423-431 (1994). gen-binding speci?city,” Proc. Natl. Acad. Sci. USA, vol. 79: 1979 Yonehara, Shin et al., “A Cell-killing Monoclonal Antibody (Anti 1983 (1982). Fas) to a Cell Surface Antigen Co-downregulated with the Receptor Sayegh, Mohamed H. et al., “CD28-B7 Blockade after Alloantigenic ofTumor Necrosis Factor,” J. Exp. Med., vol. 169:1747-1756 (1989). Challenge In Vivo Inhibits Thl Cytokines but Spares Th2,” J. Exp. Zhou, M. et al., “Real-Time Measurements of Kinetics Of EGF Med., vol. 181:1869-1874 (1995). Binding to Soluble EGF Receptor Monomers and Dimers Support Schiller, Joan H. et al., “Biological and Clinical Effects of Intrave the Dimerization Model for Receptor Activation,” Biochemistry, vol. nous Tumor Necrosis Factor-0r Administered Three Times Weekly,” 32:8193-8198 (1993). Cancer Research, vol. 51:1651-1658 (1991). International Search Report for Application No. PCT/US97/ 19436, Schoenfeld, Hans-Joachim et al., “Ef?cient Puri?cation of Recom dated May 28, 1998. binant Human Tumor Necrosis Factor [5 from Escherichia coli Yields International Search Report for Application No. PCT/US2005/ Biologically Active Protein with a Trimeric Structure That Binds to 009967, dated Aug. 3, 2005. Both Tumor Necrosis Factor Receptors,” The Journal of Biological Muppidi, J .R. et al., “Ligand-independent redistribution of Fas Chemistry, vol. 266(6):3863-3869 (1991). (CD95) into lipid rafts mediates clonotypic T cell death,” Nature Schriever, Folke et al., “The Central Role of Follicular Dendritic Immunology, vol. 5(2): 182-189 (2004). Cells in Lymphoid Tissues,” Advances in Immunology, vol. 51:243 International Preliminary Report on Patentability for Application No. 284 (1992). PCT/US2005/009967, dated Sep. 26, 2006. US. Patent Sep. 21, 2010 Sheet 1 0f 9 US 7,799,902 B2

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TRAIL-R/LTBR Bispeci?c1 (LT-BSl) Figure9 US 7,799,902 B2 1 2 RECEPTOR COUPLING AGENTS AND domain is selected from the group consisting of TNFRl COMPOSITIONS THEREOF (DR1), Fas (DR2), TRAIL-R1 (DR4), TRAIL-R2 (DR5), DR6 and p75NGF-R. RELATED APPLICATIONS The invention includes a receptor coupling agent which activates at least two distinct TNF family receptors, wherein This application is a continuation of International Patent at least one receptor does not contain a death domain. The Application Serial No. PCT/US2005/009967, ?led on Mar. invention also describes a receptor coupling agent which 23, 2005, which claims priority to US. Provisional Applica enhances receptor signaling or induces formation of hetero tion No. 60/555,805, ?led Mar. 23, 2004. The entire contents of each of these patents and patent applications are hereby meric receptor complexes, wherein at least one receptor does incorporated herein by reference. not contain a death domain. In one embodiment, the receptor does not contain a death domain and is involved in tissue BACKGROUND OF THE INVENTION differentiation. In another embodiment, the receptor which does not contain a death domain is selected from the group The ability to induce cell death by various members of the consisting of LTBR, RANK, EDAR1, XEDAR, Fn14, Troy/ TNF family has been pursued by oncologists for almost 20 Trade, and TA]. years. Originally, TNF itself was used to treat solid tumors The invention also describes a receptor coupling agent and eventually was found to be applicable to the local treat which speci?cally activates at least two distinct TNF family ment of melanoma by whole limb perfusion (Lejeune et al. receptors, wherein at least one receptor is involved in tissue (1998) Curr Opin Immunol 101573)) Most recently, activa 20 differentiation. The invention also describes a receptor cou tion of TNF receptors by ligands or anti-receptor antibodies pling agent which enhances receptor signaling or induces has sparked clinical interest. Activation of the Fas receptor, formation of heteromeric receptor complexes, wherein at for example, has shown considerable promise, although it least one receptor is involved in tissue differentiation. In one may be limited by liver toxicity. Activation of TRAILRl or embodiment, the receptor is selected from the group consist TRAILR2 by the TRAIL ligand, another member of the TNF 25 ing of LTBR, RANK, EDAR1, XEDAR, Fn14, Troy/Trade/ family, has been reported to transduce an apoptotic signal to TA], and p75NGF-R. TRAIL-sensitive cancer cells (Grif?th et al., J. Immunol. In one embodiment, the receptor coupling agent activates a 16212597, 1999; and Degli-Esposti et al., Immunity, 7:813 non-death domain containing TNF receptor and a death 820, 1997). Activation of LT-[3-R, yet another member of the domain containing receptor, e.g., LTBR/TRAIL-Rl; LTBR/ TNF family, by soluble ligands or agonistic antireceptor 30 TRAIL-R2; LTBR/p75NGF-R; Fn14/p75NGF-R; and monoclonal antibodies has also been shown to induce the p75NGF-R/TAJ . death of certain carcinomas (Lawerence et al., (2001) Nat In another embodiment, the receptor coupling agent acti Med 7:383, Ichikawa et al., (2001) Nat Med 7:954). Treat vates at least two TNF receptors which do not contain death ment with agonist TNF activating agents would thus be useful domains, e.g., LTBR/Fn14; LTBR/RANK; Fn14/TAJ; LTBR/ for treating or reducing the advancement, severity or effects 35 EDAR; LTBR/XEDAR; RANK/EDAR; RANK/XEDAR; of neoplasia in subjects (e.g., humans). and TAJ/EDAR; and TAJ/XEDAR. In still another embodiment of the invention, the receptor SUMMARY OF THE INVENTION coupling agent activates at least two death domain containing receptors. The invention describes a receptor coupling agent which 40 speci?cally activates at least two distinct TNF family recep In addition, the invention describes a receptor coupling tors. In one embodiment, the receptor coupling agent agent which speci?cally activates at least two distinct TNF enhances receptor signaling. In another embodiment, the family receptors, wherein at least one receptor is involved in receptor coupling agent induces formation of heteromeric immune regulation. In one embodiment, the receptor is receptor complexes. In one embodiment, the receptor cou 45 selected from the group consisting of TNFRII, HVEM, pling agent comprises a ?rst binding speci?city for one recep CD27, CD30, CD40, 4-1BB, 0X40, GITR, TACI, BAFF-R, tor and a second binding speci?city for the other receptor. In BCMA, and RELT. one embodiment, the ?rst binding speci?city is conferred or The invention provides a receptor coupling agent which effected by an antibody or antigen binding fragment thereof. speci?cally activates at least two distinct TNF family recep In another embodiment, the second binding speci?city is 50 tors, wherein at least one of the receptors is not overexpressed conferred or effected by an antibody or antigen binding frag on normal liver or endothelial cells. ment thereof. The binding speci?city can be conferred, for The invention also describes a receptor coupling agent example, by a single chain Fv fragment. In another embodi which speci?cally activates at least two distinct TNF family ment, the ?rst binding speci?city is effected by a natural receptors, wherein the receptor coupling agent comprises a ligand for the receptor, and the second binding speci?city is 55 ?rst binding speci?city for one receptor and a second binding derived from an antibody or antigen binding fragment speci?city for the other receptor. In one embodiment, the thereof. In still another embodiment, the ?rst binding speci receptor coupling agent enhances receptor signaling or ?city is conferred by a natural ligand for the receptor, and the induces formation of heteromeric receptor complexes. In one second binding speci?city is conferred by a natural ligand for embodiment, the ?rst binding speci?city is conferred by or the receptor. 60 derived from an anti-LTB receptor (LTBR) antibody, or anti The invention describes a receptor coupling agent which gen binding fragment thereof. An example of an anti-LTBR speci?cally activates at least two distinct TNF family recep antibody includes a humanized CBE11 antibody. In one tors, wherein at least one receptor contains a death domain. In embodiment, the second binding speci?city is conferred by or one embodiment, the receptor coupling agent enhances derived from an anti-TRAIL-R2 antibody, or antigen binding receptor signaling or induces formation of heteromeric recep 65 fragment thereof. Examples of the anti-TRAIL-R2 antibody tor complexes, wherein at least one receptor contains a death are a humanized or a chimeric 14A2 antibody. In another domain. In one embodiment, the receptor containing a death embodiment, the ?rst binding speci?city is conferred by a US 7,799,902 B2 3 4 single chain Fv fragment of a humanized CBE11 antibody ment, the receptor coupling agent is administered in the pres and the second binding speci?city is conferred by a 14A2 ence of a chemotherapeutic agent. antibody. The invention also comprises a receptor coupling agent The invention describes a receptor coupling agent which which activates at least two distinct TNF family receptors and speci?cally activates at least two distinct TNF family recep induces formation of a heteromeric receptor complex com tors, wherein the receptor coupling agent comprises a ?rst prising a ?rst binding speci?city directed to a ?rst TNF recep binding speci?city for one receptor and a second binding tor and a second binding speci?city directed to a second TNF speci?city for the other receptor, wherein the ?rst binding receptor. In one embodiment, the ?rst and second binding speci?city comprises at least two trimeric ligand-Fc con speci?cities are directed to TNF receptors, including a non structs that are commonly formed from three dimeric Fc death domain containing TNF receptor and a death domain domains and six ligand molecules. In this case, the second containing TNF receptor; two non-death domain containing binding speci?city would comprise from the three antibody TNF receptors; or two death domain containing TNF recep molecules. tors. In another embodiment, at least one binding speci?city The invention describes a receptor coupling agent which is directed to a non-death domain containing TNF receptor speci?cally activates at least two distinct TNF family recep associated with tissue differentiation. In still another embodi tors, wherein at least one of the TNF family receptors is not ment, two non-death domain containing TNF receptors are normally found in a raft environment on the cell surface. In selected from the group consisting of LTBR/Fnl4; LTBR/ one embodiment, the receptor coupling agent enhances RANK; Fnl4/TAJ; LTBR/EDAR; LTBR/XEDAR; RANK/ receptor signaling or induces formation of heteromeric recep EDAR; RANK/XEDAR; TAJ/EDAR; and TAJ/XEDAR. In tor complexes, wherein at least one of the TNF family recep 20 another embodiment of the invention, the non-death domain tors is not normally found in a raft environment on the cell containing TNF receptor and the death domain containing surface. TNF receptor is selected from the group consisting of LTBR/ The invention includes a receptor coupling agent which TRAIL-R1; LTBR/TRAIL-R2; LTBR/p75NGF-R; Fnl 4/ speci?cally activates at least two distinct TNF family recep p75NGF-R; and p75NGF-R/TAJ. tors, enhances receptor signaling or induces formation of 25 heteromeric receptor complexes, wherein at least one of the BRIEF DESCRIPTION OF THE DRAWINGS TNF family receptors is normally found in a raft environment on the cell surface. FIGS. 1a-b graphically depict results from a WiDr cell 4 The invention further describes a receptor coupling agent day proliferation assay. Results show that anti-TRAIL-R2 which speci?cally activates at least two distinct TNF family 30 antibody 14A2 and anti-LTBR antibody CBE11 were both receptors or enhances receptor signaling, wherein the signal able to induce WiDr death through agonist activity. strength is enhanced through the receptors. FIGS. 2a-b graphically depict results from a 4-day MTT The invention includes a receptor coupling agent compris growth assay in WiDr colon carcinoma cells. Results demon ing at least two antibodies or antigen binding fragments strate that the bispeci?c LTBR/TRAIL-R2 antibody (LT-BS 1) thereof, wherein each antibody binds a distinct TNF family 35 has more cell death activity (FIG. 2b) than individual parent receptor, thereby inducing formation of a heteromeric recep antibodies CBE11 and 14A2 (FIG. 2a). tor complex. In one embodiment, the antibody is derived from FIG. 3 graphically depicts results from a 4-day MTT an anti-LTBR antibody, including, for example, a humanized growth assay with 80 U/ml of IFNy in WiDr colon carcinoma CBE11 antibody. In another embodiment, the second anti cells, comparing the LTBR/TRAIL-R2 bispeci?c antibody body is derived from an anti-TRAIL-R2 antibody, including, 40 (LT-BSl) to tetravalent LTBR bispeci?c antibodies LL-BSl for example, a humanized or chimeric 14A2 antibody. (antibody CBE11 and BHAIO) and LL-MSl (antibody In one embodiment, the invention includes a method for CBEl 1). localizing a TNF family receptor to a cell membrane raft FIGS. 4a-b graphically depict results from a 4-day MTT comprising administering a receptor coupling agent compris growth assay with and without 80 U/ml of IFNY in LSl74T ing a ?rst binding speci?city for a rafted TNF family receptor 45 tumor cells. The results show the ef?cacy of receptor coupling and a second binding speci?city for a non-rafted TNF family agent LT-BSl and antibodies 14A2 and CBE11 at inhibiting receptor, wherein binding of the receptor coupling agent colon carcinoma cell growth (LSl74T tumor cells). localizes the non-rafted TNF receptor to a raft in the cell FIGS. 5a-b graphically depict results from a 4-day MTT membrane. growth assay with and without 80 U/ml of IFNY in ME180 The invention also includes a method for enhancing recep 50 tumor cells. The results demonstrate the ef?cacy of receptor tor signaling comprising administering a receptor coupling coupling agent LT-BSl and antibodies 14A2 and CBE11 at agent which speci?cally activates at least two distinct TNF inhibiting cervical carcinoma cell growth (ME180 tumor family receptors, enhances receptor signaling and induces cells). formation of heteromeric receptor complexes. FIGS. 6a-b graphically depict results from a 4-day MTT In still another embodiment, the invention describes a 55 growth assay with and without 80 U/ml of IFNy in MDA213 method of decreasing tumor volume comprising administer tumor cells. The results show the ef?cacy of receptor coupling ing to a subject a receptor coupling agent which speci?cally agent LT-BSl and antibodies 14A2 and CBE11 at inhibiting activates at least two distinct TNF family receptors, enhances breast carcinoma cell growth (MDA231 tumor cells). receptor signaling, or induces formation of heteromeric FIGS. 7a-b graphically depict results from a 4-day MTT receptor complexes. 60 growth assay with and without 80 U/ml of IFNY in Hela tumor In still another embodiment, the invention includes a cells. The results demonstrate the ef?cacy of receptor cou method of treating cancer comprising administering to a sub pling agent LT-BSl and individual antibodies 14A2 and ject a receptor coupling agent which speci?cally activates at CBE11 at inhibiting Hela cervical carcinoma cell growth. least two distinct TNF family receptors, enhances receptor FIGS. 8a-d graphically depict results from a 4-day MTT signaling, or induces formation of heteromeric receptor com 65 growth assay with 80 U/ml of IFNY in a range of tumor cell plexes. In one embodiment, the receptor coupling agent is types, including breast, cervical, and colon. The results show administered in the presence of IFNy. In another embodi the effects of receptor coupling agent LT-BSl and pentameric US 7,799,902 B2 5 6 CBEl 1 antibody at inhibiting various types of carcinoma cell The term “region” refers to a part or portion of an antibody growth, including breast (A, B), cervical (C), and colon (D). chain and includes constant or variable domains as de?ned FIG. 9 depicts a schematic drawing of the receptor cou herein, as well as more discrete parts or portions of said pling agent LT-BSl construct comprising anti-TRAIL-R2 domains. For example, light chain variable domains or antibody l4A2 and LTBR scFv antibody CBEll (striped). regions include “complementarity determining regions” or “CDRs” interspersed among “framework regions” or “FRs”, DETAILED DESCRIPTION OF THE INVENTION as de?ned herein. Immunoglobulins or antibodies can exist in monomeric or I. De?nitions polymeric form. The term “anti gen-binding fragment” refers to a polypeptide fragment of an immunoglobulin or antibody For convenience, before further description of the present binds antigen or competes with intact antibody (i.e., with the invention, certain terms employed in the speci?cation, intact antibody from which they were derived) for antigen examples and appended claims are de?ned here. binding (i.e., speci?c binding). The term “conformation” The term “administering” includes any method of delivery refers to the tertiary structure of a protein orpolypeptide (e. g., of a compound of the present invention, including but not an antibody, antibody chain, domain or region thereof). For limited to, a pharmaceutical composition or therapeutic example, the phrase “light (or heavy) chain conformation” agent, into a subject’ s system or to a particular region in or on refers to the tertiary structure of a light (or heavy) chain a subject. The phrases “systemic administration,” “adminis variable region, and the phrase “antibody conformation” or tered systemically,” “peripheral administration” and “admin “antibody fragment conformation” refers to the tertiary struc istered peripherally” as used herein mean the administration 20 ture of an antibody or fragment thereof. Binding fragments of a compound, drug or other material other than directly into are produced by recombinant DNA techniques, or by enzy the central nervous system, such that it enters the patient’s matic or chemical cleavage of intact immunoglobulins. Bind system and, thus, is subject to metabolism and other like ing fragments include Fab, Fab', F(ab')2, Fabc, Fv, single processes, for example, subcutaneous administration. chains, and single-chain antibodies. Other than “bispeci?c” “Parenteral administration” and “administered parenterally” 25 or “bifunctional” immunoglobulins or antibodies, an immu means modes of administration other than enteral and topical noglobulin or antibody is understood to have each of its administration, usually by injection, and includes, without binding sites identical. A “bispeci?c” or “bifunctional anti limitation, intravenous, intramuscular, intraarterial, intrathe body” is an arti?cial hybrid antibody having two different cal, intracapsular, intraorbital, intracardiac, intradermal, heavy/light chain pairs and two different binding sites. Bispe intraperitoneal, transtracheal, subcutaneous, subcuticular, 30 ci?c antibodies can be produced by a variety of methods intra-articular, subcapsular, subarachnoid, intraspinal and including fusion of hybridomas or linking of Fab' fragments. intrasternal injection and infusion. See, e.g., Songsivilai & Lachmann, (1990) Clin. Exp. Immu As used herein, the term “antibody” is meant to refer to nol. 791315-321; Kostelny et al., (1992) J. Immunol. 148, complete, intact antibodies, as well as Fab, Fab', F(ab)2, Fv, 1 547-1 5 53. and other fragments thereof that impart desired binding speci 35 The term “antibody construct” refers to a recombinant ?cities to the constructs of the instant invention. Antibodies molecule that comprises two or more antigen-binding frag include, for example, monoclonal antibodies such as murine ments coming from the variable domains of the heavy chain monoclonal antibodies, chimeric antibodies, anti-idiotypic and light chain of an antibody. An antibody construct may antibodies, anti-anti-idiotypic antibodies, and humanized comprise the entire or part of the constant regions of an antibodies, as well as mutivalent forms thereof. The term 40 antibody from any of the ?ve Ig classes (for example IgA, “immunoglobulin” or “antibody” (used interchangeably IgD, IgE, IgG and IgM). For example, the antibody construct herein) refers to an antigen-binding protein having a basic may be made of an antibody which heavy chains comprise at four-polypeptide chain structure consisting of two heavy and their C-terminus a single chain variable fragment. In another two light chains, said chains being stabilized, for example, by example, the antibody construct may be made of the entire or interchain disul?de bonds, which has the ability to speci? 45 part of the constant region of the two heavy chains of an cally bind antigen. Both heavy and light chains are folded into antibody which comprise at their carboxy- and amino -termini domains. The term “domain” refers to a globular region of a a single chain variable fragment. An example of antibody heavy or light chain polypeptide comprising peptide loops constructs that impart the desired binding speci?cities is (e. g., comprising 3 to 4 peptide loops) stabilized, for example, depicted schematically in FIG. 9. In yet another example, the by [3-pleated sheet and/or intrachain disul?de bond. Domains 50 antibody construct may comprise two heavy chains having are further referred to herein as “constant” or “variable”, two or more variable regions and two light chains having one based on the relative lack of sequence variation within the or more variable regions where the two heavy chains are domains of various class members in the case of a “constant” joined by a disul?de bond or other covalent linkage. In domain, or the signi?cant variation within the domains of another example, the antibody construct may comprise two various class members in the case of a “variable” domain. 55 heavy chains comprising two or more variable regions where “Constant” domains on the light chain are referred to inter the two heavy chains are joined by a disul?de bond or other changeably as “light chain constant regions”, “light chain covalent linkage. constant domains”, “CL” regions or “CL” domains). “Con stant” domains on the heavy chain are referred to interchange The term “antigen” as used herein, means a molecule which is reactive with a speci?c antibody. ably as “heavy chain constant regions”, “heavy chain constant 60 domains”, “CH” regions or “CH” domains). “Variable” The term “antigen binding site” or “antigen recognition domains on the light chain are referred to interchangeably as site” refers to a region of an antibody that speci?cally binds an “light chain variable regions”, “light chain variable epitope on an antigen. domains”, “VL” regions or “VL” domains). “Variable” The term “apoptosis”, “apoptotic cell death” or “pro domains on the heavy chain are referred to interchangeably as 65 grammed cell death” as used herein refers to any cell death “heavy chain constant regions”, “heavy chain constant that results from the cascade of cellular events that occur at domains”, “CH” regions or “CH” domains). speci?c stages of cellular differentiation and in response to US 7,799,902 B2 7 8 speci?c stimuli.Apoptotic cell death is often characterized by are biologically active and cytotoxic. Examples of plant condensation of the cytoplasm and nucleus of dying cells. alkoids include, but are not limited to, taxanes such as taxol, The term “binding speci?city” is a property of the dis docetaxel and paclitaxel and vincas such as vinblastine, vin closed receptor coupling agents that is conferred, imparted, cristine, and vinorelbine. effected or derived from a binding moiety which is directed to The term “chimeric antibody” refers to an antibody whose a speci?c TNF family receptor. A binding speci?city of the light and heavy chain genes have been constructed, typically invention may be conferred by binding moieties that include, by genetic engineering, from immuno globulin gene segments for example, an antibody, or antigen binding fragment belonging to different species. For example, the variable (V) thereof, a single chain Fv fragment soluble ligands, fc fusions segments of the genes from a mouse monoclonal antibody or the like. Those skilled in the art will appreciate that, for the may be joined to human constant (C) segments, such as IgG1 purposes of the instant application, the terms “binding speci and IgG4. Human isotype IgGl is preferred. A typical chi ?city” and “binding moiety” may be used interchangeably meric antibody is thus a hybrid protein consisting of the V or unless otherwise dictated by contextual restraints. Thus, a antigen-binding domain from a mouse antibody and the C or binding speci?city (binding moiety) may also include a TNF effector domain from a human antibody. ligand which interacts with a TNF family receptor. In one The term “death domain” refers to a cytoplasmic region of embodiment of the invention, a receptor coupling agent com a TNF family receptor which is involved TNF-mediated cell prises at least one binding speci?city (or binding moiety) for death signaling and cell-cytotoxicity induction mediated by one TNF receptor, and a second binding speci?city (or bind these receptors. This region couples the receptor to caspase ing moiety) for another TNF receptor. activation via adaptor proteins resulting in activation of the The term “cancer” or “neoplasia” refers in general to any 20 extrinsic death pathway. Examples of TNF receptors which malignant neoplasm or spontaneous growth or proliferation contain death domains include, but are not limited to, TNFRl of cells. The term as used herein encompasses both fully (DRl), Fas (DR2), TRAIL-R1 (DR4), TRAIL-R2 (DR5), developed malignant neoplasms, as well as premalignant p75NGFR, and DR6. lesions. A subject having “cancer”, for example, may have a The term “effective amount” refers to that amount of a tumor or a white blood cell proliferation such as leukemia. In 25 compound, material, or composition comprising a compound certain embodiments, a subject having cancer is a subject of the present invention which is su?icient to effect a desired having a tumor, such as a solid tumor. Cancers involving a result, including, but not limited to, for example, reducing solid tumor include but are not limited to non small cell lung tumor volume either in vitro or in vivo. An effective amount cancer (N SCLC), testicular cancer, lung cancer, ovarian can of a pharmaceutical composition of the present invention is an cer, uterine cancer, cervical cancer, pancreatic cancer, col 30 amount of the pharmaceutical composition that is suf?cient to orectal cancer (CRC), breast cancer, as well as on prostate, effect a desired clinical result, including but not limited to, for gastric, skin, stomach, esophagus and bladder cancer. example, ameliorating, stabilizing, preventing or delaying the The term “chemotherapeutic agent” refers to any small development of cancer in a patient. In either case, an effective molecule or biologic used to treat disease caused by a foreign amount of the compounds of the present invention can be cell or malignant cell, such as a tumor cell. Non-limiting 35 administered in one or more administrations. Detection and examples of chemotherapeutic agents include agents that dis measurement of these above indicators are known to those of rupt DNA synthesis, are inhibitors of topoisomerase I, are skill in the art, including, but not limited for example, reduc alkylating agents, or are plant alkaloids. Exemplary biologic tion in tumor burden, inhibition of tumor size, reduction in chemotherapeutic agents comprise rituximab, ibritumomab, proliferation of secondary tumors, expression of genes in bevacizumab and trastuzumab. Those skilled in the art will 40 tumor tissue, presence of biomarkers, lymph node involve appreciate that other chemotherapeutic agents compatible ment, histologic grade, and nuclear grade. with the teachings of the instant application are readily dis The term “epitope” refers to the region of an antigen to cernable. which an antibody or antibody construct binds preferentially The term “agent that disrupts DNA synthesis” refers to any and speci?cally. A monoclonal antibody binds preferentially molecule or compound able to reduce or inhibit the process of to a single speci?c epitope of a molecule that can be molecu DNA synthesis. Examples of agents that disrupt DNA syn larly de?ned. In the present invention, multiple epitopes can thesis include but are not limited to nucleoside analogs such be recognized by a multispeci?c antibody. as pyrimidine or purine analogs, including, for example but The term “Fv fragment” refers to the fragment of an anti not limited to, gemcitabine or alternatively anthracycline body comprising the variable domains of its heavy chain and compounds, including for example but not limited to, adria 50 light chain. The term Fc fragment refers to the fragment of an mycin, daunombicin, doxorubicin, and idambicin and epi antibody comprising the constant domain of its heavy chain. podophyllotoxins such as etoposide and teniposide. The term The term “humanized immunoglobulin” or “humanized “topoisomerase I inhibitor” refers to a molecule or compound antibody” refers to an immunoglobulin or antibody that that inhibits or reduces the biological activity of a topoi includes at least one humanized immunoglobulin or antibody somerase I enzyme. Including for example, but not limited to, 55 chain (i.e., at least one humanized light or heavy chain). The camptosar. The term “alkylating agent” refers to any mol term “humanized immunoglobulin chain” or “humanized ecule or compound able to react with the nucleophilic groups antibody chain” (i.e., a “humanized immunoglobulin light of (for examples, amines, alcohols, phenols, organic and inor chain” or “humanized immunoglobulin heavy chain”) refers ganic acids) and thus add alkyl groups (for example, ethyl or to an immunoglobulin or antibody chain (i.e., a light or heavy methyl groups) to another molecule such as a protein or chain, respectively) having a variable region that includes a nucleic acid. Examples of alkylating agents used as chemo variable framework region substantially from a human immu therapeutic agents include bisulfan, chlorambucil, cyclo noglobulin or antibody and complementarity determining phosphamide, ifosfamide, mechlorethamine, melphalan, regions (CDRs) (e.g., at least one CDR, preferably two thiotepa, various nitrosourea compounds, and platinum com CDRs, more preferably three CDRs) substantially from a pounds such as cisplatin and carboplatin. The term “plant 65 non-human immunoglobulin or antibody, and further alkaloid” refers a compound belonging to a family of alka includes constant regions (e.g., at least one constant region or line, nitrogen-containing molecules derived from plants that portion thereof, in the case of a light chain, and preferably US 7,799,902 B2 10 three constant regions in the case of a heavy chain). The term multiple antigen recognition sites, some number of which “humanized variable region” (e.g., “humanized light chain bind a ?rst epitope and some number of which bind a second variable region” or “humanized heavy chain variable region”) epitope that is different from the ?rst epitope. In one embodi refers to a variable region that includes a variable framework ment of the invention, the antibody is a multivalent, bispeci?c region substantially from a human immunoglobulin or anti antibody as shown in FIG. 9. body and complementarity determining regions (CDRs) sub A “patient” or “subject” or “host” refers to either a human stantially from a non-human immunoglobulin or antibody. or non-human animal. The term “heteromeric receptor complex” refers to a com plex comprising a receptor coupling agent and two or more The term “pharmaceutical delivery device” refers to any receptor(s) to which the receptor coupling agent is targeted. device that may be used to administer a therapeutic agent or In one embodiment, the heteromeric receptor complex of the agents to a subject. Non-limiting examples of pharmaceutical invention comprises a receptor coupling agent and at least two delivery devices include hypodermic syringes, multichamber TNF family receptors which the agent is targeted to activate. syringes, stents, catheters, transcutaneous patches, micron Preferably, signaling through the receptors is enhanced as a eedles, microabraders, and implantable controlled release result of formation of the heteromeric receptor complex. In devices. In one embodiment, the term “pharmaceutical deliv one embodiment of the invention, the heteromeric receptor ery device” refers to a dual-chambered syringe capable of complex forms on a lipidraft in the cell membrane. In another mixing two compounds prior to injection. embodiment, the heteromeric receptor complex of the inven The phrase “pharmaceutically acceptable” is employed tion forms outside of a lipid raft on the cell membrane. herein to refer to those compounds, materials, compositions, The term “inhibition of tumor volume” refers to any 20 and/or dosage forms which are, within the scope of sound decrease or reduction in a tumor volume. medical judgment, suitable for use in contact with the tissues The term “ligand” refers to any molecule which binds to a speci?c site on a protein or other molecule. A ligand is often of human beings and animals without excessive toxicity, irri a polypeptide or a compound that binds to a receptor protein tation, allergic response, or other problem or complication, in a high af?nity and speci?c manner to elicit a functional 25 commensurate with a reasonable bene?t/risk ratio. response. For example ligands of the invention include TNF The phrase “pharmaceutically-acceptable carrier” as used family receptor ligands. The term “natural ligand” refers to a herein means a pharmaceutically-acceptable material, com ligand which binds to a receptor under normal physiological position or vehicle, such as a liquid or solid ?ller, diluent, conditions. The term “receptor” refers herein to a structure, excipient, or solvent encapsulating material, involved in car usually a polypeptide, located on or in a cell, which recog 30 rying or transporting the subject compound from one organ, nizes a binding molecule, i.e., a ligand, and thereby induces a or portion of the body, to another organ, or portion of the body. cellular response. Receptors of the invention include TNF Each carrier must be “acceptable” in the sense of being com family receptors, including, for example, TRAIL-R2, patible with the other ingredients of the formulation and not HVEM, and LTBR. injurious to the patient. Some examples of materials which The term “TNF family receptor” or “TNF-R” refers to 35 can serve as pharmaceutically-acceptable carriers include: receptors which belong to the TNF receptor superfamily char acterized by disul?de bonds which form “cysteine-rich (1) sugars, such as lactose, glucose and sucrose; (2) starches, domains” or CRDs. TNF receptor family members generally such as corn starch and potato starch; (3) cellulose, and its consist of an extracellular domain, a transmembrane domain derivatives, such as sodium carboxymethyl cellulose, ethyl and an intracellular signaling domain (see Locksley et al. 40 cellulose and cellulose acetate; (4) powdered tragacanth; (5) (2001) Cell 104:487 for review). The extracellular domain is malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter built from 1 to 6 copies of a tightly disulphide-bonded domain and suppository waxes; (9) oils, such as peanut oil, cottonseed and is recognized on the basis of the unique arrangement of oil, saf?ower oil, sesame oil, olive oil, corn oil and soybean cysteine residues (Banner et al. (1993) Cell 73:431). Each oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) TNF receptor binds to a corresponding ligand, although one 45 ligand may share several receptors. esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) The term “lymphotoxin-beta receptor (LTBR) agonist” buffering agents, such as magnesium hydroxide and alumi refers to any agent which can augment ligand binding to num hydroxide; (15) alginic acid; (16) pyrogen-free water; LTBR, cell surface LTBR clustering and/or LTBR signaling. (17) isotonic saline; (18) Ringer’s solution; (19) ethyl alco The phrase “multivalent antibody” or “multivalent anti 50 hol; (20) pH buffered solutions; (21) polyesters, polycarbon body construct” refers to an antibody or antibody construct ates and/orpolyanhydrides; and (22) other non-toxic compat comprising more than one antigen recognition site. For ible substances employed in pharmaceutical formulations. example, a “bivalent” antibody construct has two antigen "Pharmaceutically-acceptable salts” refers to the relatively recognition sites, whereas a “tetravalent” antibody construct non-toxic, inorganic and organic acid addition salts of com has four antigen recognition sites. The terms “monospeci?c”, 55 pounds. “bispeci?c”, “trispeci?c”, “tetraspeci?c”, etc. refer to the The term “raft” or “lipid raft” refers to a lipid raft or a number of different antigen recognition site speci?cities (as portion thereof which is a specialized cell membrane domain opposed to the number of anti gen recognition sites) present in (see Simons et al., (2000) Nature Reviews/Molecular Cell a multivalent antibody construct of the invention. For Biology 1:31). In particular, the term “lipid raft” describes a example, a “monospeci?c” antibody construct’s antigen rec 60 cholesterol and glycosphingolipid-enriched microdomain of ognition sites all bind the same epitope. A “bispeci?c” anti any membrane of a eukaryotic cell. Lipid rafts tend to be body construct has at least one antigen recognition site that enriched in signaling molecules, with growth factor receptors binds a ?rst epitope and at least one antigen recognition site and sensor molecules having been shown to migrate to lipid that binds a second epitope that is different from the ?rst rafts after ligand binding or cross-linking. Lipid rafts are epitope. A “multivalent mono speci?c” antibody construct has 65 characterized by their resistance to solubilization at low tem multiple antigen recognition sites that all bind the same perature in nonionic detergents and can change in size and epitope. A “multivalent bispeci?c” antibody construct has composition in response to intra- or extracellular stimuli. US 7,799,902 B2 11 12 Speci?c protein-protein interactions may be favored within II. Receptor Coupling Agent Targets lipid rafts, resulting in modulation of signaling cascade A limiting factor in the treatment of tumors with TNF activities in the case of, for example, plasma membrane family receptor activating agents is that often only a subset of cytokine receptors. tumors appear to be sensitive to such therapies. Receptor The potential effects of either “rafting” (de?ned herein as coupling agents can speci?cally activate TNF family recep the incorporation of a membrane component, e.g., a receptor, tors, and enhance receptor signaling by, for example, bringing into a lipid raft) or “de-rafting” (de?ned herein as removal, the TNF family receptors into close proximity (for review on exit or barring of a membrane component, e.g., a receptor, TNF receptors and the TNF family see Locksley et al. (2001) from a lipid raft) a given receptor include modulation of cytokine receptor-mediated signaling (in some cases trigger Cell 104:487). The invention provides receptor coupling agents which can target more than one TNF family receptor ing apoptotic cell death), cellular localization of the receptor, and enhance signaling, thus providing an improved method of and receptor abundance. Sometimes, lipid rafts may cluster; treating cancer. As such, receptor coupling agents can deliver and it has been reported that such clustering is used both arti?cially and physiologically to trigger signaling cascades. stronger or more complex signaling, and are therefore, more effective on a wider range of tumors, as shown in Example 3. In one embodiment, the receptor coupling agent of the inven In one embodiment, the receptor coupling agent increases the tion brings two TNF family member receptors into a lipidraft. signal strength by increasing the number of receptors being In another embodiment, the receptor coupling agent of the invention brings a TNF family receptor out of a lipid raft. brought together (Holler N Fau-Tardivel, et al., (2003) Mol. The term “receptor coupling agent” includes any agent or Cell. Biol. 23:1428) In another embodiment, the receptor coupling agent activates two different TNF family receptors, construct which can activate at least two distinct cell surface 20 thereby increasing the signal strength and triggering two dif receptors. In one embodiment, the receptor coupling agent is ferent signal transduction cascades. a proteinaceous agent. Receptor coupling agents are used to enhance signaling capability of cell surface receptors. Recep The receptor coupling agent of the invention comprises tor coupling agents of the invention are directed to TNF binding speci?cities which are directed to at least two distinct family receptors. In some instances, activation of at least two 25 TNF family receptor members. Binding speci?cities are cho TNF family receptors by a receptor coupling agent can induce sen according to the TNF family receptor members of interest cell death. In one embodiment of the invention, the receptor which are to be targeted. For example, in one embodiment a coupling agent comprises a bispeci?c multivalent construct. receptor coupling agent comprises a ?rst binding speci?city In still another embodiment, the receptor coupling agent is a for the TNF receptor TRAIL-R2 and a second binding speci bispeci?c multivalent construct comprising an anti-LTBR 30 ?city for the TNF receptor lymphotoxin-[3receptor (LTBR). binding moiety or speci?city and an anti-TRAIL-R2 binding Examples of different types of TNF family receptors which moiety or speci?city. In another embodiment, the receptor may be targeted by a receptor coupling agent are described in coupling agent comprises binding speci?city conferred by an more detail below. anti-LTBR antibody (e.g. CBEl l) and binding speci?city A. TNF Receptors Containing a Death Domain conferred by an anti-TRAIL-R2 antibody (e.g. l4A2). 35 The term “single chain variable fragment or scFv” refers to Receptor coupling agents may target TNF family receptors an Fv fragment in which the heavy chain domain and the light containing death domains, which may be useful for the treat chain domain are linked. One or more scFv fragments may be ment of cancer. A “death domain” or “DD” refers to a protein linked to other antibody fragments (such as the constant domain of certain TNF receptors comprising six conserved alpha helices. Death domain containing TNF receptors are domain of a heavy chain or a light chain) to form antibody 40 constructs having one or more antigen recognition sites. primary targets of receptor coupling agents of the invention, The term “synergistic” refers to a combination which is and an example of such a construct is provided in the more effective than the additive effects of any two or more Examples section. single agents. In one embodiment of the invention, the term One example of a death domain receptor is Fas. Fas path synergistic includes a combination type of supra-additive 45 way molecules include any molecule involved in or related to inhibition in which both the LT-[3-R agonist and chemothera a pathway leading to apoptosis or programmed cell death peutic agent individually have the ability to inhibit tumor (PCD) induced by Fas. Fas pathway molecules include, but volume. The term “potentiation” refers to a case in which are not limited to Fas, the Fas ligand (FasL), and members of simultaneous effect of two or more agents is greater than the the TNFR superfamily of receptors. FADD, caspase 8, bid, sum of the independent effects of the agents. 50 and caspase 3 are also included as Fas pathway molecules. “Treating” cancer in a subject or “treating” a subject hav Fas pathway molecules may also be included in other groups ing cancer refers to subjecting the subject to a pharmaceutical as de?ned herein. treatment, e.g., the administration of a drug, such that the Some of the cytotoxic effects of lymphocytes are mediated extent of cancer is decreased or prevented. Treatment by interaction of a lymphocyte-produced ligand with Fas-R includes (but is not limited to) administration of a composi 55 (also known as DR-2, APO-l and CD95; GenBank GI Nos. tion, such as a pharmaceutical composition, and may be per 4507583, 23510421, 23510423, 23510425, 23510427, formed either prophylactically, or subsequent to the initiation 23510429, 23510431, and 23510434), a widely occurring of a pathologic event. cell surface receptor which has the ability to trigger cell death The term “tumor volume” refers to the total size of the (see Nagata and Golstein, (1995) Science 267: 1449-56). tumor, which includes the tumor itself plus affected lymph 60 Binding of FasL to the Fas receptor leads to aggregation of the nodes if applicable. Tumor volume may be determined by a receptor on the cell membrane and speci?c recruitment of variety of methods known in the art, such as, e.g. by measur intracellular signaling molecules known as DISC, or death ing the dimensions of the tumor using calipers, computed inducing signal complex. The adaptor protein, FADD, binds tomography (CT) or magnetic resonance imaging (MRI) to the intracellular death domain of Fas which leads to the scans, and calculating the volume using equations based on, 65 recruitment of caspase-8, also known as FLICE or MACH. for example, the Z-axis diameter, or on standard shapes such Fas-induced cell death may activate a pathway that alters as the sphere, ellipsoid, or cube. mitochondrial permeability transition. US 7,799,902 B2 13 14 Cell killing by mononuclear phagocytes involves a ligand activation of non-death domain containing TNF receptors for receptor couple, TNF and its receptor, TNFRl (also known as the treatment of solid tumors, speci?cally an anti-LTBR ago DR-1, CD120, p55-R; GenBank GI No. 4507575; see also nist monoclonal antibody (mAb), also shows potential as an US. Pat. No. 5,395,760), that is structurally related to Fas-R anti-tumor therapy (Browning, et al. (1996) JExp Med 183: and its ligand (see also Vandenabeele et al., (1995) Trends in 867, Wilson and Browning (2003) Cell Death Dz?‘ 9: 1321). Cell Biology 5:392). Like other receptor-induced effects, cell One example of a non-death domain containing TNF death induction by the TNF receptors and Fas-R occurs via a receptor family member is LTBR. LTBR is involved in the series of protein-protein interactions, leading from ligand control of the maturation status of various specialized stromal receptor binding to the eventual inactivation of enzymatic cells in the immune system and plays a critical role during the effector functions, which in the case of these particular recep development of the stromal elements of the lymph node tors results in cell death. anlagen (Mebius (2003) NalRev Immunol 3 :292). It has been Under normal circumstances, Fas receptor engagement is proposed that activation of a developmental program in epi accompanied by an in?ltration of in?ammatory cells and thelial or ?broblastoid cells in the context of a transformed secondary necrosis and also provokes in?ammation, e.g., cell is detrimental for their survival and this action may hepatic in?ammation, by inducing expression of cellular account for some of the anti-tumor activity of LTB receptor chemokines, e.g., hepatic chemokines, that recruit and acti activation. These receptors can also initiate in?ammatory vate immune cells leading to cell, e.g., hepatocyte, death in a programs that involve chemokine release or promote immu proin?ammatory milieu. In contrast, the receptor coupling nological anti-tumor responses (Yu et al. (2004) Na! Immunol agents of the current invention are designed to induce cell 5:141, Baud (2001) Trends Cell Biol 11:372). Such release death in speci?c target cells. The targeted therapy of the 20 may affect the in?ammatory status of the tumor and/ or invoke invention may be more potent due to enhanced signalling and, in?ltration of lymphoid elements promoting an immunologi therefore, may allow for treatment with lower doses of a drug. cal reaction to the tumor. Thus receptor coupling agents Such a strategy may minimize the negative consequences which activate of various TNF family receptors lacking death observed when apoptosis is systemically induced via activa domains, alone or in combination with TNF receptors con tion of a single cell-surface cytokine receptor. 25 taining death domains, are encompassed by the invention. In addition to Fas-R and TNF-Rl, other members of the In addition to LTBR, other examples of TNF receptors TNF receptor family containing death domains include DR3 which lack a death domain include Fn14 (also referred to as (also referred to as TRAMP, TR3, andApo3, see GenBank GI TWEAK-R; see Applicant’s co-pending application WO Nos. 4507569, 23200021, 23200023, 23200025, 23200027, 02/22166); RANK (see NCBI Accession Nos. AAB86809, 23200029, 23200031, 23200033, 23200035, 23200037, and 30 AF018253); TA] (also referred to as TROY, see NCBI Acces 23200039); TRAIL-R1 (also referred to as DR4 and Apo2, sion Nos. AAF71828, AAH47321, AAK28395); EDAR (see see GenBank GI No. 21361086); TRAIL-R2 (also referred to NCBI Accession Nos. AAD50076, AAD50077, AF130988); as DR5, see GenBank GI Nos. 22547116 and 22547119); XEDAR (see NCBI Accession Nos. AAG28761 ,AAH3491 9, p75NGF-R (also known as TNFRSF16; NCBI Reference AAN73210); and CD40 (also referred to as CD40L receptor, Seq. NPi002498; GenBank GI No. 4505393); and DR6 35 see NCBI Accession Nos. AAH12419, AAH64518, (TRAIL-R3, GenBAnk GI No. 22547121), each containing AAR84238). death domains that directly initiate apoptosis. A subgroup among TNF receptors lacking a death domain There are four human TRAIL receptors termed TRAIL includes TNF receptors which are involved in tissue differ R1-4. TRAIL-R1 and R2 also known as death receptors 4 and entiation, including development and wound healing. Several 5 (DR4-5) contain death domains in the intracellular region 40 TNF receptors have well-de?ned developmental roles, e.g. and are capable of triggering apoptosis (Wang and El-Deiry LTBR, RANK, EDAR and XEDAR (Mebius (2003) Nat Rev (2003) Oncogene 22:8628). TRAIL-R2 is preferred for Immunol 3:292; Theill et al., (2002) Ann Rev Immunol human tumor therapy since its activation does not trigger 20:795; Larikkala et al., (2002) Development 129:2541; Ren hepatocyte apoptosis and hence should have reduced toxicity nert (2000) J Exp Med 192: 1677). Differentiation is the pro (Ichikaw et al. (2001) Nat Med 7:954). Thus receptor cou 45 cess by which normal cells undergo physical and structural pling agents which activate various TNF family receptors changes as they develop to form different tissues of the body. containing death domains, alone or in combination with any Differentiation programs may affect tumors in several ways. other TNFR, e. g., a non-death domain TNFR such as LTBR, First, TNF receptors involved in tissue differentiation have are encompassed by the invention the potential to directly slow tumor growth by altered cell In one embodiment, a receptor coupling agent is used to 50 cycle progression. Second, the program in the context of decrease the toxic effects of death domain containing TNF transformation may lead to cell cycle con?ict and default receptors. While the activation of some death domain con apoptosis. Third, such con?icting input may render a cell taining receptors, e.g. TNFRl or Fas, has been shown to be more sensitive to chemotherapy. toxic in vivo, it is likely that tethering these receptors to other Examples of TNF receptor molecules shown to mediate TNF receptors may diminish toxicity and thus render a toxic 55 tissue differentiation which may be targeted by a receptor antibody less toxic. For example, if raft association is critical coupling agent to enhance TNF signalling include the follow for the full signaling of TNFRl, de-rafting by tethering to a ing: RANK (also known as TNFRSFl 1A; GenBank GI No. non-rafted receptor may be su?icient to reduce anti-TNFRl 4507565; Accession No. AF018523; US. Pat. Nos. 6,562, toxicity. In one embodiment, a receptor coupling agent com 948; 6,537,763; 6,528,482; 6,479,635; 6,271,349; prises a binding moiety comprising an anti-LTBR antibody, or 60 6,017,729); EDARl (also known as Downless; GenBank GI antigen binding fragment thereof, and a binding moiety No. 11641231; Accession No. AF130988; US. Pat. No. directed to an anti-TNF family receptor containing a death 6,355,782); and TAJ/Troy/Trade (also known as TNFRSF19; domain. GenBank GI Nos. 23238202 and 23238204; Accession No. AF167555). In addition, XEDAR (also known as EDA-A2R; B. Non-Death Domain Receptors 65 GenBank GI No. 11140823; Accession No. AF130988) sig Receptor coupling agents of the invention may target TNF naling is involved in the process of ectodermal differentia family receptors which do not contain the death domain. The tion. XEDAR plays a major role in the activation of the