AU2014200579B2 - Molecules with extended half-lives, compositions and uses thereof - Google Patents

Abstract

The present invention provides molecules, including IgGs, non-IgG immunoglobulins, proteins and non-protein agents, that have increased in vivo half-lives due to the presence of an IgG 5 constant domain, or a portion thereof that binds the FcRn, having one or more amino acid modifications that increase the affinity of the constant domain or fragment for FeRn, Such proteins and molecules with increased half-lives have the advantage that smaller amounts and or less frequent dosing is required in the therapeutic, prophylactic or diagnostic use of such molecules.

Description

MOLECULES WITH EXTENDED HALF-LIVES, COMPOSITIONS AND USES THEREOF The present application is a divisional application from Australian patent application number 2011253690, which in turn is a divisional application from Australia patent application number 2008201419, which in turn is a divisional application from Australian patent application number 2002248184, the entire disclosures of which are incorporated herein by reference. This application claims the benefit of United States provisional application Serial Nos. 60/254, 884, filed December 12-2000, and 60/289,760, filed May 9, 2001, both of whch are incorpofaed by reference herein in their entireties. This invention was made, in part with iefd States Government support under award number A1 39167 from the National institute of Healh. The United States Government my have certin rights in dh invention, 1. INTRODUCTION The present invention relates to molccules whose in vivo halfwlives are increased by modification of an IgG constant do min, or FeRn (Pc Recepor-neonate) binding domain thereof. Specifically, these molecules have amino acid modifications that increase the affinity of the constant domain or fragment thereof for the FcRn. Increasing the half-life of therapeutic and diagnostic s and other bioactive molecules using methods of the invTnlon has many benefits including reducing the amount and/or frequency of dosing of these molecules, for example, in vaccines, passive immunotherapy and other therapeutic and prophylactic methods. The invention further relates to fusion proteins containing all or a portion (a FcRn binding portion) of an IgG constant domain having one or more of these amino acid modifications and a nongG protein or non-protein molecule conjugated to such a modified IgG onstant domain, where thw presence of the modified igG constant domain increases the in viva half-I$ of the non-IgG protein or molecule. 2. BACKGROUND) OF THE INVENTION The use of inmrunoglobulins as therapeutic agents has increased dramticany in recent years and have expanded to different areas of medical treatments. Such uses include treatment of agannaglobuMemia and hypogammaghobulinEia, as inmmunosuppressive agents for treating au.oiune diseases and grafbvs.host (GVH) diseases, the treatment of lynphoid malignancies, and passive inmunotherapies for the treatment of various systemic and infectious diseases. Also, imnunogkbuhns are useful as in vivo diagnostic toos, ftor example, in diagnostic imaging procedures, 1 WO 02/060919 PCTIUSOI/48432 One critical issue in these therapies is the persistence of immunoglobulins in the circulation, The rate of immunoglobulin clearance directly affects the amount and frequency of dosage of the immunoglobulin. Increased dosage and frequency of dosage 5 may cause adverse effects in the patient and also increase medical costs. IgG is the most prevalent immunoglobulin class in humans and other mammals and is utilized in various types of immunotherapies and diagnostic procedwes. The mechanism of IgG catabolism in the circulation has been elucidated through studies related to the transfer of passive immunity from mother to fCtus/neonate through the 10 placenta or yolk sac or through colostrum (maternofetal transfer of IgG via transcytosis) in rodents (Brambell, Lancet, ii:1087-1093, 1966; Rodewald, J Cell Biol., 71:666-670, 1976; Morris el al., In: Antigen Absorption by the Gut, pp. 3-22, 1978, University Park Press, Baltimore; Jones et at., J Clin. Invest., 51:2916-2927, 1972). The involvement of certain receptors in the maternofetal transmission of 15 maternal IgGs was first suggested by Brambell's group in their study on the intestinal absorption of maternal antibodies from ingested milk in newborn rats (Halliday, Proc. R. Soc. B., 143:408-413, 1955; Halliday, Proc. R. Soc. B., 144:427-430, 1955; Halliday, Proc, R. Soc. B., 148:92-103, 1957; Morris, Proc. R. Soc. B., 148;84-91, 1957; Brambell et al., Proc. R. Soc. B., 149:1-11, 1958; Morris, Proc. R. Soc. B., 160:276-292, 1964). Brambell 20 et al. suggested, based on the observation that heterologous IgGs interfered with the transmission of a specific antibody, that IgG molecules from various species might have sufficiently similar structures or sequences that bind to common receptors (Brambell et al., Proc. R. Soc. B., 149:1-11, 1958). A high-affinity Fc receptor, FcRn, has been implicated in this transfer 25 mechanism. The FcRn receptor has been isolated from duodenal epithelial brush borders of suckling rats (Rodewald et al., J Cell Biol., 99:154s-164s, 1984; Simister et al, Eur. J hnmunol., 15:733-738, 1985) and the corresponding gene has been cloned (Simister et al., Nature, 337:184, 1989 and Cold Spring Harbor S'ymp. Quant. Biot, LIV, 571-580, 1989). The later clonings of FcRn-encoding genes from mice (Ahouse et al., I Immunol., 30 151:6076-6088, 1993) and humans (Story et al., T Exp. Med, 180:2377-2381, 1994) demonstrate high homology of these sequences to the rat FeRn, suggesting a similar mechanism of maternofetal transmission of IgGs involving FoRn in these species. Meanwhile, a mechanism for IgG catabolism was also proposed by Brambell's group (Brambell et al., Nature, 203:1352-1355, 1964; Brambell, Lancet, 35 ii:1087-1093, 1966). They proposed that a proportion of IgG molecules in the circulation are bound by certain cellular receptors (i.e., FRn), which are saturable, whereby the Ig~s -2- WO 02/060919 PCT[US01/48432 are protected from degradation and eventually recycled into the circulation; on the other hand, IgGs which are not bound by the receptors are degraded. The proposed mechanism was consistent with the IgG catabolism observed in hypergammaglobulinemic or 5 hypogammaglobulinemic patients, Furthermore, based on his studies as well as others (see, e.g., Spiegelberg et al, J &xp. Med, 121:323-338, 1965; Edelman et at, Proc. Nat, Acad. Sci. USA, 63:78-85, 1969), Brambell also suggested that the mechanisms involved in inaternofetal transfer of IgG and catabolism of IgG may be either the same or, at least, very closely related (Brambell, Lancet, ii:1087-1093, 1966). Indeed, it was later reported that a 10 mutation in the Fe-hinge fragment caused concomitant changes in catabolism, maternofetal transfer, neonatal transcytosis, and, particularly, binding to FeRn (Ghetie et al, Immunology Today, 18(12):592-598, 1997). These observations suggested that portions of the IgG constant domain control IgG metabolism, including the rate of IgG degradation in the serum through 15 interactions with FeRn. Indeed, increased binding affinity for FeRn increased the serum half-life of the molecule (Kim et at, Eur. J ImmunoL, 24:2429-2434, 1994; Popov et al, Mol Immunol., 33:493-5 0 2, 1996; Ghetie et aL, Eur. 3 ImmunoL, 26:690-696, 1996; Junghans et al., Proc. Nat. Acad Sci. USA, 93:5512-5516, 1996; Israel et al, Immunol, 89:573-57 8,.1996). 20 Various site-specific mutagenesis experiments in the Fc region of mouse IgGs have led to identification of certain critical amino acid residues involved in the interaction between IgG and FcRn (Kim et al, Eur. J. Immunol, 24:2429-2434, 1994; Medesan et al., Eur, I Immunol., 26:2533, 1996; Medesan et al, . Immunol., 158:2211 2217, 1997). These studies and sequence comparison studies found that isoleucine at 25 position 253, histidine at position 310, and histidine at position 435 (according to Kabat numbering, Kabat at al, In: Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991, which is hereby incorporated by reference in its entirety), are highly conserved in human and rodent IgGs, suggesting their importance in IgG-FcRn binding. 30 Additionally, various publications describe methods for obtaining physiologically active molecules whose half-lives are modified either by introducing an FeRn-binding polypeptide into the molecules (WO 97/43316; US. Patent No. 5,869,046; U.8. Patent No. 5,747,035; WO 96/32478; WO 91/14438) or by fusing the molecules with antibodies whose FcRn-binding affinities are preserved but affinities for other Fe receptors 35 have been greatly reduced (WO 99/43713) or fusing with FcRn binding domains of -3- WO 021060919 PCT/USOI/48432 antibodies (WO 00/09560; U.S. Patent No. 4,703,039). However, none of these publications disclose specific mutants in the IgG constant domain that affect half-life. Prior studies have demonstrated that certain constant domain mutations 5 actually reduce binding to FcRn and, thereby, reduce the IgG in vivo half-life. PCT publication WO 93/22332 (by Ward et al.) discloses various recombinant mouse IgGs whose in vivo half-lives are reduced by mutations between about residue 253 and about residue 434. Particularly, substitutions of isoleucine at position 253; histidine at position 310; glutanine at position 311; His at position 433; and asparagine at position 434 were 10 found to reduce IgG half-life. Modulation of Igo molecules by amino acid substitution, addition, or deletion to increase or reduce affinity for FcRn is also disclosed in WO 98/23289; however, the publication does not list any specific mutants that exhibit either longer or shorter in vivo half-lives. 15 In fact, only one mutant of mouse IgG I that actually exhibited increased half-life, the triple mutation Thr252 to Ala, Thr254 to Ser, and Thr256 to Phe, has been identified (WO 97/3463 1). In view of the pharmaceutical importance of increasing the in vivo half-lives of imunoglobulins and other bioactive molecules, there is a need to develop modified 20 IgGs and FoRn-binding fragments thereof, (particularly modified human IgGs) that confer increased in vivo half-life on immunoglobulins and other bioactive molecules, 3. SUMMARY OF THE INVENTION The present invention is based upon the inventors' identification of several 25 mutations in the constant domain of a human IgG molecule that increase the affinity of the IgG molecule for the FeRn. In particular, the present inventors have screened libraries of human IgGi constant domains with random amino acid mutations introduced into particular regions of the constant domain for increased affinity for FcRn. Such random mutations were made in the regions of residues 251-256, 285-290, and 308-314, all of which are in 30 CH2 domain, and 385-389 and 428436, which are in CH3 domain, of human IgG1 hinge Fc regions (residues as depicted in Figure 2 (SEQ ID NO:83 or analogous residues in hinge Fe regions of other IgG molecules as determined by sequence alignment). As used herein, all residues of the IgG constant domain are numbered according to Kabat et al. (Sequences of Proteins of hInmunological Interest, U.S. Department of Health and Human Services, 35 1991, which is incorporated by reference herein in its entirety) and as presented in Figure 2 (SEQ ID NO:83), and include corresponding residues in other IgG constant domains as '-4determined by sequence alignment. The in vivo half-life, or persistence in serum or other tissues of a subject, of antibodies, and other therapeutic agents and other bioactive molecules is an important clinical parameter which determines the amount and frequency of antibody (or any other pharmaceutical molecule) administration. Accordingly, such molecules, including antibodies, with 5 increased half-life are of significant pharmaceutical importance. In one aspect, the present invention provides a modified molecule comprising a protein or non-protein agent and an IgG constant domain, wherein the IgG constant domain comprises a human

CH

2 domain in which there is an amino acid substitution at amino acid residues 308, 309, and 311, numbered according to the EU index of Kabat, wherein the modified molecule has an increased half 0 life compared to the half-life of a molecule comprising the protein or the non-protein agent and a corresponding IgG constant domain comprising a wild-type human CH 2 domain. Thus, the present invention further relates to a modified molecule (preferably a protein, but may be a non-protein agent) that has an increased in vivo half-life by virtue of the presence of a modified IgG constant domain, or FcRn-binding portion thereof (preferably the Fc or hinge-Fc 5 domain) (preferably from a human IgG) wherein the IgG constant domain, or fragment thereof, is modified (e.g., by amino acid substitution, deletion or insertion) to increase the affinity for the FcRn. In a particular embodiment, the present invention relates to modified IgGs, whose in vivo half-lives are extended by the modification of amino acid residues identified to be involved in the interaction of the hinge-Fc domain with the FcRn receptor. Preferably, the constant domain or fragment thereof has .0 higher affinity for FcRn at pH 6.0 than at pH 7.4. Such modifications may also alter (i.e., increase or decrease) the bioavailabitity (e.g., transport to mucosal surfaces, or other target tissues) of the molecules. The invention also relates to other types of immunoglobulins or fragments thereof (i.e., non-IgG immunoglobulins), non-immunoglobulin proteins and non-protein agents that are fused or conjugated to, or engineered to contain, an IgG constant domain, or FcRn-binding fragment thereof, 25 having one or more such amino acid modifications, In preferred embodiments, the present invention provides molecules, particularly, immunoglobulins whose in vivo half-lives are extended by the presence of an IgG constant domain, or FcRn binding fragment thereof (preferably, Fc or hinge-Fc domain), that has modifications of one or more of amino acid residues 251-256, 285-290, 308-314, 385-389, and 428-436 that increase the 30 affinity of the constant domains or fragments thereof for FcRn. In certain embodiments, these modifications preferably exclude residues 252, 254, and 256, in particular when the IgG constant domain or fragment thereof, is murine. In particular embodiments, the modification is at one or more surface-exposed residues, and the modification is a substitution with a residue of similar charge, polarity or hydrophobicity to the residue being substituted. In preferred embodiments, the modified 35 IgG constant domain, or fragment thereof, binds with higher affinity to FcRn at pH. 6.0 than at pH 7.4. In a preferred embodiment, the constant domain, or fragment thereof, is modified by substitution of one or more of amino acid residues 251-256, 285 -5- WO 02/060919 PCT/US01/48432 290, 308-314, 385-389, and 428-436 that increase the affinity of the constant domain or FeRn-binding fragments thereof for FeRn. In certain embodiments, substitutions of residue 252 with leucine, residue 254 with serine, and/or residue 256 with phenylalanine are 5 excluded, particularly when the constant domain or fragment thereof is derived from a mouse IgG, In specific embodiments, the invention provides immunoglobulins or other bioactive molecules that contain an IgGl constant domain, or FeRn-binding fragment thereof (preferably Fe or hinge-Fo domain) (preferably human), having amino acid 10 modifications at one or more of position 308, 309, 311, 312, and 314, more specifically, having substitutions at one or more of positions 308, 309, 311, 312 and 314 with threonine, proline, serine, aspartic acid and leucine respectively. In another embodiment, residues at one or more of positions 308, 309, and 311 are substituted with isoleucine, proline, and glutamic acid, respectively. In yet another embodiment, residues at one or more of positions 15 308, 309, 311, 312, and 314, are substituted with threonine, proline, shrine, aspartic acid, and leucine, respectively. The invention further relates to combinations of these amino acid substitutions. Furthermore, the invention provides imnunoglobulins or other bioactive molecules that contain an IgG1 constant domain, or FeRn-binding fragment thereof 20 (preferably, Fc or hinge-Fc domain) (preferably human), having amino acid modifications at one or more of positions 251, 252, 254, 255, and 256, more specifically, having substitutions at one or more of these positions. in specific embodiments, residue 251 is substituted with leucine or arginine, residue 252 is substituted with tyrosine, phenylalanine, serine, tryptophan or threonine, residue 254 is substituted with threonine or serine, residue 25 255 is substituted with leucine, glycine, isoleucine or arginine, and/or residue 256 is substituted with serine, arginine, glutamine, glutamic acid, aspartic acid, alanine, asparagine or threonine. In a more specific embodiment, residue 251 is substituted with leucine, residue 252 is substituted with tyrosine, residue 254 is substituted with threonine or serine, and/or residue 255 is substituted with arginine. In yet another specific embodiment, residue 30 252 is substituted with phenylalanine and/or residue 256 is substituted with aspartic acid, In a preferred embodiment, residue 251 is substituted with leucine, residue 252 is substituted with tyrosine, residue 254 is substituted with threonine or shrine, and/or residue 255 is substituted with arginine. The invention further relates to any combination of these substitutions. 35 Furthermore, the invention provides immunoglobulins or other bioactive molecules that contain an IgG1 constant domain, or Fe&R-binding fragment thereof -6- 7 (preferably, Fc or hinge-Fe domain) (preferably human), having amino acid modifications at one or more of positions 251, 252 , 254 255, and 256, more specifically, having substitutions at one or more of these positions. In specific embodiments, residue 251 is substituted with leucine or arginine, residue 252 is 5 substituted with tyrosine, phenylalanine, serine, tryptophan or threonine, residue 254 is substituted with threonine or serine, residue 255 is substituted with leucine, glycine, isoleucine or arginine, and/or residue 256 is substituted with shrine, arginine, glutamine, glutamic acid, aspartic acid, alanine, asparagine or threonine. In a more specific embodiment, residue 251 is substituted with leucine, residue 252 is substituted 10 with tyrosine, residue 254 is substituted with threonine or serine, and/or residue 255 is substituted with arginine. In yet another specific embodiment, residue 252 is substituted with phenylalanine and/or residue 256 is substituted with aspartic acid. In a preferred embodiment, residue 251 is substituted with leucine, residue 252 is substituted with tyrosine, residue 254 is substituted with threonine or serine, and/or 15 residue 255 is substituted with arginine. The invention further relates to any combination of these substitutions. Furthermore, the invention provides immunoglobulins or other bioactive molecules that contain an IgGl constant domain, or FcRn-binding fragment thereof (preferably, Fe or hinge-Fc domain) (preferably human), having amino acid modifications at one or 20 more of positions 428, 433, 434, and 436, more specifically, having substitutions at one or more of these positions. In specific embodiments, residue 428 is substituted with methionine, threonine, leucine, phenylalanine, or serine, residue 433 is substituted with lysine, arginine, shrine, isoleucine, proline, glutamine, or histidine, residue 434 is substituted with phenylalanine, tyrosine, or histidine, and/or residue 436 is substituted 25 with histidine, asparagine, arginine, threonine, lysine, methionine, or threonine. In a more specific embodiment, residues at one or more positions 433, 434, and 436 are substituted with lysine, phenylalanine, and histidine, respectively, In a preferred embodiment, residue 428 is substituted with methionine and/or residue 434 is substituted with tyrosine. 30 Furthermore, the invention provides immunoglobulins or other bioactive molecules that contain an IgGI constant domain, or FeRn-binding fragment thereof (preferably, Fe or hinge-Fe domain) (preferably human), having amino acid modifications at one or more positions 385, 386, 387 and 389, more specifically, having substitutions at one or 7a more of these positions, In specific embodiments, residue 385 is substituted with arginine, aspartic acid, serine, threonine, histidine, lysine, or alanine, residue 386 is substituted with threonine, praline, aspartic acid, serine, lysine, arginine, isoleucine, or methionine, residue 387 is substituted with arginine, histidine, serine, threonine, 5 alanine, or praline and/or residue 389 is substituted with praline or serine, In more specific embodiments, residues at one or more positions 385, 386, 387, and 389 are substituted with arginine, threonine, arginine, and praline, respectively. In yet another specific embodiment, residues at one or more positions 385,386, and 389 are substituted with aspartic acid, praline, and serine, respectively. 10 Molecules of the invention include any combination of the above-described substitutions at one or more of residues 251, 252, 254, 255, 256, 308, 309, 311, 312, 385, 386, 387, 389, 428, 433, 434 and/or 436. In a preferred embodiment, the molecule of the invention contains a Fc region, or FcRn-binding domain thereof, having one or more of the following substitutions: leucine at residue 251, tyrosine at residue 252, 15 threonine or serine at residue 254, arginine at residue 255, threonine at residue 308, praline at residue 309, serine at residue 311, aspartic acid at residue 312, leucine at residue 314, arginine at residue 385, threonine at residue 386, arginine at residue 387, praline at residue 389, methionine at residue 428, and/or tyrosine at residue 434. Thus, in one embodiment, the present invention provides a modified IgG comprising 20 an IgG constant domain, or an FeRn-binding fragment thereof, wherein the constant domain or fragment comprises a substitution at amino acid residue 252 relative to a wild-type IgG constant domain, numbered according to the EU numbering index of Kabat, wherein the modified IgG has an increased half-life compared to the half-life of an IgG having the wild-type IgG constant domain, and wherein the substitution at 25 amino acid residue 252 is a substitution with tyrosine, phenylalanine, tryptophan, or threonine. included within the invention are pharmaceutical compositions and methods of prophylaxis and therapy using modified immunoglobulins, proteins and other bioactive molecules of the invention having extended half-lives, Also included are methods of 30 WO 02/060919 PCT/USOI/48432 diagnosis using modified immunoglobulins, proteins and other bioactive molecules of the invention having extended half-lives. In a specific embodiment, the invention provides an anti-respiratory syncytial virus (RSV) antibody useful to treat or prevent RSV infection, 5 such as SYNAGIS* (see U.S. Patent No. 5,824,307 and Johnson et al., J Infectious Disease 176:1215-1224, 1997, both of which are incorporated by reference in their entireties), and other anti-RSV antibodies, including variants of SYNAGIS* (see United States patent Application Serial No., 09/724,396, filed November 28, 2000, United States patent Application Serial No. 09/724,531, filed November 28, 2000, United States patent 10 Application Serial No. __, filed November 28, 2001 (attorney docket no. 10271-047), and United States patent Application Serial No. , filed November 28, 2001 (attorney docket no. 10271-048), all entitled "Methods of Administering/Dosing Anti-RSV Antibodies for Prophylaxis and Treatment," all by Young et al., all of which are incorporated by reference herein in their entireties, particularly the sequences of heavy and light chain variable 15 domains and CDRs of anti-RSV antibodies disclosed therein), which has one or more amino acid modifications in the constant domain that increase the affinity of the antibody for FcRn and that has an increased in vivo half-life (see also, Section 5.1 infra). 3.1 DEFINITIONS 20 The term "IgG Fe region" as used herein refers the portion of an IgG molecule that correlates to a crystallizable fragment obtained by papain digestion of an )go molecule. The Fc region consists of the C-terminal half of the two heavy chains of an IgG molecule that are linked by disulfide bonds. It has no antigen binding activity but contains the carbohydrate moiety and the binding sites for complement and Fe receptors, including 25 the FeRn receptor (see below). The Fe fragment contains the entire second constant domain CH2 (residues 231-340 of human IgG1, according to the Kabat numbering system) (e.g, SEQ ID NO:80) and the third constant domain CH3 (residues 341-447) (e.g, SEQ ID NO:81). The term "IgG hinge-Fe region" or "hinge-Fe fragment" as used herein refers 30 to a region of an IgG molecule consisting of the Fe region (residues 231-447) and a hinge region (residues 216-230; e.g., SEQ ID NO:82) extending from the N-terminus of the Fe region. An example of the amino acid sequence of the human IgG1 hinge-Fo region is SEQ ID NO:83. The term "constant domain"refers to the portion of an immunoglobulin 35 molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen binding site. The - 8- WO 02/060919 PCT/US01/48432 constant domain contains the CI1, CH2 and CH3 domains of the heavy chain and the CHL domain of the light chain. The term "FcRn receptor" or "FeRn" as used herein refers to an Fc receptor 5 ("n" indicates neonatal) which is known to be involved in transfer of maternal IgGs to a fetus through the human or primate placenta, or yolk sac (rabbits) and to a neonate from the colostrum through the small intestine. It is also known that FcRn is involved in the maintenance of constant serum IgG levels by binding the IgG molecules and recycling them into the serum, The binding of FcRn to IgG molecules is strictly p1-dependent with 10 optimum binding at p 1 H 6.0. FcRn comprises a heterodimer of two polypeptides, whose molecular weights are approximately 50 kD and 15 kD, respectively. The extracellular domains of the 50 kD polypeptide are related to major histocompatibility complex (MHC) class I a-chains and the 15 kD polypeptide was shown to be the non-polymorphic pg microglobulin (p 2 -m). In addition to placenta and neonatal intestine, FeRn is also expressed 15 in various tissues across species as well as various types of endothelial cell lines. It is also expressed in human adult vascular endothelium, muscle vasculature and hepatic sinusoids and it is suggested that the endothelial cells may be most responsible for the maintenance of serum IgG levels in humans and mice. The amino acid sequences of human FeRn and murine FeRn are indicated by SEQ ID NO:84 and SEQ ID NO:85, respectively. Homologs 20 of these sequences having FeRn activity are also included. The term "in vivo half-life" as used herein refers to a biological half-life of a particular type of IgG molecule or its fragments containing FcRn-binding sites in the circulation of a given animal and is represented by a time required for half the quantity administered in the animal to be cleared from the circulation and/or other tissues in the 25 animal. When a clearance curve of a given IgG is constructed as a function of time, the curve is usually biphasic with a rapid a-phase which represents an equilibration of the injected IgG molecules between the intra- and extra-vascular space and which is, in part, determined by the size of molecules, and a longer p-phase which represents the catabolism of the IgG molecules in the intravascular space. The term "in vivo half-life" practically 30 corresponds to the half life of the IgG molecules in the p-phase. An "isolated" or "purified" antibody or fusion protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes 35 preparations of an antibody or a fusion protein in which the antibody or the fusion protein is separated from cellular components of the cells from which it is isolated or recombinantly -9- WO 02/060919 PCT/US01/48432 produced. Thus, an antibody or a fusion protein that is substantially free of cellular material .includes preparations of antibody or fusion protein having less than about 30%, 20%, 10%, or 5% (by dry weight) of contaminating protein. When the antibody or the fusion protein is 5 recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation. When the antibody or the fusion protein is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals which are involved in the synthesis of the 10 protein. Accordingly such preparations of the antibody or the fusion protein have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the antibody or antibody fragment of interest. In a preferred embodiment of the present invention, antibodies are isolated or purified. In another preferred embodiment of the invention, fusion proteins are isolated or purified. 15 An "isolated" nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals 20 when chemically synthesized. An "isolated" nucleic acid molecule does not include cDNA molecules within a cDNA library, In a preferred embodiment of the invention, nucleic acid molecules encoding antibodies are isolated or purified. In another preferred embodiment of the invention, nucleic acid molecules encoding fusion proteins are isolated or purified. The term "host cell" as used herein refers to the particular subject cell 25 transfected with a nucleic acid molecule or infected with phagemid or bacteriophage and the progeny or potential progeny of such a cell. Progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome. 30 The names of amino acids referred to herein are abbreviated either with three-letter or one-letter symbols. To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for 35 optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are -10- WO 02/060919 PCIf0S01/48432 then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a 5 function of the number of identical positions shared by the sequences (i.e., % identity = number of identical overlapping positions/total number of positions x 100%). In one embodiment, the two sequences are the same length. The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm, A preferred, non-limiting example of a 10 mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natt. Acad Sc, USA. 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Nat. Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed with the NBLAST nucleotide 15 program parameters set, e.g., for score'=l00, wordlength-=12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the present invention. BLAST protein searches can be performed with the XBLAST program parameters set, e~g., to score-50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule of the present invention. To obtain gapped alignments for comparison purposes, Gapped BLAST 20 can be utilized as described in Altschul et at, 1997, Nucleic Acids Res, 25:3389-3402, Alternatively, PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id), When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g,, of XBLAST and NBLAST) can be used (see, e.g., http://www.ncbi.nlm.nih.gov). Another preferred, 25 non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAMI20 weight residue table, a gap length penalty of 12, and a gap penalty of 30 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted. 35 - 11 - WO 02/060919 PCTUS01/48432 4. DESCRIPTION OF THE FIGURES FIG. 1 shows the structure of the IgG hinge-Fc region indicating the locations of the residues identified to be involved in the interaction with the FeRn receptor 5 (Ghetie et al., Immunology Today, 18(12):59Z-598, 1997). FIG. 2 shows the amino acid sequence of the human IgGI hinge-Fo region (SEQ ID NO:83) containing a hinge region (SEQ ID NO:82), CH2 domain (SEQ ID NO:80), and CH3 domain (SEQ ID NO:81). FIGS. 3 (A and B) show the amino acid sequences of (A) human FcRn (SEQ 10 ID NO:84) and (B) mouse FeRn (SEQ ID NO:85), respectively. FIG. 4 shows the amino acid sequence of the human IgGi hinge-Fc region (SEQ ID NO:83), in which wild-type residues which are mutated by amino acid substitutions are indicated in underlined bold-face. FIG. 5 shows a schematic diagram of panning process for the phage 15 displayed modified hinge-Fo library. FIG. 6 shows a summary of the occurrence of selected mutant residues at the variant positions in the libraries screened. FIGS. 7 (A-D). (A) shows the binding of urine FeRn to inunmobilized IgG1 having M252Y/S254T/T256B substitutions. Murine FeRn was injected at 10 different 20 concentrations ranging from I aM to 556 nM over a surface on which 4000 resonance units (RU) of IgGI had been coupled. After equilibrium was reached, residual bound protein was eluted with a pulse of PBS, pH 7.4. (B) shows the binding of human FeRn to immobilized IgGl/M252Y/S254T/T256E. Murine FcRn was injected at 8 different concentrations ranging from 71 nM to 2.86 1 M over a surface on which 1000 RU of IgGI 25 had been coupled. After equilibrium was reached, residual bound protein was eluted with a pulse of PBS, pH 7.4. (C) and (D) show scatchard analyses of the data in (A) and (B), respectively, after correction for nonspecific binding. Rq is the corrected equilibrium response at a given concentration C, The plots are linear with correlation coefficients of 0.97 and 0.998, respectively. The apparent K,, are 24 nM and 225 nM, respectively. 30 FIGS. 8 (A-H). (A)-(D) show the results from BlAcore analysis of the binding of murine FeRn at pH 6.0 and p1 7.4 to (A) wild type human IgGI, (B) M252Y1S254T/T256E, (C) H433K/N434F/Y436H, and (U) 0385D/G3 86P/N3 898, respectively, after correction for nonspecific binding. Murine FeRn was injected at a concentration of 1. 1 pm over a surface on which 1000 RU of wild type IgG1, 1000 RU of 35 M252Y/S254T/T256E, 955 RU of H433K/N434F/Y436H, and 939 RU of G385D/Q386P/N389S had been coupled. (E)-(H) show the results from BlAcore analysis -12- WO 02/060919 PCT/USO1/48432 of the binding of human FoRn at pH 6.0 and pH 7.4 to (E) wild type human IgG1, (F) M252Y/S254T/T256E, (Q) H433K/N434F/Y436H, and (H) G385D/Q386P/N389S, respectively, after correction for nonspecific binding. Human FeRn was injected at a 5 concentration of 1.4 pm over a surface on which 1000 RU of wild type IgG1, 1000 RU of M252Y/S254T/T256B, 955 RU of H433K/N434F/Y436H, and 939 RU of G385D/Q386P/N389S had been coupled. FIG. 9 shows the space-filling model of the surface of the Fc fragment of a human IgG1 based upon the human IgGI structure of Deisenhofer, 1981, Biochemistry 10 20:2361-2370. Residues are color-coded according to the gain of free energy of stabilization of the Pc-FcRn complex; red, substitutions at these positions were found to increase affinity b a factor of at least 2.5 times in the Fc/human FcRn interaction and of at least 5 time in the Fc/mouse FcRn interaction; blue, substitutions at those positions were found to increase affinity by a factor of less than 2 times in both the Fe-human FeRn and 15 Fe-mouse FcRn interaction, The figure was drawn using Swiss pdb viewer (Guex and Peitsch, 1997, Electrophoresis 18:2714-2723). FIG, 10 shows the changes in serum concentration ([Mab) nghl) over time (in days) of antibody having a wild type constant domain (SYNAGIS*) (open squares), or constant domains with the following mutations: M252Y/S254T/T256B (open circles), 20 G385D/Q386P/N389S (solid squares), and H433K/N434F/Y436H (solid circles), Antibody concentration was determined using anti-human IgG ELISA, 5. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to molecules, particularly proteins, more 25 particularly imununoglobulins, that have an increased in vivo half-life and comprise an IgG constant domain, or fragment thereof that binds to an FcRn (preferably a Fe or hinge-Fc domain), that contains one or more amino acid modifications relative to a wild type IgG constant domain which modifications increase the affinity of the IgG constant domain, or fragment thereof, for the FeRn. In a preferred embodiment, the invention particularly 30 relates to the modification of human or humanized IgGs and other bioactive molecules containing FeRn-binding portions of human IgGs, which have particular use in human therapy, prophylaxis and diagnosis. 35 -13 - WO 02/060919 PCT/US01/48432 5.1 MOLECULES WITH INCREASED IN VIVO HALF-LIVES The present invention is based upon identification of amino acid 5 modifications in particular portions of the gG constant domain that interact with the FcRn, which modifications increase the affinity of the IgG, or fragment thereof, for the FeRn. Accordingly, the invention relates to molecules, preferably proteins, more preferably immunoglobulins, that comprise an IgG constant domain, or FcRn binding fragment thereof (preferably a Fu or hinge-Fe domain fragment), having one or more amino acid modifications (i.e, substitutions, insertions or deletions) in one or more regions that interact 10 with the FeRn, which modifications increase the affinity of the IgG or fragment thereof, for the FoRn, and also increase the in vivo half-life of the molecule. In preferred embodiments, the one or more amino acid modifications are made in one or more of residues 251-256, 285-290, 308-314, 385-389, and 428-436 of the IgG hinge-F region (for example, as in the human IgGI hinge-Fc region depicted in Figure 4, SEQ ID NO:83), or analogous residues thereof, as determined by amino acid sequence alignment, in other IgG hinge-Fe regions. In a preferred embodiment, the amino acid modifications are made in a human IgG constant domain, or FoRn-binding domain thereof, In a certain embodiment, the modifications are not made at residues 252, 254, or 256 (i.e., all are made at one or more of residues 251, 253, 255, 285-290, 308-314, 385-389, or 428-436) of the igG constant domain. In a more preferred embodiment, the amino acid modifications are not the substitution with leucine at residue 252, with serine at 254, and/or with phenylalanine at position 256, In particular, in preferred embodiments, such modifications are not made when the IgG constant domain, hinge-Fc domain, hinge-rc domain or other FeRn-binding fragment thereof is derived from a mouse. 25 The amino acid modifications may be any modification, preferably at one or more of residues 251-256, 285-290, 308-314, 3 85-389, and 428-436, that increases the in vivo half-life of the IgG constant domain, or FeRn-binding fragment thereof (e.g, Fe or hinge-Fc domain), and any molecule attached thereto, and increases the affinity of the IgG, or fragment thereof, for FoRn. Preferably, the one or more modifications also result in a 30 higher binding affinity of the constant domain, or FeRn-binding fragment thereof, for FeRn at pH 6.0 than at pH 7.4. in other embodiments, the modifications alter (I.e., increase or decrease) bioavailability of the molecule, in particular, alters (i.e., increases or decreases) transport (or concentration or half-life) of the molecule to mucosal surfaces (e.g., of the lungs) or other portions of a target tissue. In a preferred embodiment, the amino acid 35 modifications alter (preferably, increase) transport or concentration or half-life of the -14- WO 02/060919 PCTIUS01/48432 molecule to the lungs. In other embodiments, the amino acid modifications alter (preferably, increase) transport (or concentration or half-life) of the molecule to the heart, pancreas, liver, kidney, bladder, stomach, large or small intestine, respiratory tract, lymph 5 nodes, nervous tissue (central and/or peripheral nervous tissue), muscle, epidermis, bone, cartilage, joints, blood vessels, bone marrow, prostate, ovary, uterine, tumor or cancer tissue, etc. In a preferred embodiment, the amino acid modifications do not abolish, or, more preferably, do not alter, other immune effector or receptor binding functions of the constant domain, for example, but not limited to complement fixation, ADCC and binding 10 to FoyRI, FoyRIL, and FcyRM, as can be determined by methods well-known and routine in the art. In another preferred embodiment, the modified FcRn binding fragment of the constant domain does not contain sequences that mediate immune effector functions or other receptor binding. Such fragments may be particularly useful for conjugation to a non IgG or non-immunoglobulin molecule to increase the in vivo half-life thereof, In yet 15 another embodiment, the effector functions are selectively altered (e.g., to reduce or increase effector functions). In preferred embodiments, the amino acid modifications are substitutions at one or more of residues 308, 309, 311, 312 and 314, particularly a substitution with threonine at position 308, proline at position 309, serine at position 311, aspartic acid at 20 position 312, and/or leucine at position 314. Alternatively, the modification is the substitution with an isoleucine at position 308, proline at position 309, and/or a glutamic acid at position 311 In yet another embodiment, residues at one or more of positions 308, 309, 311, 312, and 314, are substituted with threonine, proline, leucine, alanine, and alanine, respectively. Accordingly, in certain embodiments the residue at position 308 is 25 substituted with threonine or isoleucine, the residue at position 309 is substituted with proline, the residue at position 311 is substituted with serine, glutamic acid or leucine, the residue at position 312 is substituted with alanine, and/or the residue at position 314 is substituted with Ieucine or alanine, In a preferred embodiment, the substitution is a threonine at position 308, a proline at position 309, a serine at position 311, an aspartic acid 30 at position 312, and/or a leucine at position 314. In preferred embodiments, the amino acid modifications are substitutions at one or more of residues 251, 252, 254, 255, and 256. In specific embodiments, residue 251 is substituted with leucine or arginine, residue 252 is substituted with tyrosine, phenylalanine, serene, tryptophan or threonine, residue 254 is substituted with threonine or 35 serine, residue 255 is substituted with arginine, leucine, glycine, or isoleucine, and/or residue 256 is substituted with serine, arginine, glutamine, glutamic acid, aspartic acid, - 15 - WO 02/060919 PCT/USO1/48432 alanine, asparagine or threonine, In a more specific embodiment, residue 251 is substituted with leucine, residue 252 is substituted with tyrosine, residue 254 is substituted with threonine or serine, residue 255 is substituted with arginine, and/or residue 256 is 5 substituted with glutamic acid. In preferred embodiments, the amino acid modifications are substitutions at one or more of residues 428, 433, 434, and 436. In specific embodiments, residue 428 is substituted with threonine, methionine, leucine, phenylalanine, or serine, residue 433 is substituted with lysine, arginine, sarins, isoleucine, proline, glutamine or histidine, residue 10 434 is substituted with phenylalanine, tyrosine, or histidine, and/or residue 436 is substituted with histidine, asparagine, arginine, threonine, lysine, or methionine. In a more specific embodiment, residues at position 428 and/or 434 are substituted with methionine, and/or histidine respectively. In preferred embodiments, the amino acid modifications are substitutions at 15 one or more of residues 385, 386, 387, and 389, more specifically, having substitutions at one or more of these positions, In specific embodiments, residue 385 is substituted with arginine, aspartic acid, serine, threonine, histidine, lysine, alanine or glycine, residue 386 is substituted with threonine, proline, aspartic acid, shrine, lysine, arginine, isoleucine, or methionine, residue 387 is substituted with arginine, praline, histidine, serine, threonine, or 20 alanine, and/or residue 389 is substituted with proline, serine or asparagine. In more specific embodiments, residues at one or more positions 385, 386, 387, and 389 are substituted with arginine, threonine, arginine, and proline, respectively. In yet another specific embodiment, residues at one or more positions 385, 386, and 389 are substituted with aspartic acid, proline, and shrine, respectively, 25 In particular embodiments, amino acid modifications are made at one or a combination of residues 251, 252, 254, 255, 256, 308, 309, 311, 312, 314, 385, 386, 387, 389, 428, 433, 434, and/or 436, particularly where the modifications are one or more of the amino acid substitutions described immediately above for these residues. In a preferred embodiment, the molecule of the invention contains a Fe 30 region, or FeRn-binding domain thereof, having one or more of the following substitutions: leucine at residue 251, tyrosine at residue 252, threonine or shrine at residue 254, arginine at residue 255, threonine at residue 308, proline at residue 309, shrine at residue 311, aspartic acid at residue 312, leucine at residue 314, arginine at residue 385, threonine at residue 386, arginine at residue 387, proline at residue 389, methionine at residue 428, and/or tyrosine at 35 residue 434. -16 - WO 02/060919 PCT/US01148432 In a preferred embodiment, the FeRn binding domain has a substitution at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16 or all 18 of residues 251, 252, 254, 255, 256, 308, 309, 311, 312, 314, 385, 386, 387, 389, 428, 433, 434, and/or 436. 5 Amino acid modifications can be made by any method known in the art and many such methods are well known and routine for the skilled artisan. For example, but not by way of limitation, amino acid substitutions, deletions and insertions may be accomplished using any well-known PCR-based technique. Amino acid substitutions may be made by site-directed mutagenesis (see, for example, Zoller and Smith, NucL. Acids Res. 10 10:6487-6500, 1982; Kunkel, Proc. Natl. Acad Sci USA 82:488, 1985, which are hereby incorporated by reference in their entireties). Mutants that result in increased affinity for FeRn and increased in vivo half-life may readily be screened using well-known and routine assays, such as those described in Section 5.11, infra. In a preferred method, amino acid substitutions are introduced at one or more residues in the IgG constant domain or FcRn 15 binding fragment thereof and the mutated constant domains or fragments are expressed on the surface of bacteriophage which are then screened for increased FeRn binding affinity (see, in particular, Section 5.2 and 5.11, infta). Preferably, the amino acid residues to be modified are surface exposed residues. Additionally, in making amino acid substitutions, preferably the amino acid 20 residue to be substituted is a conservative amino acid substitution, for example, a polar residue is substituted with a polar residue, a hydrophilic residue with a hydrophilic residue, hydrophobic residue with a hydrophobic residue, a positively charged residue with a positively charged residue, or a negatively charged residue with a negatively charged residue. Moreover, preferably, the amino acid residue to be modified is not highly or 25 completely conserved across species and/or is critical to maintain the constant domain tertiary structure or to FeRn binding. For example, but not by way of limitation, modification of the histidine at residue 310 is not preferred. Specific mutants of the Fe domain that have increased affinity for FcRn were isolated after the third-round panning (as described in Section 6) from a library of mutant 30 human IgGl molecules having mutations at residues 308-314 (histidine at position 310 and tryptophan at position 313 are fixed), those isolated after the fifth-round panning of the library for residues 251-256 isoleucinee at position 253 is fixed), those isolated after fourth round panning of the library for residues 428-436 (histidine at position 429, glutamic acid at position 430, alanine at position 431, leucine at position 432, and histidine at position 435 35 are fixed), and those isolated after sixth-round panning of the library for residues 385-389 (glutamic acid at position 388 is fixed) are listed in Table I. The wild type human IgG1 has - 17- WO 02/060919 PCT/USGl/48432 a sequence Val-Leu-His-Gln-Asp-Trp-Leu (SEQ ID NO:86) at positions 308-314, Leu-Met lle-Ser-Arg-Thr (SEQ ID NO:87) at positions 251-256, Met-His-Glu-Ala-Leu-His-Asn-His Tyr (SEQ ID NO:88) at positions 428-436, and Gly-Gin-Pro-Glu-Asn (SEQ ID NO:89) at 5 positions 385-389, Table I iITANTS ISOLATED BY PANNING LIBRARY MUTANTS* 251-256 Leu Tyr Ile Thr Arg Giu (SEQ ID NO:90) 10 Lou yr le Ser Arg Thr (SEQ ID NO:91) Leu 7yr Be Ser Arg Ser (SEQ ID NO:92) Leu Tyr le Ser Arg Arg (SBQ ID NO:93) Leu Tyr Ie Ser Arg Gin (SEQ ID NO:94) 15 Leu Trp le Ser Arg Thr (SEQ ID NO:95) Lou Tyr Ile Ser Leu GIn (SEQ ID NO:96) Leu Phe lie Ser Arg Asp (SEQ ID NO:97) Leu Phe Ile Ser Arg Thr (SEQ ID NO:98) 20 Leu Phe le Ser Arg Arg (SEQ ID NO:99) Leu Phe Ile Thr Gly Ala (SEQ D) NO:100) Leu Ser Ile Ser Arg Glu (SEQ ID NO:101) Arg Thr Ile Ser lie Ser (SEQ ID NO:102) 25 308-314 Thr Pro His Ser Asp Tr Len (SEQ ID NO:;103) Ile Pro His Glu Asp Trp Leu (SEQ ID NO:104) 385-389 Arg Thr Arg Glu Pro (SEQ ID NO: 105) Asp Pro Pro Glu Ser (SEQ ID NO:106) 30 Ser Asp Pro Glu Pro (SEQ ID NO:107) Tbr Ser His Glu Asn (SEQ ID NO: 108) Ser Lys Ser Glu Asn (SEQ ID NO:109) His Arg Ser Giu Asn (SEQ ID NO:1 10) 35 1 Lys lie Arg Glu Asn (SEQ ID NO: Il l) - 18 - WO 02/060919 PCT/US01/48432 LIBRARY I MUTANTS* Gly Ile Thr Gin Ser (SEQ ID NO: 112) 5 Ser Met Ala Glu Pro (SEQ ID NO:113) 428-436 Met His Glu Ala Leu Arg Tyr His His (SEQ ID NO:114) Met His Glu Ala Leu His Phe His His (SEQ ID NO: 115) Met His Glu Ala Leu Lys Phe His His (SEQ ID NO: 116) Met His Giu Ala Leu Ser TyrIlis Arg (SEQ ID NO: 117) 10 TM His Glu Ala Leu His Tyr His Thr (SEQ ID NO:118) Met His Giu Ala Lou His Tyr His Tyr (SEQ ID) NO: 119) * Substituting residues are indicated in bold face The underlined sequences in Table I correspond to sequences that occurred 10 to 20 times in 15 the final round of panning and the sequences in italics correspond to sequences that occurred 2 to 5 times in the final round of panning. Those sequences that are neither underlined nor italicized occurred once in the final round of panning. In one preferred embodiment, the invention provides modified immunoglobulin molecules (e.g., various antibodies) that have increased in vivo half-life 20 and affinity for FeRn relative to umnodified molecules (and, in preferred embodiments, altered bioavailabilty such as increased or decreased transport to mucosal surfaces or other target tissues). Such immunoglobulin molecules include IgG molecules that naturally contain an FeRn binding domain and other non-IgO immunoglobulins (e.g., IgE, IgM, IgD, IgA and IgY) or fragments of immunoglobulins that have been engineered to contain an 25 FcRn-binding fragment (i.e., fusion proteins comprising non-IgG immunoglobulin or a portion thereof and an FoRn binding domain). In both cases the FcRn-binding domain has one or more amino acid modifications that increase the affinity of the constant domain fragment for FRn. The modified immunoglobulins include any immunoglobulin molecule that 30 binds (preferably, immunospecifically, i.e., competes off non-specific binding), as determined by immunoassays well known in the art for assaying specific antigen-antibody binding) an antigen and contains an FcRn-binding fragment. Such antibodies include, but are not limited to, polyclonal, monoclonal, bi-specific, multi-specific, human, humanized, chimeric antibodies, single chain antibodies, Fab fragments, F(ab') 2 fragments, disulfide 35 linked Fvs, and fragments containing either a VL or Vi domain or even a complementary - 19 - WO 02/060919 PCT[USOI/48432 determining region (CDR) that specifically binds an antigen, in certain cases, engineered to contain or fused to an FcRn binding domain. The IgG molecules of the invention, and FoRn-binding fragments thereof, are 5 preferably IgGI subclass of IgGs, but may also be any other IgG subclasses of given animals. For example, in humans, the IgG class includes IgG1, IgG2, IgG3, and IgG4; and mouse IgG includes IgGI, IgG2a, IgG2b, IgG2c and IgG3. It is known that certain IgG subclasses, for example, mouse IgG2b and IgG2c, have higher clearance rates than, for example, IgGI (Medesan et al., Eur. J Immunol., 28:2092-2100, 1998). Thus, when using 10 IgG subclasses other than IgGI, it may be advantageous to substitute one or more of the residues, particularly in the CH2 and CH3 domains, that differ from the IgGI sequence with those of IgG1, thereby increasing the in vivo half-life of the other types of IgG. The immunoglobulins (and other proteins used herein) may be from any animal origin including birds and mammals. Preferably, the antibodies are human, rodent 15 (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken. As used herein, "human" antibodies include antibodies having the amino acid sequence of a human immnnoglobulin and include antibodies isolated from human imnnunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described infra and, for example, in U.S. Patent 20 No. 5,939,598 by Kucherlapati et al. The antibodies of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide or may be specific for heterologous epitopes, such as a heterologous polypeptide or solid support material. See, e.g., PCT publications WO 25 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al.,] Immunol., 147:60 69, 1991; U.S. Patent Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et a., £ Immunol., 148:1547-1553, 1992. The antibodies of the invention include derivatives that are otherwise modified, i.e., by the covalent attachment of any type of molecule to the antibody such that 30 covalent attachment does not prevent the antibody from binding antigen and/or generating an anti-idiotypic response. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of 35 numerous chemical modifications may be carried out by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of -20 - WO 02/060919 PCT/USOI/48432 tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids. Monoclonal antibodies can be prepared using a wide variety of techniques 5 known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow ot al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al, in: Monoclonal Antibodies and T-Cell 10 Hybridomas, pp. 563-681 (Elsevier, N,Y., 1981) (both of which are incorporated herein by reference in their entireties), The term "monoclonal antibody" as used herein is not limited to antibodies produced through hybridoma technology. The term monoclonall antibody" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. 15 Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art. In a non-limiting example, mice can be immunized with an antigen of interest or a cell expressing such an antigen, Once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The 20 splenocytes are then fused by well known techniques to any suitable myclona cells. Hybridomas are selected and cloned by limiting dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding the antigen. Ascites fluid, which generally contains high levels of antibodies, can be generated by inoculating mice intraperitoneally with positive hybridoma clones.' 25 Antibody fragments which recognize specific epitopes may be generated by known techniques. For example, Fab and F(ab') 2 fragments may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab'), fragments). F(ab') 2 fragments contain the complete light chain, and the variable region, the CHI region and the hinge region of the 30 heavy chain, For example, antibodies can also be generated using various phage display methods known in the art In phage display methods, fictional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular embodiment, such phage can be utilized to display antigen 35 binding domains, such as Fab and Fv or disulfide-bond stabilized Fv, expressed from a repertoire or combinatorial antibody library (e.g., human or marine). Phage expressing an -21- WO 02/060919 PCT/US01/48432 antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage, including fd and M13. The 5 antigen binding domains are expressed as a recombinantly fused protein to either the phage gene I or gene VIII protein. Alternatively, the modified FcRn binding portion of immunoglobulins of the present invention can be also expressed in a phage display system. Examples of phage display methods that can be used to make the immunoglobulins, or fragments thereof, of the present invention include those disclosed in Brinknan et al., J 10 Immunol. Methods, 182:41-50, 1995; Ames et al., , Immunol. Methods, 184:177-186, 1995; Kettleborough et al, Eur, J ImmunoL, 24:952-958, 1994; Persic et al., Gene, 187;9 18, 1997; Burton et al., Advances in Immunology, 57:191-280, 1994; PCT application No. POT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/1 1236; WO 95/15982; WO 95/20401; and U.S. Patent Nos, 5,698;426; 15 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety. As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, 20 including human antibodies, or any other desired fragments, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below, For example, techniques to recombinantly produce Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92(22324; Mullinax et al,, BioTechniques, 12(6):864 25 869, 1992; and Sawai et al.,AJJRI, 34:26-34, 1995; and Better et aL,ScIence, 240:1041 1043, 1988 (each of which is incorporated by reference in its entirety). Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S, Patent Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology, 203:46-88, 1991; Shu et al, PNAS, 90:7995-7999, 1993; and Skerra et al., 30 Science, 240:1038-1040, 1988. For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a urine 35 monoclonal antibody and a constant region derived from a human immunoglobulin. Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, - 22 - WO 02/060919 PCTIUS01/48432 Science, 229:1202, 1985; Oi et al., Bio Techniques, 4:214 1986; Gillies et al., J. Immunol. Methods, 125:191-202, 1989; U.S. Patent Nos, 5,807,715; 4,816,567; and 4,816,397, which are incorporated herein by reference in their entireties. Humanized antibodies are antibody 5 molecules from non-human species that bind the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are 10 identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions, See, e.g., Queen et at, U.S. Patent No. 5,585,089; Rieohmann et al., Nature, 332:323, 1988, which are incorporated herein by reference in their entireties. Antibodies can be humanized 15 using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Patent Nos. 5,225,539; 5,530,101 and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology, 28(4/5):489-498, 1991; Studnicka et al., Protein Engineering, 7(6):805-814, 1994; Roguska et al., Proc Natt A cad Sci USA, 91:969-973, 1994), and chain shuffling 20 (U.S. Patent No. 5,565,332), all of which are hereby incorporated by reference in their entireties. Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries 25 derived from human immunoglobulin sequences. See U.S. Patent Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645; WO 98/50433; WO 98/24893; WO 98/16654; WO 96/34096; WO 96/33735; and WO 91/10741, each of which is incorporated herein by reference in its entirety. Human antibodies can also be produced using transgenic mice which are 30 incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. For an overview of this technology for producing human antibodies, see Lonberg and Huszar, nt. Rev. ImmunoL, 13:65-93, 1995. For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT publications WO 35 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European PatentNo. 0 598 877; U.S. Patent Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; -23 - WO O2/060919 PCTIUSQI/48432 5,814,318; 5,885,793; 5,916,771; and 5,939,598, which are incorporated by reference herein in their entireties. In addition, companies such as Abgenix, Inc. (Freemont, CA), Medarex (NJ) and Genpharm (San Jose, CA) can be engaged to provide human antibodies directed 5 against a selected antigen using technology similar to that described above. Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as "guided selection." In this approach a selected non-human monoclonal antibody, e,g, a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al., Bio/technology, 10 12:899-903, 1988). In particular embodiments, the modified antibodies have in vivo therapeutic and/or prophylactic uses. Examples of therapeutic and prophylactic antibodies which may be so modified include, but are not limited to, SYNAGIS" (MedImmune, MD) which is a humanized anti-respiratory syncytial virus (RSV) monoclonal antibody for the treatment of 15 patients with RSV infection; HERCEPTIN (Trastuzumab) (Genentech, CA) which is a humanized anti-HER2 monoclonal antibody for the treatment of patients with metastatic breast cancer; REMICADE* (infliximab) (Centocor, PA) which is a chimeric anti-TNFo monoclonal antibody for the treatment of patients with Crone's disease; REOPRO* (abciximab) (Centocor) which is an anti-glycoprotein lIb/ila receptor on the platelets for 20 the prevention of clot formation; ZENAPAX* (daclizumab) (Roche Pharmaceuticals, Switzerland) which is an immunosuppressive, humanized anti-CD25 monoclonal antibody for the prevention of acute renal allograft rejection. Other examples are a humanized anti CD18 F(ab'), (Genentech); CDP860 which is a humanized anti-CD 18 F(ab') 2 (Celltech, UK); PRO542 which is an anti-HIV gp120 antibody fused with CD4 (Progenics/Genzyme 25 Transgenics); Ostavir which is a human anti Hepatitis B virus antibody (Protein Design Lab/Novartis); PROTOVIRTM which is a humanized anti-CMV IgGI antibody (Protein Design Lab/Novartis); MAK-195 (SEGARD) which is a murine anti-TNF-a F(ab')2 (Knoll Pharma/BASF); 1C14 which is an anti-CD14 antibody aCOS Pharm); a humanized anti VEGF IgGI antibody (Genentech); OVAREXTM which is a marine anti-CA 125 antibody 30 (Altarex); PANOREXTM which is a urine anti-17-IA cell surface antigen IgG2a antibody (Glaxo Wellcome/Centocor); BBC2 which is a marine anti-idiotype (GD3 epitope) IgG antibody (ImClone System); IMC-C225 which is a chimeric anti-EGFR IgG antibody (ImClone System); VITAXINTM which is a humanized anti-aVp3 integrin antibody (Applied Molecular Evolution/Medhnmune); Campath 1H/LDP-03 which is a humanized 35 anti CD52 IgOl antibody (Leukosite); Smart M195 which is a humanized anti-CD33 IgG antibody (Protein Design Lab/Kanebo); RITUXANTm which is a chimeric anti-CD20 IgOl -24- WO 02/060919 PCT/US01/48432 antibody (IDEC Pharm/Genentech, Roche/Zettyaku); LYMPHOCIDETM which is a humanized anti-CD22 IgG antibody (Immunomedics); Smart ID10 which is a humanized anti-HILA antibody (Protein Design Lab); ONCOLYTM (Lym-1) is a radiolabelled murine 5 anti-HLA DIAGNOSTIC REAGENT antibody (Techniclone); ABX-IL8 is a human anti IL8 antibody (Abgenix); anti-CD 1 Ia is a humanized IgGI antibody (Genetech/Xoma); lCM3 is a humanized anti-ICAM3 antibody (ICOS Pharm); IDEC- 114 is a primatied anti CD80 antibody ([DEC Pharm/Mitsubishi); ZEVALINTM is a radiolabelled marine anti CD20 antibody (IDEC/Schering AG); IDEC-131 is a humanized anti-CD40L antibody 10 (IDECIEisai); IDEC-151 is a privatized anti-CD4 antibody (IDEC); IDEC-152 is a primatized anti-CD23 antibody (IDEC/Seikagaku); SMART anti-CD3 is a humanized anti CD3 IgG (Protein Design Lab); 501.1 is a humanized anti-complement factor 5 (C5) antibody (Alexion Pharm); D2E7 is a humanized anti-TNF-a antibody (CAT/BASF); CDP870 is a humanized anti-TNF-a Fab fragment (Celltech); IDEC-151 is a primatized 15 anti-CD4 IgG1 antibody (IDEC Phann/SmithKline Beecham); MDX-CD4 is a human anti CD4 IgG antibody (Medarex/Eisai/Gennab); CDP571 is a humanized anti-TNF-a IgG4 antibody (Celltech); LDP-02 is a humanized anti-a4p7 antibody (LeukoSite/Genentech); OrthoClone OKT4A is a humanized anti-CD4 IgG antibody (Ortho Biotech); ANTOVATM is a humanized anti-CD40L IgG antibody (Biogen); ANTEGRENTM is a humanized anti 20 VLA-4 IgG antibody (Elan); MDX-33 is a human anti-CD64 (FoyR) antibody (Medarex/Centeon); SCH55700 is a humanized anti-IL-5 IgG4 antibody (Celltech/Schering); SB-240563 and SB-240683 are humanized anti-IL-5 and IL-4 antibodies, respectively, (SmithKline Beecham); rhuMab-E25 is a humanized anti-IgE IgG I antibody (Genentech/Norvartis/Tanox Biosystems); IDEC- 152 is a privatized anti-CD23 25 antibody (IDEC Pharm); ABX-CBL is a murine anti CD-147 IgM antibody (Abgenix); BTI 322 is a rat anti-CD2 IgG antibody (Medimmune/Bio Transplant); Orthoclone/O11T3 is a murine anti-CD3 IgG2a antibody (ortho Biotech); SIULECTrM is a chimeric anti-CD25 IgGI antibody (Novartis Pharm); LDP-01 is a humanized anti- 2 -integrin IgO antibody (LeukoSite); Anti-LFA-1 is a marine anti CD1 8 F(ab') 2 (Pasteur-Merieux/Inmunotech); 30 CAT] 52 is a human anti-TGF- 2 antibody (Cambridge Ab Tech); and Corsevin M is a chimeric anti-Factor VII antibody (Centocor). In specific embodiments, the invention provides modified antibodies having one or more of the mutations described herein and that immunospecifically bind RSV, ag, SYNAGIS*. The present invention also provides modified antibodies having one or more 35 of the mutations described herein and that comprise a variable heavy (V) and/or variable light (VL) domain having the amino acid sequence of any VH and/or VL domain listed in -25- WO 02/060919 PCT/US01/48432 Table 1I. The present invention fluther encompasses anti-RSV antibodies comprising one or more VH complementarity determining regions (CDRs) and/or one or more VL CDRs having the amino acid sequence of one or more VH CDRs and/or VL CDRS listed in Table 5 I or one or more of the CDRs listed in Table II wherein one or more of the bolded and underlined residues has an amino acid substitution, preferably that increases the affinity of the antibody for RSV. In specific embodiments, the antibody to be modified is AFFF, p12f2, p12f4, plld4, Alel09, A12a6, Al3c4, A17d4, A4B4, A8C7, IX-493L1FR, H3-3F4, M319, Y1016, DO, AFFF(1), 6H8, LI-7E5, L215B10, A13AIl, AIH5, A4B4(1), 10 A4B4L1FR-S28R, A4B4-F52S. Table H. CDR Sequences of SYNAGIS* CDR Sequence SEQ ID NO: VH1 TSGMSVG 1 15 VH2 DIWWDDKKDYNPSLKS 2 VH3 SMITNYFDV 3 VL1 KCOLSVGYM4 4 VL2 DTSKLAS 5 VL3 FQGSGYPFT 6 20 25 30 35 -26- WO 02/060919 PCT/US01/48432 a 2 £e e2 e 9 2 n n ' ed ! 0 i e R 9 I2 8 C, C Eia o2 a o R 22 2 2 R2 Cs a s, & a -2 ~> Cz 6 ZG 272 P-<a40< C:2 1 3 ,a YC ,~ r. 9o, flz 7 V~ ~ CCCa/ oo ~zz

AA

WO 02/060919 PCT/US01148432 0 0s 6 2 2 a 2' 82 x 4 tO 00' 6 Y CY Ol a a e, , a z" z z zz z z z 7 9 2' 2' 2' 2 2 2' 2 2 2' 22 Z g oo ;R ;2 1 9A ? i ZS 07 0 00 00 I C PA I Al a- Aa g'es 2' 2' a6562' se a e ow I. 0 -28- WO 02/060919 PCT/USO1/48432 aa 2 R R aa g s g- g a a a &' >5.1A 2 -29a WO 02/060919 PCT/USOI/48432 In other embodiments, the antibody is a modified anti-af3 antibody, preferably a Vitaxin antibody (see, PCT publications WO 98/33919 and WO 00/78815, both by Huse et al., and both of which are incorporated by reference herein in their entireties). 5 Modified IgGs of the present invention having longer half-lives than wild type may also include IgGs whose bioactive sites, such as antigen-binding sites, Fc-receptor binding sites, or complement-binding sites, are modified by genetic engineering to increase or reduce such activities compared to the wild type. Modification of these and other therapeutic antibodies to increase the in vivo 10 half-life permits administration of lower effective dosages and/or less frequent dosing of the therapeutic antibody. Such modification to increase in vivo half-life can also be useful to improve diagnostic immunoglobulins as well, for example, permitting administration of lower doses to achieve sufficient diagnostic sensitivity. The present invention also provides fusion proteins comprising a bioactive 15 molecule and an binge-Fe region or a fragment thereof (preferably human) having one or more modifications (i.e., substitutions, deletions, or insertions) in amino acid residues identified to be involved in the interaction between the hinge-Fe region and the FcRn receptor. In particular, the present invention provides fusion proteins comprising a bio active molecule recombinantly fused or chemically conjugated (including both covalent 20 and non-covalent conjugations) to a CH2 domain having one or more modifications in amino acid residues 251-256, 285-290, and/or amino acid residues 308-314, and/or to a C13 domain having one or more modifications in amino acid residues 385-389 and/or 428 436, in particular, one or more of the amino acid substitutions discussed above. The fusion of a bioactive molecule to a constant domain or a fragment thereof with one or more of such 25 modifications increases the in vivo half-life of the bioactive molecule. In a preferred embodiment, fusion proteins of the invention comprise a bioaetive molecule recombinantly fused or chemically conjugated to a CH2 domain having one or more amino acid residue substitutions in amino acid residues 251-256, 285-290, and/or amino acid residues 308-314, and/or to a CH3 domain having one or more 30 modifications in amino acid residues 385-389 and/or 428-436, In certain embodiments, a fusion protein comprises a C-12 domain of IgG molecule in which amino acid residues 253, 310, and 313 are not modified. In another embodiments, a fusion protein comprises a CH3 domain of IgG molecule in which amino acid residues 388,429, 430, 431, 432, and 435 are not modified. 35 A bioactive molecule can be any polypeptide or synthetic drug known to one of skill in the art. Preferably, a bioactive molecule is a polypeptide consisting of at least 5, -30- WO 02/060919 PCTIUSOI/48432 preferably at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acid residues. Examples of bioactive polypeptides include, but are not limited to, various types of antibodies, cytokines (e.g., ILr2, IL-3, IL-4, 5 IL-5, ttr6, 1l-7, IL-10, IL-12, ILA15, IFN-y, IFN-a, and IFN-p), cell adhesion molecules (e.g., CTLA4, CD2, and CD28), ligands (e.g., TNF-a,, TNF-p, and an anti-angiogenic factor such as endostatin), receptors, antibodies and growth factors (e.g, PDOF, EOF, NGF, and KGF). A bioactive molecule can also be a therapeutic moiety such as a cytotoxin 10 (e.g., a cytostatic or cytocidal agent), a therapeutic agent or a radioactive element (e.g, alpha-emitters, gamma-emitters, etc.). Examples of cytostatic or cytocidal agents include, but are not limited to, paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, calchicin, doxorubicin, daunombicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1 15 dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, 20 dibromomannitol, streptozotocin, mitomycin C, and cisdichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g, daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and antbramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine). 25 The present invention also provides polynucleotides comprising a nucleotide sequence encoding a modified IgG of the invention and fragments thereof which contain the modified FcRn binding sites with increased affinity and vectors comprising said polynucleotides. Furthermore, the invention includes polynucleotides that hybridize under stringent or lower stringent hybridization conditions to polynucleotides encoding modified 30 IgGs of the present invention. The nucleotide sequence of modified IgGs and the polynueleotides encoding the same may be obtained by any methods known in the art, including general DNA sequencing method, such as dideoxy chain termination method (Sanger sequencing), and oligonucleotide priming in combination with PCR, respectively. 35 -31- WO 02/060919 PCT/US01/48432 52. IDENTIFICATION OF MUTATIONS WITHIN THE HINGE-FC REGION OF IMMUNOGLOBIULIN MOLECULES 5 One or more modifications in amino acid residues 251-256, 285-290, 308 314, 385-389, and 428-436 of the constant domain maybe introduced utilizing any technique known to those of skill in the art. The constant domain or fragment thereof having one or more modifications in amino acid residues 251-256, 285-290, 308-314, 385 389, and 428-436 may be screened by, for example, a binding assay to identify the constant 10 domain or fragment thereof with increased affinity for the FcRu receptor (e.g, as described in section 5.11, Infra). Those modifications in the hinge-Fe domain or the fragments thereof which increase the affinity of the constant domain or fragment thereof for the FeRn receptor can be introduced into antibodies to increase the in vivo half-lives of said antibodies. Further, those modifications in the constant domain or the fragment thereof 15 which increase the affinity of the constant domain or fragment thereof for the FeRn can be fused to bioactive molecules to increase the in vivo half-lives of said bioactive molecules (and, preferably alter (increase or decrease) the bioavailability of the molecule, for example, to increase or decrease transport to mucosal surfaces (or other target tissue) (e.g., the lungs), 20 5.2.1. MUTAGENESIS Mutagenesis may be performed in accordance with any of the techniques known in the art including, but not limited to, synthesizing an oligonucleotide having one or more modifications within the sequence of the constant domain of an antibody or a fragment thereof (e.g., the CH2 or CH3 domain) to be modified. Site-specific mutagenesis 25 allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Typically, a primer of about 17 to about 75 nucleotides or more in length is preferred, with 30 about 10 to about 25 or more residues on both sides of the junction of the sequence being altered. A number of such primers introducing a variety of different mutations at one or more positions may be used to generated a library of mutants. The technique of site-specific mutagenesis is well known in the art, as exemplified by various publications (see, e.g.,. Kunkel et aL, Methods Enzynol, 35 154:367-82, 1987, which is hereby incorporated by reference in its entirety). In general, site-directed mutagenesis is performed by first obtaining a single-stranded vector or melting -32 - WO 02/060919 PCT/US0I/48432 apart of two strands of a double stranded vector which includes within its sequence a DNA sequence which encodes the desired peptide. An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically. This primer is then annealed with the 5 single-stranded vector, and subjected to DNA polymerizing enzymes such as T7 DNA polymerase, in order to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform or transfect appropriate cells, such as E. coli cells, and clones are selected which 10 include recombinant vectors bearing the mutated sequence arrangement. As will be appreciated, the technique typically employs a phage vector which exists in both a single stranded and double stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage. These phage are readily commercially available and their use is generally well known to those skilled in the art. Double stranded plasmids are 15 also routinely employed in site directed mutagenesis which eliminates the step of transferring the gene of interest from a plasmid to a phage. Alternatively, the use of PCRTM with cormnercially available thermostable enzymes such as Taq DNA polymerase may be used to incorporate a mutagenic oligonucleotide primer into an amplified DNA fragment that can then be cloned into an 20 appropriate cloning or expression vector. See, e.g., Tomic et aL, Nucleic Acids Res., 18(6):1656, 1987, and Upender etaL, Biotechniques, 18(1):29-30, 32, 1995, for PCR mediated mutagenesis procedures, which are hereby incorporated in their entireties. PCRTM employing a thermostable ligase in addition to a thermostable polymerase may also be used to incorporate a phosphorylated mutagenic oligonucleotide into an amplified DNA fragment 25 that may then be cloned into an appropriate cloning or expression vector (see e.g., Michael, Blolechniques, 16(3):410-2, 1994, which is hereby incorporated by reference in its entirety)? Other methods known to those of skill in art of producing sequence variants of the Fo domain of an antibody or a fragment thereof can be used. For example, recombinant vectors encoding the amino acid sequence of the constant domain of an 30 antibody or a fragment thereof may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants. 5.2.2. PANNING Vectors, in particular, phage, expressing constant domains or fragments 35 thereof having one or more modifications in amino acid residues 25 1-256, 285-290, 308-314, 385-389, and/or 428-436 can be screened to identify constant domains or fragments thereof -33 - WO 02/060919 PCTUSOI/48432 having increased affinity for FeRn to select out the highest affinity binders from a population of phage. Immunoassays which can be used to analyze binding of the constant domain or fragment thereof having one or more modifications in amino acid residues 251-256, 285-290, 5 308-314, 385-389, and/or 428-436 to the FcRn include, but are not limited to, radioinimunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, and fluorescent immunoassays. Such assays are routine and well known in the art (see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its 10 entirety). Exemplary immunoassays are described briefly herein below (but are not intended by way of limitation). BlIAcore kinetic analysis can also be used to determine the binding on and off rates of a constant domain or a fragment thereof having one or more modifications in amino acid residues 251-256, 285-290, 308-314, 385-389, and/or 428-436 to the FeRn. BlAcore kinetic analysis comprises analyzing the binding and dissociation of a constant 15 domain or a fragment thereof having one or more modifications in amino acid residues 251 256, 285-290, 308-314, 385-389, and/or 428-436 from chips with immobilized FoRn on their surface (see section 5.1 and the Example section infra), 5.2.3. SEQUENCING 20 Any of a variety of sequencing reactions known in the art can be used to directly sequence the nucleotide sequence encoding constant domains or fragments thereof having one or more modifications in amino acid residues 251-256, 285-290, 308-314, 385 389, and/or 428-436. Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert (Proc. Natl. Acad Sci. USA, 74:560, 1977) or Sanger 25 (Proc. Natt. A cad. Sci, USA, 74:5463, 1977). It is also contemplated that any of a variety of automated sequencing procedures can be utilized (Bio/Techniques, 19:448, 1995), including sequencing by mass spectrometry (see, e.g., PCT Publication No. WO 94/16101, Cohen at al., Adv. Chromatogr., 36:127-162, 1996, and Griffin et al., Appl. Biochem. Biotechnol, 38:147-159, 1993). 30 5.3. RECOMBINANT METHODS OF PRODUCING ANTIBODIES The antibodies of the invention or fragments thereof can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis 35 or preferably, by recombinant expression techniques. - 34 - WO 02/060919 PCT/USOI/48432 The nucleotide sequence encoding an antibody may be obtained from any information available to those of skill in the art (i.e., from Gienbank, the literature, or by routine cloning). If a clone containing a nucleic acid encoding a particular antibody or an 5 epitope-binding fragment thereof is not available, but the sequence of the antibody molecule or epitope-binding fragment thereof is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A* RNA, isolated from any tissue or cells expressing the antibody, such as hybridoma cells selected to express 10 an antibody) by PCR amplification using synthetic primers hybridizable to the 3' and 5 'ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method well known in the art. 15 Once the nucleotide sequence of the antibody is determined, the nucleotide sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al,, 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold 20 Spring Harbor, NY; and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY, which are both incorporated by reference herein in their entireties), to generate antibodies having a different amino acid sequence by, for example, introducing amino acid substitutions, deletions, and/or insertions into the epitope-binding domain regions of the antibodies and preferably, into the hinge-F regions of the antibodies which are 25 involved in the interaction with the FeRn. In a preferred embodiment, antibodies having one or more modifications in amino acid residues 251-256, 285-290, 308-314,385-389, and 428 436 are generated. Recombinant expression of an antibody requires construction of an expression vector containing a nucleotide sequence that encodes the antibody, Once a nucleotide 30 sequence encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably, but not necessarily, containing the heavy or light chain variable region) has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing an antibody encoding 35 nucleotide sequence are described herein, Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and - 3$- WO 02/060919 PCT/USOI/48432 appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The invention, thus, provides replicable vectors comprising a nucleotide 5 sequence encoding the constant region of the antibody molecule with one or more modifications in the amino acid residues involved in the interaction with the FeRn (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Patent No. 5,122,464). The nucleotide sequence encoding the heavy-chain variable region, light-chain variable region, both the heavy-chain and light-chain variable regions, an epitope-binding 10 fragment of the heavy- and/or light-chain variable region, or one or more complementarity determining regions (CDRs) of an antibody may be cloned into such a vector for expression. The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody having an increased affinity for the FoRn and an increased In vivo half-life. Thus, the 15 invention includes host cells containing a polynucleotide encoding an antibody, a constant domain or a FeRn binding fragment thereof having one or more modifications in amino acid residues 251-256, 285-290, 308-314, 385-389, and/or 428-436, preferably, operably linked to a heterologous promoter. A variety of host-expression vector systems may be utilized to express the 20 antibody molecules of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ, These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed 25 with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces and Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression 30 vectors (e.g., cauliflower mosaic virus, CaMV; and tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; and mammalian cell systems (e.g., COS, CHO, BIK, 293, 3T3 and NSO cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (eg., metallothionein promoter) or from mammalian 35 viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). Preferably, bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for -36- WO 02/060919 PCT/US01/48432 the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene 5 promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., Gene, 45:101, 1986, and Cockett et aL, Bio/Technology, 8:2, 1990). In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For 10 example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et at., EMBO, 12:1791, 1983), in which the antibody coding sequence may be ligated individually 15 into the vector in frame with the lacZ coding region so that a fusion protein is produced; and plN vectors (Inouye & Inouye, Nucleic Acids Res,, 13:3101-3109, 1985 and Van Heeke & Schuster, J. Biol. Chem, 24:5503-5509, 1989). In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera 20 frugiperda cells. The antibody coding sequence may be cloned individually into non essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter). In mammalian host cells, a number of viral-based expression systems may be utilized to express an antibody molecule of the invention. In cases where an adenovirus is 25 used as an expression vector, the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the 30 antibody molecule in infected hosts (e.g., see Logan & Shenk, Proc. Nat!. Acad. Sci. USA, 81:355-359, 1984). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These 35 exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of -37 - WO 02/060919 PCT/US01/48432 appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g,, Bitter et al, Methods in Enzymolt, 153:516-544, 1987). In addition, a host cell strain may be chosen which modulates the expression 5 of the antibody sequences, or modifies and processes the antibody in the specific fashion desired, Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the antibody, Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure 10 the correct modification and processing of the antibody expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, HeLa, COS, MDCK, 293, 3T3, W138, and in particular, myeloma cells such as NSO cells, and related cell 15 lines, see, for example, Morrison et al., U.S. Patent No. 5,807,715, which is hereby incorporated by reference in its entirety. For long-term, high-yield production of recombinant antibodies, stable expression is preferred, For example, cell lines which stably express the antibody molecule may be engineered. Rather than using expression vectors which contain viral origins of 20 replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant 25 plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the antibody molecule. Such engineered cell lines may be particularly useful in screening and evaluation of compositions that interact directly or indirectly with the antibody molecule. 30 A number of selection systems may be used, including but not limited to, the herpes simplex virus thymidine kinase (Wigler et al, Cell, 11:223, 1977), hypoxanthine guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Nat, Acad Sc. USA, 48:202, 1992), and adenine phosphoribosyltransferase (Lowy et al, Cell, 22:8-17, 1980) genes can be employed in tir, hgprt or aprr cells, respectively. Also, antimetabolite 35 resistance can be used as the basis of selection for the following genes; dhfr, which confers resistance to methotrexate (Wigler et al, Natl Acad. Sci, USA, 77:357, 1980 and O'Hare et -38- WO 02/060919 PCT/US01/48432 al., Proc. Nat?. Acad Sci. USA, 78:1527, 1981); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Nat. Acad. Scl. USA, 78:2072, 1981); neo, which confers resistance to the aminoglycoside G-418 (Wu and Wu, Biotherapy, 3:87-95, 1991; Tolstoshev, Ann Rev, Pharmacol. Toxicol., 32:573-596, 1993; Mulligan, Science, 260:926-932, 1993; and Morgan and Anderson, Ann, Rev. Biochem., 62: 191-217, 1993; and May, TIB TECH, 11 (5):155-2 15, 1993); and hygro, which confers resistance to hygromycin (Santerre et al., Gene, 30:147, 1984). Methods commonly known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such 10 methods are described, for example, in Ausubel et al (eds.), 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; in Chapters 12 and 13, Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY; and Colberre-Garapin et al., J. Mol. Biol,, 150:1, 1981, which are incorporated by reference herein in their 15 entireties. The expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and -entschel, 1987, The use ofvectors based on gene amplificarionfor the expression of cloned genes in mammalian cells in DNA cloning, Vol.3. Academic Press, New York). When a marker in the vector system expressing 20 antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et aL, Mot., Cell. Rio?., 3:257, 1983). The host cell may be co-transfected with two expression vectors of the 25 invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides or different selectable markers to ensure maintenance of both plasmids. Alternatively, a single vector may be used which encodes, and is capable of expressing, both heavy and light chain 30 polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature, 322:52, 1986; and Kohler, Proc. Natl Acad. Sci. USA, 77:2 197, 1980). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA. Once an antibody molecule of the invention has been produced by 35 recombinant expression, it may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, - 39- WO 02/060919 PCT/US01/48432 affinity, particularly by affinity for the specific antigen after Protein A purification, and sizing column chromatography), centrifugation, differential solubility, or by any other standard techniques for the purification of proteins. Further, the antibodies of the present 5 invention or fragments thereof may be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification. 5.3.1. ANTIBODY CONJUGATES The present invention encompasses antibodies recombinantly fused or 10 chemically conjugated (including both covalently and non-covalently conjugations) to heterologous polypeptides (i.e., an unrelated polypeptide; or portion thereof, preferably at least 10, at least 20, at least 30, at least 40, at least 5O, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids of the polypeptide) to generate fusion proteins. The fusion does not necessarily need to be direct, but may occur through linker sequences. 15 Antibodies fused or conjugated to heterologous polypeptides may also be used in in vitro immunoassays and purification methods using methods known in the art. See e.g, PCT Publication No. WO 93/21232; El 439,095; Naramura et al., Immunol, Lett., 39:91-99, 1994; U.S. Patent 5,474,981; Gillies et al., PNAS, 89:1428-1432, 1992; and Fell et al, J Immunol., 146:2446-2452, 1991, which are incorporated herein by reference in their 20 entireties. Antibodies can be fused to marker sequences, such as a peptide to facilitate purification. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of which are commercially available. As 25 described in Gentz et al, Proc. Nati. Acad Sc. USA, 86;821-824, 1989, for instance, hexa histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et aL, Cell, 37:767 1984) and the "flag" tag (Knappik et al, Biotechniques, 17(4):754-761, 1994). 30 The present invention also encompasses antibodies conjugated to a diagnostic or therapeutic agent or any other molecule for which in vivo half-life is desired to be increased. The antibodies can be used diagnostically to, for example, monitor the development or progression of a disease, disorder or infection as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. Detection can be 35 facilitated by coupling the antibody to a detectable substance, Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent - 40- WO 02/060919 PCT/US01/4843Z materials, bioluminescent materials, radioactive materials, positron emitting metals, and nonradioactive paramagnetic metal ions. The detectable substance may be coupled or conjugated either directly to the antibody or indirectly, through an intermediate (such as, for 5 example, a linker known in the art) using techniques known in the art. See, for example, U.S. Patent No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention, Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, p-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and 10 avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dieblorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include 1251, 131, "n1n or ""'Tc. 15 An antibody may be conjugated to a therapeutic moiety such as a cytotoxin (e.g., a cytostatic or cytocidal agent), a therapeutic agent or a radioactive element (e.g., alpha emitters, gamma-emitters, etc.). Cytotoxins or cytotoxic agents include any agent that is detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinbiastine, colchicin, 20 doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithiamycin, actinomycin D, I -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa 25 chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphanide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cisdichlorodiaminc platinum (11) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (eg., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and 30 vinblastine). Further, an antibody may be conjugated to a therapeutic agent or drug moiety that modifies a given biological response. Therapeutic agents or drug moieties are not to be construed as limited to classical chemical therapeutic agents, For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may 35 include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, a-interferon (IFN-a), p-interferon (IFN-p), -41- WO 02/060919 PCT/US01/48432 nerve growth factor (NGF), platelet derived growth factor (PDF), tissue plasminogen activator (TPA), an apoptotic agent (e.g., TNF-u, TNF-p, AIM I as disclosed in PCT Publication No. WO 97/33899), AIM II (see, PCT Publication No. WO 97/34911), Pas Ligand (Takahashi et al., J Iminunol, 6:1567-1574, 1994), and VEGI (PCT Publication No. WO 99123105), a thrombotic agent or an anti-angiogenic agent (e.g., angiostatin or endostatin); or a biological response modifier such as, for example, a lymphokine (e.g., interleukin-1 ("IL- 1"), interleukin-2 ("%-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), and granulocyte colony stimulating 10 factor ("G-CSF")), or a growth factor (e.g., growth hormone ("GHD). Techniques for conjugating such therapeutic moieties to antibodies are well known; see, e.g., Amon et al., "Monoclonal Antibodies For Imunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), 1985, pp. 243-56, Alan R. Liss, Inc.); Hellstrom el al., "Antibodies For Drug Delivery", in 15 Controlled Drug Deliveiy (2nd Ed.), Robinson et al. (eds.), 1987, pp. 623-53, Marcel Dekker, Inc. ); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), 1985, pp. 475-506); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies 20 For Caner Detection And Therapy, Baldwin et al. (eds.),1985, pp. 303-16, Academic Press; and Thorpe et al., Imnuno. Recombinant expression vector., 62:119-58, 1982. An antibody or fragment thereof, with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factor(s) and/or cytokine(s) can be used as a therapeutic. 25 Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety, Antibodies may also be attached to solid supports, which are particularly useful for imnmunoassays or purification of the target antigen. Such solid supports include, 30 but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. 5.4 1THODS OF PRODUCING FUSION PROTEINS Fusion proteins can be produced by standard recombinant DNA techniques or 35 by protein synthetic techniques, e.g., by use of a peptide synthesizer, For example, a nucleic acid molecule encoding a fusion protein can be synthesized by conventional techniques -42- WO 02/060919 PCT/USOI/48432 including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and 5 reamplified to generate a chimeric gene sequence (see, ag, Current Protocols in Molecular Biology, Ausubel et at., eds., John Wiley & Sons, 1992). Moreover, a nucleic acid encoding a bioactive molecule can be cloned into an expression vector containing the Fo domain or a fragment thereof such that the bioactive molecule is linked in-frame to the constant domain or fragment thereof. 10 Methods for fusing or conjugating polypeptides to the constant regions of antibodies are known in the art. See, e.g., U.S. Patent Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,723,125, 5,783,181, 5,908,626, 5,844,095, and 5,112,946; EP 307,434; EP 367,166; EP 394,827; PCT publications WO 91/06570, WO 96/04388, WO 96/22024, WO 97134631, and WO 99/04813; Ashkenazi et al, Proc. Nai Acad. Sc. USA, 15 88: 10535-10539, 1991; Traunecker etal., Nature, 331:84-86, 1988; Zheng et al., J Immunol, 154:5590-5600, 1995; and Vil et al., Proc. Natl, Acad Sci. USA, 89:11337 11341, 1992, which are incorporated herein by reference in their entireties. The nucleotide sequence encoding a bioactive molecule may be obtained from any information available to those of skill in the art (e.g., from Genbank, the literature, or by 20 routine cloning), and the nucleotide sequence encoding a constant domain or a fragment thereof with increased affinity for the FeRn may be determined by sequence analysis of mutants produced using techniques described herein, or may be obtained from Genbank or the literature, The nucleotide sequence coding for a fusion protein can be inserted into an appropriate expression vector, i.e,, a vector which contains the necessary elements for the 25 transcription and translation of the inserted protein-coding sequence. A variety of host-vector systems may be utilized in the present invention to express the protein-coding sequence. These include but are not limited to mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); microorganisms such as yeast containing yeast vectors; or bacteria transformed 30 with bacteriophage, DNA, plasmid DNA, or cosmid DNA. The expression elements of vectors vary in their strengths and specificities. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used, The expression of a fusion protein may be controlled by any promoter or enhancer element known in the art. Promoters which may be used to control the expression 35 of the gene encoding fusion protein include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, Nature, 290:304-310, 1981), the promoter contained in the 3' -43 - WO 02/060919 PCT/USOI/48432 long terminal repeat of Rous sarcoma virus (Yamamoto, et al, Cell, 22:787-797, 1980), the herpes thymidine kinase promoter (Wagner et al, Proc. Nat. Acad. Sci. US.A., 78:1441-1445, 1981), the regulatory sequences of the metallothionein gene (Brinster et al, 5 Nature, 296:39-42, 1982), the tetracycline (Tet) promoter (Gossen et al, Proc. Nat. Acad. Sci. USA, 89:5547-5551, 1995); prokaryotic expression vectors such as the P--lactamase promoter (Villa-Kamaroff, et a4, Proc. Natl. Acad, Sci. USA., 75:3727-3731, 1978), or the tac promoter (DeBoer, et al, Proc. Nail. A cad. Set. U.S.A., 80:21-25, 1983; see also "Useful proteins from recombinant bacteria" in Scientific American, 242:74-94, 1980); plant 10 expression vectors comprising the nopaline synthetase promoter region (Herrera-Estella et al, Nature, 303:209-213, 1983) or the cauliflower mosaic virus 35S RNA promoter (Gardner, et al., NucL Acids Res,, 9:2871, 1981), and the promoter of the photosynthetic enzyme ribulose biphosphate carboxylase (Herrera-Estrella et at, Nature, 310:115-120, 1984); promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC 15 (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter, and the following animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift et al., Cell 3 8:639-646, 1984; Ornitz et al, 50:399-409, Cold Spring Harbor Symp. Quant. Biol., 1986; MacDonald, Hepatology 20 7:425-515, 1987); insulin gene control region which is active in pancreatic beta cells (Hanahan, Nature 315:115-122, 1985), immunoglobulin gene control region which is active in lymphoid cells (Grosschedl et al., Cell, 38:647-658, 1984; Adames et at, Nature 318:533-538, 1985; Alexander et al, Mol Cell Biol., 7:1436-1444, 1987), mouse mammnary tumor virus control region which is active in testicular, breast, lymphoid and mast cells 25 (Leder et al, Cell, 45:485-495, 1986), albumin gene control region which is active in liver (Pinkrt et al, Genes and Devel,, 1:268-276, 1987), a-fetoprotein gene control region which is active in liver (Krumlauf et al, Mol. Cell. Blot., 5:1639-1648, 1985; Hanuner et at, Science, 235:53-58, 1987; z. 1-antitrypsin gene control region which is active in the liver (Kelsey et al, Genes and Devel., 1:161-171, 1987), beta-globin gene control region which is 30 active in myeloid cells (Mogram et al., Nature, 315:338-340, 1985; Kollias et al, Cell, 46:89-94, 1986; myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead et at, Cell, 48:703-712, 1987); myosin light chain-2 gene control region which is active in skeletal muscle (Sani, Nature, 314:283-286, 1985); neuronal-specific enolase (NSE) which is active in neuronal cells (Morelli et al, Gen. Virol, 35 80:571-83, 1999); brain-derived neurotrophic factor (BDNF) gene control region which is active in neuronal cells (Tabuchi et al, Blochem Blophysic. Res. Comprising., 253:818-823, - 44 - WO 02/060919 PCT/US01/4843Z 1998); glial fibrillary acidic protein (GFAP) promoter which is active in astrocytes (Gomes et al,, Braz. J. Med. Biol. Res., 32(5):619-631, 1999; Morelli et al, Gen Virol, 80:571-83, 1999) and gonadotropic releasing hormone gene control region which is active in the 5 hypothalamus (Mason et at., Science, 234:1372-1378, 1986). In a specific embodiment the expression of a fusion protein is regulated by a constitutive promoter. In another embodiment, the expression of a fusion protein is regulated by an inducible promoter, In accordance with these embodiments, the promoter may be a tissue-specific promoter. 10 In a specific embodiment, a vector is used that comprises a promoter operably linked to a fusion protein-encoding nucleic acid, one or more origins of replication, and, optionally, one or more selectable markers (e.g., an antibiotic resistance gene). In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the fusion protein 15 coding sequence may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or In vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts ( e.g., 20 see Logan & Shenk, Proc, Natl, Acad. Sc. USA, 81:355-359, 1984), Specific initiation signals may also be required for efficient translation of inserted fusion protein coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational 25 control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bitter er al., Methods in Enzymol., 153:516-544, 1987). Expression vectors containing inserts of a gene encoding a fusion protein can 30 be identified by three general approaches: (a) nucleic acid hybridization, (b) presence or absence of "marker" gene functions, and (c) expression of inserted sequences. In the first approach, the presence of a gene encoding a fusion protein in an expression vector can be detected by nucleic acid hybridization using probes comprising sequences that are homologous to an inserted gene encoding the fusion protein. In the second approach, the 35 recombinant vector/host system can be identified and selected based upon the presence or absence of certain "marker" gene functions (e.g., thymidine kinase activity, resistance to - 45 - WO 02/060919 PCT/USO1/48432 antibiotics, transformation phenotype, occlusion body formation in baculovirus, etc.) caused by the insertion of a nucleotide sequence encoding a fusion protein in the vector. For example, if the nucleotide sequence encoding the fusion protein is inserted within the marker 5 gene sequence of the vector, recombinants containing the gene encoding the fusion protein insert can be identified by the absence of the marker gene function. In the third approach, recombinant expression vectors can be identified by assaying the gene product (L.e., fusion protein) expressed by the recombinant, Such assays can be based, for example, on the physical or functional properties of the fusion protein in in vitro assay systems, e.g., binding 0 with anti-bioactive molecule antibody. In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers; thus, expression of the genetically engineered fusion protein may be controlled. 15 Furthermore, different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification (e.g., glycosylation, phosphorylation of proteins), Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed. For example, expression in a bacterial system will produce an unglycosylated product and expression in 20 yeast will produce a glycosylated product. Eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used, Such mammalian host cells include but are not limited to CHO, VERY, BHK, HeLa, COS, MDCK, 293, 3T3, W138, and in particular, neuronal cell lines such as, for example, SK-N-AS, SK-N-Fl, SK-N-DZ human neuroblastomas (Sugimoto 25 et a., J Nat. Cancer Inst, 73: 51-57, 1984), SK-N-SIH human neuroblastoma (Biochim. Biophys. Acta, 704: 450-460, 1982), Daoy human cerebellar medulloblastoma (He et al., Cancer Res,, 52: 1144-1148, 1992) DBTRG-05MG glioblastoma cells (Kruse et al, 1992, In Vitro Cell, Dev, Biot, 28A:609-614, 1992), IMR-32 human neuroblastoma (Cancer Res., 30: 2110-2118, 1970), 1321NI human astrocytoma (Proc. Natl Acad ScL USA, 74: 4816, 1997), 30 MOG-G-CCM human astrocytoma (Br, J. Cancer, 49: 269, 1984), U87MG human glioblastoma-astrocytoma (A cra Pathol. Microbiol. Scand., 74: 465-486, 1968), A172 human glioblastoma (Olopade et a., Cancer Res., 52: 2523-2529, 1992), C6 rat glioma cells (Benda et al., Science, 161: 370-371, 1968), Neuro-2a mouse neuroblastoma (Proc. Nat. Acad Sci. USA, 65: 129-136, 1970), NB41A3 mouse neuroblastoma (Proc. Nal, Acad Sci. USA, 48: 35 1184-1190, 1962), SCP sheep choroid plexus (Bolin et al., J Virol Methods, 48: 211-221, 1994), G355-5, PG-4 Cat normal astrocyte (Haapala et at, J. Virol, 53: 827-833, 1985), Mpf - 46 - WO 02/060919 PCG/US01/48432 ferret brain (Trowbridge er at, In Vitro, 18: 952-960, 1982), and normal cell lines such as, for example, CTX TNA2 rat normal cortex brain (Radany et al, Proc. NatL Acad Sco. USA, 89: 6467-6471, 1992) such as, for example, CRL7030 and Hs578Bst Furthermore, different 5 vector/host expression systems may effect processing reactions to different degrees. For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the fusion protein may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression 10 control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched medium, and then are switched to a selective medium. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid 15 into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines that express the differentially expressed or pathway gene protein. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that affect the endogenous activity of the differentially expressed or pathway gene protein. 20 A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler, et al, Cell, 11:223, 1997), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalsld, Proc. NatL. Acad. Sci. USA, 48:2026, 1962), and adenine phosphoribosyltransferase (Lowy, et al., 1980, Cell, 22:817, 1980) genes can be employed in tk-, hgprt- or aprt- cells, respectively. Also, 25 antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler, et al., Natl. Acad Sci. USA, 77:3567, 1980; O'Hare, el al., *Proc. Natl. Acad Sci, USA, 78:1527, 1981); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Nat. Acad Set USA, 78:2072, 1981); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, et at, J. Mot. Biot, 150:1, 1981); 30 and hygro, which confers resistance to hygromycin (Santerre, et al., Gene, 30:147, 1984) genes. Once a fusion protein of the invention has been produced by recombinant expression, it may be purified by any method known in the art for purification of a protein, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the 35 specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. - 47 - WO 02/060919 PCT/USO1/48432 5.5. PROPHYLACTIC AND THERAPEUTIC USES OF ANTIBODIES The present invention encompasses antibody-based therapies which involve 5 administering antibodies to an animal, preferably a mammal, and most preferably a human, for preventing, treating, or ameliorating symptoms associated with a disease, disorder, or infection, Prophylactic and therapeutic compounds of the invention include, but are not limited to, antibodies and nucleic acids encoding antibodies. Antibodies may be provided in pharmaceutically acceptable compositions as known in the art or as described herein. 10 Antibodies of the present invention that function as antagonists of a disease, disorder, or infection can be administered to an animal, preferably a mammal and most preferably a human, to treat prevent or ameliorate one or more symptoms associated with the disease, disorder, or infection. For example, antibodies which disrupt or prevent the interaction between a viral antigen and its host cell receptor may be administered to an 15 animal, preferably a mammal and most preferably a human, to treat, prevent or ameliorate one or more symptoms associated with a viral infection. In a specific embodiment, an antibody or fragment thereof prevents a viral or bacterial antigen from binding to its host cell receptor by at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at 20 least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least 10% relative to antigen binding to its host cell receptor in the absence of said antibodies. In another embodiment, a combination of antibodies prevent a viral or bacterial antigen from binding to its host cell receptor by at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, 25 at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least 10% relative to antigen binding to its host cell receptor in the absence of said antibodies. In a preferred embodiment, the antibody is used to treat or prevent RSV infection Antibodies which do not prevent a viral or bacterial antigen from binding its host cell receptor but inhibit or downregulate viral or bacterial replication can also be 30 administered to an animal to treat, prevent or ameliorate one or more symptoms associated with a viral or bacterial infection. The ability of an antibody to inhibit or downregulate viral or bacterial replication may be determined by techniques described herein or otherwise known in the art. For example, the inhibition or downregulation of viral replication can be determined by detecting the viral titer in the animal. 35 In a specific embodiment, an antibody inhibits or downregulates viral or bacterial replication by at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at - 48 - WO 021060919 PCr[US01/48432 least 75%, at least 70%, at least 60%, at least 50%, at least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least 10% relative to viral or bacterial replication in absence of said antibody. In another embodiment, a combination of 5 antibodies inhibit or downregulate viral or bacterial replication by at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least 10% relative to viral or bacterial replication in absence of said antibodies. 10 Antibodies can also be used to prevent inhibit or reduce the growth or metastasis of cancerous cells. In a specific embodiment, an antibody inhibits or reduces the growth or metastasis of cancerous cells by at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least 10% 15 relative to the growth or metastasis in absence of said antibody. In another embodiment, a combination of antibodies inhibits or reduces the growth or metastasis of cancer by at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least 10% relative to the growth or metastasis in absence of 20 said antibodies, Examples of cancers include, but are not limited to, leukemia (e.g., acute leukemia such as acute lymphocytic leukemia and acute myelocytic leukemia), neoplasms, tumors (eg., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelio sarcoma, synovioma, mesothelioma, Ewing's tumor, Icionyosarcoma, 25 rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal 30 carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymorna, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma), heavy chain disease, metastases, or any disease or disorder characterized by uncontrolled cell 35 growth. -49- WO 02/060919 PCTIUSUI/48432 Antibodies can also be used to reduce the inflammation experienced by animals, particularly mammals, with inflannatory disorders. In a specific embodiment, an antibody reduces the inflammation in an animal by at least 99%, at least 95%, at least 90%, at 5 least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least 10% relative to the inflammation in an animal in the not administered said antibody. In another embodiment, a combination of antibodies reduce the inflammation in an animal by at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, 10 at least 60%, at least 50%, at least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least 10% relative to the inflammation in an animal in not administered said antibodies. Examples of inflammatory disorders include, but are not limited to, rheumatoid arthritis, spondyloarthropathies, inflammatory bowel disease and asthma. 15 In certain embodiments, the antibody used for treatment of inflammation (or cancer) is a modified anti-a3 3 antibody, preferably a Vitaxin antibody (see, PCT publications WO 98/3 3919 and WO 00/78815, both by Huse et al., and both of which are incorporated by reference herein in their entireties). Antibodies can also be used to prevent the rejection of transplants. 20 Antibodies can also be used to prevent clot formation, Further, antibodies that function as agonists of the inuune response can also be administered to an animal, preferably a mammal, and most preferably a human, to treat, prevent or ameliorate one or more symptoms associated with the disease, disorder, or infection, One or more antibodies that immunospecifically bind to one or more antigens 25 may be used locally or systemically in the body as a therapeutic. The antibodies of this invention may also be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (such as, e.g., IL 2, IL-3 and 1L-7), which, for example, serve to increase the number or activity of effector cells which interact with the antibodies, The antibodies of this invention may also be 30 advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (such as, e.g,, IL-2, IL-3 and IL-7), which, for example, serve to increase the immune response. The antibodies of this invention may also be advantageously utilized in combination with one or more drugs used to treat a disease, disorder, or infection such as, for example anti-cancer agents, anti-inflammatory 35 agents or anti-viral agents. Examples of anti-cancer agents include, but are not limited to, isplatin, ifosfamide, paclitaxel, taxanes, topoisomerase I inhibitors (e.g., CPT-1 1, topotecan, -50- WO 02/060919 PCTIUSOI/48432 9-AC, and GG-21 1), gemeitabine, vinorelbine, oxaliplatin, 5-fluorouracil (5-FU), leucovorin, vinorelbine, temodal, and taxol. Examples of anti-viral agents include, but are not limited to, cytokines (e.g., IFN-a, IFN-P, IFN-y), inhibitors of reverse transcriptase (e.g., AZT, 3TC, 5 D4T, ddC, ddl, d4T, 3TC, adefovir, efavirenz, delavirdine, nevirapine, abacavir, and other dideoxynucleosides or dideoxyfluoronucleosides), inhibitors of viral mRNA capping, such as ribavirin, inhibitors of proteases such IV protease inhibitors (e.g., amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir,), amphotericin B, castanospermine as an inhibitor of glycoprotein processing, inhibitors of neuraninidase such as influenza virus neuraminidase 10 inhibitors (e.g., zananivir and oseltanivir), topoisomerase I inhibitors (e.g., camptothecins and analogs thereof), amantadine, and rimantadine. Examples of anti-inflammatory agents include, but are not limited to, nonsteroidal anti-inflammatory drugs such as COX-2 inhibitors (e.g., meloxicam, celecoxib, rofecoxib, flosulide, and SC-58635, and MK-966), ibuprofen and indomethacin, and steroids (e.g., deflazacort, dexamethasone and 15 methylprednisolone). In a specific embodiment, antibodies administered to an animal are of a species origin or species reactivity that is the same species as that of the animal. Thus, in a preferred embodiment, human or humanized antibodies, or nucleic acids encoding human or human, are administered to a human patient for therapy or prophylaxis. 20 In preferred embodiments, inmunoglobulins having extended In vivo half lives are used in passive immunotherapy (for either therapy or prophylaxis). Because of the extended half-life, passive immunotherapy or prophylaxis can be accomplished using lower doses and/or less frequent administration of the therapeutic resulting in fewer side effects, better patient compliance, less costly therapy/prophylaxis, etc. In a preferred embodiment, 25 the therapeutic/prophylactic is an antibody that binds RSV, for example, SYNAGIS* or other anti-RSV antibody. Such anti-RSV antibodies, and methods of administration are disclosed in U.S. patent application Serial Nos. 09/724,396 and 09/724,531, both entitled "Methods of Administering/Dosing Anti-RSV Antibodies For Prophylaxis and Treatment," both by Young et at., both filed November 28, 2000, and continuation-in-part applications of these 30 applications, Serial Nos. _ and -, respectively (attomey docket Nos. 10271-048 and 10271-047, respectively), also entitled "Methods of Administering/Dosing Anti-RSV Antibodies for Prophylaxis and Treatment," by Young et al., all which are incorporated by reference herein in their entireties. Also included are the anti-RSV antibodies described in Section 5.1, supra. 35 In a specific embodiment, fusion proteins administered to an animal are of a species origin or species reactivity that is the same species as'that of the animal. Thus, in a -51- WO 02/060919 PCT/US01/48432 preferred embodiment, human fusion proteins or nucleic acids encoding human fusion proteins, are administered to a human subject for therapy or prophylaxis. 5 5.6. PROPHYLACTIC AND THERAPEUTIC USES OF FUSION PROTEINS AND CONJUGATED MOLECULES The present invention encompasses fusion protein-based and conjugated molecule-based therapies which involve administering fusion proteins or conjugated 10 molecules to an animal, preferably a mammal and most preferably a human, for preventing, treating, or ameliorating symptoms associated with a disease, disorder, or infection. Prophylactic and therapeutic compounds of the invention include, but are not limited to, fusion proteins and nucleic acids encoding fusion proteins and conjugated molecules. Fusion proteins and conjugated molecules may be provided in pharmaceutically acceptable 15 compositions as known in the art or as described herein. Fusion proteins and conjugated molecules of the present invention that function as antagonists of a disease, disorder, or infection can be administered to an animal, preferably a mammal, and most preferably a human, to treat, prevent or ameliorate one or more symptoms associated with the disease, disorder, or infection. Further, fusion proteins 20 and conjugated molecules of the present invention that function as agonists of the immune response may be administered to an animal, preferably a mammal, and most preferably a human, to treat, prevent or ameliorate one or more symptoms associated with the disease, disorder, or infection. One or more fusion proteins and conjugated molecules may be used locally or 25 systemically in the body as a therapeutic. The fusion proteins and conjugated molecules of this invention may also be advantageously utilized in combination with monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (such as, e.g., IL 2, IL-3 and IL-7), which, for example, serve to increase the number or activity of effector cells which interact with the antibodies. The fusion proteins and conjugated molecules of 30 this invention may also be advantageously utilized in combination with monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (such as, e.g, IL 2, lL-3 and 1L-7), which, for example, serve to increase the immune response. The fusion proteins and conjugated molecules of this invention may also be advantageously utilized in combination with one or more drugs used to treat a disease, disorder, or infection such as, for 35 example anti-cancer agents, anti-inflammatory agents or anti-viral agents. Examples of anti cancer agents include, but are not limited to, isplatin, ifosfanide, paclitaxel, taxanes, - 52- WO 02/060919 PCTUS01/4843Z topoisomerase I inhibitors (e.g,, CPT-11, topotecan, 9-AC, and GG-211), gemcitabine, vinorelbine, oxaliplatin, 5-fluorouracil (5-FU), leucovorin, vinorelbine, temodal, and taxol. Examples of anti-viral agents include, but are not limited to, cytokines (e.g., IFN-a, IFN-p, 5 IFN-y), inhibitors of reverse transcriptase (e.g,, AZT, 3TC, D4T, ddC, ddl, d4T, 3TC, adefovir, efavirenz, delavirdine, nevirapine, abacavir, and other dideoxynucleosides or dideoxyfluoronucleosides), inhibitors of viral mRNA capping, such as ribavirin, inhibitors of proteases such HIV protease inhibitors (e.g., amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir,), amphotericin B, castanospermine as an inhibitor of glycoprotein processing, 10 inhibitors of neuraminidase such as influenza virus neuraminidase inhibitors (e.g., zanamivir and oseltamivir), topoisomerase I inhibitors (e.g,, camptothecins and analogs thereof), amantadine, and rimartadine, Examples of anti-inflammatory agents include, but are not listed to, nonsteroidal anti-inflammatory drugs such as COX-2 inhibitors (e.g., meloxicam, celecoxib, rofecoxib, flosulide, and SC-58635, and MK-966), ibuprofen and indomethacin, 15 and steroids (e.g., deflazacort, dexamethasone and methylprednisolone). 5.7. ADMINISTRATION OF ANTIBODIES OR FUSION PROTEINS 20 The invention provides methods of treatment, prophylaxis, and amelioration of one or more symptoms associated with a disease, disorder or infection by administrating to a subject of an effective amount of an antibody of the invention, or pharmaceutical composition comprising an antibody of the invention. The invention also provides methods of treatment, prophylaxis, and amelioration of one or more symptoms associated with a 25 disease, disorder or infection by administering to a subject an effective amount of a fusion protein or conjugated molecule of the invention, or a pharmaceutical composition comprising a fusion protein or conjugated molecules of the invention. In a preferred aspect, an antibody or fusion protein or conjugated molecule, is substantially purified (i.e., substantially free from substances that limit its effect or produce undesired side-effects). In a specific 30 embodiment, the subject is an animal, preferably a mammal such as non-primate (e.g., cows, pigs, horses, cats, dogs, rats etc.) and a primate (e.g., monkey such as a cynomolgous monkey and a human). In a preferred embodiment, the subject is a human. Various delivery systems are known and can be used to administer an antibody or fusion protein or conjugated molecule of the invention, e.g, encapsulation in 35 liposomes, microparticles, microcapsules, recombinant cells capable of expressing the -53- WO 02/060919 PCIUSO1/48432 antibody or fusion protein, receptor-mediated endocytosis (see, eg., Wu and Wu, J. Biol. Chem, 262:4429-4432, 1987), construction of a nucleic acid as part of a retroviral or other vector, etc. Methods of administering an antibody, a fusion protein or conjugated molecule, 5 or pharmaceutical composition include, but are not limited to, parenteral administration (e.g., intradenral, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural, and mucosal (e.g., intranasal and oral routes). In a specific embodiment, antibodies, fusion proteins, conjugated molecules, or pharmaceutical compositions are administered intramuscularly, intravenously, or subcutaneously. The compositions may be administered 10 by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. See, e.g., U.S. Patent 15 Nos. 6,019,968; 5,985, 320; 5,985,309; 5,934,272; 5,874,064; 5,855,913; 5,290,540; and 4,880,078; and PCT Publication Nos, WO 92/19244; WO 97/32572; WO 97/44013; WO 98/31346; and WO 99/66903, each of which is incorporated herein by reference in its entirety. In a preferred embodiment, an antibody, a fusion protein, conjugated molecules, or a pharmaceutical composition is administered using Alkermes AMTM pulmonary drug 20 delivery technology (Alkermes, Inc., Cambridge, MA). The invention also provides that an antibody, a fusion protein, or conjugated molecule is packaged in a hermetically sealed container such as an ampoule or sachette indicating the quantity of antibody, fusion protein, or conjugated molecule. In one embodiment, the antibody, fusion protein, or conjugated molecule is supplied as a dry 25 sterilized lyophilized powder or water free concentrate in a hermetically sealed container and can be reconstituted, e.g., with water or saline to the appropriate concentration for administration to a subject, Preferably, the antibody, fusion protein, or conjugated molecule is supplied as a dry sterile lyophilized powder in a hermetically sealed container at a unit dosage of at least 5 mg, more preferably at least 10 mg, at least 15 mg, at least 25 mg, at least 30 35 mg, at least 45 mg, at least 50 mg, or at least 75 mg. The lyophilized antibody, fusion protein, or conjugated molecule should be stored at between 2 and 8"C in its original container and the antibody, fusion protein, or conjugated molecules should be administered within 12 hours, preferably within 6 hours, within 5 hours, within 3 hours, or within I hour after being reconstituted. In an alternative embodiment, an antibody, fusion protein, or 35 conjugated molecule is supplied in liquid form in a hermetically sealed container indicating the quantity and concentration of the antibody, fusion protein, or conjugated molecule. -54- WO 02/060919 PCTIUS01/48432 Preferably, the liquid form of the antibody, fusion protein, or conjugated molecule is supplied in a hermetically sealed container at least I mg/ml, more preferably at least 2.5 mg/ml, at least 5 mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least 15 mg/kg, or at least 25 mg/ml. 5 In a specific embodiment, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion, by injection, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Preferably, when I0 administering an antibody or a fusion protein, care must be taken to use materials to which the antibody or the fusion protein does not absorb. In another embodiment, the composition can be delivered in a vesicle, in particular a liposome (see Langer, Science, 249:1527-1533, 1990; Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, 15 New York, pp. 353- 365 (1989); Lopez-Berestein, ibid., pp. 3 17-327; see generally ibid.). In yet another embodiment, the composition can be delivered in a controlled release or sustained release system. Any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or more antibodies, or one or more fusion proteins. See, e.g., U.S. Patent No. 4,526,938; PCT publication WO 91/05548; 20 PCT publication WO 96/20698; Ning et al., "Intratumoral Radioirmunotheraphy of a Human Colon Cancer Xenograft Using a Sustained-Release Gel," Radiotherapy & Oncology, 39:179-189, 1996; Song et al, "Antibody Mediated Lung Targeting of Long-Circulating Emulsions," PDA Journal ofPharmaceutical Science & Technology, 50:372-397, 1995; Cleek et al., "Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular 25 Application," Pro. Intl. Symp. Control Rel Bioact, Mater., 24:853-854, 1997; and Lam et al., "Microencapsulation of Recombinant Humanized Monoclonal Antibody for Local Delivery," Proc. Int'7 Symp. Control Rel Bioact. Mater., 24:759-760, 1997, each of which is incorporated herein by reference in its entirety. In one embodiment, a pump may be used in a controlled release system (see Langer, supra; Sefton, CRC Crit. Ref Biomed Eng., 14:20, 30 1987; Buchwald et al., Surgery, 88:507, 1980; and Saudek et al., N. Engl. J. Med, 321:574, 1989). In another embodiment, polymeric materials can be used to achieve controlled release of antibodies or fusion proteins (see e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bio availability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New 35 York (1984); Ranger and Peppas, J, Macronol, Sci, Rev. Macromol. Chem., 23:61, 1983; see also Levy et al, Science, 228:190, 1985; During et aL, Ann, Neurol., 25:351, 1989; - 55 - WO 02/060919 PCT[USO1/48432 Howard et al., J Neurosurg., 7 1:105, 1989); U.S. Patent No. 5,679,377; U.S. Patent No. 5,916,597; U.S. PatenitNo. 5,912,015; U.S. PatentNo. 5,989,463; U.S. PatentNo, 5,128,326; PCT Publication No. WO 99/15154; and PCT Publication No, WO 99/20253). In yet 5 another embodiment, a controlled release system can be placed in proximity of the therapeutic target (e.g., the lungs), thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)), Other controlled release systems are discussed in the review by Langer, 10 Science, 249:1527-1533, 1990). In a specific embodiment where the composition of the invention is a nucleic acid encoding an antibody or fusion protein, the nucleic acid can be administered in vivo to promote expression of its encoded antibody or fusion protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes 15 intracellular, e.g., by use of a retroviral vector (see U.S. Patent No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (see e.g, Joliot et al., Proc. Natl. Acad. Sci. USA, 88:1864-1868, 1991), etc, Alternatively, a nucleic acid can 20 be introduced intracellularly and incorporated within host cell DNA for expression by homologous recombination. The present invention also provides pharmaceutical compositions. Such compositions comprise a prophylactically or therapeutically effective amount of an antibody, fusion protein or conjugated molecule, and a pharmaceutically acceptable carrier, In a 25 specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharnacopeia or other generally recognized pharnacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant (e.g., Freund's complete and incomplete, mineral gels such as aluminum hydroxide, surface active substances such as 30 lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful adjuvants for humans such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum), excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as 35 peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and - 56 - WO 02/060919 PCT/US01/48432 aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, .5 talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral formulation can include standard carriers such as pharmaceutical grades of I imannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions will contain a prophylactically or therapeutically effective amount of the antibody or fragment thereof, or fusion protein or conjugated molecule, preferably in purified form, together with a suitable 15 amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration. In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in 20 sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection, Generally, the ingredients of compositions of the invention are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or 25 water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to 30 administration. The compositions of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric 35 hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc. - 57 - WO 02/060919 PCT/US01/4843Z The amount of the composition of the invention which will be effective in the treatment, prevention or amelioration of one or more symptoms associated with a disease, disorder, or infection can be determined by standard clinical techniques. The precise dose to 5 be employed in the formulation will depend on the route of administration, the age of the subject, and the seriousness of the disease, disorder, or infection, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model (e.g., the cotton rat or Cynomolgous monkey) test systems. 10 For fusion proteins, the therapeutically or prophylactically effective dosage administered to a subject ranges from about 0.001 to 50 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight and even more preferably about I to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. For antibodies, the therapeutically or prophylactically effective dosage 15 administered to a subject is typically 0.1 mg/kg to 200 mg/kg of the subject's body weight. Preferably, the dosage administered to a subject is between 0.1 mg/kg and 20 mg/kg of the subject's body weight and more preferably the dosage administered to a subject is between 1 mg/kg to 10 mg/kg of the subject's body weight. The dosage will, however, depend upon the extent to which the in vivo half-life of the molecule has been increased Generally, human 20 antibodies and human fusion proteins have longer half-lives within the human body than antibodies of fusion proteins from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies or human fusion proteins and less frequent administration is often possible. Further, the dosage and frequency of administration of antibodies, fusion proteins, or conjugated molecules may be reduced also 25 by enhancing uptake and tissue penetration (e.g., into the lung) of the antibodies or fusion proteins by modifications such as, for example, lipidation. Treatment of a subject with a therapeutically or prophylactically effective amount of an antibody, fusion protein, or conjugated molecule can include a single treatment or, preferably, can include a series of treatments. In a preferred example, a subject is treated 30 with an antibody, fusion protein, or conjugated molecule in the range of between about 0.1 to 30 mg/kg body weight, one time per week for between about I to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. In other embodiments, the pharmaceutical composition of the invention is administered once a day, twice a day, or three times a day. In other 35 embodiments, the pharmaceutical composition is administered once a week, twice a week, once every two weeks, once a month, once every six weeks, once every two months, twice a -58- WO 02/060919 PCT/U801/48432 year or once per year. It will also be appreciated that the effective dosage of the antibody, fusion protein, or conjugated molecule used for treatment may increase or decrease over the course of a particular treatment. 5 5.7. GENE THERAPY In a specific embodiment, nucleic acids comprising sequences encoding antibodies or fusion proteins, are administered to treat, prevent or ameliorate one or more symptoms associated with a disease, disorder, or infection, by way of gene therapy. Gene 10 therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid. In this embodiment of the invention, the nucleic acids produce their encoded antibody or fusion protein that mediates a therapeutic or prophylactic effect. Any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below. 15 For general reviews of the methods of gene therapy, see Goldspiel et a., Clinical Pharmacy, 12:488-505, 1993; Wu and Wu, Biotherapy, 3:87-95, 1991; Tolstoshev, Ann. Rev, Pharmacol. Toxicol, 32:573-596, 1993; Mulligan, Science, 260:926-932, 1993; and Morgan and Anderson, Ann. Rev. biochem. 62:191-217, 1993; TIBTECH 1i(5):155-215, 1993. Methods commonly known in the art of recombinant DNA technology which can be 20 used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990). In a preferred aspect, a composition of the invention comprises nucleic acids encoding an antibody, said nucleic acids being part of an expression vector that expresses the 25 antibody in a suitable host, In particular, such nucleic acids have promoters, preferably heterologous promoters, operably linked to the antibody coding region, said promoter being inducible or constitutive, and, optionally, tissue-specific. In another particular embodiment, nucleic acid molecules are used in which the antibody coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a 30 desired site in the genome, thus providing for intrachromosomal expression of the antibody encoding nucleic acids (Koller and Smithies, Proc. NatL. Acad Sci. USA, 86:8932-8935, 1989; and Zijlstra et al,, Nature, 342:435-438, 1989). In another preferred aspect, a composition of the invention comprises nucleic acids encoding a fusion protein, said nucleic acids being a part of an expression vector that 35 expression the fusion protein in a suitable host, In particular, such nucleic acids have promoters, preferably heterologous promoters, operably linked to the coding region of a -59- WO 02/060919 PCT/USOI/48432 fusion protein, said promoter being inducible or constitutive, and optionally, tissue-specific. In another particular embodiment, nucleic acid molecules are used in which the coding sequence of the fusion protein and any other desired sequences are flanked by regions that 5 promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the fusion protein encoding nucleic acids. Delivery of the nucleic acids into a subject may be either direct, in which case the subject is directly exposed to the nucleic acid or nucleic acid-carrying vectors, or indirect, in which case, cells are first transformed with the nucleic acids in vitro, then transplanted 10 into the subject. These two approaches are known, respectively, as in vivo or ex vivo gene therapy. In a specific embodiment, the nucleic acid sequences are directly administered in vivo, where it is expressed to produce the encoded product. This can be accomplished by any of numerous methods known in the art, e.g., by constructing them as part of an 15 appropriate nucleic acid expression vector and administering it so that they become intracellular, e.g., by infection using defective or attenuated retroviral or other viral vectors (see U.S. Patent No. 4,980,286), or by direct injection of naked DNA, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or 20 microcapsules, or by administering them in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu and Wu, J Bio. Chem., 262:4429-4432, 1987) (which can be used to target cell types specifically expressing the receptors), etc. In another embodiment, nucleic acid ligand complexes can be formed in which the ligand comprises a fusogenic viral peptide to 25 disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180; WO 92/2263 5; WO 92/20316; WO 93/14188; WO 93/20221). Alternatively, the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by 30 homologous recombination (Koller and Smithies, Proc. Natl. Acad. Sci. USA, 86:8932-8935, 1989; and Zijlstra et al, Nature, 342:435-438, 1989). In a specific embodiment, viral vectors that contain nucleic acid sequences encoding an antibody or a fusion protein are used, For example, a retroviral vector can be used (see Miller et al, Meth, Enzymol., 217:581-599, 1993). These retroviral vectors contain 35 the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding the antibody or a fusion protein to .60 - WO 02/060919 PCTUSOI/48432 be used in gene therapy are cloned into one or more vectors, which facilitates delivery of the nucleotide sequence into a subject. More detail about retroviral vectors can be found in Boesen et al., Biotherapy, 6:291-302, 1994, which describes the use of a retroviral vector to 5 deliver the mdr 1 gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et at, J Clin. Invest., 93:644-651, 1994; Klein et aL, Blood 83:1467 1473, 1994; Salmons and Gunzberg, Human Gene Therapy, 4:129-141, 1993; and Grossman and Wilson, Curr, Opin. in Genetics and DeveL, 3:110-114, 1993. 10 Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting 15 non-dividing cells. Kozarsky and Wilson, Current Opinion In Genetics and Development, 3:499-5 03, 1993, present a review of adenovirus-based gene therapy. Bout et a, Human Gene Therapy, 5:3-10, 1994, demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et at, Science, 252:431-434, 1991; Rosenfeld et at, 20 Cell, 68:143-155, 1992; Mastrangeli et al, J. Clin. Invest, 91:225-234, 1993; PCT Publication WO 94/12649; and Wang et al, Gene Therapy, 2:775-783, 1995. In a preferred embodiment, adenovirus vectors are used, Adeno-associated virus (AAV) has also been proposed for use in gene therapy (see, eg.,Walsh et al, Proc. Soc. Exp, BioL Med., 204:289-300, 1993, and U.S. Patent No. 25 5,436,146). Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. Usually, the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells 30 that have taken up and are expressing the transferred gene. Those cells are then delivered to a subject, In this embodiment, the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell, Such introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, 35 microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcellmediated gene transfer, -61- WO 02/060919 PCTIUSUI/48432 spheroplast fusion, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells (see, e.g., Loeffler and Behr, Meth Enzymol., 217:599-618, 1993; Cohen et al, Meth. Enzymol., 217:618-644, 1993; and Clin. Pharma Ther,, 29:69-92, 1985) 5 and may be used in accordance with the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not disrupted. The technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny. 10 The resulting recombinant cells can be delivered to a subject by various methods known in the art, Recombinant blood cells (e.g., hematopoictic stem or progenitor cells) are preferably administered intravenously. The amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art. 15 Cells into which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, cosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoictic 20 stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc. In a preferred embodiment, the cell used for gene therapy is autologous to the subject. In an embodiment in which recombinant cells are used in gene therapy, 25 nucleic acid sequences encoding an antibody or a fusion protein are introduced into the cells such that they are expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect. In a specific embodiment, stem or progenitor cells are used. Any stem and/or progenitor cells which can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the present invention 30 (see e.g., PCT Publication WO 94/08598; Stemple and Anderson, Cell, 7 1:973-985, 1992; Rheinwald, Merh Cell Blo., 21A:229, 1980; and Pittelkow and Scott, Mayo Clinic Proc, 61:771, 1986). In a specific embodiment, the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding region, such that 35 expression of the nucleic acid is controllable by controlling the presence or absence of the appropriate inducer of transcription. -62- WO 02/060919 PCT/USOI/48432 5.8. CHARACTERIZATION AND DEMONSTRATION OF THERAPEUTIC OR PROPHYLACTIC UTILITY Antibodies, fusion proteins, and conjugated molecules of the present 5 invention may be characterized in a variety of ways, In particular, antibodies of the invention may be assayed for the ability to immunospecifically bind to an antigen. Such an assay may be performed in solution (e.g., Houghten, Bo/Techniques, 13:412-421, 1992), on beads (Lam, Nature, 354:82-84, 1991, on chips (Fodor, Nature, 364:555-556, 1993), on bacteria (U.S. Patent No, 5,223,409), on spores (U.S. Patent Nos. 5,571,698; 5,403,484; and 10 5,223,409), on plasmids (Cull et aL, Proc. Natt. Acad. Set USA, 89:1865-1869, 1992) or on phage (Scott and Smith, Science, 249:386-390, 1990; Devlin, Science, 249:404-406, 1990; Cwirla et al, Proc. Nal. Acad. Sci. USA, 87:6378-6382, 1990; and Felici, J. Mo. Biot, 222:301-310, 1991) (each of these references is incorporated herein in its entirety by reference). Antibodies that have been identified to immunospecifically bind to an antigen or 15 a fragment thereof can then be assayed for their specificity affinity for the antigen. The antibodies of the invention or fragments thereof may be assayed for immunospecific binding to an antigen and cross-reactivity with other antigens by any method known in the art. Iumunoassays which can be used to analyze imnunospecific binding and cross-reactivity include, but axe not limited to, competitive and non-competitive assay 20 systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent inmunoassays, protein A immunoassays, to name but a few. Such assays are routine and well known in the art (see, 25 e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety). Exemplary inaaunoassays are described briefly below (but are not intended by way of limitation), Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 30 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., I to 4 hours) at 40 0 C, adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 40"C, washing the beads in lysis buffer and 35 resuspending the beads in SDS/saniple buffer, The ability of the antibody of interest to -63 - WO 021060919 PCT/USI/48432 immunoprecipitate a particular antigen can be assessed by, e,g., western blot analysis. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (e.g., pre 5 clearing the cell lysate with sepharose beads). For further discussion regarding immunoprecipitation protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol 1, John Wiley & Sons, Inc., New York at 10.16.1. Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%- 20% SDS-PAGE 10 depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in 15 washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g. 3 2 P or MI) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen. One of skill in the art would be knowledgeable as to the parameters that can 20 be modified to increase the signal detected and to reduce the background noise. For further discussion regarding western blot protocols see, eg., Ausubel ct al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1. ELISAs comprise preparing antigen, coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound 25 such as an enzymatic substrate (e.g, horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen. In ELISAs the antibody of interest does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes the antibody of interest) conjugated to a detectable compound may be added to the well. Further, instead of coating the well with the 30 antigen, the antibody may be coated to the well, In this case, a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art. For further discussion regarding ELISAs see, e.g., Ausubel et al, eds, 1994, Current 35 Protocols in Molecular Biology, Vol, , John Wiley& Sons, Inc., New York at 11,2.!L - 64 - WO 02/060919 PCT/USO1/48432 The binding affinity of an antibody to an antigen and the off-rate of an antibody-antigen interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled 5 antigen (e.g., 'H or lf1) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen. The affinity of the antibody of the present invention or a fragment thereof for the antigen and the binding off-rates can be determined from the saturation data by scatchard analysis. Competition with a second antibody can also be determined using radioimunoaasays. In 10 this case, the antigen is incubated with an antibody of the present invention or a fragment thereof conjugated to a labeled compound (e.g,, 'H or 1I) in the presence of increasing amounts of an unlabeled second antibody. In a preferred embodiment BlAcore kinetic analysis is used to determine the binding on and off rates of antibodies to an antigen. BlAcore kinetic analysis comprises 15 analyzing the binding and dissociation of an antigen from chips with immobilized antibodies on their surface (see the Example section infra). The antibodies of the invention as well as fusion proteins and conjugated molecules can also be assayed for their ability to inhibit the binding of an antigen to its host cell receptor using techniques known to those of skill in the art. For example, cells 20 expressing the receptor for a viral antigen can be contacted with virus in the presence or absence of an antibody and the ability of the antibody to inhibit viral antigen's binding can measured by, for example, flow cytometry or a scintillation counter, The antigen or the antibody can be labeled with a detectable compound such as a radioactive label (e.g., "P, S, and I5) or a fluorescent label (e.g., fluorescein isothiocyanate, rhodamine, 25 phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine) to enable detection of an interaction between the antigen and its host cell receptor. Alternatively, the ability of antibodies to inhibit an antigen from binding to its receptor can be determined in cell-free assays. For example, virus or a viral antigen (e.g., RSV F glycoprotein) can be contacted in a cell-free assay with an antibody and the ability of the antibody to inhibit the 30 virus or the viral antigen from binding to its host cell receptor can be determined. Preferably, the antibody is immobilized on a solid support and the antigen is labeled with a detectable compound. Alternatively, the antigen is immobilized on a solid support and the antibody is labeled with a detectable compound. The antigen may be partially or completely purified (e.g., partially or completely free of other polypeptides) or part of a cell lysate. Further, the 35 antigen may be a fusion protein comprising the viral antigen and a domain such as glutathionine-S-transferase. Alternatively, an antigen can be biotinylated using techniques - 65 - WO 02/060919 PCT/US01/48432 well known to those of skill in the art (e.g, biotinylation kit, Pierce Chemicals; Rockford, IL). The antibodies, fusion proteins, and conjugated molecules of the invention S can also be assayed for their ability to inhibit or dowuregulate viral or bacterial replication using techniques known to those of skill in the art. For example, viral replication can be assayed by a plaque assay such as described, eag., by Johnson et at, Journal ofInfectious Diseases, 176:1215-1224, 1997. The antibodies, fusion proteins, and conjugated molecules of the invention of the invention can also be assayed for their ability to inhibit or 10 downregulate the expression of viral or bacterial polypeptides. Techniques known to those of skill in the art, including, but not limited to, Western blot analysis, Northern blot analysis, and RT-PCR, can be used to measure the expression of viral or bacterial polypeptides. Further, the antibodies, fusion proteins, and conjugated molecules of the invention of the invention can be assayed for their ability to prevent the formation of syncytia, 15 The antibodies, fusion proteins, conjugated molecules, and compositions of the invention are preferably tested in vitro, and then In vivo for the desired therapeutic or prophylactic activity, prior to use in humans. For example, in vitro assays which can be used to determine whether administration of a specific antibody, a specific fusion protein, a specific conjugated molecule, or a composition of the present invention is indicated, include 20 in vitro cell culture assays in which a subject tissue sample is grown in culture, and exposed to or otherwise administered an antibody, a fusion protein, conjugated molecule, or composition of the present invention, and the effect of such an antibody, a fusion protein, conjugated molecule, or a composition of the present invention upon the tissue sample is observed. In various specific embodiments, in vitro assays can be carried out with 25 representative cells of cell types involved in a disease or disorder, to determine if an antibody, a fusion protein, conjugated molecule, or composition of the present invention has a desired effect upon such cell types. Preferably, the antibodies, the fusion proteins, the conjugated molecules, or compositions of the invention are also tested in in vitro assays and animal model systems prior to administration to humans, 30 Antibodies, fusion proteins, conjugated molecules, or compositions of the present invention for use in therapy can be tested for their toxicity in suitable animal model systems, including but not limited to rats, mice, cows, monkeys, and rabbits. For In vivo testing for the toxicity of an antibody, a fusion protein, a conjugated molecule, or a composition, any animal model system known in the art may be used. 35 Efficacy in treating or preventing viral infection may be demonstrated by detecting the ability of an antibody, a fusion protein, a conjugated molecule, or a composition -66- WO 02/060919 PCT/USO1/48432 of the invention to inhibit the replication of the virus, to inhibit transmission or prevent the virus from establishing itself in its host, or to prevent, ameliorate or alleviate one or more symptoms associated with viral infection. The treatment is considered therapeutic if there is, 5 for exatnple, a reduction is viral load, amelioration of one or more symptoms or a decrease in mortality and/or morbidity following administration of an antibody, a fusion protein, a conjugated molecule, or a composition of the invention. Antibodies, fusion proteins, conjugated molecules, or compositions of the invention can also be tested for their ability to inhibit viral replication or reduce viral load in in vitro and in vivo assays. 10 Efficacy in treating or preventing bacterial infection may be demonstrated by detecting the ability of an antibody, a fusion protein or a composition of the invention to inhibit the bacterial replication, or to prevent, ameliorate or alleviate one or more symptoms associated with bacterial infection. The treatment is considered therapeutic if there is, for example, a reduction is bacterial numbers, amelioration of one or more symptoms or a 15 decrease in mortality and/or morbidity following administration of an antibody, a fusion protein or a composition of the invention. Efficacy in treating cancer may be demonstrated by detecting the ability of an antibody, a fusion protein, a conjugated molecule, or a composition of the invention to inhibit or reduce the growth or metastasis of cancerous cells or to ameliorate or alleviate one 20 or more symptoms associated with cancer, The treatment is considered therapeutic if there is, for example, a reduction in the growth or metastasis of cancerous cells, amelioration of one or more symptoms associated with cancer, or a decrease in mortality and/or morbidity following administration of an antibody, a fusion protein, a conjugated molecule, or a composition of the invention. Antibodies, fusion proteins or compositions of the invention 25 can be tested for their ability to reduce tumor formation in in vitro, ex vivo, and in vivo assays. Efficacy in treating inflammatory disorders may be demonstrated by detecting the ability of an antibody, a fusion protein, a conjugated molecule, or a composition of the invention to reduce or inhibit the inflammation in an animal or to ameliorate or alleviate one 30 or more symptoms associated with an inflammatory disorder, The treatment is considered therapeutic if there is, for example, a reduction is in inflammation or amelioration of one or more symptoms following administration of an antibody, a fusion proteins, a conjugated molecule, or a composition of the invention. Antibodies, fusion proteins, conjugated molecules, or compositions of the 35 invention can be tested in vitro and in vivo for the ability to induce the expression of cytokines (e.g., IFN-a, IFN-p, IFN-y, 1L-2, IL-3, IL-4, IL-5, II-6, IL10, IL-12, and I1t5) and - 67 - WO 02/060919 PCT/USOI48432 activation markers (e.g., CD28, ICOS, and SLAM). Techniques known to those of skill in the art can be used to measure the level of expression of cytokines and activation markers. For example, the level of expression of cytokines can be measured by analyzing the level of 5 RNA of cytokines by, for example, RT-PCR and Northern blot analysis, and by analyzing the level of cytokines by, for example, immunoprecipitation followed by Western blot analysis or ELISA. Antibodies, fusion proteins, conjugated molecules, or compositions of the invention can be tested in vitro and in vivo for their ability to modulate the biological activity 10 of immune cells, preferably human immune cells (e.g, T-cells, B-cells, and Natural Killer cells). The ability of an antibody, a fusion protein, a conjugated molecule, or a composition of the invention to modulate the biological activity of immune cells can be assessed by detecting the expression of antigens, detecting the proliferation of immune cells, detecting the activation of signaling molecules, detecting the effector function of immune cells, or 15 detecting the differentiation of immune cells. Techniques known to those of skill in the art can be used for measuring these activities. For example, cellular proliferation can be assayed by 3 H-thymidine incorporation assays and trypan blue cell counts, Antigen expression can be assayed, for example, by immunoassays including, but are not limited to, competitive and non-competitive assay systems using techniques such as Western blots, 20 immunohistochemistry, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometrie assays, fluorescent immunoassays, protein A imumnoassays and FACS analysis. The activation of signaling molecules can be assayed, for example, by 25 kinase assays and electrophoretic shift assays (EMSAs). Antibodies, fusion proteins, conjugated molecules, or compositions of the invention can also be tested for their ability to increase the survival period of animals, preferably mammals and most preferably humans, suffering from a disease, disorder, or infection by at least 25%, preferably at least 50%, at least 60%, at least 75%, at least 85%, at 30 least 95%, or at least 99%. Further, antibodies, fusion proteins, conjugated molecules, or compositions of the invention can be tested for their ability reduce the hospitalization period of animals, preferably mammals and most preferably humans, suffering from a disease, disorder, or infection by at least 60%, preferably at least 75%, at least 85%, at least 95%, or at least 99%. Techniques known to those of skill in the art can be used to analyze the 35 function of the antibodies or compositions of the invention in vivo, - 68.- WO 02/060919 PCT/US01/48432 5.9. DIAGNOSTIC USES OF ANTIBODIES AND FUSION PROTEINS Labeled antibodies, fusion proteins, and conjugated molecules of the 5 invention can be used for diagnostic purposes to detect, diagnose, or monitor diseases, disorders or infections. The invention provides for the detection or diagnosis of a disease, disorder or infection, comprising: (a) assaying the expression of an antigen in cells or a tissue sample of a subject using one or more antibodies that immunospecifically bind to the antigen; and (b) comparing the level of the antigen with a control level, e.g., levels in normal 10 tissue samples, whereby an increase in the assayed level of antigen compared to the control level of the antigen is indicative of the disease, disorder or infection, The invention also provides for the detection or diagnosis of a disease, disorder or infection, comprising (a) assaying the expression of an antigen in cells or a tissue sample of a subject using one or fusion proteins or conjugated molecules of the invention that bind to the antigen; and (b) 15 comparing the level of the antigen with a control level, e.g,, levels in normal tissue samples, whereby an increase of antigen compared to the control level of the antigen is indicative of the disease, disorder or infection. Accordingly, the fusion protein or conjugated molecule comprises a bioactive molecule such as a ligand, cytokine or growth factor and the hinge-Fo region or fragments thereof, wherein the fusion protein or conjugated molecule is capable of 20 binding to an antigen being detected. Antibodies of the invention can be used to assay antigen levels in a biological sample using classical immunohistological methods as described herein or as known to those of skill in the art (eg., see Jalkanen et al, J Cell. Blot, 101:976-985, 1985; Jalkanen et al., . Cell. Biol., 105:3087-3096, 1987). Other antibody-based methods useful for detecting 25 protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, alkaline phosphatase, glucose oxidase; radioisotopes, such as iodine (.251, 133 ), carbon ("C), sulfur ("S), tritium (l), indium ( m and technetium ( 9 "'Tc); luminescent labels, such as luminol; and fluorescent labels, such as 30 fluorescein and rhodamine, Fusion proteins can be used to assay antigen levels in a biological sample using, for example, SDS-PAGE and immunoassays known to those of skill in the art. One aspect of the invention is the detection and diagnosis of a disease, disorder, or infection in a human. In one embodiment, diagnosis comprises: a) administering 35 (for example, parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a labeled antibody that immunospecifically binds to an antigen; b) waiting for a - 69 - WO 02/060919 PCT/USO1/48432 time interval following the administration for permitting the labeled antibody to preferentially concentrate at sites in the subject where the antigen is expressed (and for unbound labeled molecule to be cleared to background level); c) determining background 5 level; and d) detecting the labeled antibody in the subject, such that detection of labeled antibody above the background level indicates that the subject has the disease, disorder, or infection, In accordance with this embodiment, the antibody is labeled with an imaging moiety which is detectable using an imaging system known to one of skill in the art. Background level can be determined by various methods including, comparing the amount of 10 labeled molecule detected to a standard value previously determined for a particular system. In another embodiment, diagnosis comprises: a) administering (for example, parenterally, subcutaneously, or intraperitoncally) to a subject an effective amount of a labeled fusion protein or conjugated molecule that binds to an antigen or some other molecule; b) waiting for a time interval following the administration for permitting the 15 labeled fusion protein or conjugated molecule to preferentially concentrate at sites in the subject where the antigen or other molecule is expressed (and for unbound labeled molecule to be cleared to background level); c) determining background level; and d) detecting the labeled fusion protein or conjugated molecule in the subject, such that detection of labeled fusion protein above the background level indicates that the subject has the disease, disorder, 20 or infection, In accordance with this embodiment, the fusion protein or conjugated molecule comprises a bioactive molecule such as a ligand, cytokine or growth factor and a hinge-Fc region or a fragment thereof, wherein said fusion protein or conjugated molecule is labeled with an imaging moiety and is capable of binding to the antigen being detected. It will be understood in the art that the size of the subject and the imaging 25 system used will determine the quantity of imaging moiety needed to produce diagnostic images, In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 9 "Tc. The labeled antibody will then preferentially accumulate at the location of cells which contain the specific protein, In vivo tumor imaging is described in S.W. Burchiel et al., 30 "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments," Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982). Depending on several variables, including the type of label used and the mode of administration, the time interval following the administration for permitting the labeled 35 molecule to preferentially concentrate at sites in the subject and for unbound labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. -70- WO 02/060919 PCT/USU1/48432 In another embodiment the time interval following administration is 5 to 20 days or 5 to 10 days. In one embodiment, monitoring of a disease, disorder or infection is carried 5 out by repeating the method for diagnosing the disease, disorder or infection, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc. Presence of the labeled molecule can be detected in the subject using methods known in the art for in vivo scanning. These methods depend upon the type of label used. 10 Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods of the invention include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography. In a specific embodiment, the molecule is labeled with a radioisotope and is 15 detected in the patient using a radiation responsive surgical instrument (Thurston et al,, U.S. Patent No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive scanning instrument. In another embodiment, the molecule is labeled with a positron emitting metal and is detected in the patient using positron emission-tomography. In yet another embodiment, the 20 molecule is labeled with a paramagnetic label and is detected in a patient using magnetic resonance imaging (MRI). 5.10. KITS The invention also provides a pharmaceutical pack or kit comprising one or 25 more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. 30 The present invention provides kits that can be used in the above methods. In one embodiment, a kit comprises an antibody, fusion protein, or conjugated molecule, of the invention, preferably in a purified form, in one or more containers. In a specific embodiment, the kits of the present invention contain a substantially isolated antigen as a control. Preferably, the kits of the present invention further comprise a control antibody, 35 fusion protein, or conjugated molecule which does not react with the antigen included in the kit, In another specific embodiment, the kits of the present invention contain a means for -71- WO 02/060919 PCT/US0/48432 detecting the binding of an antibody, fusion protein, or conjugated molecule, to an antigen (e.g., the antibody, fusion protein, or conjugated molecule, may be conjugated to a detectable substrate such as a fluorescent compound, an enzymatic substrate, a radioactive compound or 5 a luminescent compound, or a second antibody which recognizes the first antibody may be conjugated to a detectable substrate), In specific embodiments, the kit may include a recombinantly produced or chemically synthesized antigen. The antigen provided in the kit may also be attached to a solid support, In a more specific embodiment the detecting means of the above-described kit includes a solid support to which antigen is attached. Such a kit 10 may also include a non-attached reporter-labeled anti-human antibody, In this embodiment, binding of the antibody to the antigen can be detected by binding of the said reporter-labeled antibody. 5.11 IN VITRO AND IN VIVO ASSAYS FOR EXTENDED HALF-LIFE OF MODIFIED lGG HINGE-FC 15 FRAGMENTS The binding ability of modified IgGs and molecules comprising an IgG constant domain of FcRn fragment thereof to FoRn can be characterized by various in vitro assays. PCT publication WO 97/34631 by Ward discloses various methods in detail and is incorporated herein in its entirety by reference. 20 For example, in order to compare the ability of the modified IgG or fragments thereof to bind to FcRn with that of the wild type IgG, the modified IgG or fragments thereof and the wild type IgG can be radio-labeled and reacted with FcRn-expressing cells in vitro, The radioactivity of the cell-bound fractions can be then counted and compared. The cells expressing FcRn to be used for this assay are preferably endothelial cell lines including 25 mouse pulmonary capillary endothelial cells (B10, D2.PCE) derived from lungs of B10,DBA/2 mice and SV40 transformed endothelial cells (SVEC) (Kim et al, J Immunol., 40:457-465, 1994) derived from C3H/HeJ mice. However, other types of cells, such as intestinal brush borders isolated from 10- to 14-day old suckling mice, which express sufficient number of FeRn can be also used. Alternatively, mammalian cells which express 30 recombinant FeRn of a species of choice can be also utilized, After counting the radioactivity of the bound fraction of modified IgG or that of wild type, the bound molecules can be then extracted with the detergent, and the percent release per unit number of cells can be calculated and compared. Affinity of modified IgGs for FoRn can be measured by surface plasmon 35 resonance (SPR) measurement using, for example, a BlAcore 2000 (BlIAcore Inc.) as -72- WO 02/060919 PCT/US01/48432 described previously (Popov et al., Mal. Immunol., 33:493-502, 1996; Karlsson et al, J Immunol. Methods, 145:229-240, 1991, both of which are incorporated by reference in their entireties). In this method, FeRn molecules are coupled to a BIAcore sensor chip (e.g., CM5 5 chip by Pharmacia) and the binding of modified IgG to the immobilized FeRn is measured at a certain flow rate to obtain sensorgrams using BlIA evaluation 2.1 software, based on which on- and off-rates of the modified IgG, constant domains, or fragments thereof, to FcRn can be calculated, Relative affinities of modified IgGs or fragments thereof, and the wild type 10 IgG for FeRn can be also measured by a simple competition binding assay. Unlabeled modified IgG or wild type IgG is added in different amounts to the wells of a 96-well plate in which FcRn is immobilize. A constant amount of radio-labeled wild type lgG is then added to each well, Percent radioactivity of the bound fraction is plotted against the amount of unlabeled modified IgG or wild type IgG and the relative affinity of the modified hinge-Fc 15 can be calculated from the slope of the curve. Furthermore, affinities of modified IgGs or fragments thereof, and the wild type IgG for FcRn can be also measured by a saturation study and the Scatchard analysis. Transfer of modified IgG or fragments thereof across the cell by FeRn can be measured by in vitro transfer assay using radiolabeled IgG or fragments thereof and FcRn 20 expressing cells and comparing the radioactivity of the one side of the cell monolayer with that of the other side. Alternatively, such transfer can be measured in vivo by feeding 10- to 14-day old suckling mice with radiolabeled, modified IgG and periodically counting the radioactivity in blood samples which indicates the transfer of the IgG through the intestine to the circulation (or any other target tissue, e.g., the lungs). To test the dose-dependent 25 inhibition of the IgG transfer through the gut, a mixture of radiolabeled and unlabeled IgG at certain ratio is given to the mice and the radioactivity of the plasma can be periodically measured (Kim et al., Eur. J Immunol., 24:2429-2434, 1994). The half-life of modified IgG or fragments thereof can be measure by pharmacokinetic studies according to the method described by Kim et al. (Eur. J ofImmuno. 30 24:542, 1994), which is incorporated by reference herein in its entirety. According to this method, radiolabeled modified IgG or fragments thereof is injected intravenously into mice and its plasma concentration is periodically measured as a function of time, for example, at 3 minutes to 72 hours after the injection, The clearance curve thus obtained should be biphasic, that is, a-phase and p-phase. For the determination of the in vivo half-life of the 35 modified IgGs or fragments thereof, the clearance rate in f-phase is calculated and compared with that of the wild type IgG. -73 - WO 02/060919 PCT/US0I/48432 6. EXAMPLES The following examples illustrate the production, isolation, and 5 characterization of modified hinge-Fc fragments that have longer in vivo half-lives. 6.1 LIBRARY CONSTRUCTON 6,1.1 REAGENTS All chemicals were of analytical grade. Restriction enzymes and DNA 10 modifying enzymes were purchased from New England Biolabs, Inc. (Beverly, MA). Oligonucleotides were synthesized by MWG Biotech, Inc. (High Point, NC). pCANTAB5E phagemid vector, anti-E-tag-horseradish peroxydase conjugate, TG1 E. Col! strain, IgG Sepharose 6 Fast Flow and H1iTrap protein A columns were purchased from APBiotech, Inc, (Piscataway, NJ). VCSM13 helper phage and the Quick change mutagenesis kit were 15 obtained from Stratagene (La Jolla, CA). CJ236 IS. coli strain was purchased from Bio-Rad (Richmond, CA), BCA Protein Assay Reagent Kit was obtained from Pierce (Rockford, IL). Lipofectamine 2000 was purchased from Invitrogen, Inc. (Carlsbad, CA). 6.1.2 EXPRESSION AND PURIFICATION OF 2/URINE AND HUMAN FCRN 20 L The amino acid sequences of human and mouse FcRn are SEQ U) NOs. 84 and 85, respectively (see also Firan et al., Intern, Immuno,, 13:993-1002, 2001 and Popov et al., Mol. mmunol., 33:521-530, 1996, both of which are incorporated herein by reference in their entireties), Human FoRn was also obtained following isolation from human placenta 25 cDNA (Clontech, Palo Alto, CA) of the genes for human p2-microglobulin (Kabat et al, 1991, Sequences of Proteins of Immunological Interest, U.S. Public Health Service, National Institutes of Health, Washington, DC) and codons -23 to 267 of the human a chain (Story et al., -. Exp. Med, 180:2377-2381, 1994) using standard FCR protocols. Light and heavy chains along with their native signal sequence (Kabat et al., 1991, supra; Story et al, supra) 30 were cloned in pFastBac DUAL and pFastBacl bacmids, respectively, and viral stocks produced in Spodopterafrugiperda cells (Sf9) according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). High-Five cells were infected at a multiplicity of infection of 3 with the baculoviruses encoding a and 02 chains using commercially available protocols (Invitrogen). Recombinant human FcRn was purified as follows supernatant of infected 35 insect cells was dialyzed into 50 mM MES (2-N-[Morpholino]ethansulfonic acid) pH 6.0 and -74 - WO 02/060919 PCT/US01/48432 applied to a 10 ml human IgG Sepharose 6 Fast Flow column (APBiotech, Piscataway, NJ). Resin was washed with 200 ml 50 mM MES pH 6.0 and FeRn elated with 0,1 M Tris-Cl pH 8.0. Purified FeRn was dialyzed against 50 mM MES pH 6.0, flash frozen and stored at 5 70"C. The purity of proteins was checked by SDS-PAGE and HPLC, 6.13 PREPARATION OF TAA-CONTAINING ssDNA URACIL TEMPLATE Construction of the libraries was based on a site directed mutagenesis strategy 10 derived from the Kunkel method (Kunkel et al., Methods Enzymol, 154:367-382, 1987). A human hinge-Fe gene spanning amino acid residues 226-478 (Kabat numbering, Kabat et al, 1991, supra) derived from MEDI-493 human IgGl (Johnson at al., J' Infect. Disease, 176:1215-1224, 1997), was cloned into the pCANTAB5E phagemid vector as an Sfil/NotI fragment. Four libraries were generated by introducing random mutations at positions 251, 15 252, 254, 255, 256 (library 1), 308, 309, 311, 312, 314 (library 2), 385, 386, 387, 389 (library 3) and 428, 433, 434, 436 (library 4). Briefly, four distinct hinge-Fc templates were generated using PCR by overlap extension (Ho et al, Gene, 15:51-59, 1989), each containing one TAA stop codon at position 252 (library 1), 310 (library 2), 384 (library 3) or 429 (library 4), so that only mutagenized phagemids will give rise to Fc-displaying phage. 20 Each TAA-containing single-stranded DNA (TAAssDNA) was then prepared as follows: a single CJ236 . coli colony harboring one of the four relevant TAA-containing phagemids was grown in 10 ml 2 x YT medium supplemented with 10 ig/ml chloramphenicol and 100 pg/mi ampicillin. At OD6 - 1, VCSM13 helper phage was added to a final concentration of 10"' pfu/ml. After 2 hours, the culture was transferred to 500 ml 25 of 2 x YT medium supplemented with 0.25 pg/ml uridine, 10 pg/mi chloramphenicol, 30 pg/inl kanamycin, 100 pg/ml ampicillin and grown overnight at 37"C. Phage were precipitated with PEG6000 using standard protocols (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York, Vols, 1-3) and purified using the Qiaprep Spin M13 Kit (Qiagen, Valencia, CA) according to 30 the manufacturer's instructions, 10 to 30 pg of each uracil-containing TAAssDNA template was then combined with 0.6 pg of the following phosphorylated oligonueleotides (randomized regions underlined) in 50 mM Tris-HCl, 10 mM MgCl 2 , pH 7.5 in a final volume of 250 pl: Library 1: 35 5'-CATGTGACCTCAGGSNNSNNSNNGATSNNSNNGGTGTCCTTGGGTTTT -75- WO 02/060919 PCT/USOI/48432 GGGGG-3' (SEQ ID NO:120) Library 2: 5'-GCACTTGTACTCCTTGCCATTMNNCCASiNSNNGTGSSNNGGTGA 5 GGACGC-3' (SEQ ID NO:121) Library 3: 5'GGTCTTGTAGIT CTCSNNSNNSNNA TTCTCTCCC-3'(SEQ ID NO: 122) Library 4: 5'-GGCTCTTCTGCGTSNNGTGSNNSNNCA.GAGCCTCATGSNNCACGGAGC 10 ATGAG-3' (SEQ ID NO:123) where N= A, C, T or G and S= G or C. 6.1.4 SYNTHESiS OF HETERODUPLEX DNA Appropriate, degenerate oligonucleotides were phosphorylated in the presence 15 of T4 polynucleotide kinase using the standard protocol. Ten to 30 xg of saDNA U template and 0.6 pg of phosphorylated oligonucleotide were combined in 50 mM Tris-HCI containing 10 mM MgCl, pH 7.5, to a final volume of 250 p1 and incubated at 90"C for 2 minutes, 50*C for 3 minutes, and 20'C for 5 minutes. Synthesis of the beteroduplex DNA was carried out by adding 30 units of both T4 DNA ligase and T7 DNA polymerase in the presence of 0.4 20 mM ATP, 1 mM dNTPs and 6 mM DTT and the mixture was incubated for 4 hours at 20'C, The heteroduplex DNA thus produced was then purified and desalted using Qiagen Qiaquick DNA purification Kit (Qiagen, CA). 6.1.5 ELECTROPORATION 25 300 p1 electrocompetent TO I. coli cells were electroporated with I to 5 pg of the heteroduplex DNA in a 2.5 kV field using 200 Q and 25 pF 1 capacitance until a library size of 1 x 108 (library 1 and 2) or 1 x 107 (library 3 and 4) was reached. The cells were resuspended in 2 ml SOC medium and the procedure was repeated 6 to 10 times. The diversity was assessed by titration of recombinant E. coli. The pulsed cells were incubated in 30 50 ml SOC medium for 30 minutes at 37*C under agitation, centrifuged, and resuspended in 500 ml 2xYT containing 100 pg/ml ampicillin and 1010 pfu/inl of VCSM13 helper phage. The culture was incubated overnight at 370C and the cells were pelleted by centrifugation. The phage in the supernatant which express mutated hinge-Fe portion on its GUI-coat protein were precipitated with PEG6000 as previously described (Sambrook et al., 1989, supra) and 35 resuspended in 5 ml of 20 mM MES, pH 6.0. - 76 - WO 02/060919 PCT/USOI/48432 6.2 PANNING OF THE LIBRARY Phage were panned using an ELISA-based approach. A 96-wel1 ELISA plate was coated with 100 IL/well of 0.01 mg/ml urine FcRn in sodium carbonate buffer, pH 5 9.0, at 4*C overnight and then blocked with 4% skimmed milk at 37*C for 2 hours, In each well of the coated plate, 100..150 [d of the phage suspension (about 10" phage in total) in 20 mM MES, p1H 6.0, containing 5% milk and 0.05% Tween 20, were placed and incubated at 37"C for two to three hours with agitation. After the incubation, the wells were washed with 20 mM MES, pH 6.0, 10 containing 0.2% Tween 20 and 0.3 M NaCl about thirty times at room temperature, The bound phage were eluted with 100 pl/well of PBS, pH1 7.4, at 37"C for 30 minutes. The eluted phage were then added to the culture of exponentially growing E. coil cells and propagation was carried out overnight at 37*C in 250 ml 2xYT supplemented with 100 pg/ml.ampicillin and 10" pfu/ml of VCSM 13 helper phage. Propagated phage 15 were collected by centrifugation followed by precipitation with PEG and the panning process was repeated up to a total of six times. For the phage library containing mutations in residues 308-314 (11310 and W313 fixed), the phage expressing hinge-Pc region with higher affinities for FeRn were enriched by each panning process as shown in Table IV. The panning results of the library 20 for the mutations in the residues 251-256 (1253 fixed) and that of the library for the mutations in the residues 428-436 (11429, E430, A431, L432, and 11435 fixed), are shown in Tables V and VI, respectively. Furthermore, the panning results of the library for the mutations in the residues 385-389 (E388 fixed) is shown in Table VI. Table IV 25 PANNING OF LIBRARY (RESIDUES 308-314; H310 AND W313 FIXED) pCANTAB5E-KUNIEL-muFeRn (MURINE FeRn) PANNING OUTPUT ENRICHMENT + FeRn -FeRn RATIO I1st Round 1.1 x 10, 0.5 x 10' 2 30 2nd Round I x 10 4 0.2 x 10 4 5 3rd Round 9 x 10 4 0.3 x I1 30 4th Round 3 x 10 5 2 x 10 4 15 35 - 77.- WO 02/060919 PCTfUSO]/48432 Table V PANNING OF LIBRARY (RESIDUES 251-256; 1253 FIXED) pCANTAB5E-KUNKEL-muFcRn 5 PANNING OUTPUT ENRICHMENT + FeRn -FeRn RATIO Ist Round 2.5 x 10 5 1 x 10 25 2nd Round 6 x 10 2 x 10 4 3.0 10 3rd Round 8x 10 5 4 x 10 4 20 4th Round 1.2 x 106 $ x4 24 5th Round 3.0 x 10' 6 x 10 4 50 15 20 25 30 35 -78- WO 02/060919 PCT/US01/48432 Table VI PANNING OF LIBRARY (RESIDUES 428-436; 11429, E430, A431, L432, AND H435 FIXED) pCANTABSE-KUNKEL-muFcRn S PANNING OUTPUT ENRICHMENT + FeRn - FeRn RATIO Ist Round 2.3 x 10 5 0.9 x 10' 2.5 10 2nd Round 3 x 10 4 1 x 10 4 3 3rd Round 2 x 10 5 2x 10 4 10 4th Round 8 x 10 5 5x 10 4 16 15 20 25 30 35 - 79 - WO 02/060919 PCT/US01/48432 Table VII PANNING OF LIBRARY (RESIDUES 385-389; E388 FIXED) pCANTABSE-KUNKEL-muFeRn PANNING OUTPUT ENRICHMENT +FeRn -FcRU RATIO Ist Rund 4.2 x 10 - 3.8 x 10 5 1.1 2nd Round 5 x 104 0,3 x 104 17 10 3rd Round 3.5 x 10' 1 x 104 35 4th Round 5.5 x 10 5 4x 10 4 14 5th Round 7.5 x 10' 5x10 4 15 6th Round 2 x 10 6 1 x 10 20 15 6.3 DENTIFCATION OF ISOLATED CLONES FROM PANNING After each panning process, phage were isolated and the nucleic acids 20 encoding the expressed peptides which bound to FcRn were sequenced by a standard sequencing method such as by dideoxynucleotide sequencing (Sanger et at, Proc. Natl, Acad Sci USA, 74:5463-5467, 1977) using a ABI3000 genomic analyzer (Applied Biosystems, Poster City, CA), As a result of panning, two mutants were isolated from the phage library containing mutations in residues 308-314 (H3 10 and W313 fixed), thirteen mutants from the 25 library for residues 251-256 (1253 fixed), six mutants from the library for residues 428-436 (H429, E430, A43 1, L432, and H435 fixed), and nine mutants from the library for residues 385-389 (E388 fixed). The mutants isolated from the libraries are listed in Table VIII. Table VIII MUTANTS ISOLATED BY PANNING LIBRARY MUTANTS* 251-256 eu Tyr Ile Thr Ar Glu (SEQ ID NO:90) Leu Tyr Pe Ser Arg Thr (SEQ ID NO:91) Leu Tyr Be Ser Arg Ser (SEQ ID NO:92) 35 Leu Tyr fie Sear Arg Arg (SEQ ID NO:93) - 80 - WO 02/060919 PCT/USO1/48432 LIBRARY MUTANTS* Leu yr e Ser Arg Gin (SEQ ID NO:94) Leu Trp Re Ser Arg Thr (SEQ ID NO:95) Leu Tyr Ile Ser Leu Gin (SEQ ID NO:96) Leu Phe Ile Ser Arg Asp (SEQ ID NO:97) Leu Phe Ile Ser Arg Thr (SEQ ID NO:98) Leu Phe Ile Ser Arg Arg (SEQ ID NO:99) 10 Lou Phe Ile Thr Gly Ala (SEQ ID NO: 100) Leu Ser Ile Ser Arg Glu (SEQ ID N0:101) Arg Thr Ile Ser le Ser (SEQ ID NO:102) 308-314 Thr Pro His Ser Asp TrM Leu (SEQ ID NO:103) 15 Ile Pro His Giu Asp Trp Leu (SEQ ID NO:104) 385-389 Arg'Thr Arg Gilu Pro (SEQ ID NO:105) Asp Pro Pro Glu Ser (SEQ ID NO:106) Ser Asp Pro Glu Pro (SEQ ID NO:107) 20 Thr Ser His Glu Asn (SEQ ID NO:108) Ser Lys Ser Glu Asn (SEQ ID NO:109) His Arg Ser Glu Asn (SEQ ID NO:110) Lys Ile Arg Glu Asa (SEQ ID NO:111) 25 Gly Ile Thr Glu Ser (SEQ ID NO: 112) Ser Met Ala Glu Pro (SEQ ID NO:113) 428-436 Met His Glu Ala Leu Arg Tyr His His (SEQ ID NO:114) Met His Glu Ala Leu His Phe His His (SEQ ID NO:115) 30 Met His Glu Ala Leou Lys Phe His His (SEQ ID NO:116) Met His Glu Ala Lou Ser Tyr His Arg (SEQ ID NO:117) Thr His Glu Ala Lou His Tyr His Thr (SEQ ID NO: 118) Met His Glu Ala Lou His Tyr His Tyr (SEQ ID NO: 119) 35 * Substituting residues are indicated in bold face -81 - WO 02/060919 PCT/US01/48432 The underlined sequences in Table VIll correspond to sequences that occurred 10 to 20 times in the final round of panning and the sequences in italics correspond to sequences that occurred 2 to 5 times in the final round of panning. Those sequences that are neither 5 underlined nor italicized occurred once in the final round of panning. 6.4 EXPRESSION AND PURIFICATION OF SOLUBLE MU TANT INGE-FC REGION The genes encoding mutated hinge-Fe fragments are excised with appropriate 10 restriction enzymes and recloned into an expression vector, for example, VppelBhis (Ward, J Mo. Biot, 224:885-890, 1992), Vectors containing any other type of tag sequence, such as c-myc tag, decapeptide tag (Huse et al., Science, 246:1275-1281, 1989), Flag" (Irmunex) tags, can be used. Recombinant clones, such as E. coli, are grown and induced to express soluble hinge-Fe fragments, which can be isolated from the culture media or cell lysate after 15 osmotic shock, based on the tag used, or by any other purification methods well known to those skilled in the art and characterized by the methods as listed below, 6.5 CONSTRUCTION, PRODUCTION AND PURIFICATION OF IgGI VARIANTS 20 Representative Fe mutations such as 1253A, M252Y/8254T/T256E, M252W, M252Y, M252Y/T256Q, M252F1T256D, V30ST/L309P/Q3 118, G3 85D/Q386P/N389S, G385R/Q386T/P387R/N389P, H433K/N434F/Y436H, and N434F/Y436 were incorporated into the human IgG1 MEDI-493 (SYNAGIS*) (Johnson et al., 1997, supra). The heavy chain was subjected to site-directed mutagenesis using a Quick Change Mutagenesis kit 25 (Stratagene, La Jolla, CA) according to the manufacturer's instructions and sequences were verified by didoxynucleotide sequencing using a ABI3000 (Applied Biosystems, Foster City, CA) sequencer. The different constructions were expressed transiently in human embryonic kidney 293 cells using a CMV immediate-early promoter and dicistronic operon in which IgGl/V 5 is cosecreted with IgGi/VL (Johnson et al., 1997, supra). Transfection was carried 30 out using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) and standard protocols. IgGs were purified from the conditioned media directly on I ml HiTrap protein A columns according to the manufacterer's instructions (APBiotech). 35 - 82 - WO 02/060919 PCT/US01/48432 6.6 CHARACTERZATION OF MUTATED RINGE-FC REGION 6.6.1 IN VITRO CHARACTERIZATION UPLC AND SDS-PAGE 5 Following the purification, general characteristics such as molecular weight and bonding characteristics of the modified hinge-Fc fragments may be studied by various methods well known to those skilled in the art, including SDS-PAGE and HPLC. FeRn binding assay 10 Binding activity of modified hinge-F fragments can be measured by incubating radio-labeled wild-type hinge-Fc or modified hinge-Fe with the cells expressing either mouse or human FcRn. Typically, endothelial cell lines such as SV40 transformed endothelial cells (SVEC) (Kim et a., J, Immunol., 40:457-465, 1994) are used. After incubation with the hinge-Fc fragments at 37'C for 16-18 hours, the cells are washed with 15 medium and then detached by incubation with 5 mM Na 2 EDTA in 50 mM phosphate buffer, pH 7,5, for 5 minutes. The radioactivity per 101 cells is measured, Then, the cells are resuspended in 2 ml of 2.5 mg/ml CHAPS, 0.1 M Tris-HCI p1-1 8.0 containing 0.3 mg/ml PMSF, 25 mg/ml pepstatin and 0.1 mg/ml aprotinin and incubated for 30 minutes at room temperature. The cell suspension is then centrifuged and 20 the supernatant separated. The radioactivity of the supernatant is measured and used to calculate the amount of the hinge-Fc fragments extracted per 10' cells. The Kd for the interaction of wild type human IgGI with murine and human FcRn (269 and 2527 nM, respectively) agree well with the values determined by others (265 and 2350 nM, respectively, Firan et al,, 2001, supra). The 1253A mutation virtually 25 abolishes binding to human and murine FcRn, as reported by others (Kim er al., Eur. J Immunol., 29:2819-2825, 1991; Shields et al., J. iol, Chem., 276:6591-6604, 200 1), This is not the result of nisfolding of the antibody as this mutant retains the same specific activity than the wild type molecule (SYNAGIS*) in a microneutralization assay (Johnson et a., 1997, supra; data not shown). 30 Human IgGI mutants with increased binding affinity towards both murine and human FcRn were generated (Table VIII). Improvements in complex stability were overall less marked for the human IgGi-human FeRn pair than for the human IgG1-murine FcRn compared to wild type IgGI were 30-(AAG =2.0 kcal/mol for N434F/Y436H) and 1 l-(AAG = 1.4 kcal/mol for M252Y/S254Y/S254T/T256E) fold, respectively. However, ranking of 35 the most critical positions remain unchanged when comparing human and murine FoRn: the largest increases in IgG I -murine FcRn complex stability (AAG > 1,3 keal/mol) occurred on - 83 - WO 02/060919 PCT/USOI/4843Z mutations at positions 252, 254, 256 (M252Y/S254T1T256E and M252W) and 433, 434, 436 (H433K/N434F/Y436H and N434F/Y436H). Likewise, the same mutations were found to have the most profound impact on the IgO 1 -human FcRn interaction and also resulted in the 5 largest increases in complex stability (AAG > 1.0 kcal/mol). Substitutions at positions 308, 309, 311, 385, 386, 387 and 389 bad little or no effect on the stability of the complexes involving human or murine FcRn (AAG < 0.5 kcal/mol). Residues at the center of the Fe FoRn combining site contribute significantly more to improvement in complex stability than residues at the periphery (FIG. 9). 10 Efficient binding of human Fe to murine FeRn apparently requires the presence of several wild type Fe residues. For example, leucine is very conserved at 251, arginine at 255, aspartic acid at 310, leucine at 314 and methionine at 428 (FIG. 6), Another specificity trend is observed when one considers positions 308, 309, and 311 where threonine, proline, and serine, respectively, are very strongly favored over the corresponding 15 wild type residues (FIG. 6). However, generation of this strong consensus sequences does not correlate with the magnitude of increase in affinity as V308T/L309P/Q3 1S binds less than 2-fold better than the wild type IgGI to both human and murine FeRn (Table IX). Increases in affinity can be strongly dependent upon residue substitution at one 'hot spot' position, For example, the single mutation M252Y causes an increase in 20 binding to murine FcRn by 9-fold, whereas additional mutations bring little (M252Y/S254T/T256E) or no (M252YF256Q) added benefit. The same trend is observed for the human receptor, although to a lesser extent, Indeed, M252Y/S254T/T256E shows a marked improvement of 2.5-fold in affinity compared to M252Y. This probably reflects the differences between the binding site of human and marine FeRn (West and Bjorkman, 25 Biochemistry, 39:9698-9708, 2000). Phage-derived IgGI mutants exhibiting a significant increase in affinity towards murine FeRn (AAG > 1.3 kcal/mol) also showed significant binding activity to the receptor at p1 7.2 when compared to wild type IgGI (FIGs. 8A-H). IgGI mutants with moderate increase in affinity (AAG < 0.3 kcal/mol) bound very poorly at pH 7.2 (data not 30 shown). In contrast, IgG1 mutants with large (AAG > 1.0 kcal/mol) increase in affinity towards human FoRn exhibited only minimal binding at pH 7.4 when compared to wild type IgG1 (FIGs. 8A-H). 35 - 84 - WO 02/060919 PCT/USOI/48432 Table IX DISSOCIATION CONSTANTS AND RELATIVE FREE ENERGY CHANGES FOR THE BINDING OF IgG1/FC MUTANTS TO MARINE AND HUMAN FcRn* 5 MUTANT Dissociation AAG Dissociation AAG Constant (keal/mol) Constant (keal/mol) Fe/Murine Fe/fluman FeRn (nM) FeRn (M) , 10 wildtype 269t1 2527 &±117 1253A NB NA NB NA M252Y/S254T/T256E 27 6 1.4 225 10 1.4 M252W 30 1 1.3 408*24 1.1 15 M252Y 41± 7 1.1 532&37 0.9 M252Y/T256Q 39±8 1.1 560± 102 0.9 M252F/T256D 52± 9 1.0 933 170 0.6 V308T/L309P/Q311S 153 23 0,3 1964 84 0.1 20 G385D/Q386P/N389S 187* 10 0.2 2164 331 0.1 0385R/Q386T/P387R/N389P 147 ± 24 0.4 1620 61 0.3 H433K/N434F/Y436H 14 ±2 1.8 399 47 1.1 N434F/Y436H 9 1 2.0 493 ±7 1.0 *Affinity measurements were carried out by BlAcore as described above. Residue numbering 25 is according to EU (Kabat et at., 1991, supra). Differences in free energy changes are calculated as the differences between the Ags of wild type and mutant reactions (AAG = AG wildtye -AGmn NB, no binding. NA, not-applicable. FeRn-mediated transfer assay This assay follows the protocol disclosed in PCT publication WO 97/3463 1. 30 Radiolabeled modified hinge-Fc fragments at various concentration (1 pg/mi-1 mg/ml) are added to the one side of the transwell and the transfer of the fragments mediated by FCRn expressing monolayer of the cells can be quantitated by measuring the radioactivity on the other side of the transwell. 35 -85 - WO 02/060919 PCT/USO1/48432 6.6.2 N VWWO PHARMACOKINETIC STUDY In order to determine the half-life of the modified IgG hinge-Fc, modified hinge-Fe fragments are radiolabelled with "'I (approximate specific activity of 10 cpm/pg) 5 and dissolved in saline (pH 7.2). The solution is injected intravenously into BALB/c mice (Harlan, Indianapolis, IN), which have been given Nat-containing water previously to block the thyroid, in a volume not more than 150 pl and with a radioactivity of 10 x I0-50 x 106 cpm. The mice are bled from the retro-orbital sinus at various time points, for example, at 3 minutes to 72 hours after the injection, into heparinized capillary tubes and the plasma 10 collected from each sample is counted for radioactivity. To generate the data provided in FIG. 10, 10 animals were used for each molecule assayed with 2.5 pg of antibody injected per animal, Antibody serum levels were determined using an anti-human IgG ELISA (FIG. 10). There seems to be an inverse correlation between affinity to mouse FcRn and persistence in serum. This might be due to 15 the significant amount of binding of the mutants observed at pH 7.2, which leads to the sequestration (i.e., lack of release in the serum) of the molecules. Preliminary data (not shown) suggests increased transport of the mutants to the lung. Additionally, since the mutants exhibit lower levels of binding to human FeRn than murine FeRn (see PIGS, 8A-H), antibody serum levels are expected to be higher in primates and humans. 20 6.6.3 SURFACE PLASMON RESONANCE ANALYSES The interaction of soluble murine and human FeRn with immobilized human IgG1 variants was monitored by surface plasmon resonance detection using a BlAcore 3000 instrument (Pharmacia Biosensor, Uppsala, Sweden). No aggregated material which could 25 interfere with affinity measurements (van der Merwe et al., EMO J., 12:4945-4954, 1993; van der Merwe et al, Biochemistry, 33:10149-10160, 1994) was detected by gel filtration. Protein concentrations were calculated by the bicinchoninic acid (BCA) method for both human and murine FcRn or using the 1% extinction coefficient at 280 nm of 1.5 for IgGI wild type and variants. The latter were coupled to the dextran matrix of a CM5 sensor chip 30 (Pharmacia Biosensor) using an Amine Coupling Kit as described (Johnson et al., supra). The protein concentrations ranged from 3-5 ag/ml in 10 mM sodium acetate, pH 5.0. The activation period was set for 7 minutes at a flow rate of 10 p1/min and the immobilization period was set to between 10 and 20 minutes at a flow rate of 10 gI/min. Excess reactive esters were quenched by injection of 70 p1 of 1.0 nethanolamine hydrochloride, pH 8,5. 35 This typically resulted in the immobilization of between 500 and 4000 resonance units (RU). Human and marine FeRn were buffer exchanged against 50 mM PBS buffer pH 6.0 -86- WO 02/060919 PCTUSJ1/48432 containing 0.05% Tween 20. Dilutions were made in the same buffer. All binding experiments were performed at 25*C with concentrations ranging from 120 to I pg/ml at a flow rate of 5 to 10 pl/min; data were collected for 25 to 50 minutes and three I-minute 5 pulses of PBS buffer pH 7,2 were used to regenerate the surfaces. PcRn was also flowed over an uncoated cell and the sensorgrams from these blank runs subtracted from those obtained with IgGi-coupled chips. Runs were analyzed using the software BlAevaluation 3.1 (Pharmacia). Association constants (KAs) were determined from Scatchard analysis by measuring the concentration of free reactants and complex at equilibrium after correction for 10 nonspecific binding. In equilibrium binding BlAcore experiments (Karlsson et aL, 1991, supra; van der Merwe et al., 1993, supra; van der Merwe et al, 1994, supra; Raghavan et al., Immunity, 1:303-315, 1994; Malchiodi et al, J. Exp. Med., 182:1833-1845, 1995), the concentration of the complex can be assessed directly as the steady-state response. The concentration of free analyte (human or urine FcRn) is equal to the bulk analyte 15 concentration since analyte is constantly replenished during sample injection. The concentration of free ligand on the surface of the sensor chip can be derived from the concentration of the complex and from the total binding capacity of the surface as K,= R,/C(R R,) where C is the free analyte concentration, Rq is the stcady-statc response, and Rm,,x is the total surface binding capacity. Rearranging, the equation reads: R, 1 /C = K, 20 R.x - KA Req A plot of R/C versus Rq at different analyte concentrations thus gives a straight line from which K, can be calculated (see Table IX). Errors were estimated as the standard deviation for two or three independent determinations and were <0%. Representative mutations identified after panning libraries 1 through 4 25 (FIG, 6, Table VIII) were introduced into the Fe portion of a human IgG1. Injection of different concentrations of human or urine FcRn over the immobilized IgG 1 variants gave concentration-dependent binding. Typical resonance profiles for equilibrium binding of the mutant M252Y/S254T/f256E to murine and human FeRn are shown in FIGs. 7A and B. To estimate apparent KAs, concentrations of FcRn ranging from 120 to I pIg/mi were used. In all 30 cases, equilibrium (or near-equilibrium) binding levels were reached within 50 minutes. To estimate the increase in RU resulting from the non specific effect of protein on the bulk refractive index, binding of FoRn to an uncoated cell was measured and the sensorgrams from these blank runs subtracted from those obtained with IgGl -coupled chips. The scatchard plots for the binding of the mutant M252Y/S254T/T256E to urine and human 35 FeRn are shown in FIGs. 7C and D. The plots were all linear, and apparent KAs were - 87 - WO 021060919 PCT/USO1/48432 calculated from the relevant slopes. Measurements were carried out in duplicate or triplicate and confirmed that the immobilized IgGs retained their original binding activity. Since there are two non-equivalent binding sites on mouse IgGI for urine 5 FeRn with affinities of < 130 nM and 6 pM (Sanchez et al., Biochemisty, 38:9471-9476, 1999; Schuck et al., Mol Immunol., 36:1117-1125, 1999; Ghetie and Ward, Ann. Rev. Immunol., 18:739-766, 2000), the receptor was used in solution to avoid avidity effects that arise when IgGI binds to immobilized FcRn. Consistent with this, systematically higher affinities are observed when FeRn, rather than IgG, immobilized on the biosensor chip 10 (Popov et al., 1996, supra; Vaughn and Bjorkman, Biochemnistry, 36:9374-9380, 1997; Martin and Bjorkman, Biochemistry, 38:12639-12647; West and 3jorkman, Blochemisty, 39:9698-9708, 2000). Under our experimental BlAcore conditions, mainly interactions corresponding to the higher-affinity association (i.e. single liganded-reeptor) are measured, according for the linearity of the scatchard plots (FIGs. 7C and D). 15 BlAcore analysis was also used to compare the affmity of wild type IgGI and IgGi mutants. Phage-derived IgG1 mutants exhibiting a significant increase in affinity towards murine FeRn at pH 6.0 (AAG 1.0 kcal/mol) also shoed significant binding to the mouse receptor at pH 7.2 with SPR signal,1 74 /SPR signal.re6> > 0.6 at saturation. IgG1 mutants with moderate increase in affinity towards marine FeRn at pH 6.0 (AAG < 0,4 20 kcal/mol) bound very poorly to the mouse receptor at pH 7.2, In contrast, IgUI mutants exhibiting large affinity increase towards human FcRn at pHl 6.0 (AAG a 1.0 kcal/mol) only showed minimal binding to the human receptor at pH 7.4 with SPR signal~ 1

,.

7 /SPR signalH 6

.

0 <0.15 at saturation. Those skilled in the art will recognize, or be able to ascertain using no more 25 routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims, All publications, patents and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually 30 indicated to be incorporated herein by reference. 35 --88 - The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the 5 priority date of each claim of this application. Where the terms "comprise", "comprises", "comprised" or "comprising" are used in this specification (including the claims) they are to be interpreted as specifying the presence of the stated features, integers, steps or components, but not precluding the presence of one or more other features, integers, steps or components, or group thereof. 88a

Claims (34)

1. A modified molecule comprising a protein or non-protein agent and an IgG constant domain, wherein the IgG constant domain comprises a human CH 2 domain in which there is an amino acid substitution at amino acid residues 308, 309 and 311, numbered according to the EU index of Kabat, wherein the modified molecule has an increased half-life compared to the half-life of a molecule comprising the protein or the non-protein agent and a corresponding IgG constant domain comprising a wild-type human CH 2 domain.

2. The modified molecule of claim 1, wherein the amino acid substitution at amino acid residue 308, is a substitution with threonine.

3. The modified molecule of claim 1 or 2, wherein the amino acid substitution at amino acid residue 309 is a substitution with proline.

4. The modified molecule of claim 1, 2 or 3, wherein the amino acid substitution at amino acid residue 311 is a substitution with serine.

5. The modified molecule according to any one of claims 1 to 4, wherein the IgG constant domain is a human IgG constant domain.

6. The modified molecule according to claim 5, wherein the human IgG constant domain is IgG 1 , IgG 2 , IgG 3 or IgG 4 .

7. The modified molecule according to claim 5 or 6, wherein the human IgG constant domain is IgG 1 .

8. The modified molecule according to claim 5, 6 or 7, wherein the IgG constant domain of the modified molecule comprises one or more amino acid substitutions at one or more of amino acid residues 251-256, 285-290, 310, 312-314, 385-389, and 428-436, relative to a wild-type human IgG constant domain. - 89 -

9. The modified molecule according to claim 8, wherein said one or more amino acid substitutions are at amino acid residues 251, 252, 254, 255, 256, 312, 314, 385, 386, 387, 389, 428, 433, 434 or 436.

10. The modified molecule according to claim 8 or 9, wherein said one or more amino acid substitutions are substitution with tyrosine, tryptophan or phenylalanine at position 252, substitution with threonine at position 254, substitution with glutamine, arginine, serine, aspartate, or glutamate at position 256, substitution with arginine, aspartate or serine at position 385, substitution with threonine or proline at position 386, substitution with arginine at position 387, substitution with proline or serine at position 389, substitution with threonine at position 428, substitution with arginine, lysine or serine at position 433, substitution with tyrosine or phenylalanine at position 434, or substitution with histidine, arginine or threonine at position 436.

11. The modified molecule according to any one of claims 5 to 10, wherein the IgG constant domain of the modified molecule has a higher affinity for FcRn than a wild-type human IgG constant domain.

12. The modified molecule according to claim 11, wherein the IgG constant domain of the modified molecule has a higher affinity for FcRn than a wild-type human IgG constant domain at pH 6.0 than at pH 7.4.

13. The modified molecule according to any one of claims 1 to 12, wherein the modified molecule is a modified IgG.

14. The modified molecule according to claim 13, wherein the modified IgG is a modified human IgG or a humanized IgG.

15. The modified molecule according to claim 14, wherein the human IgG or humanized IgG is IgG1, IgG 2 , IgG 3 or IgG 4 .

16. The modified molecule according to claim 14 or 15, wherein the human IgG or humanized IgG is IgG1. - 90 -

17. The modified molecule according to any one of claims 13 to 16, wherein the modified IgG immunospecifically binds a respiratory syncytial virus (RSV) antigen, HER2, tumor necrosis factor-alpha (TNF-a), transforming growth factor-3 (TGF-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-8 (IL-8), CD2, CD3, CD4, CD11a, CD14, CD18, CD20, CD22, CD23, CD25, CD33, CD52, CD64, CD80, CD147, CD40 ligand (CD40L), vascular endothelial growth factor (VEGF), intracellular adhesion molecule-3 (ICAM-3), epithelial growth factor receptor (EGFR), av03 integrin, a40 7 integrin, human leukocyte antigen (HLA), complement factor 5 (C5), immunoglobulin E (IgE), glycoprotein Ilb/IIa receptor, CA125, 17-IA cell surface antigen, Factor VII, GD3 epitope, human immunodeficiency glycoprotein 120 (HIV gp1120), hepatitis B virus (HBV), cytomegalovirus (CMV), VLA-4, or 02-integrin.

18. The modified molecule according to any one of claims 1 to 12, wherein the modified molecule is a fusion protein comprising a non-IgG protein covalently linked to the IgG constant domain; or wherein the non-protein agent is conjugated to the IgG constant domain.

19. A modified molecule according to any one of claims 1 to 18, for use in treating or preventing a disease or disorder in a subject; or the diagnosis of a disease or disorder.

20. The modified molecule of claim 19, wherein the subject is a human.

21. A kit comprising the modified molecule according to any one of claims 1 to 18, in a container, and instructions for use.

22. A nucleic acid comprising a nucleotide sequence encoding the constant domain of the modified molecule as defined in any one of claims 1 to 12.

23. A nucleic acid comprising a nucleotide sequence encoding the modified molecule according to any one of claims 13-17.

24. A host cell comprising the nucleic acid according to claim 22 or 23.

25. A pharmaceutical composition comprising the modified molecule of any one of claims 1 to 18, and a pharmaceutically acceptable carrier. - 91 -

26. A method for preventing or treating a disease or disorder, comprising administering to a subject a modified molecule according to any one of claims 1 to 18.

27. The method according to claim 26, wherein the subject is a human.

28. A method for diagnosing a disease or disorder, comprising: contacting the modified molecule of any one of claims 1 to 16 or 18 with a sample from the subject; and comparing the level of the antigen to which the modified molecule immunospecifically binds to a control level, wherein an increase in the level of the antigen in the sample compared to the control level is indicative of the disease or disorder.

29. An antibody conjugate comprising the modified molecule according to any one of claims 13 to 18 and a detectable substance or a therapeutic moiety.

30. A pharmaceutical composition comprising the antibody conjugate according to claim 29, and a pharmaceutically acceptable carrier.

31. A kit comprising the antibody conjugate of claim 29, in a container, and instructions for use.

32. A method for preventing or treating a disease or disorder, comprising administering to a subject the antibody conjugate according to claim 29.

33. A method for diagnosing a disease or disorder, comprising: contacting the antibody conjugate according to claim 29 with a sample from the subject; and comparing the level of the antigen to which the antibody conjugate immunospecifically binds to a control level, wherein an increase in the level of the antigen in the sample compared to the control level is indicative of the disease or disorder.

34. The method of claim 32 or 33, wherein the subject is a human. - 92 -