HK1171670A - A method of promoting dendritic spine density - Google Patents

Abstract

The invention relates to methods of increasing density of dendritic spines as a means to retain or improve cognition and to treat disorders associated with decreased dendritic spine morphology and a psychiatric disorder such as addiction and schizophrenia or a disorder associated with impaired cognition such as autism, Lett Syndrome, Tourette Syndrome, and Fragile-X Syndrome.

Description

Method for increasing density of dendritic spines

Cross reference to related applications

The present application claims benefit from U.S. provisional application No.61/260,815 filed on 12/11/2009 and U.S. provisional application No.61/294,020 filed on 11/2010, the disclosures of each of which are incorporated herein by reference in their entirety.

Technical Field

The invention relates to a method for increasing density of dendritic spines in neurons. More specifically, the present invention concerns methods of increasing synapses and treating cognitive diseases by inhibiting DR6 and/or p 75.

Background

Members of the TNFR family known as DR6 receptors (also known in the literature as "TR 9"; also known in the literature as TNF receptor superfamily members 21 or TNFRSF21) have been described as class I transmembrane receptors with four extracellular cysteine rich motif and cytoplasmic death domain structures (Pan et al, FEBS Lett. (FEBS letters), 431: 351-. Overexpression of DR6 in specific transfected cell lines was reported to result in apoptosis and activation of both NF-kB and JNK (Pan et al, FEBS Letters (FEBS Letters), 431: 351-356 (1998)).

In a mouse model deficient in DR6, T cells were essentially destroyed in JNK activation, whereas when DR6(-/-) mice were challenged with protein antigens, their T cells were found to hyperproliferate and displayed a deep polarization response to Th2 (whereas Th1 differentiation was not affected by the same) (Zhao et al, J.Exp.Med. (J.Imai. Med., 194: 1441-1448 (2001)). It was further reported that targeted disruption of DR6 resulted in enhanced T helper 2(Th2) differentiation in vitro (Zhao et al, supra). Various uses of DR6 agonists or antagonists in the modulation of B cell mediated disorders are described in US 2005/0069540 published at 31.3.2005. The DR6 receptor may play a role in modulating OVA in inducing airway inflammation in mouse models of asthma (Venkataraman et al, immunol. lett. (immunological communications), 106: 42-47 (2006)). Using the myelin oligodendrocyte glycoprotein (MOG (35-55)) induced model of experimental autoimmune encephalomyelitis, DR 6-/-mice were found to be highly tolerant to both the onset and progression of CNS disease compared to Wild Type (WT) littermates. Thus, DR6 may be involved in regulating leukocyte infiltration and play a role in the induction and progression of experimental autoimmune encephalomyelitis (Schmidt et al, J.Immunol. (J.Immunol., 175: 2286-.

Although different TNF ligand and receptor family members have been identified as having different biological activities and properties, few such ligands and receptors have been reported to be involved in neurologically-related functions. For example, WO2004/071528, published 8/26/2004, describes the inhibition of CD95(Fas) ligand/receptor complexes in a murine model to treat spinal cord injury. Currently, Nikolaev et al show that the N-terminal fragment of APP is a ligand for DR6 (Nilolaev et al (2009) Nature 457: 981-.

The nerve cells communicate with each other at synapses, which occur at "dendritic spines" on the dendrites. Dendritic spines are membrane regions of dendrites that extend from the dendrites and (usually) contact individual synapses of axons. There may be thousands of spines on a single dendrite. The spines receive both excitatory and inhibitory inputs from the axons, however, more commonly, excitatory inputs. Proximate to the tip of the dendritic spine is an electron density region known as the post-synaptic density region (PSD). Within this region is a structural protein called PSD-95, PSD-95 being a marker for PSD. The spine is rich in glutamate receptors (e.g., AMPA and NMDA receptors). Other receptors such as the TrkB receptor are believed to play a role in spine survival.

Chemical synapses connect neurons to form functional circuits that can process and store information. Loss of proper function or stability of these linkages is believed to underlie many psychiatric and neurodegenerative diseases.

Summary of The Invention

The present invention provides methods of increasing dendritic spine density in a patient having a cognitive or psychiatric disorder comprising administering to the patient an effective amount of a DR6 inhibitor and/or a p75 inhibitor. The inhibitor may be, for example, an antibody that binds to an epitope of DR6 and inhibits the function of DR6, or an antibody that binds to an epitope of p75 and inhibits the function of p 75. Examples of inhibitory anti-DR 6 antibodies include, but are not limited to, 3f4.4.8, 4b6.9.7, 1e5.5.7, and antigen-binding fragments thereof. Likewise, the antibody may be a chimeric or humanized antibody, such as, for example, a chimeric or humanized 3f4.4.8, 4b6.9.7 or 1e5.5.7 or an antibody that binds to the same epitope as 3f4.4.8, 4b6.9.7 or 1 e5.5.7. Inhibitors of DR6 reduce or prevent DR6 signaling in neurons.

The invention also provides a method of treating a cognitive or psychiatric disorder in a patient in need thereof, comprising identifying a patient having a cognitive or psychiatric disorder associated with dendritic spine reduction and administering to the patient a therapeutically effective amount of a DR6 antagonist and/or a p75 antagonist. The mental or cognitive disorder may be, for example, Rett Syndrome, tourette Syndrome, autism, schizophrenia or fragile-X mental retardation. The inhibitor may be, for example, an antibody that binds to an epitope of DR6 and inhibits the function of DR6, and/or an antibody that binds to an epitope of p75 and inhibits the function of p 75. The antibody can be, for example, 3f4.4.8, 4b6.9.7, 1e5.5.7, or an antigen-binding fragment thereof. The antibody may be a chimeric or humanized antibody, such as, for example, a chimeric or humanized 3f4.4.8, 4b6.9.7, or 1e5.5.7, or an antibody that binds to the same epitope as 3f4.4.8, 4b6.9.7, or 1 e5.5.7.

The present invention also provides a method of maintaining cognition in a subject during aging, the method comprising administering to the subject a DR6 and/or p75 inhibitor in an amount effective to increase dendritic spine density in the subject, thereby maintaining cognition in the subject. The inhibitor may be, for example, an antibody that binds to an epitope of DR6 and inhibits the function of DR6, and/or an antibody that binds to an epitope of p75 and inhibits the function of p 75. The antibody can be, for example, 3f4.4.8, 4b6.9.7, 1e5.5.7, or an antigen-binding fragment thereof. The antibody may be a chimeric or humanized antibody, such as, for example, a chimeric or humanized 3f4.4.8, 4b6.9.7, or 1e5.5.7, or an antibody that binds to the same epitope as 3f4.4.8, 4b6.9.7, or 1 e5.5.7.

Accordingly, the present invention provides the use of a DR6 antagonist and/or a p75 antagonist for the manufacture of a medicament for increasing dendritic spine density and for treating patients suffering from cognitive or psychiatric disorders associated with decreased dendritic spine density.

Brief Description of Drawings

Figure 1 shows directional labeling of excitatory neurons through a cortex 2/3 electroporated in utero. FIG. A: the E16 embryos from pregnant mice were exposed and injected with-1 μ Ι DNA into the right ventricle and applied with electrical potential; and B: layer 2/3 excitatory neuronal cell bodies and their processes can be observed histologically. Co-labeling neurons with DsRedExpress and PSD-95-paGFP; and (C) figure: an implanted cranial window; FIG. D: photon microscopy images through the cranial window (day 14 and day 44).

FIG. 2 shows DR6 at 60 days postnatal compared to control animals^-/-Increase in density and width of the animal dendritic spines (panel a). The density was calculated by averaging the total number of spines per cell/dendritic length of all animals within the same cohort. A total of 28 cells/8 animals were annotated as DR6-/-, compared to 26 cells/7 animals as DR6 +/-and 26 cells/6 animals as DR6+/+ (fig. B). Spine width and length were plotted as cumulative plots of the entire population of spines analyzed per genotype (panel C).

FIG. 3 shows the results obtained with N-APP (control) (panels A and B); 1 μ g/ml N-APP (FIG. C); 3 μ g/ml N-APP (Panel D); 10 μ g/ml N-APP (FIG. E); and E16 cortical neurons in culture after 30. mu.g/ml N-APP (panel F) treatment.

FIG. 4 shows the reduction of PSD95 points (puncta) as a result of treatment with 1, 3, 10 and 30 μ g/ml N-APP (compared to control).

Figure 5 shows that N-APP induced reduction of PSD95 dot density is dependent on DR6 function. Percentage of control in untreated neurons when 0.1, 0.3, 1.0 or 3.0ug/ml N-APP (without acidic tail) or full-length N-APP (N-APPFL) was added (dots/100 um). One group was additionally treated with 30ug/ml anti-DR 6.1 antibody (as shown).

Detailed Description

The techniques and methods described or referred to herein are generally well understood and employed by those skilled in the art using conventional methods, such as, for example, those described in Sambrook et al, Molecular Cloning: a Laboratory Manual, second edition (1989) Cold Spring Harbor Laboratory Press, N.Y. Unless otherwise indicated, the steps involving the use of commercially available kits and reagents are generally performed according to manufacturer-specified protocols and/or parameters, as appropriate.

Before the present methods and assays are described, it is to be understood that this invention is not limited to the particular methodology, protocols, cell lines, animal species or genera, constructs, and reagents described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a genetic alteration" includes a plurality of such alterations, and reference to "a probe" includes reference to one or more probes and equivalents thereof known to those skilled in the art, and so forth. Numbers recited in the specification and appended claims (e.g., amino acids 22-81, 1-354, etc.) are understood to be modified by the term "about".

All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The publications cited herein are incorporated by reference for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such publication by virtue of earlier priority date or prior date of invention. Furthermore, the actual publication date may be different from those displayed and require independent verification.

The term "amyloid precursor protein" or "APP" includes the different polypeptide isoforms encoded by APP pre-mRNA (pre-mRNA), such as shown in SEQ ID NO: APP695, APP751 and APP770 isoforms in 3-5 (isoforms translated from alternatively spliced transcripts of APP pre-mRNA), and post-translational processing portions of APP isoforms. As is known in the art, APP pre-mRNA transcribed from the APP Gene undergoes alternative exon splicingTo generate several isoforms (see, e.g., Sandbrink et al, Ann NY Acad. Sci. (proceedings of the New York academy of sciences) 777: 281-287 (1996); and information related to the PubMed NCBI protein site accession number P05067). This alternative exon splicing produces three major isoforms: 695(SEQ ID NO: 3), 751(SEQ ID NO: 4) and 770SEQ ID NO: 5) amino acids (see, e.g., Kang et al, Nature 325: 733 and 736 (1987); kitaguchi et al, Nature (Nature) 331: 530-532 (1988); ponte et al, Nature (Nature) 331: 525-527 (1988); and Tanzi et al, Nature (Nature) 331: 528-532(1988)). Two of these isoforms (App)₇₅₁And APP₇₇₀) Contains an insertion of 56 residues that are highly homologous to the Kunitz family of serine protease inhibitors (KPI) and are ubiquitously expressed. In contrast, APP, a shorter isoform lacking the KPI motif₆₉₅Expression in the nervous system (e.g., in neurons and glial cells) is predominant, and is therefore commonly referred to as "neuronal APP" (see, e.g., Tanzi et al, Science 235: 880- & 884 (1988); Neve et al, Neuron 1: 669- & 677 (1988); and Haas et al, J. Neurosci. (J. Neurosci.) 11: 3783- & 3793 (1991)). The APP isoforms, including 695, 751 and 770, undergo significant post-translational processing events (see, e.g., Esch et al 1990 Science 248: 1122 & 1124; Sisodia et al 1990 Science 248: 492 & 495). For example, each of these isoforms is cleaved by multiple secretases and/or secretase complexes, an event that produces an APP fragment that includes an N-terminal secreted polypeptide comprising the APP extracellular domains (sAPP α and sAPP β). Cleavage by alpha secretase or alternatively by beta 0 secretase results in the production and extracellular release of soluble N-terminal APP polypeptides (sAPP alpha and sAPP beta), respectively, and retention of the corresponding membrane-localized C-terminal fragments (C83 and C99). Subsequent processing of C83 by gamma secretase produces the P3 polypeptide. This is the major secretory pathway and is not amyloidogenic. Alternatively, presenilin/dulin-mediated processing of C99 by gamma-secretase releases amyloid beta polypeptide, amyloid-beta 40(a β 40) and amyloid-beta 42(a β 42) (the major component of amyloid plaques) as well as cytotoxic C-terminal fragments, gamma-CTF (50), gamma-CTF (57) and gamma-CTF (59). Certificate (certificate)The relative importance of the various cleavage events is shown to depend on the cell type. For example, non-neuronal cells preferentially process APP by cleaving the α -secretase pathway of APP within the A β sequence, thereby eliminating A β formation (see, e.g., Esch et al 1990 Science 248: 1122-1124; Sisodia et al 1990 Science 248: 492-495). In contrast, neuronal cells process a much larger portion of APP through the β -secretase pathway₆₉₅And intact a β is produced by the combined activity of at least two classes of enzymes. In neuronal cells, beta-secretase cleaves APP at the amino terminus of the A beta domain₆₉₅The different N-terminal fragment (sAPP. beta.) is released. In addition, gamma-secretase cleaves APP at alternative sites at the carboxy terminus, yielding 40(A β)₄₀) Or 42 amino acids long (A.beta.. beta.)₄₂) Of (a) beta (see, e.g., seebert et al 1993 Nature 361: 260-263; suzuki et al 1994 Science 264: 1336 and 1340; and Turner et al 1996 j.biol.chem. (journal of biochemistry) 271: 8966-8970). It is believed that nutrient deprivation triggers BACE cleavage of APP to produce a-100 kDa sAPP β, which undergoes one or more additional cleavages to produce a-55 kDa carboxy-terminal fragment (detected by anti-sAPP β antibody) and an amino-terminal-35 kDa fragment (detected by anti-N-APP (polyclonal antibody)), which we call "N-APP". The site of additional cleavage is unknown, but based on fragment size, is expected to be near the junction (amino acid 286) between the APP "acidic" and "E2" domains; in fact, recombinant APP [1-286]Is located at-35 kDa and can be detected by anti-N-APP (poly) analogously to N-APP.

As used herein, the terms "APP", "APP protein" and "APP polypeptide" include native APP sequences and APP variants and processed fragments thereof. These terms include APP expressed in a variety of mammals, including humans. APP may be expressed endogenously as occurs naturally in various human tissue lineages, or may be expressed by recombinant or synthetic methods. "native sequence APP" includes polypeptides having the same amino acid sequence as APP derived from nature (e.g., 695, 751, and 770 isoforms or processed portions thereof). Thus, the native sequence APP may have the amino acid sequence of a naturally occurring APP derived from any mammal (including a human). Such native sequence APP may be isolated from nature or may be prepared by recombinant or synthetic methods. In particular, the term "native sequence APP" includes naturally occurring processed and/or secreted forms (e.g., soluble forms comprising, for example, extracellular domain sequences), naturally occurring variant forms (e.g., alternatively spliced and/or proteolytically processed forms), and naturally occurring allelic variants. The APP variants may include fragments or deletion mutants of the native sequence APP.

APP polypeptides useful in embodiments of the invention include those described above and in the following non-limiting examples. The exemplary forms may be selected for use in different embodiments of the present invention. In some embodiments of the invention, the APP polypeptide comprises a full-length APP isoform such as the isoforms shown in SEQ ID NOs: APP in 3-5₆₉₅And/or APP₇₅₁And/or APP₇₇₀Isoforms. In other embodiments of the invention, the APP polypeptide comprises a post-translationally processed isoform of APP, e.g., an APP polypeptide (e.g., a soluble N-terminal fragment, such as sAPP α or sAPP β) that undergoes cleavage by a secretase, such as α -secretase, β -secretase, or γ -secretase. In related embodiments of the invention, APP polypeptides may be selected to include one or more specific domains such as an N-terminal extracellular domain (see, e.g., Quast et al, FASEB J. 2003; 17 (12): 1739-41), a heparin binding domain (see, e.g., Rossjohn et al, Nat.struct.biol. (Nat-structural biology) 1999 Apr; 6 (4): 327-31), a copper II type (see, e.g., Hesse et al, FEBS Letters (FEBS communications) 349 (1): 109-116(1994)), or a Kunitz protease inhibitor domain (see, e.g., Ponte et al, Nature (Nature); 331 (6156): 525-7 (1988)). In some embodiments of the invention, APP polypeptides include sequences that are observed to comprise an epitope recognized by a DR6 antagonist (such as an antibody or DR6 immunoadhesin) disclosed herein, e.g., APP₆₉₅Amino acids 22 to 81 of (a); sequences comprising an epitope bound by monoclonal antibody 22C11 (see, e.g., Hilbich et al, J.biol. chem. (J.Biol.Chem.) 268 (35): 26571-one 26577 (1993)).

In certain embodiments of the invention, the APP polypeptide does not comprise one or more specific domains or sequences, e.g., an APP polypeptide that does not comprise a Kunitz protease inhibitor domain (e.g., APP₆₉₅) Or an APP polypeptide that does not comprise the Alzheimer's beta amyloid (A beta) sequence (e.g., sAPP beta, which is a polypeptide that does not comprise A beta)₄₀And/or Abeta₄₂Polypeptides of sequence) (see, e.g., Bond et al, j.struct Biol. (journal of structural biology) 2003 Feb; 141(2): 156-70). In other embodiments of the invention, the APP polypeptides used in embodiments of the invention comprise one or more domains or sequences and no other domains or sequences, e.g., APP polypeptides comprising an N-terminal extracellular domain (or a portion thereof observed to be bound by a DR6 antagonist such as monoclonal antibody 22C11) but not a domain or sequence C-terminal to one or more secretase cleavage sites such as a beta amyloid (Α β) sequence (e.g., sappa or sappp β).

The terms "extracellular domain", "extracellular domain" or "ECD" refer to a form of APP that is substantially free of transmembrane and cytoplasmic domains. Typically, soluble ECDs will have less than 1% of such transmembrane and cytoplasmic domains, and preferably will have less than 0.5% of such domains. It is to be understood that any identified transmembrane domain of a polypeptide of the invention is identified according to the criteria commonly employed in the art for identifying hydrophobic domains of the type in question. The precise boundaries of the transmembrane domain may vary, but most likely do not exceed about 5 amino acids at either end of the initially identified domain. In a preferred embodiment, the ECD will consist of a soluble, extracellular domain sequence of the polypeptide that is free of transmembrane and cytoplasmic or intracellular domains (and does not bind to the membrane).

The term "APP variant" refers to an APP polypeptide as defined below that hybridizes to a polypeptide having the sequence shown in seq id NO: 3. 4 or 5 or a soluble fragment thereof or a lysable exodomain thereof has at least about 80%, preferably at least about 85%, 86%, 87%, 88%, 89%, more preferably at least about 90%, 91%, 92%, 93%, 94%, most preferably at least about 95%, 96%, 97%, 98% or 99% amino acid sequence identity. Such variants include, for example, APP polypeptides in which one or more amino acid residues are added to or deleted from the N-terminus or C-terminus of the full-length or mature sequence of APP, or APP polypeptides in which one or more amino acid residues are inserted or deleted from the internal sequence or domain of the polypeptide, including variants from other species, but excluding native-sequence APP polypeptides.

"DR 6" or "DR 6 receptor" includes receptors whose polynucleotide and polypeptide sequences are known in the art. The polynucleotide and polypeptide sequences of TNF receptor family members known as "DR 6" or "TR 9" have been described by Pan et al (Pan et al, FEBS Lett. (FEBS letters), 431: 351-. The human DR6 receptor is a 655 amino acid protein (SEQ ID NO: 1) having a putative signal sequence (amino acids 1-41), an extracellular domain (amino acids 42-349), a transmembrane domain (amino acids 350-369), followed by a cytoplasmic domain (amino acids 370-655). The cDNA sequence of DR6 was represented by SEQ ID NO: and 2, providing. As used herein, the term "DR 6 receptor" includes native sequence receptors and receptor variants. These terms include the DR6 receptor expressed in a variety of mammals, including humans. The DR6 receptor may be expressed endogenously as occurs naturally in various human tissue lineages, or may be expressed by recombinant or synthetic methods. "native sequence DR6 receptor" includes polypeptides having the same amino acid sequence as DR6 receptor that is derived from nature. Thus, the native sequence DR6 receptor may have the amino acid sequence of a naturally occurring DR6 receptor derived from any mammal (including a human). Such native sequence DR6 receptors may be isolated from nature or may be prepared by recombinant or synthetic methods. In particular, the term "native sequence DR6 receptor" includes naturally occurring truncated or secreted forms of the receptor (e.g., soluble forms comprising, for example, extracellular domain sequences), naturally occurring variant forms (e.g., alternatively spliced forms), and naturally occurring allelic variants. Receptor variants may include fragments or deletion mutants of the native sequence DR6 receptor.

The term "extracellular domain" or "ECD" refers to a form of the DR6 receptor that is substantially free of transmembrane and cytoplasmic domains. Typically, soluble ECDs will have less than 1% of such transmembrane and cytoplasmic domains, and preferably will have less than 0.5% of such domains. It is to be understood that any identified transmembrane domain of a polypeptide of the invention is identified according to the criteria commonly employed in the art for identifying hydrophobic domains of the type in question. The precise boundaries of the transmembrane domain may vary, but most likely do not exceed about 5 amino acids at either end of the initially identified domain. In a preferred embodiment, the ECD will consist of a soluble, extracellular domain sequence of the polypeptide that is free of transmembrane and cytoplasmic or intracellular domains (and does not bind to the membrane).

The term "DR 6 variant" refers to a DR6 polypeptide as defined below, which binds to a polypeptide having the amino acid sequence shown in seq id NO: 1 or a soluble fragment thereof or a lysable exodomain thereof has at least about 80%, preferably at least about 85%, 86%, 87%, 88%, 89%, more preferably at least about 90%, 91%, 92%, 93%, 94%, most preferably at least about 95%, 96%, 97%, 98% or 99% amino acid sequence identity. Such variants include, for example, those wherein the amino acid sequence set forth to SEQ ID NO: 1, or a full-length or mature sequence with one or more amino acid residues added to the N-terminus or C-terminus of the sequence selected from SEQ ID NO: 1, or DR6 polypeptide in which one or more amino acid residues are inserted into or deleted from an internal sequence or domain of the polypeptide, including variants from other species, but excluding native-sequence DR6 polypeptide. Typically, the DR6 variant comprises a polypeptide comprising SEQ ID NO: 1 or 42-349 and has a soluble form of DR6 receptor with up to 10 conservative amino acid substitutions. Preferably, such variants act as DR6 antagonists as defined below.

The term "DR 6 antagonist" is used in the broadest sense and includes any molecule that partially or completely blocks, inhibits or neutralizes the ability of the DR6 receptor to bind to its cognate ligand (preferably, its cognate ligand APP) or to activate one or more intracellular signaling or intracellular signaling pathways in neuronal cells or tissues in vitro, in situ, in vivo or ex vivo (ex vivo). For example, a DR6 antagonist may partially or completely block, inhibit or neutralize the ability of DR6 receptor to activate one or more intracellular signals or intracellular signaling pathways in a neuronal cell or tissue that lead to apoptosis or cell death in the neuronal cell or tissue. DR6 antagonists may function by a variety of mechanisms to partially or completely block, inhibit, or neutralize the action of DR6, including, but not limited to, by blocking, inhibiting, or neutralizing binding of cognate ligands to DR6, formation of complexes between DR6 and its cognate ligands (e.g., APP), oligomerization of the DR6 receptor, formation of complexes between the DR6 receptor and heterologous co-receptors, binding of cognate ligands to the DR6 receptor/heterologous co-receptor complex, or formation of complexes between the DR6 receptor, heterologous co-receptors, and their cognate ligands. DR6 antagonists may function in a direct or indirect manner. DR6 antagonists contemplated by the present invention include, but are not limited to: APP antibodies, DR6 antibodies, immunoadhesins, DR6 immunoadhesins, DR6 fusion proteins, covalently modified forms of DR6, DR6 variants and fusion proteins thereof, or more advanced oligomeric forms (dimeric, aggregate) of DR6 or homo-or hetero-polymeric forms of DR6, small molecules such as pharmacological inhibitors of JNK signaling cascade amplification (including small molecules and peptide inhibitors of Jun N-terminal kinase JNK activity), pharmacological inhibitors of protein kinase MLK and MKK activity that function upstream of JNK in the signal transduction pathway, pharmacological inhibitors of JNK binding to the scaffold protein jis p-1, pharmacological inhibitors of JNK binding to its substrates such as c-Jun or AP-1 transcription factor complexes, pharmacological inhibitors of JNK-mediated phosphorylation of its substrates such as JNK Binding Domain (JBD) peptides and/or phosphorylation of the substrate binding domain of JNK and/or pharmacological inhibitors of phosphorylation of peptides including JNK substrate phosphorylation sites Agents, small molecules that block ATP binding to JNK, and small molecules that block substrate binding to JNK.

To determine whether a DR6 antagonist partially or completely blocks, inhibits, or neutralizes the ability of a DR6 receptor to activate one or more intracellular signals or intracellular signaling pathways in a neuronal cell or tissue, an assay can be performed to assess the effect of a DR6 antagonist on, for example, various neuronal cells or tissues and in an in vivo model. Various assays can be performed in known in vitro or in vivo assay formats, such as those described below or known in the art and described in the literature. One embodiment of an assay to determine whether a DR6 antagonist partially or completely blocks, inhibits or neutralizes the ability of a DR6 receptor to activate one or more intracellular signals or intracellular signaling pathways in a neuronal cell or tissue comprises: binding DR6 to APP in the presence or absence of a DR6 antagonist or a potential DR6 antagonist (i.e., molecule of interest); inhibition of DR6 binding to APP in the presence of this DR6 antagonist or a potential DR6 antagonist is then tested.

By "nucleic acid" is meant to include any DNA or RNA. For example, chromosomal nucleic acids, mitochondrial nucleic acids, viral nucleic acids, and/or bacterial nucleic acids present in a tissue sample. The term "nucleic acid" includes one or both strands of a double-stranded nucleic acid molecule and includes any fragment or portion of an entire nucleic acid molecule.

"Gene" refers to any nucleic acid sequence or portion thereof that has a functional role in encoding or transcribing a protein or regulating the expression of another gene. A gene may consist of all nucleic acids responsible for encoding a functional protein or only a portion of the nucleic acids responsible for encoding or expressing a protein. The nucleic acid sequence may comprise a genetic abnormality within an exon, an intron, an initiation or termination region, a promoter sequence, other regulatory sequences, or a unique contiguous region of a gene.

The terms "amino acids" and "amino acids" refer to all naturally occurring L-alpha-amino acids. This definition is intended to include norleucine, ornithine and homocysteine. Amino acids are identified by single letter or three letter names:

Asp

D

aspartic acid

Ile

I

Isoleucine

Thr

T

Threonine

Leu

L

Leucine

Ser

S

Serine

Tyr

Y

Tyrosine

Glu

E

Glutamic acid

Phe

F

Phenylalanine

Pro

P

Proline

His

H

Histidine

Gly

G

Glycine

Lys

K

Lysine

Ala

A

Alanine

Arg

R

Arginine

Cys

C

Cysteine

Trp

W

Tryptophan

Val

V

Valine

Gln

Q

Glutamine

Met

M

Methionine

Asn

N

Asparagine

When used to describe the various peptides or proteins disclosed herein, "isolated" refers to a peptide or protein that has been identified and isolated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are substances that would ordinarily interfere with diagnostic or therapeutic uses for peptides or proteins, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the peptide or protein will be purified (1) to an extent sufficient to obtain an N-terminal or internal amino acid sequence of at least 15 residues by using a rotary cup sequencer (spinning cup sequencer), or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver staining, or (3) to homogeneity by mass spectrometry or peptide localization (mapping) techniques. The isolated substance includes an in situ peptide or protein within a recombinant cell, since at least one component of the natural environment of the peptide or protein is not present. However, isolated peptides or proteins are typically prepared by at least one purification step.

"percent (%) amino acid sequence identity" with respect to sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with amino acid residues in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignments can be made to determine percent amino acid sequence identity using various methods within the skill in the art capable of determining the appropriate parameters to measure the alignment, including the alignment algorithm required to obtain the maximum alignment over the full-length sequences compared. To this end, however, the percent amino acid identity value may be obtained using the sequence comparison computer program ALIGN-2, the author of which is Genentech, inc, and the source code of which has been submitted with the user document to the U.S. copyright office, washington d.c., 20559, and registered with U.S. copyright registration accession number TXU 510087. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, Calif. All sequence alignment parameters were set by the ALIGN-2 program and were unchanged.

The term "primer" or "primers" refers to an oligonucleotide sequence that hybridizes to a complementary RNA or DNA target polynucleotide and serves as a point of initiation of polynucleotide stepwise synthesis from a single nucleotide by nucleotide transferases as occurs, for example, in the polymerase chain reaction.

The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. For example, control sequences suitable for prokaryotes include a promoter, an optional operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

A nucleic acid is "operably linked" if it is in a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of that sequence; alternatively, if the ribosome binding site is positioned to facilitate translation, it is operably linked to a coding sequence. Generally, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in the same reading frame. However, enhancers need not be contiguous. Ligation may be achieved by ligation reactions at convenient restriction sites. If there are no such sites, synthetic oligonucleotide adaptors or linkers can be used in accordance with conventional practice.

The word "label" as used herein refers to a compound or composition that is directly or indirectly conjugated or fused to an agent, such as a nucleic acid probe or antibody, and facilitates detection of the agent conjugated or fused thereto. The label may be detectable by itself (e.g., a radioisotope label or a fluorescent label), or, in the case of an enzymatic label, may catalyze chemical alteration of a detectable substrate compound or composition.

The term "immunoadhesin" as used herein refers to antibody-like molecules that combine the binding specificity of a heterologous protein (an "adhesin") with the effector functions of immunoglobulin constant domains. Structurally, immunoadhesins comprise fusions of amino acid sequences and immunoglobulin constant domain sequences having a desired binding specificity that is different from the antigen recognition and binding site of the antibody (i.e., is "heterologous"). The adhesin part of an immunoadhesin molecule is typically a contiguous amino acid sequence comprising at least the binding site for a receptor or ligand. The immunoglobulin constant domain sequence in immunoadhesins can be obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3 or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD, or IgM.

"DR 6 receptor antibody", "DR 6 antibody" or "anti-DR 6 antibody" are used in the broad sense and refer to antibodies that bind at least one form of DR6 receptor, preferably human DR6 receptor, such as the one shown in SEQ ID NO: 1 or an extracellular domain sequence thereof. Optionally, the DR6 antibody is fused or linked to a heterologous sequence or molecule. Preferably the heterologous sequence allows or facilitates formation of higher order or oligomeric complexes of the antibody. Specifically, the term "anti-DR 6 antibody" and grammatical equivalents thereof include the DR6 monoclonal antibody described below. Optionally, the DR6 antibody binds to the DR6 receptor but does not bind to or cross-reacts with any additional receptor of the tumor necrosis factor family (e.g., DR4, DR5, TNFR1, TNFR2, Fas). Optionally, the DR6 antibody of the invention binds to the DR6 receptor at a concentration of about 0.067nM to about 0.033 μ M as measured in a BIAcore binding assay.

The terms "anti-APP antibody", "APP antibody" and grammatical equivalents are used broadly and refer to an antibody that binds at least one form of APP, preferably human APP such as the APP polypeptide isoforms specifically described herein. Preferably, the APP antibody is a DR6 antagonist antibody. For example, in methods for preparing and/or identifying DR6 antagonists as disclosed herein, one or more isoforms of APP and/or portions thereof can be used as immunogens to immunize animals (e.g., mice, as part of the process of producing monoclonal antibodies) and/or as probes to screen libraries of compounds (e.g., recombinant antibody libraries). Typical APP polypeptides useful in embodiments of the invention include the following non-limiting examples. These exemplary forms may be selected for use in various embodiments of the present invention. In some embodiments of the invention, the APP polypeptide comprises a full-length APP isoform such as the isoforms shown in SEQ ID NOs: 3. APP in 4 and 5₆₉₅And/or APP₇₅₁And/or APP₇₇₀Isoforms. In other embodiments of the invention, the APP polypeptide comprises a post-translationally processed isoform of APP, e.g., an APP polypeptide that undergoes cleavage by a secretase such as α -secretase, β -secretase or γ -secretase (e.g., a soluble N-terminal fragment such as sAPP α or sAPP β). In thatIn related embodiments of the invention, the APP polypeptide may be selected to comprise one or more specific domains such as an N-terminal extracellular domain (see, e.g., Quast et al, FASEB J.2003; 17 (12): 1739-41), a heparin binding domain (see, e.g., Rossjohn et al, Nat.Struct.biol. (Nat-structural biology) 1999 Apr; 6 (4): 327-31), a copper II type (see, e.g., Hesse et al, FEBS Letters (FEBS communications) 349 (1): 109-116(1994)), or a Kunitz protease inhibitor domain (see, e.g., Ponte et al, Nature (Nature); 331 (6156): 525-7 (1988)). In some embodiments of the invention, APP polypeptides include sequences that are observed to comprise an epitope recognized by a DR6 antagonist (such as an antibody or DR6 immunoadhesin) disclosed herein, e.g., APP₆₉₅Amino acids 22 to 81 of (a); sequences comprising an epitope bound by monoclonal antibody 22C11 (see, e.g., Hilbich et al, J.biol. chem. (J.Biol.Chem.) 268 (35): 26571-one 26577 (1993)). In certain embodiments of the invention, the APP polypeptide does not comprise one or more specific domains or sequences, e.g., APP polypeptide that does not comprise a particular N-terminal or C-terminal amino acid, APP polypeptide that does not comprise a Kunitz protease inhibitory domain (e.g., APP₆₉₅) Or an APP polypeptide that does not comprise the Alzheimer's beta amyloid (A beta) sequence (e.g., sAPP beta, which is a polypeptide that does not comprise A beta)₄₀And/or Abeta₄₂Polypeptides of sequence) (see, e.g., Bond et al, j.struct Biol. (journal of structural biology) 2003 Feb; 141(2): 156-70). In other embodiments of the invention, the APP polypeptides used in embodiments of the invention comprise one or more domains or sequences and no other domains or sequences, e.g., APP polypeptides comprising an N-terminal extracellular domain (or at least a portion of which is observed to be bound by a DR6 antagonist such as monoclonal antibody 22C11) but not a domain or sequence C-terminal to one or more secretase cleavage sites such as a beta amyloid (Α β) sequence (e.g., sappa or sappp β). Optionally, the anti-APP antibody will inhibit the binding of the N-APP polypeptide to DR6 and bind the N-APP polypeptide at a concentration of 10 μ g/ml to 50 μ g/ml, as described herein, and/or as measured in a cell-based quantitative binding assay.

The term "antibody" is used herein in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.

"antibody fragments" include portions of intact antibodies, preferably comprising antigen binding or variable regions thereof. Examples of antibody fragments include Fab, Fab ', F (ab')₂And Fv fragments; diabodies (diabodies); a linear antibody; a single chain antibody molecule; and multispecific antibodies formed from antibody fragments.

A "natural antibody" is typically a heterotetrameric glycoprotein of about 150,000 daltons, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable domain at one end (V)_H) Followed by a number of constant domains. Each light chain has a variable domain at one end (V)_L) And a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the heavy chain variable domain. Specific amino acid residues are believed to form the interface between the light and heavy chain variable domains.

The term "variable" refers to the fact that certain portions of the variable domains differ widely in sequence among antibodies and are used for the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of the antibody. It is concentrated in three segments called hypervariable regions or complementarity determining regions in the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called Framework Regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, which largely adopt a β -sheet conformation, connected by three HVRs that form loops connecting, and in some cases forming part of, the β -sheet structure. The hypervariable regions in each chain are held together in close proximity by the FR regions and together with the hypervariable regions from the other chain contribute to the formation of the antigen-binding site of the antibody (see Kabat et al, Sequences of Proteins of Immunological Interest, 5 th edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in binding of the antibody to the antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cell-mediated cytotoxicity (ADCC).

Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each having a single antigen-binding site, and a residual "Fc" fragment, the name of which reflects its ability to crystallize readily. Pepsin treatment to yield F (ab')₂A fragment having two antigen combining sites and still being capable of cross-linking antigens.

"Fv" is the smallest antibody fragment that contains the entire antigen recognition and antigen binding site. This region consists of dimers of one heavy chain variable domain and one light chain variable domain in close, non-covalent association. In this configuration, the three hypervariable regions of each variable domain interact to define V_H-V_LAn antigen binding site on the surface of the dimer. In summary, six hypervariable regions confer antigen-binding specificity to an antibody. However, even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, albeit with a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chain and the first constant domain of the heavy chain (CH 1). Fab' fragments differ from Fab fragments by the addition of several residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab '-SH is the name given herein for Fab', in which the cysteine residues of the constant domains have at least one free thiol group. F (ab')₂Antibody fragments originally asThe Fab' fragment pair with the hinge cysteine between them was generated. Other chemical couplings of antibody fragments are also known.

The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be classified into one of two clearly distinguished types, called kappa (κ) and lambda (λ), based on the amino acid sequences of the constant domains of the antibodies (immunoglobulins).

Antibodies can be classified into different classes depending on the amino acid sequence of the constant domain of their heavy chains. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA 2. The heavy chain constant domains corresponding to different classes of antibodies are referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

"Single chain Fv" or "scFv" antibody fragments comprise the V of an antibody_HAnd V_LDomains, wherein these domains are present as a single polypeptide chain. Preferably, the Fv polypeptide is at V_HAnd V_LAlso included between the domains is a polypeptide linker that allows the scFv to form the structure required for antigen binding. For reviews on scFv see, for example, Pluckthun in The Pharmacology of monoclonal antibodies (monoclonal antibody Pharmacology), Vol 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp.269-315 (1994).

The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments are in the same polypeptide chain (V)_H-V_L) Comprising a variable domain (V) with a light chain_L) Linked heavy chain variable domains (V)_H). By using a linker that is too short to pair between two domains on the same chain, the domains are forced to pair with the complementary domains of the other chain to create two antigen binding sites. Diabodies are more fully described in e.g. EP 404,097; WO 93/11161; and Hollinger et al, national science of the United statesAcademy (proc.natl.acad.sci.usa) 90: 6444- > 6448 (1993).

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, each monoclonal antibody is directed against a single determinant on the antigen, in contrast to conventional (polyclonal) antibody preparations, which typically comprise different antibodies directed against different determinants (epitopes). In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by hybridoma culture and are uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used according to the invention may be produced by a method described by Kohler et al, Nature, 256: 495(1975) or can be prepared by recombinant DNA methods (see, for example, U.S. Pat. No.4,816,567). Also useful are, for example, the following methods described in Clackson et al, Nature, 352: 624-: 581-597(1991) to isolate "monoclonal antibodies" from phage antibody libraries.

Monoclonal antibodies specifically include "chimeric" antibodies (immunoglobulins) wherein a portion of the heavy and/or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No.4,816,567; Morrison et al, Proc. Nat. Acad. Sci. USA 81: 6851-. Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen binding sequences derived from a non-human primate (e.g., Old World Monkey, such as baboon, rhesus Monkey, or cynomolgus Monkey (cynomolgus Monkey)) and human constant region sequences (U.S. patent No. 5,693,780).

"humanized" forms of non-human (e.g., murine) antibodies refer to chimeric antibodies that contain minimal sequences derived from non-human immunoglobulins. For the most part, humanized antibodies refer to human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and capacity. In some instances, Framework Region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further improve the performance of the antibody. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For more details see Jones et al, Nature 321: 522-525 (1986); riechmann et al, Nature 332: 323-329 (1988); and Presta, modern structural biology perspective (curr. op. struct. biol.) 2: 593-596(1992).

The term "hypervariable region" when used herein refers to the amino acid residues of an antibody which are responsible for binding to an antigen. The hypervariable regions comprise amino acid residues from the "complementarity determining regions" or "CDRs" (e.g.residues 24-34(L1), 50-56(L2) and 89-97(L3) in the light chain variable domain, and 31-35(H1), 50-65(H2) and 95-102(H3) in the heavy chain variable domain; Kabat et al, protein Sequences of Immunological Interest (Sequences of Proteins of biological Interest), 5 th edition. Public Health Service, National institute of Health, Bethesda, MD (1991)) and/or those residues from "hypervariable loops" (e.g.residues 26-32(L1), 50-52(L2) and 91-96(L3) in the light chain variable domain, and 26-32(H1), 53-55(H2) and 96 (H3) in the heavy chain variable domain; Bioski molecules: 901. 917J. (1987). "framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as defined herein.

An antibody that "binds" an antigen of interest is an antibody that is capable of binding to the antigen with sufficient affinity and/or avidity such that the antibody can be used as a diagnostic or therapeutic agent for targeting a protein or a cell expressing the antigen.

As used herein, "immunotherapy" shall refer to a method of treating a mammal (preferably a human patient) with an antibody, wherein the antibody may be unconjugated or "naked" or the antibody may be conjugated or fused to a heterologous molecule or agent (such as one or more cytotoxic agents), thereby producing an "immunoconjugate".

An "isolated" antibody refers to an antibody that has been identified and separated from and/or recovered from a component of its natural environment. Contaminating components of the natural environment of an antibody are substances that would interfere with its diagnostic or therapeutic use, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody is purified to (1) greater than 95% by weight of the antibody, and most preferably greater than 99% by weight of the antibody, as determined by, for example, the Lowry method, (2) to an extent sufficient to obtain an N-terminal or internal amino acid sequence of at least 15 residues by using a rotor sequencer, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using, for example, Coomassie blue or, preferably, silver staining. Isolated antibodies include antibodies in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. However, isolated antibodies are typically prepared by at least one purification step.

The term "tagged" when used herein refers to a chimeric molecule comprising an antibody or polypeptide fused to a "tag polypeptide". The tag polypeptide has enough residues to provide an epitope against which an antibody can be made, or to provide some other function, such as the ability to oligomerize (such as occurs when a peptide has a leucine zipper domain), but is short enough that it does not normally interfere with the activity of the antibody or polypeptide. The tag polypeptide is also preferably quite unique such that the tag-specific antibody does not substantially cross-react with other epitopes. Suitable tag polypeptides typically have at least 6 amino acid residues and typically between about 8 to about 50 amino acid residues (preferably between about 10 to about 20 residues).

The term "Fc receptor" or "FcR" is used to describe a receptor that binds the Fc region of an antibody. Preferably the FcR is a native sequence human FcR. In addition, a preferred FcR is one that binds an IgG antibody (gamma receptor) and includes receptors of the Fc γ RI, Fc γ RII, and Fc γ RIII subclasses, including allelic variants and alternatively spliced forms of these receptors. Fc γ RII receptors include Fc γ RIIA ("activating receptor") and Fc γ RIIB ("inhibiting receptor"), which have similar amino acid sequences, differing primarily in their cytoplasmic domains. The activating receptor Fc γ RIIA comprises in its cytoplasmic domain an immunoreceptor tyrosine-based activation motif (ITAM). The inhibitory receptor Fc γ RIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see e.g. seqAnnu.rev.immunol. (annual assessment of immunology) 15: 203-234(1997)). An overview of fcrs is found in ravatch and Kinet, annu.rev.immunol (annual assessment of immunology) 9: 457-92 (1991); capel et al, immunolmethods (immunization methods) 4: 25-34 (1994); and de Haas et al, j.lab.clin.med. (journal of laboratory clinical medicine) 126: 330-41(1995). The term "FcR" encompasses other fcrs herein, including those that will be identified in the future. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al, j. immunol. (journal of immunology) 117: 587(1976) and Kim et al, j. immunol. (journal of immunology) 24: 249 (1994)). FcR herein includes polymorphisms such as genetic polymorphisms of the gene encoding Fc γ RIIIa, which result in ammonia in the IgG1 binding region located in the receptorAt amino acid position 158 is either phenylalanine (F) or valine (V). Homozygous valine Fc γ RIIIa (Fc γ RIIIa-158V) has been shown to have higher affinity for human IgG1 and mediate increased ADCC in vitro relative to homozygous phenylalanine Fc γ RIIIa (Fc γ RIIIa-158F) or heterozygous (Fc γ RIIIa-158F/V) receptors.

The term "polyol" as used herein broadly refers to polyol compounds. The polyol may be any water-soluble poly (oxyalkylene) polymer and may, for example, have a linear or branched chain. Preferred polyols include those substituted at one or more hydroxyl positions with a chemical group such as an alkyl group having one to four carbons. Typically, the polyol is a poly (alkylene glycol), preferably poly (ethylene glycol) (PEG). However, those skilled in the art recognize that other polyols, such as, for example, poly (propylene glycol) and polyethylene-polypropylene glycol copolymers, may be employed using the conjugation techniques described herein for PEG. The polyols include those well known in the art and those available to the public, such as from commercially available sources such asCorporation。

The term "conjugated" is used herein in accordance with its broadest definition to mean bound or linked together. Molecules are "conjugated" when they behave or function as if they are bound together.

The phrase "effective amount" refers to an amount of an agent (e.g., DR6 antagonist, etc.) that is effective for preventing, ameliorating, or treating the disease or disorder in question. It is expected that DR6 antagonists of the invention will be useful in increasing dendritic spine density and maintaining PSD-95.

The terms "treating", "treatment" and "treatment", as used herein, refer to therapeutic treatment, prophylactic treatment and prophylactic therapy. Continuous treatment or administration refers to treatment on at least a daily basis, without one or more day interruptions in the treatment. Intermittent treatment or administration, or treatment or administration in an intermittent manner, refers to treatment that is not continuous but periodic in nature.

As used herein, the term "disease" generally refers to any condition that would benefit from treatment with a DR6 antagonist described herein. This includes chronic and acute diseases, as well as pathological conditions that predispose mammals to such diseases.

"neuronal cell or tissue" generally refers to motor neurons, interneurons (including but not limited to commissural neurons), sensory neurons (including but not limited to dorsal root ganglion neurons), nigral Dopamine (DA) neurons, striatal DA neurons, cortical neurons, brainstem neurons, spinal interneurons and motor neurons, hippocampal neurons (including but not limited to hippocampal CA1 pyramidal neurons), and forebrain neurons. The term neuronal cell or tissue is intended herein to refer to a neuronal cell consisting of a cell body, one or more axons and one or more dendrites, as well as to one or more axons or one or more dendrites that may form part of such a neuronal cell.

"psychiatric disorder" as used herein refers to conditions including diseases such as schizophrenia (schizophrenia) and addiction (addictions). "cognitive disorders" include diseases such as autism (autism), Tourette Syndrome (Tourette Syndrome), Rett Syndrome (Rett Syndrome) and Fragile-X Syndrome mental retardation (Fragile-X Syndrome). "

"subject" or "patient" refers to any individual subject, including humans, in need of treatment. Also intended to be included as subjects are any subjects involved in clinical study trials that do not show any clinical signs of disease, or subjects involved in epidemiological studies, or subjects used as controls.

The term "mammal" as used herein refers to any mammal classified as a mammal, including humans, cows, horses, dogs, and cats. In a preferred embodiment of the invention, the mammal is a human.

Chemical synapses connect neurons to form functional circuits that can process and store information. Loss of proper function or stability of these linkages is thought to be responsible for numerous psychiatric and cognitive disorders. Loss of or instability of dendritic spines, as well as changes in dendritic spine-associated proteins such as PSD-95, are believed to be associated with diseases such as rett syndrome, tourette syndrome, schizophrenia, autism, addiction, and fragile X syndrome.

Applicants have unexpectedly found that the TNFR family member DR6 is abundantly expressed in embryonic and mature central nervous systems, including the cerebral cortex, hippocampus, motor neurons and interneurons of the spinal cord. As described in the examples below, applicants conducted various experimental assays to examine the role of DR6 in synaptic stability in vivo.

Applicants further speculate that part of the amyloid precursor protein (N-APP) is a cognate ligand for the DR6 receptor, and thus APP also plays a role in synaptic stability. In a recent article by Bittner et al (Bittner, T. et al (2009) J. Neurosci. (J. neuroscience) 29 (33): 10405-^+/-Higher in mice than in wild-type mice, and in APP^-/-Even higher in mice. Amyloid precursor protein has previously been postulated to play a role in Alzheimer's disease (although not fully understood) (Selkoe, J.biol. chem. (J.Biol.Chem.) 271: 18295 (1996); Scheuner; et al, Nature Med. (Nature-medicine) 2: 864 (1996); Goate et al, Nature 349: 704 (1991)).

DR6 and/or APP inhibitors are believed to be particularly useful in the treatment of various psychiatric and cognitive disorders. Such inhibitors may also be used to enhance cognition or maintain cognition during the aging process.

Accordingly, the present invention provides DR6 and/or APP antagonist compositions and methods for inhibiting, blocking or neutralizing DR6 and/or APP activity in a mammal comprising administering an effective amount of a DR6 and/or APP antagonist. Preferably, the amount of DR6 and/or APP antagonist employed will be an amount effective to increase dendritic spine density and maintain healthy synapses. The amount of antagonist employed may also increase expression of PDS-95 in the dendritic spines or enhance retention of PDS-95 in the dendritic spines. In some cases, it may be beneficial to employ an antagonist of p75 in conjunction with or separate from DR6 and/or APP antagonists.

DR6 antagonists that may be employed in the methods include, but are not limited to, DR6 and/or APP immunoadhesins, fusion proteins comprising DR6 and/or APP, covalently modified forms of DR6 and/or APP, DR6 and/or APP variants, fusion proteins thereof, and DR6 and/or APP antibodies. P75 antagonists that may be employed in the methods include, but are not limited to, p75 immunoadhesins, fusion proteins comprising p75, covalently modified forms of p75, p75 variants, fusion proteins thereof, and p75 antibodies. The anti-p 75 antibody can be any known in the art. The protein sequence of p75 is provided as SEQ ID NO: 6. various techniques are described herein that can be used to prepare antagonists. For example, methods and techniques for making DR6, p75, and APP polypeptides are described. Further alterations to DR6, p75, and APP polypeptides, and antibodies to DR6, p75, and APP are also described.

There are many embodiments of the invention disclosed herein. The present invention provides methods of inhibiting binding of DR6 to APP comprising exposing a DR6 polypeptide and/or APP polypeptide to one or more DR6 antagonists under conditions wherein binding of DR6 to APP is inhibited. A related embodiment of the invention provides a method of inhibiting expression of a polypeptide comprising SEQ ID NO: 1 and a DR6 polypeptide comprising amino acids 1-655 of SEQ ID NO: 3 (e.g., sAPP β), comprising binding a DR6 polypeptide and an APP polypeptide to an isolated antagonist that binds DR6 or APP, wherein the isolated antagonist is selected from at least one of: an antibody that binds APP, an antibody that binds DR6, and a polypeptide comprising SEQ ID NO: 1, amino acids 1-354 of soluble DR6 polypeptide; and selecting the isolated antagonist according to its ability to inhibit binding of DR6 to APP; so that binding of DR6 to APP is inhibited.

The present invention also provides methods of inhibiting binding of DR6 to APP and inhibiting binding of p75 to APP comprising exposing DR6 polypeptide, p75 polypeptide and optionally APP polypeptide to one or more DR6 antagonists and one or more p75 antagonists under conditions wherein binding of DR6 and p75 to APP is inhibited. A related embodiment of the invention provides a method of inhibiting a polypeptide comprising SEQ id no: 1 and a DR6 polypeptide comprising amino acids 1-655 of SEQ ID NO: 3 (e.g., sAPP β), the method comprising binding a DR6 polypeptide and an APP polypeptide to an isolated antagonist of binding DR6 or APP and an antagonist of binding p75, wherein the isolated DR6 antagonist is selected from at least one of: an antibody that binds APP, an antibody that binds DR6, and a polypeptide comprising SEQ ID NO: 1, amino acids 1-354 of soluble DR6 polypeptide; and selecting the isolated DR6 antagonist according to its ability to inhibit binding of DR6 to APP; so that binding of DR6 to APP is inhibited. The isolated p75 antagonist is selected from at least one of: antibodies that bind p75 and soluble p75 polypeptides comprising amino acids of the extracellular domain of p75 (e.g., amino acids 29-250 of SEQ ID NO: 6); and selecting the isolated p75 antagonist based on its ability to inhibit the binding of p75 to APP; so that binding of p75 to APP is inhibited.

Optionally in such methods, the one or more DR6 antagonists are selected from antibodies that bind DR6 (e.g., an antibody that binds DR6 competitively inhibits binding of the 3f4.4.8, 4b6.9.7, or 1e5.5.7 monoclonal antibodies produced by the hybridoma cell lines deposited as ATCC accession nos. PTA-8095, PTA-8094, or PTA-8096, respectively), antibodies comprising SEQ ID NOs: 1, or a soluble DR6 polypeptide (e.g., DR6 immunoadhesin) or an antibody that binds APP (e.g., monoclonal antibody 22C 11). In certain embodiments of the invention, the DR6 antagonist is an antibody that binds DR6, an antibody that binds APP, or a soluble DR6 polypeptide linked to one or more non-proteinaceous polymers selected from the group consisting of: polyethylene glycol, polypropylene glycol and polyoxyalkylene. The p75 antagonist can also be linked to one or more non-proteinaceous polymers selected from the group consisting of: polyethylene glycol, polypropylene glycol and polyoxyalkylene.

In optional embodiments of these methods, DR6 polypeptide (alone or in combination with a p75 polypeptide) is expressed on the cell surface of one or more mammalian cells (e.g., a commissural neuron cell, a sensory neuron cell, or a motor neuron cell), and binding of the one or more DR6 antagonists and/or p75 antagonists inhibits DR6 activation or signaling and/or p75 activation or signaling.

In further embodiments of the invention, methods of inhibiting binding of DR6 (and optionally p75) to APP can be performed in vivo in a mammal having a psychiatric disease or disorder or a cognitive disease. Optionally, the psychiatric disease or disorder is schizophrenia or addiction. Alternatively, the cognitive disease or disorder comprises tourette's syndrome, retter's syndrome, fragile X syndrome, or autism.

A further embodiment of the invention provides a method for treating a mammal having a disease or disorder, comprising administering to the mammal an effective amount of one or more DR6 antagonists (alone or in combination with one or more p75 antagonists). Typically, in such methods, the one or more DR6 antagonists is selected from an antibody that binds DR6, an antibody comprising SEQ ID NO: 1 amino acids 1-354, DR6 immunoadhesin, and antibodies that bind APP. The one or more p75 antagonists is selected from the group consisting of an antibody that binds p75, a p75 immunoadhesin, and a peptide comprising SEQ ID NO: 6, amino acids 29-250. In an optional embodiment of the invention, the disorder or disease is autism, fragile X syndrome, rett syndrome, tourette syndrome, addiction and schizophrenia. In various embodiments of the invention, one or more additional therapeutic agents are administered to the mammal. In certain exemplary embodiments of the invention, the one or more additional therapeutic agents are selected from NGF, apoptosis inhibitors, EGFR inhibitors, β -secretase inhibitors, γ -secretase inhibitors, cholinesterase inhibitors, anti-a β antibodies, and NMDA receptor antagonists. Optionally, the one or more DR6 antagonists, p75 antagonists and/or additional therapeutic agents are administered to the mammal via injection, infusion or perfusion.

In addition to the full-length native sequences DR6, p75, and APP polypeptides described herein, it is also contemplated that DR6, p75, and APP polypeptide variants can be prepared. DR6, p75 and/or APP variants can be prepared by introducing appropriate nucleotide changes into the encoding DNA and/or by synthesizing the desired polypeptide. One skilled in the art will appreciate that amino acid changes may alter post-translational processing of DR6, p75, and/or APP polypeptides, such as changes in the number or position of glycosylation sites or changes in membrane anchors.

Variations of DR6, p75, and/or APP polypeptides described herein can be made, for example, using any of the techniques and guidance for conservative or non-conservative mutations described in, for example, U.S. patent No. 5,364,934. A variation may be a substitution, deletion, or insertion of one or more codons encoding a polypeptide that results in a change in the amino acid sequence compared to the native sequence polypeptide. Optionally, the variation is by replacement of at least one amino acid with any other amino acid in one or more domains of the DR6, p75, and/or APP polypeptide. Guidance in determining which amino acid residues may be inserted, substituted or deleted without adversely affecting the desired activity can be found by comparing the sequence of the DR6, p75 and/or APP polypeptide to known homologous protein molecules and minimizing the number of amino acid sequence changes made in regions of high homology. Amino acid substitutions may be the result of substituting one amino acid for another with similar structural and/or chemical properties, such as substituting leucine for serine, i.e., conservative amino acid substitutions. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. The allowed variation can be determined by systematically making amino acid insertions, deletions or substitutions in the sequence and testing the resulting variants for DR6, p75 and/or APP antagonist activity.

Provided herein are DR6, p75, and/or APP polypeptide fragments. Such fragments may be truncated at the N-or C-terminus, or intervening residues may be deleted, for example when compared to the full-length native protein. Certain fragments lack amino acid residues that are not essential for the biological activity of the desired DR6 polypeptide.

DR6, p75, and/or APP polypeptide fragments may be prepared by any of a variety of conventional techniques. The desired peptide fragment may be chemically synthesized. Alternative methods involve producing polypeptide fragments by enzymatic digestion, for example, by treating the protein with enzymes known to cleave proteins at sites defined by specific amino acid residues, or by digesting the DNA with appropriate restriction enzymes and isolating the desired fragments. Yet another suitable technique involves the isolation and amplification of a DNA fragment encoding a desired polypeptide fragment by Polymerase Chain Reaction (PCR). Oligonucleotides that define the desired ends of the DNA fragment are used in the 5 'and 3' primers in PCR.

In particular embodiments, conservative substitutions of interest are shown in the following table under the heading of preferred substitutions. If these substitutions result in a change in biological activity, further changes, designated as typical substitutions in the tables or as further described below according to amino acid classification, are introduced and the products screened.

Substantial changes in functional or immunological identity of DR6, p75 and/or APP polypeptides are achieved by selecting substitutions that differ significantly in their effect on maintaining: (a) the structure of the polypeptide backbone in the displaced region, e.g., a conformation such as a sheet or helix, (b) the charge or hydrophobicity of the molecule at the site of interest, or (c) the volume of the side chain. Based on common side chain properties, naturally occurring residues are divided into the following groups:

(1) and (3) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilicity: cys, ser, thr;

(3) acidity: asp, glu;

(4) alkalinity: asn, gln, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions would require the exchange of members of one of these classes for another. These substituted residues may also be introduced at conservative substitution sites, more preferably at the remaining (non-conservative) sites.

These variations can be performed using methods known in the art such as oligonucleotide-mediated (directed) mutagenesis, alanine scanning and PCR mutagenesis. Site-directed mutagenesis (Carter et al, Nucl. acids Res. (nucleic acids Res.), 13: 4331 (1986); Zoller et al, Nucl. acids Res. (nucleic acids Res.), 10: 6487(1987)), cassette mutagenesis (Wells et al, Gene, 34: 315(1985)), restriction selection mutagenesis (Wells et al, Philos. Trans. R. Soc. London Sera (Nature science journal A series, London SerA, Royal society of Nature science, Japan), 317: 415(1986)), or other known techniques can be performed on the cloned DNA to prepare DR6 polypeptide variant DNA.

Scanning amino acid analysis can also be used to identify one or more amino acids along adjacent sequences. In a preferred scan, the amino acids are relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine and cysteine. Generally, alanine is the preferred scanning amino acid in this group because it excludes the side chain after the beta carbon and makes it almost impossible to alter the backbone conformation of the variant (Cunningham and Wells, Science 244: 1081-1085 (1989)). Alanine is also generally preferred because it is the most common amino acid. Furthermore, it is frequently found both in buried and exposed locations (Creighton, THE PROTEINS (PROTEINS), (W.H.Freeman & Co., N.Y.); Chothia, J.mol.biol. (J.Mol.mol., 150: 1 (1976)). An iso-amino acid (isotericamine acid) may be used if the alanine substitution does not result in a sufficient amount of the variant.

Any cysteine residues not involved in maintaining the proper conformation of DR6, p75, and/or APP polypeptides may also be generally replaced by serine to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, one or more cysteine bonds may be added to DR6, p75, and/or APP polypeptide to improve its stability.

Embodiments of the invention disclosed herein are applicable to a variety of APP polypeptides. For example, in certain embodiments of the invention APP is the peptide shown in SEQ ID NO: full length 695, 750, or 770 APP isoforms in 3-5. In other embodiments of the invention, APP comprises the n-terminal portion of APP, which has the APP ectodomain and is generated from a post-translational processing event (e.g., sAPP α or sAPP β). Optionally, for example, the APP may comprise a soluble form of one of 695, 750 or 770 APP isoforms resulting from secretase cleavage, e.g., neuronal APP resulting from β -secretase cleavage₆₉₅In soluble form. In certain exemplary embodiments, the APP comprises APP₆₉₅Amino acids 20-591 (see, e.g., Jin et al, J.Neurosci., J.neuroscience, 14 (9): 5461-5470 (1994).) in another embodiment of the invention, APP comprises a polypeptide having an epitope recognized by monoclonal antibody 22C11 (e.g., available from Chemicon International Inc., Temecula, CA, U.S.A.)₆₉₅Residues 66-81 of (1), which is a region comprising the 22C11 epitope (see, e.g., Hilbrich, J.biol.chem. (J.Biol.Chem.) (268 (35): 26571-one 26577 (1993)).

The following description relates generally to the preparation of DR6, p75 and/or APP polypeptides by culturing cells transformed or transfected with a nucleic acid comprising a gene encoding DR6, p75 and/or APP polypeptides. Of course, it is contemplated that alternative methods well known in the art may be employed to prepare DR6, p75, and/or APP polypeptides. For example, suitable amino acid sequences or portions thereof can be prepared by direct peptide synthesis using SOLID phase techniques (see, e.g., Stewart et al, SOLID-PHASE PEPTIDE SYNTHESIS (SOLID phase peptide synthesis), W.H.Freeman Co., San Francisco, CA (1969); Merrifield, J.am.Chem.Soc. (J.Am.Chem., USA J.Chem., 85: 2149-. In vitro protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be accomplished, for example, using an Applied biosystems peptide Synthesizer (Foster City, Calif.) using the manufacturer's instructions. Chemical or enzymatic methods may be used, alone and in combination, to chemically synthesize different portions of DR6 and/or APP polypeptides to produce the desired DR6, p75 and/or APP polypeptides.

The methods and techniques are similarly useful for preparing DR6, p75, and/or APP variants, modified forms of DR6, p75, and/or APP; and DR6, p75, and/or APP antibodies. Isolation of DNA encoding DR6 and/or APP Polypeptides

DNA encoding DR6, p75, and/or APP polypeptide can be obtained from cDNA libraries prepared from tissues believed to have DR6, p75, and/or APP polypeptide mRNA and express it at detectable levels. Thus, human DR6, p75 and/or APP polypeptide DNA may be conveniently obtained from cDNA libraries prepared from human tissues. Genes encoding DR6, p75, and/or APP polypeptides can also be obtained from genomic libraries or by known synthetic methods (e.g., automated nucleic acid synthesis).

Libraries can be screened with probes (such as oligonucleotides having at least about 20-80 bases) designed to recognize a gene of interest or a protein encoded by a gene of interest. Standard methods such as those described in Sambrook et al, Molecular Cloning: screening of cDNA or genomic libraries with selected probes was performed as described in A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). An alternative method for isolating the gene encoding the DR6 polypeptide is to use the PCR method (Sambrook et al, supra; Dieffenbach et al, PCR Primer: A Laboratory Manual (PCR primers: A Laboratory Manual) (Cold Spring Harbor Laboratory Press, 1995)).

Techniques for screening cDNA libraries are well known in the art. Is selected asThe oligonucleotide sequence of the probe should be of sufficient length and sufficiently defined to minimize false positives. Preferably, the oligonucleotide is labeled such that it can be detected when hybridized to DNA in the library being screened. Methods of marking are known in the art and include the use of radioactive markers such as³²P-labeled ATP, biotinylated or enzyme-labeled. Hybridization conditions including medium and high stringency are provided in Sambrook et al, supra.

Sequences identified in such library screening methods can be compared and aligned with other known sequences deposited and available in public databases such as GenBank or other private sequence databases. Sequence identity (at the amino acid or nucleotide level) within a defined region of a molecule or across the entire full-length sequence can be determined using methods known in the art and as described herein.

A nucleic acid having a protein-encoding sequence can be obtained by: the selected cDNA or genomic library is screened using the deduced amino acid sequence first disclosed herein and, if necessary, conventional primer extension methods as described in Sambrook et al (supra) to detect precursors or processing intermediates of mRNA that may not be reverse transcribed into cDNA.

Selection and transformation of host cells

Host cells are transfected or transformed with expression or cloning vectors for DR6, p75, and/or APP polypeptide production as described herein and cultured in conventional nutrient media appropriately modified for induction of promoters, selection of transformants, or amplification of genes encoding sequences of interest. Culture conditions such as medium, temperature, pH, etc. can be selected by one of skill in the art without undue experimentation. Can be generally found in Mammalian Cell Biotechnology: the principles, protocols and Practical techniques for maximizing cell culture yields are found in the Practical Approach of mammalian cell biology, M.Butler editor (IRL Press, 1991) and Sambrook et al, supra.

Methods for transfection of eukaryotic cells and transformation of prokaryotic cells are known to those skilled in the art, e.g., CaCl₂、CaPO₄Liposome-mediated and electroporation. Depending on the host cell used, transformation is carried out using standard techniques appropriate for such cells. Calcium treatment or electroporation with calcium chloride as described in Sambrook et al (supra) is commonly used for prokaryotes. Infection with Agrobacterium tumefaciens (Agrobacterium tumefaciens) is used to transform certain plant cells, as described by Shaw et al, Gene, 23: 315(1983) and WO 89/05859 published on 29/6 in 1989. For mammalian cells without such cell walls, Graham and van der Eb, Virology, 52: 456-457 (1978). The general aspects of mammalian cell host system transfection have been described in U.S. Pat. No.4,399,216. Generally according to Van Solingen et al, j.bact. (journal of bacteriology), 130: 946(1977) and Hsiao et al, proc.natl.acad.sci.usa (proceedings of the american academy of sciences), 76: 3829(1979) for transformation into yeast. However, other methods for introducing DNA into cells may also be used, such as by nuclear microinjection, electroporation, fusion with bacterial protoplasts of intact cells, or polycations such as polybrene, polyornithine. For various techniques for transforming mammalian cells, see Keown et al, Methods in Enzymology, 185: 527- & 537(1990) and Mansour et al, Nature (Nature), 336: 348-352(1988).

Herein, suitable host cells for cloning or expressing the DNA in the vector include prokaryotic cells, yeast cells, or higher eukaryotic cells. Suitable prokaryotic cells include, but are not limited to, eubacteria, such as gram-negative or gram-positive organisms, e.g., Enterobacteriaceae (Enterobacteriaceae) such as e. Various E.coli strains are publicly available, such as E.coli K12 strain MM294(ATCC 31,446); escherichia coli X1776(ATCC 31,537); escherichia coli strains W3110(ATCC 27,325) and K5772 (ATCC 53,635). Other suitable prokaryotic host cells include Enterobacteriaceae such as Escherichia (Escherichia) e.g., Escherichia coli, Enterobacter, ErwiniaBacteria (Erwinia), Klebsiella (Klebsiella), proteobacteria (Proteus), Salmonella (Salmonella) such as Salmonella typhimurium, Serratia (Serratia) such as Serratia marcescens (Serratia marcescens), and Shigella (Shigella), and bacillus (bacillus) such as bacillus subtilis and bacillus licheniformis (e.g., bacillus licheniformis 41P disclosed in DD 266,710 published on 12.4.1989), Pseudomonas (Pseudomonas) such as Pseudomonas aeruginosa (p.aeruginosa), and Streptomyces (Streptomyces). These examples are illustrative and not restrictive. Strain W3110 is a particularly preferred host or parent host, since it is the usual host strain for fermentation of recombinant DNA products. Preferably, the host cell secretes a minimal amount of proteolytic enzymes. For example, strain W3110 can be altered to introduce genetic mutations in genes encoding endogenous proteins of the host, examples of such hosts include E.coli W3110 strain 1A2, which has the complete genotype tonA; coli W3110 strain 9E4, which has the complete genotype tonA ptr 3; escherichia coli W3110 strain 27C7(ATCC 55,244) having the complete genotype tonA ptr3 phoA E15(argF-lac)169degP ompT kan^r(ii) a Escherichia coli W3110 Strain 37D6, with the complete genotype tonA ptr3 phoA E15(argF-lac)169degP ompTs rbs7 ilvG kan^r(ii) a Coli W3110 strain 40B4, which is kanamycin-free and harbors a degP deletion mutant strain 37D 6; and E.coli strain with mutant periplasmic protease as disclosed in U.S. Pat. No.4,946,783 issued on 8/7/1990. Alternatively, in vitro cloning methods such as PCR or other nucleic acid polymerase reactions are suitable.

In addition to prokaryotic cells, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding DR6 polypeptides. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Others include Schizosaccharomyces pombe (Beich and Nurse (1981) Nature (Nature), 290: 140; EP 139,383 published on 5.5.2.1985); kluyveromyces (Kluyveromyces) hosts (U.S. Pat. No.4,943,529; Fleer et al, Bio/Technology (Biotechnology), 9: 968-; yarrowia (EP 402,226); pichia pastoris (Pichia pastoris) (EP183,070; Sreekrishna et al, J.basic Microbiol. (J.basic microbiology), 28: 265-278 (1988)); candida (Candida); trichoderma reesei (Trichoderma reesei) (EP 244,234); neurospora crassa (Neurospora crassa) (Case et al, Proc. Natl. Acad. Sci. USA, Proc. Sci. USA, 76: 5259-; schwanniomyces such as Schwanniomyces occidentalis (Schwanniomyces occidentalis) (published in EP394,538 at 31/10/1990); and filamentous fungi such as, for example, Neurospora (Neurospora), Penicillium (Penicillium), Beauveria bassiana (Tolypocladium) (WO 91/00357 published 1/10 1991) and Aspergillus (Aspergillus) hosts such as Aspergillus nidulans (A. nidulans) (Ballance et al (1983), biochem. Biophys. Res. Commun. (Biochemical and biophysical research communication), 112: 284. 289; Tilburn et al (1983), Gene (Gene), 26: 205. 221; Yelton et al (1984), Proc. Natl. Acad. Sci. USA (Proc. Natl. Acad. Sci., USA), 81: 1470. 1474) and Aspergillus niger (Kelly and Hynes (1985), EMBO J. ScoM.475, European molecular biology society: 479). Methanotrophic yeast (methylotrophic yeast) is suitable herein and includes, but is not limited to, yeast capable of growing on methanol selected from the group consisting of: hansenula (Hansenula), Candida, Kloeckera (Kloeckera), Pichia, Saccharomyces, Torulopsis (Torulopsis), and Rhodotorula rubra (Rhodotorula). A list of specific species that are typical of such yeasts can be found in c.anthony, The biochemistry of Methylotrophs, 269 (1982).

Host cells suitable for expression of glycosylated DR6, p75, and/or APP polypeptides are derived from multicellular organisms. Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, and plant cells such as cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco. Numerous strains and variants of baculovirus have been identified as well as corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), drosophila melanogaster (drosophila melanogaster) and Bombyx mori (Bombyx mori). Various virus strains for transfection are publicly available, for example, the L-1 variant of Autographa californica (NPV) and the Bm-5 strain of Bombycis mori NPV, and such viruses may be used according to the invention as the virus herein, particularly for transfecting Spodoptera frugiperda cells.

However, vertebrate cells are of most interest, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are SV40 transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney lines (293 cells or 293 cells subcloned for growth in suspension culture, Graham et al, J.GenVirol (J.Gen.Virol.) 36: 59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); chinese hamster ovary cells/-DHFR (CHO, Urlaub et al, Proc. Natl. Acad. Sci. USA (Proc. Sci. USA) 77: 4216 (1980)); mouse Sertoli cells (TM4, Mather, biol. reprod.23: 243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); vero cells (VERO-76, ATCC CRL-1587); human cervical tumor cells (HELA, ATCCCCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL 51); TRI cells (Mather et al (1982) Annals N.Y.Acad.Sci. (annual book of New York academy of sciences) 383: 44-68); MRC 5 cells; FS4 cells; and the human hepatoma line (Hep G2).

Host cells are transformed with the above-described expression or cloning vectors for DR6 and/or APP polypeptide production and cultured in conventional nutrient media appropriately modified for induction of promoters, selection of transformants, or amplification of genes encoding the desired sequences.

Selection and use of replicable vectors

Nucleic acids encoding DR6, p75, and/or APP polypeptides (e.g., cDNA or genomic DNA) can be inserted into replicable vectors for cloning (amplification of DNA) or expression. A variety of vectors are publicly available. The vector may, for example, be in the form of a plasmid, cosmid, viral particle or phage. The appropriate nucleic acid sequence may be inserted into the vector by several methods. Typically, the DNA is inserted into the appropriate restriction enzyme sites using techniques known in the art. Vector components generally include, but are not limited to, one or more signal sequences, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors comprising one or more of these building blocks is carried out using standard ligation techniques known to those skilled in the art.

DR6, p75 and/or APP can be recombinantly produced not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. In general, the signal sequence may be a component of the vector, or it may be part of the DR6, p75, and/or APP polypeptide-encoding DNA that is inserted into the vector. The signal sequence may be, for example, a prokaryotic signal sequence selected from the group consisting of: alkaline phosphatase, penicillinase, lpp, or a heat-stable enterotoxin II leader. For yeast secretion, the signal sequence may be, for example, a yeast invertase leader, an alpha factor leader (including yeast and kluyveromyces alpha-factor leaders, the latter described in U.S. patent No. 5,010,182) or an acid phosphatase leader, a candida albicans (c.albicans) glucoamylase leader (published EP 362,179 on 4.4.1990), or a signal as described in WO 90/13646 published on 15.11.1990. In mammalian cell expression, mammalian signal sequences can be used for direct secretion of proteins, such as signal sequences for secreted polypeptides from the same or related species, as well as viral secretory leaders.

Both expression and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Such sequences are known for a variety of bacteria, yeasts and viruses. The origin of replication from plasmid pBR322 is suitable for most gram-negative bacteria, the 2. mu. plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) can be used for cloning vectors in mammalian cells.

Expression and cloning vectors will typically contain a selection gene, also referred to as a selectable marker. Typical selection genes encode proteins that (a) provide resistance to antibiotics or other toxins (e.g., ampicillin, neomycin, methotrexate, or tetracycline), (b) complement auxotrophic deficiencies, or (c) provide important nutrients not available from complex media, e.g., the gene encoding bacillus D-alanine racemase.

Examples of suitable selectable markers for mammalian cells are those that enable the identification of cells capable of uptake of DR6, p75, and/or APP polypeptide-encoding nucleic acids, such as DHFR or thymokinase. When wild-type DHFR is used, suitable host cells are CHO cell lines deficient in DHFR activity, such as Urlaub et al, proc.natl.acad.sci.usa (proceedings of the american academy of sciences), 77: 4216 (1980). A suitable selection Gene for use in yeast is the trp1 Gene present in the yeast plasmid YRp7 (Stinchcomb et al, Nature, 282: 39 (1979); Kingsman et al, Gene, 7: 141 (1979); Tschemper et al, Gene, 10: 157 (1980)). the trp1 gene provides a selectable marker for mutant strains of yeast that are unable to grow in tryptophan, e.g., ATCC No.44076 or PEP4-1(Jones, Genetics, 85: 12 (1977)).

Expression and cloning vectors typically comprise a promoter operably linked to a nucleic acid sequence encoding DR6, p75, and/or APP polypeptide to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are known. Promoters suitable for use with prokaryotic hosts include the beta-lactamase and lactose promoter systems (Chang et al (1978) Nature (Nature), 275: 615; Goeddel et al (1979) Nature (Nature), 281: 544), alkaline phosphatase, tryptophan (trp) promoter systems (Goeddel, nucleic acids Res. (nucleic acids Res., 8: 4057 (1980); EP 36,776), and hybrid promoters such as the tac promoter (deBoer et al, Proc. Natl. Acad. Sci. USA, Proc. Sci., 80: 21-25 (1983)). Promoters for use in bacterial systems will also include Shine-Dalgarno (S.D.) sequences operably linked to DNA encoding DR6, p75, and/or APP polypeptides.

Examples of promoter sequences suitable for use with yeast hosts include the promoter of 3-phosphoglycerate kinase (Hitzeman et al, J.biol.chem. (J.Biol.Chem., 255: 2073(1980)) or the promoters of other glycolytic enzymes (Hess et al, J.Adv.enzyme Reg. (J.Adv.Enzyme Reg. (J.Admin., 7: 149 (1968)), Holland, Biochemistry (Biochemistry), 17: 4900(1978)), such as the promoters of the following enzymes: enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters (inducible promoters with the additional advantage of controlling transcription by growth conditions) are the promoter regions of: alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Vectors and promoters suitable for use in yeast expression are further described in EP 73,657.

Transcription of DR6, p75 and/or APP polypeptides from vectors in mammalian host cells is controlled by, for example, promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published on 7/5 1989), adenoviruses such as adenovirus 2, bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis b virus and monkey virus 40(SV40), promoters obtained from heterologous mammalian promoters such as the actin promoter or the immunoglobulin promoter, and promoters obtained from heat shock promoters, provided that these promoters are compatible with the host cell system.

Transcription of DNA encoding DR6, p75 and/or APP polypeptides by higher eukaryotes may be enhanced by inserting enhancer sequences into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300bp, that act on a promoter to enhance its transcription. Many enhancer sequences from mammalian genes are currently known (globin, elastase, albumin, alpha-fetoprotein, and insulin). Typically, however, an enhancer from a eukaryotic cell virus will be used. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. Enhancers may be ligated into the vector at the 5 ' or 3 ' position of the DR6, p75, and/or APP polypeptide coding sequence, but are preferably located at the 5 ' site of the promoter.

Expression vectors for use in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also contain sequences necessary for termination of transcription and stabilization of the mRNA. Such sequences are typically obtained from the 5 'and (occasionally) 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions comprise nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the DR6 polypeptide.

Other methods, vectors and host cells suitable for the synthesis of DR6, p75 and/or APP polypeptides in recombinant vertebrate cell culture are described in Gething et al, Nature, 293: 620-625 (1981); mantei et al, Nature, 281: 40-46 (1979); EP117,060; and EP117,058.

Culturing host cells

Host cells for producing DR6, p75, and/or APP polypeptides of the invention can be cultured in a variety of media. Commercially available media such as Ham's F10(Sigma), minimum essential Medium ((MEM) (Sigma), RPMI-1640(Sigma), and Dulbecco's modified Eagle Medium ((DMEM), Sigma) are suitable for culturing host cells, furthermore, any of the media described in Ham et al, meth.Enz. (methods in enzymology) 58: 44(1979), Barnes et al, anal. biochem. (analytical biochemistry) 102: 255(1980), U.S. Pat. No.4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re.30,985 may be used as the host cell culture medium any of these media may be supplemented with hormones and/or other growth factors (such as insulin, transferrin or growth factors), salts (such as sodium chloride, calcium, magnesium, and phosphate buffers (such as HEPES), adenosine nucleotides, and thymine (such as thymidine), and adenosine (thymidine (HEP), and thymidine (HEP), Antibiotics (such as GENTAMYCIN)^TMDrugs), trace elements (defined as inorganic compounds usually present in final concentrations in the micromolar range) and glucose or equivalent energy sources. Any other necessary additives may also be included in appropriate concentrations known to those skilled in the art. Culture conditions such as temperature, pH, etc. are those previously used by the host cell selected for expression, and will be apparent to those skilled in the art.

Detecting gene amplification/expression

Gene amplification and/or expression in a sample can be measured directly, for example, by conventional Southern blotting, Northern blotting to quantify mRNA transcription (Thomas, Proc. Natl. Acad. Sci. USA (Proc. Natl. Acad. Sci., USA), 77: 5201-. Alternatively, antibodies may be employed which can recognize specific duplexes including DNA duplexes, RNA duplexes and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibody may in turn be labelled and the assay may be carried out while the duplex is bound to the surface, so that when the duplex is formed at the surface, the presence of antibody bound to the duplex can be detected.

Alternatively, gene expression can be measured by the following method: immunological methods such as immunohistochemical staining of cells or tissue sections and measurement of cell cultures or body fluids to directly quantify gene product expression. Antibodies useful for immunohistochemical staining and/or assaying sample fluids may be monoclonal or polyclonal and may be prepared in any mammal. Antibodies may be conveniently prepared against the native sequence DR6 polypeptide, or against synthetic peptides based on the DR6 sequences provided herein, or against foreign sequences fused to the DR6DNA and encoding specific antibody epitopes.

Purification of DR6 polypeptide

Various forms of DR6, p75, and/or APP polypeptides can be recovered from the culture medium or host cell lysate. If membrane bound, it may be released from the membrane using a suitable detergent solution (e.g.Triton-X100) or by enzymatic cleavage. Cells for expression of DR6 polypeptides can be disrupted by a variety of physical or chemical methods, such as freeze-thaw cycles, sonication, mechanical disruption, or cell lysis reagents.

It may be desirable to purify DR6, p75, and/or APP polypeptides from recombinant cellular proteins or polypeptides. The following methods are examples of suitable purification methods: separation by fractionation on an ion exchange column; ethanol precipitation; reversed phase HPLC; chromatography on silica or on a cation exchange resin such as DEAE; carrying out chromatographic focusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; protein a Sepharose column to remove contaminants such as IgG; and a metal chelating column that binds an epitope-tagged form of DR6 and/or APP polypeptide. A variety of protein purification Methods can be used and are known in the art and described, for example, in Deutscher, Methods in enzymology, 182 (1990); scopes, Protein Purification: principlsand Practice (protein purification: principles and practices), Springer-Verlag, New York (1982). The purification step or steps selected will depend, for example, on the production method used and the nature of the particular DR6 polypeptide produced.

Soluble forms of DR6, p75, and/or APP may be used as DR6 antagonists or p75 antagonists in the methods of the invention. Such soluble forms of DR6, p75, and/or APP may comprise modifications, as described below (such as by fusion with an immunoglobulin, epitope tag, or leucine zipper). Immunoadhesin molecules are also contemplated for use in the methods herein. DR6, p75 and/or APP immunoadhesins may include DR6, p75 and/or APP in different forms, such as full-length polypeptides and DR6, p75 and/or APP or fragments thereof in the form of a lysable ectodomain. In particular embodiments, the molecule may comprise a fusion of the DR6 polypeptide with an immunoglobulin or a particular region of an immunoglobulin. For the bivalent form of immunoadhesin, such fusion may be to the Fc region of an IgG molecule. Ig fusion preferably involves the replacement of at least one variable region in an Ig molecule with a soluble (transmembrane domain deleted or inactivated) form of the polypeptide. In particularly preferred embodiments, the immunoglobulin fusion comprises the hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3 regions of an IgG1 molecule. For the preparation of immunoglobulin fusions see also U.S. Pat. No. 5,428,130 issued 6, 27 of 1995 and Chamow et al, TIBTECH, 14: 52-60(1996).

An optional immunoadhesin design binds one or more binding domains of an adhesin (e.g., DR6, p75, and/or APP extracellular domain) to the Fc region of an immunoglobulin heavy chain. Typically, when preparing an immunoadhesin of the present invention, the nucleic acid encoding the binding domain of the adhesin will be fused via the C-terminus to the nucleic acid encoding the N-terminus of the immunoglobulin constant domain sequence, however N-terminal fusions are also possible.

Typically, in such fusions, the encoded chimeric polypeptide will retain at least functionHinge of the constant region of an immunoglobulin heavy chain with activity, C_H2 and C_H3 domain. Fusion also occurs at the C-terminus of the Fc portion of the constant domain, or so that the N-terminus is immediately adjacent to the C of the heavy chain_H1 or the corresponding region of the light chain. The exact site at which fusion occurs is not critical; specific sites are known and can be selected to optimize the biological activity, secretion or binding properties of the immunoadhesin.

In a preferred embodiment, the adhesin sequence is fused to immunoglobulin G₁(IgG₁) N-terminal to the Fc region of (a). It is possible to fuse the entire heavy chain constant region to the adhesin sequence. More preferably, however, sequences are used in the fusion that start at the hinge region immediately upstream of the following sites: the papain cleavage site of IgG Fc (i.e., residue 216, the first residue of the heavy chain constant region being 114), or other immunoglobulin-like sites, is chemically defined. In a particularly preferred embodiment, the adhesin amino acid sequence is fused to (a) the hinge region and C of the IgG heavy chain_H2 and C_H3, or (b) C_H1. Hinge, C_H2 and C_H3 domain.

For bispecific immunoadhesins, the immunoadhesins are assembled into multimers, and in particular into heterodimers or heterotetramers. Typically, these assembled immunoglobulins will have a known unit structure. The basic four-chain building block is formed when IgG, IgD and IgE are present. The four-chain unit repeats in higher molecular weight immunoglobulins; IgM typically exists as a pentamer of four basic units held together by disulfide bonds. IgA globulin and occasionally IgG globulin can also be present in multimeric form in the serum. In the case of multimers, each of the four units may be the same or different.

Various exemplary assembled immunoadhesins within the scope of this disclosure are schematically illustrated below:

(a)AC_L-AC_L；

(b)AC_H-(AC_H、AC_L-AC_H、AC_L-V_HC_Hor V_LC_L-AC_H)；

(c)AC_L-AC_H-(AC_L-AC_H、AC_L-V_HC_H、V_LC_L-AC_HOr V_LC_L-V_HC_H)

(d)AC_L-V_HC_H-(AC_HOr AC_L-V_HC_HOr V_LC_L-AC_H)；

(e)V_LC_L-AC_H-(AC_L-V_HC_HOr V_LC_L-AC_H) (ii) a And

(f)(A-Y)_n-(V_LC_L-V_HC_H)₂，

wherein each A represents the same or different adhesin amino acid sequence;

V_Lis an immunoglobulin light chain variable domain;

V_His an immunoglobulin heavy chain variable domain;

C_Lis an immunoglobulin light chain constant domain;

C_His an immunoglobulin heavy chain constant domain;

n is an integer greater than 1;

y indicates the residue of the covalent cross-linker.

For the sake of brevity, the above-described structure shows only the main features; they do not show the linkage (J) or other domains of immunoglobulins, nor disulfide bonds. However, when such domains are required for binding activity, they should be considered to be present at the usual positions they occupy in immunoglobulin molecules.

Alternatively, the adhesin sequence may be inserted between the immunoglobulin heavy and light chain sequences, so that an immunoglobulin comprising a chimeric heavy chain is obtained. In this embodiment, the adhesin sequence is fused to the 3' end of the immunoglobulin heavy chain in each arm of the immunoglobulin, at the hinge and C_H2 between domains, or at C_H2 and C_H3 domain. Hoogenboom et al, mol. immunol. (molecular immunology), 28: 1027-1037(1991) reported similar constructs.

Although the presence of an immunoglobulin light chain in the immunoadhesin of the present invention is not required, the immunoglobulin light chain may be present as follows: covalently bound to an adhesin-immunoglobulin heavy chain fusion polypeptide, or directly fused to an adhesin. In the former case, the DNA encoding the immunoglobulin light chain is typically co-expressed with the DNA encoding the adhesin-immunoglobulin heavy chain fusion protein. When secreted, the hybrid heavy and light chains will covalently associate to provide an immunoglobulin-like structure comprising two disulfide-linked immunoglobulin heavy chain-light chain pairs. Methods suitable for producing such structures are disclosed, for example, in U.S. Pat. No.4,816,567 issued on 28.3.1989.

Immunoadhesins are most conveniently constructed by in-frame fusion of a cDNA sequence encoding the adhesin moiety to an immunoglobulin cDNA sequence. However, fusions to genomic immunoglobulin fragments may be used (see, e.g., Aruffo et al, Cell, 61: 1303-. The latter type of fusion requires the presence of Ig regulatory sequences for expression. cDNAs encoding IgG heavy chain constant regions can be isolated by hybridization or by Polymerase Chain Reaction (PCR) techniques based on published sequences from cDNA libraries derived from spleen or peripheral blood lymphocytes. The cdnas encoding the immunoglobulin portions of the "adhesins" and immunoadhesins are inserted in tandem into a plasmid vector for efficient expression in a selected host cell.

In another embodiment, the DR6 antagonist can be covalently modified by: as described in U.S. patent nos. 4,640,835; 4,496,689, respectively; 4,301,144, respectively; 4,670,417, respectively; 4,791,192 or 4,179,337 to link the receptor polypeptide to one of a variety of non-protein polymers (e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylene), or other similar molecules such as polyglutamic acid. Such pegylated forms may be prepared using techniques known in the art.

Leucine zipper versions of these molecules are also contemplated by the present invention. In the art, the term "leucine zipper" is used to denote a leucine-rich sequence that enhances, promotes, or drives dimerization or trimerization of its fusion partner (e.g., a sequence or molecule fused or linked to a leucine zipper). A variety of leucine zipper polypeptides have been described in the art. See, e.g., Landschulz et al, Science, 240: 1759 (1988); us patent 5,716,805; WO 94/10308; hoppe et al, FEBS Letters (FEBS communications), 344: 1991 (1994); maniatis et al, Nature (Nature), 341: 24(1989). One skilled in the art will appreciate that the leucine zipper sequence may be fused at the 5 'or 3' end of the DR6 or p75 molecules.

The DR6, p75 and/or APP polypeptides of the invention may also be modified by fusing the polypeptides with another, heterologous polypeptide or amino acid sequence to form chimeric molecules. Preferably, such heterologous polypeptide or amino acid sequence is a heterologous polypeptide or amino acid sequence that oligomerizes the chimeric molecule. In one embodiment, such chimeric molecules include fusions of DR6, p75, and/or APP polypeptides with tag polypeptides that provide epitopes to which anti-tag antibodies can selectively bind. Typically, the epitope tag is placed at the amino or carboxy terminus of the polypeptide. Antibodies to the tag polypeptide can be used to detect the presence of such epitope tagged forms of the polypeptide. Also, the provision of an epitope tag enables the polypeptide to be easily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag. A variety of tag polypeptides and their respective antibodies are known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; influenza HA tag polypeptide and its antibody 12CA5(Field et al, mol.cell.biol. (molecular cell biology), 8: 2159-; the C-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies directed against it (Evan et al, mol. cell. biol., 5: 3610-3616 (1985)); and the herpes simplex virus glycoprotein D (gD) tag and antibodies thereto (Paborsky et al, Protein Engineering, 3 (6): 547-553 (1990)). Other tag polypeptides include Flag-peptide (Hopp et al, Biotechnology (Biotechnology), 6: 1204-1210 (1988)); KT3 epitope peptide (Martin et al, Science (Science), 255: 192-; alpha tubulin epitope peptide (Skinner et al, J.biol.chem. (J.Biol.Chem., 266: 15163-15166 (1991)); and the T7 gene 10 protein peptide tag (Lutz-Freymeruth et al, Proc. Natl. Acad. Sci. USA, 87: 6393-.

anti-DR 6, anti-p 75, and anti-APP antibodies

In other embodiments of the invention, DR6, p75 and/or APP antibodies are provided. Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies. In some embodiments, the anti-DR 6, p75, and/or APP antibodies are preferably DR6 antagonist antibodies.

Polyclonal antibodies

The antibodies of the invention may include polyclonal antibodies. Methods for preparing polyclonal antibodies are well known to the skilled person. Polyclonal antibodies can be raised in mammals, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant is injected into the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may include DR6, p75, and/or APP polypeptides (e.g., DR6, p75, and/or APP ECD) or fusion proteins thereof. It may be useful to conjugate an immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include, but are not limited to, hemocyanin (keyhole limpet hemocyanin), serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants that can be used include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate). The person skilled in the art can select an immunization regimen without undue experimentation. The mammal is then bled and the serum is assayed for DR6 and/or APP antibody titer. If desired, the mammal is given a booster injection (boost) until the antibody titer increases or plateaus.

Monoclonal antibodies

Alternatively, the antibody of the invention may be a monoclonal antibody. Hybridoma methods such as those described by Kohler and Milstein, Nature, 256: 495(1975) to produce monoclonal antibodies. In the hybridoma method, a mouse, hamster, or other suitable host animal, is typically immunized with an immunizing agent to induce lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes may be immunized in vitro.

The immunizing agent will typically include DR6, p75, and/or APP polypeptides (e.g., DR6, p75, and/or APP ECD) or fusion proteins thereof, such as DR6ECD-IgG, p75ECD-IgG, and/or appapp-IgG fusion proteins.

Typically, peripheral blood lymphocytes ("PBLs") are used if cells of a human organ are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice), Academic Press, (1986) pp.59-103). Immortalized cell lines are generally transformed mammalian cells, in particular myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are used. The hybridoma cells may be cultured in a suitable medium, which preferably comprises one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of cells lacking HGPRT.

Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. A more preferred immortalized cell line is a murine myeloma cell line, which is available from, for example, the Salk Institute cell distribution Center, San Diego, California and American Type Culture Collection, Manassas, Virginia. An example of such a murine myeloma cell line is P3X63Ag8U.1, (ATCC CRL 1580). The use of human myeloma and murine-human hybrid myeloma cell lines for the Production of human Monoclonal antibodies has also been described (Kozbor, J.Immunol. (J.Immunol., 133: 3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp.51-63).

The presence or absence of monoclonal antibodies directed against DR6, p75, and/or APP in the medium in which the hybridoma cells are cultured can then be determined. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. The concentration of the compound can be determined, for example, by Munson and Pollard, anal. 220(1980) or by BiaCore analysis to determine the binding affinity of the monoclonal antibody.

After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution means and grown by standard methods (Goding, supra). Suitable media for this purpose include, for example, Dulbecco's Modified Eagle's Medium (DMEM Medium) and RPMI-1640 Medium. Alternatively, the hybridoma cells may be grown in a mammal as ascites fluid.

Monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification methods such as, for example, protein a-sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

Monoclonal antibodies can also be prepared by recombinant DNA methods such as those described in U.S. patent No.4,816,567. DNA encoding the monoclonal antibody can be readily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into an expression vector, which is then transfected into a host cell such as an E.coli cell, a monkey COS cell, a Chinese Hamster Ovary (CHO) cell, or a myeloma cell that does not otherwise produce immunoglobulin protein, to obtain synthesis of the monoclonal antibody in the recombinant host cell. DNA may also be altered, for example, by replacing the homologous murine sequences with the coding sequences for the human heavy and light chain constant domains (Morrison et al, Proc. Nat. Acad. Sci. USA 81, 6851(1984)) or by covalent linkage to all or part of the coding sequence for an immunoglobulin coding sequence, a non-immunoglobulin polypeptide. "chimeric" or "hybrid" antibodies are prepared in such a manner as to have binding specificity to the anti-DR 6 monoclonal antibody herein.

Typically, such non-immunoglobulin polypeptides are used to replace the constant domains of an antibody of the invention, or they are used to replace the variable domains of one antigen binding site of an antibody of the invention, to produce a chimeric bivalent antibody comprising one antigen binding site specific for DR6 and another antigen binding site specific for a different antigen.

Chimeric or hybrid antibodies can also be prepared using known methods in synthetic protein chemistry, including those involving cross-linking agents. For example, immunotoxins may be constructed using a disulfide interchange reaction or by forming a thioether bond. Examples of suitable reagents for use herein include iminothiolate and methyl-4-mercaptobutylimidate (methyl-4-mercaptobutyrimidate).

Single chain Fv fragments can also be prepared, such as those described in Iliades et al, FEBS Letters (FEBS Letters), 409: 437-441 (1997). In Kortt et al, Protein Engineering, 10: the use of various linkers for coupling such single-stranded fragments is described in 423-433 (1997). Various techniques for recombinant production and antibody manipulation are known in the art. Illustrative examples of such techniques commonly used by the skilled artisan are described in more detail below.

Humanized antibodies

Typically, humanized antibodies have one or more amino acid residues introduced into them from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be performed by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody essentially following the method of Winter and colleagues (Jones et al, Nature (Nature), 321: 522-525 (1986); Riechmann et al, Nature (Nature), 332: 323-327 (1988); Verhoeyen et al, Science (Science), 239: 1534-1536 (1988)).

Thus, such "humanized" antibodies are chimeric antibodies in which sequences substantially less than an intact human variable domain are replaced by corresponding sequences from a non-human species. In fact, humanized antibodies are typically human antibodies in which certain CDR residues and possibly certain FR residues are substituted by residues from analogous sites in rodent antibodies.

It is important that the humanized antibody retains high affinity for the antigen and other favorable biological properties. To achieve this, according to a preferred method, humanized antibodies are prepared by a process of analyzing the parental sequences and various conceptual humanized products (using three-dimensional models of the parental and humanized sequences). Three-dimensional immunoglobulin models are generally available and familiar to those skilled in the art. Computer programs are available that illustrate and display the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. The observation of these displays allows the analysis of the likely role of residues in the functional performance of candidate immunoglobulin sequences, i.e., the analysis of residues that affect the ability of a candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected from the consensus and input sequences and bound to obtain desired antibody properties, such as increased affinity for one or more target antigens. Generally, CDR residues are directly and most fully involved in the effect on antigen binding.

Human antibodies

Human monoclonal antibodies can be prepared by the hybridoma method. For example, human myeloma and mouse-human heteromyeloma cell lines for the Production of human monoclonal antibodies have been described by Kozbor, J.Immunol. (J.Immunol.) 133, 3001(1984), and Brodeur et al, monoclonal antibody Production Techniques and Applications (monoclonal antibody Production Techniques and Applications), pp.51-63(Marcel Dekker, Inc., New York, 1987).

It is now possible to prepare transgenic animals (e.g., mice) that, when immunized, are capable of producing a full panel of human antibodies in the absence of endogenous immunoglobulin production. For example, it is described that homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. Transfer of a human germline immunoglobulin gene array into such germline mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al, Proc. Natl. Acad. Sci. USA (Proc. Natl. Acad. Sci.) 90, 2551-; jakobovits et al, Nature 362, 255-258 (1993).

Mendez et al (Nature Genetics 15: 146-156(1997)) further improved the technology and produced what is known as "XeA transgenic mouse of nomouse II "that produces high affinity fully human antibodies when challenged with an antigen. This was achieved by germline integration of megabase human heavy and light chain loci into endogenous J as described above_HSegment deletion in mice. Xenomouse II has a1,020 kb human heavy chain locus, which contains approximately 66V_HGene, complete D_HAnd J_HThe regions and three different constant regions (μ, δ and χ), and also has an 800kb human κ locus, which contains 32 vk genes, jk segments and ck genes. The antibodies produced in these mice are very similar to those seen in humans in all respects including gene rearrangement, assembly and profiling (repotoreire). Due to endogenous J_HDeletion of the segments (which prevents gene rearrangement at the murine locus) the human antibody is preferentially expressed relative to the endogenous antibody.

Alternatively, phage display technology (McCafferty et al, Nature 348, 552 and 553(1990)) can be used to prepare human antibodies and antibody fragments in vitro from the genomic repertoire of immunoglobulin variable (V) domains from non-immunized donors. According to this technique, antibody V domain genes are cloned in-frame into the major or minor coat protein genes of filamentous phage (such as M13 or fd) and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle comprises a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody also results in selection of the gene encoding the antibody displaying those properties. Thus, the phage mimics certain properties of B cells. Phage display can be performed in a variety of formats; for a review of these, see, for example, Johnson, Kevin S. and Chiswell, David J., Current Opinion in structural biology 3, 564-571 (1993). V-gene segments of various origins can be used for phage display. Clackson et al, Nature 352, 624-628(1991) isolate multiple antibodies against a small random combinatorial library of V genes derived from the spleen of immunized miceAn oxazolone antibody. Can be used forA full set of V genes from non-immunized human donors was constructed and antibodies against a variety of antigens, including self-antigens, could be isolated substantially following the techniques described by Marks et al, J.mol.biol. (J.Mol. biol.) 222, 581-597(1991) or Griffith et al, EMBO J. (J.European Megaku) 12, 725-734 (1993). In the innate immune response, antibody genes accumulate mutations at a high rate (somatic hypermutations). Some of the changes introduced will provide higher affinity and B cells displaying high affinity surface immunoglobulins are preferentially replicated and differentiated during subsequent antigen challenge. Can be prepared by using a technique known as "Strand replacement" (Marks et al, Bio/Technol. (Biotechnology) 10, 779-]) To mimic this natural process. In this method, the affinity of a "primary" human antibody obtained by phage display can be improved by: the heavy and light chain V region genes were continued to be replaced with a full set of naturally occurring variants (repertoires) of V domain genes obtained from non-immunized donors. This technique allows the preparation of antibodies and antibody fragments with affinities in the nM range. Waterhouse et al, nucleic acids Res. (nucleic acids research) 21, 2265. 2266(1993) have described strategies for preparing very large antibody profiles (also known as "mothers-of-all libraries"). Gene replacement can also be used to derive human antibodies from rodent antibodies, where the human antibodies have similar affinity and specificity to the starting rodent antibody. According to this approach (which is also referred to as "epitopic imprinting"), either the heavy or light chain V domain genes of rodent antibodies obtained by phage display technology are replaced with a full set of human V domain genes, resulting in a rodent-human chimera. Selection of the antigen results in a variable separation of the human that is able to restore a functional antigen binding site, i.e. epitope control (imprinting) selection of partners. When this process is repeated to replace the remaining rodent V domains, human antibodies are obtained (see PCT patent application WO 93/06213, published at 1/4 1993). Unlike conventional humanization of rodent antibodies by CDR grafting, this technique provides fully human antibodies that do not have rodent-derived framework or CDR residues.

As detailed below, the antibodies of the invention may optionally include monomeric, dimeric, and multivalent forms of the antibody. These dimeric or multivalent forms can be constructed by those skilled in the art by techniques known in the art and using the DR6 and/or APP antibodies herein. Methods for making monovalent antibodies are also known in the art. For example, one approach involves recombinant expression of immunoglobulin light chains and modified heavy chains. The heavy chain is typically truncated at any point in the Fc region to prevent heavy chain cross-linking. Alternatively, the relevant cysteine residue is replaced with another amino acid residue or deleted to prevent cross-linking.

Bispecific antibodies

Bispecific antibodies are monoclonal (preferably) human or humanized antibodies having binding specificity for at least two different antigens. In this example, one of the binding specificities is for the DR6 receptor, the other is for any other antigen, and preferably for another receptor or receptor subunit. In one embodiment, the other antigen is p 75. Methods for making bispecific antibodies are known in the art. Conventionally, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature (Nature), 305: 537-539 (1983)). Due to the random assignment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a possible mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome and the product yield is low. Similar methods are disclosed in PCT application publication No. WO 93/08829 (published on 3.5.1993) and Traunecker et al, EMBO J, (journal of the european society for molecular biology), 10: 3655-3659 (1991).

According to a different and more preferred method, antibody variable domains with the desired binding specificity (antibody-antigen binding site) are fused to immunoglobulin constant domain sequences. The fusion is preferably to an immunoglobulin heavy chain constant domain (which comprises at least part of the hinge, CH2, and CH3 regions). Preferably, in at least one of the fusions there is a first heavy chain constant region (CH1) comprising the site required for light chain binding. The DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain are inserted into separate expression vectors and co-transfected into a suitable host organism. This provides great flexibility in adjusting the mutual proportions of the three polypeptide fragments in an embodiment, as unequal ratios of the three polypeptide chains used for construction provide optimal yields. However, it is possible to insert the coding sequences for two or all three polypeptide chains into one expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratio is not particularly important. In a preferred embodiment of this method, the bispecific antibody is composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the isolation of the desired bispecific compound from the undesired immunoglobulin chain combinations, since the presence of the immunoglobulin light chain in only half of the bispecific molecule provides an easy way of isolation. This method is disclosed in PCT publication No. WO 94/04690 published 3/3 in 1994.

For further details on the preparation of bispecific antibodies see, e.g., Suresh et al, meth.enzymol. (methods in enzymology) 121, 210 (1986).

Heteroconjugate antibodies

Heterologous conjugate antibodies are also within the scope of the invention. The heteroconjugate antibody is composed of two covalently bound antibodies. Such antibodies have been proposed, for example, to target unwanted cells of the immune system (U.S. Pat. No.4,676,980), and for the treatment of HIV infection (PCT application publication Nos. WO 91/00360 and WO 92/200373; EP 03089). Heteroconjugated antibodies can be prepared using any convenient crosslinking method. Suitable crosslinking agents are known in the art and are disclosed in U.S. Pat. No.4,676,980, along with numerous crosslinking techniques.

Antibody fragments

In certain embodiments, the anti-DR 6, anti-p 75, and/or anti-APP antibodies (including murine, human, and humanized antibodies, and antibody variants) are antibody fragments. Various techniques have been developed for the preparation of antibody fragments. These fragments are routinely obtained by proteolytic digestion of intact antibodies (see, e.g., Morimoto et al, J.biochem.Biophys.methods (J.Biochem.Biophys.methods) 24: 107-117(1992) and Brennan et al, Science 229: 81 (1985)). However, these fragments can now be produced directly by recombinant host cells. For example, Fab '-SH fragments can be recovered directly from E.coli and chemically coupled to form F (ab') 2 fragments (Carter et al, Bio/Technology 10: 163-167 (1992)). In another embodiment, the leucine zipper GCN4 is used to form F (ab')₂To promote F (ab')₂And (4) assembling molecules. According to another method, Fv, Fab or F (ab')₂And (3) fragment. Various techniques for preparing antibody fragments will be apparent to those skilled in the art. For example, digestion can be performed using papain. Examples of papain digestion are disclosed in WO 94/29348 and U.S. patent No.4,342,566, published at 12/22/94. Papain digestion of antibodies typically produces two identical antigen-binding fragments, called Fab fragments, each of which has a single antigen-binding site and a residual Fc fragment. Pepsin treatment to yield F (ab')₂A fragment having two antigen binding sites and still being capable of cross-linking antigens.

The Fab fragments produced in the antibody digestion also contain the constant domain of the light chain and the first constant domain of the heavy Chain (CH)₁). Fab' fragment is due to heavy chain CH₁The carboxy terminus of the domain is differentiated from the Fab fragment by the addition of residues, including one or more cysteines from the antibody hinge region. Fab' -SH is a cysteine for which the constant domains are referred to hereinThe designation Fab' in which the amino acid residue carries a free thiol group. F (ab')₂Antibody fragments were originally produced as pairs of Fab 'fragments with hinge cysteines between the Fab' fragments. Other chemical couplings of antibody fragments are also known. Glycosylation variants of antibodies

Antibodies are glycosylated at conserved positions in their constant regions (Jefferis and Lund, chem. Immunol. (chemical immunology) 65: 111-128 (1997); Wright and Morrison, TibTECH 15: 26-32[1997 ]). The oligosaccharide side chains of immunoglobulins influence the function of the protein (Boyd et al, mol. Immunol. (molecular immunology) 32: 1311- & 1318 (1996); Wittwe and Howard, Biochem. (biochemistry) 29: 4175- & 4180(1990)), and the intramolecular interactions between parts of the glycoprotein that are capable of influencing the conformation of the glycoprotein and the parts of the three-dimensional surface presented (Hefferis and Lund, supra; Wyss and Wagner, Current Opin. Biotech. (Biotechnology New) 7: 409- & 416 (1996)). Oligosaccharides can also be used to target a given glycoprotein to a particular molecule based on a specific recognition structure. For example, it has been reported that in galactosylated IgG, the oligosaccharide moiety "flips" out from between the CH2 spaces, while the terminal N-acetylglucosamine residue becomes available for binding to the mannose binding protein (Malhotra et al, Nature (Nature) Med.1: 237-. Removal of oligosaccharides from CAMPATH-1H (recombinant humanized murine monoclonal IgG1 antibody recognizing the CDw52 antigen of human lymphocytes) prepared in Chinese Hamster Ovary (CHO) cells by a glycosylpeptidase resulted in a complete reduction of complement-mediated lysis (CMCL) (Boyd et al, mol. Immunol. (molecular immunology) 32: 1311-1318[1996]), whereas selective removal of sialic acid residues using neuraminidase did not result in loss of DMCL. It has also been reported that glycosylation of antibodies affects antibody-dependent cellular cytotoxicity (ADCC). In particular, CHO cells in which tetracycline regulates expression of β (1, 4) -N-acetylglucosaminyltransferase III (GnTIII), glycosyltransferase catalysis, etc., and which bisect GlcNAc formation have been reported to have improved ADCC activity (Umana et al, Mature Biotech.17: 176-180 (1999)).

A glycosylation variant of an antibody is a variant in which the glycosylation pattern of the antibody is altered. Altered refers to deletion of one or more carbohydrate moieties found in the antibody, addition of one or more carbohydrate moieties to the antibody, alteration of the composition of glycosylation (glycosylation pattern), degree of glycosylation, and the like. Glycosylation variants can be prepared, for example, by removing, altering, and/or adding one or more glycosylation sites in a nucleic acid sequence encoding an antibody.

Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates potential glycosylation sites. By O-linked glycosylation is meant that one of the sugars N-acetylgalactosamine, galactose or xylose is linked to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

The addition of glycosylation sites to antibodies (for N-linked glycosylation sites) is conventionally achieved by altering the amino acid sequence such that it comprises one or more of the above-described tripeptide sequences. Changes (for O-linked glycosylation sites) can also be made by adding one or more serine or threonine residues to or replacing the sequence of the original antibody with one or more serine or threonine residues.

Glycosylation (including glycosylation patterns) of antibodies can also be altered without altering the underlying nucleotide sequence. Glycosylation is largely dependent on the host cell used to express the antibody. Since the cell type used to express recombinant glycoproteins (e.g., antibodies) as potential therapeutic agents is rarely a native cell, significant changes in the glycosylation pattern of the antibody can be expected (see, e.g., Hse et al, J.biol.chem. (J.Biol.Chem.) 272: 9062-9070 (1997)). In addition to the choice of host cell, factors that influence glycosylation during recombinant production of antibodies include growth patterns, media formulations, culture density, oxygenation, pH, purification protocols, and the like. Various methods have been proposed for effecting changes in glycosylation patterns in specific host organisms, including the introduction or overexpression of certain enzymes involved in oligosaccharide production (U.S. Pat. Nos. 5,047,335; 5,510,261 and 5.278,299). Endoglycosidase h (endo h) can be used, for example, to enzymatically remove glycosylation or certain types of glycosylation from glycoproteins. In addition, recombinant host cells can be genetically engineered, for example, to create defects in the processing of certain types of polysaccharides. These and similar techniques are known in the art.

The glycosylation structure of an antibody can be readily analyzed by conventional techniques of carbohydrate analysis including lectin chromatography, NMR, mass spectrometry, HPLC, GPC, monosaccharide composition analysis, sequential enzyme digestion, and HPAEC-PAD, using high pH anion exchange chromatography to separate oligosaccharides based on charge. Methods of liberating oligosaccharides for analysis are also known and include, but are not limited to, enzymatic treatment (typically performed using peptide-N-glycosidase F/endo- β -galactosidase), removal using an extremely basic environment to liberate primarily O-linked structures, and chemical methods using anhydrous hydrazine to liberate N-linked and O-linked oligosaccharides.

Representative antibodies

As described in the examples below, anti-DR 6 monoclonal antibodies have been identified. In alternative embodiments, the DR6 antibodies of the invention will have the same biological properties as any of the anti-DR 6, anti-p 75 and/or anti-APP antibodies specifically disclosed herein.

The term "biological property" is used to refer to an in vitro and/or in vivo activity or property of a monoclonal antibody, such as the ability to specifically bind DR6 or block, inhibit or neutralize DR6 activation. The properties and activities of DR6, p75, and/or APP antibodies are further described in the examples below.

Optionally, the monoclonal antibodies of the invention will have the same biological properties as any of the antibodies specifically characterized in the examples below, and/or bind to the same epitope or epitopes as those bound by these antibodies. This can be determined by performing a variety of assays, such as those described herein and in the examples. For example, to determine whether a monoclonal antibody has the same specificity as the DR6, p75, and/or APP antibodies specifically referred to herein, its activity can be compared in a competitive binding assay. Furthermore, which epitope a particular anti-DR 6, p75 and/or APP antibody binds to can be determined by crystallographic studies of the complex between DR6, p75 and/or APP and the antibody.

As described herein, DR6, p75, and/or APP antibodies will preferably have the desired DR6, p75, or APP antagonistic activity. Such antibodies may include, but are not limited to, chimeric antibodies, humanized antibodies, human antibodies, and affinity matured antibodies. As described above, DR6, p75, and/or APP antibodies can be constructed or engineered using a variety of techniques to obtain these desired activities or properties.

Additional embodiments of the invention include anti-DR 6 receptor, anti-p 75 and/or anti-APP ligand antibodies disclosed herein linked to one or more non-protein polymers selected from the group consisting of polyethylene glycol, polypropylene glycol and polyoxyalkylene. Optionally, the anti-DR 6 receptor, anti-p 75, and/or anti-APP ligand antibodies disclosed herein are glycosylated or alternatively are not glycosylated.

Antibodies of the invention include "cross-linked" DR6, p75, and/or APP antibodies. The term "crosslinked" as used herein refers to at least two IgG molecules bound together to form one (or single) molecule. A variety of linker molecules may be used to cross-link DR6, p75 and/or APP antibodies, preferably anti-IgG molecules, complements, chemical modifications or molecular alterations are used to cross-link DR6, p75 and/or APP antibodies. It is understood by those skilled in the art that complement has a relatively high affinity for antibody molecules once the antibody binds to the cell surface membrane. Thus, for example, it is believed that complement can be used as a cross-linking molecule to link two or more anti-DR 6 antibodies bound to the cell surface membrane.

The invention also provides isolated nucleic acids encoding DR6, p75, and/or APP antibodies as disclosed herein, vectors and host cells comprising the nucleic acids, and recombinant techniques for making the antibodies.

For recombinant production of the antibody, the nucleic acid encoding the antibody is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or expression. The DNA encoding the antibody can be readily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes that are capable of binding specifically to the gene encoding the antibody). Many vectors are available. Carrier components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

The methods herein include methods for making chimeric or recombinant anti-DR 6 and/or APP antibodies, comprising the steps of: providing a vector comprising a DNA sequence encoding anti-DR 6, anti-p 75, and/or anti-APP antibody light or heavy chain (or both light and heavy chains), transfecting or transforming a host cell with the vector, and culturing the host cell under conditions sufficient to produce recombinant anti-DR 6 antibody, anti-p 75 antibody, and/or anti-APP antibody product.

Formulations of DR6 antagonists

In the preparation of typical formulations herein, it is noted that the recommended quality or "grade" of the components used will depend on the end use of the formulation. For therapeutic use, it is preferred that the one or more components be of a grade that is acceptable as an additive to a pharmaceutical product (such as "GRAS").

In certain embodiments, compositions are provided comprising DR6 and optionally one or more p75 antagonists, and one or more excipients that provide sufficient ionic strength to enhance the solubility and/or stability of the DR6 antagonist, wherein the composition has a pH of 6 (or about 6) to 9 (or about 9). DR6 and p75 antagonists may be prepared by any suitable method to achieve the desired protein purity, for example, according to the above methods. In certain embodiments, the antagonist is recombinantly expressed in a host cell or is prepared by chemical synthesis. The concentration of DR6 or p75 antagonist in the formulation may vary depending, for example, on the intended use of the formulation. The concentration of DR6 or p75 antagonist required can be determined by one skilled in the art without undue experimentation.

The composition of one or more excipients in the formulation that provide sufficient ionic strength to enhance the solubility and/or stability of the DR6 or p75 antagonist is optionally a polyionic organic or inorganic acid, aspartate, sodium sulfate, sodium succinate, sodium acetate, sodium chloride, Captisol^TMTris, arginine salts or other amino acids, sugars and polyols such as trehalose and sucrose. Preferably the excipient or excipients in the formulation that provide sufficient ionic strength is a salt. Salts that may be employed include, but are not limited to, sodium and arginine salts. Preferably, the type of salt and the concentration of salt used are such that the formulation has a relatively high ionic strength which allows the DR6 antagonist in the formulation to be stable. Optionally, the salt is present in the formulation at a concentration of about 20mM to about 0.5M.

The composition preferably has a pH of 6 (or about 6) to 9 (or about 9), more preferably has a pH of about 6.5 to about 8.5, and even more preferably has a pH of about 7 to about 7.5. In a preferred aspect of this embodiment, the composition will further comprise a buffering agent to maintain the pH of the composition at least about 6 to about 8. Examples of useful buffers include, but are not limited to, Tris, HEPES, and histidine. When Tris is used, the pH may optionally be adjusted to about 7 to 8.5. When Hepes or histidine is used, the pH may optionally be adjusted to about 6.5 to 7. Optionally, the buffer is used in the formulation at a concentration of about 5mM to about 50 mM.

Particularly for liquid formulations (or reconstituted lyophilized formulations), it may be desirable to include one or more surfactants in the composition. Such surfactantsMay, for example, include a nonionic surfactant such as TWEEN^TMOr PLURONICS^TM(e.g., a polysorbate or poloxamer). Preferably, the surfactant comprises polysorbate 20 ("Tween 20"). The surfactant will optionally be used at a concentration of about 0.005% to about 0.2%.

In addition to one or more DR6 antagonists and those components described above, the formulations of the present invention may also include a variety of other excipients or components. Optionally, for parenteral administration, the formulations may contain a pharmaceutically or parenterally acceptable carrier, i.e., a carrier that is non-toxic to the recipient at the dosages and concentrations employed and that is compatible with the other ingredients of the formulation. Optionally, the carrier is a parenteral carrier, such as a solution that is isotonic with the blood of the recipient. Examples of such carriers include water, saline or buffered solutions such as Phosphate Buffered Saline (PBS), Ringer's solution and dextrose solution. Various optional Pharmaceutical carriers, excipients or stabilizers are further described in Remington's Pharmaceutical Sciences, 16 th edition, Osol, a. editor (1980).

The formulations herein may also contain one or more preservatives. Examples include octadecyl dimethyl benzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkyl benzyl dimethyl ammonium chlorides, where the alkyl group is a long chain compound), and benzethonium chloride. Other types of preservatives include aromatic alcohols, alkyl parabens such as methyl or propyl paraben, and m-cresol. Antioxidants include ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride; butanol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, or other carbohydrates including glucose, mannose, or dextrins; sugars such as sucrose, mannitol, trehalose, or sorbitol; or polyethylene glycol (PEG).

The compositions of the present invention may comprise liquid formulations (liquid solutions or liquid suspensions), and lyophilized formulations, as well as suspension formulations.

If the final formulation is a liquid, it is preferably frozen at ≦ 20 deg.C. Alternatively, the formulation may be lyophilized and provided as a powder that can be stored at 2-30 ℃ for regeneration with water for injection.

Formulations for therapeutic administration must be sterile. Sterility can be readily achieved by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). The therapeutic composition is typically placed into a container having a sterile access end, for example, an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle.

The compositions are typically stored in a single unit or multi-dose container, e.g., a sealed ampoule or vial, as an aqueous solution or as a lyophilized formulation for reconstitution. The container may be any available container in the art and is filled using conventional methods. Optionally, the formulation may be contained in an injection pen device (or cartridge adapted pen device) suitable for therapeutic delivery of the formulation, such as those available in the art (see, e.g., U.S. patent 5,370,629). Injectable solutions can be prepared by reconstituting lyophilized formulations of DR6 antagonist using, for example, water for injection.

Treatment with DR6 antagonists

The DR6 antagonists of the invention have a variety of uses. The DR6 antagonists are useful in the diagnosis and treatment of psychosis. Diagnosis of a mammal having a variety of pathological conditions as described herein can be made by a skilled artisan. Diagnostic techniques are available in the art that allow, for example, the diagnosis or detection of various psychiatric disorders in a mammal.

Psychiatric disorders contemplated for treatment by the present invention include addiction and schizophrenia. Cognitive diseases contemplated for treatment by the present invention include tourette's syndrome, retter's syndrome, fragile X syndrome, and autism. It is contemplated that the compositions and methods of the present invention may be used in normal, elderly patients to maintain and possibly improve cognition during the aging process.

In the methods of the invention, the DR6 antagonist is preferably administered to the mammal in a vehicle; preferably a pharmaceutically acceptable carrier. Suitable carriers and their formulations are described in Remington's Pharmaceutical Sciences, edited by Osol et al, 16 th edition, 1980, Mack Publishing Co. Generally, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to make the formulation isotonic. Examples of the carrier include saline, Ringer's solution and glucose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Other carriers include sustained release formulations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those skilled in the art that certain carriers may be more preferred depending on, for example, the route of administration and the concentration of DR6 antagonist being administered.

The DR6 antagonist can be administered to the mammal by injection (e.g., intravenous, intraperitoneal, subcutaneous, intramuscular, intraportal), oral, or other methods such as infusion that ensures its delivery to the bloodstream in an effective form. The DR6 antagonist can also be administered for local therapeutic effect by a barrier perfusion technique such as barrier tissue perfusion or by intrathecal injection, intraocular injection or lumbar puncture. DR6 antagonists that do not readily cross the blood-brain barrier may be administered directly, e.g., intracerebrally or to the spinal cord space or otherwise, which will transport them across the barrier. Effective dosages and schedules for administering the DR6 antagonist can be determined empirically, and making such determinations is within the skill of the art. One skilled in the art will appreciate that the dosage of DR6 antagonist that must be administered will vary depending upon, for example, the mammal to receive the antagonist, the route of administration, the particular type of antagonist used, and other drugs being administered to the mammal. Guidance in selecting the appropriate dosage can be found in the literature, for example, regarding the therapeutic use of antibodies, e.g., Handbook of monoclonal antibodies, edited by Ferrone et al, Nos. Publications, ParkRidge, N.J. (1985) ch.22 and pp.303-357; smith et al, Antibodies in human diagnostics and Therapy, edited by Haber et al, ravenPress, New York (1977) pp.365-389. Depending on the factors mentioned above, a typical daily dose of DR6 antibody used alone may range from about 1 μ g/kg body weight to over 100mg/kg body weight per day.

The DR6 antagonist can also be administered to the mammal in combination with one or more other therapeutic agents. APP was shown to bind p75 (EC) to a lesser extent as well₅₀300nM, determined by ELISA). Thus, it may be advantageous to treat mental and cognitive disorders with a combination of a DR6 antagonist and a p75 antagonist. Other therapeutic agents may be further combined with DR6 antagonists, optionally with p75 antagonists. Examples of such other therapeutic agents include Epidermal Growth Factor Receptor (EGFR) inhibitors, e.g., compounds that bind or otherwise interact directly with EGFR and prevent or attenuate its signaling activity, such as Tarceva, antibodies such as C225 (also known as cetuximab), and(erbitux) (immunocone Systems Inc.), fully human ABX-EGF (panitumumab, Abgenix Inc.), and fully human antibodies designated E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6.3, and E7.6.3 and described in US6,235,883; MDX-447 (Metarex Inc) and EGFR small molecule inhibitors such as described in US5616582, US5457105, US5475001, US5654307, US5679683, US6084095, US6265410, US6455534, US6521620, US6596726, US6713484, US5770599, US6140332, US5866572, US6399602, US6344459, US6602863, US6391874, WO9814451, WO9850038, WO9909016, WO9924037, US6344455, US 5760060060060060041, US5747498,000A compound; particular small molecule EGFR inhibitors include OSI-774(CP-358774, erlotinib, OSI Pharmaceuticals); PD 183805(CI1033, 2-propenamide, N- [4- [ (3-chloro-4-fluorophenyl) amino)]-7- [3- (4-morpholinyl) propoxy]-6-quinazolinyl]Dihydrochloride, Pfizer Inc.); iressa (Iressa) (ZD1839, gefitinib (gefitinib), 4- (3 '-chloro-4' -fluoroaniline) -7-methoxy-6- (3-morpholinopropoxy) quinazoline, AstraZeneca); ZM 105180 ((6-amino-4- (3-methylphenyl-amino) -quinazoline, Zeneca); BIBX-1382(N8- (3-chloro-4-fluoro-phenyl) -N2- (1-methyl-piperidin-4-yl) -pyrimido [5, 4-d)]Pyrimidine-2, 8-diamine, Boehringer Ingelheim); PKI-166((R) -4- [4- [ (1-phenylethyl) amino)]-1H-pyrrolo [2, 3-d]Pyrimidin-6-yl]-phenol); (R) -6- (4-hydroxyphenyl) -4- [ (1-phenylethyl) amino group]-7H-pyrrolo [2, 3-d]Pyrimidines); CL-387785(N- [4- [ (3-bromophenyl) amino)]-6-quinazolinyl]-2-butanamide); and EKB-569(N- [4- [ (3-chloro-4-fluorophenyl) amino group]-3-cyano-7-ethoxy-6-quinoline]-4- (dimethylamino) -2-butenamide). Other therapeutic agents that may be employed include apoptosis inhibitors, particularly intracellular apoptosis inhibitors, for example caspase inhibitors such as caspase-3, caspase-6 or caspase-8 inhibitors, Bid inhibitors, Bax inhibitors or any combination thereof. Examples of suitable inhibitors are caspase inhibitors (in general), dipeptide inhibitors, carbamate inhibitors, substituted aspartic acetals, heterocyclic p-urazines, quinoline- (di-, tri-, tetrapeptide) derivatives, substituted 2-aminobenzamide caspase inhibitors, substituted a-hydroxy acid caspase inhibitors, inhibition by nitration; CASP-1; CASP-3: protein inhibitors, antisense molecules, nicotinyl-aspartyl-ketones, y-keto acid dipeptide derivatives, CASP-8: antisense molecules, interacting proteins CASP-9, CASP 2: an antisense molecule; CASP-6: an antisense molecule; CASP-7: an antisense molecule; and CASP-12 inhibitors. Other examples are mitochondrial inhibitors such as Bcl-2-modulator; bcl-2 mutant peptides derived from Bad, BH 3-interacting domain death agonists, Bax inhibitor proteins, and BLK genes and gene products. In addition, suitable intracellular apoptosis modulators have a CASP9/Apaf-1 associationModulators, antisense modulators of Apaf-1 expression, apoptosis inhibiting peptides, anti-apoptotic compositions comprising the subunit of herpes simplex virus R1, MEKK1 and fragments thereof, modulators of survivin, modulators of apoptosis inhibitors, and HIAP 2. Further examples of such agents include minocycline (neuro-apositis Laboratory) which inhibits cytochrome c release from mitochondria and blocks the up-regulation of caspase-3 mRNA, pifizon alpha (UIC) as a p53 inhibitor, CEP-1346 (cephalolon Inc.) as a JNK pathway inhibitor, TCH346(Novartis) which inhibits GAPDH signaling prior to apoptosis, IDN6556(Idun Pharmaceuticals) as a pan-caspase inhibitor; AZQs (AstraZeneca) as a caspase-3 inhibitor, HM-3480(Aventis Pharma) as a caspase-1/-4 inhibitor, and activated enzyme/TPA (genentech) for clot lysis.

In addition, suitable agents that may be administered in addition to DR6 antagonists include BACE inhibitors, cholinesterase inhibitors (such as Donepezil (Donepezil), Galantamine (Galantamine), Rivastigmine (Rivastigmine), Tacrine (Tacrine)), NMDA receptor antagonists (such as Memantine (Memantine)), Abeta aggregation antagonists, antioxidants, gamma-secretase modulators, NGF mimetics or NGF gene therapy, PPAR γ agonists, HMG-CoA reductase antagonists (statins), ampakines (ampakines), calcium channel blockers, GABA receptor antagonists, glycogen synthase kinase antagonists, intravenous immunoglobulins, muscarinic receptor agonists, nicotinic receptor modulators, active or passive Abeta immunization, phosphodiesterase antagonists, 5-hydroxytryptamine receptor antagonists, and anti Abeta antibodies (see, e.g., WO 2007/062852; WO 2007/064972; WO 2003/040183; WO 1999/06066; WO 2006/081171; WO 1993; WO 2007/062852; WO 3648; WO 3; beta-mediated therapy, beta-mediated conditions, and antagonists 21526; EP 0276723B 1; WO 2005/028511; WO 2005/082939).

The DR6 antagonist can be administered sequentially or simultaneously with one or more other therapeutic agents. The amount of DR6 antagonist and therapeutic agent depends, for example, on the type of drug employed, the pathological condition being treated, and the timing and route of administration, but will generally be less than if each were used alone.

After administration of the DR6 antagonist and optionally the p75 antagonist to a mammal, the pathological state of the mammal can be monitored in a variety of ways well known to the skilled artisan.

The efficacy of DR6 antagonists of the invention, and optionally p75 antagonists, can be examined in vitro assays as well as using in vivo animal models.

Kits and articles of manufacture

In further embodiments of the invention, articles of manufacture and kits comprising substances useful for the treatment of psychiatric and cognitive disorders are provided. The article comprises a container having a label. Suitable containers include, for example, bottles, vials, and test tubes. The container may be made of a variety of materials, such as glass or plastic, and is preferably sterile. The container contains a composition having an active agent effective for treating mental and cognitive disorders. The active agent in the composition is a DR6 antagonist and preferably includes an anti-DR 6 monoclonal antibody or an anti-APP monoclonal antibody. In some embodiments, the other active agent in the composition is a p75 antagonist and preferably includes an anti-p 75 monoclonal antibody or an anti-APP monoclonal antibody. The label on the container indicates that the composition is used to treat mental and cognitive disorders, and may also indicate instructions for use in vivo or in vitro applications such as those described above. The article of manufacture or kit optionally further comprises a package insert, which refers to instructions for use, typically contained in a commercial package of therapeutic products, containing information about: indications, usage, dosage, administration, contraindications, other therapeutic products in combination with the packaged product and/or warnings regarding the use of such therapeutic products, and the like.

The kit of the present invention comprises the above container and a second container comprising a buffer. It may also include other materials as desired from a commercial and user perspective, including other buffers, diluents, filters, needles and syringes.

Examples

Various aspects of the invention are further described and exemplified by the following examples, which are not intended to limit the scope of the invention.

Synapses may be visualized in vivo using a two-photon microscope through a long-term cranial window (see fig. 1). Using this approach, we can (1) assess the density of dendritic spines as morphologically related terms of excitatory synapses, and (2) assess the morphology of dendritic spines.

Example 1: monitoring in vivo PSD-95 Retention

Materials and methods

Electroporation is carried out in utero. L2/3 progenitors were transfected by intrauterine electroporation (Saito T. and N.Nakatsuji (2001) Dev.biol. (developmental biology) 240: 237-. E16 time-timed pregnant C57BL/6J mice (Charles River, Wilmington, Massachusetts, United States) were deeply anesthetized with an isoflurane-oxygen cocktail. The uterine horn was exposed and approximately 1 μ l of DNA solution (containing plasmid expressing DsRed-Express, 1ug/ul) and Fast Green [ Sigma, St. Louis, Missouri, United States ]) were injected into the right ventricle of each embryo by pulled glass capillary pressure. The head of each embryo was placed between custom forceps electrodes, and the anode plate contacted the right side of the head. Electroporation was achieved with five rectangular pulses (duration 50ms, frequency 1Hz, 40V). The co-transfection efficiency was 60-70%. See fig. 1.

And (5) performing an operation. The imaging window was mounted above the somatosensory cortex at P8 or behind P60. Mice were deeply anesthetized with isoflurane-oxygen mixtures. Craniotomy (diameter: 4-5mm) was performed above the right somatosensory cortex (0.5/1.5 mm posterior to the pre-halogen spot and 3.0/3.5mm medial for pups/adults, respectively), keeping the dura intact. The dura was covered with 1% agarose (Type-IIIA, Sigma) dissolved in HEPES buffered artificial cerebrospinal fluid and with a 5mm custom cover glass (No.1) which was sealed in place with dental acrylic. Animals were also given injections of 20- μ l of 4% dexamethasone (dexamethasone) (Phoenix Scientific, St. Joseph, Missouri, United States of America). After a 1-h recovery period, adult mice were placed in cages, and pups were housed with littermates and surrogate mothers.

And (6) imaging. High resolution images were collected by a custom made dual laser two photon laser scanning microscope (2 PLSM). The light source used for imaging was solid Ti: sapphire lasers (λ -1020; 100mW in the objective back focal plane) (Spectra Physics, Fremont, California, United States); a band pass filter (610/90; Chroma Technology) is used to separate the red fluorescence photons. Signals were collected using a photomultiplier tube (3896; Hamamatsu, Hamamatsu City, Japan). Objective lens (40, 0.8NA) and trioc were from Olympus (Tokyo, Japan).

We used the vascular system and dendritic branching pattern daily to identify the region of interest. The imaging session consists of a series of image stacks within 90 min. The image stack consists of individual segments (512x512 pixels; pixel size, 0.08 μm) spaced 1 μm apart in the axial direction. After the imaging session, the mice were allowed to recover on the heat blanket for approximately 30min before dwelling with the littermate mice and the surrogate mothers; the adult animals were returned to their cages.

And (6) analyzing the data. Individual spines were identified, annotated, and tracked at multiple time points using a custom analysis routine in matlab (mathworks). Spike longevity, density and length were measured using custom software (Holtmaat A.J. et al (2005) Neuron 45: 279-.

Results

We observed that: and DR6^+/-In comparison with DR6+/+ animals, at DR6^-/-Increase in density and width of dendritic spines in animals (figure 2). The density was calculated by averaging the total number of spines per cell/dendritic length of all animals within the same population. Compared to 26 cells/7 animals annotated as DR6 +/-and 26 cells/6 animals annotated as DR6+/+,a total of 28 cells/8 animals were annotated as DR 6-/-. Spine width and length were plotted as cumulative plots of the entire population of spines analyzed per genotype.

Together with our earlier finding that APP is a cognate ligand for DR6, the results of Bittner et al (supra) indicate that APP also plays a role in dendritic spine density.

Example 2: effect of N-APP on dendritic spines in vitro

Materials and methods

Cell culture: PDL/laminin coated 8-well glass slides (Becton, Dickinson and Company) were filled with the following: 500 μ l/well Neurobasal Medium (Invitrogen) plus 50ng/ml of each of recombinant BDNF and NT-3(Chemicon), plus B-27 supplement X50 (Invitrogen); plus Pen Strep glutamine X100(Cat. No. 10378-016; Gibco) plus glucose X100. E16 cortical neuron explants were placed into each well and placed in a 37 ℃ incubator for 21 days. On day 21, cultures were treated with 0,1, 3, 10 or 30 μ g/ml N-APP. The culture was cultured for 24 hours. Then fixing and using the tape with AlexaMouse anti-PSD 95 antibody of 488(Molecular Probes) conjugated secondary antibody (goat anti-mouse IgG) treated neurons for microscopy. The results are shown in fig. 3.

The points per mm neuron (punca) were quantified and plotted as the change in point/mm versus the percent change in untreated cortical neurons (control). The results are shown in fig. 4.

In separate experiments, cortical cells in culture were exposed to 0ug/ml N-APP (control) or N-APP without an acidic tail (N-APP (-) acidic tail) or full-length N-APP (N-APP FL) at concentrations of 0.1, 0.3, 1.0, or 3.0ug/ml, with or without 30ug/ml of the anti-DR 6 antibody aDR6.1. The results are shown in fig. 5.

Results

FIG. 3 shows that N-APP induced PSD95 point reduction. As shown in fig. 4, the reduction in PSD95 point is concentration dependent. This result is consistent with N-APP and DR6 interacting to cause nerve degeneration and/or a reduction in neurite (axon or dendrite) length and branching thus causing loss of dendrite spines (indicated by a reduction in PSD95 points). FIG. 5 shows that the N-APP induced reduction in PSD95 point is dependent on DR6.

Claims (23)

1. A method of increasing dendritic spine density in neurons of a patient suffering from a cognitive or psychiatric disorder comprising administering to said patient an effective amount of a DR6 inhibitor or a p75 inhibitor.

2. The method of claim 1, wherein said DR6 inhibitor is an antibody that binds to an epitope of DR6 and inhibits the function of DR6.

3. The method of claim 1, wherein the p75 inhibitor is an antibody that binds to an epitope of p75 and inhibits the function of p 75.

4. The method of claim 2, wherein the antibody is selected from the group consisting of 3f4.4.8, 4b6.9.7, 1e5.5.7, and antigen binding fragments thereof.

5. The method of claim 4, wherein the antibody is a chimeric or humanized 3F4.4.8, 4B6.9.7, or 1E5.5.7, or an antibody that binds to the same epitope as 3F4.4.8, 4B6.9.7, or 1 E5.5.7.

6. The method of claim 1, wherein the DR6 inhibitor reduces or prevents DR6 signaling in the neuron.

7. The method of claim 1, wherein the p75 inhibitor reduces or prevents p75 signaling in the neuron.

8. A method of treating a cognitive or psychiatric disorder in a patient in need of such treatment, the method comprising identifying a patient having a cognitive or psychiatric disorder associated with reduction of dendritic spines, and administering to the patient a therapeutically effective amount of a DR6 antagonist or a p75 antagonist.

9. The method of claim 1 or 8, wherein the mental or cognitive disorder is selected from the group consisting of: rett syndrome, tourette syndrome, autism, schizophrenia, and fragile X mental retardation.

10. The method of claim 8, wherein said DR6 inhibitor is an antibody that binds to an epitope of DR6 and inhibits the function of DR6.

11. The method of claim 8, wherein the p75 inhibitor is an antibody that binds to an epitope of p75 and inhibits the function of p 75.

12. The method of claim 10, wherein the antibody is selected from the group consisting of 3f4.4.8, 4b6.9.7, 1e5.5.7, and antigen binding fragments thereof.

13. The method of claim 12, wherein the antibody is a chimeric or humanized 3f4.4.8, 4b6.9.7, or 1e5.5.7, or an antibody that binds to the same epitope as 3f4.4.8, 4b6.9.7, or 1 e5.5.7.

14. A method of maintaining cognition in a subject during aging, the method comprising administering to the subject a DR6 inhibitor or a p75 inhibitor in an amount effective to increase dendritic spine density in the subject, thereby maintaining cognition in the subject.

15. The method of claim 14, wherein said DR6 inhibitor is an antibody that binds to an epitope of DR6 and inhibits the function of DR6.

16. The method of claim 15, wherein the antibody is selected from the group consisting of 3f4.4.8, 4b6.9.7, 1e5.5.7, and antigen binding fragments thereof.

17. The method of claim 16, wherein the antibody is a chimeric or humanized 3f4.4.8, 4b6.9.7, or 1e5.5.7, or an antibody that binds to the same epitope as 3f4.4.8, 4b6.9.7, or 1 e5.5.7.

18. The method of claim 14, wherein the p75 inhibitor is an antibody that binds to an epitope of p75 and inhibits the function of p 75.

Use of a DR6 antagonist in the manufacture of a medicament for a patient suffering from a cognitive or psychiatric disorder, wherein the antagonist inhibits DR6 activity.

20. The use of the DR6 antagonist of claim 15, wherein the DR6 antagonist is an antibody that binds to an epitope of DR6.

21. The use of the DR6 antagonist of claim 16, wherein the antibody is selected from the group consisting of 3f4.4.8, 4b6.9.7, 1e5.5.7, and antigen-binding fragments thereof.

22. The use of a DR6 antagonist as claimed in claim 16, wherein the antibody is a chimeric or humanized 3f4.4.8, 4b6.9.7 or 1e5.5.7, or an antibody that binds to the same epitope as 3f4.4.8, 4b6.9.7 or 1 e5.5.7.

Use of a p75 antagonist in the manufacture of a medicament for use in a patient suffering from a cognitive or psychiatric disorder wherein the antagonist inhibits p75 activity.