TW200950808A - Anti-PirB antibodies - Google Patents

Abstract

The present invention relates generally to neural development and neurological disorders. The invention specifically concerns identification of novel modulators of the myelin-associated inhibitory system and various uses of the modulators so identified.

Description

200950808 VI. Description of the invention: [Technical field to which the invention pertains] The present invention relates generally to neurodevelopment and neurological disorders. In particular, the invention relates to the identification of novel modulators of myelin-related inhibitory systems and the various uses of such modulators as identified herein. [Prior Art] Wei phospholipid and Zhao phospholipid related protein

V It is known that the axons of adult mammalian CNS neurons regenerate after injury and the energy is extremely limited, while the axons in the peripheral nervous system (PNS) can be rapidly regenerated. It is known that the limited regenerative capacity of CNS neurons is due in part to the intrinsic properties of CNS axons, but also due to environmental tolerances. CNS myelin is not the only source of neurite outgrowth signals' and it contains a variety of inhibitory molecules that are effective in blocking axonal growth, and thus constitute a significant remodeling barrier. Three of these myelin-related maps have been identified: Nogo (also known as NogoA) is a member of the Reticulon family of proteins that has two transmembrane domains; myelin-associated glycoprotein (MAG) is a superfamily of Ig ® family of transmembrane proteins; and OMGp has a glycosylated phospholipid inositol (GPI) anchored leucine-rich repeat (LRR) protein. Chen et al, Nature 403: 434-- 39 (2000); GrandPre et al, Nature 417: 439-444 (2000); • Prinjha et al, Nature 403: 383-384 (2000); McKerracher et al., Neuron 13 :805-1 1 (1994) ; Wang et al, Nature 417:941-4 (20020: Kottis et al, J. Neurochem 82: 1566-9 (2002). One of the NogoA parts of Nogo66 has been described as having 66 The extracellular polypeptide of amino acid is present in all three subtypes of Nogo. 139862.doc 200950808 Despite the differences in structure, it has been shown that all three inhibitory proteins (including Nogo66) bind to the same GPI anchoring receptor (called It is the Nogo receptor) (NgR; also known as Nogo receptor-1 or NgRl), and it has been suggested that NgR may be required to mediate the inhibition of Nogo, MAG and OMgp. Fournier et al, Nature 409:341- 346 (2001). Two NgR1 homologs (NgR2 and NgR3) have also been identified. US 2005/0048520 Al (Strittmatter et al.) published on March 3, 2005. In view of the NgR-based GPI-anchored cell surface proteins, It is not possible to use a direct signal transconductor (Zheng et al, Proc. Natl. Acad. Sci. USA 102: 1205-1210 (2005)). It has been suggested that the neurotrophin receptor p75NTR can be used as a co-receptor for NgR and can provide a signal transduction moiety in the receptor complex (Wang et al, Nature 420: 74-78 (2002); Wong et al. Nat. Neurosci. 5: 1302-1308 (2002)).

PirB and human homologs Class I major histocompatibility complexes (MHCs) were originally identified as segments encoding a family of molecules important for the immune system. Recent evidence indicates that class I MHC molecules have other functions in developing and adult CNS. Boulanger and Shatz, Nature Rev Neurosci. 5:521-53 1 (2004); US 2003/0170690 (Shatz and Syken), September 11, 2003. It has been found that multiple class I MHC members and their binding partners are expressed in CNS neurons. In recent years, genetic and molecular studies have focused on the physiological functions of CNS class I MHC, and initial results indicate that class I MHC molecules can play a role in activity-dependent synaptic plasticity, during which the intensity of synaptic connections has been established. Increased or decreased in response to neuronal activity, followed by long-term structural changes in the 139862.doc 200950808 circuit. In addition, class I MHC coding regions are genetically associated with a wide variety of neurological symptoms, and it is believed that the abnormal function of class I MHC molecules contributes to disrupting normal brain development and plasticity. A known class I MHC receptor system PirB in the immune environment is the murine polypeptide first described by Kubagawa et al., Proc. Nat. Acad. Sci. USA 94:5261-6 (1997). Mouse PirB has several human homologs that are members of the leukocyte immunoglobulin-like receptor subfamily B (LILRB) and are also referred to as "immunoglobulin-like transcripts" (ILT). Human homologs exhibit significant homology to the murine sequences, from highest to lowest order: LILRB3/ILT5, LILRB1/ILT2, LILRB5/ILT3, LILRB2/ILT4, and they are inhibitory receptors like PirB . LILRB3/ILT5 (NP_006855) and LILRB 1/ILT2 (NP_006660) were first described by Samaridis and Colonna in Eur. J. Immunol. 27(3): 660-665 (1997). LILRB5/ILT3 (NP-006831) has been determined by Borges et al., J. Immunol. 159(11): 5192-5196 (1997). LILRB2/ILT4 (also known as MIR10) is determined by Colonna et al., J. Exp. Med. 186: 1809-18 (1997). PirB and its human homologues exhibit a large degree of structural difference. Sequences of various alternative splicing formats are available from EMBL/GenBank, including, for example, the human accession number of human ILT4 cDNA: ILT4-cll AF009634, ILT4-cll7 AF1 1566, ILT4-C126 AF1 1565. As noted above, PirB/LILRB polypeptides are class I MHC (MHCI) inhibitory receptors and their role in modulating immune cell activation is well known in the art (Kubagawa et al., supra; Hayami et al, J) Biol. Chem. 272:7320 (1997); Takai et al. 139862.doc 200950808, Immunology 1 15:433 (2005); Takai et al, Immunol. Rev. 181:215 (2001); Nakamura et al. Nat·Imml 5:623 (2004); Liang et al., Eur. J. Immunol. 32:2418 (2002)).

A recent study by Syken et al. (Science 313: 1795-800 (2006)) reports that pirB is expressed in a subset of neurons throughout the brain. In mutant mice lacking functional PirB, the cortical-dominant column (〇D) plasticity was significantly enhanced at all ages. This suggests that PirB has a function of limiting activity-dependent plasticity in the visual cortex. SUMMARY OF THE INVENTION The present invention is based, at least in part, on the discovery that the use of a functional blocker anti-PirB antibody to interfere with PirB activity contributes to rescue of neurite outgrowth inhibition by Nogo66 and medullary lipids, while blocking PirB and NgR activity can result in Myelin inhibition is almost completely relieved. In one aspect, the invention relates to the same table of an isolated anti-PirB/LILRB antibody that binds substantially to an antibody selected from the group consisting of YW259.2, YW259.9, and YW259.12, binding to human PirB (LILRB). Bit. In another aspect, the invention relates to an isolated anti-PirB/LILRB antibody that competes for binding to human PirB (LILRB) with an antibody selected from the group consisting of YW259.2, YW259.9, and YW259.12. In another aspect, the invention relates to an isolated anti-pirB/LILRB antibody comprising one, two or three hypervariable region sequences derived from a heavy chain selected from the group consisting of: YW259.2 heavy chain (SEQ ID NO: 4 or 11), YW259.9 heavy chain (SEQ ID NO: 5 or 12), and YW259.12 heavy chain (SEQ ID NO: 6 or 13). 139862.doc 200950808 In one embodiment, the antibody comprises all of the hypervariable region sequences of the YW259.2 antibody heavy chain (SEQ ID NO: 4 or 11). In another embodiment, the antibody comprises all of the hypervariable region sequences of the YW259.9 antibody heavy chain (SEQ ID NO: 5 or 12). In another embodiment, the antibody comprises all of the hypervariable region sequences of the YW259.12 antibody heavy chain (SEQ ID NO: 6 or 13). In another embodiment, the antibody comprises a light chain. In another embodiment, the antibody comprises one, two or three hypervariable region sequences from the light chain of the polypeptide sequence of SEQ ID NO: 7. In another embodiment, the antibody comprises all of the hypervariable region sequences of the light chain comprising the polypeptide sequence of SEQ ID NO: 7 or 15. In a specific embodiment, the antibody comprises both a heavy chain and a light chain, wherein the heavy chain comprises one, two or three hypervariable region sequences from a heavy chain selected from the group consisting of: YW259.2 heavy chain (SEQ ID NO: 4), YW259.9 heavy chain (SEQ ID NO: 5) and YW259.12 heavy chain (SEQ ID NO: 6), and/or light chain comprising one, two or three: one from SEQ ID NO: 7 The hypervariable region sequence of the light chain of the polypeptide sequence. In another embodiment, the anti-system is selected from the group consisting of YW259.2, YW259.9, and YW259.12. In another aspect, the invention relates to an isolated anti-PirB antibody, wherein the full length IgG form of the antibody specifically binds to human PirB with a binding affinity of 5 nM or greater, or 1 nM or greater. In one embodiment, the antibody promotes axonal regeneration, such as regeneration of CNS neurons. 139862.doc 200950808 In another embodiment, the antibody at least partially rescues neurite outgrowth inhibition caused by N〇g〇66 and myelodys. In all aspects, the antibody is preferably a monoclonal antibody, which may be, for example, a chimeric antibody, a humanized antibody, an affinity matured antibody, a human antibody, or a bispecific antibody, antibody fragment or immunoconjugate. In another aspect the invention relates to a polynucleotide encoding an anti-pirB antibody described herein. In other aspects, the invention relates to vectors and host cells comprising a polynucleotide encoding an antibody described herein, including a coding sequence for one or more antibody chains. Host cells include prokaryotic, eukaryotic, and mammalian hosts. In another aspect, the invention relates to a method of making an anti-PirB antibody comprising (a) expressing a vector comprising a nucleic acid encoding an antibody in a suitable host cell' and (b) recovering the antibody. In another aspect, the invention is directed to a composition comprising an anti-PirB/LILRB antibody described herein and a pharmaceutically acceptable excipient. If desired, the composition comprises a second drug wherein the anti-pirB/ULRB resistant system is the first piece of music. For example, the second drug can be an NgR inhibitor, such as an anti-NgR antibody. In a different aspect, the invention relates to a kit comprising an anti-anti-PirB/LILRB antibody as described herein. In another aspect, the invention relates to a method of promoting axonal regeneration comprising administering to an individual in need thereof an effective amount of an anti-pirB/LILRB antibody described herein. Preferably, the individual is a human patient. In various embodiments, the methods of treatment described herein promote neuronal survival 139862.doc -8 - 200950808 and/or induce neuronal growth. In another state, A, i >#明系方法 on the treatment of neurodegenerative diseases, including the need for it! The side of the disease

PirB/LILRB antibody. For example, a physical cast and an effective amount of a physical 2 transgenic disease caused by the anti-insertion nervous system described herein may be characterized by a brain damage to the middle.胄 且 且 且 在一 在一 在一 在一 在一 在一 在一 在一 在一 在一 在一 在一 在一 在一 在一 在一 在一 在一 在一 在一 ' ' ' ' ' ' ' ' ' ' ' ' 神经 神经 神经 神经 神经 神经 神经 神经 神经 神经 神经 神经 神经 神经 神经 神经 神经 神经 神经 神经 神经: myasthenia gravis, muscular dystrophy, amyotrophic lateral sclerosis (MS), progressive muscle atrophy, invasive medullary hereditary muscular atrophy, physical injury (eg burn, illusion) or such as diabetes, kidney Dysfunction (4) Disease state or toxic effects caused by chemotherapy for AIDS and AIDS cause peripheral nerve injury m, rupture, or prolapsed disc syndrome, cervical spondylosis, plexus disease, thoracic discharge syndrome 1 Neuropathy (eg, caused by erroneous, amphetamine )), porphyrin, etc., chlorpheniramine, Gullain-Barre ─, Alzheimer's disease (Al-, s disease), Huntington Huntington's Disease, and parkin s disease ° The present invention further relates to anti-idiotypic antibodies that specifically bind to the anti-PirB antibodies described herein. [Embodiment] A. Definitions As used herein, the terms "paired immunoglobulin-like receptor B" and "pirB" 139862.doc 200950808 are used interchangeably and refer to the native sequence (841 amino acids of SEQ ID NO: 1). Mouse inhibitory protein (Fig. 1) (NP_035225)) and its natural sequence homologs in rats and other non-human mammals, including all naturally occurring variants (eg, alternative splicing and allelic variants and Subtype), as well as its soluble form. See Kubagawa ί/α 94, 5261 (1997) for additional details. The terms "LILRB", "ILT" and "MIR" are used interchangeably herein and refer to all members of the human "leukocyte immunoglobulin-like receptor subfamily", including all naturally occurring variants (eg, alternatives) Sexual splicing and allelic variants and subtypes), as well as soluble forms thereof. Each member of the LR-type subfamily of the LILR receptor is named by an abbreviation followed by a number, such as LILRB3/ILT5, LILRB1/ILT2, LILRB5/ILT3, and ILRIB2/ILT4, unless otherwise stated, no, J All naturally occurring variants (eg, alternative splicing and allelic variants and subtypes), as well as soluble forms thereof, are also included when referring to any individual member. Thus, for example, "LILRB2", "LIR2" and "MIR10" are used interchangeably herein and refer to the polypeptide of 598 amino acids of SEQ ID ΝΟ: 2 (Figure 1) (ΝΡ_005 865), and their natural presence. Variants (eg, alternative splicing and allelic variants and subtypes), as well as soluble forms thereof. For further details see Martin % K 'Trends Immunol. 23, 81 (2002) The term "PirB/LILRB" as used herein refers to the corresponding mouse and human proteins and natural sequence homologs in other non-human mammals, including all Naturally occurring variants (eg, alternative splicing and allelic variants and subtypes), as well as soluble forms thereof. 139862.doc -10- 200950808 has the broadest meaning and includes all proteins in the fat, including the term "myelin-related protein" used to inhibit neuronal regeneration of CNS myeloids Nogo, MAG and OMgp.

❹Isolated" is defined and separated and/or used in the composition of the natural environment in which the various proteins described herein are described. Contaminant components of their natural environment typically interfere with the diagnostic or therapeutic application of the protein' and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In a preferred embodiment, (1) the protein can be purified by using a rotary cup sequencer to obtain at least 15 residues of the alpha terminal or internal amino acid sequence, or (2) not light or (d) The protein is purified to homogeneity by sds_PAGE using Coomassie blue (c〇_ssie blue) or preferably silver staining, or (7) the protein is purified to homogeneity by mass spectrometry or peptide mapping techniques. The isolated protein includes the original T protein in the recombinant cell, since at least one of the groups n flb in the natural environment of the protein is absent. However, usually the isolated protein can be prepared by at least one purification step. A nucleic acid molecule is a nucleic acid molecule that is identified and isolated from at least one π-stained nucleic acid, and which is typically associated with a 6-helogram contaminating nucleic acid molecule in the natural source of the nucleic acid. The shape of the isolated nucleic acid molecule or the ring in which it is located is different from that in nature. Thus, the isolated nucleic acid fraction = is different from the nucleic acid molecule present in the natural cell. However, an isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally expresses the nucleic acid, wherein, for example, the nucleic acid molecule is located at a different chromosomal location than the native cell. The term "r PirB/LILRB antagonist" as used herein, is used to mean a reagent that inhibits, neutralizes, inhibits, eliminates, reduces or interferes with PirB/LILRB activity in 139S62.doc 200950808. In particular, PirB/LILRB antagonists interfere with myelin-associated inhibitory activity, thereby promoting neurite outgrowth and/or promoting neuronal growth, repair and/or regeneration. In a preferred embodiment, the PirB/LILRB antagonist inhibits binding of PirB/LILRB to Nogo66 and/or MAG and/or OMgp by binding to PirB/LILRB. PirB/LILRB antagonists include, for example, antibodies against PirB/LILRB and antigen-binding fragments thereof, can be isolated between PirB/LILRB and Nogo 66, or between PirB/LILRB and MAG, or

I

A truncated or soluble fragment of PirB/LILRB, Nogo 66, MAG or OMgp that binds between PirB/LILRB and OMgp, and a small molecule inhibitor of the PirB/LILRB-related inhibitory pathway. PirB/LILRB antagonists also include short interfering RNA (siRNA) molecules that inhibit or reduce the expression of PirB/LILRB mRNA. Preferably, the PirB/LILRB antagonist is an anti-PirB/LILRB antibody. The term "antibody" as used herein has the broadest meaning and specifically encompasses intact antibodies, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies) formed from at least two intact antibodies, and antibody fragments ( As long as it exhibits the desired biological activity). The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homologous antibodies, i.e., the individual antibodies comprising the population are identical except for the possible naturally occurring mutations which may be present in minor amounts. Individual antibodies are highly specific and target a single antigenic site. Moreover, in contrast to multiple antibody preparations comprising different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to specificity, the advantage of monoclonal antibodies is that they are not contaminated by other antibodies during synthesis. The modifier "single plant" table 139862.doc -12- 200950808 The non-antibody is characterized as being obtained from a substantially homologous antibody population and should not be construed as requiring any particular method to produce the antibody. For example, a monoclonal antibody to be used in accordance with the present invention can be produced by a hybridoma method first described by Köhler et al., 256:495 (1975) or by recombinant DNA methods (eg, see U.S. Patent No. 4,816,567). For example, 'single antibody can also be used by claeks〇n et al. (iVaiwre, 352: 624-628 (1991)) and Marks et al. (". Mo/. , 222··581-5 97 (1991)) The technique described is isolated from a phage antibody library. Antibodies include, inter alia, "chimeric" antibodies in which one of the heavy and/or light chains is identical or homologous to the corresponding sequence of an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain is derived from The corresponding sequence of another species or antibody belonging to another antibody class or subclass (and fragments of such antibodies, as long as they exhibit the desired biological activity) is identical or homologous (U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Aca.d. Sci. USA, 81:6851-6855 (1984)). The chimeric antibodies of interest herein include primatized antibodies comprising variable domain antigen-binding sequences derived from non-human primates (eg, Old World Monkey, sputum, etc.) and human constant region sequences. . An "antibody fragment" comprises a portion of an intact antibody, preferably comprising an antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab, F(ab,)2 and Fv fragments; bispecific antibodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. The "intact" anti-system comprises antibodies to the antigen binding variable region as well as the light chain constant domain (Cl) and the heavy chain constant domains (CH1, Ch2 and CH3).悝 139862.doc • 13· 200950808 The domain may be a native sequence constant domain (eg, a human native sequence constant domain) or an amino acid sequence variant thereof. Preferably, the intact antibody has one or more effector functions. A "humanized" form of a non-human (e.g., rodent) antibody is a chimeric antibody comprising a minimal sequence derived from a non-human immunoglobulin. Most humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from the hypervariable region of the recipient are subjected to hypervariable regions from non-human species (eg, mouse, rat, rabbit, or non-human primate). (donor antibody) and residue substitution with the desired specificity, affinity and ability. In some cases, human immunoglobulin framework region (FR) residues are substituted with corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These changes were implemented to further improve antibody performance. In general, a humanized antibody can comprise substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab')2, Fabc, Fv), wherein all or substantially all of the hypervariable loops All correspond to non-human immunoglobulins, and all or substantially all of the FRs are those of human immunoglobulin sequences. Humanized antibodies may also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin constant region. Further details can be found in Jones et al, Nature 321:522-525 (1986); Riechmann et al, Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992) ). The term "hypervariable region" as used herein, refers to a region of the variable domain of an antibody that has hyperdenaturation and/or can form a structurally defined ring. The hypervariable region comprises amino acid residues from the "complementarity determining region" or "CDR" 139862.doc 14· 200950808 (ie residues 24-34, 50-56 and 89-97 in the light chain variable domain and 31-3 5, 50-65, and 95-102 in the heavy chain variable domain; Kabat et al, Sequences of Proteins of Immunological Interest, % 5 edition, Public Health Service > National Institutes of Health » Bethesda ' MD. (1991)) and/or their residues from the "hypervariable loop" (eg residues 26-32, 50-52 and 91-96 in the light-bond variable domain and heavy chain variable domains) 26-32, 53-5 5 and 96-101; 〇1〇如3和1^让J. Μσ/·出〇/· 196:901-917 (1987)). In both cases, variable domain residues are numbered according to Kabat et al. (sug.) as described in more detail below. The "framework region" or "FR" residues are those variable domain residues other than the hypervariable region residues defined herein. A "parent antibody" or "wild type" anti-system comprises an antibody that lacks one or more amino acid sequence changes compared to the antibody variants disclosed herein. Thus, a parent antibody typically has at least one hypervariable region, the amino acid sequence of which differs from the amino acid sequence of the corresponding hypervariable region of the antibody variants disclosed herein. The parent polypeptide may comprise native (and naturally occurring) antibodies (including naturally occurring allelic variants), or existing amino acid sequence changes (eg, insertions, deletions, and/or other changes) in naturally occurring sequences. ) antibodies. Throughout the disclosure, "wild-type", "WT", "wt" and "parent" or "parental" antibodies are used interchangeably. As used herein, "antibody variant" or "variant antibody" refers to an antibody having an amino acid sequence that differs from the amino acid sequence of the parent antibody. Preferably, the antibody variant comprises an amine group that is not found in nature. A heavy chain variable domain or a light chain variable domain of an acid sequence. The sequence of these variants and the parent antibody 139862.doc -15- 200950808 is necessarily less than 100%. In a preferred embodiment, the amino acid sequence of the antibody variant has an identity or similarity to the amino acid sequence of the amino acid sequence of the heavy or light chain variable domain of the parent antibody of from about 75% to less than 100°/. More preferably, it is from about 80% to less than 100%, more preferably from about 85% to less than 100%, still more preferably from about 90% to less than 100%, and most preferably from about 95% to less than 100%. Antibody variants typically comprise one or more amino acid changes in or adjacent to one or more of the hypervariable regions. "Amino acid change" means a change in the amino acid sequence of a predetermined amino acid sequence. Exemplary changes include insertions, substitutions, and deletions. "Amino acid substitution" refers to the replacement of an existing amino acid residue in a predetermined amino acid sequence with another different amino acid residue. By "alternative" amino acid residue is meant an amino acid residue in place of or substituted for another amino acid residue in the amino acid sequence. The replacement residue may be a naturally occurring or non-naturally occurring amino acid residue. . "Amino acid insertion" refers to the introduction of one or more amino acid residues into a predetermined amino acid sequence. The amino acid insertion may comprise a "peptide insertion", in which case a peptide comprising two or more peptide-linked amino acid residues is introduced into the predetermined amino acid sequence. If amino acid insertion involves peptide insertion, the inserted peptide can be generated by random mutagenesis such that it has an amino acid sequence that is not found in nature. The amino acid change "adjacent to the hypervariable region" refers to the introduction or substitution of one or more amino acid residues at the N-terminus and/or C-terminus of the hypervariable region, thereby allowing the inserted or substituted amine. At least one of the acid residue forms a peptide bond with the N-terminal or C-terminal amino acid residue of the hypervariable region. The "naturally occurring amino acid residue" is encoded by the genetic code, and its 139862.doc -16- 200950808 is selected from the group consisting of alanine (Ala), arginine (Arg), and winter brewing. Amine (Asn), aspartic acid (ASp), cysteine (Cys), face amine (Gin), glutamic acid (Glu), glycine (Gly), histidine (His), Isoleucine (lie), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (pro), serine (ser), Threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and proline (Val). As used herein, "non-naturally occurring amino acid residues" are amino acid residues other than those naturally occurring amino acid residues listed above, which are covalently bonded adjacent in the polypeptide chain. Amino acid residue. Examples of non-naturally occurring amino acid residues include norleucine, uric acid, n-proline, homoserine, and other amino acid residue analogs, such as Ellman et al. at Meth. Enzym. 202: They are described in 301-336 (1991). Such non-naturally occurring amino acid residues can be generated using the procedures of Noren et al., Science 244: 182 (1989) and Ellman et al. (supra). Briefly, the procedures involve chemically activating an inhibitory tRNA having a non-naturally occurring amino acid residue, followed by transcription and translation of the RNA in vitro. Throughout this disclosure, reference is made to the numbering system of Kabat, E. A. et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)). In these summaries, Kabat lists the various amino acid sequences of each subclass of antibodies and lists the most common amino acids at each residue position in the subclass. Kabat uses a method of assigning residue numbers to each of the amino acids listed, and this method of assigning residue numbers has become the industry standard. The Kabat numbering scheme is followed in this description. For the purposes of the present invention, to assign the residue 139862.doc -17-200950808 base number to the candidate antibody amino acid sequence not included in the Kabat outline, the following procedure can be followed. In general, the candidate sequence is aligned with any of the immunoglobulin sequences or any consensus sequences in Kabat. The comparison can be performed manually or by a computer using a generally accepted computer program. An example of this program is the Align 2 program. Alignment can be aided by the use of certain amino acid residues common to most Fab sequences. For example, the 'light and heavy chains typically each have two cysteine acids with the same residue number; in the VL domain the two cysteine acids typically have residue numbers 23 and 88, and in the VH structure The two cysteine residues in the domain are typically numbered 22 and 92. The framework residues are generally, but not always, substantially identical in residue number' however, the size of the CDRs is variable. For example, if the CDRs from the candidate sequence are longer than the CDRs of the Kabat sequence to which they are aligned, a suffix is typically added to the residue number to indicate insertion of additional residues (see, for example, residue lOOabc in Figure j B). No. 35 is assigned to any residue for, for example, a candidate sequence that aligns residues 34 and 36 with a Kabat sequence but no residues are contiguous with residue 35 between the residues Just fine. As used herein, an anti-system with a "high affinity" (or dissociation constant) is a nanomolar (nM) or better antibody. The size of "Nemo or better" can be expressed as Ζ nM ' where X is less than about 丨〇. An "affinity mature" anti-system has one or more altered antibodies in one or more of its CDRs that result in an improvement in the affinity of the antibody to the antigen relative to the parent antibody that does not have such a change. Preferably, the affinity matured antibody has a Namrol-level or even Pimor-level affinity for the target antigen. Affinity mature anti-systems are prepared by procedures known in the art. Marks et al., 139862.doc -18- 200950808 Price σ/TVc/mo/og less 10:779-783 (1992) illustrates affinity rejuvenation by VH and VL domain shuffling. The following literature describes random mutagenesis of CDR and/or framework residues: Barbas et al, 91: 3809-3813 (1994); Schier et al, Gene 169: 147-155 (1995); Yelton et al, // Immunol 155: 1994-2004 (1995); Jackson et al., /. /wmw(10)/· 154(7):3310- 9(1995); and Hawkins et al., J. Mo/.price/. 226:889- 896 (1992). The "functional antigen binding site" of an antibody binds to the site of the target antigen. The antigen binding affinity of the antigen binding site is not necessarily stronger than the parent antibody producing the nucleophilic site of the antigen, but the ability to bind the antigen can be performed using any of various methods known to be useful for assessing the binding of the antibody to the antigen. Measure. An anti-system having a "biological identity" of a given antibody has one or more biological characteristics of the antibody that distinguish the antibody from other antibodies that bind to the same antigen. To screen for antibodies that bind to an antigenic epitope to which the antibody of interest binds, conventional cross-blocking assays can be performed, such as those described in J Manual, Cold Spring Harbor Laboratory, Harlow and David Lane, eds. (1988). The term "filamentous phage" refers to viral particles capable of displaying a heterologous polypeptide on the surface and includes, but is not limited to, fl, fd, Pfl and M13. The filamentous phage may contain an optional marker such as tetracycline (e.g., ":fd-tet"). Various filamentous phage display systems are well known to those skilled in the art (see, for example, 139862.doc -19- 200950808)

Zacher et al., Gene 9: 127-140 (1980); Smith et al.

Science 228: 1315-1317 (1985); and Parmley and Smith, Gene 73: 305-318 (1988)). The term "panning" is used to mean a plurality of rounds of screening processes performed when identifying and isolating phage carrying a compound having high affinity and specificity for a target such as an antibody. The term "short interfering RNA (siRNA)" refers to a small double-stranded RNA that interferes with the expression of a gene. siRNA is an intermediate of RNA interference, and RNA interference is a process in which double-stranded RNA silences homologous genes. siRNA typically comprises two single-stranded RNAs that are about 15-25 nucleotides in length and form diploids, which may include single-stranded overhangs. Treatment of double-stranded RNA by an enzyme complex (e.g., a polymerase) cleaves the double-stranded RNA to produce siRNA. The use of the antisense strand of siRNA directs mRNA cleavage by RNA interference (RNAi) silencing of the complex, thereby promoting mRNA degradation. For example, using siRNA to silence specific genes in mammalian cells, base pairing regions are selected to avoid the chance of complementing irrelevant mRNAs. RNAi silencing complexes have been identified in the industry, such as Fire et al, Nature 391: 806-81 1 (1998) and McManus et al., Nat. Rev.

Genet. 3(10): 737-47 (2002). The term "interfering RNA (RNAi)" as used herein refers to a double-stranded RNA which can cause catalytic degradation of a particular mRNA, and thus can be used to inhibit/reduce the performance of a particular gene. The term "polymorphism" as used herein refers to more than one form of a gene or a portion thereof (e.g., an allelic variant). The portion of the gene that has at least two different forms is called the "polymorphic region" of the gene. The specific genetic sequence of the polymorphic region of the gene 139862.doc -20- 200950808 is listed as "allele." The polymorphic region may be a single nucleotide, differing in different alleles, or may be several nucleotides in length. The term 'disorder' as used herein refers to any condition that can benefit from treatment with a (iv) /ULRB2 antagonist (eg, an anti-PirB antibody), including antibiotics expected to benefit from axonal regeneration therapy and/or synapses in the nervous system. Any condition that improves plasticity. Non-limiting examples of treats in this article include, but are not limited to, diseases and conditions that may benefit from promoting neurite outgrowth, promoting neuronal growth, repair or regeneration, including nerves. Conditions such as physical damage to nerves and neurodegenerative diseases. Such conditions include, inter alia, t-injury of the middle turble nervous system (eg, vertebral injury and head trauma); brain damage associated with stroke; and neurological disorders associated with neurodegeneration, such as trigeminal neuralgia, glossopharyngealgia, Bell pruritus, myasthenia gravis, muscular dystrophy, muscle atrophy:,: rs), progressive muscular atrophy, progressive extension of hereditary muscle atrophy, rheumatoid arthritis (MS) 'dog-like, rupture, or off Pelvic discs = scars, plexus disorders, thoracic outlet destruction syndrome, peripheral nerve damage caused by physical (four) or disease states such as diabetes, peripheral neuropathy (eg, lead, phenyl sulfone mr~ - 5) ), Yulin disease, Green-Baline syndrome, Alzheimer's disease. f is Huntington's disease, or Parkinson The term "treatment" as used herein refers to therapeutic, Λ 麻 麻 box and prevention (7). Continuous treatment or administration refers to a person who does not have one or more days of treatment in Tianyi and intermittently, “^ Μ 性 性 或 投 “ “ “ “ “ “ “ “ 而 而 而 而 而 而 而 而 而 而 而 而 而 而 而 而 而 而 而 而 而 而It is periodic. The term "preventing neurodegeneration" includes (1) the ability to inhibit or prevent dementia or have neurodegenerative degeneration in a newly diagnosed 139862.doc 200950808 with neurodegenerative diseases. The ability of a patient to have a degenerative condition in a patient at risk of developing a new neurodegenerative disease, and (2) to inhibit or prevent further feeding in a patient already suffering from a symptom of a neurodegenerative disease. 1 "Mammal" means any classification The second mammals include humans, higher non-human primates, rodents, livestock, such as cattle' horses, dogs and cats. In the preferred embodiment of the invention, the mammal is a human. "Combination" with one or more other therapeutic agents includes simultaneous (together) administration and continuous administration in any order. The "effective amount" of the society is sufficient to achieve beneficial or desired therapeutic (including preventive). The amount of fruit. An effective amount can be administered by one or more administrations. The terms "cell", "cell line" and "cell culture" as used herein are used interchangeably and all such expressions include the progeny thereof. Therefore, the words "transformants" and "transformed cells" include primary individual cells and derived therefrom.

Cultures without regard to the number of transfers. It should also be understood that the DNA contained in all offspring may not be exactly the same due to intentional or unintentional mutations. The term "progeny" refers to any and all progeny of each generation after the original transformed cell or cell line. It includes mutant progeny that have the same function or biological activity as those screened in the original transformed cell. If you want to use a unique name, you can understand it according to the context. The "amino acid sequence identity percent (%)" for the sequences identified herein is defined as: in the candidate sequence and in the reference sequence after aligning the sequences and, if necessary, introducing a gap to achieve a maximum percent sequence identity. Amino group 139862.doc • 22· 200950808 Percentage of amino acid residues identical to acid residues, and does not treat any conservative substitution as part of sequence identity. Alignment for the purpose of determining the percentage of amino acid sequence percent can be achieved in a variety of ways known in the art to determine the appropriate parameters for the alignment, including specifying the maximum alignment required for the full length sequence being compared. Nasal method. For the purposes of this paper, the sequence-ratio computer-based ALIGN-2 can be used to obtain the amino acid identity percentile value, which was designed by Genentech and whose source code has been linked to the user documentation to the US Copyright Office. (us Copyright Office) (Washington, DC, 20559), the registration number is US copyright registration number TXU51〇〇87. The ALIGN-2 program is publicly available through Genentech, Inc. (South San Francisco, C A). All sequence comparison parameters are set by the ALiGN 2 program and are immutable. Those skilled in the art can readily determine the "stringency" of the hybridization reaction and are generally based on empirical calculations of probe length, wash temperature, and salt concentration. In general, longer probes require higher temperatures to achieve proper annealing, and shorter probes require lower temperatures. Hybridization generally depends on the ability of the denatured DNA to reanneal when the temperature of the environment in which the complementary strand is located is below its melting temperature. The higher the degree of expectation between the material and the hybridizable sequence, the higher the relative temperature that can be used. It can therefore be concluded that a higher relative temperature tends to make the reaction strip 2 more stringent. The reaction conditions required for lower temperatures are less stringent. For additional details on the rigor of the hybrid reaction, see Ausubei et al.

Protocols in Molecular Biology, Wiley

Interscience Publishers, (1995). The 4 π stringency conditions in this paper can be confirmed by the following methods: 139862.doc -23- 200950808 Determination: (1) Low ionic strength and high temperature are used in washing; Under the armpit, 0.015 bismuth sodium sulphate/0.0015 μ sodium citrate/0.1% sodium lauryl sulfate was used; (2) a denaturant was used during the hybridization; 5〇0/〇(ν/ν) guanamine and 0.1% bovine serum albumin 01% sucrose (Ficoll) / 〇 1 〇 / 〇 polyvinyl ketone / 750 mM 50 mM sodium sulphate buffer (pH 6.5) with sodium sulphate, 75 mM sodium citrate; or (3) 50% guanamine, 5 X SSC ((K75 M NaCl, 0.075 柠檬 lemon) at 42 °C Sodium sulphate, 50 mM sodium phosphate (pH 6.8), 0.1% sodium sulphate, 5xDenhardt, s solution, ultrasonically treated drum sperm DNA (50 eg/ml), 〇tl% SDS, and 10% sulphuric acid Glucan' and washed in 0.2x SSC (sodium sulphate/sodium citrate) at 42 ° C and in 50% amylamine at 55 C, followed by 55. Under the OD containing EDTA. High stringency washing is implemented in lxSSC.

"Moderate stringency conditions" can be determined according to Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and is included at 37. Incubate overnight in a solution containing: 20% guanamine, 5xSSC (15 mM mM)

NaCl, 15 mM sodium citrate), 50 ηηΜ sodium phosphate (pH 7.6), 5 X

Denhardt's solution, 10% dextran sulfate, and 2 〇mg/ml denatured cut salmon sperm DNA, followed by washing the filter at about 37_5 (rc) in lxSSC. Those skilled in the art can understand how to adjust the temperature as needed, Ionic strength, etc. to adapt factors such as probe length and the like. The term "control sequence" refers to the DNA sequence required to express an operably linked coding sequence in a particular host organism. For example, a control sequence suitable for prokaryotes Including promoter, (as needed) operator sequence, and core 139862.doc -24· 200950808 Known eukaryotic cell-producible promoter, polyadenylation Φ When making a nucleic acid functionally related to another nucleic acid sequence, The nucleic acid is "operably linked." For example, if the DNA of the pre-sequence or secretion leader sequence is expressed as the egg involved in the secretion of the polypeptide, the DNA of the (4) pre-sequence or secretion leader sequence is operably linked to the polypeptide. a promoter or enhancer operably linked to the coding sequence if it affects the transcription of the coding sequence; or if the ribosome binds The location of the point facilitates translation, and the ribosome binding site is operably linked to the coding sequence. In general, "operably linked" means that the ligated dna sequence is contiguous and in the case of a secretory leader sequence It is in the contiguous sequence of the reading phase. However, the 'enhancer does not need to be contiguous. The ligation can be accomplished by ligation at a convenient restriction site. If the site is not present, the synthetic oligonucleotide can be used according to customary conventions. Child or connector.

Glycosome Binding Site Signals and Enhancers In this context, "small molecules" are defined as having a molecular weight of less than about 丨〇〇〇Dalton, preferably less than about 500 Daltons. B. Generation of anti-PirB/LILRB anti-PirB antibodies The anti-PirB antibodies of the invention can be produced by methods known in the art, including recombinant DNA techniques. (i) Preparation of antigen A soluble antigen or a fragment thereof which is conjugated to other molecules as needed may be used as an immunogen for generating an antibody. For transmembrane molecules such as receptors, fragments thereof (e.g., the extracellular domain of the receptor) can be used as the immunogen. Alternatively, cells expressing transmembrane molecules can be used as immunogens. Such cells 139862.doc -25- 200950808 may be derived from natural sources (e.g., cancer cell lines) or may be cells that have been transmembrane molecules that have been transformed by recombinant techniques. Those skilled in the art will be aware of other antigens and the forms in which they can be used to prepare antibodies. (ii) Multiple antibodies Preferably, multiple antibodies are produced in an animal by multiple subcutaneous (SC) or intraperitoneal (ip) injections of the relevant antigen and adjuvant. It is useful to use a bifunctional reagent or a derivatizing agent in an organism to be immunized (for example, 'Malay-bromide phenyl sulfosuccinimide (via a cysteine residue), N_ Hydroxy sulphon

Perinimide (via an lysine residue), glutaraldehyde, succinic anhydride, socl2 or RlN=C=NR, wherein the ruthenium is different from the R1 group) to associate the antigen with an immunogenic protein (eg, keyhole) Hemoglobin, serum albumin, bovine thyroglobulin or soybean trypsin inhibitor).

Immunization of antigens, immunogenic conjugates or derivatives by means of (for example, bark or 5 protein or conjugate (for) in rabbits or mice) and 3 volumes of Freund's (- 3) Complete adjuvant combination," the solution was injected intradermally at multiple sites. After one month, the booster is performed by a multi-spot _ = shot of 1/5 of the initial dose to the peptide or ^ =: in the complete adjuvant of Freund's complete adjuvant... After the day, the animal is inserted" Perform antibody titer analysis. Add anti-stable: state to the animal. Preferably, 'splicing to different proteins and plagues. The conjugate of the antigen is used to artificially prepare the animal. Similarly, such as M cell culture towel as protein. Fusion 4 (4) Single anti-purine aggregating agent _ to enhance the immune response. 139862.doc * 26 - 200950808 Monoclonal antibodies can be prepared using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975). Alternatively, it can be prepared by a recombinant DNA method (U.S. Patent No. 4,816,567). In the hybridoma method, a mouse or other suitable host animal (such as a hamster or a macaque) is immunized according to the above to induce Lymphocytes that produce or produce antibodies that specifically bind to proteins used for immunization. Alternatively, lymphocytes can be immunized in vitro, and then lymphocytes can be fused with bone marrow tumor cells using a suitable fusion agent (eg, polyethylene glycol). 'To form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). The hybridoma cells thus prepared are seeded in a suitable medium and grown therein, the medium Preferably, it contains one or more substances which inhibit the growth or survival of the unfused parental myeloma cells. For example, if the parental myeloma cells lack hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), Bay I hybridization The culture medium of the tumor usually contains hypoxanthine, aminopterin and thymidine (HAT medium), which can prevent the growth of HGPRT-deficient cells. Preferred myeloma cell lines can effectively fuse and support selected antibody-producing cells. A cell that stably produces a large amount of antibody and is sensitive to a medium such as HAT medium. Among these cells, a preferred myeloma cell line is a murine myeloma line, for example, available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA obtained MOPC-21 and MPC-11 mouse tumors, and can be obtained from American Type Culture Collection, Rockvi Rp, Md. USA obtained SP-2 or X63-Ag8-653 cells. Also 139862.doc -27- 200950808 Describes that human myeloma and mouse-human hybrid myeloma cell lines can be used to produce human monoclonal antibodies (Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)). The production of a monoclonal antibody against the antigen was analyzed in a medium in which hybridoma cells were grown. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or in vitro binding assays (e.g., radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)). After identifying hybridoma cells that produce antibodies with the desired specificity, affinity, and/or activity, the pure lines can be sub-selected by limiting dilution procedures and grown by standard methods (Goding, Mono clonal) Anti bo dies · ' Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can be grown as ascites tumors in animals. The monoclonal antibody secreted by the meptoline can be separated from the culture medium, ascites, or serum by a conventional immunoglobulin purification program, for example, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, Dialysis or affinity chromatography. The DNA encoding the monoclonal antibody can be easily isolated and sequenced using a conventional procedure (for example, by using a nucleotide probe capable of specifically binding to a gene encoding a heavy chain and a light chain of a monoclonal antibody). Hybridoma cells can be used as a preferred source of such DNA. After isolation, the DNA can be immediately placed in the expression vector 139862.doc -28 - 200950808, and then transfected into host cells (eg, E. coli cells, 猿COS cells, which are not originally immunoglobulin-producing, In Chinese hamster ovary (CHO) cells, or myeloma cells, synthesis of monoclonal antibodies is achieved in recombinant host cells. Recombinant production of antibodies is set forth in more detail below. In another embodiment, the antibody or antibody fragment can be isolated from an antibody phage library generated using the techniques set forth in McCafferty et al., Nature 3 48:552-5 54 (1990).

The use of phage libraries to isolate murine and human antibodies is described in Clackson et al, Nature, 352: 624-628 (1991) and Marks et al, J. Mol. Biol, 222: 581-597 (1991), respectively. Subsequent publications describe the production of high-affinity (nM-class) human antibodies by chain shuffling (Marks et al, Bio/Technology 10:779-783 (1992)), and a combination of strategies as a strategy for constructing maximal phage libraries. And in vivo reorganization (Waterhouse et al., Nuc.

Acids·Res., 21: 2265-2266 (1993)). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma technology that separates individual antibodies. The homologous murine sequence can also be replaced by, for example, the coding sequences of the human heavy and light chain constant domains (U.S. Patent No. 4,8,567; M〇rHs〇n et al., /Vm. #如/. Scz·· ί/α , 81:6851 (1984)) or modification of DNA by covalently joining all or part of the coding sequence of a non-immunoglobulin polypeptide to an immunoglobulin coding sequence. Typically, such non-immunoglobulin polypeptides can be substituted for the constant domain of an antibody or a variable domain thereof that can replace one of the antigen binding sites of the antibody, thereby producing a chimeric bivalent antibody comprising __specific to the antigen The antigen binding site and another antigen binding site specific for different antigens 139862.doc •29· 200950808 (iv) Introduction of one or more non-human sources from humanized antibodies and human anti-humanized antibodies Amino acid residue. These non-human amino acid residues are often referred to as "input" residues, which are typically taken from the "input" variable domain. Humanization can basically be performed according to the method of Winter and co-workers by substituting the corresponding sequences of human antibodies with rodent CDR or CDR sequences (Jones et al, Nature, 321:522-525 (1986); Riechmann et al., Nature , 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)). Thus, such "humanized" anti-system chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantially less than a portion of the entire human variable domain has been substituted with the corresponding sequence of 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 at analogous sites in rodent antibodies. The choice of human variable domains (both light and heavy) to be used to make humanized antibodies is of great importance for reducing antigenicity. The sequence of the variable domain of the rodent antibody is screened against a complete library of known human variable domain sequences according to the so-called "best fit" method. The human sequence closest to the rodent sequence is then accepted as the human framework region (FR) for humanized antibodies (Sims et al., /. /mww„〇/· 151: 2296 (1993); Chothia et al., 乂Mo/ 〇 / 196 : 901 (1987)). Another method uses a specific framework derived from the consensus sequence of all human antibodies with a specific light chain or heavy chain subgroup. The same framework can be used in several different humanized antibodies. (Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); 139862.doc • 30- 200950808

Presta et al., immunol. 151: 2623 (1993)). More importantly, humanized antibodies should retain high affinity for antigens and other beneficial biological properties. In order to achieve this, humanized antibodies can be prepared according to a preferred method by using parental and humanized three-form analysis of parental sequences and various conceptual humanized products. Three-dimensional immunoglobulin models are commonly commercially available and are well known to those skilled in the art. Computer programs can be used which elucidate and display possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. Observation of such displays allows for the possible role of the analyzable residues in the functioning of the candidate immunoglobulin sequences, i.e., the analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this manner, FR residues can be selected from the recipient and the input sequence and combined to achieve the desired antibody characteristics, e.g., high affinity for the target antigen. In general, CDR residues are directly and most substantially involved in affecting antigen binding.

Alternatively, transgenic animals (e. g., mice) can now be produced that, upon immunization, produce all of the components of a human antibody without producing endogenous immunoglobulin. For example, it has been described in the literature that homozygous deletion of the antibody re-ligated region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. The degraded transfer of human germline immunoglobulin genes into these germline mutant mice can result in the production of human antibodies following antigen challenge. For example, 'See Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551 (1993); Jakobovits et al, Nature 362:255-258 (1993); Bruggermann et al., Year in Immunol. 7:33 (1993) ); and Duchosal et al, Nature 355: 258 (1992). Human resistance 139862.doc 31 200950808 The body can also be derived from the sputum display library (Hoogenboom et al., J. Mol

Biol. 227:381 (1991); Marks et al. 'J. MoL. Biol. 222:581-597 (1991); Vaughan et al.' Nature Biotech 14:309 (1996)). The production of human antibodies from antibody phage display libraries is further described below. (v) Antibody Fragments A variety of techniques have been developed for the preparation of antibody fragments. Traditionally, these fragments were obtained by proteolytic digestion of intact antibodies (see, for example, M〇rim〇t〇 et al., journal 〇f Bi〇chemical and 〇

Biophysical Methods 24: 107-1 17 (1992); and Brennan et al.

Science, 229:81 (1985)). However, such fragments can now be prepared directly by recombinant host cells. For example, antibody fragments can be isolated from the above-described antibody phage library. Alternatively, Fab, _SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab,) 2 fragments (Carter et al,

Bio/Technology 10: 163_167 (1992)). In another embodiment described in the Examples below, F(ab')2 was formed using the leucine zipper gCN4 to facilitate assembly of the F(ab,)2 molecule. According to another method, the F(ab,)2 fragment can be isolated directly from the recombinant host © cell culture. Those skilled in the art should be aware of other methods of preparing antibody fragments. In other embodiments, the selected anti-system is a single-chain Fv fragment (scFv). See w〇 93/16185. (vi) Multispecific antibodies Multispecific antibodies have binding specificity for at least two different epitopes where the epitopes are usually derived from different antigens. Although these molecules typically only bind to two different epitopes (ie, bispecific antibodies, BsAb), this expression also covers antibodies with additional specificity, such as trispecific, when used in 139862.doc •32·200950808 herein. antibody. Examples of BsAbs include those with one arm for pirB/LILRB2 and the other for Nogo or MAG or OMgp. Other examples of BsAB include those with one arm for PirB/LILRB2 and the other for NgR. Methods of making bispecific antibodies are known in the art. The traditional method of producing full length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, wherein the two chains have different specificities (MiUstein et al., Nature, 305:537-539 (1983)) ). Due to the random association of immunoglobulin heavy and light chains, such hybridomas (quaternary hybridomas) can produce a potential mixture of one different antibody molecules, of which only one has the correct bispecific structure. Correct molecular to purification is usually carried out by affinity chromatography steps which are cumbersome and have low product yields. A similar procedure is disclosed in w〇 93/08829 A Traunecker # A > EMBO J.5 l〇: 3655-3659 (1991). According to a different method, an antibody variable domain (antibody/antigen binding site) having a desired binding specificity can be fused to an immunoglobulin constant domain sequence. The fusion preferably has an immunoglobulin heavy chain constant domain' which comprises at least a portion of the hinge region, the CH2 region and the CH3 region. Preferably, the first heavy chain constant region (CH1) containing the site required for light chain binding is present in at least one of the fusions. 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. When the unequal ratios of the three polypeptide chains used for construction provide optimal yields, this co-transfection provides greater flexibility in adjusting the mutual ratio of the three polypeptide fragments in the examples. However, when at least two polypeptide chains are expressed in equal ratios, which may result in high yields or when the ratios are not significant, 139862.doc •33-200950808, the coding sequences of two or all three polypeptide bonds may be inserted into one In the performance vector. In the method - a preferred embodiment, the bispecific anti-system consists of a heterozygous immunoglobulin heavy bond in one arm and a first binding specificity and a hybrid immunoglobulin heavy chain in the other arm. Constituent (providing a second binding specificity). It has been found that this asymmetric structure facilitates the separation of the desired bispecific compound from the undesired immunoglobulin chain, since the immunoglobulin light chain is present in only half of the bispecific molecule to facilitate separation. This method is disclosed in Solitary 94/04690. Further details regarding the generation of bispecific antibodies can be found, for example, in Suresh et al., Meth〇ds in Enzym〇 〇gy, i2i 2i〇 (1986). According to another method described in WO 96/2701 1, the interface between pairs of antibody molecules can be designed to maximize the percentage of heterodimers recovered from recombinant cell culture. Preferably, the interface comprises at least a portion of the CH3 domain of the antibody constant domain. In this method, one or more small amino acid side chains from the first antibody molecule interface are replaced with a larger side chain (e.g., tyrosine or tryptophan). Compensatory "cavities" having the same or similar size as the large side chain on the second antibody molecule interface by replacing the larger amino acid side chain with a smaller amino acid side chain (eg, alanine or threonine) "." This provides a mechanism to increase the heterodimer yield relative to other undesirable end products (e.g., homodimers). Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin while the other antibody can be coupled to biotin. It has been suggested in the literature that such antibodies can, for example, target immune cells 139862.doc • 34- 200950808 system cells to undesirable cells (U.S. Patent No. 4,676,980) and can be used to treat HIV infection (WO 91/00360, WO 92/). 200373). Heterologous conjugated antibodies can be prepared using any convenient cross-linking method. Suitable crosslinking agents are well known in the art and are disclosed in U.S. Patent No. 4,676,980, the disclosure of which is incorporated herein by reference. Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, chemical binding can be used to prepare bispecific antibodies. Brennan et al. (Science, 229: 81 (1985)) describe procedures for cleavage of intact antibodies by protein hydrolysis to generate F(ab')2 fragments. The fragments are reduced in the presence of the dithiol miscicide sodium arsenite to stabilize adjacent dithiols and prevent the formation of intermolecular disulfide bonds. The resulting Fab' fragment is then converted to a thionitrobenzoate (TNB) derivative. A Fab'-TNB derivative is then reconverted to a Fab'-thiol by reduction with mercaptoethylamine and mixed with other molar amounts of other Fab'-TNB derivatives to form a bispecific antibody. . The bispecific antibody produced can be used as a reagent for selective immobilization of the enzyme.

The Fab'-SH fragment can also be directly recovered from E. coli and can be chemically coupled to form a bispecific antibody. Shalaby et al, J. Exp. Med. '175: 217-225 (1992) describe the production of fully humanized bispecific antibody F(ab.)2 molecules. Each Fab' fragment can be separately secreted from E. coli and subjected to in vitro directed chemical coupling to form a bispecific antibody. Various techniques for directly preparing and isolating bispecific antibody fragments from recombinant cell cultures have also been described in the literature. For example, a leucine zipper can be used to generate a bispecific antibody. Kostelny et al., j Immun〇 1, 148(7): 1547· 139862.doc * 35 - 200950808 1553 (1992). The leucine zipper peptide from F〇s and Jun proteins was ligated to the Fab portion of two different antibodies by gene fusion. The antibody homodimer is reduced in the hinge region to form a monomer, which is then reoxidized to form an antibody heterodimer. This method can also be used to generate antibody homodimers.

The "bispecific antibody" technique set forth by Hollinger et al.' proc. Nati. Acad. Sci. USA, 90:6444- 6448 (1993) provides an alternative mechanism for the preparation of bispecific antibody fragments. The fragments comprise a heavy chain variable domain (VH) linked to the light chain variable domain (VL) via a linker that is too short to be paired between the two domains on the same strand. Thus, the VH and VL. domains of one fragment are forced to align with the complementary vl and VH domains of another fragment' thereby forming two antigen binding sites. Another strategy for preparing bispecific antibody fragments by using single-chain Fv (sFv) dimers has also been reported. See

Gruber et al, J. Immunol, 152: 5368 (1994). The invention encompasses antibodies above two valences. For example, a trispecific antibody can be prepared. Tuft et al, J. Immunol. 147: 60 (1991). (vii) Effector Function Design It may be desirable to modify the antibody of the present invention in terms of effector function to enhance the efficacy of the antibody. For example, a cysteine residue can be introduced in the Fc region, thereby forming an interchain disulfide bond in the region. The homodimeric antibody thus produced may have improved internalization ability and/or enhanced complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., /. Med. 2922 (1992). Homologous dimer antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers, such as Wolff et al., Cancer 139862.doc • 36- 200950808

Research 53: 2560-2565 (1993). Alternatively, an antibody having a dual Fc region can be designed which can have enhanced complement cleavage and ADCC capabilities. See Stevenson et al. 'Anti-Cancer Drug Dehgw 3: 219-230 (1989). (viii) Antibody-Remedy Receptor Binding Epitope Fusion. For example, in certain embodiments of the invention, it may be desirable to use antibody fragments rather than intact antibodies to enhance luminal tumor penetration. In this case, it may be desirable to modify the antibody fragment to extend its half-life. For example, this can be achieved by incorporating a remedy for the conjugated epitope into the antibody fragment (eg, by mutation of a suitable region in the antibody fragment, or by incorporating the epitope into the peptide tag, followed by (for example) DNA or peptide synthesis allows the label to be incorporated into the end or middle of the antibody fragment. The salvage receptor binding epitope preferably constitutes a segment in which any one or more amino acid residues from one or both of the Fc domains are transferred to analogous positions on the antibody fragment. 4 to more money, transferring three or more residues from one or two loops in the & domain. More preferably, the epitope is taken from the CH2 domain of the Fc region (e.g., of IgG) and transferred to chi, (10), or ^" or more than one such segment of the antibody. Alternatively, the epitope is taken from the CH2 domain of the Fc region and transferred to the region of the antibody fragment or the VL region or both regions. (ix) Other covalent changes in antibodies Covalent changes in antibodies are included in the scope of the invention. It can be prepared by chemical synthesis or by enzymatic or chemical cleavage of the antibody, if applicable. Covalently altering other types of antibodies into a molecule by reacting the dried amino acid residue of the antibody with an organic derivatizing agent capable of reacting with a selected side chain or a terminal residue 139862.doc •37·200950808 An example of a covalent change is set forth in U.S. Patent No. 5,534,61, the disclosure of which is expressly incorporated herein by reference in its entirety in its entirety herein in Connecting to one of a variety of non-proteinaceous polymers such as polyethylene glycol, polypropylene glycol or polyalkylene oxides: U.S. Patent Nos. 4,64,835, 4,496,689, 4,301,144, 4,67 〇, 417, 4, 791, 192 or 4, 179, 337. (X) Generation of antibodies from synthetic antibody phage libraries In a preferred embodiment, the invention provides methods for generating and selecting novel antibodies using unique phage display methods. Generating a synthetic antibody phage library based on a single framework template, designing a sufficient diversity within the variable domain, displaying a polypeptide having a diverse variable domain, selecting a target antigen High-affinity candidate antibodies, and isolation of selected antibodies. For details of the method of displaying the bacillus, see, for example, W003/102157, published February 2, 丨 February, the entire disclosure of which is incorporated by reference. It is expressly incorporated herein. In one aspect, the antibody library used in the present invention can be generated by mutating a solvent accessible position and/or a high diversity position in at least one CDR of an antibody variable domain. Mutation of some or all of the CDRs using the methods provided herein. In certain embodiments, various antibody libraries are preferably generated by morphing the positions of CDRH1, CDRH2, and CDRH3 to form a single library, Or can be mutated in positions in CDRL3 and CDRH3 to form a single library' or mutate positions in CDRL3 and CDRH1, 139862.doc 200950808 CDRH2 and CDRH3 to form a single library. For example, CDRH1, CDRH2 and A library of CDRH3 accessible to a variable domain of a variable antibody at a position and/or a high diversity position. Another gene having mutations in CDRL1, CDRL2 and CDRL3 can be generated The libraries can also be used in combination with each other to generate a binding having the desired affinity. For example, the light chain library can be re-introduced after one or more rounds of selection of the heavy chain library for binding to the target antigen. The heavy chain conjugate population is additionally subjected to several rounds of selection to enhance the affinity of the conjugates. Preferably, the original amine group is replaced by a variant amino acid in the CDRH3 region of the variable region of the heavy chain sequence. The acid is used to create a library. The resulting library may contain a plurality of antibody sequences in which the sequence diversity is predominantly found in the CDRH3 region of the heavy chain sequence. In one aspect, a library is created in the context of a humanized antibody 4D5 sequence, or a framed amino acid sequence of a humanized antibody 4D5 sequence. Preferably, the library is created by replacing at least residues 95-100a in the heavy chain with a codon-encoded amino acid, wherein the DFA: codon set is used to encode a variant amine at each of these positions. Group of acid groups. An example of a group of oligonucleotides that can be used to generate such substitutions comprises the sequence (DFiC)7. In certain embodiments, the library is created by substituting residues 95-1 00a with an amino acid encoded by both the DFK and WA: codon sets. Examples of sets of nucleotides that can be used to generate such substitutions include sequence access (WiViO. In another embodiment, the library is at least amino acid encoded by both the DM and iViV/C codon sets. Substituting residues 95-1 00a to create an example package that can be used to generate such substituted nucleotide sets 139862.doc -39- 200950808 contains sequences (DF Magic 5 (WiViQ. can be used to generate such substituted oligonucleotides) Another example of a glucuronide group comprises a sequence (^#尺) 6. Those skilled in the art can determine other examples of suitable oligonucleotide sequences according to the criteria described herein. In another embodiment, different CDRH3 designs are employed. To isolate high affinity binders and to isolate bindings to different epitopes. The length of CDRH3 produced in this library ranges from 11 to 13 amino acids, but can also be generated with a length different from this range. Use ulnar, DFi (and codon sets to expand H3 diversity and more restricted diversity at the N and / or C-terminus. Also produce diversity in CDRH1 and CDRH2. CDR-H1 and H2 diversity The design follows a strategy of targeting the library of simulated natural antibodies, and The improvement of this strategy compared to the previous design focuses on the diversity that closely matches the natural diversity. For the diversity in CDRH3, multiple libraries can be constructed separately for different H3 lengths, and then combined to select for target antigens. Combining bodies. Multiple libraries can be pooled and sorted using solid support selection and solution sorting methods as described in the previous literature and hereinafter. Multiple sorting strategies can be employed. For example, a variant involves The target bound to the solid is sorted, then sorted for a label (eg, an anti-gD tag) that may be present on the fusion polypeptide, and then sorted again according to the target bound to the solid. Alternatively, it may be first based on the solid surface The library is subjected to sorting by combining the target, and then the eluted combination is sorted using a solution phase that binds to a low concentration of the target antigen. The combination of different sorting methods can only minimize the selection of high performance sequences, and Provides a selection of a variety of different high-affinity pure lines. 139862.doc -40- 200950808 Can be isolated from the library for high affinity binding of antigens Limiting the diversity in the H1/H2 region can reduce degeneracy by about 1〇4 to 1.5 times, and allowing larger H3 diversity to provide a more affinity-combined combination with different diversity in CDRH3 Types of libraries (eg, using DVK or NVT) allow for the isolation of conjugates that bind to different epitopes of the target antigen. Among the conjugates isolated from the above confluent libraries, it has been found that by providing limited diversity in the light chain To further improve the affinity. In this example, the light chain diversity is produced according to the following: in CDRL1: amino acid position 28 is encoded by RDT; amino acid position 29 is encoded by RKT; The amino acid position 30 is encoded by RVW; the amino acid position 31 is encoded by ANW; the amino acid position 32 is encoded by THT; if desired, the amino acid position 33 is encoded by CTG In CDRL2: amino acid position 50 is encoded by KBG; amino acid position 53 is encoded by AVC; and amino acid position 55 is encoded by GMA, if desired; in CDRJL3: amine The acid acid position 91 is encoded by TMT or SRT or both; amino acid position 9 2 is encoded by DMC; amino acid position 93 is encoded by RVT; amino acid position 94 is encoded by NHT; and amino acid position 96 is encoded by TWT or YKG or both. In another embodiment, a library having diversity in the CDRH1, CDRH2, and CDRH3 regions is generated. In this example t, H3 regions of various lengths and major uses and AWA: or codon sets are used to generate diversity in CDRH3. A separate oligonucleotide can be used to form and pool the libraries, or the oligonucleotides can be pooled to form a subset of libraries. The library of this example can be sorted for targets that bind to solids. The pure line isolated from the multiple sorting can be screened using ELISA for specificity 139862.doc •41 _ 200950808 and affinity. For specificity, pure lines can be screened for the desired target antigen as well as other non-target antigens. The binding of their target antigens can then be screened for affinity in a solution binding competition ELISA assay or a point competition assay. High affinity binders can be isolated from the libraries prepared using the codon set above. Such a combination can be easily prepared as an antibody or antigen-binding fragment in a high yield in cell culture. In certain embodiments, it may be desirable to generate a library with greater diversity in the length of the CDRH3 region. For example, it may be desirable to generate a library of CDRH3 regions between about 7 and 19 amino acids. The high affinity binding system isolated from the libraries of these examples was readily produced in high yields in bacterial and eukaryotic cell cultures. The vector can be designed to facilitate removal of sequences such as gD tags, viral coat protein component sequences, and/or to be readily added to constant region sequences to achieve full-length antibody or antigen-binding fragments in high yield. A library having a mutation in CDRH3 can be combined with a library containing other CDR variant forms (e.g., CDRL1, CDRL2, CDRL3, CDRH1, and/or CDRH2). Thus, for example, in one embodiment, a CDRH3 library is combined with a CDRL3 library having a variant amino acid at positions 28, 29, 30, 31, and/or 32 using a predetermined codon set. The humanized 4D5 antibody sequence was created in the context of the sequence. In another embodiment, a library having a CDRH3 mutation can be combined with a library comprising a variant CDRH1 and/or CDRH2 heavy chain variable domain. In one embodiment, the CDRH1 library is created using a humanized antibody 4D5 sequence having a variant amino acid at positions 28, 30, 31, 32 and 33. The CDRH2 library can be created using a predetermined codon set 139862.doc -42. 200950808 to humanized antibody 4D5 sequences with variant amino acids at positions 50, 52, 53, 54, 56 and 58. (xi) Antibody Mutants Novel antibodies produced by phage libraries can be further modified to produce antibody mutants that are physically, chemically and/or biologically modified over parental antibodies. If the assay used is a biological activity assay, the biological activity of the antibody mutant in the selected assay is preferably at least about 10 times greater than the biological activity of the parent antibody in the assay, preferably at least about 20 fold greater, more preferably stronger At least about 50 times, sometimes at least about 100 times or 200 times stronger. For example, the binding affinity of the anti-pirB/LILRB antibody mutant to PirB/LILRB is preferably at least about 10 times stronger than the parent anti-antibody, preferably at least about 20 times stronger, and more preferably at least about 5 〇 times, sometimes at least about 100 times or 200 times stronger. To generate an antibody mutant, one or more amino acid changes (eg, substitutions) are introduced into one or more hypervariable regions of the parent antibody. Alternatively, or in addition, one or more alterations (e. g., substitutions) of the framework region residues can be introduced into the parent antibody, wherein such changes result in enhanced binding affinity of the antibody mutant to the antigen of the second mammal. Examples of residues in the framework regions to be modified include those that directly bind non-covalent antigens (Amit et al. (1986), 233: 747-753), and conformations of interference/jing 〇〇11 (chothia et al. (1987). ), J. Mol. Β1〇1· 196:901-917); and/or participate in the Vl_Vh interface (Ep 239 400B 1). In certain embodiments, modification of one or more of these framework region residues can result in an antibody affixing to a second mammalian antigen 乂 binding affinity enhancement example 5, which can be varied in this embodiment of the invention. Up to about 5 framework residues. Sometimes, even if there are no changes in the hypervariable region, this is sufficient to produce an antibody mutant suitable for preclinical testing. However, antibody mutants typically contain other hypervariable region changes. The altered hypervariable region residues can be randomly altered, particularly when the initial binding affinity of the parent antibody allows such randomly generated antibody mutants to be readily screened. One of the procedures for generating such antibody mutants is called "Cunningham and Wells (1989) 244:1081-1085). Here, one or more hypervariable region residues are replaced with alanine or polyalanine residues.

To affect the interaction of the amino acid with the antigen from the second mammalian species. The hypervariable region residues that are sensitive to the substitution display function are then modified by introducing additional or partial mutations at the substitution sites or against the substitution sites. Although the site in which the amino acid sequence variation is introduced is predetermined, the mutation itself I·living is not & Screening was performed on the ala-mutants produced in this manner for the biological activities described herein. It is usually a conservative substitution to start the sequel, such as the ones titled "Better Desire" below. The bitter stems and the right ones cause biological activity (such as dancing)

The change in affinity is Μ , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , Better substitution:

139862.doc •44· 200950808

Asp (D) glu glu Cys (C) ser ser Gin (Q) asn asn Glu (E) asp asp Gly (G) pro ' ala ala His (H) asn, gin, lys, arg arg He (I) leu, Val, met, ala, phe, leucine leu Leu (L) leucine, ile, val, met, ala, phe ile Lys (K) arg, gin, asn arg Met (M) leu, phe, Ile leu Phe (F) leu, val, ile, ala, tyr leu Pro (P) ala ala Ser(S) thr thr Thr(T) ser ser Trp(W) tyr ' phe tyr Tyr (Y) trp, phe, Thr,ser phe Val (V) ile, leu, met, phe, ala, white amine 酉man leu

Even more substantial changes in the biological properties of an antibody are achieved by selecting substitutions that maintain significantly different effects of the following characteristics: (a) the structure of the polypeptide backbone in the substitution region, eg, in a sheet or helical conformation, (b) The charge or hydrophobicity of the molecule at the target site, or (c) the size of the side chain. Naturally occurring residues can be classified into the following categories according to the characteristics of common side chains: (1) Hydrophobic amino acids: n-leucine, met, ala, val, leu, ile; (2) neutral hydrophilic amine groups Acid: cys, ser, thr, asn, gin; (3) Acidic amino acid: asp ' glu; (4) Amine amino acid: his, lys, arg; 139862.doc -45- 200950808 (5) Influence Chain oriented residues: gly, pro; and (6) aromatic amino acids: trp, tyr, phe. Non-conservative substitutions necessitate the exchange of members of one of these categories for another. In another embodiment, the affinity of the selected alteration site is matured using a sputum display (see above). Nucleic acid molecules encoding amino acid sequence mutants are prepared by a variety of methods known in the art. Such methods include, but are not limited to, performing nucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis on the preparation of mutant or non-variant forms of the parent antibody. The preferred method of preparing the mutant is a fixed point

Mutagenesis (see, for example, Kunkel (1985) 7Va" Jed. 5W. USA 82:488) 〇 In certain embodiments, only a single hypervariable region residue may be substituted in an antibody mutant. In other embodiments, two or more hypervariable region residues of the parent antibody can be substituted, e.g., from about 2 to about 1 超 hypervariable region. The amino acid sequence of an antibody mutant having improved biological properties typically has an amino acid sequence of at least 75 °/ with the parent antibody heavy or light chain variable domain. The amino acid sequence identity or similarity is more preferably at least 8%, more preferably at least 85%, more preferably at least 90%' and most preferably at least 95%. The identity or similarity of this sequence is defined herein as: the candidate sequence is identical to the parent antibody residue (ie, the same residue) after aligning the sequence and, if necessary, introducing a gap to achieve a maximum percent sequence identity. Or a similar (i.e., a percentage of amino acid residues from the same class of amino acids according to general side chain characteristics, see above). The N-terminal 139862.doc-46·200950808 end of the variable sequence in the antibody sequence should not be considered to be a c-terminal or internal extension, deletion or insertion of the 庠丨v詈 sequence-likeness or similarity. After the production of the antibody mutant, the biological activity of the molecule is determined relative to the parent antibody. As described above, this may involve determining the binding affinity of the antibody and/or other biological activity. In a preferred embodiment of the invention, a panel of broad mutants is prepared and screened for binding affinity to an antigen or fragment thereof. One or more other biological activity assays selected from one or more of the antibody mutants selected from this initial screen, as necessary, to demonstrate that the antibody affinity-enhanced antibody mutants are indeed useful, for example, in preclinical studies. Further changes can be made to the antibody mutants thus selected, for the intended use of the antibody. Such alterations may involve other changes in the amino acid sequence, fusions with and/or covalent changes to the heterologous polypeptide, such as those detailed below. With regard to amino acid sequence changes, exemplary changes are detailed above. For example, any cysteic acid residue that does not participate in maintaining the correct conformation of the antibody mutant can generally be substituted with serine to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine linkages can be added to the antibody to increase its stability (especially when against antibody fragments such as Fv fragments). Another type of amino acid mutant has a modified glycosylation pattern. This can be accomplished by deleting one or more carbohydrate moieties present in the antibody' and/or by adding one or more glycosylation sites that are not present in the antibody. Glycosylation of antibodies is typically N-linked or 〇-linked. N-linkage refers to the attachment of a carbohydrate moiety to the side chain of an aspartate residue. Tripeptide sequence aspartame-X-serine and aspartame _x_threonine (in which any amino acid other than valeric acid) is a carbohydrate moiety and an aspartic acid side chain Enzymatic 139862.doc •47- 200950808 Linked recognition sequence. Thus, the presence of any of these tripeptide sequences in a polypeptide can result in a potential glycosylation site. 0-linked glycosylation refers to a sugar (N-ethyl galactosamine, galactose or xylose) and a hydroxyl amino acid (most commonly serine or threonine) but may also use 5-hydroxyl The linkage of proline or 5-hydroxy lysine. The glycosylation site can be readily added to the antibody by altering the amino acid sequence such that the antibody contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). It is also possible to add one or more serine or threonine residues (or substitution with one or more serine or threonine residues) to the original antibody sequence (for 〇-linked glycosylation) Bit)) to achieve the change 13 changes. (xii) Recombinant production of antibodies For recombinant production of antibodies, the nucleic acid encoding the antibody can be isolated and inserted into a replicable vector for further selection (amplification of DNA) or expression. The DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using a nucleotide probe that specifically binds to the gene encoding the heavy and light chains of the antibody). A variety of carriers can be used. Vector group injury generally includes, but is not limited to, one or more of the following: signal sequences, retinoesis, digested or multiple marker genes, enhancer elements, promoters, and transcription termination sequences (eg, such as US patents) It is described in U.S. Patent No. 5,534,615, the disclosure of which is incorporated herein by reference. In this context, a suitable host cell line that selects or expresses DNA in a vector is a prokaryotic, cell, yeast cell or higher eukaryotic cell. Suitable prokaryotes for this purpose include, for example, Gram (9) (four) negative or Gram-positive organic bacteria such as Enterobacteriaceae, such as Ai 139862.doc -48· 200950808 Helicobacter (heart ί ^ ^ / π·β) (eg E. coli), Enterobacter genus r), Erwinia, Klebsiella (A7eWe//a), Proteus (iv (10)), Salmonella (heart/foot) "e//a" (eg Salmonella typhimurium (5W(4)(10)//βmWm/w)), Serratia (&"α/ι·β) (eg Serratia marcescens • β (10)〇 ), and Shigella (from seven dk), and rods of the genus (5^///) (such as Bacillus subtilis (5. out (4) and Bacillus licheniformis (anti-heart / orwzO (for example, in 1989) DD 266 710 published on April 12, 2009

B. licheniformis 41))), Pseudomonas (/^Wi/〇m〇«like) (for example, Pseudomonas aeruginosa (P_ aerMgh(10)(9), and Streptomyces (5> The E. coli colonization host is Escherichia coli 294 (ATCC 3 1,446), but other strains such as E. coli b, E. coli 776 (ATCC 3 1,537) and E. coli W3110 (ATCC 27, 325) are also suitable. Sexuality and not limitation. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable colonization or expression hosts for ❿ antibody-encoding vectors. Saccharomyces cerevisiae cereWhae), or common baker's yeast (baker, s yeast) The most commonly used lower eukaryotic host microorganisms. However, a variety of other genus, species, and strains are available in a general manner and can be used herein, for example • Schizosaccharomyces (Sc/n'zosacc/zarom less ces;?〇 ?~~); Kluyveromyces host, such as Kluyveromyces lactis (L. lactis), Kluyveromyces cerevisiae (R. /ragz7&) (ATCC 1 2, 424), Kluyveromyces cerevisiae (尺.Zm/garz'cws) (ATCC 1 6,045), K. wickwmi! (ATCC 24, 178), Kluyverom solutii 139862.doc -49- 200950808 (K. waltii) (ATCC 56,500), fruit fly Kluyveromyces (N. drosophilarum) (ATCC 36, 906), and 克 Kluyveromyces cerevisiae (I. iermoio/eraw), and R. cerevisiae (French. Marxiam/·?); Ascomycetes (3;arro>Wa) (EP 402,226); Pichia pastoris (P/c/na (EP 183,070); Candida (Ca«i/zda); Trichoderma reesei (TWcAoc/erwa rees) ^'a) (EP 244,234); iVewrospora crawa; iSc/zwanm'owj/ces, such as the western xylobacter occidentalis; and filamentous fungi such as Streptomyces (iVewrc^pora), Penicillium (Pem'ci/nwm), B. genus, and Aspergillus host (eg, Aspergillus nidulans (Children (1) Wa"·5) and Aspergillus niger (Children (1)'ger). Suitable host cells for glycosylated antibodies are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. A variety of baculovirus strains and variants have been identified and corresponding licensed aphid host cells from hosts such as: grass worms (in the case of locusts) (caterpillars), Aedes aegypti (jWa (9) illusory " ) (mosquito), Aedes albopictus (Jec/a mosquito), and black-bellied fruit (Nan Guang 0*50/7^2"*3 (Drosophila), and silkworm wori)). A variety of transfection virus strains can be obtained from publicly available routes, such as the L-1 variant of Aut〇graPha calif〇rnica NPV and the Bm-5 strain of Bombyx mori NPV, and according to the present invention Used as a virus in this paper, specifically for transfection of grass mucus cells. Plant cell cultures of cotton 'corn, horse yam, soybean, petunia, tomato and tobacco can also be used as hosts. However, it is a routine procedure to proliferate vertebrate cells in cultures (tissue 139862.doc -50-200950808 culture). Examples of mammalian host cell lines available are monkey kidney CV1 cell lines transfected with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell lines (293 or 293 subcloned to grow in suspension culture) Cells, Graham et al, 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 77:4216 (1980)); mouse testis-supporting cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney Cells (乂110-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo liver cells (BRL 3A, ATCC CRL 1442); Human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumors (MMT 060562, ATCC CCL5 1); TRI cells (Mather et al., Annals NY Acad. Sci. 383: 44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatoma cell line (Hep G2). The host cells are transformed with the above-described expression or selection vector for antibody production, and cultured in a conventional nutrient medium, and the medium is appropriately modified to induce a promoter, select a transformant, or amplify a gene encoding a desired sequence. The host cells used to produce the antibodies of the invention can be cultured in a variety of media. Commercially available media are suitable for culturing host cells such as Ham's F10 (Sigma), Minimal Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (Dulbecco's 139862.doc - 51 - 200950808

Modified Eagle’s MediUm) ((DMEM), Sigma). Alternatively, any of the media described in the following literature can be used as the host cell culture medium:

Ham et al., Meth. Enz. 58:44 (1979); Bames et al., Oct. Biochem. 102:255 (1980); US Patent 4 767 7〇4, 4'657'866, 4' 927 '762 '4, 56, 655, or 5' 122, 469; WO 90/03430, WO 87/00195; or U.S. Patent No. 30,985. Hormone and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium, calcium, magnesium, and phosphate) may be added to any of these media as needed, Buffered sputum (eg HEPES), nucleotides (eg adenosine and thymidine), antibiotics (eg GENTAMYCINTM), trace elements (defined as inorganic compounds usually present in the final concentration in the micromolar range), and glucose or Equivalent energy. Any other desired supplement may also be introduced at an appropriate concentration known to those skilled in the art. Culture conditions such as temperature, pH, and the like are conditions previously used for the host cell of choice for use' and are readily apparent to those skilled in the art. When using recombinant techniques, antibodies can be produced intracellularly, in the interstitial space, or directly into the culture medium. If the 4-charge system is produced intracellularly, J is used as a first step to remove particulate debris (host cells or lysed cells) by, for example, centrifugation or ultrafiltration. If the anti-system is secreted into the culture medium, the supernatant from the performance systems is typically first concentrated using a commercially available protein concentration filter (e.g., Amic〇n or Millipore Pellicon ultrafiltration unit). In any of the foregoing steps, a protease inhibitor (e.g., pMsF) may be added to inhibit proteolysis, and an antibiotic may be added to prevent the exogenous contaminant from growing 139862.doc -52- 200950808. The antibody composition prepared from the cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, wherein affinity chromatography is a preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and the isoform of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human gamma, gamma 2, or gamma 4 heavy chains (Lindmark et al. 'J. Immunol. Meth. 62: 1-13 (1983)). Protein G is recommended for all mouse isotypes and humans (Guss et al, EMBO J. 5: 1567-1575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices may also be utilized. Mechanically stable matrices (e.g., controlled apertured glass or poly(ethylidene-ethyl) benzene) allow for faster flow rates and shorter processing times than rumose. If the antibody contains a CH3 domain, Bakerbond ABXTM resin (J T Baker,

Purification was carried out by Phillipsburg, N.J.). To recover the antibody, other protein purification techniques can also be used, such as fractionation on an ion exchange column, ethanol precipitation, reversed phase HPLC, chromatography on cerium oxide, chromatography on heparin SEPHAROSETM, Chromatography, chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation were performed on an anion or cation exchange resin such as a polyaspartic acid column. C. Use of anti-pirB/LILRB antibodies It is believed that the anti-PirB/LILRB antibodies of the invention are useful as agents that aid in the survival or induction of growth of nerve cells. It can therefore be used to treat degenerative disorders of the nervous system ("neurodegenerative diseases"), including, for example, physical impairment of the middle sacral nervous system (spinal cord and brain); brain damage associated with stroke; and with God 139862.doc -53- 200950808 Neurological disorders related to degeneration, such as tri-analgia, glossopharyngeal neuralgia, Bell itch, myasthenia gravis, muscular dystrophy, amyotrophic lateral sclerosis =), multiple sclerosis (10), progressive Muscular atrophy, progressive medullary hereditary muscular atrophy, peripheral nerve injury due to physical damage (eg, burns, illusion, or disease states such as diabetic nephropathy or toxic effects due to chemotherapy used to treat cancer and s , prominent rupture, or prolapsed disc syndrome, cervical spondylosis, plexus disorders, thoracic outlet destruction syndrome, peripheral neuropathy (eg, by mistake, amine benzene hard, sputum, their Bu Lin, Green Balinese syndrome , Alzheimer's disease, T-Town's disease, or Parkinson's disease. - Anti-PirB/LILRB antibodies can also be used to culture nerve cells in vitro. Components of the medium. Finally, formulations containing anti-PirB/LILRB antibodies herein can be used as standards in competitive binding assays upon radioiodine, enzymes, fluorophores, spin labels, and the like. Means for the preparation of a therapeutic formulation of an anti-PirB/ULRB antibody herein for storage: mixing an identified compound (eg, an antibody) of a desired purity with an optional physiologically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceuticai Seiences As stated above, the formulations are in the form of a dry cake or an aqueous solution. Acceptable carriers, excipients or stabilizers are non-toxic to the recipient at the dosages and concentrations employed, and include buffers such as phosphoric acid. Salts, citrates and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 1 residue) polypeptide; proteins such as serum albumin, gelatin or immunoglobulin; hydrophilic polymers such as 139862. Doc •54- 200950808 polyvinylpyrrolidone; amino acids such as glycine, glutamine, aspartame, arginine or lysine; monosaccharides Disaccharides, and other carbohydrates, including glucose, mannose or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salts forming counterions, such as sodium; and/or nonionic surfaces The active agent, such as Tween, Pluronics or PEG. The anti-PirB/LILRB antibody to be administered in vivo must be sterile. This can be easily accomplished by filtration through a sterile filtration membrane before or after lyophilization and reconstitution. The therapeutic composition is placed in a container having a sterile access port, such as an intravenous solution bag or vial having a stopper pierceable by a hypodermic needle. The anti-PirB/LILRB antibodies of the invention may optionally include NGF, NT-3 and/or The neurotrophic factors of BDNF are administered in combination or together and can be used with other conventional therapeutic agents for degenerative neurological disorders. Furthermore, the anti-PirB/LILRB antibodies of the present invention are preferably administered together with NgR inhibitors (e.g., antibodies, small molecules or peptides) which block the binding of Nogo-66, MAG and/or OMgp to NgR. The route of administration is consistent with known methods, such as by injection, infusion, local administration, or by sustained release system, by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial or intralesional routes. And, as described below. For intracerebral applications, the compound can be administered continuously to the fluid chamber of the CNS by infusion, but a bolus can also be accepted. Preferably, the compound is administered to 139862.doc -55-200950808 and to the ventricles or otherwise introduced into the CNS or cerebrospinal fluid. Administration can be performed by indwelling catheters in a continuous administration such as a pump, or by implantation of a sustained release vehicle (e.g., intracerebral implantation). More specifically, the compound can be injected via a long-term implanted cannula or a long-term infusion of a compound with the aid of a osmotic micro pump. A subcutaneous pump can be used that delivers protein into the cerebral ventricle via a small infusion tube. The extremely complex pump can be refilled via the skin and its delivery rate can be set without surgical intervention. Examples of suitable administration protocols and delivery systems involving subcutaneous pump devices or continuous intraventricular infusion via a total implantable drug delivery system are used to administer dopamine, dopamine agonists and cholinergic agonists For Alzheimer's patients and animal models of Parkinson's disease, such as Harbaugh, J. Neural Transm. Suppl., 24:271 (1987); and DeYebenes et al., 'Mov· Disord. 2:143 (1987) Said. Suitable examples of sustained release formulations include semipermeable polymeric matrices in the form of shaped articles such as films or microcapsules. Sustained release matrices include polyesters, hydrogels, polylactic acid compounds (U.S. Patent No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and γ-L-glutamate (Sidman et al. '1983, Biopolymers 22: 547), poly(2-hydroxyethyl-mercapto acrylate) (Langer et al., 1981, J. Biomed. Mater. Res. 15: 167; Langer, 1982, Chem. Tech. 12: 98) Ethylene vinyl acetate (Langer et al., Id.) or poly-D-(-)-3-hydroxybutyric acid (EP 133, 988A). Sustained release compositions also include liposomally embedded compounds which can be prepared by methods which are known per se. (Epstein et al., Proc. Natl. Acad. Sci. 82:3688 (1985); Hwang et al., Proc. Natl. 139862.doc •56- 200950808

Acad. Sci. USA 77:4030 (1980); U.S. Patents 4,485,045 and 4,544,545; and £102,324). Liposomes are typically small (about 200-800 angstroms) monolayers having a lipid content greater than about 30 mol.% cholesterol, and the selected ratio can be adjusted to achieve optimal treatment. The effective amount of active compound to be employed in the treatment may depend, for example, on the therapeutic target, the route of administration, and the condition of the patient. Therefore, the therapist should perform a titration of the dose as needed and change the route of administration to achieve optimal treatment. Depending on the above factors, a typical daily dose may range from about 1 gg/kg to 100 pg/kg or more. Typically, the clinician can administer the active compound until a dose that repairs, maintains, and (optimally) rebuilds neuronal function. The progress of the therapy can be easily monitored by routine analysis. The invention is further detailed in the following non-limiting examples. Example 1 Characterization of the selection of LILRB2 To identify novel receptors for inhibitory myelin proteins, the expression selection method was employed. As a bait, a construct that fused the alkaline phosphatase (AP) to the N- and/or C-terminus of the following qualitative myelin inhibitor (human cDNA used) was produced: two Nobs and NogoA two anti-suppressive structures Domains (NiR < 8 > D2 and NiG < 6 > 20) (Oertle T, J Neurosci. 2003, 23(13): 5393-406), MAG, and OMgp. The constructs were transfected into 293 cells to produce a conditioned medium containing bait protein (stored in DMEM/2% FBS). The cDNA library used for screening includes a full length human cDNA pure line deposited in a ready-to-use expression vector manufactured by Origene. Edit, array, and confluence of the cDNAs. A confluence of approximately 100 cDNAs was transiently transfected into COS7 139862.doc -57-200950808 cells. Specifically, on day 1, COS7 cells were plated at a density of 85,000 cells/well in a 12-well plate. On day 2, 1 mg confluent cDNA/well was transfected with the fat-based transfection agent FuGENE 6 (Roche). On day 4, screening was performed. Briefly, medium was removed from the cells and replaced with 0.5 ml of 293 cell-conditioned medium containing AP-fusion bait protein (20-50 nM). The cells were incubated for 90 minutes at room temperature. The cells were then washed 3 times with phosphate buffered saline (PBS), fixed with 4% paraformaldehyde for 7 minutes, washed 3 times with HEPES buffered saline (HBS), and inactivated by heating at 65 °C for 90 minutes to destroy endogenous sources. AP activity. The cells were washed once in AP buffer (100 mM NaCl, 5 mM MgCl2, 100 mM Tris, pH 9.5), and cultured in a chromogenic substrate (Western Blue, Promega), and the reaction product was analyzed after one hour of culture. The presence and subsequent analysis of this assay after overnight incubation" identified positive cells by the presence of a dark blue precipitate on the surface of the membrane. The positive pool was further subdivided to identify each positive pure line by subsequent rounds of screening. The following positive hits were identified by screening: MAG-AP baits obtained 4 positive hits. One was previously referred to as the Nogo receptor (Fournier et al. ' Nature 409, 342-346 (2001)). Both of these hits are glycolysis processing enzymes' and are believed to be unlikely to be related. The fourth is called "smart person hypothetical protein from pure line 643 (LOC57228), mRNA." A more detailed analysis of the cDNA revealed an alternative ORF homologous to the aforementioned protein SMAG. The AP-Nogo66 bait obtained 2 positive hits. One was previously known as the N 〇 g 〇 receptor. Another series "Homo sapiens immunoglobulin-like receptor Aa 139862.doc -58- 200950808 Family B (with TM and ITIM domains), member 2 (LILRB2), mRNA" (SEQ ID NO: 2) . This gene also has multiple alternative names, including MIG10, ILT4, and LIR2 (Kubagawa et al., proc. Natl. Acad. Sei. USA 94: 5261-6 (1997); Colonna et al., J. Exp. Med. 186 :1809-18 (1997)) ° Example 2

Preparation and testing of PirB functional blocking antibody

PirB Functional Blocking Antibodies Anti-systems against PirB are generated by panning synthetic phage antibody libraries against the PirB extracellular domain (W. C. Liang et al., / Mo/ 〇/366, 815 (2007)). The ability of the antibody-derived line (10(tetra)^^ and AP-Nogo66 (50 nM) to bind to cpirS7 cells expressing pirB was then tested in vitro. The heavy and light chain sequences of various YW259 anti-mouse PirB (anti-mPirB) antibodies The nucleotide and amino acid sequences are shown in Figures 6-16 and 17 and 18. Figures 17 and 18 also show the hypervariable region sequences in the heavy and light chains of YW259.2, YW250.9 and YW259.12, respectively. Neurite growth assays were coated with myelin (0.75 pg/ml) in 96-well plates pre-coated with poly-D_ lysine (Biocoat, BD) overnight or with AP-Nogo66 or MAG-Fc (150-300). Ng/dot) was coated for two hours and then treated with laminin (1 〇pg/ml in F-12) for 2 hours (CGN culture) or 4 hours (DRG culture). Murine P7 cerebellar neurons (B. Zheng et al, Proc 102, 1205 (2005)) and plated at approximately 2χ104 cells/well. Cultured mouse PI〇DRG neurons as previously described 139862.doc •59 · 200950808 (Zheng et al., 2005, supra) and tiling it at approximately 5χ1〇3 cells/well. Cultures were grown for 22 hours at 37° C. and 5% C〇2, and subsequently 4% polyfurfural /1〇〇/0 sucrose was fixed and stained with anti-tubulin (TuJl, Covance). For each experiment, all conditions were performed in six replicate wells, where the maximum neurite length was measured and The average between the six wells was determined. Each experiment was performed at least three times to obtain similar results. The values were determined using the Student's t test (Student, sr test). Growth crown atrophy analysis was performed from 3 week old mice. DRG was excised to isolate the DRG explants and cut into three equal portions. Then coated with PD1 (1〇〇pg/ml) and coated with laminin (10 pg/ml) in an 8-well plate. Each DRG explant was cultured in a separate well. The explants were incubated with Ap-Nogo66 (100 nM) or myelin (3 pg/ml) for 30 minutes after tiling to stimulate atrophy. % poly-furfural/10% sucrose-fixed culture, followed by staining with Rhodamine-Phalloidin (molecular probe) to visualize the growth crown and record atrophy by averaging in at least 3 replicate wells Value to determine the average growth crown atrophy. The result is to clarify whether PirB is a functional receptor for Nogo66, and we are concerned about childhood ( P7) Cerebellar granule neurons (CGN), which are inhibited by growth during growth on AP-Nogo66 (K.C. Wang et al., iVaiwre 420, 74 (2002)). It has been shown that adult CGN can express PirB (J. Syken et al., Science 3 13, 1795 (2006)), and in the case of RT-PCR, immunohistochemistry and in situ hybridization analysis, we found that young CGN is also the same (data Not 139862.doc -60· 200950808 show). First, the ability of the soluble extracellular domain of PirB (PirB-His) to interfere with AP-Nogo66 inhibition was tested in vitro. As shown in Figure 2A, 'AP_Nogo66 inhibited neurite outgrowth of P7 CGN to about 66% of the untreated control. In this assay, PirB-His was included to reverse AP-Nogo66 inhibition, in which the growth of the sacral process was substantially restored to the control level. These results are similar to those reported for the inhibition of Nogo66 by the extracellular domain of NgR (B Zgeng et al., /Voc. iVa". dcW. Scz.. t/M 102,1205 (2005); A Four· • Fournier et al, J. Neurosci. 22, 8876 (200); ZL He et al, 38, 177 (2003)), and shows that PirB binds to the functional inhibitory domain of Nogo66, but does not address CGN Whether the endogenous PirB can mediate inhibition by AP-Nogo66. Therefore, an antibody against PirB (anti-PirB) capable of interfering with PirB-Nogo66 interaction was generated. A phage display platform targeting the PirB extracellular domain (WC Liang et al., Mo/. 〇/. 366, 815 (2007)) was screened according to the ability of multiple pure lines to block the binding of AP-Nogo66 to PirB. 9 selected. The pure line YW259.2 (hereinafter referred to as aPB1) which has the strongest interference with AP-Nogo66-PirB binding has a Kd of 5 nM for PirB (see Figs. 13-16).

aPBl is ineffective for baseline axon growth of CGN. However, in cultured CGN • aPBl significantly reduced AP-Nogo66 or myelin-induced inhibition (Fig. 2B), rescued neurite outgrowth from 41% to 59% for AP-Nogo66 and 47% to 62 for myelin %. Similar results were observed when using MAG as an inhibitory matrix or using different cell types (dorsal root ganglion (DRG) neurons) (Figure 5). These results demonstrate that the PirB line mediates long-term inhibition of neurite outgrowth. 139862.doc •61 · 200950808 Functional receptors. To confirm this result, it was tested whether the genetically removed cell surface PirB could also reverse the inhibition by AP-Nogo66 or myelin by culturing neurons from PirBTM mice, four of which were removed The transmembrane domain and part of the PirB intracellular domain exon are encoded (J. Syken et al., Sckwe 313, 1795 (2006)). CGN from PirBTM mice or wild type (WT) siblings were cultured on control matrices, AP-Nogo66 or myelin. On the control matrix (PDL/laminin), PirBTM neurons were similar in characteristics to WT neurons (Fig. 2C). However, inhibition of neurite outgrowth in PirBTM neurons on AP-Nogo66 or myelin was significantly lower than in WT neurons. On AP-Nogo66, the growth of WT neurons was inhibited to 50% of the control, while PirBTM neurons were only inhibited to 66%. Similarly, on myelin, WT neurons were inhibited to 52% of the control, while PirBTM neurons were only inhibited to 70%. Similarly, we observed a similar partial de-inhibition effect on PirBTM DRG neurons on both myelin and AP-Nogo66 (Fig. 5). These findings indicate that PirB is actually a functional receptor for neurite outgrowth mediated by AP-Nogo66 and myeloid lipids. However, the loss of PirB activity does not completely rescue growth. Since NgR has previously been described as a receptor for myelin inhibitors, PirB and NgR may act together to mediate inhibition of neurite outgrowth. To clarify this situation, the function of both PirB and NgR in CGN was blocked by culturing neurons from NgR-deficient mice in the presence of anti-PirB. As previously reported (B. Cheng et al., PNAS 2005, supra), NgR-/- CGN neurite outgrowth was inhibited by AP-Nogo66 or myelin 139862.doc -62- 200950808 to the same as in WT neurons The extent (50% and 49%; Figure 3). Administration of aPB1 antibody to NgR+/- neurons partially reversed the inhibition by AP-Nogo66 or myelin, as observed above for the aPB 1 treatment of WT neurons. Similarly, aPB 1 treatment of NgR-/- neurons partially reverses the inhibition by AP-Nogo66, but does not provide rescue levels beyond the aPB 1 treatment of NgR+/- neurons or WT neurons. Observed. In contrast, aPB1 treatment of NgR-/- neurons restored neurite outgrowth on myelin to a control level. Therefore, matrix inhibition by APNogo66 in CGN appears to require PirB rather than NgR, but only partially. In addition, both PirB and NgR can together promote matrix inhibition by myelin. It is considered to be more acute because it is believed that NgR is required for growth crown atrophy in response to various myelin inhibitors (JE Kim et al., 44, 439 (2004); O. Chivatakarn et al., */· iVewrosci. 27, 7117 (2007)). PirB may also be involved in the reaction. Sensory neurons from the 3 week old mouse dorsal root ganglia (DRG) and which have been shown to exhibit PirB have been used in this experiment. Growth crowns in this culture system have been found to have a higher degree of atrophic baseline (about 30%), which can be further enhanced by incubation with APNogo66 or myelin (Figure 4). This atrophy is largely eliminated in NgR-/- neurons. Furthermore, blocking PirB function with aPB 1 is also sufficient to reverse the growth crown atrophy caused by such inhibitors. Inhibition of both PirB and NgR pathways together (using aPB 1 treatment on neurons from NgR-/- mice) can also completely reverse growth crown atrophy, but since either treatment alone can result in complete isolation Rescue, so this result does not provide any information. 139862.doc -63 - 200950808 In another experiment, 'C1QTNF5 inhibited the growth of cisterna granule neurons (CGN), and this inhibition was reversed by the PirB function-blocking antibody Yw259 2 . The results are shown in Figure 19. The results, etc., together support that PirB has a novel role as a receptor required for myelosuppression by myelin extract, and more specifically by the myelin-related inhibitor N〇g〇66 & mag. In fact, pirB appears to be a more important matrix inhibitory medium than NgR, because the pirB function alone (either genetically or with antibodies) allows the growth of both myelin extract and myelin inhibitor to go. Inhibition, while genetically removed NgR alone cannot be inhibited on any of these vesicles. However, NgR appears to play a subsidiary role in mediating inhibition by myelin extract (rather than N〇g〇66) 'This is due to genetic removal of NgR which is enhanced on myelin (rather than N〇g) 〇66) is inhibited by anti-chemical antibodies. Our findings may help to explain the fact that although regeneration or growth has been reported in rodents that infuse the extracellular domain of NgR (S. Li et al., J. 24, 1051 1 (2004)), Surprisingly, CST regeneration was not enhanced in NgR knockout mice (JE Kim et al., et al., 'B. Zheng et al., Λ^/·, eg j〇2, 1205 (2005)). Therefore, it may be desirable to remove both pirB and NgR to achieve significant regeneration in vivo. Furthermore, since the genetic removal of NgR on the Nogo66 matrix does not further enhance the partial de-inhibition effect of PirB removal, Nogo66 may have other binding receptors. Although PirB appears to be a more important matrix inhibitory receptor than NgR, the early inactivation of pirB or NgR is sufficient to block acute growth crown atrophy due to the addition of radix inhibitor. This observation suggests that the atrophy requires a more stringent process of 139862.doc •64-200950808, which requires the activity of both PirB and NgR to act simultaneously or together. In this context, 'attribute' has recently shown that pirB and NgR receptors have a similar role in limiting synaptic connectivity in the visual cortex: in mice lacking either receptor, closed eyes can be excessive during critical developmental periods Strengthening the connection via blink (J. Syken et al., 2006, as previously described; AW McGee et al., 3, 9, 2222 (2〇〇5), as previously described). The mechanism by which the two receptors mediate the effects of growth crown atrophy may also be the basis for their interaction in the dominance of the dominant column of the eye. The failure of adult axons to recover after injury is a major obstacle to recovery after traumatic injury in the CNS. It is speculated that the potential for regeneration declines due to the limited ability of synaptic plasticity to age-admitted to limit the development of excessive or excessive synaptic connections. This hypothesis can be supported by the discovery that pirB, previously involved in limiting synaptic plasticity during development and adulthood (J. Syken et al., 2006, supra), is also a mediator of axonal inhibition by sewn phospholipids; It was also found that the NgR originally involved in axonal inhibition also regulates synaptic plasticity (S. Li et al., / 24, 1051 1 (2004)). Our findings also extend the range of potential PirB ligands beyond the molecular range to include neuronal regrowth inhibitors. Conversely, since the genetic deletion of the myelin inhibitor Nogo or MAG is known to cause only a modest reduction in inhibition by myelin - indicating the presence of other inhibitors - our findings suggest the following possibilities: usually expressed by a small amount of oligodendrocytes The MHC I molecules can be up-regulated after injury and promote growth inhibition in the central myelin with Nogo and MAG.

The mechanism by which PirB signaling inhibits axon growth in response to myelin inhibitors is not yet known 139862.doc -65- 200950808. However, PirB has been shown to antagonize the function of integrin receptors (S_

Pereira et al, J. 173:5757 (2004)), and can supplement both SHP-1 and SHP-2 phosphatase; one or both of these events can attenuate normal neurite outgrowth. Blocking PirB activity using the anti-pirB antibodies herein or by other means may provide an important new goal for therapeutic intervention to stimulate axonal regeneration. All references cited throughout the disclosure are hereby expressly incorporated by reference in their entirety. Although the present invention has been described with reference to the specific embodiments, it is to be understood that the invention is not limited to the embodiments. On the contrary, the invention is intended to cover various modifications and equivalents BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A and 1B show the mouse PirB sequence (S]Eq ID NO: 1) and the human LILRB2 sequence (SEQ ID NO: 2). Figures 2A and 2B block PirB reversible inhibition of CGN growth based on AP_N〇g〇66 or myelin. Will the dissociated mice? 7 CGN was plated on pDL/layer adhesion protein (control), AP-Nogo66, or Zhao fat to test for inhibition by these receptors. (A) Representative photomicrographs, (B) From a representative experiment. A graph of mean neurite length (±SE) was measured. Neurons grown on PDL/laminin, Ap_N〇g〇66, or myelin in the presence or absence of a functional blocking antibody against PirB (aPB1; 5〇 μ§/ιη1). aPBl significantly reduces inhibition by either host. Fpui; ratio, 50 μηι) 139862.doc -66- 200950808 Figure 3A-3D Blocking PirB reversible inhibition of CGN growth based on AP-Nogo66 or myelin. Dissociated mouse P7 CGN was plated on PDL/laminin (control), AP-Nogo66, or myelin to test for inhibition by these receptors. Representative micrographs are shown in Figures 3A and 3C, and a graph of mean neurite length 〇SE from a representative experiment is shown in Figures 3B and 3D. Neurons grown on PDL/laminin, AP-Nogo66, or Wei scales in the presence or absence of a functional blocking antibody against PirB (aPB1; 50 pg/ml). aPB 1 significantly reduces the inhibition caused by any of the substrates. (*ρ<〇·〇1; scale bar, 50 μηι) Figures 4Α and 4Β require both PirB and NgR to mediate growth crown atrophy caused by myelin inhibitors. The growth crown of the postnatal DRG axons was treated with medium (control), myelin (3 pg/ml), or AP-Nogo66 (100 nM) alone for 30 minutes to stimulate atrophy, and rhodamine-glum pentapeptide was used ( Rhodamine-phalloidin) was stained to visualize the growth crown. (A) Representative photomicrographs, (B) Graphs of growth crown atrophy percentage (shen SEM) from the accumulation test. The genetic loss of NgR or the inhibition of PirB achieved only by anti-PirB treatment is sufficient to prevent the growth crown atrophy activity of myelin or AP-Nogo66. Inhibition of both pathways can also completely block atrophy. (Scale bar, 50 μηι) Figure 5 A-5D blockade of PirB can de-suppress the growth of neurons in DRG neurons and on the recipient MAG. Representative photomicrographs are shown in Figures 5A and 5C; a graph showing the average neurite length (JS) from a representative experiment is shown in Figures 5B and 5D. (A) and (B) Dissociation of dissociated Pl〇DRG neurons in PDL/laminin, 139862.doc -67- 200950808 AP-Nogo66, or myelin in the presence or absence of anti-PirB . aPBl significantly reduced the inhibition by AP-Nogo66 and myelin. (C) and (D) Dissociate the dissociated P7 CGN on PDL/laminin or MAG-Fc in the presence or absence of aPBl. Antibodies against PirB reduce the inhibition of neurite outgrowth by MAG-Fc. (* ρ<0·01; scale bar, 200 μιη Α, Β; 50 μπι C). Figure 6. Anti-PirB antibody YW259.2 Heavy chain (SEQ ID NO: 8) DNA sequence. Figure 7. Anti-PirB antibody YW259.9 heavy chain (SEQ ID NO..9) DNA sequence. Figure 8. Anti-PirB antibody YW259.12 heavy chain (SEQ ID NO: 3) DNA sequence. Figure 9 is a protein sequence of the anti-PirB antibody YW259.2 heavy chain (SEQ ID NO: 4). Figure 10 is a protein sequence of the anti-PirB antibody YW259.9 heavy chain (SEQ ID NO: 5). Figure 11 is a protein sequence of the anti-PirB antibody YW259.12 heavy chain (SEQ ID NO: 6). Figure 12 is a protein sequence of the light chain of all YW259 antibodies (SEQ ID NO: 7). Figure 13. The ability of the anti-PirB antibody YW259.2 (IgG) to inhibit the activity of His-tagged mouse PirB. Figure 14. The ability of the anti-PirB antibody YW25 9.9 (IgG) to inhibit the activity of His-tagged mouse PirB. Figure 15 Anti-PirB antibody YW259.12 (IgG) inhibits His-tagged mice 139862.doc -68- 200950808

The ability of PirB to be active. Figure 16 includes the relative AP-Nogo66 binding of a panel of anti-PirB antibodies, including YW259.2, YW25 9.9 and YW259.12. Figure 17A-17C Alignment of the heavy chain sequences of the anti-PirB antibodies YW259.2 (SEQ ID NO: 11), YW259.9 (SEQ ID NO: 12) and YW259.12 (SEQ ID NO 13). The CDR HI, CDR H2 and CDR H3 sequences are framed with the CDR H domain of Kabat, Chothia and the CDR H domain. Hum III is shown as SEQ ID NO: 10. ® Figure 18A-18C Alignment of the light chain sequences of the anti-PirB antibodies YW259.2 (SEQ ID NO: 15), YW259.9 (SEQ ID NO.15) and YW259.12 (SEQ ID NO.15). The CDR L domains along Kabat, Chothia, and the CDR L domain are framed for CDR LI, CDR L2, and CDR L3 sequences. HuKI is shown as SEQ ID NO: 14. Figure 19 C1QTNF5 (CTRP5; NP_05646) inhibits neurite outgrowth of the dorsal root ganglia, and this inhibition is reduced when PirB is blocked by the PirB function blocking antibody ^YW259.2. 139862.doc -69- 200950808 [Sequence List] <11〇>US-based Nandek Company<120>Anti-PirB Antibody<130> GNE-0324 <140>098115945 <141> 2009-05 -13 <150> US 61/052,949 US 12/208,883 US 12/316,130 <151> 200^05-13 2008*09-11 2008-12-09 <160> 15 <!?0> Patemla version 3,5

<2l〇> I <2Π> 841 <212> PRT <213> Mouse Genus <4〇〇> 1

Met Ser Cys Thr Phc Thr Ala Leu Leu Arg Leu Gly Leu Thr Leu Scr 1 5 10 15

Leu Trp lie Pro VaJ Leu Thr Gly Scr Leu Pro Lys Pro lie Lea Arg 20 25 30

Vai Gin Pro Asp Ser Val Val Ser Arg Trp Tlir Lys Val Thr Fhe Phe 35 40 45

Cys Glu Glu Thr l!c Gly Ala Asn GIu Tyr Arg Leu Tyr Ly& Asp Gly 50 55 60

Lys Leu Tyr Lys TTir Val Tbr Lys Asn Lys Gin Lys Pro Ala Asn Lys 65 70 75 80

Ala Glu Phe Ser Leu Scr Asn Val Asp Leu Arg Asn Ata Cly Gin Tyr 85 90 95

Arg Cys Ser Tyr Ser Thr Gin Tyr Lys Ser Scr Gly Tyr Ser Asp Pro 100 105 no

Uru Giu Leu Val Va丨 Thr Gly Asp Tyr Trp Thr Pm Ser Leu Leu 115 120 125

Gin Ala Ser Pro Val Vai Thr Ser Gly Gly Tyr Val Thr Leu Gin Cys 130 135 140 G\u Ser Trp His Asn A^p Ly$ Phe fie Leu Thr Val 〇]u Gly Pro 145 150 155 160

Gin Lys Leu Ser Trp Thr Gin Asp Ser Gin Tyr Asn Tyr Ser Thr Arg 165 170 175

Lys Tyr His Ala Leu Fhe Sef Vat Gly Pro Va] Thr Pro Asn Gin Arg m 185 190 139862.doc 200950808

Trp ik Cys Arg Cys Tyr Ser Tyr Asu Arg Asn Afg Pro Tyr Va丨 Trp 13⁄4 200 205

Ser Pro Pro Ser Glu Ser Val Gtu LeH Leu Va, Ser Gly Asn Leu Gin 210 215 220 ^ Pro mile Lys Ala Glu Pro Glv Pr〇 Vaille A) a Ser Lys Arg

Ala Sfct Thr tie Trp Cys Gin Gly k%n Leu Asp Ala Gin Val Tyr Phe 245 ISO 255

Leu Ηϊχ Asn Glu Gly Ser Gin Lys Thr G n Ser Thr G]u Thr Leti Gin 260 265 270

Gin Pro 〇Iy Ash Lys Gly Lys Phe Phe lie Pro Ser Met Thr Arg Gin 275 280 285

His Ala Gly G\n Tyr Arg Cys Tyr C>fs Tyr Gly Scr Ala Gly Trp Ser 290 295 300 ΰίη Pro Scr Asp Thr Leu Gla Leu Vsl Val Ώιτ Gly He Tyr Glu His 30S 310 315 320

Tyr Lys Fro Arg leu Scr Vai Lea Pro Ser Pro Val Val Thr Ala 〇]y 325 330 3J5

Gly Ash Met Thr Uu His Cys Ala Ser Asp Phe His Tyr Asp Lys Phe 340 345 35D lie Leu Thr Ly$ G3u Asp Lys Lys Pile Qly Asn Scr As^i Thr GH 355 360 365

His lie Stt Str &r Arg Gin Tyr Arg Ala L·^ Phe He He Giy Pro 370 375 380

Thr Thr Pro Thr His Thr Gb Thr Pbe Arg Cys Tyr Gly T.yr Phe Lyt 385 390 395 400

Asn Ala Pro Gin Uu Trp Scr Val Pro Ser Asp Lem Gk Gin He Leu 405 410 41$

Jlc Ser Gfy Leu Ser Ly Technique Lys Pro Ser Leu L«u Tlir His Girt H·" 420 425 430 lie lxu Mp Pro Cly itet Thr Leu Tbr Ixu Gin Cys Tyr Scr Asp lit 435 440 445

Asn Tyr Asp Arg Phe A] a Leu His Lys Va] Gty Gly Ala Asp lie Mei ^30 4SS HU Ser S$r Slit ϋ丨n Thr Asp Hr Gly Phe Ser \%丨 Ala Aim Phe 465 470 475 480

Thr Leu Gly Tyr Vjii Scr Scr Ser Thr G]y Gly Gin Tyr An Cy§ Tyr 4S5 490 495 , 2· 139862.doc 200950808

Gly Ala His Asn Ser Ser Glu Trp Scr Ala Scr Ser G!a Pro Leu 500 505 510

Asp He Leu. I!a thr Gly Oln Leu Pro leu Thr Pro Ser Lea S«r Val 515 520 525

Lys Pro A.St> His Thr Va丨 His Sef Gty Glu Thr Va.] Ser leu Leu Cys 53ύ 535 540

Trp S«r Met Asp Sfir Val Asp Thr Phe lie Uu Ser lys Glu Gly Ser 545 550 S55 560

Ala Gift Gin Pro Leu Arg Leu Lyss Scr ly» Ser His Asp Glu Oln Scr 565 $70 575

Girt A!a Gia Phe Ser Met Ser Ala Val Thr Scr His lee Ser Cly Thr 580 585 590 e

Tyr Arg Cys Tyr Cly Alt Gin Asri Ser Ser Phe Tyr lea Leu Sir Ser 595 6£M) 605

Ala Ser Ala Pro Val fi5u Uu iht Val Ser Gly Ρΐ〇 \U 0!u Thr Ser 610 615 620

Hu Pro Pro Pro Thr Met Sef Mst Pro Leu GSy Gly Ua Hb Met Tyr 625 630 635 640

Lea Lys A!a Leu He Gly Val Ser Val Aia i^c lie L«m Phe Lew Pbe 645 650 655 lie Uu lie Phe lie Leu IjCU Arg Arg Afg His Arg Oly Lys Phe Arg 660 665 670

Lys Asp Val Gin Lys Glu Lys Asp Leu Gin Leu Ser Ser G!y Ala Glu 675 680 685 G!b Pro lie Thr Arg Lys Gly Gla Lea Gift Lys Afg Fro Asn Pro Ala 690 695 700

Ala Ala Thr Cla Olu Glu Ser Leu Tyr A!a Ser Val G1« Asp Met Gin 705 710 715 720 T.r Gi, ASP Oly Vg Glu Uu AS〇 Sar Trp Thr ^ Pro Oiu G«u ASp

Pro Oln Cly GJu Thr Tyr Ala Gin Vsl Lys Pro $cr Arg Leu Arg Lys 740 745 7S0

Ata Giy His Val &r Pro Ser Val Met Ser Afg Gin Leu Asn Thr 755 760 765

Gl^ Ty^ Glu Gin Ain <31u dlu Gly Gin Gly Ala A.sn Asn Gin Ala Ala 770 775 780

Gl, S,r Oi, G«u || Asp Val T1,r Tyr Al, Gl« ^ C,SS,r ^ 139862.doc 200950808 T1,rl,a Arg OI„ Gly Ala Ala ^la Ser Pro Le« Ser AU |Ιξ Gl«

Aia Pro Glu Glu Pro Set Val Tyr Ala Thr Leu Ala Ala A!a Arg Pro 820 S25 830 G!a Aia inpro Lys Asp Val ai g|r <210> 2 <211> 598 <212> PRT <213> Homo sapiens <400> 2

Met Thr Pro 11« Val Thr Lea 11« Cys Leu Gb Leu Ser Leu Gly 1 5 10 Ϊ5

Pro Arg Thr His Val Gin Tiir G!y Thr He Pro Lys Pfo Thr Leu Trp 20 25 30

Ata G!u Pro Asp Scr Val lie Thr Gla Cly Ser Pro Vat Thr Leu Ser 35 40 45

Cys Gin C3y Set Leu G(u Ala Glis 013⁄4 T^r Arg Leu Tyr Arg Q]u Lys 50 S5 60

S<.ys Ser A]a Ser Trp lie Thr Arg lie Arg Pro Glu Leu Val Lys Asn 65 70 75 SO

Gly Gin Phe His lie Pro Ser lie THr Tep Glu His Thr Cly Are Tyr 85 90 95 CSy Cats G3n Tyr Tyr Scr hti Ala Atg Tfp Ser Glu Leu Scr Asp Pro 100 105 110

Le, V3, L!?Vai,ct ^GI. AJaTyr Pro Ly, Pro ^ Leu Ser Λ,3

Gin Pro Ser Pro Vai Val Ί^γ Scr Gly Gly Arg Val Thr Leu Gin Cys BO ]3$ 140

Gin Ser Gin Val Ala Pbe Gly Gty Phe Jfe Leu Cys Lys Glu Gly Glu 145 150 155 160

Asp Oiy Hss Pro Gin Cys Leu Asn Ser Oiu Pro His Ala Arg Gly Scr 165 170 175

Scr Arg ASa lie Phe Ser Vai Gly Pro Vai Scr Pro Asa Arg Arg Trp 180 185 190

Ser His Are Cn Tyr Oly TVr Asp Lea Asu Ser Pro Tyr V»1 Trp Scr 1S5 200 20S

Ser Ser Asp Leu Leu G3u Leu Leu Val Pro GSy Val Ser Lys Lys 2i〇 215 220 -4- 139862.doc 200950808 yo 15 G2 va p As f se *1va s $· y4 02 n Gl f Th u Le

Le ln 01 065 f 6 A2 u ¢- LpAs close to uclfys G2u G s Ly ΓTy pos Γ A p Γ Ty u«5 V2 β rs reg A27 o pr .0 Γ csu Le y5 17 G2 8 A1ΏG1 r8 M fe sw,orp y-Suu c ph . leopard o iss A 2 ss Γ cs Γ € su Le ns B. Al y 5 Γ ns vrf c 85 Γ9 A2 Γ Ty Π 0 ymy G, ro V9 T2 Γ cs no I 2 03 ym 6 1 1 u Le c5 n 41 ϋ' p M uuofpp As ro cl & 3 0 fp β Al 1 csp Tr U5 n G^ .c [ — 3 G3 汪 V Γ se ei I c ph 05 ^-2 i % s ly Gt r0 2 IK 15 v433 ❿ s Hi c ph n must i ^ G3 Γ Γ A p Γ - Γ Γ 1 sn 1 G s 5 su Lc u Lc Γ Th val Ro s times. A3 u 0 y G, Γ csa 0 M uu t065 P3 & A, p £# A 3 A ce y<5 53⁄4 s A, s Ly .IMW h π u Le 85 ΛΛ5 L· 3 c ph Th

Arg Ser i ie 01« Tyr Pro Lys Tyr Gin Ala Glu Phe Pro «ec Ser

Pro W] Tlif Ser Ala His Als G】y Hr Tyr Arg C^s Tyr Gly Ser ku 385 390 31⁄2 400 A$n Ser Asp Pro Tyr Leu Leu Scr His Pro Ser Giu Pro Leu Glu 405 410 415 V^l Str Gly Pro Ser Net Cly Scr Ser Pro Pro Pro Ihr Giy Pro 420 425 430

He Ser TTir Pro Ala Gly Pro Glu Asp Gin Pro Leo Iht Pro Thr Gly 435 440 445

Ser Asp Pro G^n Str Gly Leu Gly Arg His Uu Gty Va! Val lie Gly 450 43.5 460 lie Uy V^l Als Val Val Leu lxu Lcti Ixb Leu Leu Leu Lets Leu Fht 465 470 475 4S0 lie Lea Arg His Arg Arg Gin Gly Lys His Trp Thf Ser Thr Glis 485 490 495 Λγ2 U, Ala As, ^ mn His Pro m Oly Ala Va, G,y Pro Of« Fro

Thr A^p Ar^ Gly Lea filn Trp Arf Ser Scr Pro Aia Ala Asp Ala Oln 51$ 520 525

Glu G1, ASB 1,« Tyr AU Alf Val Lys Asp Gin Fro Gi, Asp Oi, 139862.doc 200950808

Va! Glu Met Asp Tlir Arg Ala Ala Ala Scr Glu Ala Pro Gin Asp Val 545 550 555 560 THt Tyr Ala Ola Leu His Set Lta Tbr Leu Arg kxg Lys Ala rtht G!u 565 570 575

Pro Pro Pro Ser Olti Glu Arg Glu Pro Pro Ala Glu Pro Scr lie Tyt 5S0 585 590 A] a Thr Uu lie His 595 <210 3 <2li> 3419 <2!2> mk <2丨3> Sequence <220><223> artificial sequence description: synthetic polynucleotide <400> 3 atgggaiggt catguicat cttmicu giagcaactg caaciggagc gitegetiag gatti^ctgg tg^agicigg cggi^cclg gigcajicc^g gfgfctcact ccfitugtcc 】20 igigcagcct ciggcticac cttcactaai tccuii* ita gctgggtgcg tcaggccccg] g〇gguagggcc tggaatgggt tggigggait tctccitctg gcggiictac tuctatgec 240 gais ^ cgtca aeggeegut cactaiMgc gcagacacai ccaaaaacac agcciaccta 3¾} caaaigaaca Ectisagigc tgag ^ acact gccgtctati atigtgcaaa aicggtctgg 3¾) gcgticgcu acigg £ gtca aggaacccig gicaccgict cci ^ ggccic caccaagggc 420 ccatcggtct tccccciggc accctcctcc aagagcacci cig | Gggcac agcgficccig 4g〇ggcigcctgg tcaaggaci^ cttccccgaa ccggtgaegg tgicgiggaa cicaggcgcc 540 ctgaccagcg gcgtgcacac eucecggct gicctacagi cclcafgact ctaciccctc 600 a£cagc£tgg tgacifigcc ctciagcagc ugggcaccc agaccuca t ctgcaacgig 660 aiucacaagc ecage rolled into cac € a ^ ggiggae aagaastgitg ugtgaeaaa 720 acicacacai gcccaccglg cccagcacct gaactcctgg ggggaccgte agicttccic 7¾) ttccccccaa aacccaagga caccctcaig atciccegga ccccigaggt cacaigcgig g4〇gtgsugacg tgagccacga agaccctgag gtcaagitca aciggiacgt ggaeggegig 9〇〇gaggigeata atgccaagac aaageegegg gaggageagi acaacagcac gtaccgggig 960 gtcagcgicc tcaccgicct gcaccisggac tggctgaaig gcaaggagta caagtgcaag 1020 gictccaaca aagccctccc agcccccaic gagaaaacca tctccaaagc caangggcag i〇M) ccccgagaac cac ^ gitgu caccctgccc ccaicccgsg aagagatgac caagaaccag 1140 gtcagcciga ccigcciggt caaaggcuc tatcecagcg acatcgccgt ggagigg ^ ag 1200 agcaatg £ gc ageeggagaa caacucaag accacgcctc ccgi $ ctgga ctccgacggc 1260 iccticttcc tciac ^ gcaa gcicaccgig gacaagagea ggtgstigca Gg^gaacB^c 1320 itetealget ccgtgaigca tgaggctci£. cacasccaci acacgcagaa gagcctctcc 1380 cigiciccgg giaaatga&t fcgacggccc ugagtega 1419 139862.doc 200950808 <210> 4 <2Π> 465 <212> PRT <213> Sequence of <22€><2'23> Artificial Sequence Description: Synthetic Peptide <4〇〇> 4

Met Gly Trp Ser Ci>s He He Leu Phe lea Val Ala Thr Ala 'Dir Gly I 5 10 15

GtnUuVal | u Ser Gly Gl, GIy Uu Va« Gin

Pro Gly Gly Ser Uu Arg Lea Ser Cys Ala Ala Ser Giy Phe Thr Pht 35 40 45 seinSerTyilSeSergpvat_GlnAla^GlyLysGlyu„ ❿

Glu Trp Val Gly G«y lie Tyr Pro Ser Gly G,y Λ,η Br AS« Tyr Ala

Asp Ser Yai Lys Gly Arg Hie Thr Ik Ser Ala Asp Thr Ser Lys Asn 85 90 95

Thr Ala Tyr L·^ Gin Mei Asn Ser Lea Arg Ala Glu A$p Thr Als Val 100 105 110

Tyr Tyr Cys Ala Lys Scr Aia Trp Cln Phe Ala Tyr Trp Gly Gin Gly 115 120 ns

Hu Leu Va! Tbr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Va丨 Phe 130 135 !40

Pro Leu Ala. Pro Ser Ser Lys S«r Thr Scr (ily Gly Thr Ala Ala Leu 145 150 155 160

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 165 170 175

Asa Ser Gly Ala. Leu ΊΪΓ Ser Gly Vlt His Th;r Phe Pro A]a VaS Leu 180 185 190

Gh Ser Ser Gly Leu Tyr Ser Ser Ser Val Val Thr Yal Pro Ser 195 200 205

Ser Ser Leu Gly Tlir Gin Hu Tyr lie Cys Asn Val Asa His Lys Pro 210 215 220

Ser Asn Thi Lys Val A$p Lys Lys Vat Gla Pro Lys Ser Cys Asp Lys 225 230 235 240

Thr HU Thr Cys Pro Pro Cys Pro Ala Fro Clu Uu Leu CSy Giy Pro 245 250 255

Ser Val Phe Leu Phe Pro Pro Lys Pro lys Asp Thr lei* ifci lie Ser 260 265 27D •Ί · 139862.doc 200950808

Arg TTir Pro Gly Val Thr Cys Val Vsl Val Asp Val $tr His Glu Asj> 2?5 280 285

Pro Glu Val Lys Phc Asn Trp Tyr Val Asp Cly Va] Gin Val Hb Asn 290 295 300

Ala Lys Thr Ly$ Pro Arg GJu Glu Gin Tyr Asii Str Thr Tyr Atg V^l 305 310 315 320

Scr V8l Uu Thr Val LeU His Gin Mp Trp Leu Mn Giy G1a

Tyr Lys Cys Lys Val Ser Asn Lys Ala Uu Pro Ala Pro lie Clu Lys 340 J4S 350

Tbr lie Ser Lys Ak Lys Gfy Gin Pro Arg Glu Pio Gin Va! Tyr Thr 355 360 365

Leu Pro Pro Ser Arg GIu Gi« Met Thr Lys Asn Gin Val Ser Leu Tlr 370 375 380 illYal Lys Gly ?hc Tyr Pro Set Asp lie Ala Vs! Glu Trp 390 395 400

Ser Ash Gly 〇lji Pro Glu Asn Asn Tyr lys Thr Thr Pro Pro Val Leu 405 410 4}5

Λ,ρ Ser Asp G,y Ser Phe Tyr Ly, Leu Vai Asp LyS

Ser Arg Trp Gin Gin Gfy A^n Val Fhe Ser Cys Ser Vat Met His Glu 435 44〇 445

Ala Leu His Asn His Tyr Tlu Glti Lys Ser Leu Ser Ixu Ser Pro G]y 4S0 455 460

Lys 465 <2W> 5 七11> 465 <2)2> m <2i3> artificial sequence <220><223> artificial sequence description: synthetic polypeptide <400>

Met Gly Trp Scr He lit Leu Rie htu Val Ala rHir Ala rfhr Oiy 1 5 10 15

Ala TK Ala 〇tu Va, Leu Vaf gU Ser Gty GI, Gly L,, Cln

Pro Gl, Gly S, r Lca Arg Leu S^r CyS Ala Ala Ser Gly Ph, Br P^e

Scr Scr Am Jyt lie Ser Trp Vat ktg Gin Ala Pro Gly Lys Qly Leu ^ 55 60 139862.doc 200950808 G|« Trp V91 〇ly Oly He Asn Pro TT. 〇Iy G,y Tyr l,r Tyr Tyf Ma

Ser Val Lys | y Arg Phe TTtr lie Ser Ala Asp Π, γ Ser Lf Asn H,r ΛΙ, Tyr Uu Oln «cl Asn Ser U« Arg Ala Olu Asp ^ Ala V,)

Tyx Tyr Cys Ala Arg Ser Va] Trp Val Phe Asp Tyr Tri> Gly Gin Oly 115 120 125

Br^Var^VatS.S.AI.^TIu^^P.S.ValP.e

Pro .U P. Ser fs L. SCr ^Ser?riyH, AiaAlaU5

Oly Cys Leu Va] Lys Asp TVr Phe Pro Glu Pro Va] Tlir T Print 165 170 175

Asn Ser Gly Ala Lea Thr Set Gl^ Va] His Tlir Phe Pro Ala Vsl Leu sao 】85 m

Gin Ser Stt flly Leu Tyr Ser Leu Sor Str V^I Val Iht Val Pro Ser 195 200 205

Ser Sef Uu G昼y ΤΉγ Oin Thr Tyr lie Cys Asn \fa! Asn His Lys Pro 210 215 220

Ser Asn TTir Lys 225

Asp tys Lys Va! Qlu Pro Lys Ser Cys Asp Ly$ 230 235 240

Thr His Thr Cy$ Pro Pro Cyx Pro Ala Pro Glu Leu Leu Gly Gty Pro 245 250 255

Scr Val Phe Uu Phe Pro Pro Lys Pro Lys Asp Tbr Leu Met lit Ser 260 265 270

Arg Thr Pro Glu %丨 Tlrr Cys Val Va! Val Asp VaS Sej* His Glu Asp 275 tm 285

Pro Giu Val Lys Rie Aim Ding rp Tyr Val Asp G】y Yal GJu Val His Asn 290 295 300

Ala Lys Thf Lys Pro Arg Glu Glu Gin Tyr Asr Ser Thr Tyr Arg Val 305 310 315 320

Val S^r Vgl Leu TNr Val Lcii His Gin Asp Trp Leu Asn Giy Lys G]y 325 330 335

Tyr Lys Cys Lys Val Ser Asn Lys Ala Uu Pro Ala Pro lie Glu Lys 340 345 35a llr He |e, L,S Ala Ly, Glv Gin Pro Arg Clu Fro 0|« Vs, T>. -9- 139862.doc 200950808

Lett Pro Pro Ser Ar Rong Glu Glu Me! Tfcr Lys Asn CHn Val Ser Leu Thr 370 375 380

Cy| Uu Val LSS Gly Tyr Pro Ser Λ,ρ He A,a Val 01, Trp

Ser Asrt Gly Gtn Pm Gl.u Asn Asn Tyr Lys Thr Tlir Pio Pro Val Leu 405 410 415

Asp Ser A&p Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Mp Lys 420 425 430

Scr Arg Trp Gtn Gin Gly Asn Va] Phe Ser Cys Ser Val Uti His Gla 435 440 445 A]a Leu His Asti His Tyr Thr Gin Lys Set Uti Ser Leu Ser Pro Giy 45Θ 455 460

Ly$ 465

<210> 6 <211> 465 <212> PRT <2丨3> artificial sequence <223> artificial sequence description: synthetic polypeptide <4Q0> 6

Mel Oly Trp Ser Cys lie lie Leu Phe Im Val Ala Thr Ab Thr Gly 15 10 15

Ala Tyr Ala Clu Val Gin Lau Val Glu Ser Gly Gly Gly Ley Val Q]n 20 2S 30

Pro Gty Giy Ser Ua Arg Leu Sei Ala Ala Ser Gly Phe TTir Phe 35 40 45

Tbr M$n Scr Tyr He Ser Trp Vai Arg Gin Ala Pro Gty Lys Ciy Lea 50 55 60

Giu Trp Val Oly Gly lie Ser Pro Ser Gly Gly Ser Iht Jyt Tyr Ala 65 70 75 80

Asp Ser Va] Lys Cty Arg Phc Thr tic $er AU Asp Thr Ser Lys Asn 85 90 95 ΤΤιτ A)a Tyr Leu Gin Mel A$n Ser Leu Arg AU Clu Asp Thr Aiu Va] 100 105 110

Tyt Tyr Cys Ala Lys Ser Val Trp Ala Phe Ala Tyr Trp Gly Gin Gly 115 Γ20 125

Thr lc« Val Thr Val Set Ser Ala Ser Thr Lys G!y Pro Ser Val Phc 130 I3S 140

Pro leu Ala Pro Ser Ser Lys Ser TKr Scr Cly Gly Thr Ala Ala leu -10- 139862.doc 200950808 145 150 15S 160 C3y Cys Leu Va] Lys Asp Tyr Phe Pro Glu Pro Yai Thf Val Ser Ding 165 170 175

Asn Ser Gly Ala Leu tlu Ser Gly Kis Tfer Phe Pn> Ala Va3 Icy 180 1S5 m m, Ser S,r G,y 1,. T^r Ser Ser Ser V8i Val TJr Val Pro Ser

Ser Ser Leu Gly Tbr Gin Tlu T>r Ik Cys Asn Val Asn His Lp Pio 210 215 220 &r A$n Hir Lys Val Asp Lys Lys Vai Glu Pro Ser Cvs hsp Lys 225 230 235 ' 240

Thr His Thr Cys Pro Fro Cys Pro Ala Pro GIu Leu Leu Gly Gly Pro Ref. 245 250 255

Ser Val Phe Leu Phe Pro Fro Lys Pro Lys Asp Thr ]^c« Met Uc Ser 260 265 210

Arg Thr Pro Glu Val Thr Cys Vd Val Asp %] Ser HU Glu λ: φ 275 280 285

Pro Gk Val Lys Phe Asn Trp· Tyr Office] Private p Giy Vd (Ha Val His Asn 29Q 295 300

Ala Lys Thr Lys Pro Arg 0]u Gili Gin Tyr Α§β Ser TTir Tyr Arg Val 305 310 315 320

Val Ser Va) Lea Utr Val Leu Nis Gin Asp Trp Leu Asa Gly Lys G!u 325 330 335

Tyr Lys Cys Lys Ser Asn Lys Ala Leu Pro A] a Pro He Gh Lys MO 345 350

Thr Ik Ser Lys Ala Lys Gly G】n Pro Arg Glu Pro Gin Va丨 Tyr Utr 355 360 365

Leu Pro Pro Ser krg Gin 013⁄4 Met Thr Lys ksn Gin Val Ser Leu IJit m 375 380

Cys Leu Vai Ly$ Gly fht Tyr Pro Ser Asp fie Ala Val Glu Trp GIu 385 33⁄4 395 400

Ser Ash Cly Gin Pro Glu km Ase Tyr lys Thr Thr Pro Pro Val Lea Shed 430 435

Asp Scr Asp Cly Set Phe Plie Le§i Tyr S-er Lys Leu Hir Vat Asp Lys 420 425 430

Ser Arg Τφ G!n G_n Gly Asn Va] Rie Ser Cy$ Ser says! G]u 435 440 445

Ala Leti His km His Tyr The Gin Lys Ser Lea Ser &r Pro Civ -11 - 139862.doc 200950808 450 455 460

Lys 465 <2J〇> 7 <in> m <2n> m artificial array _ <22 ft><M3> artificial sequence description: synthetic polypeptide <400>

Me ding Gly butyl Scr Cys He He Leu Phe L«ii V5I Afa Thr Ala Hu Gly 】 5 丨〇 15

Va Xin His Ser Asp He Gla Mbt Tbr Gin Ser Ρτο Ser Ser Ua &r Ala 20 25 30

Scr Val Gty Asp Arg Val Thr 11c Thr CVs ^tg Ala Ser Gin Asp Val 35 40 45

Ser Thr Ala Val Ala Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys 50 55 60

Leu Leu Ik Tyr Ser Ala Ser Phe Leu Tyr Scr Gly Vai Pro Ser Arg 6$ 70 75 80 cs .Γ ¢5 S9 t 1 ..tm§ Γ Th u~ u T ^ c BI90 p As Γ Thr y 5 1m· Cs y- •tt G& Γ sc y G, ser cn Γ o -_c Js i ϋ G, η» G1 s cy f Ty rt 5 yo T1 f Th This A, c fl ps luoo G 1 o pr n 0 V Le ot OS Γ n G, € ph f ^ D 5 F 1 £ Γ $ -y C5 nua 3 V sn y20 Gl12 01 u .0 p M ΓΟ ^4 o fa o Γ pcn Av fB -C5 ^ 3 HI vai Γ & o f> p 0 A so ΙΛ4 A 1» va

Th y G, Γ & v^~4 I·:1"«i. sl A a As a 5 Le5 Q Le s 1 va wes oo 崄6 PI Γ τϊ po Asn vsl $ Ly rp Play G1 15 vale s Ly a I s cl Γ 5 ύ s ϋ Le Λα stt

TJ n, Γ o _^9 s 1J p s A sLy i €s p s A Π 5 G18 u Q Γ n a, V Γ .280 ϋ ] 0 Gi k* €s Π s A

Ly Γ- se uo .320 Γ Th ΰ ra 13⁄4 Γ se er95 s i u Le scr f 5 yo T 2pAs

Lv' I» Λυ ft I V 2 s Ly

Ly f*5 s

Tyrser Γ {5 15 va2i 1 G s cy 3 V o £ c s c s αΰ ϋ 2 cy) u 3 y Gt Γ ^ A2 n As c phi -12- 139862.doc

200950808 <210> 8 <2Π> 1419 <212> DNA <213> Artificial Sequence <220><223> Artificial Sequence Description: Synthetic Polynucleotide <400> 8 sigggatggt catgutcat ixtmuu gtagcaaeig caaciggagc it buckle gcigag gttc $ gcttg tggagtctgg cggtggccig stgcagccag ggg ^ ctcact ccgmgtcc igtgcagcu ctggcticac cttcagtaat tcctautta gcijgggtscg tcaggccccg ggxmgggcc igsaatgggt tggtgggatt tatccttcig gcgitaaigc laactaigcc gatag ^ gtca ag ^ ccgm cactataagc gcagacacai ccaaaaacae agcctaccia c ^ itaigaaca ictta ^ Eagc tgagpcact gccgicnn altitEcaaa aagcgcctgg cagucfcu aciggigica aggaaccctg gtcaccgtci ccicggcctc caccaagggc ccatcggtci xcccctxggc acccicctcc sagagcacct ctgggggeac agc ^ gcccig ggctgcctgg ccaag ^ acia ctrccccgaa ccggcgacgg tgtcgtg £ 3a ctcaggcgcc ctgaccagcg gcgtgcacac cticccggct gtcctacagt cctcaggact ctacicccic agcagcgtgg tgaciitgcc ctctafic ^ gc ttgggcaccc agacctacat ctgtaacgig aatcacaagc ccagcsacac caaggtggac aagaaagug agcccaaatc Ugtgac ^ aa acicacacat gcccaccgtfi cccagcacct gaactcct £ g ggggaccgtc ^ gtcttcc te uccccccas aaccc ^ agp caccctcaig aictcccgga cccsiga ^ t cscatgcgti j £ tggiggac £ ^ iagccacp agaccctgag iicaaguca ggacggcgtg gsggigcaia atgceaagac aaagccgcgg gaggagcagt ^ caacagcac giaccg ^ gtg gtcagcgtcc tcaccgicct gcaccagsac tsgetgaatg gcaaggagia caagtgcaai gtctccaaca aagccctccc agcccccatc gd £ aaaacca iciccaaagc caaagggcag ccccgagaac cacaggtgta caccctgccc ccaicccggg aagagatgac caagaaccag gtcsgcciga cctgcctggt caaafgcttc tatcccagcg acstcgccgt gg ^ gtgggag agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctg ^ ciccgacggc tccttcrtcc ictacagm gctcaccgig gacaagagca ggtggcage ^ ggggaaogtc ttctcargct ccgigatgca taagictctg cacaaccact acacjgcsgan ga «ccicicc ctgtctccgg £ Maalga £ i gcgacggccc iagagtcga <! 2 square > 9 < 2ii > im <m>DNA<2I3>Artificialsequence<220> Λ c223> Artificial sequence description: synthetic polynucleotide <shed> 9

Wgggatggi eiHgiatea! Cc ^ Uttcta giagcr hereby acig caiimg eggs ge guegOi this g gttcagctgg ijgsgtctgg cggiEgccti gigcagccag g ^ Sictcaa ccgutgtcc iltficagct! Ciggcttciae cttcagiagi aactatatia gctgggtgcg icaggccccg ggt ^ aEIECc tfgaatgggt tg £ t £ g £ au. AaEcctacig gcggttsiac Etact »^ gcc calagcgtca agggccgtu Ciictata^^c ^cagacacat ccaaaaacac agcctaccia 139862.doc 60 120 ISO 240 3<X) 360 420 480 540 60D 660 720 7S0 840 900 960 1020 1080 U4Q 1200 1260 1320 B80 3419 60 120 180 240 •13- 300 200950808 ca«atgaac ^ gcttaagagc tgaggacaci gccgtctatt attgigcaag aagcglgttg J60 gigttcgail actggggica aggaaccctg gicaccgict cctcggcctc caccaassgc 420

ccatcggtct tccccctggc accctccecc aagagcacct ctgggggc ^ c agcggccctg 4SO ggctgcctgg tcaaggacta citccccs ^ ccg £ tgacgg tgicgtgiaa ctcaggcgcc 540 ci ^ accagcg gcgtgcacac citcccggct gtcctacagt cctcaggsci cUctccctc 600 agcagcgtgg tgactgtgcc ctciageagc uggicaccc agacctacat crgcaacgtg 660 aatcacaagc ccsgcaacac caaggtgg ^ c agcccaaatc tcitgaca ^ a 720 actcacacat gcccaecgtg cccagcacct gaacwigg ggggaccilc Agtcticctc 780

Uccccccaa aacccaag^a caccctcalg atctcccgga ccccigaggt cacatgcgtg 840 gV^tggacg tgagccacga itgiccctgag gicaa^uca accggtacgt ggacgicgtg 9(3⁄4

Eaggtgcaia atgccaagac a ^ agccgcgg gaggagcagt aca ^ cagcac £ iaccgggtg 96 € glca £ C £ tcc tc ^ ccgtcct gcaccaggac tggctgaatg gcaaggagia caagtgcaag 1020 giciccaaca aagccctccc agcccccatc gagaaaacca ictccaaagc caaagggcag 10S0 ceccgagaac cacaggtgi ^ c ^ cccigccc ccatcccgfg aagagaigac caagaaccag 1140 gtcagcclgs cctgcctg £ t caaaggcitc iatcccagcg acatc £ ccgi £ .gagtgggag 1200 agcaatgggc agceggagaa caactscaag accacgeete eatgeigga e eegacgge 1260 tccttatcc tctacagcaa gctciccgtg gBcs ^ iBgca ggrggcagca 1320 tUKruigct ccgtgatgca tg &! agctctg cacsaccact acacgcagaa gagccicicc B80 cigtctccgg giaaaigagt gcgacggccc tagagicgii 141θ swsiiolG 0 > l > 2 > 3 > »> va Tsf tt 11 Λν u <2<2<2<2<4ΰι 0 v^· 15 GI o pr ln G va uu Glyio y G, V- G, Γ se Vi G va5 uu ln

Ty Γ € s Γ 60 S3 c 01 Γ Th c ph ys Γ ¢5 S2 A, s cy F € s L 2 rB A u le Γ sc A1 y! L y G, DJ -a: I o A45 n 0 ε Γ A va! pnf ¢5 S3 u £5 ru4

Va p T va w sc p .3⁄4 A Λ y yQ Η·" Γ y τ Γ Ώ Γ QJ S y G! y Gl55 ps A y Gl Γ sc c V- G, 50 Γ cs -yo T segment 3 A, Γ nt Π s A s Ly f ¢5 s? Γ |S ps A A- se c *1 Λν J Γ € hpsf A y G1 s y5 L6 cy fr y$ T9 f Ty ¥ 3 ΓΛ wp lso A9 V 0, Λ» Al r8 A uuf e5 S8 a As ic an ,nu

AtaArgGIyffiAspT>TTrp0lyl?C!yThr !i〇 -14- 139862.doc 200950808 <2i2> mr <213>Artificial sequence <22〇>

<223> Artificial sequence description: synthetic polypeptide <400> II

Ctn Val Cli5 Lea Val Glu Ser Gty GSy G!y Leu Val 01 n Pro 01 y 01 y 1 5 10 15

Ser fjen A.rg Leu Scr Cys Ala Ala Gt^ Pte Thr Phe Ser ksn Set 20 25 30

Tyr lie Ser TrP Val Arg Gin Ata Pro Giy Ip 〇]y Leq GIu Trp Val 35 40 45

Oiy Gly He Tyr Pro Ser Gly Gt^ hin Thr Asti Tyr Ala Asp Ser Vai 50 SS 60

Lys CJIy Arg Phe Thr lie Ser Ala Asp Thr Ser Lys Asu Thr Ala Tyr 65 70 75 SO ben Gin Asn Ser Leu Arg Ala Gtn Asp Thr Ala Val Tyr Tyr Cp 85 90 95

Ala Lys Ser Afa Trp Gin Phe Ala Tyr Trp Gly Gln<Gly Thr 100 105 110 <2IO> 32 <2ii> m <2I2> m <2I3> Artificial Sequence <220><223> Artificial Sequence Description : Synthetic Peptide <4〇〇> 12 GIu Va] Gin Uu Val Gly Ser Gly G) y Gly leu Val Gin Fro Gly Giy ϊ 5 10 15

Ser Leu Arg Uu Ser Cy$ Ala Ala Ser Oly Ptss Thr Phe Ser Ser km 20 25 30

Tyr IJe Scr Trp Vai Arg Gin Ala Pro 〇b Lys Gly Uu Glu Trp Val 35 40 45 GJy Cly Ik Asn Pro Thr Gly GSy T^r Thr Tyr Tyr Ala His Ser Vai 50 55 60

Lys Arg Thr Ue Ser Wa A Print Thr Scr Lys Ma Leu 65 70 75 g〇

G!^ ilet km $cr Leu Arg Ala Gl^i Asp Thr Ala Val Tyr Tyr Oj% Ala S5 90 9S

Ar, Ser ^ Pha Asp ^ Tg Ciy Gly ^ <210> 13 <2Π> Π0 <2]2> PRT <2]3> Artificial sequence -15-139862.doc 200950808 <220>Θ23> Sequence Description: Synthetic Peptide <m> 13

Glu Vai GIs Leu Val 〇]» Ser G)y Gly G!y Leu Val 0in Pro Gly Gly 1 5 10 15

Ser Lea Atg ku Ser Cys Ala Ala Ser Gly Phe Thr Rie Tttr Asit Ser 20 25 30

Tyr He Ser Trp Va] Arg OSn Ala Pro Gly Lys Gly leu Ciu Trp Va! 35 40 45 G1y Glv lie Ser Pro Ser GSy Gly Ser Thr Tyr Tyr Ala Asp Ser Val 5€' S5 60

Lys Arg Phe Thr lie Ssr A!a Asp Thr Ser Lys Asn Tbr Ala Tyr 65 70 75 SO t^uGl, MetAsn |r Uu Arg Ala ON Af Br Ala Val Tp T|t Cys

Ala lys Ser Vai Trp Ala Btc Ala Tyr Trp Gly Cln Giy Thr 100 105 110 <210> 14 <211> 108 <212> PRT <2!3> Homo sapiens <4(K)> 14

Asp lie Gin Met Thr G!b Scr Pro Scr Ser Uu Ser Ala Scr Val Gly 1 5 10 IS

Asp Arg Val Hir He Utr Cys Arg Als Ser GJn Ser lie See Asn Tyi 20 25 30 L€u Ala trp Tyr Gin Gin Lys Pro Cly Lvs Ala Pro Lys Leu Leu fie 3S 40 ' 45

Tyr Ala ^Ja Ser Ser Leu Gius Ser Qly Val Pro Ssr Arf Phc Scr Gly 50 55 60

Ser Gly Ser Gly Thr Asp Pfec Thr Leu Thr lie Ser Sir Leu Girt Pro 65 70 7$ 80

Giu k%p File Ala Thr Tyr Tyr Cys Gin Gin Tyr Asn Ser Leu Pro Trp 85 90 95

Tiir Phe Gly Gin Gly ITir Lys Val Glu He Lys Arg 100 105 <2ΐϋ> 1$ <21i> m <212> mt <213>Artificial Sequence <320>

<223> Artificial sequence description: synthetic polypeptide <400> IS •16- 139862.doc 200950808

Asp He Gin Met Tht Gin Ser Pro Ser Ser Lea Ser Ais Ser VaJ Oly } 5 10 15

Asp Arf Val Hr lie Thr Arg Ala Ser Qlti Asp Va! Ser Thr Ala 2ϋ 25 30

Val Ala Trp Tyr Gin Gin Lys Pro Oly Lys Ala Pro Lys Leu Leu lie 35 40 45

Tyr Sef Als Ser Hie Leu Tyr Ser Gly Val Pro Ser Arg Fhe Ser G)y 50 55 60

Ser Gly Ser Gly Hir Asp Phe Thr Leu TTir lie Scr Scr Leu Gin Pro 65 70 15 80

Glu Asp Pbe AU Thr T>r Tyr Cp Gin Gin Ser Tyr Thr Tlir Pro Pro 85 90 95

Thr Phe Gly Gin Gly Iht Lys Va! 〇]u lie Lys Arg just 105 e -17- 139862.doc

Claims (1)

200950808 VII. Patent Application Range: 1 · An isolated anti-PirB/LILRB antibody that binds to the same epitope on human PirB (LILRB) as an antibody selected from the group consisting of YW259.2, YW259.9 and YW259.12 ( Episode). 2. An isolated anti-PirB/LILRB antibody that competes with human PirB (LILRB) in competition with an antibody selected from the group consisting of YW259.2, YW259.9 and YW25 9.12. 3. An isolated anti-PirB/LILRB antibody comprising one, two or three of the hypervariable region sequences from a heavy chain selected from the group consisting of: YW259.2 heavy chain (SEQ ID NO: 4 or 11) ), YW259.9 heavy chain (SEQ ID NO.. 5 or 12), and YW259.12 heavy chain (SEQ ID NO: 6 or 13). 4. The antibody of claim 3, wherein the antibody comprises all of the hypervariable region sequences of the YW259.2 antibody heavy chain (SEQ ID NO.. 4 or 11). 5. The antibody of claim 3, wherein the antibody comprises all of the hypervariable region sequences of the YW259.9 antibody heavy chain (SEQ ID NO: 5 or 12). 6. The antibody of claim 3, wherein the antibody comprises all of the hypervariable region sequences of the YW259.12 antibody heavy chain (SEQ ID NO: 6 or 13). The antibody of any one of claims 3 to 6, which additionally comprises a light chain. 8. The antibody of claim 7, wherein the light chain comprises one, two or three. The hypervariable sequence of the polypeptide sequence of SEQ ID NO: 15. 9. The antibody of claim 7, wherein the light chain comprises all of the hypervariable region sequences of the SEQ ID NO: 7 or 15 polypeptide sequence. 10. The antibody of claim 3, which is selected from the group consisting of antibodies YW259.2, YW 259.9, and YW259.12. 139862.doc 200950808 11. An isolated anti-PirB/LILRB antibody, wherein the eight forms of the antibody specifically bind to human p B (LILRB) with a binding affinity of 5 nM or greater. 12. An isolated anti-PirB/LILRB antibody, wherein the full length form of the antibody specifically binds to human pirB (LILRB) with a binding affinity of 1 nM or greater. 13. The antibody of any one of claims 1 to 6 and 9 to 12 which promotes axonal regeneration. 14. The antibody of any one of claims 1 to 6 and 9 to 12 which promotes regeneration of the cns god. The antibody according to any one of claims 1 to 6 and 9 to 12, which at least partially rescues neurite outgrowth caused by Nog 〇 66 and medullary lipid. 16. The antibody of any one of claims 6 to 9 to 12, wherein the anti-system early antibody. The antibody according to any one of claims 6 to 9 wherein the anti-system is selected from the group consisting of chimeric antibodies, humanized antibodies, affinity matured antibodies, human antibodies, and bispecific Sexual antibodies. The antibody according to any one of claims 1 to 9 wherein the anti-system antibody fragment. 19. The antibody of any one of clauses 1 to 6 and 9 to 12, wherein the anti-system immunoconjugate. 20. The genus of the genus, which is the antibody of any one of the items 12 or the heavy or light key thereof. 21. A vector comprising 'polynucleic acid as claimed in claim 2'. 139862.doc -2- 200950808 22. The carrier of claim 21 wherein the vector is a representation vector. 23. A host cell 'which comprises the vector of claim 21. 24. The host cell of claim 23, wherein the host cell is a prokaryotic cell. 25. The host cell of claim 23, wherein the host cell is a eukaryotic cell. 26. The host cell of claim 25, wherein the host cell is a mammalian cell. 27. A method of making an anti-PirB/ULRB antibody, the method comprising (a) rendering the vector of claim 22 in a suitable host cell, and recovering the antibody. 28. The method of claim 27, wherein the host cell is a prokaryotic cell. 29. The method of claim 27, wherein the host cell is a eukaryotic cell. A composition comprising the anti-PirB/LILRB antibody of any one of claims 1 to 12 and a pharmaceutically acceptable excipient. 31. The composition of claim 3, wherein the composition further comprises a second drug, wherein the anti-PirB/LILRB anti-system first drug. Φ 32. The composition of claim 31, wherein the second drug is a NgR inhibitor. 33. The composition of claim 32, wherein the NgR inhibitor is an anti-NgR antibody. 34. A kit comprising an anti-degradation/apos. lilrb antibody as claimed in any one of claims M2. 35. An anti-PirB/LILR system for use in any of the items of the invention, for use in the manufacture of a medicament for promoting axonal regeneration in an individual in need thereof. 36. The use of claim 35, wherein the system is a human patient. 139862.doc 200950808 37. In the case of claim 36, which promotes the survival of neurons. 38. The use of claim 36, wherein the growth of the neuron is induced. 39. Use of an anti-pirB/LILR system according to any one of claims -12, for the manufacture of a medicament for treating a neurodegenerative disease in an individual in need thereof. 40. The use of claim 39, wherein the neurodegenerative disease is characterized by physical damage to the central nervous system. 41. The use of claim 40, wherein the neurodegenerative disease is a brain injury associated with a stroke. 42. The use of claim 39, wherein the neurodegenerative disease is selected from the group consisting of: tri-and neuralgia, glossopharyngeal neuralgia, Bell (sell), myasthenia gravis, muscular dystrophy, muscle Atrophic lateral sclerosis (ALS), multiple sclerosis (MS), progressive muscular atrophy, progressive medullary hereditary muscular atrophy, physical injury (eg burns, trauma) or disease states such as diabetes, renal dysfunction, or Toxic effects of chemotherapy for the treatment of cancer and Ams cause peripheral nerve injury, prominent, ruptured, or prolapsed disc syndrome, cervical spondylosis, plexus disorders, thoracic outlet destruction syndrome, peripheral neuropathy (eg, by lead, amine) DipSone, ticks, porphyria, Gullain-Barre syndrome, Alzheimer's disease, Huntingt〇n, s sease), and Parkinson's disease (parkinson, s disease). 43. An anti-idiotype antibody which specifically binds to an anti-PirB/LILRB antibody according to any one of claims 12. 139862.doc