KR20110011676A - Anti-pirb antibodies - Google Patents

Abstract

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

Description

Anti-PiRB antibody {ANTI­PirB ANTIBODIES}

The present invention relates generally to neural development and nervous system disorders. The present invention specifically relates to the identification of novel modulators of myelin-associated suppression systems and the various uses of such modulators so identified.

Myelin and Myelin-Related Proteins

Axons in adult mammalian CNS neurons are very limited in their ability to regenerate after injury, while axons in the peripheral nervous system (PNS) are known to regenerate rapidly. The limited regenerative capacity of CNS neurons is known in part because of the inherent properties of CNS axons, but also due to unacceptable circumstances. CNS myelin is not the only source of inhibitory signals for neurite growth, but contains numerous inhibitory molecules that actively block axon growth and thus constitute a significant barrier to regeneration. Three such myelin-associated proteins (MAPs) have been identified: Nogo (also known as NogoA) is a member of the Reticulon family protein with two transmembrane domains, and myelin-associated glycoprotein (MAG) is Ig A transfamily transmembrane protein, OMgp is a leucine rich repeat (LRR) protein with a glycosylphosphatidylinositol (GPI) anchor. Chen et al., Nature 403: 434-39 (2000); Grand Pre et al., Nature 417: 439-444 (2000); Prinjha et al., Nature 403: 383-384 (2000); McKerracher et al., Neuron 13: 805-11 (1994); Wang et al., Nature 417: 941-4 (2002): Kottis et al., J. Neurochem 82: 1566-9 (2002). Nogo66, part of NogoA, has been described as a 66 amino acid extracellular polypeptide found in all three isotypes of Nogo.

Despite structural differences, all three inhibitory proteins (including Nogo66) were found to bind to the same GPI-anchor receptor (NgR; also known as Nogo receptor-1 or NgR1) called the Nogo receptor, and NgR was Nogo, MAG It has been suggested that it may be necessary to mediate the inhibitory action of OMgp. Fournier et al., Nature 409: 341-346 (2001). Two NgR1 homologues (NgR2 and NgR3) were also identified. US 2005/0048520 A1 (Strittmatter et al.), Published March 3, 2005. Given that NgR is a GPI-anchor cell surface protein, it will not be a direct signal transducer (Zheng et al., Proc. Natl. Acad. Sci. USA 102: 1205-1210 (2005)). It has been suggested that the neurotropin receptor p75 ^NTR acts as a co-receptor for NgR and provides a signal-transforming 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 ortholog

Major histocompatibility complex (MHC) class I was originally identified as a region encoding an important molecular family in the immune system. Recent evidence has shown that MHC class I molecules have additional functions in developmental and adult CNS. Boulanger and Shatz, Nature Rev Neurosci. 5: 521-531 (2004); US 2003/0170690 (Shatz and Syken) (published September 11, 2003). Many MHC class I members and their binding partners have been found to be expressed in CNS neurons. Recent genetic and molecular studies have focused on the physiological function of CNS MHC class I, and early results indicate that long-term structural changes to the circuit are followed by an increase or decrease in the strength of existing synaptic connections in response to neuronal activity. It was suggested that phosphorus activity-dependent synaptic plasticity may be accompanied by MHC class I molecules. Moreover, the MHC class I coding region is also genetically linked to a wide range of disorders with neurological symptoms, and the abnormal function of MHC class I molecules is believed to contribute to normal brain development and destruction of plasticity.

One of the known MHC class I receptors in the immune environment is described by Kubagawa et al., Proc. Nat. Acad. Sci. USA 94: 5261-6 (1997), PirB, the first murine pol peptide described. Mouse PirB has several human orthologs, which are members of the leukocyte immunoglobulin-like receptor, subfamily B (LILRB), and are also referred to as "immunoglobulin-like transcripts" (ILTs). Human orthologs show significant homology to the murine sequence in the order of highest to lowest LILRB3 / ILT5, LILRB1 / ILT2, LILRB5 / ILT3, LILRB2 / ILT4, and are all inhibitory receptors, like PirB. LILRB3 / ILT5 (NP_006855) and LILRB1 / ILT2 (NP_006660) are described in Samaridis and Colonna, Eur. J. Immunol. 27 (3): 660-665 (1997). LILRB5 / ILT3 (NP_006831) is described by Berges et al., J. Immunol. 159 (11): 5192-5196 (1997). LILRB2 / ILT4 (also known as MIR10) is described by Colonna et al., J. Exp. Med. 186: 1809-18 (1997). PirB and its human orthologs exhibit a high degree of structural variability. Various alternatively spliced forms of sequences are available from EMBL / GenBank and include, for example, the following registration numbers for human ILT4 cDNA: ILT4-c11 AF009634; ILT4-c117 AF11566; ILT4-c126 AF11565. As noted above, PirB / LILRB polypeptides are MHC class I (MHCI) inhibitory receptors and their role in regulating immune cell activation is known (Kubagawa et al., Supra); Hayami et al. , J. Biol. Chem. 272: 7320 (1997); Takai et al., Immunology 115: 433 (2005); Takai et al., Immunol. Rev. 181: 215 (2001); Nakamura et al., Nat. Immunol. 5: 623 (2004); Liang et al., Eur. J. Immunol. 32: 2418 (2002)).

A recent study by Syken et al. (Science 313: 1795-800 (2006)) reported that PirB is expressed in a subset of neurons throughout the brain. In mutant mice lacking functional PirB, cortical eye dominance (OD) plasticity is significantly enhanced at all ages, suggesting the ability of PirB to limit activity-dependent plasticity in the visual cortex.

The present invention provides that at least in part, interfering with the activity of PirB using a function-blocking anti-PirB antibody deviates from neurite growth inhibition by Nogo66 and myelin, and simultaneously blocking PirB and NgR activity from myelin inhibition. It is based on the finding of almost total deviation.

In one aspect, the invention relates to an isolated anti-PirB / LILRB antibody which essentially 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.

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 present invention provides a kit for 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); An isolated anti-PirB / LILRB antibody comprising two, or three hypervariable region sequences.

In one embodiment, the antibody comprises all hypervariable region sequences of the YW259.2 antibody heavy chain (SEQ ID NO: 4 or 11).

In another embodiment, the antibody comprises all hypervariable region sequences of the YW259.9 antibody heavy chain (SEQ ID NO: 5 or 12).

In another embodiment, the antibody comprises all hypervariable region sequences of the YW259.12 antibody heavy chain (SEQ ID NO: 6 or 13).

In further embodiments, the antibody comprises a light chain.

In yet further embodiments, the antibody comprises one, two or three hypervariable region sequences of the light chain from the polypeptide sequence of SEQ ID NO: 7.

In another embodiment, the antibody comprises all hypervariable region sequences of the light chain comprising the polypeptide sequence of SEQ ID NO: 7 or 15.

In certain embodiments, the antibody comprises both heavy and light chains, wherein the heavy chain is 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) comprises one, two, or three hypervariable region sequences in the heavy chain selected from the group consisting of 13) and / or the light chain comprises one, two or three hypervariable region sequences of the light chain from the polypeptide sequences of SEQ ID NO: 7 or 15. do.

In further embodiments, the antibody is selected from the group consisting of antibody YW259.2, YW259.9, and YW259.12.

In a further aspect, the invention relates to an isolated anti-PirB antibody wherein the full length IgG form of the antibody specifically binds human PirB with a binding affinity of at least 5 nM, or at least 1 nM.

In one embodiment, the antibody promotes axon regeneration, such as regeneration of CNS neurons.

In another embodiment, the antibody is at least partially out of neurite growth inhibition by Nogo66 and myelin.

In all aspects, the antibody is preferably a monoclonal antibody, and may be, for example, a chimeric antibody, humanized antibody, affinity matured antibody, human antibody or bispecific antibody, antibody fragment or immunoconjugate.

In a further aspect, the present invention relates to a polynucleotide encoding an anti-PirB / LILRB antibody herein.

In another aspect, the invention relates to vectors and host cells comprising a polynucleotide encoding an antibody of the present disclosure (including the coding sequence of one or more antibody chains). Host cells include prokaryotes, eukaryotes and mammalian hosts.

In a further aspect, the invention provides an anti-PirB / LILRB antibody comprising (a) expressing a vector comprising a nucleic acid encoding an antibody in a suitable host cell, and (b) recovering the antibody. It is about a method.

In yet a further aspect, the present invention relates to a composition comprising an anti-PirB / LILRB antibody herein and a pharmaceutically acceptable excipient. Optionally, the composition comprises a second medicament, wherein the anti-PirB / LILRB antibody is the first medicament. The second medicament can be, for example, an NgR inhibitor such as an anti-NgR antibody.

In various aspects, the present invention relates to a kit comprising an anti-PirB / LILRB antibody herein.

In another aspect, the invention relates to a method for promoting axon regeneration comprising administering an effective amount of an anti-PirB / LILRB antibody of the present disclosure to a subject in need thereof. Preferably, the subject is a human patient.

In embodiments, the methods of treatment herein improve the survival of neurons and / or induce the growth of neurons.

In another aspect, the invention relates to a method of treating a neurodegenerative disease, comprising administering an effective amount of an anti-PirB / LILRB antibody herein to a subject in need thereof. Neurodegenerative diseases can be characterized, for example, by physical damage to the central nervous system and include, but are not limited to, brain damage associated with stroke.

In certain embodiments, the neurodegenerative disorders include trigeminal neuralgia, Yeti neuralgia, Bell's Palsy, Myasthenia Gravis, Muscular Dystrophy, Amyotrophic Lateral Sclerosis (ALS), Multiple Sclerosis (MS), Progressive Muscular Dystrophy, Progressive Training Atrophy, Physical Atrophy Peripheral nerve injury, intervertebral hernia, rupture, or escape invertebrae caused by injury (eg burns, wounds) or disease states such as diabetes, renal dysfunction or toxic effects of chemotherapeutic agents used to treat cancer and AIDS Disc syndrome, cervical spondylosis, plexus disorders, thoracic outlet destruction syndrome, lead neuropathy, such as caused by lead, tadpoles, ticks, porphyrins, Gullain-Barre syndrome, Alzheimer's disease, Huntington's disease, and Parkinson's disease It is selected from the group consisting of.

The present invention further relates to anti-idiotype antibodies that specifically bind to anti-PirB antibodies herein.

1A and 1B show the mouse PirB sequence (SEQ ID NO: 1) and human LILRB2 sequence (SEQ ID NO: 2).
2A and 2B. Blocking PirB reverses CGN growth inhibition on AP-Nogo66 or myelin. Isolated mouse P7 CGN was plated on PDL / laminin (control), AP-Nogo66, or myelin to test inhibition by these substrates. (A) Representative photomicrographs, (B) Graph measuring average neurite length (± SE) from one representative experiment. Neurons grown on PDL / laminin, AP-Nogo66, or myelin were cultured in the presence or absence of a function-blocking antibody against PirB (aPB1; 50 μg / ml). aPB1 significantly reduced the inhibition by the substrate. (* p <0.01; scale bar, 50 μm).
3A-3D. Blocking PirB reverses CGN growth inhibition on AP-Nogo66 or myelin. Isolated mouse P7 CGN was plated on PDL / laminin (control), AP-Nogo66, or myelin to test inhibition by these substrates. Representative micrographs are shown in FIGS. 3A and 3C and graphs measuring average neurite length (± SE) from one representative experiment are shown in FIGS. 3B and 3D. Neurons grown on PDL / laminin, AP-Nogo66, or myelin were cultured in the presence or absence of a function-blocking antibody against PirB (aPB1; 50 μg / ml). aPB1 significantly reduced the inhibition by the substrate. (* p <0.01; scale bar, 50 μm).
4A and 4B. PirB and NgR are both required to mediate growth cone collapse by myelin inhibitors. Post-natal DRG axon growth cones were treated with medium alone (control), myelin (3 μg / ml), or AP-Nogo66 (100 nM) for 30 minutes to stimulate disintegration, and stained with rhodamine-palloydine to grow The cone was visualized. (A) Representative micrograph, (B) Graph of measuring growth cone collapse rate (± SEM) from cumulative experiments. Inhibition of PirB by genetic loss of NgR or by anti-PirB treatment alone is sufficient to prevent growth cone disruption activity of myelin or AP-Nogo66. In addition, inhibition of both pathways completely blocks the collapse. (Scale bar, 50 μm).
Figures 5A-5D. Blocking PirB partially desuppresses neurite outgrowth in DRG neurons and on matrix MAG. Representative micrographs are shown in FIGS. 5A and 5C; Graphs showing mean neurite length (± SE) from one representative experiment are shown in FIGS. 5B and 5D. (A) and (B) Separated P10 DRG neurons were plated on PDL / laminin, AP-Nogo66, or myelin in the presence or absence of anti-PirB. With aPB1, inhibition by AP-Nogo66 and myelin was significantly reduced. (C) and (D) Separated P7 CGN cultures were plated on PDL / laminin or MAG-Fc with or without aPB1. Antibodies against PirB reduced neurite outgrowth by MAG-Fc. (* p <0.01; scale bar, 200 μm A, B; 50 μm C).
6. DNA sequence of anti-PirB antibody YW259.2 heavy chain (SEQ ID NO: 8).
7. DNA sequence of anti-PirB antibody YW259.9 heavy chain (SEQ ID NO: 9).
8. DNA sequence of anti-PirB antibody YW259.12 heavy chain (SEQ ID NO: 3).
9. Protein sequence of anti-PirB antibody YW259.2 heavy chain (SEQ ID NO: 4).
10. Protein sequence of anti-PirB antibody YW259.9 heavy chain (SEQ ID NO: 5).
11. Protein sequence of anti-PirB antibody YW259.12 heavy chain (SEQ ID NO: 6).
12. Protein sequence of the light chain of all YW259 antibodies (SEQ ID NO: 7).
13. Ability to inhibit the activity of His-tagged mouse PirB of anti-PirB antibody YW259.2 (IgG).
14. Ability to inhibit the activity of His-tagged mouse PirB of anti-PirB antibody YW259.9 (IgG).
15. Ability to inhibit the activity of His-tagged mouse PirB of anti-PirB antibody YW259.12 (IgG).
16. Relative AP-Nogo66 binding of anti-PirB antibody panels, including YW259.2, YW259.9, and YW259.12.
17A-17C. Anti-PirB antibody YW259.2 (SEQ ID NO: 11); Alignment of the heavy chain sequences of YW259.9 (SEQ ID NO: 12) and YW259.12 (SEQ ID NO: 13). CDR H1, CDR H2 and CDR H3 sequences are boxed together with the CDR H domains according to the Kabat, Chothia and contact CDR H domains. Hum III is disclosed as SEQ ID NO: 10.
18A-18C. Anti-PirB antibody YW259.2 (SEQ ID NO: 15); Alignment of the light chain sequences of YW259.9 (SEQ ID NO: 15) and YW259.12 (SEQ ID NO: 15), and HuκI (SEQ ID NO: 14). CDR L1, CDR L2 and CDR L3 sequences are boxed together with the CDR L domains according to the Kabat, Chothia and contact CDR L domains. Hum III is disclosed as SEQ ID NO: 10.
19. C1QTNF5 (CTRP5; NP_05646) inhibits neurite outgrowth of dorsal root ganglion neurons, and this inhibition is reduced when PirB is blocked by PirB function-blocking antibody YW259.2.

Justice

The terms "paired immunoglobulin-like receptor B" and "PirB" are used interchangeably herein and include the native-sequence of SEQ ID NO: 1 (FIG. 1) (NP_035225), 841-amino acid mouse inhibitory protein, and rats and other. It refers to its naturally-sequential homologs in non-human mammals, including all naturally occurring variants such as alternatively spliced variants and allelic variants and isoforms, as well as soluble forms thereof. For further details, see Kubagawa et al., Proc Natl Acad Sci USA 94, 5261 (1997).

The terms "LILRB", "ILT" and "MIR" are used interchangeably herein and refer to all members of the human "leukocyte immunoglobulin-like receptor, subfamily B", including all naturally occurring variants such as stars. Legally spliced variants and allelic variants and isoforms, as well as soluble forms thereof. Individual members within the B-type subfamily of such LILR receptors are designated by numbers following the acronyms such as LILRB3 / ILT5, LILRB1 / ILT2, LILRB5 / ILT3, and LILRB2 / ILT4, with reference to any individual member Unless otherwise indicated, it also includes all naturally occurring variants, such as alternatively spliced variants and allelic variants and isoforms, as well as soluble forms thereof. Thus, for example, "LILRB2", "LIR2", and "MIR10" are used interchangeably herein, and the 598-amino acid polypeptide of SEQ ID NO: 2 (FIG. 1) (NP_005865), and naturally occurring variants thereof, such as alternatives Splice variants and allelic variants and isoforms, as well as soluble forms thereof. For further details, see Martin et al., Trends Immunol. 23, 81 (2002).

The term “PirB / LILRB” is used herein to refer congruently to native sequence homologues in corresponding mouse and human proteins and other non-human mammals, where all naturally occurring variants, such as alternatively spliced Variants and allelic variants and isoforms, as well as soluble forms thereof.

The term “myelin-related protein” is used in its broadest sense and includes all proteins present in CNS myelin that inhibit neuronal regeneration, including Nogo, MAG and OMgp.

“Isolated”, when used to describe the various proteins disclosed herein, means proteins that have been identified and isolated and / or recovered from components of their natural environment. Contaminant components of its natural environment are materials that will typically interfere with diagnostic or therapeutic uses for proteins, which may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In a preferred embodiment, the protein is sufficient to (1) obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a spinning cup sequencer, or (2) Coomassie blue or preferably silver staining. It will be purified to homogeneity by SDS-PAGE under reducing or non-reducing conditions using or (3) homogeneity by mass spectrometry or peptide mapping techniques. Isolated protein includes proteins in situ within recombinant cells, since at least one component of the protein's natural environment will not be present. Ordinarily, however, isolated protein will be prepared by one or more purification steps.

An “isolated” nucleic acid molecule is a nucleic acid molecule identified and separated from one or more contaminating nucleic acid molecules that are typically associated within the natural source of the nucleic acid in question. Isolated nucleic acid molecules are other than in the form or environment found in nature. Thus, isolated nucleic acid molecules are distinguished from nucleic acid molecules present in natural cells. However, isolated nucleic acid molecules include nucleic acid molecules contained within cells that normally express such nucleic acid, for example when the nucleic acid molecule is at a chromosomal location different from that of the natural cell.

As used herein, the term “PirB / LILRB antagonist” is used to refer to an agent that can block, neutralize, inhibit, discard, reduce or interfere with PirB / LILRB activity. In particular, PirB / LILRB antagonists interfere with myelin related inhibitory activity, thereby improving neurite growth and / or promoting neuronal growth, repair and / or regeneration. In a preferred embodiment, the PirB / LILRB antagonist inhibits the binding of PirB / LILRB to Nogo66 and / or MAG and / or OMgp by binding to PirB / LILRB. PirB / LILRB antagonists can, for example, sequester binding to antibodies against PirB / LILRB and antigen-binding fragments thereof, between PirB / LILRB and Nogo 66, or between PirB / LILRB and MAG, or between PirB / LILRB and OMgp. Truncated or soluble fragments of PirB / LILRB, Nogo 66, MAG or OMgp which are present and small molecule inhibitors of PirB / LILRB related inhibitory pathways. PirB / LILRB antagonists also include short interfering RNA (siRNA) molecules that can inhibit or reduce the expression of PirB / LILRB mRNA. Preferred PirB / LILRB antagonists are anti-PirB / LILRB antibodies.

The term "antibody" as used herein is used in its broadest sense and specifically intact, monoclonal antibody, polyclonal antibody, multispecific formed from two or more intact antibodies, so long as they exhibit the desired biological activity. Antibodies (eg bispecific antibodies), and antibody fragments.

As used herein, the term “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies that make up the population are identical except for possible naturally occurring mutations that may be present in trace amounts. . Monoclonal antibodies are highly specific for a single antigenic site. In addition, unlike polyclonal antibody preparations that include different antibodies against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on an antigen. In addition to its specificity, monoclonal antibodies are advantageous in that they can be synthesized without being contaminated with other antibodies. The modifier “monoclonal” characterizes the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the preparation of the antibody in any particular manner. For example, monoclonal antibodies to be used in accordance with the present invention can be prepared by the hybridoma method first described in Kohler et al., Nature, 256: 495 (1975), or recombinant DNA methods (eg For example, US Pat. No. 4,816,567). “Monoclonal antibodies” are also described, eg, in Clackson et al., Nature, 352: 624-628 (1991) and in Marks et al., J. Mol. Biol. 222: 581-597 (1991). )] Can be isolated from phage antibody libraries using the techniques described.

Antibodies specifically include a portion of the heavy and / or light chain, as long as they exhibit the desired biological activity, while the chain is identical or homologous to the corresponding sequence of an antibody derived from a particular species or an antibody belonging to a particular antibody class or subclass, The remainder of the (s) includes "chimeric" antibodies and fragments of such antibodies that are identical or homologous to the corresponding sequence of an antibody derived from another species or an antibody belonging to another antibody class or subclass (US Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). Chimeric antibodies of interest herein include primatized antibodies comprising variable domain antigen-binding sequences and human constant region sequences derived from non-human primates (eg, Old World Monkey, apes, etc.). do.

"Antibody fragments" comprise a portion of an intact antibody, and preferably comprise an antigen-binding region or variable region thereof. Examples of antibody fragments include Fab, Fab ', F (ab') 2 and Fv fragments; Diabody; Linear antibodies; Single chain antibody molecules; And multispecific antibodies formed from antibody fragment (s).

An "intact" antibody is one comprising an antigen-binding variable region, as well as a light chain constant domain (C _L ) and heavy chain constant domains C _H 1, C _H 2 and C _H 3. The constant domains may be native sequence constant domains (eg human native sequence constant domains) or amino acid sequence variants thereof. Preferably, the intact antibody has one or more effector functions.

A “humanized” form of a non-human (eg rodent) antibody is a chimeric antibody containing a minimal sequence derived from a non-human immunoglobulin. In most cases, humanized antibodies are those of non-human species (donor antibodies) such as mice, rats, rabbits or non-human primates whose residues from the hypervariable region of the recipient have the desired specificity, affinity and ability. Human immunoglobulin (conferring antibody) replaced with a residue from the variable region. In some cases, framework region (FR) residues of human immunoglobulins are replaced with corresponding non-human residues. Humanized antibodies may also include residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. In general, humanized antibodies will comprise substantially all of one or more, typically two, variable domains (Fab, Fab ', F (ab') ₂ , Fabc, Fv), where all or substantially all hypervariable loops are Corresponding to the hypervariable loops of non-human immunoglobulins, all or substantially all FRs are FRs of human immunoglobulin sequences. Humanized antibodies will also optionally include at least some immunoglobulin constant regions (Fc), typically the constant regions of human immunoglobulins. For more details, see 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).

As used herein, the term “hypervariable region” refers to a region of an antibody variable domain that is hypervariable in sequence and / or forms a structurally defined loop. Hypervariable regions include amino acid residues from “complementarity determining regions” or “CDRs” (ie, residues 24-34, 50-56 and 89-97 in the light chain variable domain and 31-35, 50-65 and 95 in the heavy chain variable domain). -102; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or such residues from "hypervariable loops". (Ie residues 26-32, 50-52 and 91-96 in the light chain variable domain and 26-32, 53-55 and 96-101 in the heavy chain variable domain; Chothia and Lesk J. Mol. Biol. 196: 901 -917 (1987)). In both cases, variable domain residues are numbered according to Kabat et al., Supra, as discussed in more detail below. “Framework” or “FR” residues are those variable domain residues other than those in the hypervariable regions as defined herein.

A “parent antibody” or “wild type” antibody is an antibody comprising an amino acid sequence that does not have one or more amino acid sequence changes compared to the antibody variants disclosed herein. Thus, parental antibodies generally have one or more hypervariable regions that differ in terms of amino acid sequence and amino acid sequence of the corresponding hypervariable region of the antibody variant as disclosed herein. Parental polypeptides may include native sequences (ie, naturally occurring) antibodies (including naturally occurring allelic variants), or antibodies with pre-existing amino acid sequence modifications (eg, insertions, deletions, and / or other alterations) of the naturally occurring sequences. Throughout the specification, “wild type”, “WT”, “wt”, and “parental” or “parental” antibodies are used interchangeably.

As used herein, “antibody variant” or “variant antibody” refers to an antibody whose amino acid sequence differs from the amino acid sequence of the parent antibody. Preferably, the antibody variant comprises a heavy chain or light chain variable domain having an amino acid sequence not found in nature. Such variants necessarily have less than 100% sequence identity or similarity with the parent antibody. In a preferred embodiment, the amino acid sequence of the antibody variant has about 75% to less than 100% amino acid sequence identity or similarity with the amino acid sequence of one of the heavy or light chain variable domains of the parent antibody, more preferably about 80% to 100% Less than%, more preferably from about 85% to less than 100%, more preferably from about 90% to less than 100%, and most preferably from about 95% to less than 100%. In general, antibody variants are those comprising one or more amino acid alterations within or adjacent to one or more hypervariable regions thereof.

"Amino acid alteration" refers to a change in the amino acid sequence of a predetermined amino acid sequence. Exemplary alterations include insertions, substitutions, and deletions. "Amino acid substitution" refers to the replacement of an existing amino acid residue within a predetermined amino acid sequence with another different amino acid residue.

"Alternative" amino acid residues refer to amino acid residues that replace or substitute for other amino acid residues in the amino acid sequence. Alternative residues may be naturally occurring amino acid residues or non-naturally occurring amino acid residues.

"Amino acid insertion" refers to the introduction of one or more amino acid residues into a predetermined amino acid sequence. Amino acid insertions may include “peptide insertions” where a peptide comprising two or more amino acid residues joined by peptide bond (s) is introduced into a predetermined amino acid sequence. If amino acid insertion involves the insertion of a peptide, the inserted peptide may be generated by random mutagenesis to have an amino acid sequence that does not exist in nature. An amino acid alteration “adjacent to the hypervariable region” may result in one or more amino acid residues being hypervariable such that at least one of the insertion or replacement amino acid residue (s) forms a peptide bond with the N-terminal or C-terminal amino acid residue of the hypervariable region in question. Refers to being introduced or substituted at the N-terminal and / or C-terminal end of the region.

“Naturally occurring amino acid residues” include alanine (Ala); Arginine (Arg); Asparagine (Asn); Aspartic acid (Asp); Cysteine (Cys); Glutamine (Gln); Glutamic acid (Glu); Glycine (Gly); Histidine (His); Isoleucine (Ile): Leucine (Leu); Lysine (Lys); Methionine (Met); Phenylalanine (Phe); Proline (Pro); Serine (Ser); Threonine (Thr); Tryptophan (Trp); Tyrosine (Tyr); And valine (Val), which is generally encoded by a genetic code.

A “non-naturally occurring amino acid residue” herein is an amino acid residue other than the naturally occurring amino acid residues listed above capable of covalently binding to adjacent amino acid residue (s) in a polypeptide chain. Examples of non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine, and other amino acid residue analogues, such as Ellman et al., Meth. Enzym. 202: 301-336 (1991). To generate such non-naturally occurring amino acid residues, the procedures of Noren et al., Science 244: 182 (1989) and Ellman et al., Supra can be used. Briefly, these procedures include chemically activating inhibitor tRNA with non-naturally occurring amino acid residues, followed by in vitro transcription and translation of RNA.

Throughout this disclosure, reference is made to the numbering system from Kabat, EA et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991). Kabat lists a plurality of amino acid sequences for antibodies of each subclass, and lists the most commonly occurring amino acids for each residue position in this subclass. This method of assigning residue numbers to each amino acid and assigning residue numbers has become a standard in the art, which follows the Kabat numbering scheme, for purposes of the present invention, not included in the Introduction to Kabat. To assign a residue number to a candidate antibody amino acid sequence that is not present, the following steps are generally followed: In general, the candidate sequence is subjected to any immunization in Kabat. Align with globulin sequences or any consensus sequences, manually or by computer using commonly accepted computer programs; examples of such programs are Align 2 programs. Some amino acid residues common to the Fab sequence of may facilitate alignment, for example light and heavy chains each have two cysteines which typically have the same residue number; in the V _L domain two cysteines typically Two cysteine residues are typically numbered at residues 23 and 88 and in the V _H domain are 22 and 92. Although not always, framework residues generally have approximately the same number of residues, but CDRs vary in size. For example, if the CDRs from the candidate sequences are longer than the CDRs of the sequences in the aligned Kabat, Typically, a subscript is added to the residue number to indicate the insertion of additional residues (see, eg, residue 100abc in Figure 1B), eg, for residues 34 and 36 aligned with the Kabat sequence, but with residue 35 between them. For candidate sequences without residues to be added, number 35 is not simply assigned to the residues.

As used herein, an antibody with "high affinity" is an antibody with a K _D or dissociation constant in the nanomolar (nM) range or greater. K _{D which} is “above the nanomolar range” may be represented by X nM, where X is a number less than about 10.

An “affinity matured” antibody is one that has one or more alterations in one or more CDRs thereof, with improved affinity of the antibody for antigen as compared to a parent antibody that does not carry the alteration (s). Preferred affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are prepared by procedures known in the art. Marks et al., Bio / Technology 10: 779-783 (1992) describe affinity maturation by V _H and V _L domain shuffling. Random mutagenesis of CDR and / or framework residues is described by: Barbas et al., Proc Nat. Acad. Sci, USA 91: 3809-3813 (1994); Chier et al., Gene 169: 147-155 (1995); Yelton et al., J. Immunol. 155: 1994-2004 (1995); Jackson et al., J. Immunol. 154 (7): 3310-9 (1995); And Hawkins et al., J. Mol. Biol. 226: 889-896 (1992).

The "functional antigen binding site" of an antibody is one capable of binding a target antigen. The antigen binding affinity of the antigen binding site does not necessarily have to be as strong as the parent antibody from which the antigen binding site is derived, but the ability to bind the antigen can be determined using any of a variety of methods known to assess antibodies that bind antigen. Should be measurable.

An antibody having the "biological characteristics" of a designated antibody possesses one or more biological characteristics of the antibody that distinguishes it from other antibodies that bind the same antigen.

For screening antibodies that bind epitopes on antigens to which the antibody of interest has bound, routine cross-blocking assays are described, such as in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988). Can be done.

The term “filamentous phage” refers to viral particles capable of displaying heterologous polypeptides on their surface, including but not limited to f1, fd, Pf1, and M13. The filamentous phage may contain a selectable marker such as tetracycline (eg "fd-tet"). Various filamentous phage display systems are well known to those skilled in the art (see, eg, Zacher et al., Gene 9: 127-140 (1980)), Smith et al., Science 228: 1315-1317 (1985). And Parley and Smith Gene 73: 305-318 (1988).

The term “panning” is used to refer to multiple screening processes in identifying and isolating phages carrying compounds, such as antibodies, having high affinity and specificity for a target.

The term “short-interfering RNA (siRNA)” refers to small double-stranded RNA that interferes with gene expression. siRNAs are mediators of RNA interference, a process in which double-stranded RNA silences homologous genes. siRNAs typically consist of two single-stranded RNAs of about 15-25 nucleotides in length, forming a duplex, which may comprise single-strand overhang (s). When double-stranded RNA is processed by an enzyme complex, such as a polymerase, the double-stranded RNA is cleaved to produce siRNA. Antisense strands of siRNA are used by RNA interference (RNAi) silencing complexes to promote mRNA cleavage, thereby promoting mRNA degradation. For example, to silence specific genes using siRNAs in mammalian cells, base pairing regions are selected to eliminate accidental complementarity to irrelevant mRNAs. RNAi silent complexes are known in the art, for example, in Fire et al., Nature 391: 806-811 (1998) and McManus et al., Nat. Rev. Genet. 3 (10): 737-47 (2002).

The term “interfering RNA (RNAi)” is used herein to refer to double-stranded RNA that results in catalytic degradation of a particular mRNA and thus can be used for inhibiting / lowering the expression of a particular gene.

The term “polymorphism” is used herein to refer to more than one form of a gene or portion thereof (eg an allelic variant). A portion of a gene that has two or more different forms is referred to as the "polymorphic region" of the gene. The particular gene sequence in the polymorphic region of the gene is an “allele”. Polymorphic regions may be different single nucleotides in different alleles, or may be several nucleotides in length.

As used herein, the term “disorder” generally refers to any condition that would benefit from treatment with an antagonist of PirB / LILRB2, such as an anti-PirB antibody, which may benefit from axon regeneration therapy and / or synaptic plasticity. Any state in the nervous system where improvement is expected is included. Non-limiting examples of disorders to be treated herein include, but are not limited to, neurological disorders such as physically impaired neurological and neurodegenerative diseases, including those that would benefit from improved neurite growth, neuronal growth, repair, or regeneration. Limited inclusion. Such disorders specifically include physical damage to the central nervous system (eg spinal cord injury and head trauma); Brain damage associated with stroke; And nervous system disorders associated with neurodegeneration, such as trigeminal neuralgia, Yeti neuralgia, Bell's palsy, myasthenia gravis, muscular dystrophy, amyotrophic lateral sclerosis (ALS), progressive atrophy, progressive medullary muscular dystrophy, multiple sclerosis (MS), intervertebral hernia, Rupture, or detachment Invertebrate disc syndrome, cervical spondylosis, plexus disorders, thoracic outlet destruction syndrome, peripheral neuropathy such as caused by physical damage or disease state such as peripheral nerve damage caused by diabetes mellitus, lead, hand loss, ticks, Porphyria, Guillain-Barré syndrome, Alzheimer's disease, Huntington's disease, or Parkinson's disease.

As used herein, the terms “treating”, “treatment” and “treatment” refer to healing, prophylactic, and prophylactic therapies. Continuous treatment or administration refers to treatment at least daily, without stopping treatment for more than one day. Intermittent treatment or administration, or intermittent treatment or administration, is not continuous, but in fact refers to periodic treatment.

As used herein, the term "anti-neurodegeneration" refers to (1) the ability to inhibit or prevent neurodegeneration in patients newly diagnosed with neurodegenerative disease or at risk of developing a new neurodegenerative disease and (2) neurodegenerative disease And the ability to inhibit or prevent further neurodegeneration in patients already suffering from or with symptoms of neurodegenerative disease.

As used herein, the term "mammal" refers to any mammal classified as a mammal, including humans, non-human higher primates, rodents, livestock and farm animals such as cattle, horses, dogs, and cats. In a preferred embodiment of the invention, the mammal is a human.

“Combination” administration with one or more additional therapeutic agents includes simultaneous (simultaneous) and sequential administration in any order.

An “effective amount” is an amount sufficient to produce a beneficial result or a desired treatment (including prevention). An effective amount can be administered in one or more administrations.

As used herein, the expressions "cell", "cell line" and "cell culture" are used interchangeably and all such names include progeny. Thus, the words “transformer” and “transformed cell” include primary subject cells and cultures derived therefrom, regardless of the number of times of delivery. In addition, it is understood that all progeny may not exactly match DNA content due to intentional or unintentional mutations. The term “progeny” refers to any and all descendants of all subsequent generations for the original transformed cell or cell line. Mutant progeny that have the same function or biological activity as screened for in the original transformed cell are included. If a separate name is intended, it will be apparent from the context.

The "% amino acid sequence identity rate" for a sequence identified herein refers to an amino acid in the candidate sequence that is identical to the amino acid residue in the reference sequence after introducing a gap to align the sequence and achieve a maximum sequence identity rate if necessary. As defined herein as a percentage of residues, any conservative substitutions are not considered part of the sequence identity. Alignment to determine the percentage of amino acid sequence identity is within the skill of the art in a variety of ways to determine appropriate parameters for measuring alignment, including the assignment of algorithms necessary to achieve maximal alignment over the full length of the sequences being compared. Can be achieved. For purposes of the present invention, amino acid identity rate values are Genentech, Inc. Owned by Genentech, Inc., whose source code is filed as a user submission in the US Copyright Office, Washington, DC, 20559 and registered under US Copyright No. TXU510087, ALIGN-2. Can be obtained using The ALIGN-2 program is Genentech, Inc. (South San Francisco, Calif., USA). All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

The "stringency" of the hybridization reaction can be readily determined by one skilled in the art and is generally calculated experimentally depending on probe length, wash temperature and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes require lower temperatures. Hybridization generally relies on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The greater the degree of identity required between the probe and the hybridizable sequence, the higher the relative temperature that can be used. As a result, higher relative temperatures tend to make the reaction conditions more stringent, while lower temperatures tend to be less stringent. For further details and explanations on the stringency of the hybridization reaction, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).

“High stringency conditions” as defined herein include (1) using low ionic strength and high temperature for washing; 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50 ° C .; (2) using denaturant during hybridization; 50% (v / v) formamide + 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer (pH 6.5) + 750 mM sodium chloride, 75 mM sodium citrate (42 ° C.); Or (3) 50% form with a high stringency wash consisting of 0.2 x SSC (sodium chloride / sodium citrate) at 42 ° C. and 50% formamide at 55 ° C. followed by 0.1 × SSC containing EDTA at 55 ° C. Amide, 5 × SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 × Denhardt solution, sonicated salmon sperm DNA (50 μg / ml), 0.1% SDS, and 10% dextran sulfate (42 ° C.).

“Moderate stringency conditions” can be identified as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, 20% formamide, 5 × SSC (150 mM). NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 × Denhardt's solution, 10% dextran sulfate, and 20 mg / ml denatured shear treated salmon sperm DNA at 37 ° C. Incubation overnight followed by washing the filter in 1 × SSC at about 37-50 ° C. Those skilled in the art will know how to adjust the temperature, ionic strength, and the like required to adjust factors such as probe length and the like.

The term "regulatory sequence" refers to the DNA sequence required for expression of a coding sequence operably linked in a particular host organism. Suitable control sequences for prokaryotes include, for example, promoters, optionally operator sequences, and ribosomal binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

A nucleic acid is "operably linked" when it is in a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide when expressed as a proprotein that participates in the secretion of the polypeptide; A promoter or enhancer is operably linked to a coding sequence when it affects the transcription of the sequence; Or the ribosome binding site, when positioned to facilitate translation, is operably linked to a coding sequence. In general, "operably linked" means that the linked DNA sequences are contiguous, in the case of a secretory leader, contiguous and in the reading phase. However, enhancers do not necessarily have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice.

A “small molecule” is defined herein as having a molecular weight of less than about 1000 Daltons, preferably less than about 500 Daltons.

B. Production of Anti-PirB / LILRB Antibodies

Anti-PirB antibodies of the invention can be produced by methods known in the art, including techniques of recombinant DNA technology.

i) antigen production

Soluble antigens or fragments thereof (optionally conjugated to other molecules) can be used as immunogens to generate antibodies. In the case of transmembrane molecules such as receptors, fragments thereof (eg the extracellular domain of the receptor) can be used as immunogens. Alternatively, cells expressing the transmembrane molecule can be used as the immunogen. Such cells may be derived from natural sources (eg cancer cell lines) or may be cells transformed by recombinant techniques to express the transmembrane molecule. Other antigens and forms thereof useful for the preparation of antibodies will be apparent to those skilled in the art.

(ii) polyclonal antibodies

Polyclonal antibodies can be produced in animals preferably by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant. Difunctional or derivatizing agents such as maleimidobenzoyl sulfosuccinimide esters (conjugation via cysteine residues), N-hydroxysuccinimide (conjugation via lysine residues), glutaraldehyde, succinic anhydride, SOCI ₂ , or R ₁ N = C = NR (wherein R, R and R ₁ are different alkyl group) was, immunogenic proteins in the species to be immunized using, for example, keyhole rimpet not H. ramie, serum albumin, bovine thyroglobulin, or soybean It may be useful to conjugate related antigens to trypsin inhibitors.

Animals can be infected, for example, by combining 100 μg or 5 μg of protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution into multiple sites subcutaneously. , Immunogenic conjugates, or derivatives. After one month, the animal is boosted by subcutaneous injection of the peptide or conjugate in Freund's complete adjuvant into multiple sites at 1/5 to 1/10 the original amount. After 7-14 days, animals are bled to assay antibody titers in serum. Animals are boosted up to titer plateau. Preferably, the animal is boosted with conjugates of the same antigen, but this is conjugated to different proteins and / or through different crosslinking reagents. Conjugates can also be prepared in recombinant cell culture as protein fusions. In addition, flocculants such as alum are suitably used to improve the immune response.

(iii) monoclonal antibodies

Monoclonal antibodies can be prepared using the hybridoma method first described in Kohler et al., Nature, 256: 495 (1975), or by recombinant DNA methods (US Pat. No. 4,816,567). It can manufacture. In the hybridoma method, mice or other suitable host animals, such as hamsters or macaque monkeys, are immunized as described above to produce lymphocytes capable of producing or producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro. Lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)]).

The hybridoma cells thus prepared are preferably grown by seeding in a suitable culture medium containing one or more substances that inhibit the growth or survival of unfused parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthin guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium of hybridoma typically includes hypoxanthine, aminopterin and thymidine (HAT medium). And these substances prevent the growth of HGPRT-deficient cells.

Preferred myeloma cells are cells that fuse efficiently, support stable high-level production of antibodies by selected antibody-producing cells, and are sensitive to media, such as HAT media. Among these, preferred myeloma cell lines are derived from murine myeloma cell lines, such as the MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center (San Diego, CA, USA), and the American type SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection (Rockville, MD). Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol. 133: 3001 (1984); and Broeur et al., Monoclonal Antibody Production Techniques). and Applications, pp. 51-63 (Marcel Dekker, Inc, New York, 1987)].

Culture medium in which hybridoma cells are grown is assayed for production of monoclonal antibodies that act on the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Decide

After hybridoma cells producing antibodies of the desired specificity, affinity and / or activity have been identified, the clones are subcloned by restriction dilution procedures and standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59). -103 (Academic Press, 1986)]. Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can be grown in vivo as ascites tumors in an animal.

Monoclonal antibodies secreted by the subclone may be cultured, ascites, by conventional immunoglobulin purification procedures, eg, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. Isolate appropriately from fluid or serum.

Easily isolate and sequence DNA encoding monoclonal antibodies using conventional procedures (e.g., using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of monoclonal antibodies). Analyze Hybridoma cells serve as a preferred source of the DNA. Once isolated, the DNA can be placed in an expression vector, after which host cells such as E. coli do not produce immunoglobulin proteins. Monoclonal antibodies were synthesized in recombinant host cells by transfection into E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells. Recombinant production of antibodies will be described in more detail below.

In further embodiments, the antibody or antibody fragment can be isolated from the antibody phage library generated using the techniques described in McCafferty et al., Nature, 348: 552-554 (1990).

Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol. 222: 581-597 (1991) describes the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent literature describes the generation of high affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology, 10: 779-783 (1992)), and for preparing very large phage libraries. As a strategy, combinatorial infection and in vivo recombination (Waterhouse et al., Nuc. Acids. Res. 21: 2265-2266 (1993)) are described. That is, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for monoclonal antibody isolation.

DNA can also substitute, for example, human heavy and light chain constant domain coding sequences in place of homologous murine sequences (US Pat. No. 4,816,567; Morrison, et al., Proc. Natl. Acad. Sci. USA , 81: 6851 (1984))), may be modified by covalently linking an immunoglobulin coding sequence with all or part of a coding sequence of a non-immunoglobulin polypeptide.

Typically, the non-immunoglobulin polypeptide is substituted with the constant domain of the antibody or with the variable domain of one antigen-binding site of the antibody, thereby allowing one antigen-binding site having specificity for the antigen and specificity for different antigens to be present. Chimeric bivalent antibodies comprising other antigen-binding sites having

(iv) humanized and human antibodies

Humanized antibodies incorporate one or more amino acid residues from a non-human source into the antibody. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization consists essentially of the method of Winter and co-researchers (Jones et al., Nature, 321: 522-525 (1986); Riechmann by substituting the corresponding sequences of human antibodies with rodent CDRs or CDR sequences); et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988). Thus, such “humanized” antibodies are chimeric antibodies (US Pat. No. 4,816,567) in which substantially fewer intact human variable domains are substituted with corresponding sequences from non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from similar sites in rodent antibodies.

The choice of both human variable domain light and heavy chains used to prepare humanized antibodies is of great importance for antigenicity reduction. According to the so-called "best-fit" method, the sequences of the variable domains of rodent antibodies are screened against the entire library of known human variable-domain sequences. Subsequently, the human sequence closest to the rodent sequence is accepted as the human framework (FR) for humanized antibodies (Sims et al., J. Immunol. 151: 2296 (1993); Chothia et al., J. Mol. Biol., 196: 901 (1987)]. Another method utilizes a specific framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad Sci. USA, 89: 4285 (1992); Presta et al., J. Immnol. 151: 2623 (1993)).

It is even more important that the antibody be humanized to possess high affinity for the antigen and other desirable biological properties. To achieve this, according to a preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are well known to those skilled in the art. Computer programs are available that show and display possible three-dimensional geometric structures of selected candidate immunoglobulin sequences. Examining these displays allows for the analysis of possible roles of residues in the function of candidate immunoglobulin sequences, ie, residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the accepting sequence and the introducing sequence to achieve the desired antibody properties, eg, increased affinity for the target antigen (s). In general, CDR residues are directly and most substantially involved in influencing antigen binding.

Alternatively, it is presently possible to produce transgenic animals (eg mice) that, upon immunization, can produce the entire repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, homozygous deletions of the antibody heavy chain linkage region (J _H ) genes of chimeric and germ-line mutant mice are described as completely inhibiting endogenous antibody production. Delivery of the human germ-line immunoglobulin gene array into such germ-line mutant mice will produce human antibodies upon antigen challenge inoculation. See, eg, Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature, 362: 255-258 (1993); Bruggermann et al., Year in Immuno. 7:33 (1993); And Duchusal et al., Nature 355: 258 (1992). Human antibodies can also be derived from phage-display libraries (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 generation of human antibodies from antibody phage display libraries is further described below.

(v) antibody fragments

Various techniques have been developed for generating antibody fragments. Traditionally, these fragments have been derived through digestion by proteolytic digestion of intact antibodies (eg, Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992); and Brennan et. al., Science, 229: 81 (1985). However, these fragments can now be produced directly by recombinant host cells. For example, antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, the Fab'-SH fragments are E. coli. It can be recovered directly from E. coli and chemically coupled to form F (ab ') ₂ fragments (Carter et al., Bio / Technology 10: 163-167 (1992)). In other embodiments described in the Examples below, F (ab ') ₂ is formed using leucine zipper GCN4 to facilitate assembly of F (ab') ₂ molecules. According to another method, F (ab ') ₂ fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to those skilled in the art. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185.

(vi) multispecific antibodies

Multispecific antibodies have binding specificities for at least two different epitopes, wherein the epitopes are generally from different antigens. Such molecules will generally only bind to two different epitopes (ie bispecific antibodies, BsAb), but are included in this expression when antibodies with additional specificities such as trispecific antibodies are used herein. Examples of BsAb include having one arm acting on PirB / LILRB2 and the other arm acting on Nogo or MAG or OMgp. Further examples of BsAB include having one cancer that acts on PirB / LILRB2 and the other cancer that acts on NgR.

Methods of making bispecific antibodies are known in the art. Traditional production of full length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al., Nature, 305: 537-539 (1983). )]). Because of the random collection of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of precise molecules, usually by affinity chromatography steps, is rather cumbersome and yields low. Similar procedures are disclosed in WO 93/08829 and in Traunecker et al., EMBO J. 10: 3655-3659 (1991). According to different methods, antibody variable domains with the desired binding specificities (antibody-antigen binding sites) are fused to immunoglobulin constant domain sequences. Preferably, the fusion uses an immunoglobulin heavy chain constant domain comprising at least a portion of the hinge, CH2 and CH3 regions. Preferably, the first heavy chain constant region (CH1) containing the site necessary for light chain binding is present in at least one of the fusions. The DNA encoding the immunoglobulin heavy chain fusion and, if necessary, the immunoglobulin light chain, is inserted into a separate expression vector and cotransfected into a suitable host organism. This provides great flexibility in adjusting the mutual ratios of the three polypeptide fragments in embodiments where the uneven proportions of the three polypeptide chains used in the production provide the optimum yield. However, when the expression yield of at least two polypeptide chains in equal proportions is high or the ratio does not have a particular significance, it is possible to insert the coding sequences of two or all three polypeptide chains into a single expression vector.

In a preferred embodiment of the method, the bispecific antibody consists of a hybrid immunoglobulin heavy chain having a first binding specificity in one cancer, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other cancer. . The presence of immunoglobulin light chains in only half of the bispecific molecules provides an easy method of separation, and thus the asymmetric structure has been found to facilitate the separation of the desired bispecific compounds from unwanted immunoglobulin chain combinations. The method is disclosed in WO 94/04690. For more details on the production of bispecific antibodies, see, eg, Suresh et al., Methods in Enzymology, 121: 210 (1986).

According to another method described in WO96 / 27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. Preferred interfaces comprise at least a portion of the CH3 domain of the antibody constant domains. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (eg tyrosine or tryptophan). By replacing the large amino acid side chain with a smaller amino acid side chain (eg alanine or threonine), a compensating "cavity" of the same or similar size for the large side chain (s) is created on the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers over other undesired end products, such as homodimers.

Bispecific antibodies include crosslinked or "heteroconjugate" antibodies. For example, one of the heteroconjugate antibodies may be coupled to avidin and the other may be coupled to biotin. Such antibodies have been proposed, for example, for targeting immune system cells to unwanted cells (US Pat. No. 4,676,980), and for the treatment of HIV infection (WO 91/00360, WO 92/200373). Heteroconjugate antibodies can be prepared using any conventional crosslinking method. Along with many crosslinking techniques, suitable crosslinkers are well known in the art and are disclosed in US Pat. No. 4,676,980.

Techniques for generating bispecific antibodies from antibody fragments are also described in the literature. For example, bispecific antibodies can be prepared using chemical linkers. Brennan et al., Science 229: 81 (1985) describe a procedure in which intact antibodies are cleaved proteolytically to generate F (ab ') ₂ fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize adjacent dithiols and prevent intermolecular disulfide formation. The Fab 'fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of another Fab'-TNB derivative to form a bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

Fab'-SH fragments to E. coli. It can also be recovered directly from E. coli and can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175: 217-225 (1992) describes the production of fully humanized bispecific antibody F (ab ') ₂ molecules. Each Fab 'fragment is E. coli. Separated from E. coli and induced chemical coupling reaction in vitro to form bispecific antibodies.

Various techniques for preparing and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. 148 (5): 1547-1553 (1992). Leucine zipper peptides from Fos and Jun proteins were linked to the Fab 'portion of two different antibodies by gene fusion. The antibody homodimer was reduced in the hinge region to form a monomer and then reoxidized to form the antibody heterodimer. The method can also be used for the production of antibody homodimers. Hollinger et al., Proc. Nati. Acad. Sci. USA, 90: 6444-6448 (1993), provided that the "diabody" technique provided an alternative mechanism for the preparation of bispecific antibody fragments. This fragment comprises a heavy chain variable domain (VH) linked to the light chain variable domain (VL) by a linker that is too short to form a pair between two domains on the same chain. Thus, the VH and VL domains of one fragment are paired with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Other fabrication strategies for bispecific antibody fragments using single chain Fv (sFv) dimers have also been reported. See Gruber et al., J. Immunol, 152: 5368 (1994).

Antibodies greater than divalent are contemplated. For example, trispecific antibodies can be prepared. Tuft et al., J. Immunol. 147: 60 (1991).

(vii) Effector Function Operation

In order to improve the effectiveness of the antibodies, it may be desirable to modify the antibodies of the invention with respect to effector function. For example, by introducing cysteine residue (s) into the Fc region, interchain disulfide bonds can be formed in this region. Homodimeric antibodies thus produced may have improved internalization capacity and / or increased complement-mediated cell death and antibody-dependent cell type cytotoxicity (ADCC). Caron et al., J. Exp Med. 176: 1191-1195 (1992) and Shopes, B. J. Immunol. 148: 2918-2922 (1992). Homodimeric antibodies with improved anti-tumor activity can also be prepared using heterobifunctional crosslinkers as described in Wolfff et al., Cancer Research 53: 2560-2565 (1993). Alternatively, antibodies with double Fc regions can be engineered, thereby improving the complement lysis and ADCC ability of the antibody. See Stevenson et al., Anti-Cancer Drug Design 3: 219-230 (1989).

(viii) antibody-salvage receptor binding epitope fusions

In certain embodiments of the invention, it may be desirable to use antibody fragments rather than intact antibodies, for example to increase tumor permeation. In such cases, it may be desirable to modify the antibody fragment to increase its serum half life. This can be accomplished, for example, by incorporating salvage receptor binding epitopes into the antibody fragment (eg by mutation of a suitable region in the antibody fragment, or after incorporation of the epitope into the peptide tag, either end of the antibody fragment Or by fusion in between (eg by DNA or peptide synthesis).

Preferably, the salvage receptor binding epitope constitutes a region in which any one or more amino acid residues from one or two loops of the Fc domain have been moved to similar positions of the antibody fragment. Even more preferably, at least three residues from one or two loops of the Fc domain are transferred. Even more preferably, the epitope is taken from the CH2 domain of the Fc region (eg the Fc region of IgG) and transferred to the CH1, CH3, or V _H region, or more than one such region of the antibody. Alternatively, epitopes are taken from the CH2 domain of the Fc region and transferred to the CL region or the VL region or both of the antibody fragments.

(ix) other covalent modifications of the antibody

Covalent modifications of antibodies are included within the scope of the present invention. This may be by chemical synthesis or by enzymatic or chemical cleavage of the antibody, if applicable. Another type of covalent modification of an antibody is introduced into the molecule by reacting the targeted amino acid residue of the antibody with an organic derivatizing agent that can react with a selected side chain or N- or C-terminal residue. Examples of covalent modifications are described in US Pat. No. 5,534,615, which is expressly incorporated herein by reference. Preferred covalent modifications of the antibody are described in US Pat. No. 4,640,835; 4,496,689; 4,496,689; 4,301,144; 4,301,144; 4,670,417; 4,670,417; Linking the antibody to one of a variety of non-proteinaceous polymers, such as polyethylene glycol, polypropylene glycol, or polyoxyalkylene, in the manner described in US Pat. No. 4,791,192 or 4,179,337.

(x) Generation of Antibodies from Synthetic Antibody Phage Library

In a preferred embodiment, the present invention provides methods for generating and selecting novel antibodies using unique phage display methods. This method produces a synthetic antibody phage library based on a single framework template, design of sufficient diversity within variable domains, display of polypeptides with diversified variable domains, and screen for candidates with high affinity targeting antigens. , And isolation of selected antibodies.

Details of the phage display method can be found, for example, in WO03 / 102157 (December 11, 2003 publication), the entire disclosure of which is expressly incorporated herein by reference.

In one aspect, the antibody libraries used in the present invention can be generated by mutating solvents in one or more CDRs of the antibody variable domains that are accessible and / or highly diverse. Some or all CDRs can be mutated using the methods provided herein. In some embodiments, a single library is mutated by mutating a position in CDRH1, CDRH2 and CDRH3 to form a single library or by mutating a position in CDRL3 and CDRH3 to form a single library or by mutating a position in CDRL3 and CDRH1, CDRH2 and CDRH3. It may be desirable to generate various antibody libraries by forming.

For example, libraries of antibody variable domains may be generated in which the solvents of CDRH1, CDRH2 and CDRH3 are accessible and / or have mutations in a wide variety of locations. Another library with mutations in CDRL1, CDRL2 and CDRL3 can be generated. These libraries can also be used together with each other to produce binders of the desired affinity. For example, after one or more rounds of selection of the heavy chain library for binding to a target antigen, the light chain library can be replaced into a population of heavy chain binders for further rounds of selection to increase the affinity of the binder.

Preferably, the library is generated by replacing the original amino acid with variant amino acids in the CDRH3 region of the variable region of the heavy chain sequence. The resulting library may contain multiple antibody sequences whose sequence diversity is predominantly within the CDRH3 region of the heavy chain sequence.

In one aspect, a library is generated in the context of a humanized antibody 4D5 sequence, or a sequence of framework amino acids of a humanized antibody 4D5 sequence. Preferably, a library is created by substituting at least residues 95-100a of the heavy chain with amino acids encoded by the DVK codon set, wherein the DVK codon set is used to encode a set of variant amino acids for all of these positions. Examples of oligonucleotide sets useful for making such substitutions include sequence (DVK) ₇ . In some embodiments, a library is generated by replacing residues 95-100a with amino acids encoded by both DVK and NNK codon sets. Examples of oligonucleotide sets useful for making such substitutions include the sequence (DVK) ₆ (NNK). In another embodiment, a library is generated by replacing at least residues 95-100a with amino acids encoded by both DVK and NNK codon sets. Examples of oligonucleotide sets useful for making such substitutions include the sequence (DVK) ₅ (NNK). Another example of a set of oligonucleotides useful for making such substitutions includes the sequence (NNK) ₆ . Those skilled in the art can determine another example of a suitable oligonucleotide sequence according to the criteria described herein.

In another embodiment, different CDRH3 designs are used for isolation of high affinity binders and for binders for various epitopes. The length of CDRH3 generated in this library ranges from 11 to 13 amino acids, although other lengths may also be generated. H3 diversity can be extended by using NNK, DVK and NVK codon sets, as well as more limited diversity at the N and / or C-terminus.

Diversity may also be generated in CDRH1 and CDRH2. The design of the CDR-H1 and H2 diversity follows a targeting strategy to mimic the natural antibody repertoire as described, with modifications focused on diversity that more closely matches natural diversity than previous designs.

For diversity in CDRH3, multiple libraries of different lengths of H3 can be separately constructed and combined to select a binder for the target antigen. Multiple libraries can be pooled and sorted using the solid support sorting and solution sorting methods previously described and described herein below. Multiple classification strategies can be used. For example, one modification involves sorting on a target bound to a solid, then sorting for a tag (eg anti-gD tag) that may be present on the fusion polypeptide, and classifying it differently on a target bound to the solid. do. Alternatively, the library can first be sorted on a target bound to a solid surface, and then the eluted binder is sorted using solution phase binding while reducing the concentration of the target antigen. Using a combination of different sorting methods minimizes the selection of only highly expressed sequences and allows a large number of different high affinity clones to be selected.

High affinity binders for the target antigen can be isolated from the library. Limiting the diversity in the H1 / H2 region reduces the degeneracy by about 10 ⁴ to 10 ⁵ times, and allowing more H3 diversity provides higher affinity binders. Using libraries of different types of diversity in CDRH3 (eg, using DVK or NVT) allows the binding of binders capable of binding to different epitopes of the target antigen.

Among the binders isolated from pooled libraries as described above, it has been found that affinity can be further improved by providing limited diversity in the light chain. In this embodiment the light chain diversity is generated as follows: In CDRL1, amino acid position 28 is encoded by RDT; Amino acid position 29 is encoded by RKT; Amino acid position 30 is encoded by RVW; Amino acid position 31 is encoded by ANW; Amino acid position 32 is encoded by THT; Optionally, 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; Optionally, amino acid position 55 is encoded by GMA; In CDRL3, amino acid position 91 is encoded by TMT or SRT or both; Amino acid position 92 is encoded by DMC; Amino acid position 93 is encoded by RVT; Amino acid position 94 is encoded by NHT; Amino acid position 96 is encoded by TWT or YKG or both.

In another embodiment, a library or libraries are created with diversity in the CDRH1, CDRH2 and CDRH3 regions. In this embodiment, H3 regions of varying lengths are used, mainly using codon sets XYZ and NNK or NNS to generate diversity in CDRH3. Individual oligonucleotides can be used to form and pool a library, or oligonucleotides can be pooled to form a subset of a library. Libraries of these embodiments can be classified for targets bound to a solid. Clones isolated from multiple classifications can be screened using ELISA assays for specificity and affinity. For specificity, clones can be screened for the desired target antigen as well as other non-target antigens. The binder for the target antigen can then be screened for affinity in a solution binding competition ELISA assay or spot competition assay. XYZ codon sets prepared as described above can be used to isolate high affinity binders from the library. Such binding agents can be readily produced as antibodies or antigen binding fragments in high yield in cell culture.

In some embodiments, it may be desirable to create libraries with greater diversity in the length of the CDRH3 region. For example, it may be desirable to generate libraries with CDRH3 regions ranging from about 7 to 19 amino acids.

High affinity binders isolated from the libraries of these embodiments are readily produced in high yields in bacterial and eukaryotic cell cultures. Vectors can be designed to include constant region sequences to easily remove sequences such as gD tags, viral coat protein component sequences, and / or provide for the production of high yield full length antibodies or antigen binding fragments.

Libraries with mutations in CDRH3 can be combined with libraries containing variant versions of other CDRs, eg, CDRL1, CDRL2, CDRL3, CDRH1 and / or CDRH2. Thus, for example, in one embodiment, the CDRH3 library is a CDRL3 library generated in the context of a humanized 4D5 antibody sequence having variant amino acids at positions 28, 29, 30, 31, and / or 32 using a predetermined codon set; Combined. In another embodiment, a library with mutations in CDRH3 can be combined with a library comprising variant CDRH1 and / or CDRH2 heavy chain variable domains. In one embodiment, a CDRH1 library is generated with humanized antibody 4D5 sequence having variant amino acids at positions 28, 30, 31, 32, and 33. A CDRH2 library can be generated with the sequence of humanized antibody 4D5 with variant amino acids at positions 50, 52, 53, 54, 56 and 58 using a predetermined set of codons.

(xi) antibody mutants

New antibodies generated from phage libraries can be further modified to generate antibody mutants with improved physical, chemical and / or biological properties compared to parental antibodies. If the assay used is a biological activity assay, preferably, the biological activity of the antibody mutants in the selected assay is at least about 10 times better than the biological activity of the parent antibody in this assay, preferably at least about 20 times Better, more preferably at least about 50 times better, often at least about 100 times or 200 times better. For example, preferably, the anti-PriB / LILRB antibody mutant has a binding affinity for PriB / LILRB at least about 10 times stronger, preferably at least about 20 times stronger than the parental antibody's binding affinity, More preferably at least about 50 times stronger, often at least about 100 times or 200 times stronger.

To generate antibody mutants, one or more amino acid alterations (eg substitutions) are introduced into one or more of the hypervariable regions of the parent antibody. Alternatively, or in addition, one or more alterations (eg, substitutions) of framework region residues may be introduced into the parent antibody, which improves the binding affinity of the antibody mutant to the antigen from the second mammalian species. Let's do it. Examples of framework region residues to be modified include non-covalently binding directly to an antigen and / or (Amit et al., (1986) Science 233: 747-753); Interact with and / or cause the shape of the CDRs (Chothia et al., (1987) J. Mol. Biol. 196: 901-917); Those participating in the V _L -V _H interface (EP 239 400B1) are included. In certain embodiments, modification of one or more of these framework region residues improves the binding affinity of the antibody for antigens from the second mammalian species. For example, about 1 to about 5 framework residues may be altered in this embodiment of the invention. Sometimes this may be sufficient to generate antibody mutants suitable for use in preclinical experiments, even if the hypervariable region residues have not been altered. In general, however, antibody mutants will include additional hypervariable region alteration (s).

The hypervariable region residues that are altered can be randomly altered, particularly if the starting binding affinity of the parental antibody allows antibody mutants produced at random to be easily screened.

One useful procedure for generating such antibody mutants is called "alanine scanning mutagenesis" (Cunningham and Wells (1989) Science 244: 1081-1085). Here, one or more of the hypervariable region residue (s) is replaced with alanine or polyalanine residue (s) to affect the interaction of amino acids with antigens from the second mammalian species. Thereafter, the hypervariable region residue (s) exhibiting functional sensitivity to substitutions are refined at the substitution site or by introducing additional or other mutations to the substitution site. Thus, the site for introducing amino acid sequence variation is predetermined, but the nature of the mutation itself does not need to be determined in advance. The ala-mutants produced in this manner are screened for their biological activity as described herein.

Typically, substitutions will begin with conservative substitutions such as those set forth below under the heading of “preferred substitutions”. If such substitutions result in a change in biological activity (eg, binding affinity), then more substantial changes, which are termed “exemplary substitutions” in the table below or as further described below in connection with amino acid classes, are introduced. And the product is screened.

Preferred Substitutions:

More substantial modifications in the biological properties of the antibody include (a) the structure of the polypeptide backbone in the substitution region, such as, for example, sheet or helix, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the side chain The effect on maintaining the bulk of is achieved by choosing significantly different substitutions. Naturally occurring residues are classified into the following groups based on common side chain properties:

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

(2) neutral hydrophilic: cys, ser, thr, asn, gln;

(3) acidic: asp, glu;

(4) basic: his, lys, arg;

(5) residues affecting chain orientation: gly, pro; And

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging one member of this class for another class.

In another embodiment, the site selected for modification is affinity matured using phage 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, previously prepared mutant or non-mutant versions of oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis. A preferred method for preparing mutants is site directed mutagenesis (see, eg, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82: 488).

In certain embodiments, only a single hypervariable region residue will be substituted in the antibody mutant. In another embodiment, two or more of the hypervariable region residues of the parent antibody, eg, from about 2 to about 10 hypervariable regions, will be substituted.

Typically, antibody mutants with improved biological properties have at least 75%, more preferably at least 80%, more preferably at least 85% amino acid sequence identity or similarity with the amino acid sequences of the heavy or light chain variable domains of the parent antibody. , More preferably at least 90%, and most preferably at least 95%. Identity or similarity with respect to such sequences is herein referred to herein as amino acid residues (ie, identical residues) or similar candidate sequences within the same candidate sequence as the parent antibody residues after alignment of the sequences and introduction of gaps to achieve maximum sequence identity ratios as necessary. It is defined as the percentage of amino acid residues within (ie, amino acid residues from the same group based on common side chain properties, see above). N-terminal, C-terminal, or internal extension, deletion or insertion in the outer antibody sequence of the variable domain should not be interpreted as affecting sequence identity or similarity.

After production of antibody mutants, the biological activity of these molecules is determined in comparison to parental antibodies. As mentioned above, this may involve determining the binding affinity and / or other biological activity of the antibody. In a preferred embodiment of the invention, a panel of antibody mutants is prepared to screen binding affinity for the antigen or fragment thereof. Optionally applying one or more additional biological activity assays to one or more of the antibody mutants selected from this initial screening to determine whether the antibody mutant (s) with improved binding affinity is actually useful, for example, in preclinical studies. Check it.

Additional modifications may be applied to the antibody mutant (s) thus selected, often depending on the intended use of the antibody. Such modifications may involve additional amino acid sequence alterations, fusion to heterologous polypeptide (s) and / or covalent modifications such as those described in detail below. Regarding amino acid sequence alterations, exemplary modifications are described in detail above. For example, any cysteine residue not involved in maintaining the proper shape of the antibody mutant can also be substituted (usually substituted with serine) to improve the oxidative stability of the molecule and prevent abnormal crosslinking. Conversely, cysteine bond (s) can be added to the antibody to improve its stability (especially when the antibody is an antibody fragment, eg an Fv fragment). In another type of amino acid mutant, the glycosylation pattern is altered. This can be accomplished by deleting one or more carbohydrate moieties found in the antibody and / or adding one or more glycosylation sites that are not present in the antibody. Glycosylation of antibodies is typically either N-linked or O-linked. N-linking refers to a carbohydrate moiety attached to the side chain of an asparagine residue. Tripeptide sequences, asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are recognition sequences for enzymatic attachment of carbohydrate moieties to asparagine side chains. Thus, the presence of the tripeptide sequence in the polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of sugar N-aceylgalactosamine, galactose or xylose to hydroxyamino acids, most commonly serine or threonine, but 5-hydroxyproline or 5-hydroxylysine May also be used. The addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence to contain one or more of the above described tripeptide sequences (for N-linked glycosylation sites). The alteration can also be accomplished by addition or substitution of one or more serine or threonine residues on the sequence of the original antibody (for O-linked glycosylation sites).

(xii) recombinant production of antibodies

For recombinant production of an antibody, the nucleic acid encoding it is isolated and inserted into a replicable vector for further cloning (amplification of DNA) or expression. Using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody), the DNA encoding the monoclonal antibody is easily isolated and sequenced. . Multiple vectors can be used. Vector components generally include, but are not limited to, one or more of signal sequences, origins of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences (e.g., in the United States expressly incorporated herein by reference). Patent number 5,534,615).

Suitable host cells for cloning or expression of DNA in a vector herein are the prokaryote, yeast, or higher eukaryotic cells described above. Prokaryotes suitable for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, such as Escherichia, for example E. coli. E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, for example Salmonella typhimurium, Serratia Serratia marcescans, and Shigella, as well as Bacillus, such as B. subtilis and B. licheniformis, for example. (See, eg, B. rickenformis 41P, Pseudomonas, such as P. aeruginosa, and Streptomyces, disclosed in DD 266,710, published April 12, 1989). Streptomyces). One desirable tooth. E. coli cloning host. E. coli 294 (ATCC 31,446), but other strains such as E. coli. E. coli B. Lee. E. coli X1776 (ATCC 31,537), and two. E. coli W3110 (ATCC 27,325) is also suitable. These examples are illustrative rather than limiting.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-coding vectors. Among lower eukaryotic host microorganisms, Saccharomyces cerevisiae, or conventional baker's yeast, is most commonly used. However, many other genera, species and strains, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. Thermo Tolerance (K) thermotolerans) and K. marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; And filamentous fungi such as neurospora, penicillium, tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger (A. niger) is commonly available and useful herein.

Suitable host cells for the expression of glycosylated antibodies are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains and variants, and Spodoptera frugiperda (larvae), Aedes aegypti (mosquitoes), Aedes albopictus ( Corresponding permitted insect host cells from hosts such as mosquitoes, Drosophila melanogaster (Drosophila) and Bombyx mori have been identified. Various viral strains for transfection, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, are publicly available, and such viruses are described herein, in particular It can be used as a virus according to the present invention for the transfection of Spododotera pruperifera cells. Plant cell cultures of cotton, corn, potatoes, soybeans, petunias, tomatoes, and tobacco may also be used as hosts.

However, vertebrate cells are of most interest, and proliferation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines include monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); Human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture (Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL) 10); Chinese Hamster Ovarian Cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); Mouse Sertoli Cells (TM4, Mather, Biol) Reprod. 23: 243-251 (1980)]); Monkey Kidney Cells (CV1 ATCC CCL 70); African Green Monkey Kidney Cells (VERO-76, ATCC CRL-1587); Human Cervical Carcinoma Cells (HELA, ATCC CCL) 2); dog kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75), human hepatocytes (Hep G2, HB 8065); mouse breast tumors (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. 383: 44-68 (1982)); MRC 5 cells; FS4 cells; and human liver cancer lines (Hep G2) to be.

The host cell is transformed into an expression or cloning vector for the production of the antibodies described above, followed by appropriate modification of conventional nutrient media to induce a promoter, select a transformant, or amplify the gene encoding the desired sequence. Incubate in.

Host cells used to produce the antibodies of the invention can be cultured in a variety of media. Commercially available media such as Ham F10 (Sigma), Minimum Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma) and Dulbecco's Modified Eagle's Medium (DMEM) ( Sigma) is suitable for culturing host cells. See also, Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102: 255 (1980), US Pat. No. 4,767,704; 4,657,866; US Pat. 4,927,762; 4,927,762; 4,560,655; 4,560,655; Or 5,122,469; WO 90/03430; WO 87/00195; Or US Patent No. Re. Any of the media described in 30,985 can be used as the culture medium for host cells. Hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thyme if needed in any such medium) Dine), antibiotics (such as GENTAMYCIN®), trace elements (defined as inorganic compounds typically present in final concentrations in the micromolar range), and glucose or equivalent energy sources. Any other necessary supplements may also be included at appropriate concentrations well known to those skilled in the art. Culture conditions, such as temperature, pH, etc., have been used previously with host cells selected for expression and will be apparent to those skilled in the art.

When using recombinant technology, antibodies can be produced in the space around the plasma membrane in the cell or secreted directly into the medium. When antibodies are produced intracellularly, as a first step, particulate debris, which is a host cell or lysed cells, is removed, for example by centrifugation or ultrafiltration. When the antibody is secreted into the medium, the supernatant from the expression system is generally first purified using a commercially available protein enrichment filter, such as Amicon or Millipore Pellicon ultrafiltration unit. Concentrate. Protease inhibitors, such as PMSF, may be included in any preceding step to inhibit proteolysis, and antibiotics may be included to prevent accidental increases in contaminants.

Antibody compositions prepared from cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of Protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human γ1, γ2 or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983)). Protein G is recommended for all mouse isotypes and human γ3 (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 are available. Mechanically stable matrices such as glass or poly (styrenedivinyl) benzene with controlled pores allow for faster flow rates and shorter processing times than can be achieved with agarose. If the antibody comprises a CH3 domain, Bakerbond ABX ™ resin (J. T. Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification, for example fractionation on ion-exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin Sepharose ™, anion or cation exchange resins (e.g. polyaspartic acid columns) Chromatography on chromium), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation can also be used depending on the antibody to be recovered.

C. Use of anti-PirB / LILRB antibodies

The anti-PirB / LILRB antibodies of the present invention are believed to be of use as agents for improving the survival of neurons or inducing their growth. Thus, this may include, for example, physical damage to the central nervous system (the spinal cord and the brain); Brain damage associated with stroke; And nervous system disorders associated with neurodegeneration, such as trigeminal neuralgia, Yeti neuralgia, Bell's palsy, myasthenia gravis, muscular dystrophy, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), progressive muscular atrophy, progressive training hereditary muscular atrophy, physical injury ( Peripheral nerve damage, intervertebral hernia, rupture, or dislodged invertebrate disc syndrome caused by, for example, burns, wounds) or disease states such as diabetes, renal dysfunction or toxic effects of chemotherapeutic agents used to treat cancer and AIDS Degenerative disorders of the nervous system, including cervical spondylosis, plexus disorders, thoracic outlet destruction syndrome, peripheral neuropathy, such as caused by lead, daphnia, ticks, porphyria, guillain-barre syndrome, Alzheimer's disease, Huntington's disease, or Parkinson's disease ( Useful in the treatment of "neurodegenerative diseases").

Anti-PirB / LILRB antibodies herein are also useful as components of a culture medium for use in culturing neurons in vitro.

Finally, agents comprising the anti-PirB / LILRB antibodies herein are useful as standards in competitive binding assays when labeled with radioactive iodine, enzymes, fluorophores, spin labels, and the like.

Anti-PirB / LILRB antibodies herein by mixing identified compounds (such as antibodies) with the desired degree of purity with any physiologically acceptable carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences, supra) The therapeutic formulation of is prepared for storage in the form of a lyophilized cake or aqueous solution. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; Antioxidants including ascorbic acid; Low molecular weight (less than about 10 residues) polypeptide; Proteins such as serum albumin, gelatin or immunoglobulins; Hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine or lysine; Monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; Chelating agents such as EDTA; Sugar alcohols such as mannitol or sorbitol; Salt-forming counter ions such as sodium; And / or nonionic surfactants such as Tween, Pluronics or PEG.

Anti-PirB / LILRB antibodies used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes before or after lyophilization and reconstitution.

The therapeutic composition may be placed in a container with a sterile access port, eg, an intravenous solution bag or vial with a stopper through which a hypodermic needle can penetrate.

Anti-PirB / LILRB antibodies of the invention can optionally be combined with or administered concurrently with neurotrophic factors (including NGF, NT-3, and / or BDNF), and with other conventional therapies for degenerative neurological disorders. Can be used together. In addition, the anti-PirB / LILRB antibodies of the invention can be advantageously administered with NgR inhibitors such as antibodies, small molecules or peptides to block the binding of Nogo-66, MAG and / or OMgp to NgR.

The route of administration is in accordance with known methods, for example by injection or infusion by intravenous, intraperitoneal, intracranial, intramuscular, intraocular, intraarterial or intralesional routes, topical administration, or sustained release systems as mentioned below. By

For intracranial use, the compound may be administered continuously by infusion of the CNS into the fluid reservoir, but bolus injection is acceptable. Preferably the compound is administered into the ventricle or otherwise introduced into the CNS or spinal fluid. Administration may be carried out by an indwelling catheter using a continuous means of administration, such as a pump, or may be administered by implantation of a sustained release vehicle, such as intracranial implantation. More specifically, the compound may be injected through a chronically implanted cannula or chronically infused with the aid of an osmotic minipump. Subcutaneous pumps are available that deliver proteins to the ventricles via small tubing. Highly sophisticated pumps can be refilled through the skin and their delivery rates can be set without surgical intervention. Examples of suitable dosing protocols and delivery systems involving continuous intraventricular infusion via subcutaneous pump devices or fully implantable drug delivery systems are described in Harbaugh, J. Neural Transm. Suppl. 24: 271 (1987); And De Yebenes, et al., Mov. Disord. 2: 143 (1987), which are used to administer dopamine, dopamine agonists, and cholinergic agonists to animal models for Alzheimer's patients and Parkinson's disease.

Suitable examples of sustained-release preparations include semipermeable polymer matrices in the form of shaped articles, eg films, or microcapsules. Sustained release matrices include polyesters, hydrogels, polylactides (US Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman, et al., 1983, Biopolymers 22: 547), poly (2-hydroxyethyl-methacrylate) (Langer, et al., 1981, J. Biomed. Mater. Res. 15: 167; Langer, 1982, Chem. Tech 12:98]), ethylene vinyl acetate (Langer, et al., Supra), or poly-D-(-)-3-hydroxybutyric acid (EP 133,988A). Sustained release compositions also include compounds encapsulated in liposomes, which can be prepared by methods known per se (Epstein, et al., Proc. Natl. Acad. Sci. 82: 3688 (1985)). Hwang, et al., Proc. Natl. Acad. Sci. USA 77: 4030 (1980); US Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324A. It is a small (about 200-800 angstroms) monolayer type whose content exceeds about 30 mol% cholesterol, and the proportion selected is adjusted for optimal treatment.

The effective amount of active compound to be used therapeutically will depend, for example, on the therapeutic purpose, the route of administration, and the condition of the patient. Therefore, it will be necessary for the therapist to titrate the dosage and modify the route of administration as necessary to obtain the optimal therapeutic effect. Typical daily dosages will range from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. Typically, the clinician will administer the active compound until a dosage is reached that restores, maintains and optimally reestablishes neuronal function. The progress of this therapy is easily monitored by conventional assays.

Further details of the invention are illustrated by the following non-limiting examples.

Example 1

Expression Cloning LILRB2

To identify new receptors for inhibitory myelin proteins, expression cloning methods were used. As a bait, constructs were constructed in which alkaline phosphatase (AP) was fused to the N- and / or C-terminus of the following characterized myelin inhibitors (using human cDNA): Nogo66, two additional inhibitions of NogoA Domains (NiR <delta> D2 and NiG <delta> 20) (Oertle T, J Neurosci. 2003, 23 (13): 5393-406), MAG, and OMgp. These constructs were transfected into 293 cells to prepare conditioned medium containing the exemplary protein (in DMEM / 2% FBS). The cDNA library used for screening consisted of full length human cDNA clones in the expression-preparation vector generated by Origene. These cDNAs were compiled, sorted and pooled. A pool of about 100 cDNAs was transiently transfected into COS7 cells.

In particular, on day 1, COS7 cells were plated in 12-well plates at a density of 85,000 cells / well. On day 2, each well was transfected with 1 mg of pooled cDNA using the lipid-based transfection reagent FuGENE 6 (Roche). On day 4, screening was performed. Briefly, culture medium was removed from the cells and replaced with 0.5 ml of 293 cell-conditioned medium (20-50 nM) containing the AP-fusion exemplary protein. Cells were incubated for 90 minutes at room temperature. Cells are then washed three times with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 7 minutes, washed three times in HEPES-buffered saline (HBS), and heated at 65 ° C. for 90 minutes. Inactivation disrupted endogenous AP activity. Cells are washed once in AP buffer (100 mM NaCl, 5 mM MgCl ₂ , 100 mM Tris pH 9.5), incubated in chromogenic substrate (Western Blue, Promega), incubated and It was again incubated overnight and analyzed for the presence of reaction product after 1 hour. Positive cells were identified by the presence of dark blue precipitates on the membrane surface. Positive pools were further disrupted to identify individual positive clones by subsequent rounds of screening.

From the screening, the following positive hits were identified:

The MAG-AP example generated four positive hits. One was a previously characterized Nogo receptor (Fournier et al., Nature 409, 342-346 (2001)). Two of the hits were glycolytic processing enzymes and were considered irrelevant. The fourth was described as "Clone 643 (LOC57228), Homo sapiens hypothetical protein from mRNA". A closer analysis of the cDNA showed another ORF homologous to the previously described protein SMAG.

The AP-Nogo66 example generated two positive hits. One was a previously characterized Nogo receptor. The other was “homo sapiens leukocyte immunoglobulin-like receptor, subfamily B (with TM and ITIM domain), member 2 (LILRB2), mRNA” (SEQ ID NO: 2). The gene is also known by several other names, including MIG10, ILT4, and LIR2 (Kubagawa et al., Proc. Natl. Acad. Sci. USA 94: 5261-6 (1997); Colonna); et al., J. Exp. Med. 186: 1809-18 (1997)].

Example 2

Preparation and Testing of PirB Function-Blocking Antibodies

PirB function-blocking antibody

An antibody against PirB was generated by panning the synthetic phage antibody library against the PirB extracellular domain (W. C. Liang et al., J Mol Biol 366, 815 (2007)). The antibody clone (10 μg / ml) was then tested in vitro for the ability to block the binding of AP-Nogo66 (50 nM) to PirB-expressing COS7 cells. Nucleotide and amino acid sequences of the heavy and light chain sequences of various YW259 anti-mouse PirB (anti-mPirB) antibodies are shown in FIGS. 6-16, and 17 and 18. 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 test

96-well plates precoated with poly-D-lysine (Biocoat (BD)) overnight with myelin (0.75 μg / ml), or AP-Nogo66 or MAG-Fc (150-300 ng / spot) Coated for 2 hours and then treated with laminin (10 μg / ml in F-12) for 2 hours (CGN culture) or 4 hours (DRG culture). Mouse P7 cerebellar neurons were cultured as previously described (B. Zheng et al., Proc Natl Acad Sci USA 102, 1205 (2005)) and plated at about 2 × 10 ⁴ cells per well. Mouse P10 DRG neurons were cultured as previously described (Zheng et al., 2005, supra) and plated at about 5 × 10 ³ cells per well. Cultures are grown at 37 ° C. (with 5% CO ₂ present) for 22 hours, then fixed with 4% paraformaldehyde / 10% sucrose, anti-βIII-tubulin (TuJ1, Covance). ). For each experiment, all conditions were performed in six replicate wells from which the maximum neurite length was measured and the average between the six wells determined. Each experiment was performed three or more times and similar results were obtained. p-values were determined using Student's t test.

Growth cone collapse black

DRG explants were isolated by detaching DRG from 3-week old mice and slicing it into three pieces. Each DRG explant was then incubated in individual PDL (100 μg / ml)-and laminin (10 μg / ml) -coated wells from eight well plates. After 72 hours of plating, explants were incubated with AP-Nogo66 (100 nM) or myelin (3 μg / ml) for 30 minutes to stimulate disruption. Cultures were fixed with 4% paraformaldehyde / 10% sucrose, then growth cones were visualized by rhodamine-palloidine (Molecular Probes) staining and the disruption was measured. Mean growth cone collapse was determined by averaging three or more replicate wells.

result

To determine if PirB is a functional receptor of Nogo66, we focused on cerebellar granule neurons (CGN) in adolescents (P7), and neurite outgrowth was inhibited when grown on AP-Nogo66 (KC Wang et al., Nature 420, 74 (2002)]. Adult CGN has been found to express PirB (J. Syken et al., Science 313, 1795 (2006)), and we also found it by evaluation by RT-PCR, immunohistochemistry and in situ hybridization. Also confirmed in juvenile CGN cases (data not shown).

First, the ability to interfere with AP-Nogo66 inhibition of soluble ectodomain of PirB (PirB-His) was tested in vitro. As shown in FIG. 2A, AP-Nogo66 inhibited neurite outgrowth of P7 CGN to approximately 66% of untreated control levels. Inclusion of PirB-His in this assay reverses AP-Nogo66 inhibition, while reverting neurite growth to essentially control levels. These results are similar to those reported using ectodomains of NgR to block inhibition by Nogo66 (B Zgeng et al., Proc. Natl. Acad. Sci. USA 102, 1205 (2005)); AE Fournier, et al., J. Neurosci. 22, 8876 (200); ZL He et al., Neuron 38, 177 (2003)), which allows PirB to bind to the functional inhibitory domain of Nogo66 However, it does not confirm whether the endogenous PirB of CGN mediates inhibition by AP-Nogo66.

This produced antibodies against PirB (anti-PirB) that could interfere with PirB-Nogo66 interaction. Using the phage display platform for the extracellular domain of PirB (WC Liang et al., J. Mol. Biol. 366, 815 (2007)), AP-Nogo66 binds to PirB for multiple clones. The ability to block was screened. The clone YW259.2 (hereinafter referred to as aPB1) that most interfered with AP-Nogo66-PirB binding had a Kd of 5 nM for PirB (see Figures 13-16).

aPB1 had no effect on baseline axon growth of CGN. However, aPB1 significantly reduced inhibition by AP-Nogo66 or myelin in cultured CGN (FIG. 2B) and restored neurite outgrowth on AP-Nogo66 from 41% to 59% and 47% to 62% on myelin I was. Similar results were observed when using MAG as an inhibitory substrate or using other cell types (dorsal root ganglion (DRG) neurons) (FIG. 5). These results demonstrate that PirB is a functional receptor that mediates long-term inhibition of neurite growth.

To confirm this result, genetic removal of cell surface PirB was also induced in AP-Nogo66 or myelin by culturing neurons from PirBTM mice and removing four exons encoding the transmembrane domain and part of the PirB intracellular domain. Was tested to reverse the inhibition (J. Syken et al., Science 313, 1795 (2006)). CGN was cultured from PirBTM mice or wild type (WT) litters on control substrates, AP-Nogo66 or myelin. On control substrates (PDL / laminin), PirBTM neurons behaved similarly to WT neurons (FIG. 2C). However, neurite growth from PirBTM neurons was significantly less inhibited than neurite growth from WT neurons on AP-Nogo66 or myelin. On AP-Nogo66, growth from WT neurons was inhibited to 50% of control levels, whereas only 66% of PirBTM neurons were inhibited. Similarly, WT neurons on myelin were inhibited at 52% of the control level, while only 70% of PirBTM neurons were inhibited. In addition, we observed similar partial deinhibition of PirBTM DRG neurons in both myelin and AP-Nogo66 (FIG. 5). This finding indicates that PirB is in fact a functional receptor for AP-Nogo66 and a myelin-mediated inhibitor of neurite growth. However, loss of PirB activity does not completely recover growth.

Since NgR has previously been described as a receptor for myelin inhibitors, it is possible for both PirB and NgR to function to mediate the inhibition of neurite growth. To confirm this, neurons were cultured from NgR-deficient mice in the presence of anti-PirB, blocking the function of both PirB and NgR together in CGN. As we previously reported (B. Cheng et al., PNAS 2005, supra), NgR-/-CGN neurite outgrowth is the same as that in WT neurons by AP-Nogo66 or myelin. Suppressed to a degree (50% and 49%; FIG. 3). Treatment with aPB1 antibodies of NgR +/− neurons partially reversed inhibition by AP-Nogo66 or myelin as observed in aPB1 treatment of WT neurons. Similarly, aPB1 treatment of NgR − / − neurons partially reversed inhibition by AP-Nogo66, but did not provide any further recovery than observed in aPB1 treatment of NgR +/− neurons or WT neurons. In contrast, aPB1 treatment of NgR − / − neurons restored neurite outgrowth on myelin to nearly control levels. Thus, PirB, but not NgR, is required for substrate inhibition by APNogo66 in CGN, but only partially. In addition, both PirB and NgR together contribute to substrate inhibition imparted by myelin.

Since NgR is believed to be required for growth cone collapse in response to various myelin inhibitors (JE Kim et al., Neuron 44, 439 (2004)), O. Chivatakarn et al., J. Neurosci. 27 , 7117 (2007)]), PirB may also be involved in more acute reactions. Sensory neurons from the dorsal root ganglion (DRG) of three week old mice identified to express PirB were used in this experiment. The growth cones in this culture system had a high baseline level of decay (about 30%), which was found to be further increased by incubation with APNogo66 or myelin (FIG. 4). This decay rarely occurred in NgR-/-neurons. In addition, blocking PirB function by aPB1 was sufficient to reverse growth cone collapse by these inhibitors. Blocking the pathways of both PirB and NgR together (using treatment of aPB1 in neurons from NgR − / − mice) also completely reversed growth cone collapse, but treatment alone resulted in complete recovery in this assay. This result is not beneficial.

In another experiment, C1QTNF5 inhibited neurite outgrowth of cerebellar granule neurons (CGN), which was reversed by PirB function-blocking antibody YW259.2. This result is shown in FIG.

In addition, these results support the novel role of PirB as a receptor required for neurite inhibition by myelin extracts, and more specifically by the myelin-associated inhibitors Nogo66 and MAG. In fact, removal of PirB function alone (genetically or with an antibody) partially inhibits growth in both myelin extract- and myelin inhibitors, whereas genetic removal of NgR alone does not depress any of these substrates. PirB appears to be a more significant mediator of substrate inhibition than NgR. However, NgR mediates inhibition by myelin extracts (but not Nogo66) because genetic removal of NgR may increase deinhibition induced by anti-PirB antibodies to myelin (but not Nogo66). Seems to play an additional role. Our findings were found in NgR knockout mice, despite the reported regeneration or germination observed in rodents fused with NgR ectodomains (S. Li et al., J. Neurosci. 24, 10511 (2004)). It may be helpful to explain the surprising lack of improved CST regeneration (JE Kim et al., Supra), B. Zheng et al., Proc. Natl. Acad. Sci. USA 102, 1205. (2005)]. Thus, it may be necessary to remove both PirB and NgR to achieve significant regeneration in vivo. In addition, for Nogo66 substrates, there appears to be additional binding receptor (s) for Nogo66, since genetic clearance of NgR does not further increase the partial deinhibition effect of PirB removal.

Although PirB appears to be a more significant receptor for substrate inhibition than NgR, inactivating only PirB or NgR is sufficient to block the acute growth cone collapse caused by the addition of myelin inhibitors. This observation suggests that decay is a more difficult process that requires the activity of both PirB and NgR to work in parallel or together. In this regard, it is interesting to note that the PirB and NgR receptors have recently been found to play a similar role in limiting the plasticity of synaptic connections in the visual cortex: in mice lacking receptors, eye colds during critical developmental periods can Too much linking (J. Syken et al., 2006, supra, AW McGee et al., Science 309, 2222 (2005), supra). The mechanisms associated with the effects of both receptors in mediating growth cone collapse may also be based on the commonality of their role in eye predominance plasticity.

Inability to regenerate adult axons after injury is a major obstacle to regaining function after traumatic injury to the CNS. Reduction of regenerative potential due to synaptic plasticity capacity is expected to be age-limited in an effort to limit the development of excessive or lush synaptic connections. This prediction, along with the finding that PirB (J. Syken et al., 2006, supra), which has been previously implicated in limiting synaptic plasticity in both developing and adulthood, is also a mediator of axon inhibition by myelin And support from the discovery that NgR, which has been involved in axon inhibition from the outset, similarly regulates synaptic plasticity (S. Li et al., J. Neurosci. 24, 10511 (2004)).

The inventors' findings also extend the repertoire of potential PirB ligands to include neuronal regrowth inhibitors beyond the scope of class I MHC molecules. Conversely, since genetic clearance of known myelin inhibitor Nogo or MAG reduces to some extent inhibition by myelin—which suggests that other inhibitors are present—the inventors' findings are typically at low levels by oligodendrocytes. It raises the possibility that the expressed MHCI molecules can be upregulated after injury and cooperate with Nogo and MAG in central myelin to contribute to growth inhibition.

The mechanism by which PirB signals the inhibition of axon growth in response to myelin inhibitors is not clear. However, PirB antagonizes the function of integrin receptors (S. Pereira et al., J. Immunol. 173: 5757 (2004)) and found to use both SHP-1 and SHP-2 phosphatase. Lost; These events can each or both attenuate normal neurite growth. Blocking PirB activity using the anti-PirB antibodies herein or by other means provides an important new target for therapeutic intervention to stimulate axon regeneration.

All references cited throughout the specification are expressly incorporated herein in their entirety.

While the invention has been described with reference to what are considered to be specific embodiments, it is to be understood that the invention is not limited to such embodiments. On the contrary, the invention is intended to cover various modifications and equivalents falling within the spirit and scope of the appended claims.

                               SEQUENCE LISTING <110> GENENTECH, INC.       Jasvinder Atwal       Marc Tessier-Lavigne       Yan wu   <120> ANTI-PIRB ANTIBODIES <130> GNE-0324PCT <140> Not yet Assigned <141> Herewith <150> 61 / 052,949 <151> 2008-05-13 <150> 12 / 316,130 <151> 2008-12-09 <150> 12 / 208,883 <151> 2008-09-11 <160> 15 <170> PatentIn version 3.5 <210> 1 <211> 841 <212> PRT <213> Mus sp. <400> 1 Met Ser Cys Thr Phe Thr Ala Leu Leu Arg Leu Gly Leu Thr Leu Ser 1 5 10 15 Leu Trp Ile Pro Val Leu Thr Gly Ser Leu Pro Lys Pro Ile Leu Arg             20 25 30 Val Gln Pro Asp Ser Val Val Ser Arg Trp Thr Lys Val Thr Phe Phe         35 40 45 Cys Glu Glu Thr Ile Gly Ala Asn Glu Tyr Arg Leu Tyr Lys Asp Gly     50 55 60 Lys Leu Tyr Lys Thr Val Thr Lys Asn Lys Gln Lys Pro Ala Asn Lys 65 70 75 80 Ala Glu Phe Ser Leu Ser Asn Val Asp Leu Arg Asn Ala Gly Gln Tyr                 85 90 95 Arg Cys Ser Tyr Ser Thr Gln Tyr Lys Ser Ser Gly Tyr Ser Asp Pro             100 105 110 Leu Glu Leu Val Val Thr Gly Asp Tyr Trp Thr Pro Ser Leu Leu Ala         115 120 125 Gln Ala Ser Pro Val Val Thr Ser Gly Gyr Tyr Val Thr Leu Gln Cys     130 135 140 Glu Ser Trp His Asn Asp His Lys Phe Ile Leu Thr Val Glu Gly Pro 145 150 155 160 Gln Lys Leu Ser Trp Thr Gln Asp Ser Gln Tyr Asn Tyr Ser Thr Arg                 165 170 175 Lys Tyr His Ala Leu Phe Ser Val Gly Pro Val Thr Pro Asn Gln Arg             180 185 190 Trp Ile Cys Arg Cys Tyr Ser Tyr Asp Arg Asn Arg Pro Tyr Val Trp         195 200 205 Ser Pro Pro Ser Glu Ser Val Glu Leu Leu Val Ser Gly Asn Leu Gln     210 215 220 Lys Pro Thr Ile Lys Ala Glu Pro Gly Pro Val Ile Ala Ser Lys Arg 225 230 235 240 Ala Met Thr Ile Trp Cys Gln Gly Asn Leu Asp Ala Glu Val Tyr Phe                 245 250 255 Leu His Asn Glu Gly Ser Gln Lys Thr Gln Ser Thr Gln Thr Leu Gln             260 265 270 Gln Pro Gly Asn Lys Gly Lys Phe Phe Ile Pro Ser Met Thr Arg Gln         275 280 285 His Ala Gly Gln Tyr Arg Cys Tyr Cys Tyr Gly Ser Ala Gly Trp Ser     290 295 300 Gln Pro Ser Asp Thr Leu Glu Leu Val Val Thr Gly Ile Tyr Glu His 305 310 315 320 Tyr Lys Pro Arg Leu Ser Val Leu Pro Ser Pro Val Val Thr Ala Gly                 325 330 335 Gly Asn Met Thr Leu His Cys Ala Ser Asp Phe His Tyr Asp Lys Phe             340 345 350 Ile Leu Thr Lys Glu Asp Lys Lys Phe Gly Asn Ser Leu Asp Thr Glu         355 360 365 His Ile Ser Ser Ser Arg Gln Tyr Arg Ala Leu Phe Ile Ile Gly Pro     370 375 380 Thr Thr Pro Thr His Thr Gly Thr Phe Arg Cys Tyr Gly Tyr Phe Lys 385 390 395 400 Asn Ala Pro Gln Leu Trp Ser Val Pro Ser Asp Leu Gln Gln Ile Leu                 405 410 415 Ile Ser Gly Leu Ser Lys Lys Pro Ser Leu Leu Thr His Gln Gly His             420 425 430 Ile Leu Asp Pro Gly Met Thr Leu Thr Leu Gln Cys Tyr Ser Asp Ile         435 440 445 Asn Tyr Asp Arg Phe Ala Leu His Lys Val Gly Gly Ala Asp Ile Met     450 455 460 Gln His Ser Ser Gln Gln Thr Asp Thr Gly Phe Ser Val Ala Asn Phe 465 470 475 480 Thr Leu Gly Tyr Val Ser Ser Ser Thr Gly Gly Gln Tyr Arg Cys Tyr                 485 490 495 Gly Ala His Asn Leu Ser Ser Glu Trp Ser Ala Ser Ser Glu Pro Leu             500 505 510 Asp Ile Leu Ile Thr Gly Gln Leu Pro Leu Thr Pro Ser Leu Ser Val         515 520 525 Lys Pro Asn His Thr Val His Ser Gly Glu Thr Val Ser Leu Leu Cys     530 535 540 Trp Ser Met Asp Ser Val Asp Thr Phe Ile Leu Ser Lys Glu Gly Ser 545 550 555 560 Ala Gln Gln Pro Leu Arg Leu Lys Ser Lys Ser His Asp Gln Gln Ser                 565 570 575 Gln Ala Glu Phe Ser Met Ser Ala Val Thr Ser His Leu Ser Gly Thr             580 585 590 Tyr Arg Cys Tyr Gly Ala Gln Asn Ser Ser Phe Tyr Leu Leu Ser Ser         595 600 605 Ala Ser Ala Pro Val Glu Leu Thr Val Ser Gly Pro Ile Glu Thr Ser     610 615 620 Thr Pro Pro Pro Thr Met Ser Met Pro Leu Gly Gly Leu His Met Tyr 625 630 635 640 Leu Lys Ala Leu Ile Gly Val Ser Val Ala Phe Ile Leu Phe Leu Phe                 645 650 655 Ile Leu Ile Phe Ile Leu Leu Arg Arg Arg His Arg Gly Lys Phe Arg             660 665 670 Lys Asp Val Gln Lys Glu Lys Asp Leu Gln Leu Ser Ser Gly Ala Glu         675 680 685 Glu Pro Ile Thr Arg Lys Gly Glu Leu Gln Lys Arg Pro Asn Pro Ala     690 695 700 Ala Ala Thr Gln Glu Glu Ser Leu Tyr Ala Ser Val Glu Asp Met Gln 705 710 715 720 Thr Glu Asp Gly Val Glu Leu Asn Ser Trp Thr Pro Pro Glu Glu Asp                 725 730 735 Pro Gln Gly Glu Thr Tyr Ala Gln Val Lys Pro Ser Arg Leu Arg Lys             740 745 750 Ala Gly His Val Ser Pro Ser Val Met Ser Arg Glu Gln Leu Asn Thr         755 760 765 Glu Tyr Glu Gln Ala Glu Glu Gly Gln Gly Ala Asn Asn Gln Ala Ala     770 775 780 Glu Ser Gly Glu Ser Gln Asp Val Thr Tyr Ala Gln Leu Cys Ser Arg 785 790 795 800 Thr Leu Arg Gln Gly Ala Ala Ala Ser Pro Leu Ser Gln Ala Gly Glu                 805 810 815 Ala Pro Glu Glu Pro Ser Val Tyr Ala Thr Leu Ala Ala Ala Arg Pro             820 825 830 Glu Ala Val Pro Lys Asp Val Glu Gln         835 840 <210> 2 <211> 598 <212> PRT <213> Homo sapiens <400> 2 Met Thr Pro Ile Val Thr Val Leu Ile Cys Leu Gly Leu Ser Leu Gly 1 5 10 15 Pro Arg Thr His Val Gln Thr Gly Thr Ile Pro Lys Pro Thr Leu Trp             20 25 30 Ala Glu Pro Asp Ser Val Ile Thr Gln Gly Ser Pro Val Thr Leu Ser         35 40 45 Cys Gln Gly Ser Leu Glu Ala Gln Glu Tyr Arg Leu Tyr Arg Glu Lys     50 55 60 Lys Ser Ala Ser Trp Ile Thr Arg Ile Arg Pro Glu Leu Val Lys Asn 65 70 75 80 Gly Gln Phe His Ile Pro Ser Ile Thr Trp Glu His Thr Gly Arg Tyr                 85 90 95 Gly Cys Gln Tyr Tyr Ser Arg Ala Arg Trp Ser Glu Leu Ser Asp Pro             100 105 110 Leu Val Leu Val Met Thr Gly Ala Tyr Pro Lys Pro Thr Leu Ser Ala         115 120 125 Gln Pro Ser Pro Val Val Thr Ser Gly Gly Arg Val Thr Leu Gln Cys     130 135 140 Glu Ser Gln Val Ala Phe Gly Gly Phe Ile Leu Cys Lys Glu Gly Glu 145 150 155 160 Asp Glu His Pro Gln Cys Leu Asn Ser Gln Pro His Ala Arg Gly Ser                 165 170 175 Ser Arg Ala Ile Phe Ser Val Gly Pro Val Ser Pro Asn Arg Arg Trp             180 185 190 Ser His Arg Cys Tyr Gly Tyr Asp Leu Asn Ser Pro Tyr Val Trp Ser         195 200 205 Ser Pro Ser Asp Leu Leu Glu Leu Leu Val Pro Gly Val Ser Lys Lys     210 215 220 Pro Ser Leu Ser Val Gln Pro Gly Pro Val Val Ala Pro Gly Glu Ser 225 230 235 240 Leu Thr Leu Gln Cys Val Ser Asp Val Gly Tyr Asp Arg Phe Val Leu                 245 250 255 Tyr Lys Glu Gly Glu Arg Asp Leu Arg Gln Leu Pro Gly Arg Gln Pro             260 265 270 Gln Ala Gly Leu Ser Gln Ala Asn Phe Thr Leu Gly Pro Val Ser Arg         275 280 285 Ser Tyr Gly Gly Gln Tyr Arg Cys Tyr Gly Ala Tyr Asn Leu Ser Ser     290 295 300 Glu Trp Ser Ala Pro Ser Asp Pro Leu Asp Ile Leu Ile Thr Gly Gln 305 310 315 320 Ile His Gly Thr Pro Phe Ile Ser Val Gln Pro Gly Pro Thr Val Ala                 325 330 335 Ser Gly Glu Asn Val Thr Leu Leu Cys Gln Ser Trp Arg Gln Phe His             340 345 350 Thr Phe Leu Leu Thr Lys Ala Gly Ala Ala Asp Ala Pro Leu Arg Leu         355 360 365 Arg Ser Ile His Glu Tyr Pro Lys Tyr Gln Ala Glu Phe Pro Met Ser     370 375 380 Pro Val Thr Ser Ala His Ala Gly Thr Tyr Arg Cys Tyr Gly Ser Leu 385 390 395 400 Asn Ser Asp Pro Tyr Leu Leu Ser His Pro Ser Glu Pro Leu Glu Leu                 405 410 415 Val Val Ser Gly Pro Ser Met Gly Ser Ser Pro Pro Thr Thr Gly Pro             420 425 430 Ile Ser Thr Pro Ala Gly Pro Glu Asp Gln Pro Leu Thr Pro Thr Gly         435 440 445 Ser Asp Pro Gln Ser Gly Leu Gly Arg His Leu Gly Val Val Ile Gly     450 455 460 Ile Leu Val Ala Val Val Leu Leu Leu Leu Leu Leu Leu Leu Leu Phe 465 470 475 480 Leu Ile Leu Arg His Arg Arg Gln Gly Lys His Trp Thr Ser Thr Gln                 485 490 495 Arg Lys Ala Asp Phe Gln His Pro Ala Gly Ala Val Gly Pro Glu Pro             500 505 510 Thr Asp Arg Gly Leu Gln Trp Arg Ser Ser Pro Ala Ala Asp Ala Gln         515 520 525 Glu Glu Asn Leu Tyr Ala Ala Val Lys Asp Thr Gln Pro Glu Asp Gly     530 535 540 Val Glu Met Asp Thr Arg Ala Ala Ala Ser Glu Ala Pro Gln Asp Val 545 550 555 560 Thr Tyr Ala Gln Leu His Ser Leu Thr Leu Arg Arg Lys Ala Thr Glu                 565 570 575 Pro Pro Pro Ser Gln Glu Arg Glu Pro Pro Ala Glu Pro Ser Ile Tyr             580 585 590 Ala Thr Leu Ala Ile His         595 <210> 3 <211> 1419 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 3 atgggatggt catgtatcat cctttttcta gtagcaactg caactggagc gtacgctgag 60 gttcagctgg tggagtctgg cggtggcctg gtgcagccag ggggctcact ccgtttgtcc 120 tgtgcagctt ctggcttcac cttcactaat tcctatatta gctgggtgcg tcaggccccg 180 ggtaagggcc tggaatgggt tggtgggatt tctccttctg gcggttctac ttactatgcc 240 gatagcgtca agggccgttt cactataagc gcagacacat ccaaaaacac agcctaccta 300 caaatgaaca gcttaagagc tgaggacact gccgtctatt attgtgcaaa atcggtctgg 360 gcgttcgctt actggggtca aggaaccctg gtcaccgtct cctcggcctc caccaagggc 420 ccatcggtct tccccctggc accctcctcc aagagcacct ctgggggcac agcggccctg 480 ggctgcctgg tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc 540 ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact ctactccctc 600 agcagcgtgg tgactgtgcc ctctagcagc ttgggcaccc agacctacat ctgcaacgtg 660 aatcacaagc ccagcaacac caaggtggac aagaaagttg agcccaaatc ttgtgacaaa 720 actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttcctc 780 ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 840 gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 900 gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgggtg 960 gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 1020 gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 1080 ccccgagaac cacaggtgta caccctgccc ccatcccggg aagagatgac caagaaccag 1140 gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 1200 agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 1260 tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 1320 ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 1380 ctgtctccgg gtaaatgagt gcgacggccc tagagtcga 1419 <210> 4 <211> 465 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 4 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Ala Tyr Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln             20 25 30 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe         35 40 45 Ser Asn Ser Tyr Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu     50 55 60 Glu Trp Val Gly Gly Ile Tyr Pro Ser Gly Gly Asn Thr Asn Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn                 85 90 95 Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val             100 105 110 Tyr Tyr Cys Ala Lys Ser Ala Trp Gln Phe Ala Tyr Trp Gly Gln Gly         115 120 125 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe     130 135 140 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 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 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu             180 185 190 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser         195 200 205 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro     210 215 220 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 225 230 235 240 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro                 245 250 255 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser             260 265 270 Arg Thr Pro Glu Val Thr Cys Val Val Asp Val Ser His Glu Asp         275 280 285 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn     290 295 300 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 305 310 315 320 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu                 325 330 335 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys             340 345 350 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr         355 360 365 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr     370 375 380 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 385 390 395 400 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu                 405 410 415 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys             420 425 430 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu         435 440 445 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly     450 455 460 Lys 465 <210> 5 <211> 465 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 5 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Ala Tyr Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln             20 25 30 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe         35 40 45 Ser Ser Asn Tyr Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu     50 55 60 Glu Trp Val Gly Gly Ile Asn Pro Thr Gly Gly Tyr Thr Tyr Tyr Ala 65 70 75 80 His Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn                 85 90 95 Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val             100 105 110 Tyr Tyr Cys Ala Arg Ser Val Trp Val Phe Asp Tyr Trp Gly Gln Gly         115 120 125 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe     130 135 140 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 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 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu             180 185 190 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser         195 200 205 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro     210 215 220 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 225 230 235 240 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro                 245 250 255 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser             260 265 270 Arg Thr Pro Glu Val Thr Cys Val Val Asp Val Ser His Glu Asp         275 280 285 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn     290 295 300 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 305 310 315 320 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu                 325 330 335 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys             340 345 350 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr         355 360 365 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr     370 375 380 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 385 390 395 400 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu                 405 410 415 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys             420 425 430 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu         435 440 445 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly     450 455 460 Lys 465 <210> 6 <211> 465 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 6 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Ala Tyr Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln             20 25 30 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe         35 40 45 Thr Asn Ser Tyr Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu     50 55 60 Glu Trp Val Gly Gly Ile Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn                 85 90 95 Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val             100 105 110 Tyr Tyr Cys Ala Lys Ser Val Trp Ala Phe Ala Tyr Trp Gly Gln Gly         115 120 125 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe     130 135 140 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 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 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu             180 185 190 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser         195 200 205 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro     210 215 220 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 225 230 235 240 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro                 245 250 255 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser             260 265 270 Arg Thr Pro Glu Val Thr Cys Val Val Asp Val Ser His Glu Asp         275 280 285 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn     290 295 300 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 305 310 315 320 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu                 325 330 335 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys             340 345 350 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr         355 360 365 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr     370 375 380 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 385 390 395 400 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu                 405 410 415 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys             420 425 430 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu         435 440 445 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly     450 455 460 Lys 465 <210> 7 <211> 233 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 7 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala             20 25 30 Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val         35 40 45 Ser Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys     50 55 60 Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg 65 70 75 80 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser                 85 90 95 Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr             100 105 110 Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr         115 120 125 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu     130 135 140 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 145 150 155 160 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly                 165 170 175 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr             180 185 190 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His         195 200 205 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val     210 215 220 Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 8 <211> 1419 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 8 atgggatggt catgtatcat cctttttcta gtagcaactg caactggagc gtacgctgag 60 gttcagctgg tggagtctgg cggtggcctg gtgcagccag ggggctcact ccgtttgtcc 120 tgtgcagctt ctggcttcac cttcagtaat tcctatatta gctgggtgcg tcaggccccg 180 ggtaagggcc tggaatgggt tggtgggatt tatccttctg gcggtaatac taactatgcc 240 gatagcgtca agggccgttt cactataagc gcagacacat ccaaaaacac agcctaccta 300 caaatgaaca gcttaagagc tgaggacact gccgtctatt attgtgcaaa aagcgcctgg 360 cagttcgctt actggggtca aggaaccctg gtcaccgtct cctcggcctc caccaagggc 420 ccatcggtct tccccctggc accctcctcc aagagcacct ctgggggcac agcggccctg 480 ggctgcctgg tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc 540 ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact ctactccctc 600 agcagcgtgg tgactgtgcc ctctagcagc ttgggcaccc agacctacat ctgcaacgtg 660 aatcacaagc ccagcaacac caaggtggac aagaaagttg agcccaaatc ttgtgacaaa 720 actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttcctc 780 ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 840 gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 900 gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgggtg 960 gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 1020 gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 1080 ccccgagaac cacaggtgta caccctgccc ccatcccggg aagagatgac caagaaccag 1140 gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 1200 agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 1260 tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 1320 ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 1380 ctgtctccgg gtaaatgagt gcgacggccc tagagtcga 1419 <210> 9 <211> 1419 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 9 atgggatggt catgtatcat cctttttcta gtagcaactg caactggagc gtacgctgag 60 gttcagctgg tggagtctgg cggtggcctg gtgcagccag ggggctcact ccgtttgtcc 120 tgtgcagctt ctggcttcac cttcagtagt aactatatta gctgggtgcg tcaggccccg 180 ggtaagggcc tggaatgggt tggtgggatt aatcctactg gcggttatac ttactatgcc 240 catagcgtca agggccgttt cactataagc gcagacacat ccaaaaacac agcctaccta 300 caaatgaaca gcttaagagc tgaggacact gccgtctatt attgtgcaag aagcgtgtgg 360 gtgttcgatt actggggtca aggaaccctg gtcaccgtct cctcggcctc caccaagggc 420 ccatcggtct tccccctggc accctcctcc aagagcacct ctgggggcac agcggccctg 480 ggctgcctgg tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc 540 ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact ctactccctc 600 agcagcgtgg tgactgtgcc ctctagcagc ttgggcaccc agacctacat ctgcaacgtg 660 aatcacaagc ccagcaacac caaggtggac aagaaagttg agcccaaatc ttgtgacaaa 720 actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttcctc 780 ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 840 gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 900 gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgggtg 960 gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 1020 gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 1080 ccccgagaac cacaggtgta caccctgccc ccatcccggg aagagatgac caagaaccag 1140 gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 1200 agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 1260 tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 1320 ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 1380 ctgtctccgg gtaaatgagt gcgacggccc tagagtcga 1419 <210> 10 <211> 107 <212> PRT <213> Homo sapiens <400> 10 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr             20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ser Gly Ile Ser Gly Asp Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Phe Asp Tyr Trp Gly Gln Gly Thr             100 105 <210> 11 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 11 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ser             20 25 30 Tyr Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Gly Gly Ile Tyr Pro Ser Gly Gly Asn Thr Asn Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Lys Ser Ala Trp Gln Phe Ala Tyr Trp Gly Gln Gly Thr             100 105 110 <210> 12 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 12 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Asn             20 25 30 Tyr Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Gly Gly Ile Asn Pro Thr Gly Gly Tyr Thr Tyr Tyr Ala His Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala                 85 90 95 Arg Ser Val Trp Val Phe Asp Tyr Trp Gly Gln Gly Thr             100 105 <210> 13 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 13 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asn Ser             20 25 30 Tyr Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Gly Gly Ile Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Lys Ser Val Trp Ala Phe Ala Tyr Trp Gly Gln Gly Thr             100 105 110 <210> 14 <211> 108 <212> PRT <213> Homo sapiens <400> 14 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Asn Tyr             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile         35 40 45 Tyr Ala Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Leu Pro Trp                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg             100 105 <210> 15 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 15 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala             20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile         35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Thr Pro Pro                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg             100 105

Claims (43)

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. 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. 1, 2, or 3 from the 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) An isolated anti-PirB / LILRB antibody comprising a hypervariable region sequence. The antibody of claim 3, comprising all hypervariable region sequences of the YW259.2 antibody heavy chain (SEQ ID NO: 4 or 11). The antibody of claim 3, comprising all hypervariable region sequences of the YW259.9 antibody heavy chain (SEQ ID NO: 5 or 12). The antibody of claim 3, comprising all hypervariable region sequences of the YW259.12 antibody heavy chain (SEQ ID NO: 6 or 13). The antibody according to any one of claims 3 to 6, further comprising a light chain. 8. The antibody of claim 7, wherein the light chain comprises one, two, or three hypervariable region sequences of the polypeptide sequence of SEQ ID NO: 15. 8. The antibody of claim 7, wherein the light chain comprises all hypervariable region sequences of the polypeptide sequence of SEQ ID NO: 7 or 15. The antibody of claim 3, wherein the antibody is selected from the group consisting of antibody YW259.2, YW259.9, and YW259.12. An isolated anti-PirB / LILRB antibody wherein the full length IgG form of the antibody specifically binds human PirB (LILRB) with a binding affinity of at least 5 nM. An isolated anti-PirB / LILRB antibody wherein the full length IgG form of the antibody specifically binds human PirB (LILRB) with a binding affinity of at least 1 nM. The antibody of claim 1, which promotes axon regeneration. The antibody of claim 1, which promotes regeneration of CNS neurons. The antibody of any one of claims 1 to 12, which at least partially restores neurite outgrowth inhibition by Nogo66 and myelin. The antibody according to any one of claims 1 to 12, which is a monoclonal antibody. The antibody of claim 1, wherein the antibody is selected from the group consisting of chimeric antibodies, humanized antibodies, affinity matured antibodies, human antibodies and bispecific antibodies. The antibody of claim 1, wherein the antibody is an antibody fragment. The antibody according to any one of claims 1 to 12, which is an immunoconjugate. The polynucleotide encoding the antibody of any one of claims 1 to 12, or a heavy or light chain thereof. A vector comprising the polynucleotide of claim 20. The vector of claim 21 which is an expression vector. A host cell comprising the vector of claim 21. The host cell of claim 23, which is a prokaryotic cell. The host cell of claim 23, which is a eukaryotic cell. The host cell of claim 25, which is a mammalian cell. A method of making an anti-PirB / LILRB antibody comprising (a) expressing the vector of claim 22 in a suitable host cell, and (b) recovering the antibody. The method of claim 27, wherein the host cell is a prokaryotic cell. The method of claim 27, wherein the host cell is a eukaryotic cell. A composition comprising the anti-PirB / LILRB antibody of claim 1, and a pharmaceutically acceptable excipient. 31. The composition of claim 30, further comprising a second medicament, wherein the anti-PirB / LILRB antibody is the first medicament. 32. The composition of claim 31, wherein the second medicament is an NgR inhibitor. 33. The composition of claim 32, wherein the NgR inhibitor is an anti-NgR antibody. A kit comprising the anti-PirB / LILRB antibody of any one of claims 1-12. A method of promoting axon regeneration comprising administering to a subject in need thereof an effective amount of the anti-PirB / LILRB antibody of claim 1. The method of claim 35, wherein the subject is a human patient. The method of claim 36, wherein survival or neurons are improved. The method of claim 36, wherein the growth of neurons is induced. A method of treating a neurodegenerative disease, comprising administering to a subject in need thereof an effective amount of the anti-PirB / LILRB antibody of claim 1. The method of claim 39, wherein the neurodegenerative disease is characterized by physical damage to the central nervous system. 41. The method of claim 40, wherein the neurodegenerative disease is brain injury associated with stroke. The neurodegenerative disorder of claim 39, wherein the neurodegenerative disorder is trigeminal neuralgia, Yeti neuralgia, Bell's Palsy, Myasthenia Gravis, Muscular Dystrophy, Amyotrophic Lateral Sclerosis (ALS), Multiple Sclerosis (MS), Progressive Muscular Dystrophy, Progressive Training Atrophy, Peripheral nerve injury, intervertebral hernia, rupture, or departure caused by physical injury (eg burns, wounds) or disease states such as diabetes, renal dysfunction or toxic effects of chemotherapeutic agents used to treat cancer and AIDS Invertebrate disc syndrome, cervical spondylosis, plexus disorders, thoracic outlet destruction syndrome, lead, answer, peripheral neuropathy such as caused by ticks, porphyrins, Guillain-Barre syndrome, Alzheimer's disease, Huntington's disease, and Parkinson's And selected from the group consisting of diseases. An anti-idiotype antibody that specifically binds to the anti-PirB / LILRB antibody of any one of claims 1 to 12.

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