RU2788122C2 - Chimeric protein composed of an ngf antagonist domain and a tnfα antagonist domain - Google Patents

Abstract

FIELD: biotechnology and medicine.

SUBSTANCE: invention relates to biotechnology and medicine. The proposed is a binding molecule containing an NGF antagonist domain (nerve growth factor) and a TNFα antagonist domain (tumor necrosis factor alpha), where the NGF antagonist is an anti-NGF antibody or its antigen-binding fragment, and where the anti-NGF antibody or its fragment contains a severe variable domain. chain (VH domain) containing a set of hypervariable regions (CDR) HCDR1, HCDR2, HCDR3, and a light chain variable domain (VL domain) containing a set of CDRs LCDR1, LCDR2 and LCDR3, and HCDR1 has the amino acid sequence of SEQ ID NO: 4 , HCDR2 has the amino acid sequence of SEQ ID NO: 5, HCDR3 has the amino acid sequence of any of SEQ ID NO: 6, 11, or 12, LCDR1 has the amino acid sequence of SEQ ID NO: 8, LCDR2 has the amino acid sequence of SEQ ID NO: 9, and LCDR3 has the amino acid sequence the sequence of SEQ ID NO: 10.

EFFECT: administration of an NGF antagonist and a TNFα antagonist of the present invention can control pain in a subject more effectively.

17 cl, 17 dwg, 1 tbl, 8 ex

Description

RELATED APPLICATIONS

This application claims priority of U.S. Provisional Application Serial No. 61/934828, filed February 2, 2014. All principles of the above application are incorporated into this description by reference.

SEQUENCE LIST

This application contains the Sequence Listing, which is in electronic ASCII format and is hereby incorporated by reference in its entirety. The specified ASCII document created on January 29, 2015 is named 110421-0054-WOl_SL.txt and is 134721 bytes in size.

PRIOR ART

Pain is one of the most common symptoms that seek medical attention and is the main complaint of half of all patients visiting a doctor. Despite the availability and widespread use of numerous pain relievers, success in the management of pain, especially chronic pain, has not been achieved. As a result, the burden of costs borne by society remains high. Various studies have estimated that pain is responsible for 50 million lost work days and $61.2 billion in lost productivity each year. For patients with chronic pain, only about half of them are able to manage their pain with the prescribed treatment options available. The total turnover of the market for prescription painkillers is approximately 25 billion dollars a year. These data suggest that there remains a strong need for safe and effective new pain relievers.

Therapeutic agents that reduce tissue levels or inhibit the effects of secreted nerve growth factor (NGF or beta-NGF) could be just such new painkillers. It is well known that NGF plays a key role in the development of the nervous system; however, NGF is also a recognized target for pain management as it causes pain in animals and humans. In adults, NGF specifically stimulates the viability and survival of a subpopulation of central and peripheral neurons (Huang & Reichardt, Ann. Rev. Neurosci., 24: 677-736 (2001)). NGF also contributes to modulating the functional characteristics of these neurons and exerts tonic control of the sensitivity or excitability of sensory nociceptors called nociceptors (Priestley et al ., Can. J. Physiol. Pharmacol., 80: 495-505 (2002); Bennett, Neuroscientist , 7:13-17 (2001)). Nociceptors perceive and transmit various pain stimuli to the central nervous system, which cause the perception of pain (nociception). NGF receptors are located on nociceptors. NGF expression is upregulated in injured and inflamed tissue and is upregulated in human pain conditions. Thus, due to the involvement of NGF in nociception, NGF-binding agents that reduce NGF levels find use as analgesic therapeutic agents.

Subcutaneous injections of NGF itself cause pain in humans and animals. Injected NGF leads to rapid thermal hyperalgesia followed by delayed thermal hyperalgesia and mechanical allodynia (Petty et al ., Ann. Neurol., 36: 244-46 (1994); McArthur et al ., Neurology, 54: 1080-88 (2000 )). Similarly, endogenously secreted NGF is pronociceptive. Tissue injury-induced release of NGF and its subsequent action in the periphery plays an important role in inducing thermal hyperalgesia through a process of "peripheral sensitization" (Mendell & Arvanian, Brain Res. Rev., 40: 230-39 (2002)). Tissue injury stimulates the release of pronociceptive and pro-inflammatory cytokines, which in turn induce the release of NGF from keratinocytes and fibroblasts. This released NGF acts directly on nociceptors to induce pain or nociceptive states within minutes of the onset of pain. At the same time, NGF also acts indirectly, inducing and maintaining nociceptive/pain states with preemptive release. It triggers mast cell degranulation to release pronociceptive agents such as histamine and serotonin and, importantly, even more NGF, and can also stimulate sympathetic nerve endings to release pronociceptive neurotransmitters such as norepinephrine (Ma & Woolf, Neuroreport., 8: 807-10 (1997)).

Tissue levels of NGF are elevated in animals injected with complete Freund's adjuvant (CFA) and carrageenan (Ma & Woolf, Neuroreport., 8: 807-10 (1997); Amann & Schuligoi, Neurosci. Lett., 278: 173-78 (2000 )). NGF potentiates the response to capsaicin in the dorsal root ganglia (DRG) in the rat. Elevated NGF levels have been documented in patients suffering from rheumatoid arthritis (Aloe & Tuveri, Clin. Exp. Rheumatol. 15: 433-38 (1997)) or cystitis (Lowe et al ., Br. J. Urol. 79: 572-77 (1997)). Injury to peripheral nerves in rodents enhances NGF messenger RNA (mRNA) expression in macrophages, fibroblasts, and Schwann cells (Heumann et al ., J. Cell Biol. 104: 1623-31 (1987)). Overexpression of NGF in transgenic mice results in a greater increase in neuropathic pain behavior after nerve injury than in wild-type mice (Ramer et al ., Pain, Supp. 6: S111-20 (1998)). Over a period of time from several hours to 15 days, elevated levels of NGF are involved in the stimulation of "central sensitization" - an increase in neurotransmission in the synapses of the nociceptive pathways of the spinal cord. Central sensitization leads to the development of persistent and chronic hyperalgesia and allodynia. The internalization of complexes of NGF and its high affinity receptor, receptor tyrosine kinase A (trkA), is thought to be involved in this process. Retrograde transport of these complexes to nociceptive cell bodies in the DRG enhances secretion of nociceptive neuropeptides, such as substance P or calcitonin genetically related peptide (CGRP), activation of protein kinase C (PKC), and activation of N-methyl-D-aspartate (NMDA) receptors in the posterior horn. spinal cord (Sah et al ., Nat. Rev. Drug Disc., 2: 460-72 (2003)) are all processes that stimulate sensitization of nociceptive pathways. NGF also plays a role in the activation and redistribution of voltage-gated and ligand-gated ion channels, including sodium channels of various subtypes and the capsaicin receptor, transient receptor potential cation channel, subfamily member 1 V (TRPV1) (Mamet et al ., J. Biol. Chem., 278: 48907-13 (1999), Fjell et al ., J. Neurosci. Res., 57: 39-47 (1999), Priestley et al ., Can. J. Physiol. Pharmacol., 80: 495 -505 (2002)). Alterations in the activities and/or expression of transmitters, receptors, and ion channels underlie the increased sensitivity and excitability of nociceptors associated with neuropathic pain conditions.

NGF-induced nociception/pain is mediated by the high affinity NGF receptor, receptor tyrosine kinase A (trkA) (Sah, et al ., Nat. Rev. Drug Disc. 2: 460-72 (2003)). Approximately 40-45% of nociceptive cell bodies in DRG express trkA. They are cell bodies of small diameter fibers or C-fibers that also express secreted pronociceptive peptides, substance P and CGRP. These fibers end in plates I and II of the posterior horn, from where they transmit pain stimuli perceived by peripheral nociceptors to the central nervous system. Mutations or deletions in the trkA gene result in a loss of pain phenotype in both humans (Indo, Clin. Auton. Res., 12 (Supp 1): I20-I32 (2002)) and trkA knockout mice ( de Castro et al ., Eur. J. Neurosci., 10: 146-52 (1998)). Importantly, trkA expression is upregulated in animals tested in models of arthritic pain (Pozza et al ., J. Rheumatol. 27: 1121-27 (2000)) or bladder pain (Qiao & Vizzard, J. Comp Neurol., 454: 200-11 (2002)), or inflammatory pain induced by injection of CFA or carrageenan into the paw (Cho et al ., Brain Res., 716: 197-201 (1996)).

NGF also binds to the p75 neurotrophin receptor (p75NTR). The task of p75NTR depends on its cellular environment and the presence of other receptors with which it is thought to function as an accessory receptor or co-receptor. As a result of the interaction of trkA with p75NTR, high-affinity NGF-binding sites are formed. The importance of such receptor interactions in NGF-mediated pain signaling has not been elucidated, but recent studies have noted the involvement of p75NTR in cellular processes that may be relevant (Zhang & Nicol, Neurosci. Lett., 366: 187-92 (2004)) . However, while p75NTR knockout mice exhibit elevated pain thresholds, they remain susceptible to the hyperalgesic effects of NGF, suggesting that trkA receptors alone are sufficient to mediate these effects (Bergmann et al ., Neurosci. Lett., 255: 87-90 (1998)).

Blocking NGF leads to a significant increase in efficacy compared to NSAIDs (non-steroidal anti-inflammatory drugs) in chronic nociceptive pain, such as osteoarthritis (OA) and chronic low back pain. A number of candidate therapeutic antibodies targeting NGF are at various stages of preclinical and clinical development. Such antibodies include, for example, tanezumab (PF-4383119; Pfizer), which is a humanized antibody in IgG2 format; SAR164877/REGN475 (Sanofi-Aventis/Regeneron Pharmaceuticals), which is a human antibody in IgG4 format; AMG 403 (Amgen/Johnson & Johnson), which is a human antibody in IgG2 format; PG110 (PanGenetics/Abbott), which is a humanized antibody in IgG4 format. Another candidate therapeutic antibody is described in WO 2006/077441 concerning anti-NGF antibodies and methods of treating diseases or disorders in which the interaction of NGF with the disclosed antibodies is important. MEDI-578 is a human IgG4 antibody. Despite the development of these candidates, there remains a need to provide an analgesic effect for a wide range of pain conditions through the use of an NGF-binding agent with enhanced efficacy and an improved safety profile.

Tumor necrosis factor-alpha (TNFα), also called cachectin, is a pleiotropic cytokine with a wide variety of biological activities affecting cytotoxicity, immune cell proliferation, inflammation, tumorigenesis, and viral replication. Kim et al ., J. Mol. Biol., 374, 1374 (2007). Initially, TNFα is produced as a transmembrane protein (tmTNFα), which is then cleaved by metalloproteinase to a soluble form (sTNFα). Wallis, Lancet Infect. Dis., 8(10): 601 (2008). TNFα (approximately 17 kDa) exists as a rigid homotrimeric molecule that binds to cell surface TNF receptor 1 or TNF receptor 2, inducing receptor oligomerization and signal transduction.

It is known that inflammatory cytokines, and in particular TNFα, play an important role in the occurrence of hyperalgesia. Leung, L., and Cahill, C.M.,J. Neuroinflammation,7: 27 (2010). Based on some preliminary data, it has been shown that TNFα inhibitors may be useful in the control of neuropathic pain. See, for example, Summer C.et al.,J. Peripher. Nerv. Syst., 6:67-72 (2001); Cohen et al., A&A, Feb 2013, 116, 2, 455-462; Genevayet al., Ann. Rheum. Dis., 2004, 63, 1120-1123. Results from clinical trials testing TNFα inhibitors as monotherapy for the treatment of neuropathic pain remain inconclusive. See Leung and Cahill (2010).

Despite the development of NGF-targeting and TNFα-targeting candidates for the treatment of pain, there remains a need to provide analgesic effect in various pain conditions through the use of agents that are more effective than the current standard of care. The present invention provides combined treatment methods targeting both NGF and TNFα that can increase efficacy and have the potential to reduce both the number and frequency of administrations for pain patients.

BRIEF DESCRIPTION OF THE INVENTION

The present invention provides methods for controlling pain in a subject comprising administering to a subject in need thereof an effective amount of a nerve growth factor (NGF) antagonist and a tumor necrosis factor-alpha (TNFα) antagonist or a binding molecule comprising an NGF antagonist domain and a TNFα antagonist domain. In some embodiments, such administration controls pain in a subject more effectively than administration of an equivalent amount of an NGF antagonist or a TNFα antagonist alone. In some embodiments, the method includes the co-administration of a TNFα antagonist and an NGF antagonist. In some embodiments, the TNFα antagonist and the NGF antagonist are administered sequentially or simultaneously.

In some embodiments, the method is sufficient to prevent, alleviate, lessen, or eliminate pain in a subject. In some embodiments, the pain is acute pain, transient pain, persistent or chronic nociceptive pain, or persistent or chronic neuropathic pain. In some embodiments, the method is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% more effective in controlling pain in a subject, than administering an equivalent amount of an NGF antagonist or a TNFα antagonist alone.

In some embodiments, the TNFα antagonist portion of the binding molecule binds to a polypeptide containing the amino acid sequence of SEQ ID NO: 2. In some embodiments, the NGF antagonist binds to a polypeptide containing the amino acid sequence of SEQ ID NO: 1.

In some embodiments, the NGF antagonist portion of the binding molecule is an anti-NGF antibody or an antigen-binding fragment thereof. In some embodiments, the anti-NGF antibody or fragment thereof can inhibit the binding of NGF to TrkA, p75NTR, or both TrkA and p75NTR. In some embodiments, the anti-NGF antibody preferentially blocks NGF binding to TrkA over NGF binding to p75NTR. In some embodiments, the anti-NGF antibody, or fragment thereof, binds to human NGF with an affinity of about 0.25-0.44 nM. In some embodiments, the anti-NGF antibody, or fragment thereof, binds to the same epitope as MEDI-578. In some embodiments, the anti-NGF antibody or fragment thereof competitively inhibits the binding of MEDI-578 to human NGF.

In some embodiments, the anti-NGF antibody or fragment thereof comprises an antibody heavy chain variable domain (VH domain) comprising a constellation of heavy chain hypervariable regions (CDRs) HCDR1, HCDR2, HCDR3, and an antibody light chain variable domain (VL domain) comprising a constellation of Light chain CDRs of LCDR1, LCDR2 and LCDR3, wherein HCDR1 has the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 4 with up to two amino acid substitutions, HCDR2 has the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 5 c up to and including two amino acid substitutions, HCDR3 has the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 6 with up to and including two amino acid substitutions, SSRIYDFNSALISYYDMDV (SEQ ID NO: 11) or SSRIYDMISSLQPYYDMDV (SEQ ID NO: 12), LCDR1 has amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 8 with up to two amino acid substitutions, LCDR2 has the amino acid sequence of SEQ ID NO: 9 or S EQ ID NO: 9 with up to two amino acid substitutions, and LCDR3 has the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 10 with up to two amino acid substitutions. In some embodiments, the anti-NGF antibody or fragment thereof comprises a VH domain having the amino acid sequence of SEQ ID NO: 3. In some embodiments, the anti-NGF antibody or fragment thereof contains a VL domain having the amino acid sequence of SEQ ID NO: 7. In some embodiments, the antibody to NGF, or a fragment thereof, contains a VH domain having an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the anti-NGF antibody or fragment thereof contains a VL domain having an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 7. In some embodiments, an anti-NGF antibody or fragment thereof contains a VH domain, having the amino acid sequence of SEQ ID NO: 94. In some embodiments, the anti-NGF antibody or fragment thereof contains a VL domain having the amino acid sequence of SEQ ID NO: 95. In some embodiments, the anti the anti-NGF body or fragment thereof contains a VH domain having an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 94. In some embodiments, the anti-NGF antibody or its the fragment contains a VL domain having an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 95.

In some embodiments, the anti-NGF antibody or fragment thereof is a full length antibody in H ₂ L ₂ format, a Fab fragment, a Fab' fragment, an F(ab) ₂ fragment, or a single chain Fv (scFv) fragment. In some embodiments, the anti-NGF antibody, or fragment thereof, is humanized, chimeric, primatized, or fully human. In some embodiments, the NGF antagonist is an anti-NGF scFv antibody fragment. In some embodiments, the scFv is SS stabilized. In some embodiments, the scFv fragment of an anti-NGF antibody contains, N-terminally to C-terminally, a VH domain having the amino acid sequence of SEQ ID NO: 3, a 15 amino acid linker sequence (GGGGS) ₃ (SEQ ID NO : 15), and a VL domain having the amino acid sequence of SEQ ID NO: 7. In some embodiments, the scFv fragment of an anti-NGF antibody comprises, N-terminally to the C-terminus, a VH domain having the amino acid sequence of SEQ ID NO: : 94, a 20 amino acid linker sequence (GGGGS) ₄ (SEQ ID NO: 19), and a VL domain having the amino acid sequence of SEQ ID NO: 95.

In certain aspects, the method includes administering a TNFα antagonist that inhibits TNFα binding to a TNF receptor (TNFR), thereby blocking TNFα activity. In some embodiments, the TNFα antagonist comprises an anti-TNFα antibody or an antigen-binding fragment thereof. In some embodiments, the anti-TNFα antibody or fragment thereof comprises an antibody VH domain containing a set of HCDR1, HCDR2, and HCDR3 CDRs and an antibody VL domain containing a set of LCDR1, LCDR2, and LCDR3 CDRs, where said CDRs are identical to HCDR1, HCDR2, HCDR3, LCDR1 , LCDR2 and LCDR3 of infliximab or HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of adalimumab.

In some embodiments, the binding molecule comprises a full-length anti-TNFα antibody and an anti-NGF scFv fragment fused to the C-terminus of the heavy chain of the anti-TNFα antibody. Such a binding molecule may contain a light chain containing the amino acid sequence of SEQ ID NO: 20 and a heavy chain containing the amino acid sequence of SEQ ID NO: 22.

In some embodiments, the TNFα antagonist comprises a soluble TNFα-binding TNFR fragment. In some embodiments, TNFR is TNFR-2 or a soluble fragment thereof. In other embodiments, TNFR is TNFR-1 or a soluble fragment thereof. In some embodiments, the soluble fragment of TNFR-1 is a fragment with a molecular weight of 55 kDa. In other embodiments, the soluble fragment of TNFR-2 is a fragment with a molecular weight of 75 kDa. In some embodiments, the TNFR fragment is fused to the Fc domain of an immunoglobulin. In some embodiments, the immunoglobulin Fc domain is a human IgG1 Fc domain. In some embodiments, the TNFα antagonist has the amino acid sequence shown in SEQ ID NO: 13, or a functional fragment thereof.

In some embodiments, the binding molecule is a fusion protein containing an NGF antagonist fused to a TNFα antagonist via a linker. In some embodiments, the binding molecule is a fusion protein homodimer.

In some embodiments, the NGF antagonist is the scFv domain of an anti-NGF antibody and the TNFα antagonist is a soluble TNFα binding fragment of TNFR-2 fused at its carboxy terminus to an immunoglobulin Fc domain. In some embodiments, the scFv is fused to the carboxy terminus of an immunoglobulin Fc domain via a linker.

In some embodiments, the binding molecule comprises a homodimer of a fusion polypeptide comprising, in N-terminal to C-terminal direction, a TNFα binding fragment (75 kDa) of TNFR-2, a human IgG1 Fc domain, a 10 amino acid linker (GGGGS) ₂ , a VH domain containing the amino acid sequence of SEQ ID NO: 3, a 15 amino acid linker sequence (GGGGS) ₃ (SEQ ID NO: 15), and a VL domain containing the amino acid sequence of SEQ ID NO: 7. In some in embodiments, the binding molecule comprises a fusion polypeptide homodimer comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the binding molecule comprises a fusion polypeptide homodimer comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical amino acid sequence of SEQ ID NO: 14.

In some embodiments, the binding molecule comprises a homodimer of a fusion polypeptide comprising, in N-terminal to C-terminal direction, a TNFα binding fragment (75 kDa) of TNFR-2, a human IgG1 Fc domain, a 10 amino acid linker (GGGGS) ₂ , a VH domain containing the amino acid sequence of SEQ ID NO: 94, a linker sequence of 20 amino acids (GGGGS) ₄ (SEQ ID NO: 19), and a VL domain containing the amino acid sequence of SEQ ID NO: 95. In some in embodiments, the binding molecule comprises a fusion polypeptide homodimer comprising the amino acid sequence of SEQ ID NO: 17. In some embodiments, the binding molecule comprises a fusion polypeptide homodimer comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical amino acid sequence of SEQ ID NO: 17.

In some embodiments, the binding molecule comprises a homodimer of a fusion polypeptide comprising, in N-terminal to C-terminal direction, a TNFα-binding fragment (75 kDa) of TNFR-2, a human IgG1 Fc domain, a linker sequence, and an anti-NGF antibody scFv domain. .

The invention also provides methods for inhibiting p38 phosphorylation in a cell, said method comprising contacting the cell with any of the polypeptides described herein (e.g., any of the binding molecules comprising an NGF antagonist domain and a TNFα antagonist domain described herein). ). The invention also provides methods for inhibiting extracellular signal-regulated kinase (ERK) phosphorylation in a cell, which method comprises contacting the cell with any of the polypeptides described herein (for example, with any of the binding molecules containing an NGF antagonist domain and TNFα antagonist domain described in this application). In some embodiments, the cell is a neuronal cell. In other embodiments, the cell is a peripheral neuronal cell. In the following embodiments, the cell is a central neuronal cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the mammal is a human. In some embodiments, the cell is in cell culture.

The invention also provides polynucleotide sequences encoding the binding molecules described in this application, vectors containing these polynucleotide sequences, and host cells containing these polynucleotides or vectors.

The invention also provides methods for preparing the binding molecules described in this application.

The invention also provides compositions, pharmaceutical compositions and kits containing the binding molecules described in this application.

BRIEF DESCRIPTION OF GRAPHICS/SHAPES

Fig. 1. Schematic representation of a TNFR2-Fc fusion protein (panel A) and an exemplary multispecific binding molecule, TNFR2-Fc_VH#4, containing a TNFR2-Fc domain fused to an anti-NGF scFv domain (panel B).

On FIG. 2A shows the results of the analysis, using gel permeation high performance liquid chromatography (SEC-HPLC), levels of aggregates, monomers and protein fragments in a batch of purified TNFR2-Fc_VH#4.

On FIG. 2B shows the results of SDS-PAGE analysis of purified TNFR2-Fc_VH#4 protein and purified TNFR2-Fc protein under reducing and non-reducing conditions. Gel loading order: 1. TNFR2-Fc_VH#4, 2. TNFR2-Fc_VL-VH (TNFR2-Fc fused to the scFv domain of an anti-NGF antibody, reversed variable domain gene), 3. TNFR2-Fc with an irrelevant scFv 1, 4. TNFR2-Fc, 5. TNFR2-Fc with irrelevant scFv 2.

On FIG. 3A shows the purity of TNFR2-Fc_VH#4 after purification on a protein A immobilized column. FIG. 3B shows the purity of TNFR2-Fc_VH#4 after a second purification step on a sulfopropyl(SP)-Sepharose column.

On FIG. 4 shows the results of stability analysis of TNFR2-Fc_VH#4 using differential scanning calorimetry.

On FIG. 5 shows the binding of TNFR2-Fc_VH#4 to TNFα and NGF, either alone or together, as determined by ELISA. On FIG. 5A shows binding to NGF, FIG. 5B shows binding to TNFα and FIG. 5C shows binding simultaneously to TNFα and NGF.

On FIG. 6 shows a sensogram of surface plasmon resonance binding analysis for TNFR2-Fc_VH#4. Competitive binding of antigens to the multispecific antibody TNFR2-Fc_VH#4 was performed using BIAcore 2000. Simultaneous binding to antigens was assessed by binding a series of TNFα and NGF to TNFR2-Fc_VH#4 attached to the surface of the sensor chip. The first part of the sensogram shows the binding of saturating amounts of TNFα to the multispecific antibody, the second part of this sensogram shows binding when a second antigen is applied, either TNFα again, indicating complete surface saturation, or an equimolar mixture of TNFα and NGF. An increase in the number of resonance units (RU) corresponded to the binding of NGF to a multispecific molecule and, therefore, spoke of the joint participation of antigens. The analysis was also carried out under the conditions of adding antigens in reverse order, which confirmed these data.

On FIG. 7 shows inhibition of NGF-mediated TF-1 cell proliferation. A. NGF-mediated proliferation in the absence of an added NGF antagonist. B. Inhibition of human NGF (hNGF) response by TNFR2-Fc_VH#4. C. Inhibition of mouse NGF (muNGF) response by TNFR2-Fc_VH#4. NGF activity is usually expressed in RLU - relative luminescence units, and % NGF-mediated proliferation is calculated as % response to NGF ligand alone using the following formula:

100*(RLU per well - RLU background)/(total RLU - RLU background),

where RLU background denotes the average value for the environments used as controls, and the total number of RLU denotes the average value for the controls, representing only the ligand. D. Inhibition of response to human NGF by TNFR2-Fc_VarB and ndimab VarB. E. Inhibition of mouse NGF response by TNFR2-Fc_VarB and ndimab VarB.

On FIG. 8 shows inhibition of TNFα-induced caspase 3 activity in U937 cells. A. TNFα-induced caspase 3 activity in U937 cells in the absence of added TNFα antagonist. B. Inhibition of TNFα-induced caspase 3 activity in U937 cells, shown as percent response in the absence of added antagonist. TNF activity is usually expressed in RFU, relative fluorescence units, and % TNF-mediated caspase 3 release is calculated as % response to TNF ligand alone, using the formula described above for FIG. 7C. C. Similar results are shown for the related molecule TNFR2-Fc_VarB and ndimab VarB.

On FIG. 9 shows the effect of combined treatment with etanercept and MEDI-578 on mechanical hyperalgesia induced by partial ligation of the sciatic nerve. Results are shown as ratio between ipsilateral/contralateral paws. N = 9-10 per group. The data were analyzed using two-way analysis of variance (2-way ANOVA), using time and method of treatment as dependent factors. Next, statistical significance was determined using Bonferroni's post hoc test. ***p<0.001 relative to control Op. (operated animals) + CAT-251.

On FIG. 10A shows the effect of TNFR2-Fc_VH#4 on mechanical hyperalgesia induced by partial sciatic nerve ligation. Results are shown as ratio between ipsilateral/contralateral paws. N = 10 per group. Data were analyzed using two-way ANOVA using time and mode of treatment as dependent factors. Next, statistical significance was determined using Bonferroni's post hoc test. ***p<0.001 vs. bispecific isotype control (0.03 mg/kg, sc). On FIG. 10B shows similar results for the related molecule TNFR2-Fc_VarB.

On FIG. 11 shows the effect of co-administration of MEDI-578 and etanercept on pain relief in a mechanical hypersensitivity model using joint pain as an example. N = 9-10 per group. Data were analyzed using two way ANOVA. Next, statistical significance was determined using Bonferroni's post hoc test. *p>0.05; ***p<0.001 vs. CAT-251 (0.03 mg/kg, i.v.) + PBS (10 ml/kg, i.p.).

On FIG. 12 shows the effect of TNFR2-Fc_VH#4 on pain relief in a mechanical hypersensitivity model. N = 9-10 per group. Data were analyzed using two way ANOVA. Next, statistical significance was determined using Bonferroni's post hoc test. ***p<0.001 vs. bispecific isotype control (0.01 mg/kg, sc).

On FIG. 13 shows the effect of five different doses of TNFR2-Fc_varB on CFA-induced hyperalgesia in a rat model. Statistical analysis: repeated measures ANOVA, then test for planned comparisons using InVivoStat. Significant elimination of hyperalgesia compared to filler at each time point.

Fig. 14: Heat map showing HTRF (time-resolved homogeneous fluorescence) ratios for p38 phosphorylation reactions.

Fig. 15: Dose-response curves showing the effect of TNFα, NGF, or a combination of TNFα and NGF on p38 phosphorylation.

Fig. 16: Heat map showing HTRF ratios for ERK phosphorylation reactions.

Fig. 17: Dose-response curves showing the effect of TNFα, NGF, or a combination of TNFα and NGF on ERK phosphorylation.

DETAILED DESCRIPTION

Definitions

It should be noted that the term "object" refers to one or more such "object". As such, the terms "one/one/one or more" and "at least one/one/one" may be used interchangeably throughout this specification.

In addition, when used in this description, the expression "and/or" should be interpreted as a specific description of each of two specific features or components, with or without the second. Thus, the term "and/or" as used herein in a phrase such as "A and/or B" is intended to include "A and B", "A or B", (only) "A ' and (only) 'B'. Likewise, the term "and/or" as used in a phrase such as "A, B and/or C" is intended to cover each of the following: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; (only) A; (only) B; and (only) C.

Obviously, in addition to cases where aspects in this application are described using the word "comprising", other similar aspects are also proposed, described in terms of "consisting of" and/or "consisting essentially of".

Unless otherwise indicated, all technical and scientific terms used in this specification have the meaning generally understood by one of ordinary skill in the art to which this invention pertains. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press, provide the skilled artisan with a vocabulary of many of the commonly used terms used in this specification.

Units of measurement, prefixes and symbols are given in the form adopted by the International System of Units (SI). Numeric ranges contain the numbers that define the range. Unless otherwise indicated, amino acid sequences are written from left to right in the direction from the amino end to the carboxy end. The headings provided in this specification do not limit the various aspects of the invention, which may be defined by reference to the specification as a whole. Accordingly, the terms described immediately below are more fully defined by reference to the description in its entirety.

Used in this description, the term "binding molecule" refers in its broadest sense to a molecule that specifically binds to an antigenic determinant, such as an antigen. Non-limiting examples of a binding molecule include antibodies or fragments thereof, soluble receptor fusion proteins or fragments thereof, non-immunoglobulin scaffolds or fragments thereof, all of which retain antigen-specific binding.. Exemplary non-immunoglobulin scaffolds include Tn3 (Koide et al., J. Mol. Biol., 2012, Jan 13, 415(2): 393-405), DARPin (engineered ankyrin repeat protein) (Boersma & Pluckthun, Curr. Opin. Biotechnol., 2011, 22(6): 849-57), anticalin (Gebauer & Skerra, Methods Enzymol., 2012, 503: 157-88). Typical soluble receptor-based fusion proteins as well as antibodies are shown below. In some embodiments, the binding molecule may be designed as a molecule containing combinations of such antibodies or fragments thereof, soluble receptor fusion proteins or fragments thereof, and non-immunoglobulin scaffolds or fragments thereof.

A binding molecule, or any portion of a binding molecule that recognizes an antigen, is referred to herein as a "binding domain". In addition to specifically referring to full-length binding molecules, such as natural antibodies, the term "binding molecule" includes, without limitation, full-length antibodies or other non-antibody binding molecules, as well as antigen-binding fragments, variants, analogs, or derivatives of such binding molecules, for example , natural antibody or immunoglobulin molecules, or engineered binding molecules or fragments that bind to an antigen in a manner similar to a full-length binding molecule.

In some embodiments, the invention provides some multispecific binding molecules, eg, bispecific, trispecific, tetraspecific, etc. binding molecules or antigen-binding fragments, variants or derivatives thereof. As used herein, a multispecific binding molecule may include one or more antibody binding domains, one or more non-antibody binding domains, or a combination thereof.

As used herein, the term "nerve growth factor" ("NGF"), also referred to in the literature as beta nerve growth factor, refers to a secreted protein that is involved in the growth and survival of various neurons. Human NGF (humNGF) is represented by Genbank Accession Number NP_002497.2 and is referred to herein as SEQ ID NO: 1. The term "NGF" as used herein is not limited to human NGF and includes all species orthologues of human NGF. The term "NGF" encompasses pro-NGF, pro-NGF, full-length NGF, as well as any form of NGF that results from cellular processing. The term also encompasses naturally occurring variants of NGF, such as splice variants, allelic variants, and isoforms. NGF can bind to two receptors: the p75 neurotrophin receptor (p75(NTR)) and TrkA, a transmembrane tyrosine kinase. NGF is a well established target for pain relief and is known to mediate nociceptor sensitization.

A number of agents are currently being tested as antagonists of NGF activity. One such anti-NGF agent is trkA-Fc, which acts as a decoy or scavenger by binding to endogenous NGF and thereby inactivating it. TrkA-Fc is a fusion protein consisting of the NGF-binding site of trkA fused to a constant domain (Fc) fragment of an IgG antibody. TrkA-Fc leads to the development of hypoalgesia in previously unexplored animals, attenuates nociceptive responses and reduces the outgrowth of unmyelinated pain-sensing neurons (Bennett, DL et al . (1998) Eur. J. Neurosci. , 10 : 1282-91).

The binding molecules described in this application are particularly well suited for the safe and effective treatment of NGF-mediated pain, since NGF levels increase in the periphery in response to pain stimuli, and antibodies have low permeability through the blood-brain barrier. A number of antibodies to NGF and their antigen-binding fragments that can be used in therapeutic regimens and compositions described in this invention can be found in the literature; see, for example, PCT Publication Nos. WO02/096458 and WO04/032870.

The term "MEDI-578" refers to an antibody that specifically binds to NGF, which is the subject of International Application No. PCT/GB2006/000238 and US Patent Application Publication No. 2008/0107658 A1; both are incorporated herein by reference in their entirety. The MEDI-578 heavy and light chain sequences are shown in SEQ ID NOs: 3 and 7, respectively.

The term NGF-NG refers to an antibody that specifically binds to NGF. The NGF-NG heavy and light chain sequences are shown in SEQ ID NOs: 24 and 26, respectively.

The term "tumor necrosis factor alpha"("TNFα"), also referred to in the literature as cachectin, is an APC1 protein; tumor necrosis factor; TNF; or TNF ligand superfamily member 2, as used herein, refers to a specific TNFα protein and not to a TNF ligand superfamily. Human TNFα is listed in Genbank with accession number NP_000585.2 and is listed as SEQ ID NO: 2. The term "TNFα" as used herein is not limited to human TNF but includes all human TNFα species orthologues. The term "TNFα" encompasses pro-form TNFα, pro-TNFα, full-length TNFα, as well as any form of TNFα that results from cellular processing. The term also encompasses natural and non-natural variants of TNFα, such as splice variants, allelic variants and isoforms. TNFα can bind to two receptors, TNFR1 (TNF receptor type 1; CD120a; p55/60) and TNFR2 (TNF receptor type 2; CD120b; p75/80). TNFα acts as a pro-inflammatory cytokine, for example, functioning in neuroinflammation. For example, TNFα is thought to be functionally involved in neuropathic pain (Leung, L. and Cahill, CM., J. Neuroinflammation , 7:27 ( 2010)).

A large number of TNFα antagonists are known in the art, and many of them are marketed as therapeutic agents. Examples of commercially available TNF-alpha antagonists that may be used in the therapeutic regimens and compositions of this invention include etanercept (ENBREL®, Amgen/Pfizer), infliximab (eg, REMICADE®, Centocor), certolizumab pegol (eg, CIMZIA ®, UCB), golimumab (eg SIMPONI™, Centocor) and adalimumab (eg HUMIRA®/TRUDEXA®, Abbott).

The term "isolated" binding molecule, polypeptide, antibody, polynucleotide, vector, host cell, or compound refers to a binding molecule, polypeptide, antibody, polynucleotide, vector, host cell, or compound that is not in the form found in nature. Isolated binding molecules, polypeptides, antibodies, polynucleotides, vectors, host cells, or compounds include those that have been altered, adapted, combined, rearranged, engineered, or otherwise processed to the extent that they are no longer in the form in which they occur. in nature. In some aspects, the "isolated" binding molecule, antibody, polynucleotide, vector, host cell, or compound is "recombinant".

As used herein, the terms "multifunctional polypeptide" and "bifunctional polypeptide" refer to a non-naturally occurring binding molecule designed to target two or more antigens. The multifunctional polypeptide described in this application is usually a genetically engineered fusion protein designed to combine two different desired biological functions in a single binding molecule. For example, a multifunctional polypeptide may be a multifunctional binding molecule. A typical multifunctional polypeptide described in this application is a multifunctional binding molecule containing an NGF antagonist domain, for example, a peptide domain that blocks, reduces or inhibits one or more of the natural functions of NGF, and a TNFα antagonist domain, for example, a peptide domain that blocks , impairs or inhibits one or more of the natural functions of TNFα.

One group of multifunctional polypeptides provided by this invention are multispecific binding molecules, e.g., binding molecules that contain one or more antibody binding domains, e.g., a "multispecific antibody", one or more non-antibody binding domains, e.g. trap, or a combination of both. Multispecific binding molecules, for example, containing one or more antibody binding domains, one or more non-antibody binding domains, or a combination thereof, are molecules containing binding domains capable of specifically recognizing and binding to at least two different epitopes. Different epitopes can either be on the same molecule (e.g., the same NGF) or on different molecules, so that, for example, multispecific binding molecules can specifically recognize and bind NGF as well as a molecule containing a different epitope, e.g. , TNFα, thus the multispecific binding molecule specifically recognizes NGF and TNFα.

Methods for producing multispecific binding molecules, for example, containing one or more antibody binding domains, one or more non-antibody binding domains, or a combination thereof, are known in the art (Dimasi, N., et al. , 2009, J. Mol. Biol. , 393 :672-92; Milstein et al ., 1983, Nature , 305 :537-539; Brennan et al ., 1985, Science , 229 :81; Suresh et al ., 1986, Methods in Enzymol. , 121 : 120, Traunecker et al ., 1991, EMBO J. , 10 : 3655-3659, Shalaby et al ., 1992, J. Exp. Med. , 175 : 217-225, Kostelny et al ., 1992, J. Immunol . , 148 :1547-1553; Gruber et al ., 1994, J. Immunol., 152 :5368; and U.S. Patent 5,731,168). Also covered are antibodies with more than two valences. For example, trispecific antibodies can be generated (Tutt et al ., J. Immunol., 147:60 (1991)).

The term "antibody" means an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing, through at least one antigen recognition site in the variable region of the immunoglobulin molecule. The term "antibody" as used herein includes intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab') ₂ and Fv fragments), single chain Fv (scFv) mutants, multispecific antibodies such as bispecific, trispecific, tetraspecific, etc. antibodies formed from at least two intact antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing an antigen-defining portion of an antibody, and any other modified immunoglobulin molecule that contains an antigen-recognition site, provided that these antibodies exhibit the desired biological activity. An antibody can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the identification of its constant domains heavy chain, called alpha, delta, epsilon, gamma, and mu, respectively. Different classes of immunoglobulins have different and well known subunit structures and spatial configurations.

In some embodiments, a "blocking" binding molecule, such as a blocking antibody, or an "antagonistic" binding molecule, such as, for example, an antagonistic antibody or fusion protein, is a molecule that inhibits or reduces the biological activity of the antigen to which it binds, such like NGF or TNFα. In certain aspects, blocking antibodies or antagonistic binding molecules substantially or completely inhibit the biological activity of a given antigen. For example, biological activity can be reduced by 0.01%, 0.1%, 0.5%, 1%, 5%, 10%, 20%, 30%, 50%, 70%, 80%, 90%, 95% or even 100%. "Antagonists" and "antagonist domains", as used herein, include polypeptides or other molecules that bind to their target (eg, TNFα or NGF), thereby blocking or inhibiting that target's interaction with the receptor. Thus, NGF and/or TNFα antagonists include molecules that block or inhibit the interaction of NGF with trkA or p75 neurotrophin, or the interaction of TNFα with TNFR-1 or TNFR-2. NGF and/or TNFα antagonists also include molecules that attenuate p38 phosphorylation and/or ERK phosphorylation. Exemplary antagonists include, but are not limited to, antibodies or antigen-binding fragments thereof and target-specific peptides of soluble non-signaling receptors ("decoy receptors" or ligand-binding fragments thereof).

The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigenicity-determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab') ₂ and Fv fragments, linear antibodies, single chain antibodies, and multispecific antibodies formed from antibody fragments. Antigen-binding fragments of non-antibody binding molecules described elsewhere in this specification are also provided in accordance with this invention.

"Monoclonal antibody" refers to a homogeneous population of antibodies involved in the highly specific recognition and binding of one antigenic determinant or one epitope. This distinguishes them from polyclonal antibodies, which usually include different antibodies directed against different antigenic determinants. The term "monoclonal antibody" encompasses both intact and full-length monoclonal antibodies, as well as antibody fragments (such as Fab, Fab', F(ab') ₂ , Fv), mutant forms of single-chain Fv (scFv), fusion proteins containing part of the antibody, and any other modified immunoglobulin molecule containing an antigen recognition site. In addition, "monoclonal antibody" refers to those antibodies that are produced by any of a number of methods, including, but not limited to, the use of hybridomas, selection using phages, recombinant expression, and the use of transgenic animals.

The term "humanized antibody" refers to forms of non-human (e.g., murine) antibodies, which are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof, that contain a minimal amount of non-human (e.g., murine) sequences. Generally, humanized antibodies are human immunoglobulins in which residues in a hypervariable region (CDR) are replaced with residues from a CDR of a non-human species (e.g., mouse, rat, rabbit, or hamster) that have the desired specificity, affinity, and potency (Jones et al . , 1986, Nature , 321: 522-525; Riechmann et al ., 1988, Nature , 332: 323-327; Verhoeyen et al ., 1988, Science , 239: 1534-1536). In some instances, Fv framework region (FR or FW) residues of a human immunoglobulin are replaced with corresponding residues found in a non-human antibody that has the desired specificity, affinity, and potency. The humanized antibody can be further modified by substitution of additional residues either in the Fv framework region and/or among such non-human antibody substitution residues to improve and optimize the specificity, affinity and/or performance of the antibody. In general, a humanized antibody will contain substantially all of at least one, and usually two or three, variable domains containing all or substantially all of the CDR regions corresponding to a non-human immunoglobulin, while all or substantially all of the FRs are regions of the human immunoglobulin consensus sequence. The humanized antibody may also comprise at least a portion of the constant region or constant domain (Fc) of an immunoglobulin, typically a human immunoglobulin. Examples of methods used to make humanized antibodies are described in US Pat. No. 5,225,539 or 5,639,641.

The "variable region" of an antibody refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, either alone or in combination. The variable regions of each of the heavy and light chains consist of four framework regions (FR or FW) connected by three complementarity determining regions (CDRs), also known as hypervariable regions. The CDRs in each chain are held together in close proximity by the FR and, together with the CDRs from the other chain, contribute to the formation of the antibody antigen-binding site. There are at least two methods for determining CDRs: (1) an approach based on interspecies sequence variability (i.e. Kabat et al ., Sequences of Proteins of Immunological Interest (5th ed., 1991, National Institutes of Health of Health), Bethesda Md.)); and (2) an approach based on the results of crystallographic studies of antigen-antibody complexes (Al-lazikani et al. (1997) J. Molec. Biol. , 273: 927-948)). In addition, combinations of the two approaches are sometimes used in the art to determine CDR.

The Kabat numbering system is usually used with reference to a residue in the variable domain (approximately light chain residues 1-107 and heavy chain residues 1-113) (e.g., Kabat et al ., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

Amino acid position numbering according to the Kabat approach refers to the numbering system used for heavy chain variable domains or light chain variable domains when compiling antibodies according to Kabat et al ., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991). When using this numbering system, the true linear sequence of amino acid residues may contain fewer amino acids or contain additional amino acids in accordance with the reduction in the length of the FR or CDR of the variable domain, or with the insertion into the FR or CDR of the variable domain. For example, a heavy chain variable domain may contain an insertion of one amino acid residue (residue 52a according to Kabat) after residue 52 in H2 and inserted residues (e.g., residues 82a, 82b and 82c, etc. according to Kabat) after residue 82 in heavy chain FR . Kabat numbering of residues can be done for a given antibody by aligning regions of homology in the sequence of that antibody with a "standard" Kabat numbered sequence. In contrast, Chothia refers to the arrangement of structural loops (Chothia and Lesk, J. Mol. Biol., 196: 901-917 (1987)). Chothia's CDR-H1 loop ending, when numbered using the Kabat numbering rule, varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme allows inserts at positions H35A and H35B; if neither position 35A and no insert in position 35B, then the loop ends at position 32; if the insert is only present at position 35A, then the loop ends at position 33; if there are inserts at both positions 35A and 35B, then the loop ends at position 34). The AbM hypervariable regions represent a compromise between Kabat CDRs and Chothia structural loops and are used by Oxford Molecular's AbM software for antibody modeling. The comparison is shown in Table 1 below.

Table 1. Comparison of numbering systems for antibodies

A loop Kabat AbM Chothia L1 L24-L34 L24-L34 L24-L34 L2 L50-L56 L50-L56 L50-L56 L3 L89-L97 L89-L97 L89-L97 H1 H31-H35B H26-H35B H26-H32..34 (Numbering system according to Kabat) H1 H31-H35 H26-H35 H26-H32 (Numbering system according to Chothia) H2 H50-H65 H50-H58 H52-H56 H3 H95-H102 H95-H102 H95-H102

The term "human antibody" means a native human antibody or an antibody having an amino acid sequence corresponding to a native human antibody, obtained using any method known in the art. Such definition of a human antibody includes intact or full-length antibodies, fragments thereof, and/or antibodies comprising at least one human immunoglobulin heavy and/or light chain polypeptide, such as, for example, an antibody comprising mouse immunoglobulin light chain and heavy chain polypeptides. human immunoglobulin.

The term "chimeric antibodies" refers to antibodies in which the amino acid sequence of an immunoglobulin molecule is derived from two or more species. Typically, the variable region of both the light and heavy chains corresponds to the variable region of antibodies derived from the same mammalian species (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and potency, while the sequences of the constant regions are homologous to the sequences in antibodies derived from another species (usually human) to avoid inducing an immune response in that species. Multispecific binding molecules, e.g., containing one or more antibody binding domains, one or more non-antibody binding domains, or a combination thereof, e.g., TNFα antagonists and/or NGF antagonists of the present invention, may contain antibody constant regions (e.g., Fc regions) in which at least a portion of one or more domains of the constant region is deleted or otherwise altered in order to provide the desired biochemical characteristics, such as increased localization in tumors or reduced serum half-life compared to an antibody of approximately the same immunogenicity, containing a native or unmodified constant region. The modified constant regions described herein may contain changes or modifications to one or more of the three heavy chain constant domains (CH1, CH2 or CH3) and/or the light chain constant domain (CL). In certain aspects, one or more constant domains may be partially or completely deleted. In certain aspects, the entire CH2 domain can be deleted (ΔCH2 constructs). See, for example, Oganesyan V., et al. , 2008, Acta Crystallogr. D Biol. Crystallogr., 64: 700-4; Oganesyan V., et al. , Mol. Immunol. , 46: 1750-5; Dall'Acqua, WF, et al. , 2006, J. Biol. Chem. , 281: 23514-23524; and Dall'Acqua, et al. , 2002, J. Immunol. , 169: 5171-5180.

The terms "epitope" and "antigenic determinant" are used interchangeably herein and refer to that portion of an antigen that a particular antibody can recognize and specifically bind to. If the antigen is a polypeptide, then epitopes can be formed both from successive amino acids and from non-consecutive amino acids brought together as a result of the formation of the protein's tertiary structure. Epitopes formed from successive amino acids are usually retained after protein denaturation, while epitopes formed as a result of the formation of tertiary structure are usually lost after protein denaturation. Typically, an epitope comprises at least 3 and usually more, at least 5 or 8-10 amino acids, in a unique spatial conformation. The epitope described in this application need not be defined down to the specific amino acids that form this epitope. In certain aspects, an epitope can be identified by examining the binding of an antibody to peptide subunits of a polypeptide antigen, or by examining competitive binding to a given antigen of a group of antigen-specific antibodies.

By "subject" or "individual" or "animal" or "patient" or "mammal" is meant any subject, in particular a mammal, for which diagnosis, prognosis or therapy is desired. Mammals include humans, domestic animals, farm animals, sporting animals, and zoo animals, including, for example, humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears and so on.

The terms "composition" and "pharmaceutical composition" refer to a preparation in a form that effectively exhibits the biological activity of the active ingredient and which does not contain any additional components that are unacceptably toxic to the subject to whom this composition will be administered. Such compositions may be sterile.

As used herein, the terms "effective amount" and "therapeutically effective amount" refer to the amount of one or more therapeutic compositions effective to control pain in a subject. The term "pain control" and its grammatical equivalents are used in this application to describe any beneficial or desirable effect in relation to a subject in need of pain control. For example, an effective amount of one or more of the therapeutic compositions described herein may, for example, prevent the onset of pain, maintain a tolerable level of pain, reduce the intensity of pain, alleviate pain, minimize pain, or eliminate pain in a subject.

The term "administering" as used herein refers to administering to a subject one or more of the therapeutic compositions described herein, for example, a bifunctional polypeptide comprising an NGF antagonist domain and a TNFα antagonist domain, a therapeutic composition comprising a combination of an NGF antagonist and a TNFα antagonist, or individual therapeutic compositions, one containing an NGF antagonist and the other containing a TNFα antagonist. The term "co-administration" refers to the administration to a subject of two or more therapeutic compositions, for example, one containing an NGF antagonist and one containing a TNFα antagonist. As used herein, co-administration includes, but does not require, that two or more therapeutic compositions be administered to a subject at the same time. Such two or more therapeutic compositions may be administered to the subject sequentially, for example, thirty minutes apart, one hour apart, two hours apart, three hours apart, four hours apart, or five or more hours apart. . The sequence and timing of co-administration, as set forth herein, may be fixed or may be changed based on the judgment of the healthcare professional.

The terms "polynucleotide" or "nucleic acid" refer to a polymeric compound containing covalently linked nucleotide residues. The polynucleotides can be DNA, cDNA, RNA, single or double stranded polynucleotides, vectors, plasmids, phage or viral polynucleotides.

The term "vector" means a construct that is capable of delivering to and expressing in a host cell one or more genes or one or more sequences of interest. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA encapsulated in liposomes and some eukaryotic cells such as producer cells.

The terms "polypeptide", "peptide" and "protein" are used interchangeably in this specification and refer to polymers of amino acids of any length. The polymer may be linear or branched and may contain modified amino acids which may alternate with non-amino acid residues. The terms also cover a polymer composed of amino acids that has been modified naturally or through targeted action; for example, by disulfide bonding, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification such as conjugation with a label. Also included in this definition are, for example, polypeptides containing one or more analogs of amino acids (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.

A "conservative amino acid substitution" is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains ( e.g. asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g. glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine). For example, substitution of phenylalanine for tyrosine is a conservative substitution. In certain aspects, conservative substitutions in the sequences of the polypeptides and antibodies proposed in this invention do not inhibit the binding or other functional activity of the polypeptide containing such an amino acid sequence. Methods for identifying nucleotide and amino acid conservative substitutions that do not affect function are well known in the art (see, for example, Brummell et al ., Biochem. , 32: 1180-1187 (1993); Kobayashi et al . , Protein Eng. , 12: 879-884 (1999); and Burks et al ., Proc. Natl. Acad. Sci. USA , 94: 412-417 (1997)).

Binding molecule containing an NGF antagonist domain and a TNFα antagonist domain

The present invention provides a bifunctional polypeptide comprising an NGF antagonist domain and a TNFα antagonist domain. In certain aspects, administration of an effective amount of a bifunctional polypeptide of the invention can control pain in a subject in need thereof more effectively than administration of an equivalent amount of an NGF antagonist or TNFα antagonist alone. The bifunctional polypeptides of the invention may include an NGF antagonist domain and a TNFα antagonist domain in any order, structure, or conformation. All suitable NGF antagonists or TNFα antagonists may be part of a bifunctional polypeptide according to the invention. Exemplary NGF antagonists and TNFα antagonists are described elsewhere in this application.

In certain aspects, an NGF antagonist is a non-antibody molecule or binding domain thereof capable of inhibiting NGF activity, for example, a soluble NGF binding fragment of TrkA. In certain aspects, the NGF antagonist is an anti-NGF antibody or an antigen-binding fragment thereof. Suitable anti-NGF antagonists, for example antagonist antibodies, can inhibit NGF binding to TrkA, p75NTR, or both TrkA and p75NTR. In certain aspects, an anti-NGF antagonist, e.g., an antagonist antibody or fragment thereof for use in a bifunctional molecule provided herein, e.g., a multispecific binding molecule, can preferably block NGF binding to TrkA compared to NGF binding to p75NTR.

Exemplary antibodies or fragments thereof for use in the bifunctional polypeptides, eg, multispecific binding molecules described herein, are provided in US Application Publication No. 2008/0107658, which is incorporated herein by reference in its entirety. In certain aspects, an anti-NGF antibody or fragment thereof binds to the same epitope, can competitively inhibit, or can bind to NGF with higher affinity than an anti-NGF antibody MEDI-578. In some embodiments, the anti-NGF antibody, or fragment thereof, binds to human and/or rat NGF with an affinity equal to or less than 1, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, or 0. .2 nM. For example, an anti-NGF antibody, or fragment thereof, can bind to human NGF with an affinity of about 0.2-0.8, 0.2-0.7, 0.2-0.6, 0.2-0.5, and/or 0.25-0.44 nM and with rat NGF with an affinity of about 0.2-0.9, 0.2-0.8 and/or 0.25-0.70 nM.

In certain aspects, the anti-NGF antibody or fragment thereof is MEDI-578. The MEDI-578 antibody is described in US Publication No. 2008/0107658 as clone 1252A5. In other aspects, the anti-NGF antibody or fragment thereof is tanezumab (RN-624), a humanized anti-NGF monoclonal antibody (mAb) (Pfizer; described in Kivitz et al. (2013) Pain, 154, 9, 1603-161), fullranumab, a fully human anti-NGF mAb (Amgen; described in Sanga et al., Pain, volume 154, issue 10, October 2013, pp. 1910-1919); REGN475/SAR164877, fully human anti-NGF mAb (Regeneron/Sanafi-Aventis); ABT-110 (PG110), humanized anti-NGF mAb (Abbott Laboratories). An anti-NGF antibody or fragment thereof incorporated into a bifunctional polypeptide, for example a multispecific binding molecule of the invention, may be, for example, humanized, chimeric, primatized, or fully human.

In certain aspects, an anti-NGF antibody or fragment thereof comprises an antibody VH domain comprising the HCDR1, HCDR2, and HCDR3 domains of MEDI-578, MEDI-578 heavy chain CDR variants with one, two, three, four, five, or more amino acid substitutions, for example, conservative amino acid substitutions. For example, an anti-NGF antibody or fragment thereof may comprise HCDR1 with the amino acid sequence of SEQ ID NO: 4 as such or with the amino acid sequence of SEQ ID NO: 4 with one or more, for example, one, two, three, four, five or more amino acid substitutions. . Similarly, an anti-NGF antibody or fragment thereof may comprise HCDR2 with the amino acid sequence of SEQ ID NO: 5 as such or with the amino acid sequence of SEQ ID NO: 5 with one or more, for example, one, two, three, four, five or more amino acids. substitutions. Similarly, an anti-NGF antibody or fragment thereof may comprise HCDR3 with the amino acid sequence of SEQ ID NO: 6 as such or with the amino acid sequence of SEQ ID NO: 6 with one or more, for example, one, two, three, four, five or more amino acids. substitutions. In certain aspects, HCDR3 may comprise the amino acid sequence SSRIYDFNSALISYYDMDV (SEQ ID NO: 11) or SSRIYDMISSLQPYYDMDV (SEQ ID NO: 12).

In certain aspects, an anti-NGF antibody, or fragment thereof, comprises an antibody VL domain comprising the LCDR1, LCDR2, and LCDR3 domains of MEDI-578, MEDI-578 light chain CDR variants with one, two, three, four, five, or more amino acid substitutions, for example, conservative amino acid substitutions. In certain aspects, an anti-NGF antibody, or fragment thereof, may comprise an LCDR1 with the amino acid sequence of SEQ ID NO: 8 as such, or with the amino acid sequence of SEQ ID NO: 8 with one or more, e.g., one, two, three, four, five, or more amino acid substitutions. Similarly, an anti-NGF antibody or fragment thereof may comprise an LCDR2 with the amino acid sequence of SEQ ID NO: 9 as such or with the amino acid sequence of SEQ ID NO: 9 with one or more, e.g., one, two, three, four, five or more amino acids. substitutions. Similarly, an anti-NGF antibody or fragment thereof may comprise an LCDR3 with the amino acid sequence of SEQ ID NO: 10 as such or with the amino acid sequence of SEQ ID NO: 10 with one or more, for example, one, two, three, four, five or more amino acids. substitutions.

In certain aspects, an anti-NGF antibody or fragment thereof comprises an antibody VH domain containing an amino acid sequence of VH that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid of SEQ ID NO: 3. In certain aspects, an anti-NGF antibody or fragment thereof comprises an antibody VH domain comprising the amino acid sequence of VH of SEQ ID NO: 3.

In certain aspects, an anti-NGF antibody or fragment thereof comprises an antibody VL domain containing an amino acid sequence of VL that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences of SEQ ID NO: 7. In certain aspects, an anti-NGF antibody or fragment thereof comprises an antibody VL domain comprising the amino acid sequence VL of SEQ ID NO: 7.

In certain aspects, an anti-NGF antibody or fragment thereof comprises an antibody VH domain containing an amino acid sequence of VH that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid of SEQ ID NO: 94. In certain aspects, an anti-NGF antibody or fragment thereof comprises an antibody VH domain comprising the amino acid sequence of VH of SEQ ID NO: 94.

In certain aspects, an anti-NGF antibody or fragment thereof comprises an antibody VL domain containing an amino acid sequence of VL that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid of SEQ ID NO: 95. In certain aspects, an anti-NGF antibody, or fragment thereof, comprises an antibody VL domain comprising the VL amino acid sequence of SEQ ID NO: 95.

In certain aspects, an anti-NGF antibody or fragment thereof comprises an antibody VH domain comprising the HCDR1, HCDR2, and HCDR3 domains of any of SEQ ID NOs: 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86 and 96 or variants with one, two, three, four , five or more amino acid substitutions inclusive, for example, conservative amino acid substitutions.

In certain aspects, an anti-NGF antibody or fragment thereof comprises an antibody VL domain comprising the LCDR1, LCDR2, and LCDR3 domains of any of SEQ ID NOs: 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87 and 97 or variants with one, two, three, four , five or more amino acid substitutions inclusive, for example, conservative amino acid substitutions.

In certain aspects, an anti-NGF antibody or fragment thereof comprises an antibody VH domain containing an amino acid sequence of VH that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences with any of SEQ ID NOs: 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, and 96. In certain aspects, an anti-NGF antibody or fragment thereof comprises an antibody VH domain comprising a VH amino acid sequence of any of SEQ ID NOs: 30, 32, 34 , 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84 , 86 and 96.

In certain aspects, an anti-NGF antibody or fragment thereof comprises an antibody VL domain containing an amino acid sequence of VL that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences with any of SEQ ID NOs: 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, and 97. In certain aspects, an anti-NGF antibody or fragment thereof comprises an antibody VL domain comprising a VL amino acid sequence of any of SEQ ID NOs: 31, 33, 35 , 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85 , 87 and 97.

In certain aspects, an anti-NGF antibody, or fragment thereof, comprises an antibody VH domain comprising the HCDR1, HCDR2, and HCDR3 domains of NGF-NG, NGF-NG heavy chain CDR variants with one, two, three, four, five, or more amino acid substitutions, for example, conservative amino acid substitutions. For example, an anti-NGF antibody or fragment thereof may comprise HCDR1 with the amino acid sequence of SEQ ID NO: 88 as such or with the amino acid sequence of SEQ ID NO: 88 with one or more, for example, one, two, three, four, five or more amino acid substitutions. . Similarly, an anti-NGF antibody or fragment thereof may comprise HCDR2 with the amino acid sequence of SEQ ID NO: 89 as such or with the amino acid sequence of SEQ ID NO: 89 with one or more, for example, one, two, three, four, five or more amino acids. substitutions. Similarly, an anti-NGF antibody or fragment thereof may comprise HCDR3 with the amino acid sequence of SEQ ID NO: 90 as such or with the amino acid sequence of SEQ ID NO: 90 with one or more, e.g., one, two, three, four, five or more amino acids. substitutions.

In certain aspects, an anti-NGF antibody or fragment thereof comprises an antibody VL domain comprising the LCDR1, LCDR2, and LCDR3 domains of NGF-NG, NGF-NG light chain CDR variants with one, two, three, four, five, or more amino acid substitutions, for example, conservative amino acid substitutions. In certain aspects, an anti-NGF antibody, or fragment thereof, may comprise an LCDR1 with the amino acid sequence of SEQ ID NO: 91 as such, or with the amino acid sequence of SEQ ID NO: 91 with one or more, e.g., one, two, three, four, five, or more amino acid substitutions. Similarly, an anti-NGF antibody or fragment thereof may comprise an LCDR2 with the amino acid sequence of SEQ ID NO: 92 as such or with the amino acid sequence of SEQ ID NO: 92 with one or more, for example, one, two, three, four, five or more amino acids. substitutions. Similarly, an anti-NGF antibody or fragment thereof may comprise an LCDR3 with the amino acid sequence of SEQ ID NO: 93 as such or with the amino acid sequence of SEQ ID NO: 93 with one or more, for example, one, two, three, four, five or more amino acids. substitutions.

In certain aspects, an anti-NGF antibody or fragment thereof comprises an antibody VH domain containing an amino acid sequence of VH that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid of SEQ ID NO: 24. In certain aspects, an anti-NGF antibody or fragment thereof comprises an antibody VH domain comprising the amino acid sequence of VH of SEQ ID NO: 24.

In certain aspects, an anti-NGF antibody or fragment thereof comprises an antibody VL domain containing an amino acid sequence of VL that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid of SEQ ID NO: 26. In certain aspects, an anti-NGF antibody, or fragment thereof, comprises an antibody VL domain comprising the VL amino acid sequence of SEQ ID NO: 26.

A multifunctional polypeptide, such as a multispecific binding molecule of the invention, may comprise a full-length anti-NGF antibody, i. e. an antibody containing two full length heavy chains and two full length light chains in H ₂ L ₂ format. If the anti-NGF antibody is a full-length antibody, then one or more TNFα antagonist domains may be fused to the N-terminus or C-terminus of one or more heavy chains of the anti-NGF antibody, or to the N-terminus or C-terminus of one or more light chains. chains of anti-NGF antibodies. Alternatively, a multifunctional polypeptide, eg, a multispecific binding molecule, of the invention may comprise an antigen-binding fragment of an anti-NGF antibody. In certain aspects, an anti-NGF antibody fragment may contain any portion of the antibody's constant domains, or may contain only the variable domains. Exemplary anti-NGF antibody fragments for inclusion in a bifunctional polypeptide, such as a multispecific binding molecule, include, but are not limited to, Fab fragments, Fab' fragments, F(ab) ₂ fragments, or single chain Fv (scFv) fragments.

In some exemplary compositions provided herein, the anti-NGF antibody is an scFv fragment, such as a MEDI-578 scFv fragment or an NGF-binding variant thereof. In some exemplary compositions provided herein, the anti-NGF antibody is an scFv fragment, eg, an NGF-NG scFv fragment or an NGF-binding variant thereof. An anti-NGF scFv fragment polypeptide may contain VH and VL domains in any order, either N-VH-VL-C or N-VL-VH-C. ScFv molecules are typically designed such that the VH and VL domains are connected via a flexible linker. Exemplary scFv structures, including various linkers, can be found in Dimasi, N., et al. , J. Mol. Biol. , 393 : 672-92 (2009) and PCT Publication No. WO 2013/070565, both of which are incorporated herein by reference in their entirety. As will be appreciated by one of skill in the art, scFv antibody fragments may have reduced stability compared to variable domains existing in the standard Fab conformation. In certain aspects, the scFv fragment can be structurally stabilized by introducing stabilizing mutations or by introducing interchain(s) disulfide(s) bond(s) (eg, SS-stabilize). However, stabilizing mutations and/or introduced interstrand disulfide bonds are not required and, in certain aspects, are not present. A number of methods recognized in the art are available for stabilizing scFv polypeptides.

Linkers can be used to connect domains/regions of the bifunctional polypeptides of the invention. Linkers can be used to connect the NGF antagonist domain and the TNFα antagonist domain in a bifunctional molecule, and can also be used to interconnect the scFv heavy and light chain variable regions. A typical non-limiting example of a linker is a polypeptide chain containing at least 4 residues. Portions of such linkers may be flexible, hydrophilic and have little or no secondary structure of their own (linker portions or flexible linker portions). Linkers of at least 4 amino acids can be used to connect domains and/or regions that are close to each other after assembly of the bifunctional polypeptide molecule. You can also use longer linkers. Thus, linkers may be composed of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 , 35, 40, 45, 50 residues. In addition, linkers may consist of, for example, about 100-175 residues. When multiple linkers are used to interconnect the parts of a bifunctional polypeptide molecule, such linkers may be the same or different (eg, the same and different lengths and/or have the same and different amino acid sequence).

The linker(s) in the bifunctional polypeptide molecule contribute to the formation of the desired structure. Linkers may contain residues (Gly-Ser) _n (where n is an integer of at least 1, 2 and up to, for example, 3, 4, 5, 6, 10, 20, 50, 100 or more), when In this case, some Glu or Lys residues are distributed along the entire length to increase solubility. Alternatively, some linkers do not contain serine residues at all, for example, in cases where this linker is the object of O-glycosylation. In certain aspects, the linkers may contain cysteine residues, for example, if dimerization of the linkers is used to give domains of the bifunctional polypeptide a properly folded configuration. According to certain aspects, the bifunctional polypeptide may contain at least one, two, three, four or more polypeptide linkers that connect the domains of this polypeptide.

In some aspects, the polypeptide linker may contain 1-50 residues, 1-25 residues, 25-50 residues, or 30-50 residues. In some aspects, the polypeptide linker may comprise a portion of an Fc moiety. For example, in certain aspects, the polypeptide linker may comprise a portion of the immunoglobulin hinge domain of an IgG1, IgG2, IgG3 and/or IgG4 antibody, or a variant thereof.

In some aspects, the polypeptide linker may be or consist of a gly-ser linker. Used in this description, the term "gly-ser-linker" refers to a peptide, which consists of residues of glycine and serine. A typical gly-ser linker has an amino acid sequence of the formula (Gly ₄ Ser) _n , where n is an integer of at least 1, 2 and up to, for example, 3, 4, 5, 6, 10, 20, 50, 100 or more. In certain aspects, a polypeptide linker may comprise at least a portion of a hinge region (e.g., derived from an IgG1, IgG2, IgG3, or IgG4 molecule) and a number of gly-ser amino acid residues (e.g., a gly-ser linker such as (Gly ₄ Ser) _n ).

If a multifunctional polypeptide, eg, a multispecific binding molecule, contains an scFv, then a flexible linker can connect the heavy and light chains of the scFv. Such a flexible linker typically does not include part of the hinge region, but rather is a gly-ser linker or other flexible linker. The length and amino acid sequence of the flexible linker connecting the scFv domains to each other can be easily selected and optimized.

In certain aspects, a multifunctional polypeptide, e.g., a multispecific binding molecule, may comprise an anti-NGF antibody scFv fragment that contains, N-terminally to C-terminally, VH, a 15 amino acid linker sequence (GGGGS) ₃ . and V.L. In some embodiments, the linker connecting the VH and VL of the scFv is a 20 amino acid linker sequence (GGGGS) ₄ . In certain aspects, the VH contains the amino acid sequence of SEQ ID NO: 3. In certain aspects, the VL contains the amino acid sequence of SEQ ID NO: 7. In some embodiments, the VH contains an amino acid sequence with any of SEQ ID NO: 24, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 94, and 96. In some embodiments, VL comprises an amino acid sequence with any of SEQ ID NOs: 26, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 95, and 97. In certain aspects, the VH domain contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of an amino acid sequence with any of SEQ ID NOs: 3, 24, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 94 and 96. According to certain aspect m, the VL domain contains an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence with any of SEQ ID NO: 7, 26, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 95 and 97.

In other aspects, the stability of the polypeptide can be improved by adding an interchain disulfide bond between the VH domain and the VL domain by replacing some residues in the VH and VL domain with cysteine residues. See, for example, Michaelson, JS, et al. (2009) MAbs , 1, 128-41; Brinkmann, U., et al., (1993) Proc. Natl. Acad. sci. USA , 90, 7538-42; Young, NM, et al., (1995) FEBS Lett. , 377, 135-9. For example, the glycine residue at positions 100, 101, or 102 VL can be changed to a cysteine residue, and the glycine residue at position 44 VH can be changed to a cysteine residue.

A multifunctional polypeptide, such as a multispecific binding molecule of the invention, may include a TNFα antagonist domain. In certain aspects, the TNFα antagonist domain can inhibit TNFα binding to the TNF receptor (TNFR) on the cell surface, thereby blocking TNF activity.

In certain aspects, the TNFα antagonist domain of the multifunctional polypeptide of the invention is an anti-TNFα antibody or an antigen-binding fragment thereof. In certain aspects, the anti-TNFα antibody is infliximab, adalimumab, certolizumab pegol, golimumab, or an antigen-binding fragment of any of these antibodies.

In certain aspects, an anti-TNFα antibody, or fragment thereof, binds to the same epitope, can competitively inhibit, or can bind to TNFα with a higher affinity than any of the anti-TNFα antibodies infliximab, adalimumab, certolizumab pegol, or golimumab, or an antigen-binding fragment. any of these antibodies. In certain aspects, the anti-TNFα antibody is infliximab, adalimumab, certolizumab pegol, or golimumab, or an antigen-binding fragment of any of these antibodies. The structure and sequence information of such anti-TNFα antibodies is publicly available to those skilled in the art, and they can be incorporated into a multifunctional polypeptide, such as the multispecific binding molecule described herein, without undue experimentation. An anti-TNFα antibody or fragment thereof incorporated into a multifunctional polypeptide may be, for example, humanized, chimeric, primatized, or fully human.

In certain aspects, an anti-TNFα antibody or fragment thereof comprises an antibody VH domain comprising the HCDR1, HCDR2, and HCDR3 domains of infliximab, adalimumab, certolizumab pegol, or golimumab, or heavy chain CDR variants of infliximab, adalimumab, certolizumab pegol, or golimumab with one, two, three , four, five or more amino acid substitutions, eg conservative amino acid substitutions.

In certain aspects, an anti-TNFα antibody or fragment thereof comprises an antibody VL domain comprising the LCDR1, LCDR2, and LCDR3 domains of infliximab, adalimumab, certolizumab pegol, or golimumab, or light chain CDR variants of infliximab, adalimumab, certolizumab pegol, or golimumab with one, two, three , four, five or more amino acid substitutions, eg conservative amino acid substitutions.

In certain aspects, an anti-TNFα antibody or fragment thereof comprises an antibody VH domain containing an amino acid sequence of VH that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid the VH sequences of infliximab, adalimumab, certolizumab pegol, or golimumab. In certain aspects, the anti-TNFα antibody, or fragment thereof, comprises an antibody VH domain comprising the amino acid sequence of the VH of infliximab, adalimumab, certolizumab pegol, or golimumab.

In certain aspects, an anti-TNFα antibody or fragment thereof comprises an antibody VL domain containing an amino acid sequence of VL that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid the VL sequences of infliximab, adalimumab, certolizumab pegol, or golimumab. In certain aspects, the anti-TNFα antibody, or fragment thereof, comprises an antibody VL domain comprising the VL amino acid sequence of infliximab, adalimumab, certolizumab pegol, or golimumab.

In certain aspects, an anti-TNFα antibody or fragment thereof comprises an antibody VH domain containing an amino acid sequence of VH that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid SEQ ID NO: 28. In certain aspects, the anti-TNFα antibody, or fragment thereof, comprises an antibody VH domain comprising the VH amino acid sequence of SEQ ID NO: 28.

In certain aspects, an anti-TNFα antibody or fragment thereof comprises an antibody VL domain containing an amino acid sequence of VL that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid SEQ ID NO: 29. In certain aspects, the anti-TNFα antibody, or fragment thereof, comprises an antibody VL domain comprising the VL amino acid sequence of SEQ ID NO: 29.

A multifunctional polypeptide, such as a multispecific binding molecule of the invention, may comprise a full-length anti-TNFα antibody, i.e. an antibody containing two full length heavy chains and two full length light chains in H ₂ L ₂ format. If the anti-TNFα antibody is a full-length antibody, then one or more NGF antagonist domains may be fused to the N-terminus or C-terminus of one or more heavy chains of the anti-TNFα antibody, or to the N-terminus or C-terminus of one or more light chains. anti-TNFα antibody chains. Alternatively, a multifunctional polypeptide, eg, a multispecific binding molecule of the invention, may comprise an antigen-binding fragment of an anti-TNFα antibody. In certain aspects, an anti-TNFα antibody fragment may contain any portion of the antibody constant domains, or may contain only variable domains. Exemplary anti-TNFα antibody fragments for inclusion in a multifunctional polypeptide, such as a multispecific binding molecule, include, but are not limited to, Fab fragments, Fab' fragments, F(ab) ₂ fragments, or single chain Fv (scFv) fragments.

In certain aspects, the multifunctional molecule is varB ndimab, which is a molecule containing a full-length anti-TNFα antibody, i. an antibody containing two full-length heavy chains and two full-length light chains in H ₂ L ₂ format, with a MEDI-578 scFv fragment fused to the C-terminus of the anti-TNFα antibody heavy chain. Ndimab varB contains a light chain containing the amino acid sequence of SEQ ID NO: 20 and a heavy chain containing the amino acid sequence of SEQ ID NO: 22. According to certain aspects, the bifunctional molecule contains a light chain containing an amino acid sequence that is at least 80% identical, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the sequence of SEQ ID NO: 20, and a heavy chain containing an amino acid sequence at least 80%, 85%, 90%, 95 identical %, 96%, 97%, 98% or 99% of the sequence of SEQ ID NO: 22.

In certain aspects, the anti-TNFα antibody is an scFv fragment, eg, an scFv fragment derived from infliximab, adalimumab, certolizumab pegol, or golimumab, or a TNFα binding variant thereof. The polypeptide representing the scFv fragment of an anti-TNFα antibody may contain the VH and VL domains in any order, either N-VH-VL-C or N-VL-VH-C. ScFv molecules are typically designed such that the VH and VL domains are connected via a flexible linker and can take on a variety of different structures as described above. An anti-TNFα scFv fragment polypeptide can be stabilized in the same manner as described above.

In certain aspects, the TNFα antagonist is a TNFα-binding soluble fragment of a TNF receptor, such as TNFR-1 or TNFR-2, or a variant or soluble fragment thereof. In certain aspects, the soluble fragment of TNFR-1 is a fragment with a molecular weight of 55 kDa. In some embodiments, the soluble fragment of TNFR-2 is a fragment with a molecular weight of 75 kDa. In certain aspects, the TNF receptor fragment is linked to a heterologous polypeptide, eg, an immunoglobulin Fc fragment, eg, an IgG1 Fc domain. In certain aspects, the TNFα antagonist comprises the amino acid sequence shown in SEQ ID NO: 13 or a TNFα binding fragment thereof. The TNFR-2-related portion contains amino acids 1-235 of SEQ ID NO: 13. In certain aspects, a variant of the TNFα-binding soluble fragment of TNFR-2 contains an amino acid sequence that is at least 80%, 85%, 90%, 95 identical %, 96%, 97%, 98%, 99%, or 100% amino acids 1-235 of SEQ ID NO: 13. In certain aspects, the TNFα-binding soluble fragment variant of TNFR-2 contains amino acids 1-235 of SEQ ID NO: 13, excluding, for example, 1, 2, 3, 4, 5, 10, 20, 20, 40, or 50 amino acid insertions, substitutions, or deletions. The Fc portion of IgG1 comprises amino acids 236-467 of SEQ ID NO: 13. In certain aspects, the TNFα-binding TNFR-2 soluble fragment may be fused to the Fc portion of any human or non-human antibody, or any other protein or non-protein origin, which will provide stability, for example, albumin or polyethylene glycol. In certain aspects, a variant TNFα-binding TNFR-2 soluble fragment contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 236 -467 of SEQ ID NO: 13. In certain aspects, the TNFα-binding TNFR-2 soluble fragment variant comprises amino acids 236-467 of SEQ ID NO: 13, except for, for example, 1, 2, 3, 4, 5, 10 , 20, 20, 40 or 50 amino acid insertions, substitutions or deletions. In certain aspects, a variant TNFα-binding TNFR-2 soluble fragment contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the SEQ sequence. ID NO: 13. In certain aspects, a variant TNFα-binding TNFR-2 soluble fragment contains the sequence of SEQ ID NO: 13, except for, for example, 1, 2, 3, 4, 5, 10, 20, 20, 40, or 50 amino acid insertions, substitutions or deletions.

In certain aspects, the TNFα-binding TNFR-2 soluble fragment is a single-stranded fusion protein. In certain aspects, the TNFα-binding TNFR-2 soluble fragment is a dimer of two fusion proteins that form an associate, for example, via disulfide bonds between two Fc domains.

The multifunctional polypeptide, eg, the multispecific binding molecule of the present invention, can take on a wide variety of structures and conformations. In one aspect, the multifunctional polypeptide of the present invention comprises a fusion protein wherein the NGF antagonist domain described above is connected to the TNFα antagonist domain described above via a flexible linker. Examples of linkers are described elsewhere in this application. In certain aspects, the multifunctional polypeptide comprises a fusion protein homodimer.

In an exemplary aspect, a multifunctional polypeptide is provided wherein the NGF antagonist is an anti-NGF scFv domain derived from e.g. the immunoglobulin domain. The scFv domain of an anti-NGF antibody may, in certain aspects, be connected to the carboxy terminus of an immunoglobulin Fc domain via a linker. In certain aspects, the monomers of this multifunctional polypeptide form a homodimer, with each subunit containing, N-terminally to C-terminally, a 75 kDa TNFα-binding fragment of TNFR-2, a human IgG1 Fc domain, a linker consisting 10 amino acids (GGGGS) ₂ (SEQ ID NO: 98), VH domain of an anti-NGF antibody containing the amino acid sequence of SEQ ID NO: 3, a 15 amino acid linker sequence (GGGGS) ₃ (SEQ ID NO: 15), and the VL domain of an anti-NGF antibody comprising the amino acid sequence of SEQ ID NO: 7. In one aspect, the multifunctional polypeptide is TNFR2-Fc_VH#4, which comprises a fusion polypeptide homodimer comprising the amino acid sequence of SEQ ID NO: 14. In certain aspects, the multifunctional polypeptide contains a homodimer of a fused polypeptide containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical the sequences of SEQ ID NO: 14.

In another exemplary aspect, the multifunctional polypeptide comprises, N-terminus to C-terminus, a 75 kDa TNFα binding fragment of TNFR-2, a human IgG1 Fc domain, a 10 amino acid linker (GGGGS) ₂ (SEQ ID NO: 98), an anti-NGF antibody VH domain containing the amino acid sequence of SEQ ID NO: 94, a 20 amino acid linker sequence (GGGGS) ₄ (SEQ ID NO: 19), and an anti-NGF antibody VL domain containing the amino acid sequence of SEQ ID NO: 95. In certain aspects, the multifunctional polypeptide is a TNFR2-Fc_varB that contains a fusion polypeptide homodimer comprising the amino acid sequence of SEQ ID NO: 17. In certain aspects, the multifunctional polypeptide comprises a fusion polypeptide homodimer comprising the amino acid sequence, at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to the sequence of SEQ ID NO: 17.

Polypeptides

The polypeptides of this invention, for example, multifunctional polypeptides containing an NGF antagonist domain and a TNFα antagonist domain, or individual polypeptides containing an NGF antagonist domain and a TNFα antagonist domain, respectively, may be recombinant polypeptides derived from natural polypeptides or synthetic polypeptides . It is recognized in the art that certain amino acid sequences may vary without significantly affecting protein structure or function. Thus, the invention further provides variants of the polypeptides of the invention that have significant activity or that include NGF antagonist domains and TNFα antagonist domains. Such mutants include deletions, insertions, inversions, repeats and substitutions.

Polypeptides and analogs may be further modified to contain additional chemical moieties not normally part of the protein. These modified moieties can improve solubility, biological half-life, or absorption of the protein. These groupings can also reduce or eliminate any undesirable side effects of proteins and the like. A review of such groupings can be found in REMINGTON'S PHARMACEUTICAL SCIENCES, 20th ed., Mack Publishing Co., Easton, PA (2000).

In certain aspects, a multifunctional polypeptide of the invention may include an NGF or TNFα binding domain that is not an antibody. A number of methods are known in the art for identifying and generating non-antibody polypeptides that bind with high affinity to a target protein. See, for example, Skerra, Curr. Opin. Biotechnol ., 18:295-304 (2007), Hosse et al ., Protein Science , 15:14-27 (2006), Gill et al ., Curr. Opin. Biotechnol ., 17: 653-658 (2006), Nygren, FEBS J. , 275: 2668-76 (2008) and Skerra, FEBS J. , 275: 2677-83 (2008), each of which is incorporated herein by links in their entirety. In certain aspects, phage display can be used to identify/generate suitable multifunctional polypeptides. In certain aspects, a multifunctional polypeptide, e.g., a multispecific binding molecule, of the present invention may comprise a protein scaffold of a type selected from the group consisting of protein A, lipocalin, a fibronectin domain, an ankyrin consensus repeat domain, and thioredoxin.

Polynucleotides, vectors and host cells

The present invention provides nucleic acid molecules containing polynucleotides that encode a multifunctional polypeptide comprising an NGF antagonist domain and a TNFα antagonist domain. The invention also provides nucleic acid molecules containing polynucleotides that encode individual polypeptides containing, respectively, an NGF antagonist and a TNFα antagonist. In certain aspects, such polynucleotides encode a peptide domain that specifically binds to NGF or a fragment thereof and also binds to TNFα or a fragment thereof. For example, the present invention provides a polynucleotide that encodes a polypeptide domain comprising an anti-NGF antibody, or an antigen-binding fragment thereof, and a polypeptide domain comprising a TNFα antagonist, such as an anti-TNFα antibody, or an antigen-binding fragment thereof, or a soluble TNFα-binding portion. a TNF receptor, such as TNFR2. Polynucleotides may be in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA and synthetic DNA; and may be double-stranded or single-stranded, and in the case of single-stranded, may be a coding strand or a non-coding (antisense) strand.

In some embodiments, an isolated polynucleotide that encodes a multifunctional polypeptide described in this application contains the nucleotide sequence of SEQ ID NO: 16, 18, or 99, or fragments or a sequence identical to at least 80%, 85%, 90%, 95% , 96%, 97%, 98% or 99% of the sequence of SEQ ID NO: 16, 18 or 99 or fragments thereof.

The isolated polypeptides described in this application can be obtained by any suitable method known in the art. Such methods range from direct protein synthesis methods to designing a DNA sequence encoding isolated polypeptide sequences and expressing those sequences in a suitable transfected host. In certain aspects, the DNA sequence is constructed using recombinant technology by isolating or synthesizing a DNA sequence encoding a multifunctional polypeptide comprising an NGF antagonist domain and a TNFα antagonist domain, or individual polypeptides comprising an NGF antagonist domain and a TNFα antagonist domain, respectively. Accordingly, the present invention provides an isolated polynucleotide that encodes a bifunctional polypeptide comprising an NGF antagonist domain and a TNFα antagonist domain, as described in detail above. Also provided are isolated polynucleotides that encode single polypeptides containing, respectively, an NGF antagonist domain and a TNFα antagonist domain.

In certain aspects, a DNA sequence encoding a multifunctional polypeptide, e.g., a multispecific binding molecule of interest, or individual polypeptides containing an NGF antagonist domain and a TNFα antagonist domain, respectively, can be constructed by chemical synthesis using an oligonucleotide synthesizer. Such oligonucleotides can be designed based on the amino acid sequence of the desired multifunctional polypeptide and the choice of those codons that are preferred by the host cell in which the recombinant polypeptide of interest will be produced. Standard techniques can be used to synthesize an isolated polynucleotide sequence encoding a multifunctional polypeptide of interest. For example, the complete amino acid sequence can be used to construct a gene with a possible sequence recovered from a polypeptide. In addition, it is possible to synthesize a DNA oligomer containing a nucleotide sequence encoding a particular multifunctional polypeptide or individual polypeptides. For example, several small oligonucleotides encoding portions of the desired polypeptide can be synthesized and then ligated. Typically, individual oligonucleotides contain sticky 5' or 3' ends for complementary assembly.

In certain aspects, the polynucleotides of the present invention may contain a mature polypeptide coding sequence fused in the same reading frame with a marker sequence, which helps, for example, to purify the encoded polypeptide. For example, the marker sequence may be a hexa-histidine tag delivered by the pQE-9 vector to effect purification of the mature polypeptide fused to the marker in the case of a bacterial host, or the marker sequence may be a haemagglutinin (HA) tag derived from the hemagglutinin protein of the virus. influenza, in the case of using a host isolated from a mammal (for example, COS-7 cells).

The polynucleotides of the present invention may further comprise changes in coding regions, non-coding regions, or both. In certain aspects, polynucleotide variants contain changes that result in silent substitutions, insertions or deletions, but do not change the properties or activities of the encoded polypeptide. In certain aspects, nucleotide variants are generated due to the presence of silent substitutions due to the degeneracy of the genetic code. Polynucleotide variants can be generated for a variety of reasons, such as to optimize codons for expression in a particular host (to change codons in human mRNA to those that would be preferred by a bacterial host such as E. coli ).

Also proposed are vectors and cells containing the polynucleotides described in this application. Once assembled (by synthesis, site-directed mutagenesis, or other method), polynucleotide sequences encoding a particular isolated polypeptide of interest can be inserted into an expression vector and operably linked to an expression control sequence suitable for expression of the protein in the desired host. Such vectors are provided in accordance with the present invention. Nucleic acid sequencing, restriction mapping, and expression of the biologically active polypeptide in a suitable host can confirm proper assembly. As is well known in the art, in order to achieve high levels of expression of a transfected gene in a host, the gene must be operably linked to transcriptional and translational expression control sequences that function in the host of choice for expression.

In certain aspects, recombinant expression vectors can be used to amplify and express DNA encoding multifunctional polypeptides, for example, multispecific binding molecules containing an NGF antagonist domain and a TNFα antagonist domain, or single polypeptides containing an NGF antagonist domain and a TNFα antagonist domain, respectively. Recombinant expression vectors are replicable DNA constructs that have synthetic or cDNA-derived DNA fragments encoding a multifunctional polypeptide or single polypeptides containing an NGF antagonist domain and a TNFα antagonist domain, respectively, operably linked to suitable transcriptional or translational regulatory elements derived from genes of mammals, microbes, viruses or insects. A transcription unit typically contains a collection of (1) a genetic element(s) that plays a regulatory role in gene expression, such as transcriptional promoters or enhancers, (2) a structural or coding sequence that is transcribed into mRNA and translated into protein, and (3) appropriate transcription and translation initiation and termination sequences, as detailed below. Such regulatory elements may include an operator sequence to regulate transcription. In addition, an origin of replication can be included, usually providing the ability to replicate in the host, and a gene that promotes the recognition of transformants in selection. Regions of DNA are considered functionally related when they are related to each other in function. For example, the DNA for a signal peptide (the leader sequence of a secreted peptide) is operably linked to the DNA for a polypeptide if it is expressed as a precursor that participates in the secretion of that polypeptide; a promoter is operably linked to a coding sequence if it regulates the transcription of that sequence; or a ribosome binding site is operably linked to a coding sequence if it is located in such a way as to allow translation to take place. Structural elements intended for use in yeast expression systems include a leader sequence that ensures the secretion of the translated protein by the host cell into the external environment. Alternatively, if the recombinant protein is expressed without a leader or transport sequence, it may include a methionine residue at the N-terminus. This residue may subsequently be cleaved from the expressed recombinant protein to obtain the final product.

The choice of expression control sequences and expression vector will depend on the choice of the host. A wide variety of expression host/vector combinations can be used. Expression vectors useful in eukaryotic hosts include, for example, vectors containing expression control sequences from simian virus 40 (SV40), bovine papillomavirus, adenovirus, and cytomegalovirus. Expression vectors useful for bacterial hosts include known bacterial plasmids such as plasmids from E. coli including pCR 1, pBR322, pMB9 and their derivatives, plasmids for a wide range of hosts such as M13 based plasmids containing single stranded DNA of filamentous bacteriophages.

The invention also provides host cells containing polynucleotides encoding polypeptides of the invention. Suitable host cells for expression of the polypeptides of the invention include prokaryotes, yeast, insects or higher eukaryotes under the control of appropriate promoters. Prokaryotes include gram-negative or gram-positive microorganisms, such as E. coli or bacilli. Higher eukaryotic cells include the commonly known mammalian cell lines described below. Cell-free translation systems can also be used. Appropriate cloning and expression vectors for use in bacterial, fungal, yeast, and mammalian hosts are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, NY, 1985), the relevant description of which is hereby incorporated by reference. . Additional information regarding methods for producing proteins, including producing antibodies, can be found, for example, in US Patent Application Publication No. 2008/0187954, US Patent Nos. 6,413,746 and 6,660,501 and International Patent Application Publication No. WO 04009823; all are hereby incorporated herein by reference in their entirety.

Various mammalian or insect cell culture systems can also advantageously be used to express the recombinant protein. The expression of recombinant proteins in mammalian cells can be carried out due to the fact that in them such proteins, as a rule, are correctly folded, appropriately modified and fully functional. Examples of suitable host mammalian cell lines include HEK-293 and HEK-293T, the COS-7 monkey kidney cell lines described by Gluzman (Cell, 23: 175, 1981) and other cell lines including, for example, L cells, C127, 3T3, Chinese Hamster Ovary (CHO), HeLa, and Syrian Hamster Kidney (BHK) cell lines. Mammalian expression vectors may contain non-transcribed elements such as an origin of replication, an appropriate promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking non-transcribed sequences and 5' or 3' non-translated sequences, such as essential ribosome binding sites, polyadenylation site, donor and acceptor splicing sites, and transcription termination sequences. For a review of baculovirus systems used to produce heterologous proteins in insect cells, see Luckow and Summers, Bio/Technology, 6: 47 (1988).

The invention also provides a method for the production of a multifunctional polypeptide as described herein or for the production of individual polypeptides containing an NGF antagonist and a TNFα antagonist, respectively. The method includes culturing a host cell as described above under conditions that promote expression of the multifunctional polypeptide or individual polypeptides and recovering the multifunctional polypeptide or individual polypeptides.

For long-term production of recombinant proteins in high yield, stable expression is desirable. For example, cell lines can be designed that stably express a multifunctional polypeptide. Instead of using expression vectors that contain viral origins of replication, host cells can be transfected with DNA under the control of appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.) and DNA encoding selectable marker. After the introduction of foreign DNA, the engineered cells can be left to grow for 1-2 days in an enriched medium and then transferred to a selective medium. The selectable marker present in the recombinant plasmid confers selection resistance and encourages cells to stably integrate this plasmid into their chromosomes and grow to form plaques, which in turn can be cloned and distributed into cell lines. This method can be used to construct cell lines that express a multifunctional polypeptide.

In some embodiments, the multifunctional polypeptides provided herein are expressed in a cell line characterized by transient expression of the multifunctional polypeptide. Transient transfection is a process in which a nucleic acid introduced into a cell is not integrated into the cell's genomic or chromosomal DNA, but is maintained in the cell as an extrachromosomal element, such as an episome. The processes of transcription of the nucleic acid of this episome are not affected, and the protein encoded by the nucleic acid of this episome is produced.

The cell line, either stably or transiently transfected, is maintained in cell culture medium and under conditions known in the art to result in expression and production of polypeptides. In some embodiments, the mammalian cell culture medium is based on commercially available media formulations, including, for example, Dulbecco's Modified Eagle's Medium (DMEM) or Ham's F12 Nutrient Formula. In some embodiments, the cell culture medium formulations are modified to promote enhancement of both cell growth and protein expression in a biological system. As used herein, the terms "cell culture medium", "culture medium" and "media composition" refer to a nutrient solution for maintaining, growing, propagating or spreading cells in an in vitro artificial environment outside of a multicellular organism or tissue. The cell culture medium can be optimized for a particular use of the cell culture, including, for example, a cell culture growth medium that is prepared to stimulate cell growth or a cell culture production medium that is prepared to stimulate the production of a recombinant protein. The terms "nutrient", "ingredient" and "component" can be used interchangeably and refer to elements that are part of the cell culture medium.

In various embodiments, the cell lines are maintained using a fed-batch method. As used herein, "fed-batch method" refers to a method in which a fed-batch cell culture is fed additional nutrients after it is initially incubated in a basic nutrient medium. For example, a fed-batch method may include adding a support medium according to a specific feeding schedule within a predetermined period of time. Thus, “fed-batch cell culture” refers to a cell culture where cells, usually mammalian cells, and culture medium are first loaded into a culture vessel and during culture, additional culture nutrients are supplied to the culture, continuously or stepwise, with periodic withdrawal of cells and/or product, or without such diversion, until the end of cultivation.

In some embodiments, the cell culture medium comprises a basic nutrient medium and at least one hydrolysate, such as soy hydrolyzate, a yeast hydrolyzate, or a combination of the two types of hydrolysates, resulting in a modified basic nutrient medium. Additional nutrients may sometimes include only the main nutrient medium, such as a concentrated basic nutrient medium, or may include only hydrolysates or concentrated hydrolysates. Suitable basic culture media include, but are not limited to, Dulbecco's Modified Eagle's Medium (DMEM), DME/F12, Minimal Maintenance Medium (MEM), Eagle's Basic Medium (BME), Roswell Park Memorial Institute (RPMI) 1640 Medium, F -10, F-12, alpha-modified minimum maintenance medium (alpha-MEM), Glasgow minimum maintenance medium (G-MEM), PF CHO (see e.g. protein-free (PF) CHO cell medium (Sigma) or serum-free EX-CELL™ 325 PF CHO medium for protein-free medium for CHO cells (SAFC Bioscience)) and Iscove's modified Dulbecco's medium. Other examples of basic culture media that can be used in the process of this invention include BME basic culture medium (GibcoInvitrogen), Dulbecco's Modified Eagle's Medium (DMEM, powder) (GibcoInvitrogen (No. 31600)).

In some embodiments, the base culture medium may be a serum-free medium, meaning that the medium does not contain any serum (e.g., fetal bovine serum (FBS), horse serum, goat serum, or serum derived from any other animal known to those skilled in the art. of this technical field), or be media that do not contain proteins of animal origin, or media with a given chemical composition.

The basic culture medium can be modified to eliminate some of the non-nutritional components found in the standard basic culture medium, such as buffers based on various inorganic and organic compounds, surfactant(s) and sodium chloride. The exclusion of such components from the main nutrient medium for cells allows you to increase the concentration of the remaining nutrients and can optimize overall cell growth and protein expression. In addition, the withdrawn components can be added back to the cell culture medium containing the modified basic cell nutrient medium according to the requirements of the cell culture conditions. In some embodiments, the cell culture medium comprises a modified basic cell nutrient medium and at least one of the following nutrients: an iron source, a recombinant growth factor, a buffer, a surfactant, an osmolarity regulator, an energy source, and non-animal derived hydrolysates. In addition, the modified basic cell culture medium may optionally contain amino acids, vitamins, or a combination of both amino acids and vitamins. In some embodiments, the modified basic nutrient medium additionally contains glutamine, such as L-glutamine, and/or methotrexate.

In some embodiments, a bioreactor method is used to produce a protein in large quantities, using methods using a fed-batch bioreactor, a batch bioreactor, a continuous-fed bioreactor, or a continuous-feed bioreactor known in the art. The capacity of large-scale bioreactors is at least 50 liters, sometimes a little over 500 liters, or 1,000 to 100,000 liters. In such bioreactors paddles can be used to distribute oxygen and nutrients. Small scale bioreactors are generally concerned with culturing cells in a volume of no more than about 100 liters, and the volumetric capacity may vary from about 1 liter to about 100 liters. Alternatively, single use bioreactors (SUBs) can be used for both large scale and small scale cultivation.

The temperature, pH, mixing conditions, aeration and density of the inoculum may vary depending on the host cells used and the recombinant protein to be expressed. For example, the cell culture used to express the recombinant protein can be maintained at 30 to 45 degrees Celsius. During cultivation, the pH of the culture medium can be monitored to maintain the pH at an optimal level, which for some host cells may be in the pH range of 6.0-8.0. In such cultivation methods, paddle wheel agitation can be used for agitation. The speed of rotation of such a stirrer may be, in terms of blade tip circumferential velocity, approximately 50-200 cm/s, however, depending on the type of cultured host cell, other air supply systems or other agitation/aeration systems known in the art may be used. Sufficient aeration is provided to maintain the dissolved oxygen concentration in the culture at about 20% to 80% air saturation, again depending on the host cell selected for culture. Alternatively, the bioreactor may be provided for bubbling air or oxygen directly into the culture medium. Other methods of oxygen supply exist, including the use of bubble-free aeration systems that use hollow fiber membrane aerators.

Protein Purification

Proteins produced by the transfected host described above can be purified according to any suitable method. Such standard methods include chromatography (eg, ion exchange, affinity and gel permeation column chromatography (sizing column chromatography)), centrifugation, use of differences in solubility, or any other standard protein purification method. To facilitate purification by passing through an appropriate affinity chromatography column, affinity labels such as hexahistidine, maltose binding domain, influenza envelope protein sequence, and glutathione S-transferase can be attached to the protein. Isolated proteins can also be characterized using physical methods such as proteolysis, nuclear magnetic resonance, and X-ray diffraction crystallography.

For example, supernatants from systems that secrete recombinant protein into culture media can first be concentrated using a commercially available protein concentration filter, such as an Amicon or Pellicon ultrafiltration cell from Millipore. After the concentration step, the concentrate can be applied to a suitable matrix for purification. Alternatively, an anion exchange resin, such as a matrix or carrier, having pendent diethylaminoethyl (DEAE) groups can be used. Such matrices can be matrices based on acrylamide, agarose, dextran, cellulose, or other types of matrices commonly used in protein purification. Alternatively, a cation exchange step can be used. Suitable cation exchangers include various insoluble matrices containing sulfopropyl or carboxymethyl groups. Finally, one or more reverse phase high performance liquid chromatography (RP-HPLC) steps can be performed to further purify the NGF binder using a hydrophobic carrier for RP-HPLC, such as silica gel having pendant methyl or other aliphatic groups. Some or all of the above purification steps may also be used in various combinations to obtain a homogeneous recombinant protein.

The recombinant protein produced by the bacterial culture can be isolated, for example, by first extracting from pellets after cell centrifugation, then performing one or more steps of concentration, salting out, ion exchange or gel permeation chromatography under aqueous conditions. For final purification steps, high performance liquid chromatography (HPLC) can be used. The microbial cells used in the expression of the recombinant protein may be disrupted by any convenient method, including freeze-thaw cycle, ultrasonication, mechanical disruption, or the use of cell-lysing agents.

Methods known in the art for purifying recombinant polypeptides also include, for example, the methods described in U.S. Patent Application Publication Nos. 2008/0312425, 2008/0177048, and 2009/0187005, each of which is hereby incorporated herein by reference. in its entirety.

Methods of administration and pharmaceutical compositions

The present invention provides methods for controlling or treating pain in a subject comprising administering a therapeutically effective amount of a multifunctional TNFα and NGF antagonist polypeptide, such as a multispecific binding molecule as provided herein, or comprising co-administration of a TNFα antagonist and an NGF antagonist. According to certain aspects, such a subject is a person.

The invention also provides pharmaceutical compositions comprising a multifunctional TNFα and NGF antagonist polypeptide, such as a multispecific binding molecule as provided herein, or comprising a combination of a TNFα antagonist and an NGF antagonist as provided herein. In certain aspects, the pharmaceutical compositions further comprise a pharmaceutically acceptable excipient. These pharmaceutical compositions are useful in the treatment of pain, such as neuropathic and inflammatory (eg, due to osteo- or rheumatoid arthritis) pain.

The multifunctional polypeptides and compositions comprising an NGF antagonist and a TNFα antagonist provided herein may be useful in a variety of applications, including, but not limited to, the control or treatment of pain, such as neuropathic pain. Methods for such use may be in vitro, ex vivo or in vivo methods.

In certain aspects, the disease, disorder, or condition being treated with an NGF-binding agent (eg, antibody or polypeptide) is associated with pain. In certain aspects, the pain is associated with chronic nociceptive pain, chronic low back pain, neuropathic pain, cancer pain, postherpetic neuralgia (PHN) pain, or visceral pain conditions.

The present invention provides a method for controlling pain in a subject, comprising administering to a subject in need of pain control an effective amount of a nerve growth factor (NGF) antagonist and a tumor necrosis factor (TNFα) antagonist, which administration can control the subject's pain more effectively than administering an equivalent amount of an NGF antagonist or a TNFα antagonist as monotherapy.

In certain aspects, the administration is the co-administration of an NGF antagonist and a TNFα antagonist as a combination therapy. As discussed elsewhere in this specification, the individual components may be administered simultaneously or sequentially. The individual NGF antagonist or TNFα antagonist may be any of the NGF or TNFα antagonists of the present invention, e.g., a soluble NGF-binding fragment of the TrkA receptor, an anti-NGF antibody or antigen-binding fragment thereof, a soluble TNFα-binding fragment of the TNF receptor, e.g., TNFR -2, or an anti-TNFα antibody or fragment thereof, for example, infliximab, adalimumab, certolizumab pegol, golimumab, or an antigen-binding fragment of any of these antibodies.

When co-administered, different doses of each of the antagonists may be used as needed to control pain. In certain aspects, co-administration may use lower doses of each component or administer doses at a lower frequency than conventional administration as single agent therapy, thereby providing additional safety, convenience, and economy.

In certain aspects, the method of controlling pain of the present invention comprises administering a multifunctional polypeptide, for example, a multispecific binding molecule comprising an NGF antagonist domain and a TNFα antagonist domain. Typical multifunctional polypeptides for use in this method are detailed in this description. Given this description, one of ordinary skill in the art will recognize additional multifunctional polypeptides useful for use in this method.

By "more effective" pain control compared to administering the components as monotherapy is meant that the combination treatment is more effective in controlling pain than administering equivalent amounts of either the NGF antagonist or the TNFα antagonist alone. In certain aspects, and as described in more detail below, the pain control method of the present invention may provide synergistic efficacy, for example, the effect of administering both an NGF antagonist and a TNFα antagonist may provide more than an additive effect, or such administration may be effective in where neither the NGF antagonist nor the TNFα antagonist is effective alone. In certain aspects, such a combination may provide for a sparing dosing regimen, for example, the effective dosages of the individual components when administered together may be less than the effective dosages of either component alone.

According to certain aspects, the pain control method proposed in this application is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% 80%, 90% or 100% more effective. in terms of controlling pain in a subject than administering an equivalent amount of an NGF antagonist or a TNFα antagonist as monotherapy. In certain aspects, the dosages of the NGF antagonist or TNFα antagonist alone when co-administered to a subject may be lower, or the amount of relative dose of NGF antagonist or TNFα antagonist provided when a bifunctional polypeptide of the invention is administered may be lower, for example by 5%, 10%, 20%, 30%, 40%, 50% 60%, 70%, 80% or 90% below the dosages required when these components are administered as monotherapy.

The effectiveness of pain control can be assessed by questioning the patient in order to classify the nature and intensity of the pain experienced in accordance with a number of different scales. In the case of the verbal pain scale, verbal ratings are used to describe the pain in the following range: "no pain", "mild pain", "moderate pain" and "severe pain" with scores from 0 to 3 assigned to each condition. Alternatively, the patient may be asked to classify their pain according to a quantitative pain rating scale ranging from 0 (no pain) to 10 (worst pain). The vertical or horizontal line of the Visual Analogue Scale (VAS) contains words describing pain in terms of "no pain" to "maximum possible pain", and patients are asked to mark the point on this line that corresponds to their current level of pain. The McGill Pain Score allows patients to describe both the nature and intensity of pain by selecting the words that best describe their pain from a set of short lists, such as throbbing, burning, squeezing pain. Other pain rating scales can be used for adults who have difficulty using the VAS or quantitative scales, such as the FACES Facial Pain Rating Scale, or for patients with non-verbal behavior, such as the Behavioral Rating Scale. To assess functional activity in relation to the extent to which the pain experienced by patients burdens them, you can ask them to perform a task related to this painful area. An improvement in pain scores detected using these types of scale would theoretically indicate an improvement in the effectiveness of the pain reliever.

According to the pain control method of the present invention, such administration is sufficient to control pain in a subject in need of pain control, for example, co-administration of an NGF antagonist and a TNFα antagonist, or administration of a multifunctional polypeptide, for example, a multispecific binding molecule containing an NGF antagonist domain and a a TNFα antagonist can prevent, alleviate, reduce or eliminate pain in a subject. In certain aspects, the pain may be acute pain, transient pain, persistent or chronic nociceptive pain, or persistent or chronic neuropathic pain.

In certain aspects, compositions are prepared for storage and use by combining a multifunctional TNFα and NGF antagonist polypeptide, e.g., a multispecific binding molecule of the invention, or a combination of a TNFα antagonist and an NGF antagonist of the invention, with a pharmaceutically acceptable excipient (e.g., carrier , excipient) (Remington, The Science and Practice of Pharmacy, 20th Edition, Mack Publishing, 2000). Suitable pharmaceutically acceptable excipients include, but are not limited to, non-toxic buffers such as buffers based on phosphoric, citric and other organic acids; salts such as sodium chloride; antioxidants, including ascorbic acid and methionine; preservatives (for example, ammonium octadecyldimethylbenzyl chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and meta -cresol); low molecular weight polypeptides (eg, containing less than about 10 amino acid residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; carbohydrates such as monosaccharides, disaccharides, glucose, mannose or dextrins; chelating agents such as ethylenediaminetetraacetic acid (EDTA); sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium ion; metal complexes (eg Zn-protein complexes); and non-ionic surfactants such as tween or polyethylene glycol (PEG).

Based on the multifunctional polypeptides of the present invention, compositions can be prepared in liquid, semi-solid or solid forms, depending on the physico-chemical properties of the molecule and the route of delivery. Such compositions may include excipients or combinations of excipients such as sugars, amino acids and surfactants. Liquid compositions may include polypeptides in a wide range of concentrations and pH. Solid compositions can be prepared by lyophilization, spray drying or drying using, for example, supercritical fluid technology. In some embodiments, any of the compositions described in this application is a lyophilized composition.

In a specific embodiment, the multifunctional polypeptide of the invention is formulated in 20 mM sodium phosphate, 50 mM L-arginine-HCL, 150 mM sucrose, 0.03% (w/v) polysorbate 80, pH 6.5.

The pharmaceutical composition of this invention can be administered by any of a number of routes used either for local or systemic treatment options. Administration may be topical (eg, mucosal, including vaginal and rectal delivery), eg, via transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders; pulmonary (for example, by inhalation or insufflation of powders or aerosols, including by means of a nebulizer; intratracheal, intranasal, epidermal and transdermal); oral; or parenteral, including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial (eg, intrathecal or intraventricular) administration.

A multifunctional TNFα and NGF antagonist polypeptide of the present invention or a combination of a TNFα antagonist and an NGF antagonist of the present invention may further be combined in a pharmaceutical combination composition, or in a combination therapy regimen, with a second (or third) compound with antinociceptive properties.

With respect to the treatment of pain, the appropriate dosage of a multifunctional TNFα and NGF antagonist polypeptide, such as the multispecific binding molecule of this invention, or the combination of a TNFα antagonist and an NGF antagonist of this invention, depends on the type of pain being treated, the severity, and the source. pain, pain susceptibility, whether the multifunctional polypeptide or combination of polypeptides is administered for therapeutic or prophylactic purposes, previous therapy, patient's clinical history, and so on; All this is at the discretion of the attending physician. To maintain effective pain control, a multifunctional polypeptide or combination of polypeptides can be administered once or as a course of treatment lasting from several days to several months. Optimal dosing schedules can be calculated based on measurements of drug accumulation in the patient's body and will vary depending on the relative potency of the individual antibody or polypeptide. The physician in charge of administration can readily determine optimal dosages, administration methodologies, and repetition rates.

Administration of a multifunctional polypeptide, such as a multispecific binding molecule, or combination of polypeptides as provided herein can provide "synergism" and exhibit a "synergistic character", i.e. the effect achieved by the combined use of active ingredients exceeds the sum of the effects obtained as a result of the use of these compounds separately. A synergistic effect can be achieved when: (1) the active ingredients are administered as a single multifunctional fusion polypeptide; (2) these active ingredients are formulated into a combined unit dosage formulation and administered or delivered simultaneously in its composition; (3) the active ingredients are delivered alternately or in parallel as separate formulations; or (4) using some other mode (introduction). When delivered using interleaved therapy, a synergistic effect can be achieved when the compounds are administered or delivered sequentially, for example, through different injections using separate syringes. In general, in alternating therapy, an effective dosage of each active ingredient is administered sequentially, while in the case of combination therapy, effective dosages of two or more active ingredients are administered together.

Pain

In its broadest sense, the term "pain" refers to an experienced symptom that is highly subjective to the individual experiencing it and is influenced by the individual's mental state, including the environment and cultural background. "Physical" pain can usually be associated with an externally felt stimulus that causes actual or potential tissue damage. In this sense, pain can be thought of as "a sensory and emotional experience associated with or described in terms of actual or potential tissue damage" according to the International Association for the Study of Pain (IASP). However, some cases of pain have no identifiable cause. For example, psychogenic pain, including exacerbation of previously experienced physical pain under the influence of psychogenic factors, or syndromes of sometimes persistent, felt pain in individuals with psychological disorders without any evidence of an identifiable cause of pain.

Types of pain

Pain includes nociceptive pain, neuropathic/neurogenic pain, breakthrough pain, allodynia, hyperalgesia, hyperesthesia, dysesthesia, paresthesia, hyperpathia, phantom pain, psychogenic pain, pain anesthesia, neuralgia, neuritis. Other categories include cancer pain, angina and/or idiopathic pain, complex regional pain syndrome I, complex regional pain syndrome II. The types and symptoms of pain need not be mutually exclusive. These terms are intended to be defined in the IASP.

Nociceptive pain is initiated in response to noxious stimuli by specialized sensory nociceptors in peripheral nerves that convert nociceptive stimuli into action potentials. Nociceptors, typically on Aδ fibers and (polymodal) C fibers, are free nerve endings that terminate just below the skin, in tendons, joints, and in body organs. Spinal ganglion neurons (DRGs) provide a connection point between the periphery and the spinal cord. This signal is processed as it travels through the spinal cord to the brainstem and sites in the thalamus and finally to the cerebral cortex, where it usually (but not always) produces the sensation of pain. Nociceptive pain can be caused by a wide range of chemical, thermal, biological (e.g., inflammatory) or mechanical events that can lead to irritation or damage to body tissue, usually above a certain minimum threshold of intensity necessary to induce nociceptive activity in nociceptors.

Neuropathic pain is usually the result of abnormal functioning in the peripheral or central nervous system, leading to the onset of peripheral or central neuropathic pain, respectively. Neuropathic pain is defined by the IASP as pain initiated or caused by a primary lesion or dysfunction in the nervous system. Neuropathic pain often involves actual damage to the nervous system, especially in chronic cases. Inflammatory nociceptive pain is typically the result of tissue damage and the resulting inflammatory process. Neuropathic pain may persist for a considerable time after (eg months or years) apparent recovery from any observable tissue damage.

In cases of neuropathic pain, sensory processing along the way from the affected area may become abnormal, and neutral stimuli (eg, thermal stimuli, touch/pressure) that do not normally cause pain may cause pain (i.e., allodynia), or noxious stimuli may cause an abnormally heightened perception of pain (ie, hyperalgesia) in response to a normal noxious stimulus. In addition, conventional stimuli may elicit sensations similar to electrical tingling or electric shocks or "tingling in the extremities" (i.e., paresthesia) and/or may elicit sensations with characteristic unpleasant features (i.e., numbness). dysesthesia). Breakthrough pain is an exacerbation of pre-existing chronic pain. Hyperpathy is a pain syndrome characterized by an abnormally painful reaction to stimulus. In most cases, this irritation is repeated with increasing pain threshold, which can be considered as the smallest pain perception that the patient can recognize as pain.

Examples of neuropathic pain include tactile allodynia (eg, induced by nerve injury), neuralgia (eg, postherpetic neuralgia (or neuralgia after herpes zoster), trigeminal neuralgia), reflex sympathetic dystrophy/causalgia (nerve injury), cancer pain components (eg, pain due to the cancer itself or associated conditions such as inflammation, or pain due to treatments such as chemotherapy, surgery, or radiation therapy), phantom pain in amputees, compression neuropathy (eg, carpal tunnel), and neuropathies such as peripheral neuropathy (eg, due to diabetes, human immunodeficiency virus (HIV), chronic alcohol use, exposure to other toxins (including many chemotherapeutic agents), vitamin deficiency, and a wide range of other health disorders). Neuropathic pain includes pain induced by the manifestation of abnormal functioning of the nervous system after nerve injury due to various causes, such as surgery, injury, herpes zoster, diabetic neuropathy, leg or arm amputation, cancer, and the like. Health disorders associated with neuropathic pain include traumatic nerve injury, stroke, multiple sclerosis, syringomyelia, spinal cord injury, and cancer.

The pain-producing stimulus often induces an inflammatory response, which itself may contribute to the sensation of pain. In some conditions, pain appears to be caused by a combination of nociceptive and neuropathic factors. For example, chronic pain often includes inflammatory nociceptive pain or neuropathic pain, or a mixture of both. Initial dysfunction or damage to the nervous system can trigger the neuronal release of inflammatory mediators and subsequent neuropathic inflammation. For example, migraine-like headaches can be a mixture of neuropathic and nociceptive pain. In addition, myofascial pain is likely secondary to nociceptive signal from muscles, but abnormal muscle activity may be the result of neuropathic conditions.

Kits containing TNFα and NGF antagonists

According to this invention, kits containing a multifunctional TNFα and NGF antagonist polypeptide, for example, a multispecific binding molecule proposed in this invention, or a combination of a TNFα antagonist and an NGF antagonist proposed in this invention, which can be used to implement the methods described in this application . In certain aspects, the kit comprises at least a multifunctional fusion polypeptide comprising a TNFα antagonist and an NGF antagonist, e.g., a polypeptide comprising the amino acid sequence of SEQ ID NO: 14 or 17, in one or more containers, or a combination of an NGF antagonist, e.g., MEDI-578 , and a TNFα antagonist, eg, an anti-TNFα antibody, such as infliximab or adalimumab, or a TNFα-binding soluble TNF receptor fragment, eg, TNFR2-Fc. One of ordinary skill in the art will readily appreciate that the described TNFα and NGF antagonists of the present invention can easily be included in one of the established kit formats that are well known in the art.

EXAMPLES

Now the invention as a whole is described, it will be more understandable with reference to the following examples, which are included mainly for the purpose of illustrating certain aspects and embodiments set forth in the description of the present invention, and are not intended to limit this description.

Example 1 Construction and Characterization of a Multi-Specific Binding Molecule (anti-NGF Antibody ScFv Fragment)/TNFR2-Fc

A multifunctional molecule, specifically, a multispecific binding molecule containing an anti-NGF antibody domain and a TNFR2-Fc domain, was prepared as follows. Attachment of the anti-NGF antibody scFv fragment to the C-terminus of the TNFR2-Fc fusion protein (SEQ ID NO: 13) was via the heavy chain CH3 domain according to the Bs3Ab format (bispecific antibodies) described in Dimasi, N., et al. , J. Mol. Biol. , 393 : 672-92 (2009) and PCT Publication No. WO 2013/070565. A graphic representation of the structure is shown in Fig. 1. DNA constructs encoding a TNFR2-Fc polypeptide and a multispecific binding molecule were synthesized by GeneArt (Invitrogen). To obtain such a multispecific binding molecule, an anti-NGF scFv fragment was constructed containing the VH (SEQ ID NO: 3) and VL (SEQ ID NO: 7) domains of MEDI-578 linked together via a 15 amino acid linker sequence (GGGGS ) ₃ (SEQ ID NO: 15). Attachment of the N-terminus of the scFv to the C-terminus of SEQ ID NO: 13 was done via a 10 amino acid linker sequence (GGGGS) ₂ . This multispecific binding molecule is referred to herein as TNFR2-Fc_VH#4. A DNA construct encoding such a multispecific binding molecule was designed to contain a stop codon and an EcoRI restriction site at the 3' end for cloning into a Bs3Ab expression vector. The DNA sequence encoding TNFR2-Fc_VH#4 is shown as SEQ ID NO: 16 and its corresponding amino acid sequence as SEQ ID NO: 14.

The thermal stability of the TNF-NGF multispecific binding molecule was improved by adding an interchain disulfide bond between the VH and VL domains of the scFv portion of MEDI-578 in the multispecific binding molecule. This was done by introducing a G→C mutation at amino acid 44 of the VH domain (SEQ ID NO: 94) and at amino acid 102 of the VL domain (SEQ ID NO: 95). This clone was named TNFR2-Fc_varB. The amino acid sequence of TNFR2-Fc_varB is shown as SEQ ID NO: 17. The DNA sequence encoding TNFR2-Fc_varB is shown as SEQ ID NO: 18. The codon-optimized DNA sequence encoding TNFR2-Fc_varB is shown as SEQ ID NO: 99 TNFR2-Fc_varB further differs from TNFR2-Fc_VH#4 in that the 15 amino acid linker sequence (GGGGS) ₃ connecting the VH and VL scFv portions is changed to a 20 amino acid linker (GGGGS) ₄ (SEQ ID NO : nineteen). Differential scanning fluorometry (DSF) was used to measure Tm for TNFR2- _{Fc_VH} #4 and TNFR2-Fc_varB. This method evaluates the incorporation of the fluorescent dye Sypro Orange (Invitrogen), which binds to hydrophobic surfaces exposed during protein domain unfolding when exposed to elevated temperatures. In the analysis using DSF, T _pl for TNFR2-Fc_VH#4 was 62°C, while T _pl for TNFR2-Fc_varB was 66°C. Thus, adding an interchain disulfide bond to the scFv portion of the MEDI-578 multispecific molecule improved the thermal stability of the molecule by 4°C.

The TNFR2-Fc and TNFR2-Fc_VH#4 proteins were transiently expressed in CHO cell suspension using polyethyleneimine (PEI) (Polysciences) as a transfection reagent. Cells were maintained in CD-CHO medium (Life Technologies). Culture harvests from small-scale transfections were purified using affinity chromatography on a HiTrap MabSelect SuRe™ column (1 ml) according to the manufacturer's protocol (GE Healthcare) and then the buffer was changed to the following buffer: 1% sucrose, 100 mM NaCl, 25 mM L-arginine hydrochloride and 25 mM sodium phosphate (pH 6.3). The purity of the recombinant proteins was analyzed using SDS-PAGE under reducing conditions and using analytical gel permeation chromatography (see method below), and concentrations were determined by measuring absorbance at 280 nm using theoretically determined extinction coefficients.

Small-scale transient expression and purification of the TNFR2-Fc fusion protein and the TNF-NGF multispecific construct, TNFR2-Fc_VH#4, on a protein A immobilized column resulted in yields of 36.6 and 79.9 mg L ^-1 , respectively. .

A larger batch of TNFR2-Fc_VH#4 was prepared as follows. The crude culture harvest after large-scale transfection (up to and including 6 L) was filtered using depth filtration and loaded onto a 1.6 x 20 cm column of immobilized protein A agarose (GE Healthcare) pre-equilibrated with buffer A (phosphate buffered saline, pH 7, 2). The column was then washed with buffer A and the product was eluted using a stepwise gradient of buffer B (50 mM sodium acetate, pH less than 4.0). The product was further purified by loading onto a 1.6 x 20 cm Poros HS 50 column (Applied Biosystems) pre-equilibrated with buffer C (50 mM sodium acetate buffer, pH less than 5.5), washed with buffer C and then the product was eluted using a linear gradient from 0 to 1 M NaCl in 50 mM sodium acetate buffer, pH less than 5.5. The resulting eluates were analyzed using gel permeation HPLC. Protein concentration was determined by A ₂₈₀ spectroscopy on a Beckman DU520 spectrophotometer using a calculated extinction coefficient of 1.36.

TNFR2-Fc_VH#4 Characterization Methods

Western blot analysis was performed using standard protocols. Proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Life Technologies) using the Xcell SureLock™ system (Invitrogen) according to the manufacturer's instructions. The membrane was blocked with 3% (w/v) skimmed milk powder in phosphate buffered saline (PBS) for 1 hour at room temperature. Western blots were developed using standard protocols using a horseradish peroxidase (HRP) conjugated antibody specific for the human IgG Fc fragment (Sigma).

Gel permeation HPLC was performed using a Gilson HPLC system (307 Isocratic Pump, 151 Ultraviolet/Visible (UV/Vis) Detector, 215 Liquid Dispenser, and 819 Injection Module) with a BioSep column. -SEC-S3000 (300 x 7.8 mm) from Phenomenex and D-PBS (Dulbecco's PBS buffer; Life Technologies) as mobile phase at a flow rate of 1 ml/min. Twenty-five μl of the sample solution was injected into the column and the separation of the protein components was monitored by A _{280 nm} .

Enzymatic deglycosylation of the small batch purified TNFR2-Fc_VH#4 protein was performed using the EDGLY kit (Sigma-Aldrich) according to the manufacturer's protocols. Proteins were subjected to deglycosylation under both denaturing and native conditions. For denatured proteins, deglycosylation of 30 µg of protein was performed for 3 h at 37°C using peptide:N-glycosidase (PNGase; peptide:N-glycosidase) F, O-glycosidase, α-(2→3.6, 8,9)-neuraminidase, β-N-acetylglucosaminidase and β-(1→4)-galactosidase. Using the same set of enzymes as described above, deglycosylation of 35 µg of protein was performed under native conditions for 3 days at 37°C. Deglycosylated proteins were analyzed by SDS-PAGE using Coomassie staining and Western blotting using standard analysis protocols.

N-terminal sequencing of the amino acid sequence of TNFR2-Fc_VH#4 was performed as follows. Approximately 2 μg of TNFR2-Fc_VH#4 was analyzed on SDS-PAGE gel using standard protocols. Proteins were transferred to a PVDF membrane using the Xcell SureLock™ system (Invitrogen) according to the manufacturer's instructions. The membrane was stained with 0.1% (w/v) amido black for approximately 15 minutes on an orbital shaker, then washed with distilled water (dH ₂ O) to reduce background staining of the PVDF membrane. The membrane was dried in air, after which N-terminal sequencing was performed. Zones of interest were excised and sequencing of the N-terminus of the multispecific binding molecule was performed on an Applied Biosystems 494 HT sequencer (Applied Biosystems, San Francisco, CA, USA) using a real-time phenylthiohydantoin method using a micro-HPLC system, model 140A , from Applied Biosystems.

Characterization results

Purified TNFR2-Fc_VH#4 and TNFR2-Fc proteins were analyzed by SEC-HPLC for levels of aggregates, monomers and protein fragments (FIGS. 2A and 2B). The main monomer-containing peak represented approximately 90% of the total protein present, with the remaining approximately 10% corresponding to the protein mass with a lower retention time in the column, indicating the presence of higher order structures or aggregates. However, according to SEC-HPLC, the monomer peak had two distinct arms, indicating that the protein in this peak is not homogeneous. SDS-PAGE analysis with Coomassie staining showed two distinct bands for TNFR2-Fc_VH#4 (at approximately 100 and 75 kDa) and similarly also two distinct bands for the TNFR2-Fc fusion protein (at approximately 70 and 45 kDa) under reducing conditions (FIG. 2B). Under non-reducing conditions, three major bands for TNFR2-Fc_VH#4 (150 to 250 kDa) and one major and one minor band for the TNFR2-Fc fusion protein were found at approximately 150 and 120 kDa, respectively. Since the difference in molecular weights for these two zones under reducing conditions was approximately equivalent to the size of the scFv fragment (about 26.5 kDa), further analysis was performed to understand in what forms the multispecific binding molecule was obtained. Mass spectroscopic analysis under native conditions confirmed the SDS-PAGE data that in the case of two separate preparations of the purified protein, three proteins with molecular masses approximately corresponding to 125, 152 and 176 kDa were present in the purified TNFR2-Fc_VH#4 preparation (Fig. 2C ).

If the zone pattern observed in the gel after SDS-PAGE was due to different glycosylation of TNFR2-Fc_VH#4, then after deglycosylation there should be a return back to one zone. However, this zone pattern was maintained under both reducing and non-reducing conditions when TNFR2-Fc_VH#4 was deglycosylated either as a native protein or as a denatured protein (data not shown). Western blot staining of both glycosylated and deglycosylated TNFR2-Fc_VH#4 protein using a polyclonal antibody specific for the human IgG Fc fragment showed that both the expected full-length protein zone and the lower molecular weight zone were reactive with respect to an antibody specific for the Fc fragment (data not shown).

Final identification of the truncated product was performed using N-terminal amino acid protein sequencing. It was found that the first 8 amino acids from the N-terminus of the truncated protein are SMAPGAVH, which corresponds to amino acids 176-183 in the sequence TNFR2-Fc_VH#4 (SEQ ID NO: 14). This corresponded to a 175 amino acid shortening from the N-terminus of TNFR2-Fc_VH#4, leaving only 42 amino acids of the TNFR2 domain. This allows the inventors to accurately interpret mass data from SDS-PAGE, mass spectroscopy and SEC-HPLC analysis. Three combinations of TNFR2-Fc_VH#4 dimers are possible, and all were present in the purified protein preparations: (1) full-length homodimer, (2) full-length and truncated heterodimer, and (3) truncated homodimer. To accurately measure both in vitro and in vivo biological activity, a full length homodimer preparation was prepared using a two step column chromatography process. In the first stage, after purification on a protein A immobilized column, the product contained 80.5% monomer (Fig. 3A), and after the second stage purification on a column (SP Sepharose), the percentage of monomer was 97.8% (Fig. 3B) . The yield after the entire process was 7.3%.

Example 2 Thermal Stability Analysis Using Differential Scanning Calorimetry (DSC)

For calorimetric measurements, an automated system for DSC MicroCal VP-Capillary (GE Healthcare, USA) was used. Protein samples were tested at 1 mg/ml in 25 mM histidine/histidine-HCl buffer, pH 6.0. Protein samples and buffer were subjected to a temperature ramp from 25°C to 100°C at a rate of 95°C in one hour. Reference buffer data was subtracted from protein sample data using Origin 7 software and temperature transition regions were determined.

The thermogram for TNFR2-Fc_VH#4 (FIG. 4) shows three distinct unfolding transitions with denaturation temperatures ( _Tm ) of 64, 67 and 84°C. The inventors concluded that _Mp 64°C corresponded to denaturation of both the TNFR2 domain and the scFv domain of the anti-NGF antibody, with _Tm 67°C and 84°C representing typical Tm corresponding to denaturation for _CH2- and CH3- IgG1 domains, respectively (e.g., Dimasi, N., et al. , J. Mol. Biol. , 393 : 672-92 (2009) and PCT Publication No. WO 2013/070565). Without being bound by theory, scFvs generally have lower denaturation temperatures compared to other antibody domains and their unfolding is characterized by a single junction (Roberge et al., 2006, Jung et al., 1999, Tischenko et al., 1998).

Example 3 Confirmation of Antigen Binding to TNFR2-Fc_VH#4

A. Single and dual antigen binding by ELISA

Wells of Nunc Maxisorp plates were coated overnight at 4°C using 50 μl of TNFα solution (R&D Systems) diluted to 5 μg ml ^-1 in PBS (pH 7.4). The next day, the coating solution was removed and the wells were blocked with 150 μl of blocking buffer (3% skimmed milk in PBS) for 1 hour at room temperature. The wells were washed three times in PBS, after which 50 µl of solutions from a series of dilutions of the TNFR2-Fc_VH#4 protein prepared in blocking buffer were added. After 1 hour incubation at room temperature, the wells were washed three times in PBS-tween 20 (0.1% v/v; PBS-T). Fifty microliters of biotinylated NGF solution was then added to the wells and incubated for another hour at room temperature, after which they were washed as above and 50 μl of streptavidin-HRP solution (1:100) was added. After 1 hour of incubation at room temperature, the wells were washed with PBS-T, 50 μl of 3,3',5,5'-tetramethylbenzidine substrate solution was added and set aside for color development. The reaction was stopped by adding 1 M H ₂ SO ₄ solution and the absorbance at 450 nm was measured using a titration microplate reader. The data obtained was analyzed using Prism 5 software (GraphPad, San Diego, CA). To perform a single antigen binding ELISA, wells were coated with either TNFα or NGF-biotin as described above and antibody binding detection was performed using an HRP-conjugated antibody specific for human IgG Fc fragment (1:5000) and staining was performed. as described above.

The ELISA results are shown in FIG. 5. The TNFR2-Fc_VH#4 protein was designed to bind to both TNFα and NGF antigens. Single antigen binding was performed by first immobilizing a single antigen in a 96-well microtiter plate, then adding serial dilutions of TNFR2-Fc_VH#4. Specific binding detection was performed using a horseradish peroxidase (HRP) conjugated antibody specific for the IgG Fc fragment. To perform a dual antigen binding ELISA, the first antigen, TNFα, was immobilized on an ELISA plate and then serial dilutions of TNFR2-Fc_VH#4 were added, followed by the addition of the second biotinylated antigen, NGF, at a fixed concentration. Then, specific binding was detected using HRP-conjugated streptavidin. The TNFR2-Fc_VH#4 protein bound to TNFα and NGF in a single antigen binding ELISA (FIGS. 5A and B). In a dual antigen binding ELISA, TNFR2-Fc_VH#4 bound simultaneously to both TNFα and NGF (FIG. 5C).

B. Simultaneous Antigen Binding Using Surface Plasmon Resonance

Simultaneous antigen binding experiments were performed essentially as described in Dimasi, N., et al. , J. Mol. Biol. , 393 : 672-92 (2009) using a BIAcore 2000 instrument (GE Healthcare). Briefly, a CM5 sensor chip was used to immobilize approximately 1500 resonant units of TNFR2-Fc_VH#4 at a concentration of 100 nM. The sensor chip surfaces were then used to study competitive binding to TNFα and NGF. Antigen samples were prepared in HBS-EP buffer (10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (pH 7.4), 150 mM NaCl, 3 mM ethylenediaminetetraacetic acid (EDTA), 0.005% polysorbate P20) . A flow rate of 30 μl/min was used for all binding measurements. To assess the simultaneous binding of the multispecific antibody to TNFα and NGF, a 1 μM solution of TNFα (molecular weight 17.5 kDa) was injected over the surface of the sensor chip, and after completion of the injection, a mixture of TNFα and NGF (molecular weight 13.5 kDa), both at a concentration of 1 μM. TNFα was included in the NGF mixture to prevent signal loss due to TNFα dissociation during the NGF binding phase. As a control, a similar binding procedure was carried out and only TNFα was added at the last injection; the absence of any increase in the number of resonant units for this introduction indicated that binding to TNFα occurred at saturation levels. Conducted similar experiments on binding and control experiments, in which the order of introduction of TNFα and NGF was reversed.

The simultaneous binding of TNFR2-Fc_VH#4 to antigens was characterized using surface plasmon resonance. Qualitative analysis of binding events was carried out sequentially one after the other. TNFR2-Fc_VH#4 was covalently immobilized on the sensor chip surface using amino coupling chemistry. After that, the first antigen was injected, obtaining saturating levels of binding to TNFR2-Fc_VH#4, then the second antigen was injected as an equimolar mixture with antigen 1. The binding sensorogram clearly showed that the TNFR2-Fc_VH#4 protein bound simultaneously to TNFα and NGF (Fig. 6). Simultaneous binding to these two antigens occurred regardless of the order in which the antigens were administered.

Example 4 Inhibition of NGF-Induced TF-1 Cell Proliferation

TF-1 cells (ECACC, cat# 93022307) were seeded at 1.5×10 ⁴ cells/well in 50 µl of serum-free culture media in a 96-well tissue culture plate (Corning Costar) and incubated for 18 h at 37 °C in an atmosphere with 5% CO ₂ . Recombinant human (Sigma) or mouse (R&D Systems) NGF was preincubated with dilutions of TNFR2-Fc_VH#4, MEDI-578 as IgG1 TM YTE, non-binding isotype control for MEDI-578 IgG1 TM YTE or non-binding bispecific isotype control R347 Bs3Ab for 30 min at 37°C in a 96-well round bottom plate (Greiner). Then, fifty microliters of each sample was added to the cell plate and incubated for 48 h at 37°C. At the end of the incubation period, 100 μl of CellTiter-GLO® Cell Assay Buffer (Promega) was added and the plate was incubated for 10 min at 37° C. in a 5% CO ₂ atmosphere. The luminescence was then measured using a standard luminescence protocol. Standard NGF-induced TF-1 cell proliferation in the absence of antibody is shown in FIG. 7A.

The functional activity of TNFR2-Fc_VH#4 was determined using NGF-induced proliferation of TF-1 cells. The TNFR2-Fc_VH#4 protein is able to completely inhibit both human and mouse NGF-induced proliferation (FIGS. 7B and 7C, respectively). Fig. 7B: TF-1 cells were stimulated with recombinant human NGF at a concentration corresponding to EC ₈₀ . Cells were incubated with ligand in the presence of serial dilutions of the antibody for 48 hours, after which cell proliferation was quantified by culturing for 10 minutes using CellTiter-GLO® cell assay buffer (Promega). Fig. 7C: TF-1 cells were stimulated with recombinant mouse NGF at a concentration corresponding to EC ₈₀ . Cells were incubated with ligand in the presence of serial dilutions of the antibody for 48 h, after which cell proliferation was quantified by culturing for 10 min using CellTiter-GLO® cell assay buffer (Promega). These data demonstrate that the NGF-inhibitory portion of TNFR2-Fc_VH#4 is biologically active and inhibits NGF-induced proliferation with similar potency to MEDI-578 as IgG1TM. Similar data were also observed for TNFR2-Fc_varB and another TNF-NGF multispecific binding molecule, ndimab varB (FIGS. 7D and 7E). Ndimab varB contains a full-length anti-TNFα antibody, i. an antibody containing two full heavy chains and two full light chains in H ₂ L ₂ format, with a MEDI-578 scFv fragment fused to the C-terminus of the heavy chain of an anti-TNFα antibody. Ndimab varB light chain is shown in SEQ ID NO: 20 and ndimab varB heavy chain is shown in SEQ ID NO: 22.

Example 5 Inhibition of TNFα-Induced U937 Cell Apoptosis

U937 cells (ECACC cat# 85011440) were plated in a 96-well black walled tissue culture plate (Corning Costar) at 8 x 10 ⁵ cells/well in 50 μl of culture media. U937 cells were stimulated with recombinant human TNFα at a concentration corresponding to EC ₈₀ . Cells were incubated with ligand in the presence of serial dilutions of the antibody for 2 h, after which caspase 3 activity was quantified by culture for 2 h using caspase 3 assay buffer. Bs3Ab and etanercept were preincubated with cells for 30 min at 37°C. Thereafter, 50 μl of recombinant human TNFα (R&D Systems) was added to give a final concentration in the assay of 20 ng/ml, and further incubated for 2 hours at 37°C. At the end of the incubation period, 50 μl of caspase 3 activity buffer solution (0.2% (w/v) CHAPS (3-[(3-cholamidopropyl)dimethyl ammonio]-1-propanesulfonate) was added; 0.5% (v/v) igepal CA-630; 200 mM NaCl; 50 mM HEPES; 20 μM substrate DEVD-R110 (Invitrogen)) and the cells were incubated for 2.5 h at 37°C. Fluorescence was measured with excitation at 475 nm and emission at 512 nm. Caspase activity in the absence of a TNFα antagonist is shown in FIG. 8A.

The functional activity of TNFR2-Fc_VH#4 was determined using the analysis of TNFα-induced caspase 3 activity in U937 cells. The TNFR2-Fc_VH#4 protein completely inhibited TNFα-induced caspase 3 activity in the same way as etanercept (FIG. 8B). This clearly shows that the TNFα-inhibitory portion of TNFR2-Fc_VH#4 is biologically active and has similar potency to etanercept. Similar data was also observed for TNFR2-Fc_varB and ndimab varB (see Fig. 8C).

Example 6 In Vivo Assays

All in vivo procedures were performed in accordance with the UK Home Office Animals (Scientific Procedures) Act (1986), approved by the local ethics committee. Female C57Bl/6 mice (Charles River, UK) were used for all steps. Mice were housed in groups of 5-6 per cage in individual ventilated cages (IVC) with free access to food and water under a 12-hour light/dark cycle (light: 07:00-19:00). The temperature in the vivarium and experiment room was maintained at 24°C, and a constant background noise was provided through a conventional radio unit. For identification purposes, transponders were inserted in all mice at least 5 days before the start of each study under anesthesia (3% isoflurane in oxygen).

A. Seltzer Model of Neuropathic Pain

Mechanical hyperalgesia was assessed using an analgesimeter (Randall LO, Selitto JJ, Arch. Int. Pharmacodyn. Ther . , 111: 409-19 (1957)) (Ugo Basile). Increasing force was applied to the dorsal surface of each hind paw in turn until a withdrawal response was observed. At this point, the force application was stopped and the load in grams was recorded. Data are expressed as withdrawal threshold in grams for the ipsilateral and contralateral paws. After establishing the baseline, the mice were divided into 2 groups with approximately the same ratio between the ipsilateral/contralateral paws and underwent surgery. Mice were anesthetized using 3% isoflurane. Approximately 1 cm of the left sciatic nerve was then exposed by blunt dissection through an incision at mid-thigh level. A suture (10/0 natural silk:ethicon) was then passed through the dorsal third of the nerve and tightly tied. The incision was closed with glue and the mice were allowed to recover for at least seven days prior to the study. Sham operated mice were treated according to the same protocol, but after the nerve was exposed, the wound was sealed and the mice were allowed to recover. Mice were tested for hyperalgesia on days 7 and 10 after surgery. On the 10th day of testing, the operated mice were further subdivided into groups that received the isotype control CAT251 IgG1 (0.03 mg/kg, subcutaneously (sc)), etanercept (0.01 mg/kg, sc) , MEDI-578 (0.03 mg/kg, sc) or a combination of etanercept (0.01 mg/kg, sc) and MEDI-578 (0.03 mg/kg, sc) . All sham-operated mice received CAT251 (0.03 mg/kg, sc). Mechanical hyperalgesia was measured at 4 hours, 1st, 2nd, 3rd, 4th and 7th days post-dose.

Co-administration of etanercept and MEDI-578 in a model of mechanical hyperalgesia manifested as a significant decrease in the ratio between the ipsilateral/contralateral paws on the 10th day after surgery compared with sham-operated controls (Fig. 9). A single dose of either etanercept (0.01 mg/kg, sc) or MEDI-578 (0.03 mg/kg, sc) failed to significantly eliminate this hyperalgesia. Co-administration of etanercept (0.01 mg/kg, sc) with MEDI-578 (0.03 mg/kg, sc) resulted in a significant resolution of mechanical hyperalgesia 4 hours post-dose, and this effect was maintained within 7 days after dosing.

The second study evaluated the effect of TNFR2-Fc_VH#4. After the establishment of mechanical hyperalgesia, mice on the 13th day after surgery were injected with the isotype control R347 Bs3Ab (0.03 mg/kg, sc), etanercept (0.01 mg/kg, sc), MEDI-578 (0.03 mg/kg, sc) or TNFR2-Fc_VH#4 (0.01 mg/kg or 0.03 mg/kg, sc). Sham-operated animals received the isotype control R347 Bs3Ab (0.03 mg/kg, sc). Mice were tested for mechanical hyperalgesia 4 hours after administration and on days 1, 2, 4 and 7 after administration, as described above.

The introduction of TNFR2-Fc_VH#4 resulted in a significant decrease in the ratio between the ipsilateral/contralateral paws on the 10th day after surgery compared with sham-operated controls (Fig. 10A). Administration of either etanercept (0.01 mg/kg, sc) or MEDI-578 (0.03 mg/kg, sc) failed to significantly eliminate mechanical hyperalgesia. Meanwhile, administration of TNFR2-Fc_VH#4 (0.01 and 0.03 mg/kg, s.c.) resulted in a significant elimination of mechanical hyperalgesia 4 hours post-dose, an effect that persisted for 6 days post-dose. No effect was observed after administration of the R347 Bs3Ab control. Similar results were obtained with the introduction of TNFR2-Fc_varB (see Fig. 10B). These data confirm that TNFR2-Fc_VH#4 at very low doses can provide significant pain relief, while showing that equivalent doses of MEDI-578 or etanercept alone were ineffective or marginally effective.

B. Model of chronic joint pain

Hypersensitivity to mechanical stimuli was determined using an incapacitance tester for mice (Linton Instrumentation). Mice were placed in this device with their hind legs on separate sensors and body weight distribution was calculated for 4 s. The data were expressed as the ratio of the weight load between the ipsilateral and contralateral paws in grams.

After establishing the baseline, mice were divided into 2 groups with approximately the same ratio between the ipsilateral/contralateral paws. Intra-articular injections were performed using the following method: Animals were anesthetized using 3% isoflurane in oxygen and the left knee was shaved and wiped. The knee joint of each mouse was injected with either 10 µl of complete Freund's adjuvant (FCA) (10 mg/ml) or vehicle (light mineral oil) using a 25 gauge needle mounted on a 100 µl Hamilton syringe. Injections were performed directly into the synovial space of the knee joint. Mice were allowed to recover and retested for changes in hypersensitivity to mechanical stimuli on days 7 and 10 post-injection as described above. On day 10 of testing, FCA-treated mice were further randomized into groups and on day 13, mice were treated with etanercept (0.01 mg/kg, ip) or vehicle, after which they received a dose of MEDI-578 ( 0.03 mg/kg, intravenously (i.v.)) or isotype control CAT251 (0.03 mg/kg, i.v.). Mice were tested for hypersensitivity to mechanical stimuli 4 hours after administration and on the 1st, 2nd, 4th and 7th days after administration, as described above.

The effect of co-administration of etanercept and MEDI-578 was evaluated using an intra-articular FCA-induced inflammatory pain model. Intra-articular administration of FCA caused hypersensitivity to mechanical stimuli, which manifested itself as a significant decrease in the ratio between the ipsilateral/contralateral paws on the 7th and 10th day compared with the introduction of the filler as a control (Fig. 11). No reduction in the ipsilateral/contralateral paw ratio was observed in the sham-treated groups compared to pre-treatment baseline levels. Administration of etanercept (0.01 mg/kg, i.p.) + CAT251 (0.03 mg/kg, i.v.) or PBS (10 ml/kg, i.p.) + MEDI-578 (0, 03 mg/kg, i.v.) caused a slight elimination of FCA-induced hypersensitivity to mechanical stimuli after 4 hours and on the 1st, 2nd, 4th and 7th days after dosing, but it was not possible to reach statistical significance. Meanwhile, administration of etanercept (0.01 mg/kg, i.v.) + MEDI-578 (0.03 mg/kg, i.v.) significantly eliminated FCA-induced hypersensitivity to mechanical stimuli at all testing times. after the introduction.

The second study evaluated the effect of TNFR2-Fc_VH#4. After the establishment of FCA-induced hypersensitivity to mechanical stimuli, on the 13th day after the administration of FCA, mice were injected with: isotype control R347 Bs3Ab (0.01 mg/kg, sc), etanercept (0.01 mg/kg, sc .), MEDI-578 (0.01 mg/kg, sc) or TNFR2-Fc_VH#4 (0.003 mg/kg or 0.01 mg/kg, sc). Mice were again tested for hypersensitivity to mechanical stimuli 4 hours after administration and on the 1st, 2nd, 4th and 7th days after administration, as described above.

The action of TNFR2-Fc_VH#4 ("bispecific") compared to the actions of etanercept and MEDI-578 alone is shown in FIG. 12. Neither etanercept (0.01 mg/kg, sc) nor MEDI-578 (0.01 mg/kg, sc) significantly abolished FCA-induced mechanical hypersensitivity at any time point after the introduction. At the same time, the introduction of TNFR2-Fc_VH#4 contributed to a significant elimination of FCA-induced hypersensitivity to mechanical stimuli. The higher dose of TNFR2-Fc_VH#4 (0.01 mg/kg, sc) showed significant activity throughout the study, while the lower dose (0.003 mg/kg, sc) reached the level of significance. on the 1st day after administration, which remained similar to the level for the higher dose throughout the study.

B. Validated model of FCA-induced hypersensitivity to mechanical stimuli in rats

Intraplantar injection of complete Freund's adjuvant (FCA) elicits an inflammatory response that induces hypersensitivity and edema and mimics some aspects of the clinical manifestation of inflammatory pain. These effects can be investigated using weight-bearing equipment. To assess the possible antihyperalgesic properties of TNFR2-Fc_VH#4 in FCA-induced hypersensitivity, the weight loading method was used. Previously unexposed (naive) rats distribute their body weight equally between their two hind legs. However, when the hind (left) foot becomes inflamed and/or painful after the injection, the weight is redistributed so that less weight is placed on the affected foot (less weight bearing on the injured foot). The weight bearing on each hind limb was measured using a Temporary Incapacitation Monitor (hereinafter referred to as "Monitor") (Linton Instruments, UK) for rats. Rats are placed in this tester with their hind legs on separate sensors and the average force applied to both hind legs is recorded for 4 seconds.

For this study, naive rats (male Sprague Dawley rats (Harlan, UK), 198-258 g) were acclimatized in a treatment room in their own home cages with free access to food and water. Getting used to the control and measuring device was carried out within a few days. Before the introduction of the damaging factor, the initial weight load was recorded. Hypersensitivity to inflammation was induced by intraplantar injection of FCA (supplied by Sigma, 100 μl of a 1 mg/ml solution) into the left hindpaw. 23 hours after FCA administration, pre-treatment weight-bearing was measured to assess hypersensitivity.

Animals were then ranked and randomized into treatment groups according to the FCA weight-bearing range in the Latin square trial. 24 hours after FCA injection, animals were treated with either TNFR2-Fc_VH#4 ("bispecific") administered iv. at a dose of 0.003, 0.01, 0.03, 0.3 and 3 mg/kg, or NIP228 antibody as a negative control (an antibody developed to bind to the nitrophenol hapten) administered i.v. at a dose of 3 mg / kg, or filler (1% methylcellulose solution), administered orally (p.o.) at a dose of 2 ml / kg, or indomethacin, administered p.o. at a dose of 10 mg/kg.

Weight loading was assessed 4 and 24 hours after antibody/drug treatment. Data for each time point was analyzed by comparing the treated groups with the vehicle control group. Statistical analysis included repeated measures ANOVA followed by a planned comparison test using InVivoStat (invivostat.co.uk) (p<0.05 indicates statistical significance). The results are shown in FIG. 13. Significant elimination of hypersensitivity at time points 4 and 24 hours was observed with indomethacin (10 mg/kg). TNFR2-Fc_VH#4 at doses of 0.3 and 3 mg/kg showed significant elimination of hypersensitivity for both 4 and 24 hours, TNFR2-Fc_VH#4 at doses of 0.003 and 0.03 mg/kg also showed significant elimination of hypersensitivity but only for 24 hours. The NIP228 isotype control did not have any significant effect on the FCA response at any time point.

Example 7 Phosphorylation of p38 by TNFα and NGF

The scientific literature suggests that p38 phosphorylation plays an important role in neuropathic pain. For example, treatment with p38 inhibitors has been shown to prevent symptoms of neuropathic pain in a partial nerve injury model (Wen Y.R. et al., Anesthesiology, 2007, 107: 312-321) and in a model of sciatic inflammatory neuropathy (Milligan E.D. et al., J Neurosci., 2003, 23: 1026-1040). In the experiment presented in this application, investigated the effect of TNFα, NGF and the combination of TNFα and NGF on p38 phosphorylation in the analysis using cell cultures. Briefly, Neuroscreen-1 cells (a subclone of rat neuroendocrine PC-12 cells) were incubated with TNFα, NGF, or a combination of TNFα and NGF in increasing amounts. At the end of the 20 minute incubation period, phosphorylated p38 (phospho-p38) was quantitated using time-resolved homogeneous fluorescence (HTRF) analysis (Cisbio).

HTRF assay : After stimulation with TNFα, NGF, or a combination of TNFα and NGF, cell supernatants were quickly removed and cells were lysed in lysis buffer. The detection of phosphorylated mitogen-activated protein kinase phospho-p38 MAPK (Thr180/Tyr182) was performed in lysates in a sandwich assay format using two antibodies with different specificities: an antibody to phospho-p38 conjugated with europium cryptate (donor fluorophore) and an antibody to p38 (total) conjugated to d2 (acceptor fluorophore). Antibodies were incubated with cell lysates and HTRF ratios were calculated from fluorescence measurements at 665 nm and 620 nm using an EnVision Multilabel (Perkin Elmer) plate reader.

Data are presented as HTRF ratios, which are calculated as the ratio between emission at 665 nm and emission at 620 nm. A heat map showing HTRF relationships for p38 phosphorylation reactions is shown in FIG. 14. Dose-response curves showing the effect of TNFα, NGF, or a combination of TNFα and NGF are shown in FIG. 15. As can be seen from FIG. 15, the combined effect of higher concentrations of TNFα and NGF on p38 phosphorylation exceeds the predicted sum of phospho-p38 signals induced by either factor alone. These data suggest that TNFα and NGF may act together to induce p38 phosphorylation and that these two pathways may be involved in the molecular mechanism of pain signaling.

Example 8 ERK Phosphorylation by TNFα and NGF

Like p38, ERK is also activated during the development of neuropathic pain. In the experiment presented in this application, investigated the effect of TNFα, NGF and the combination of TNFα and NGF on ERK phosphorylation in the analysis using cell cultures. Briefly, Neuroscreen-1 cells (a subclone of rat neuroendocrine PC-12 cells) were incubated with TNFα, NGF, or a combination of TNFα and NGF in increasing amounts. At the end of the 20 minute incubation period, phospho-ERK was quantitated using HTRF analysis (Cisbio).

HTRF analysis : After stimulation, cell supernatants were quickly removed and cells were lysed in lysis buffer. Detection of phospho-ERK MAPK (Thr202/Tyr204) was performed in lysates in a sandwich assay format using two antibodies with different specificities: an antibody to phospho-ERK conjugated with europium cryptate (donor fluorophore) and an antibody to ERK (total) conjugated with d2 (acceptor fluorophore). Antibodies were incubated with cell lysates and HTRF ratios were calculated from fluorescence measurements at 665 nm and 620 nm using an EnVision Multilabel (Perkin Elmer) plate reader.

Data are presented as HTRF ratios, which are calculated as the ratio between emission at 665 nm and emission at 620 nm. A heat map showing HTRF relationships for ERK phosphorylation reactions is shown in FIG. 16. Dose-response curves showing the effect of TNFα, NGF, or a combination of TNFα and NGF are shown in FIG. 17. As can be seen from FIG. 17, small amounts of TNFα alone did not induce ERK phosphorylation, however, higher amounts increased NGF-induced ERK phosphorylation. These data suggest that TNFα and NGF may co-operate on ERK phosphorylation and that these two pathways may be involved in the molecular mechanism of pain signaling.

Sequence listing

SEQ ID NO: 1 - NP_002497.2| precursor of nerve growth factor beta (Homo sapiens)

1 MSMLFYTLIT AFLIGIQAEP HSESNVPAGH TIPQAHWTKL QHSLDTALRR ARSAPAAAIA

61 ARVAGQTRNI TVDPRLFKKR RLRSPRVLFS TQPPREAADT QDLDFEVGGA APFNRTHRSK

121 RSSSSHPIFHR GEFSVCDSVS VWVGDKTTAT DIKGKEVMVL GEVNINNSVF KQYFFETKCR

181 DPNPVDSGCR GIDSKHWNSY CTTTHTFVKA LTMDGKQAAW RFIRIDTACV CVLSRKAVRR

241A

SEQ ID NO: 2 - NP_000585.2| tumor necrosis factor (Homo sapiens)

1 MSTESMIRDV ELAEEALPKK TGGPQGSRRC LFLSLFSFLI VAGATTLFCL LHFGVIGPQR

61 EEFPRDLSLI SPLAQAVRSS SRTPSDKPVA HVVANPQAEG QLQWLNRRAN ALLANGVELR

121 DNQLVVPSEG LYLIYSQVLF KGQGCPSTHV LLTHTISRIA VSYQTKVNLL SAIKSPCQRE

181 TPEGAEAKPW YEPIYLGGVF QLEKGDRLSA EINRPDYLDF AESGQVYFGI IAL

SEQ ID NO: 3 - VH MEDI-578 (VH 1256A5)

1 QVQLVQSGAE VKKPGSSVKV SCKASGGTFS TYGISWVRQA PGQGLEWMGG IIPIFDTGNS

61 AQSFQGRVTI TADESTSTAY MELSSLRSED TAVYYCARSS RIYDLNPSLT AYYDMDVWGQ

121 GTMVTVSS

SEQ ID NO: 4 - VHCDR1 MEDI-578

1 TYGIS

SEQ ID NO: 5 - VHCDR2 MEDI-578

1 GIIPIFDTGN SAQSFQG

SEQ ID NO: 6 - VHCDR3 MEDI-578

1 SSRIYDLNPS LTAYYDMDV

SEQ ID NO: 7 - VL MEDI-578 (VL 1256A5)

1 QSVLTQPPSV SAAPGQKVTI SCSGSSSNIG NNYVSWYQQL PGTAPKLLIY DNNKRPSGIP

61 DRFSGSKSGT SATLGITGLQ TGDEADYYCG TWDSSLSAWV FGGGTKLTVL

SEQ ID NO: 8 - VLCDR1 MEDI-578

1 SGSSSNIGNN YVS

SEQ ID NO: 9 - VLCDR2 MEDI-578

1 DNNKRPS

SEQ ID NO: 10 - VLCDR3 MEDI-578

1 GTWDSSLSAW V

SEQ ID NO: 11

1 SSRIYDFNSA LISYYDMDV

SEQ ID NO: 12

1 SSRIYDMISS LQPYYDMDV

SEQ ID NO: 13 - Soluble TNFR2 amino acid sequence

1 LPAQVAFTPY APEPGSTCRL REYYDQTAQM CCSKCSPGQH AKVFCTKTSD TVCDSCEDST

61 YTQLWNWVPE CLSCGSRCSS DQVETQACTR EQNRICTCRP GWYCALSKQE GCRLCAPLRK

121 CRPGFGVARP GTETSDVVCK PCAPGTFSNT TSSTDICRPH QICNVVAIPG NASMDAVCTS

181 TSPTRSMAPG AVHLPQPVST RSQHTQPTPE PSTAPSTSFL LPMGPSPPAE GSTGDEPKSC

241 DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD

301 GVEVHNAKTK PREQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK

361 GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS

421 DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK

SEQ ID NO: 14 - TNFR2-Fc_VH#4 - amino acid sequence

1 LPAQVAFTPY APEPGSTCRL REYYDQTAQM CCSKCSPGQH AKVFCTKTSD TVCDSCEDST

61 YTQLWNWVPE CLSCGSRCSS DQVETQACTR EQNRICTCRP GWYCALSKQE GCRLCAPLRK

121 CRPGFGVARP GTETSDVVCK PCAPGTFSNT TSSTDICRPH QICNVVAIPG NASMDAVCTS

181 TSPTRSMAPG AVHLPQPVST RSQHTQPTPE PSTAPSTSFL LPMGPSPPAE GSTGDEPKSC

241 DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD

301 GVEVHNAKTK PREQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK

361 GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS

421 DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK GGG GSGGGGS QVQ

481 LVQSGAEVKK PGSSVKVSCK ASGGTFSTYG ISWVRQAPGQ GLEWMGGIIP IFDTGNSAQS

541 FQGRVTITAD ESTSTAYMEL SSLRSEDTAV YYCARSSRIY DLNPSLTAYY DMDVWGQGTM

601 VTVSS GGGGS GGGGSGGGGS AQSVLTQPPS VSAAPGQKVT ISCSGSSSNI GNNYVSWYQQ

661 LPGTAPKLLI YDNNKRPSGI PDRFSGSKSG TSATLGITGL QTGDEADYYC GTWDSSLSAW

721 VFGGGTKLTV L

SEQ ID NO: 15 - 15 amino acid (a.a.) linker sequence (Gly ₄ Ser) ₃

1 GGGGSGGGGS GGGGS

SEQ ID NO: 16 - TNFR2-Fc_VH#4 - nucleotide sequence

1 CTGCCCGCCC AGGTGGCCTT TACCCCTTAT GCCCCCGAGC CCGGCAGCAC CTGTCGGCTG

61 AGAGAGTACT ACGACCAGAC CGCCCAGATG TGCTGCAGCA AGTGCTCTCC TGGCCAGCAT

121 GCCAAGGTGT TCTGCACCAA GACCAGCGAC ACCGTGTGCG ACAGCTGCGA GGACAGCACC

181 TACACCCAGC TGTGGAACTG GGTGCCCGAG TGCCTGAGCT GCGGCAGCAG ATGCAGCAGC

241 GACCAGGTGG AAACCCAGGC CTGCACCAGA GAGCAGAACC GGATCTGCAC CTGTAGACCCC

301 GGCTGGTACT GCGCCCTGAG CAAGCAGGAA GGCTGCAGAC TCTGCGCCCC TCTGCGGAAG

361 TGCAGACCCG GCTTTGGCGT GGCCAGACCC GGCACCGAGA CAAGCGACGT GGTCTGTAAG

421 CCCTGCGCTC CTGGCACCTT CAGCAACACC ACCAGCAGCA CCGACATCTG CAGACCCCAC

481 CAGATCTGCA ACGTGGTGGC CATCCCCGGC AACGCCAGCA TGGATGCCGT CTGCACCAGC

541 ACTAGCCCCA CCAGAAGTAT GGCCCCTGGC GCCGTGCATC TGCCCCAGCC TGTGTCCACC

601 AGAAGCCAGC ACACCCAGCC CACCCCTGAG CCTAGCACCG CCCCCTCCAC CAGCTTTCTG

661 CTGCCTATGG GCCCTAGCCC TCCAGCCGAG GGAAGCACAG GCGACGAGCC CAAGAGCTGC

721 GACAAGACCC ACACTGTCC CCCCTGCCCT GCCCCTGAAC TGCTGGGCGG ACCCAGCGTG

781 TTCCTGTTCC CCCCAAAGCC CAAGGACACC CTGATGATCA GCCGGACCCC CGAAGTGACC

841 TGCGTGGTGG TGGACGTGTC CCACGAGGAC CCTGAAGTGA AGTTCAATTG GTACGTGGAC

901 GGCGTGGAAG TGCACAACGC CAAGACCAAG CCCAGAGAGG AACAGTACAA CTCCACCTAC

961 CGGGTGGTGT CCGTGCTGAC CGTGCTGCAC CAGGACTGGC TGAACGGCAA AGAGTACAAG

1021 TGCAAGGTCT CCAACAAGGC CCTGCCTGCC CCCATCGAGA AAACCATCAG CAAGGCCAAG

1081 GGCCAGCCCC GCGAGCCTCA GGTGTACACA CTGCCCCCCA GCCGGGAAGA GATGACCAAG

1141 AACCAGGTGT CCCTGACCTG CCTGGTCAAA GGCTTCTACC CCAGCGATAT CGCCGTGGAA

1201 TGGGAGAGCA ATGGCCAGCC CGAGAACAAC TACAAGACCA CCCCCCCTGT GCTGGACAGC

1261 GACGGCTCAT TCTTCCTGTA CAGCAAGCTG ACCGTGGACA AGAGCCGGTG GCAGCAGGGC

1321 AACGTGTTCA GCTGCAGCGT GATGCACGAG GCCCTGCACA ACCACTACAC CCAGAAGTCC

1381 CTGAGCCTGA GCCCCGGAAA GGGCGGTGGC GGATCCGGAG GTGGGGGATC TCAGGTGCAG

1441 CTGGTGCAGT CTGGCGCCGA AGTGAAGAAA CCCGGCTCTA GCGTGAAGGT GTCCTGCAAG

1501 GCCAGCGGCG GCACCTTCTC CACCTACGGC ATCAGCTGGG TCCGCCAGGC CCCTGGACAG

1561 GGCCTGGAAT GGATGGGCGG CATCATCCCC ATCTTCGACA CCGGCAACAG CGCCCAGAGC

1621 TTCCAGGGCA GAGTGACCAT CACCGCCGAC GAGAGCACCT CCACCGCCTA CATGGAACTG

1681 AGCAGCCTGC GGAGCGAGGA CACCGCCGTG TACTACTGCG CCAGAAGCAG CCGGATCTAC

1741 GACCTGAACC CCAGCCTGAC CGCCTACTAC GACATGGACG TGTGGGGCCA GGGCACCATG

1801 GTCACAAGTGT CTAGCGGAGG CGGCGGATCT GGCGGCGGAG GAAGTGGCGG GGGAGGATCT

1861 GCCCAGAGCG TGCTGACCCA GCCCCCTTCT GTGTCTGCCG CCCCTGGCCA GAAAGTGACC

1921 ATCTCCTGCA GCGGCAGCAG CAGCAACATC GGCAACAACT ACGTGTCCTG GTATCAGCAG

1981 CTGCCCGGCA CCGCCCCTAA GCTGCTGATC TACGACAACA ACAAGCGGCC CAGCGGCATC

2041 CCCGACCGGT TTAGCGGCAG CAAGAGCGGG ACTTCTGCTA CACTGGGCAT CACAGGCCTG

2101 CAGACCGGCG ACGAGGCCGA CTACTACTGC GGCACCTGGG ACAGCAGCCT GAGCGCTTGG

2161 GTGTTCGGCG GAGGCACCAA GCTGACAGTG CTG

SEQ ID NO: 17 - TNFR2-Fc_varB - amino acid sequence

1 LPAQVAFTPY APEPGSTCRL REYYDQTAQM CCSKCSPGQH AKVFCTKTSD TVCDSCEDST

61 YTQLWNWVPE CLSCGSRCSS DQVETQACTR EQNRICTCRP GWYCALSKQE GCRLCAPLRK

121 CRPGFGVARP GTETSDVVCK PCAPGTFSNT TSSTDICRPH QICNVVAIPG NASMDAVCTS

181 TSPTRSMAPG AVHLPQPVST RSQHTQPTPE PSTAPSTSFL LPMGPSPPAE GSTGDEPKSC

241 DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD

301 GVEVHNAKTK PREQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK

361 GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS

421 DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK GGG GSGGGGS QVQ

481 LVQSGAEVKK PGSSVKVSCK ASGGTFSTYG ISWVRQAPGQ CLEWMGGIIP IFDTGNSAQS

541 FQGRVTITAD ESTSTAYMEL SSLRSEDTAV YYCARSSRIY DLNPSLTAYY DMDVWGQGTM

601 VTVSS GGGGS GGGGSGGGGS GGGGS QSVLT QPPSVSAAPG QKVTISCSGS SSNIGNNYVS

661 WYQQLPGTAP KLLIYDNNKR PSGIPDRFSG SKSGTSATLG ITGLQTGDEA DYYCGTWDSS

721 LSAWVFGCGT KLTVL

SEQ ID NO: 18 - TNFR2-Fc_varB - nucleotide sequence

1 CTGCCCGCCC AGGTGGCCTT TACCCCTTAT GCCCCCGAGC CCGGCAGCAC CTGTCGGCTG

61 AGAGAGTACT ACGACCAGAC CGCCCAGATG TGCTGCAGCA AGTGCTCTCC TGGCCAGCAT

121 GCCAAGGTGT TCTGCACCAA GACCAGCGAC ACCGTGTGCG ACAGCTGCGA GGACAGCACC

181 TACACCCAGC TGTGGAACTG GGTGCCCGAG TGCCTGAGCT GCGGCAGCAG ATGCAGCAGC

241 GACCAGGTGG AAACCCAGGC CTGCACCAGA GAGCAGAACC GGATCTGCAC CTGTAGACCCC

301 GGCTGGTACT GCGCCCTGAG CAAGCAGGAA GGCTGCAGAC TCTGCGCCCC TCTGCGGAAG

361 TGCAGACCCG GCTTTGGCGT GGCCAGACCC GGCACCGAGA CAAGCGACGT GGTCTGCAAG

421 CCCTGCGCTC CTGGCACCTT CAGCAACACC ACCAGCAGCA CCGACATCTG CAGACCCCAC

481 CAGATCTGCA ACGTGGTGGC CATCCCCGGC AACGCCAGCA TGGATGCCGT GTGCACCAGC

541 ACCAGCCCCA CCAGAAGTAT GGCCCCTGGC GCCGTGCATC TGCCCCAGCC TGTGTCCACC

601 AGAAGCCAGC ACACCCAGCC CACCCCTGAG CCTAGCACCG CCCCCTCCAC CAGCTTTCTG

661 CTGCCTATGG GCCCTAGCCC TCCAGCCGAG GGAAGCACAG GCGACGAGCC CAAGAGCTGC

721 GACAAGACCC ACACTGTCC CCCCTGCCCT GCCCCTGAAC TGCTGGGCGG ACCCAGCGTG

781 TTCCTGTTCC CCCCAAAGCC CAAGGACACC CTGATGATCA GCCGGACCCC CGAAGTGACC

841 TGCGTGGTGG TGGACGTGTC CCACGAGGAC CCTGAAGTGA AGTTCAATTG GTACGTGGAC

901 GGCGTGGAAG TGCACAACGC CAAGACCAAG CCCAGAGAGG AACAGTACAA CTCCACCTAC

961 CGGGTGGTGT CCGTGCTGAC CGTGCTGCAC CAGGACTGGC TGAACGGCAA AGAGTACAAG

1021 TGCAAAGTCT CCAACAAGGC CCTGCCTGCC CCCATCGAGA AAACCATCAG CAAGGCCAAG

1081 GGCCAGCCCC GCGAGCCTCA gGTGTACACA CTGCCCCCCA GCCGGGAAGA GATGACCAAG

1141 AACCAGGTGT CCCTGACCTG CCTGGTCAAA GGCTTCTACC CCAGCGATAT CGCCGTGGAA

1201 TGGGAGAGCA ACGGCCAGCC CGAGAACAAC TACAAGACCA CCCCCCCTGT GCTGGACAGC

1261 GACGGCTCAT TCTTCCTGTA CAGCAAGCTG ACCGTGGACA AGAGCCGGTG GCAGCAGGGC

1321 AATGTCTTCA GCTGTAGCGT GATGCACGAG GCCCTGCACA ACCACTACAC CCAGAAGTCC

1381 CTGAGCCTGA GCCCCGGAAA GGGCGGAGGC GGATCCGGAG GTGGGGGATC TCAGGTGCAG

1441 CTGGTGCAGT CTGGCGCCGA AGTGAAGAAA CCCGGCTCTA GCGTGAAGGT GTCCTGCAAG

1501 GCCAGCGGCG GCACCTTCTC CACCTACGGC ATCAGCTGGG TCCGCCAGGC CCCTGGACAG

1561 TGTCTGGAAT GGATGGGCGG CATCATCCCC ATCTTCGACA CCGGCAACAG CGCCCAGAGC

1621 TTCCAGGGCA GAGTGACCAT CACCGCCGAC GAGAGCACCT CCACCGCCTA CATGGAACTG

1681 AGCAGCCTGC GGAGCGAGGA CACCGCCGTG TACTACTGCG CCAGAAGCAG CCGGATCTAC

1741 GACCTGAACC CCAGCCTGAC CGCCTACTAC GACATGGACG TGTGGGGCCA GGGCACCATG

1801 GTCACAAGTGT CTAGCGGAGG CGGAGGCAGC GGAGGTGGTG GATCTGGTGG CGGAGGAAGT

1861 GGCGGCGGAG GCTCTCAGAG CGTGCTGACC CAGCCCCCTT CTGTGTCTGC CGCCCCTGGC

1921 CAGAAAGTGA CCATCTCCTG CAGCGGCAGC AGCAGCAACA TCGGCAACAA CTACGTGTCC

1981 TGGTATCAGC AGCTGCCCGG CACCGCCCCT AAGCTGCTGA TCTACGACAA CAACAAGCGG

2041 CCCAGCGGCA TCCCCGACCG GTTTAGCGGC AGCAAGAGCG GGACTTCTGC TACACTGGGC

2101 ATCACAGGCC TGCAGACCGG CGACGAGGCC GACTACTACT GCGGCACCTG GGACAGCAGC

2161 CTGAGCGCTT GGGTGTTCGG CTGCGGCACC AAGCTGACAG TGCTG

SEQ ID NO: 19 - consisting of 20 a.a. linker sequence (Gly ₄ Ser) ₄

1 GGGGSGGGGS GGGGSGGGGS

SEQ ID NO: 20 - ndimab varB - L chain amino acid sequence

1 EIVLTQSPAT LSLSPGERAT LSCRASQSVY SYLAWYQQKP GQAPRLLIYD ASNRAIGIPA

61 RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWPPFTFG PGTKVDIKRT VAAPSVFIFP

121 PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDSK DSTYSLSSTL

181 TLSKADYEKH KVYACEVTHQ GLSSPVTKSF NRGEC

SEQ ID NO: 21 - ndimab varB - L chain nucleotide sequence

1 GAAATCGTGC TGACCCAGAG CCCCGCCACC CTGTCTCTGA GCCCTGGCGA GAGAGCCACC

61 CTGAGCTGCA GAGCCAGCCA GAGCGTGTAC TCCTACCTGG CTTGGTATCA GCAGAAGCCC

121 GGCCAGGCCC CCAGACTGCT GATCTACGAC GCCAGCAACC GGGCCATCGG CATCCCTGCC

181 AGATTTTCTG GCAGCGGCAG CGGCACCGAC TTCACCCTGA CCATCAGCAG CCTGGAACCC

241 GAGGACTTCG CCGTGTACTA CTGCCAGCAG CGGAGCAACT GGCCCCCCTT CACCTTCGGC

301 CCTGGCACCA AGGTGGACAT CAAGCGTACG GTGGCTGCAC CATCTGTCTT CATCTTCCCG

361 CCATCTGATG AGCAGTTGAA ATCTGGAACT GCCTCTGTTG TGTGCCTGCT GAATAACTTC

421 TATCCCAGAG AGGCCAAAGT ACAGTGGAAG GTGGATAACG CCCTCCAATC GGGTAACTCC

481 CAGGAGAGTG TCACAGAGCA GGACAGCAAG GACAGCACCT ACAGCCTCAG CAGCACCCTG

541 ACGCTGAGCA AAGCAGACTA CGAGAAACAC AAAGTCTACG CCTGCGAAGT CACCCATCAG

601 GGCCTGAGCT CGCCCGTCAC AAAGAGCTTC AACAGGGGAG AGTGT

SEQ ID NO: 22 - ndimab varB - H chain amino acid sequence

1 QVQLVESGGG VVQPGRSLRL SCAASGFIFS SYAMHWVRQA PGNGLEWVAF MSYDGSNKKY

61 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDR GISAGGNYYY YGMDVWGQGT

121 TVTVSSASTK GPSVFPLAPS SKSTSGGTAA LGCLVKDYFP EPVTVSWNSG ALTSGVHTFP

181 AVLQSSGLYS LSSSVVTVPSS SLGTQTYICN VNHKPSNTKV DKRVEPKSCD KTHTCPPCPA

241 PELLGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP

301 REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL

361 PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT

421 VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK GGGG SGGGGS QVQL VQSGAEVKKP

481 GSSVKVSCKA SGGTFSTYGI SWVRQAPGQC LEWMGGIIPI FDTGNSAQSF QGRVTITADE

541 STSTAYMELS SLRSEDTAVY YCARSSRIYD LNPSLTAYYD MDVWGQGTMV TVSS GGGGSG

601 GGGSGGGGSG GGGS QSVLTQ PPSVSAAPGQ KVTISCSGSS SNIGNNYVSW YQQLPGTAPK

661 LLIYDNNKRP SGIPDRFSGS KSGTSATLGI TGLQTGDEAD YYCGTWDSSL SAWVFGCGTK

721 LTL

SEQ ID NO: 23 - ndimab varB - H chain nucleotide sequence

1 CAGGTGCAGC TGGTGGAAAG CGGCGGAGGC GTGGTGCAGC CCGGCAGAAG CCTGAGACTG

61 AGCTGCGCTG CCAGCGGCTT CATCTTCAGC AGCTACGCCA TGCACTGGGT CCGCCAGGCC

121 CCTGGCAACG GACTGGAATG GGTGGCCTTC ATGAGCTACG ACGGCAGCAA CAAGAAGTAC

181 GCCGACAGCG TGAAGGGCCG GTTCACCATC AGCCGGGACA ACAGCAAGAA CACCCTGTAC

241 CTGCAGATGA ACAGCCTGCG GGCTGAGGAC ACCGCCGTGT ACTACTGCGC CAGAGACCGA

301 GGCATCAGTG CTGGCGGCAA CTACTACTAC TACGGCATGG ACGTGTGGGG CCAGGGCACC

361 ACCGTGACCG TGTCTAGCGC GTCGACCAAG GGCCCATCCG TCTTCCCCCT GGCACCCCTCC

421 TCCAAGAGCA CCTCTGGGGG CACAGCGGCC CTGGGCTGCC TGGTCAAGGA CTACTTCCCC

481 GAACCGGTGA CGGTGTCCTG GAACTCAGGC GCTCTGACCA GCGGCGTGCA CACCTTCCCG

541 GCTGTCCTAC AGTCCTCAGG ACTCTACTCC CTCAGCAGCG TGGTGACCGT GCCCTCCAGC

601 AGCTTGGGCA CCCAGACCTA CATCTGCAAC GTGAATCACA AGCCCAGCAA CACCAAGGTG

661 GACAAGAGAG TTGAGCCCAA ATCTTGTGAC AAAACTCACA CATGCCCACC GTGCCCAGCA

721 CCTGAACTCC TGGGGGGACC GTCAGTCTTC CTCTTCCCCC CAAAACCCAA GGACACCCTC

781 ATGATCTCCC GGACCCCCTGA GGTCACATGC GTGGTGGTGG ACGTGAGCCA CGAAGACCCT

841 GAGGTCAAGT TCAACTGGTA CGTGGACGGC GTGGAGGTGC ATAATGCCAA GACAAAGCCG

901 CGGGAGGAGC AGTACAACAG CACGTACCGT GTGGTCAGCG TCCTCACCGT CCTGCACCAG

961 GACTGGCTGA ATGGCAAGGA GTACAAGTGC AAGGTCTCCA ACAAAGCCCT CCCAGCCCCC

1021 ATCGAGAAAA CCATCTCCAA AGCCAAAGGG CAGCCCCGAG AACCACAGGT CTACACCCTG

1081 CCCCCATCCCC GGGAGGAGAT GACCAAGAAC CAGGTCAGCC TGACCTGCCT GGTCAAAGGC

1141 TTCTATCCCA GCGACATCGC CGTGGAGTGG GAGAGCAATG GGCAGCCGGA GAACAACTAC

1201 AAGACCACGC CTCCCGTGCT GGACTCCGAC GGCCTCCTTCT TCCTCTATAG CAAGCTCACC

1261 GTGGACAAGA GCAGGTGGCA GCAGGGGAAC GTCTTCTCAT GCTCCGTGAT GCATGAGGCT

1321 CTGCACAACC ACTACACGCA GAAGAGCCTC TCCCTGTCTC CGGGTAAAGG CGGAGGGGGA

1381 TCCGGCGGAG GGGGCCTCTCA GGTGCAGCTG GTGCAGTCTG GCGCCGAAGT GAAGAAACCC

1441 GGCCTCTAGCG TGAAGGTGTC CTGCAAGGCC AGCGGCGGCA CCTTCTCCAC CTACGGCATC

1501 AGCTGGGTCC GCCAGGCCCC TGGACAGTGT CTGGAATGGA TGGGCGGCAT CATCCCCATC

1561 TTCGACACCG GCAACAGCGC CCAGAGCTTC CAGGGCAGAG TGACCATCAC CGCCGACGAG

1621 AGCACCTCCA CCGCCTACAT GGAACTGAGC AGCCTGCGGA GCGAGGACAC CGCCGTGTAC

1681 TACTGCGCCA GAAGCAGCCG GATCTACGAC CTGAACCCCA GCCTGACCGC CTACTACGAC

1741 ATGGACGTGT GGGGCCAGGG CACCATGGTC ACAGTGTCTA GCGGAGGCGG AGGCAGCGGA

1801 GGTGGTGGAT CTGGTGGCGG AGGAAGTGGC GGCGGAGGCT CTCAGAGCGT GCTGACCCAG

1861 CCCCCTTCTG TGTCTGCCGC CCCTGGCCAG AAAGTGACCA TCTCCTGCAG CGGCAGCAGC

1921 AGCAACATCG GCAACAACTA CGTGTCCTGG TATCAGCAGC TGCCCGGCAC CGCCCCTAAG

1981 CTGCTGATCT ACGACAACAA CAAGCGGCCC AGCGGCATCC CCGACCGGTT TACGGGCAGC

2041 AAGAGCGGGA CTTCTGCTAC ACTGGGCATC ACAGGCCTGC AGACCGGCGA CGAGGCCGAC

2101 TACTACTGCG GCACCTGGGA CAGCAGCCTG AGCGCTTGGG TGTTCGGCTG CGGCACCAAG

2161 CTGACAGTGC TG

SEQ ID NO: 24 - VH NGF-NG amino acid sequence

QVQLVQSGAEVKKPGSSVKVSCKASGGTFWFGAFTWVRQAPGQGLEWMGGIIPIFGLTNLAQNFQGRVTITADESTSTVYMELSSLRSEDTAVYYCARSSRIYDLNPSLTAYYDMDVWGQGTMVTVSS

SEQ ID NO: 25 - VH NGF-NG nucleotide sequence

caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60

tcctgcaagg cctctggagg caccttctgg ttcggcgcgt tcacctgggt gcgacaggcc 120

cctggacaag gacttgagtg gatgggaggg attattccta tcttcgggtt gacgaacttg 180

gcacagaact tccagggcag agtcacgatt accgcggacg aatccacgag cacagtctac 240

atggagctga gcagcttgag atctgaagac acggccgtat attattgtgc acgttcaagt 300

cgtatctacg atctgaaccc gtccctgacc gcctactacg atatggatgt ctggggccag 360

gggacaatgg tcaccgtctc gagt 384

SEQ ID NO: 26 - Amino acid sequence of VL NGF-NG

QSVLTQPPSVSAAPGQKVTISCSGSSSDIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTVL

SEQ ID NO: 27 - VL NGF-NG nucleotide sequence

cagtctgtgc tgactcagcc gccatcagtg tctgcggccc caggacagaa ggtcaccatc 60

tcctgctctg gaagcagctc cgacattggg aataattatg tatcgtggta ccagcagctc 120

cggaacag cccccaaact cctcatttat gacaataata agcgaccctc agggattcct 180

gaccgattct ctggctccaa gtctggcacg tcagccaccc tgggcatcac cggactccag 240

actggggacg aggccgatta ttactgcgga acatgggata gcagcctgag tgcttgggtg 300

ttcggcggag ggaccaagct gaccgtccta 330

SEQ ID NO: 28 - VH amino acid sequence of ndimab

1 QVQLVESGGG VVQPGRSLRL SCAASGFIFS SYAMHWVRQA PGNGLEWVAF MSYDGSNKKY

61 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDR GISAGGNYYY YGMDVWGQGT

121 TVTVSS

SEQ ID NO: 29 - VL amino acid sequence of ndimab

1 EIVLTQSPAT LSLSPGERAT LSCRASQSVY SYLAWYQQKP GQAPRLLIYD ASNRAIGIPA

61 RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWPPFTFG PGTKVDIK

SEQ ID NO: 30 - Amino acid sequence of VH 1126F1

EVQLVQTGAEVKKPGSSVKVSCKASGGTFSTYGISWVRQAPGQGLEWIGGIIPIFDTGNSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDANRQAVPYYDMDVWGQGTMVTVSS

SEQ ID NO: 31 - Amino acid sequence of VL 1126F1

QAVLTQPSSVSTPPGQMVTISCSGSSSDIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTVL

SEQ ID NO: 32 - Amino acid sequence of VH 1126G5

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGISWVRQAPGQGLEWIGGIIPIFDTGNSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDFTSGLAPYYDMDVWGQGTMVTVSS

SEQ ID NO: 33 - Amino acid sequence of VL 1126G5

QAVLTQPSSVSTPPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPPGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSTWVFGGGTKLTVL

SEQ ID NO: 34 - Amino acid sequence of VH 1126H5

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGISWVRQAPGQGLEWIGGIIPIFDAGNSAQSFQGRVTITADESTSTAHMEVSSLRSEDTAVYYCASSSRIYDHHIQKGGYYDMDVWGQGTMVTVSS

SEQ ID NO: 35 - Amino acid sequence of VL 1126H5

QAVLTQPSSVSTPPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTVL

SEQ ID NO: 36 - Amino acid sequence of VH 1127D9

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGISWVRQAPGQGLEWIGGIIPIFDTGNSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDYHTIAYYD

SEQ ID NO: 37 - Amino acid sequence of VL 1127D9

QAVLTQPSSVSTPPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTVL

SEQ ID NO: 38 - Amino acid sequence of VH 1127F9

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGISWVRQAPGQGLEWIGGIIPIFDTGNSAQSFQGRVTITADESTSTAYMKVSSLRSDDTAVYYCASSSRIYDYIPGMRPYYDMDVWGQGTMVTVSS

SEQ ID NO: 39 - Amino acid sequence of VL 1127F9

QAVLTQPSSVSTPPGQKVTISCSGNSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSRSGTLATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTVL

SEQ ID NO: 40 - Amino acid sequence of VH 1131D7

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGISWVRQAPGQGLEWIGGIIPIFDTGNSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDFNSSLIAYYDMDVWGQGTMVTVSS

SEQ ID NO: 41 - Amino acid sequence of VL 1131D7

QAVLTQPSSVSTPPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDETDYYCGTWDSSLSAWVFSGGTKLTVL

SEQ ID NO: 42 - Amino acid sequence of VH 1131H2

EVQLVQSGAEVKKPGSTVKVSCKASGGTFSTYGISWVRQAPGQGLEWIGGIIPIFDTGNSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDLNPSLTAYYDMDVWGQGTMVTVSS

SEQ ID NO: 43 - Amino acid sequence of VL 1131H2

QAVLTQPSSVSTPPGQKVTISCSGTSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTVL

SEQ ID NO: 44 - Amino acid sequence of VH 132A9

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGISWVRQAPGQGLEWIGGIIPIFGTGNSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDFEPSLIYYYDMDVWGQGTMVTVSS

SEQ ID NO: 45 - Amino acid sequence of VL 132A9

QAVLTQPSSVSTPPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTVL

SEQ ID NO: 46 - Amino acid sequence of VH 1132H9

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGISWVRQAPGQGLEWIGGIIPIFDTGNSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDLNPSLTAYYDMDVWGQGTMVTVSS

SEQ ID NO: 47 - Amino acid sequence of VL 1132H9

QAVLTQPSSVSTPPGQKVTISCSGSSSDIGNNYVSWYQQLPGTAPKLLIYDNNKRPTGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTVL

SEQ ID NO: 48 - Amino acid sequence of VH 1133C11

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGISWVRQAPGQGLEWIGGIIPIFDTGNSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDLNPSLTAYYDMDVWGQGTMVTVSS

SEQ ID NO: 49 - Amino acid sequence of VL 1133C11

QAVLTQPSSVSTPPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTVL

SEQ ID NO: 50 - Amino acid sequence of VH 1134D9

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGISWVRQAPGQGLEWIGGIIPIFDTGNSAQSFQGRVAITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDLNPSLTAYYDMDVWGQGTMVTVSS

SEQ ID NO: 51 - Amino acid sequence of VL 1134D9

QAVLTQPSSVSTPPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSGLSAWVFGGGTKLTVL

SEQ ID NO: 52 - Amino acid sequence of VH 1145D1

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGISWVRQAPGQGLEWIGGIIPIFDTSNSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDFRTLYSTYYDMDVWGQGTMVTVSS

SEQ ID NO: 53 - Amino acid sequence of VL 1145D1

QAVLTQPSSVSTPPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGISDRFSGSKSGTSATLGIAGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTVL

SEQ ID NO: 54 - Amino acid sequence of VH 1146D7

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGISWVRQAPGQGLEWIGGIIPIFDTGNSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDLNPSLTAYYDMDVWGQGTMVTVSS

SEQ ID NO: 55 - Amino acid sequence of VL 1146D7

QAVLTQPSSVSTPPGQEVTISCSGSSTNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTVL

SEQ ID NO: 56 - Amino acid sequence of VH 1147D2

EVQLVQSGAEVKKPGSSVRISCKASGGTFSTYGVSWVRQAPGQGLEWIGGIIPIFDTGNSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDLNPSLTAYYDMDVWGQGTMVTVSS

SEQ ID NO: 57 - Amino acid sequence of VL 147D2

QAVLTQPSSVSTPPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGVPDRFSSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTVL

SEQ ID NO: 58 - Amino acid sequence of VH 1147G9

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSAYGISWVRQAPGQGLEWIGGIIPIFNTGNSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDLNPSLTAYYDMDVWGQGTMVTV

SEQ ID NO: 59 - Amino acid sequence of VL 1147G9

QAVLTQPSSVSTPPGQKVTVSCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTVL

SEQ ID NO: 60 - Amino acid sequence of VH 1150F1

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGISWVRQAPGQGLEWIGGIIPIFDTGNSAQSFQDRVTITADESTSTAYMEVGSLRSDDTAVYYCASSSRIYDLNPSLTAYYDMDVWGHGTMVTVSS

SEQ ID NO: 61 - Amino acid sequence of VL 1150F1

QAVLTQPSSVSTPPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTVL

SEQ ID NO: 62 - Amino acid sequence of VH 1152H5

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGISWVRQAPGQGLVWIGGIIPIFDTGNSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDMISSLQPYYDMDVWGQGTMVTVSS

SEQ ID NO: 63 - Amino acid sequence of VL 1152H5

QAVLTQPSSVSTPPGQKATISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTVL

SEQ ID NO: 64 - Amino acid sequence of VH 1155H1

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGISWVRQAPGQGLEWIGGIIPIFDTGNSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDFHLANKGYYDMDVWGQGTMVTVSS

SEQ ID NO: 65 - Amino acid sequence of VL 1155H1

QAVLTQPSSVSTPPGQKATISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLDITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTVL

SEQ ID NO: 66 - Amino acid sequence of VH 1158A1

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGISWVRQAPGQGLEWIGGIIPIFGTGNSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDHHNHVGGYYDMDVWGQGTMVTVSS

SEQ ID NO: 67 - Amino acid sequence of VL 1158A1

QAVLTQPSSVSTPPGQKVTISCSGSSSNIGNNYASWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDGSLSAWVFGGGTKLTVL

SEQ ID NO: 68 - Amino acid sequence of VH 1160E3

EVQLVQSGAEVKKPGSSAKVSCKASGGTFSTYGISWVRQAPGQGLEWIGGIIPIFDTGNSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDLNPSLTAYYDMDVWGQGTMVTVSS

SEQ ID NO: 69 - Amino acid sequence of VL 1160E3

QAVLTQPSSVSTPPGQKVTISCSGSNSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTV

SEQ ID NO: 70 - Amino acid sequence of VH 1165D4

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGISWVRQAPGQGLEWIGGIIPIFDTGNSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDLNPSLTAYYDMDVWGQGTMVTVSS

SEQ ID NO: 71 - Amino acid sequence of VL 1165D4

QAVLTQPSSVSTPPGQKVTISCSGSSSNIENNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTVL

SEQ ID NO: 72 - Amino acid sequence of VH 1175H8

EVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGISWVRQAPGQRLEWIGGIIPIFDTGNSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDATTGLTPYYDMDVWGQGTMVTVSS

SEQ ID NO: 73 - Amino acid sequence of VL 1175H8

QAVLTQPSSVSTPPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLRTGDEADYYCGTWDSSLSAWVFGGGTKLTVL

SEQ ID NO: 74 - Amino acid sequence of VH 1211G10

EVQLVQSGAEVRKPGSSVKVSCKAYGGTFSTYGISWVRQAPGQGLEWVGGIIPIFDTRNSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDMVSTLIPYYDMDVWGQGTMVTVSS

SEQ ID NO: 75 - Amino acid sequence of VL 1211G10

QAVLTQPSSVSTPPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTVL

SEQ ID NO: 76 - Amino acid sequence of VH 1214A1

EVQLVQSGAEVKKPGSSVRVSCKASGGTFSTYGISWVRQAPGQGLEWIGGIIPIFDTGNSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDAHLQAYYDMDVWGQGTMVTVSS

SEQ ID NO: 77 amino acid sequence of VL 1214A1

QAVLTQPSSVSTPPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPPGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTRDSSSLSAWVFGGGTKLTVL

SEQ ID NO: 78 - Amino acid sequence of VH 1214D10

EVQLVQSGAEAKKPGSSVKVSCKASGGTFSTYGISWVRQAPGRGLEWIGGIIPIFDTGNSAQSFQGRVAITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDAHLNHHGYYDMDVWGQGTMVTVSS

SEQ ID NO: 79 - Amino acid sequence of VL 1214D10

QAVLTQPSSVSTPPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQAGDEADYYCGTWDSSLSAWVFGGGTKLTVL

SEQ ID NO: 80 - Amino acid sequence of VH 1218H5

EVQLVQSGAVVKKPGSSVKVSCKASGGTFSTYGISWVRQAPGQGLEWIGGIIPIFDTGSSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDLNPSLTAYYDMDVWGQGTMVTVSS

SEQ ID NO: 81 - Amino acid sequence of VL 1218H5

QAVLTQPSSVSTPPGQKVTISCSGSSSNTGNNYVSWYQQLSGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTVL

SEQ ID NO: 82 - Amino acid sequence of VH 1230H7

EMQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGISWVRQAPGQGLEWIGGIIPIFDTGNSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDFNSALISYYDMDVWGQGTMVTVSS

SEQ ID NO: 83 - Amino acid sequence of VL 1230H7

QAVLTQPSSVSTPPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTV

SEQ ID NO: 84 - Amino acid sequence of VH 1083H4

QMQLVQSGAEVKKTGSSVKVSCKASGYTFAYHYLHWVRQAPGQGLEWMGGIIPIFGTTNYAQRFQDRVTITADESTSTAYMELSSLRSEDTAVYYCASADYVWGSYRPDWYFDLWGRGTMVTVSS

SEQ ID NO: 85 - Amino acid sequence of VL 1083H4

QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQRLPGAAPQLLIYNNDQRPSGIPDRFSGSKSGTSGSLVISGLQSEDEADYYCASWDDSLNGRVFGGGTKLTVL

SEQ ID NO: 86 - Amino acid sequence of VH 1227H8

QMQLVQSGAEVKKTGSSVKVSCKASGHTFAYHYLHWVRQAPGQGLEWMGGIIPIFGTTNYAQRFQDRVTITADESTSTAYMELSSLRSEDTAVYYCASADYAWESYQPPQINGVWGRGTMVTVSS

SEQ ID NO: 87 - Amino acid sequence of VL 1227H8

QSVLTQPPSVSAAPGQKVTITCSGSTSNIGNNYVSWYQQHPGKAPKLMIYDVSKRPSGVPDRFSGSKSKSGNSASLDISGLQSEDEADYYCAAWDDSLSEFFFGTGTKLTVL

SEQ ID NO: 88 - HCDR1 NGF-NG

FGAFT

SEQ ID NO: 89 - HCDR2 NGF-NG

GIIPIFGLTNLAQNFQG

SEQ ID NO: 90 - HCDR3 NGF-NG

SSRIYDLNPSLTAYYDMDV

SEQ ID NO: 91 - LCDR1 NGF-NG

SGSSSDIGNNYVS

SEQ ID NO: 92 - LCDR2 NGF-NG

DNNKRPS

SEQ ID NO: 93 - LCDR3 NGF-NG

GTWDSSLSAWV

SEQ ID NO: 94 - VH MEDI-578 amino acid sequence with G→C substitution

QVQLVQSGAE VKKPGSSVKV SCKASGGTFS TYGISWVRQA PGQCLEWMGG IIPIFDTGNS

AQSFQGRVTI TADESTSTAY MELSSLRSED TAVYYCARSS RIYDLNPSLT AYYDMDVWGQ

GTMVTVSS

SEQ ID NO: 95 - VL amino acid sequence of MEDI-578 with G→C substitution

QSVLTQPPSV SAAPGQKVTI SCSGSSSNIG NNYVSWYQQL PGTAPKLLIY DNNKRPSGIP

DRFSGSKSGT SATLGITGLQ TGDEADYYCG TWDSSLSAWV FGCGTKLTVL

SEQ ID NO: 96 - Amino acid sequence of VH 1230D8

QMQLVQSGAEVKKTGSSVKVSCKASGYTFPYHYLHWVRQAPGQGLEWMGGIIPIFGTTNYAQRFQDRVTITADESTSTAYMEFSSLRSEDTAVYYCASADYVWESYHPATSLSLWGRGTMVTVSS

SEQ ID NO: 97 - Amino acid sequence of VL1230D8

QSVLTQPPSVSAAPGQKVTISCPGSTSNIGNNYVSWYQQRPGKAPKLMIYDVSKRPSGVPDRFSGSKSGNSASLDISELQSEDEADYYCAAWDDSLSEFLFGTGTKLTVL

SEQ ID NO: 98

GGGGSGGGGS

SEQ ID NO: 99 - TNFR2-Fc_varB - codon-optimized nucleotide sequence

1 CTGCCCGCCC AGGTGGCCTT TACCCCTTAT GCTCCTGAGC CCGGCTCTAC CTGCCGGCTG

61 AGAGAGTACT ACGACCAGAC CGCCCAGATG TGCTGCTCCA AGTGCTCTCC TGGCCAGCAC

121 GCCAAGGTGT TCTGCACCAA GACCTCCGAT ACCGTGTGCG ACTCCTGCGA GGACTCCACC

181 TACACCCAGC TGTGGAACTG GGTGCCCGAG TGCCTGTCCT GCGGCTCCAG ATGTTCCTCC

241 GACCAGGTGG AAACCCAGGC CTGCACCAGA GAGCAGAACC GGATCTGCAC CTGTCGGCCT

301 GGCTGGTACT GCGCCCTGTC TAAGCAGGAA GGCTGCAGAC TGTGCGCCCC TCTGCGGAAG

361 TGTAGACCTG GCTTTGGCGT GGCCAGACCC GGCACCGAGA CATCTGATGT CGTGTGCAAG

421 CCTTGCGCCC CTGGCACCTT CTCCAACACC ACCTCCTCCA CCGACATCTG CCGGCCTCAC

481 CAGATCTGCA ACGTGGTGGC CATCCCTGGC AACGCCTCTA TGGACGCCGT GTGCACCTCT

541 ACCTCCCCCA CCAGAAGTAT GGCCCCTGGC GCTGTGCATC TGCCCCAGCC TGTGTCTACC

601 AGATCCCAGC ACACCCAGCC CACCCCTGAG CCTTCTACCG CCCCTTCTAC CAGCTTCCTG

661 CTGCCTATGG GCCCTAGCCC TCCTGCTGAG GGATCTACAG GCGACGAGCC CAAGTCCTGC

721 GACAAGACCC ACACTGTCC CCCTTGTCCT GCCCCTGAAC TGCTGGGCGG ACCTTCCGTG

781 TTCCTGTTCC CCCCAAAGCC CAAGGACACC CTGATGATCA GCCGGACCCC TGAAGTGACC

841 TGCGTGGTGG TGGATGTGTC CCACGAGGAT CCCGAAGTGA AGTTCAATTG GTACGTGGAC

901 GGCGTGGAAG TGCACAACGC CAAGACCAAG CCCAGAGAGG AACAGTACAA CTCCACCTAC

961 CGGGTGGTGT CCGTGCTGAC CGTGCTGCAC CAGGATTGGC TGAACGGCAA AGAGTACAAG

1021 TGCAAGGTGT CCAACAAGGC CCTGCCTGCC CCCATCGAAA AGACCATCTC CAAGGCCAAG

1081 GGCCAGCCCC GGGAACCCCA GGTGTACACA CTGCCCCCTA GCCGGGAAGA GATGACCAAG

1141 AACCAGGTGT CCCTGACCTG TCTCGTGAAG GGCTTCTACC CCTCCGATAT CGCCGTGGAA

1201 TGGGAGTCCA ACGGCCAGCC TGAGAACAAC TACAAGACCA CCCCCCCTGT GCTGGACTCC

1261 GACGGCTCAT TCTTCCTGTA CTCCAAGCTG ACAGTGGACA AGTCCCGGTG GCAGCAGGGC

1321 AACGTGTTCT CCTGCTCCGT GATGCACGAG GCCCTGCACA ACCACTACAC CCAGAAGTCC

1381 CTGTCCCTGA GCCCTGGAAA AGGCGGCGGA GGATCTGGCG GAGGCGGATC TCAGGTGCAG

1441 CTGGTGCAGT CTGGCGCTGA AGTGAAGAAA CCCGGCTCCT CCGTGAAGGT GTCCTGCAAG

1501 GCTTTCTGGCG GCACCTTCTC TACCTACGGC ATCTCCTGGG TGCGACAGGC CCCTGGCCAG

1561 TGCCTGGAAT GGATGGGCGG CATCATCCCC ATCTTCGACA CCGGCAACTC CGCCCAGAGC

1621 TTCCAGGGCA GAGTGACCAT CACCGCCGAC GAGTCTACCT CCACCGCCTA CATGGAACTG

1681 TCCTCCCTGC GGAGCGAGGA CACCGCCGTG TACTACTGCG CCCGGTCCTC TCGGATCTAC

1741 GACCTGAACC CTTCCCTGAC CGCCTACTAC GACATGGACG TGTGGGGCCA GGGCACAATG

1801 GTCACCGTGT CATCTGGTGG TGGCGGCTCT GGTGGCGGAG GAAGTGGGGG AGGGGGTTCT

1861 GGGGGGGGAG GATCTCAGTC TGTGCTGACC CAGCCTCCTT CCGTGTCTGC TGCCCCAGGC

1921 CAGAAAGTGA CAATCTCCTG CAGCGGCTCC AGCTCCAACA TCGGCAACAA CTACGTGTCC

1981 TGGTATCAGC AGCTGCCCGG CACCGCTCCC AAACTGCTGA TCTACGATAA CAACAAGCGG

2041 CCCTCCGGCA TCCCCGACAG ATTCTCCGGC TCTAAGTCCG GCACCTCTGC CACCCTGGGC

2101 ATCACCGGAC TGCAGACAGG CGACGAGGCC GACTACTACT GTGGCACCTG GGACTCCTCC

2161 CTGTCCGCTT GGGTGTTCGG CTGCGGCACC AAACTGACTG TGCTG

The scope of this invention should not be limited to the specific aspects described, which are intended merely as illustrations of certain aspects of the invention, and any compositions or methods that are functionally equivalent are included within the scope of this description. Indeed, various modifications of the invention, other than those shown and set forth herein, will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

All publications and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication or patent application were specifically and separately indicated as being incorporated by reference.

SUBSTITUTE SHEET

--->

SEQUENCE LIST

<110> MEDIMMUNE LIMITED

<120> COMPOSITIONS AND METHODS FOR THE TREATMENT OF PAIN

<130> 110421-0054-WO1

<140>

<141>

<150> 61/934.828

<151> 2014-02-02

<160> 99

<170> PatentIn version 3.5

<210> 1

<211> 241

<212> PRT

<213> Homo sapiens

<400> 1

Met Ser Met Leu Phe Tyr Thr Leu Ile Thr Ala Phe Leu Ile Gly Ile

1 5 10 15

Gln Ala Glu Pro His Ser Glu Ser Asn Val Pro Ala Gly His Thr Ile

20 25 30

Pro Gln Ala His Trp Thr Lys Leu Gln His Ser Leu Asp Thr Ala Leu

35 40 45

Arg Arg Ala Arg Ser Ala Pro Ala Ala Ala Ile Ala Ala Arg Val Ala

50 55 60

Gly Gln Thr Arg Asn Ile Thr Val Asp Pro Arg Leu Phe Lys Lys Arg

65 70 75 80

Arg Leu Arg Ser Pro Arg Val Leu Phe Ser Thr Gln Pro Pro Arg Glu

85 90 95

Ala Ala Asp Thr Gln Asp Leu Asp Phe Glu Val Gly Gly Ala Ala Pro

100 105 110

Phe Asn Arg Thr His Arg Ser Lys Arg Ser Ser Ser His Pro Ile Phe

115 120 125

His Arg Gly Glu Phe Ser Val Cys Asp Ser Val Ser Val Trp Val Gly

130 135 140

Asp Lys Thr Thr Ala Thr Asp Ile Lys Gly Lys Glu Val Met Val Leu

145 150 155 160

Gly Glu Val Asn Ile Asn Asn Ser Val Phe Lys Gln Tyr Phe Phe Glu

165 170 175

Thr Lys Cys Arg Asp Pro Asn Pro Val Asp Ser Gly Cys Arg Gly Ile

180 185 190

Asp Ser Lys His Trp Asn Ser Tyr Cys Thr Thr Thr His Thr Phe Val

195 200 205

Lys Ala Leu Thr Met Asp Gly Lys Gln Ala Ala Trp Arg Phe Ile Arg

210 215 220

Ile Asp Thr Ala Cys Val Cys Val Leu Ser Arg Lys Ala Val Arg Arg

225 230 235 240

Ala

<210> 2

<211> 233

<212> PRT

<213> Homo sapiens

<400> 2

Met Ser Thr Glu Ser Met Ile Arg Asp Val Glu Leu Ala Glu Glu Ala

1 5 10 15

Leu Pro Lys Lys Thr Gly Gly Pro Gln Gly Ser Arg Arg Cys Leu Phe

20 25 30

Leu Ser Leu Phe Ser Phe Leu Ile Val Ala Gly Ala Thr Thr Leu Phe

35 40 45

Cys Leu Leu His Phe Gly Val Ile Gly Pro Gln Arg Glu Glu Phe Pro

50 55 60

Arg Asp Leu Ser Leu Ile Ser Pro Leu Ala Gln Ala Val Arg Ser Ser

65 70 75 80

Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val Val Ala Asn Pro

85 90 95

Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu

100 105 110

Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser

115 120 125

Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly

130 135 140

Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile Ser Arg Ile Ala

145 150 155 160

Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro

165 170 175

Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu

180 185 190

Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu

195 200 205

Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly

210 215 220

Gln Val Tyr Phe Gly Ile Ile Ala Leu

225 230

<210> 3

<211> 128

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 3

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Thr Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Ile Phe Asp Thr Gly Asn Ser Ala Gln Ser Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Ser Arg Ile Tyr Asp Leu Asn Pro Ser Leu Thr Ala Tyr

100 105 110

Tyr Asp Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 4

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 4

Thr Tyr Gly Ile Ser

15

<210> 5

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 5

Gly Ile Ile Pro Ile Phe Asp Thr Gly Asn Ser Ala Gln Ser Phe Gln

1 5 10 15

gly

<210> 6

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 6

Ser Ser Arg Ile Tyr Asp Leu Asn Pro Ser Leu Thr Ala Tyr Tyr Asp

1 5 10 15

Met Asp Val

<210> 7

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 7

Gln Ser Val Leu Thr Gln Pro Ser Val Ser Ala Ala Pro Gly Gln

1 5 10 15

Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln

65 70 75 80

Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu

85 90 95

Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 8

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 8

Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn Tyr Val Ser

1 5 10

<210> 9

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 9

Asp Asn Asn Lys Arg Pro Ser

15

<210> 10

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 10

Gly Thr Trp Asp Ser Ser Leu Ser Ala Trp Val

1 5 10

<210> 11

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 11

Ser Ser Arg Ile Tyr Asp Phe Asn Ser Ala Leu Ile Ser Tyr Tyr Asp

1 5 10 15

Met Asp Val

<210> 12

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 12

Ser Ser Arg Ile Tyr Asp Met Ile Ser Ser Leu Gln Pro Tyr Tyr Asp

1 5 10 15

Met AspVal

<210> 13

<211> 467

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 13

Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser

1 5 10 15

Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys

20 25 30

Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr

35 40 45

Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu

50 55 60

Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser

65 70 75 80

Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys

85 90 95

Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys

100 105 110

Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala

115 120 125

Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro

130 135 140

Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His

145 150 155 160

Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala

165 170 175

Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val

180 185 190

His Leu Pro Gln Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr

195 200 205

Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly

210 215 220

Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly Asp Glu Pro Lys Ser Cys

225 230 235 240

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

245 250 255

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

260 265 270

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

275 280 285

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

290 295 300

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

305 310 315 320

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

325 330 335

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

340 345 350

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

355 360 365

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

370 375 380

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

385 390 395 400

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

405 410 415

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

420 425 430

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

435 440 445

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

450 455 460

Pro Gly Lys

465

<210> 14

<211> 731

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 14

Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser

1 5 10 15

Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys

20 25 30

Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr

35 40 45

Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu

50 55 60

Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser

65 70 75 80

Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys

85 90 95

Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys

100 105 110

Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala

115 120 125

Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro

130 135 140

Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His

145 150 155 160

Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala

165 170 175

Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val

180 185 190

His Leu Pro Gln Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr

195 200 205

Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly

210 215 220

Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly Asp Glu Pro Lys Ser Cys

225 230 235 240

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

245 250 255

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

260 265 270

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

275 280 285

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

290 295 300

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

305 310 315 320

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

325 330 335

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

340 345 350

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

355 360 365

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

370 375 380

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

385 390 395 400

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

405 410 415

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

420 425 430

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

435 440 445

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

450 455 460

Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln

465 470 475 480

Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser Val Lys

485 490 495

Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Thr Tyr Gly Ile Ser

500 505 510

Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Gly Ile

515 520 525

Ile Pro Ile Phe Asp Thr Gly Asn Ser Ala Gln Ser Phe Gln Gly Arg

530 535 540

Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met Glu Leu

545 550 555 560

Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser

565 570 575

Ser Arg Ile Tyr Asp Leu Asn Pro Ser Leu Thr Ala Tyr Tyr Asp Met

580 585 590

Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly

595 600 605

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Gln Ser Val

610 615 620

Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln Lys Val Thr

625 630 635 640

Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn Tyr Val Ser

645 650 655

Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Asp

660 665 670

Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser Lys

675 680 685

Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln Thr Gly Asp

690 695 700

Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu Ser Ala Trp

705 710 715 720

Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

725 730

<210> 15

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 15

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 16

<211> 2193

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<400> 16

ctgcccgccc aggtggcctt taccccttat gcccccgagc ccggcagcac ctgtcggctg 60

agagagtact acgaccagac cgccgatg tgctgcagca agtgctctcc tggccagcat 120

gccaaggtgt tctgcaccaa gaccagcgac accgtgtgcg acagctgcga ggacagcacc 180

tacacccagc tgtggaactg ggtgcccgag tgcctgagct gcggcagcag atgcagcagc 240

gaccaggtgg aaacccaggc ctgcaccaga gagcagaacc ggatctgcac ctgtagaccc 300

ggctggtact gcgccctgag caagcaggaa ggctgcagac tctgcgcccc tctgcggaag 360

tgcagacccg gctttggcgt ggccagaccc ggcaccgaga caagcgacgt ggtctgtaag 420

ccctgcgctc ctggcacctt cagcaacacc accagcagca ccgacatctg cagaccccac 480

cagatctgca acgtggtggc catccccggc aacgccagca tggatgccgt ctgcaccagc 540

actagcccca ccagaagtat ggcccctggc gccgtgcatc tgccccagcc tgtgtccacc 600

agaagccagc acacccagcc cacccctgag cctagcaccg ccccctccac cagctttctg 660

ctgcctatgg gccctagccc tccagccgag ggaagcacag gcgacgagcc caagagctgc 720

gacaagaccc acacctgtcc cccctgccct gcccctgaac tgctgggcgg acccagcgtg 780

ttcctgttcc ccccaaagcc caaggacacc ctgatgatca gccggacccc cgaagtgacc 840

tgcgtggtgg tggacgtgtc ccacgaggac cctgaagtga agttcaattg gtacgtggac 900

ggcgtggaag tgcacaacgc caagaccaag cccagagagg aacagtacaa ctccacctac 960

cgggtggtgt ccgtgctgac cgtgctgcac caggactggc tgaacggcaa agagtacaag 1020

tgcaaggtct ccaacaaggc cctgcctgcc cccatcgaga aaaccatcag caaggccaag 1080

ggccagcccc gcgagcctca ggtgtacaca ctgcccccca gccgggaaga gatgaccaag 1140

aaccaggtgt ccctgacctg cctggtcaaa ggcttctacc ccagcgatat cgccgtggaa 1200

tgggagagca atggccagcc cgagaacaac tacaagacca ccccccctgt gctggacagc 1260

gacggctcat tcttcctgta cagcaagctg accgtggaca agagccggtg gcagcaggggc 1320

aacgtgttca gctgcagcgt gatgcacgag gccctgcaca accactacac ccagaagtcc 1380

ctgagcctga gccccggaaa gggcggtggc ggatccggag gtgggggatc tcaggtgcag 1440

ctggtgcagt ctggcgccga agtgaagaaa cccggctcta gcgtgaaggt gtcctgcaag 1500

gccagcggcg gcaccttctc cacctacggc atcagctggg tccgccaggc ccctggacag 1560

ggcctggaat ggatgggcgg catcatcccc atcttcgaca ccggcaacag cgccgagc 1620

ttccagggca gagtgaccat caccgccgac gagagcacct ccaccgccta catggaactg 1680

agcagcctgc ggagcgagga caccgccgtg tactactgcg ccagaagcag ccggatctac 1740

gacctgaacc ccagcctgac cgcctactac gacatggacg tgtggggcca gggcaccatg 1800

gtcacagtgt ctagcgggagg cggcggatct ggcggcggag gaagtggcgg gggaggatct 1860

gccgagcg tgctgaccca gcccccttct gtgtctgccg cccctggcca gaaagtgacc 1920

atctcctgca gcggcagcag cagcaacatc ggcaacaact acgtgtcctg gtatcagcag 1980

ctgcccggca ccgcccctaa gctgctgatc tacgacaaca acaagcggcc cagcggcatc 2040

cccgaccggt ttagcggcag caagagcggg acttctgcta cactgggcat cacaggcctg 2100

cagaccggcg acgaggccga ctactactgc ggcacctggg acagcagcct gagcgcttgg 2160

gtgttcggcg gaggcaccaa gctgacagtg ctg 2193

<210> 17

<211> 735

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 17

Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser

1 5 10 15

Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys

20 25 30

Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr

35 40 45

Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu

50 55 60

Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser

65 70 75 80

Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys

85 90 95

Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys

100 105 110

Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala

115 120 125

Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro

130 135 140

Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His

145 150 155 160

Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala

165 170 175

Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val

180 185 190

His Leu Pro Gln Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr

195 200 205

Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly

210 215 220

Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly Asp Glu Pro Lys Ser Cys

225 230 235 240

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

245 250 255

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

260 265 270

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

275 280 285

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

290 295 300

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

305 310 315 320

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

325 330 335

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

340 345 350

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

355 360 365

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

370 375 380

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

385 390 395 400

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

405 410 415

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

420 425 430

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

435 440 445

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

450 455 460

Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln

465 470 475 480

Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser Val Lys

485 490 495

Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Thr Tyr Gly Ile Ser

500 505 510

Trp Val Arg Gln Ala Pro Gly Gln Cys Leu Glu Trp Met Gly Gly Ile

515 520 525

Ile Pro Ile Phe Asp Thr Gly Asn Ser Ala Gln Ser Phe Gln Gly Arg

530 535 540

Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr Met Glu Leu

545 550 555 560

Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser

565 570 575

Ser Arg Ile Tyr Asp Leu Asn Pro Ser Leu Thr Ala Tyr Tyr Asp Met

580 585 590

Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly

595 600 605

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

610 615 620

Ser Gln Ser Val Leu Thr Gln Pro Ser Val Ser Ala Ala Pro Gly

625 630 635 640

Gln Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn

645 650 655

Asn Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu

660 665 670

Leu Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe

675 680 685

Ser Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu

690 695 700

Gln Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser

705 710 715 720

Leu Ser Ala Trp Val Phe Gly Cys Gly Thr Lys Leu Thr Val Leu

725 730 735

<210> 18

<211> 2205

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<400> 18

ctgcccgccc aggtggcctt taccccttat gcccccgagc ccggcagcac ctgtcggctg 60

agagagtact acgaccagac cgccgatg tgctgcagca agtgctctcc tggccagcat 120

gccaaggtgt tctgcaccaa gaccagcgac accgtgtgcg acagctgcga ggacagcacc 180

tacacccagc tgtggaactg ggtgcccgag tgcctgagct gcggcagcag atgcagcagc 240

gaccaggtgg aaacccaggc ctgcaccaga gagcagaacc ggatctgcac ctgtagaccc 300

ggctggtact gcgccctgag caagcaggaa ggctgcagac tctgcgcccc tctgcggaag 360

tgcagacccg gctttggcgt ggccagaccc ggcaccgaga caagcgacgt ggtctgcaag 420

ccctgcgctc ctggcacctt cagcaacacc accagcagca ccgacatctg cagaccccac 480

cagatctgca acgtggtggc catccccggc aacgccagca tggatgccgt gtgcaccagc 540

accagcccca ccagaagtat ggcccctggc gccgtgcatc tgccccagcc tgtgtccacc 600

agaagccagc acacccagcc cacccctgag cctagcaccg ccccctccac cagctttctg 660

ctgcctatgg gccctagccc tccagccgag ggaagcacag gcgacgagcc caagagctgc 720

gacaagaccc acacctgtcc cccctgccct gcccctgaac tgctgggcgg acccagcgtg 780

ttcctgttcc ccccaaagcc caaggacacc ctgatgatca gccggacccc cgaagtgacc 840

tgcgtggtgg tggacgtgtc ccacgaggac cctgaagtga agttcaattg gtacgtggac 900

ggcgtggaag tgcacaacgc caagaccaag cccagagagg aacagtacaa ctccacctac 960

cgggtggtgt ccgtgctgac cgtgctgcac caggactggc tgaacggcaa agagtacaag 1020

tgcaaagtct ccaacaaggc cctgcctgcc cccatcgaga aaaccatcag caaggccaag 1080

ggccagcccc gcgagcctca ggtgtacaca ctgcccccca gccgggaaga gatgaccaag 1140

aaccaggtgt ccctgacctg cctggtcaaa ggcttctacc ccagcgatat cgccgtggaa 1200

tgggagagca acggccagcc cgagaacaac tacaagacca ccccccctgt gctggacagc 1260

gacggctcat tcttcctgta cagcaagctg accgtggaca agagccggtg gcagcaggggc 1320

aatgtcttca gctgtagcgt gatgcacgag gccctgcaca accactacac ccagaagtcc 1380

ctgagcctga gccccggaaa gggcgggaggc ggatccggag gtgggggatc tcaggtgcag 1440

ctggtgcagt ctggcgccga agtgaagaaa cccggctcta gcgtgaaggt gtcctgcaag 1500

gccagcggcg gcaccttctc cacctacggc atcagctggg tccgccaggc ccctggacag 1560

tgtctggaat ggatgggcgg catcatcccc atcttcgaca ccggcaacag cgccgagc 1620

ttccagggca gagtgaccat caccgccgac gagagcacct ccaccgccta catggaactg 1680

agcagcctgc ggagcgagga caccgccgtg tactactgcg ccagaagcag ccggatctac 1740

gacctgaacc ccagcctgac cgcctactac gacatggacg tgtggggcca gggcaccatg 1800

gtcacagtgt ctagcgggagg cggaggcagc ggaggtggtg gatctggtgg cggaggaagt 1860

ggcggcggag gctctcagag cgtgctgacc cagccccctt ctgtgtctgc cgcccctggc 1920

cagaaagtga ccatctcctg cagcggcagc agcagcaaca tcggcaacaa ctacgtgtcc 1980

tggtatcagc agctgcccgg caccgcccct aagctgctga tctacgacaa caacaagcgg 2040

cccagcggca tccccgaccg gtttagcggc agcaagagcg ggacttctgc tacactgggc 2100

atcacaggcc tgcagaccgg cgacgaggcc gactactact gcggcacctg ggacagcagc 2160

ctgagcgctt gggtgttcgg ctgcggcacc aagctgacag tgctg 2205

<210> 19

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 19

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<210> 20

<211> 215

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 20

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Tyr Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Ile Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro

85 90 95

Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 21

<211> 645

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<400> 21

gaaatcgtgc tgacccagag ccccgccacc ctgtctctga gccctggcga gagagccacc 60

ctgagctgca gagccagcca gagcgtgtac tcctacctgg cttggtatca gcagaagccc 120

ggccaggccc ccagactgct gatctacgac gccagcaacc gggccatcgg catccctgcc 180

agattttctg gcagcggcag cggcaccgac ttcaccctga ccatcagcag cctggaaccc 240

gaggacttcg ccgtgtacta ctgccagcag cggagcaact ggcccccctt caccttcggc 300

cctggcacca aggtggacat caagcgtacg gtggctgcac catctgtctt catcttcccg 360

ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgct gaataacttc 420

tatccgag aggccaaagt acagtggaag gtggataacg ccctccaatc gggtaactcc 480

540

acgctgagca aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag 600

ggcctgagct cgcccgtcac aaagagcttc aacaggggag agtgt 645

<210> 22

<211> 724

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 22

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Ser Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Asn Gly Leu Glu Trp Val

35 40 45

Ala Phe Met Ser Tyr Asp Gly Ser Asn Lys Lys Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 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 Asp Arg Gly Ile Ser Ala Gly Gly Asn Tyr Tyr Tyr Tyr Gly

100 105 110

Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser

115 120 125

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

130 135 140

Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

145 150 155 160

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

165 170 175

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

180 185 190

Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile

195 200 205

Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val

210 215 220

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

225 230 235 240

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

245 250 255

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

260 265 270

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

275 280 285

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

290 295 300

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

305 310 315 320

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

325 330 335

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

340 345 350

Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Glu Glu Met Thr

355 360 365

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

370 375 380

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

385 390 395 400

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

405 410 415

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

420 425 430

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

435 440 445

Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly

450 455 460

Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro

465 470 475 480

Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser

485 490 495

Thr Tyr Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Cys Leu Glu

500 505 510

Trp Met Gly Gly Ile Ile Pro Ile Phe Asp Thr Gly Asn Ser Ala Gln

515 520 525

Ser Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr

530 535 540

Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr

545 550 555 560

Tyr Cys Ala Arg Ser Ser Arg Ile Tyr Asp Leu Asn Pro Ser Leu Thr

565 570 575

Ala Tyr Tyr Asp Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val

580 585 590

Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

595 600 605

Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro Ser Val

610 615 620

Ser Ala Ala Pro Gly Gln Lys Val Thr Ile Ser Cys Ser Gly Ser Ser

625 630 635 640

Ser Asn Ile Gly Asn Asn Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly

645 650 655

Thr Ala Pro Lys Leu Leu Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly

660 665 670

Ile Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu

675 680 685

Gly Ile Thr Gly Leu Gln Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly

690 695 700

Thr Trp Asp Ser Ser Leu Ser Ala Trp Val Phe Gly Cys Gly Thr Lys

705 710 715 720

Leu Thr Val Leu

<210> 23

<211> 2172

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<400> 23

60

agctgcgctg ccagcggctt catcttcagc agctacgcca tgcactggggt ccgccaggcc 120

cctggcaacg gactggaatg ggtggccttc atgagctacg acggcagcaa caagaagtac 180

gccgacagcg tgaagggccg gttcaccatc agccgggaca acagcaagaa caccctgtac 240

ctgcagatga acagcctgcg ggctgaggac accgccgtgt actactgcgc cagagaccga 300

ggcatcagtg ctggcggcaa ctactactac tacggcatgg acgtgtgggg cggggcacc 360

accgtgaccg tgtctagcgc gtcgaccaag ggcccatccg tcttccccct ggcaccctcc 420

tccaagagca cctctggggg cacagcggcc ctgggctgcc tggtcaagga ctacttcccc 480

gaaccggtga cggtgtcctg gaactcaggc gctctgacca gcggcgtgca caccttcccg 540

gctgtcctac agtcctcagg actctactcc ctcagcagcg tggtgaccgt gccctccagc 600

agcttgggca cccagaccta catctgcaac gtgaatcaca agcccagcaa caccaaggtg 660

gacaagagag ttgagcccaa atcttgtgac aaaactcaca catgcccacc gtgcccagca 720

cctgaactcc tggggggacc gtcagtcttc ctcttccccc caaaacccaa ggacaccctc 780

atgatctccc ggacccctga ggtcacatgc gtggtggtgg acgtgagcca cgaagaccct 840

gaggtcaagt tcaactggta cgtggacggc gtggaggtgc ataatgccaa gacaaagccg 900

cgggaggagc agtacaacag cacgtaccgt gtggtcagcg tcctcaccgt cctgcaccag 960

gactggctga atggcaagga gtacaagtgc aaggtctcca acaaagccct cccagccccc 1020

atcgagaaaa ccatctccaa agccaaaggg cagccccgag aaccacaggt ctacaccctg 1080

cccccatccc gggaggagat gaccaagaac caggtcagcc tgacctgcct ggtcaaaggc 1140

ttctatccca gcgacatcgc cgtggagtgg gagagcaatg ggcagccgga gaacaactac 1200

aagaccacgc ctcccgtgct ggactccgac ggctccttct tcctctatag caagctcacc 1260

gtggacaaga gcaggtggca gcaggggaac gtcttctcat gctccgtgat gcatgaggct 1320

ctgcacaacc actacacgca gaagagcctc tccctgtctc cgggtaaagg cggaggggga 1380

tccggcggag ggggctctca ggtgcagctg gtgcagtctg gcgccgaagt gaagaaaccc 1440

ggctctagcg tgaaggtgtc ctgcaaggcc agcggcggca ccttctccac ctacggcatc 1500

agctgggtcc gccaggcccc tggacagtgt ctggaatgga tgggcggcat catccccatc 1560

ttcgacaccg gcaacagcgc ccagagcttc cagggcagag tgaccatcac cgccgacgag 1620

agcacctcca ccgcctacat ggaactgagc agcctgcgga gcgaggacac cgccgtgtac 1680

tactgcgcca gaagcagccg gatctacgac ctgaacccca gcctgaccgc ctactacgac 1740

1800

ggtggtggat ctggtggcgg aggaagtggc ggcggaggct ctcagagcgt gctgacccag 1860

cccccttctg tgtctgccgc ccctggccag aaagtgacca tctcctgcag cggcagcagc 1920

agcaacatcg gcaacaacta cgtgtcctgg tatcagcagc tgcccggcac cgcccctaag 1980

ctgctgatct acgacaacaa caagcggccc agcggcatcc ccgaccggtt tagcggcagc 2040

aagagcggga cttctgctac actgggcatc acaggcctgc agaccggcga cgaggccgac 2100

tactactgcg gcacctggga cagcagcctg agcgcttggg tgttcggctg cggcaccaag 2160

ctgacagtgc tg 2172

<210> 24

<211> 128

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 24

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Trp Phe Gly

20 25 30

Ala Phe Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Leu Thr Asn Leu Ala Gln Asn Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Ser Arg Ile Tyr Asp Leu Asn Pro Ser Leu Thr Ala Tyr

100 105 110

Tyr Asp Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 25

<211> 384

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<400> 25

caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60

tcctgcaagg cctctggagg caccttctgg ttcggcgcgt tcacctgggt gcgacaggcc 120

cctggacaag gacttgagtg gatgggaggg attattccta tcttcgggtt gacgaacttg 180

gcacagaact tccagggcag agtcacgatt accgcggacg aatccacgag cacagtctac 240

atggagctga gcagcttgag atctgaagac acggccgtat attattgtgc acgttcaagt 300

cgtatctacg atctgaaccc gtccctgacc gcctactacg atatggatgt ctggggccag 360

gggacaatgg tcaccgtctc gagt 384

<210> 26

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 26

Gln Ser Val Leu Thr Gln Pro Ser Val Ser Ala Ala Pro Gly Gln

1 5 10 15

Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asp Ile Gly Asn Asn

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln

65 70 75 80

Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu

85 90 95

Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 27

<211> 330

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<400> 27

cagtctgtgc tgactcagcc gccatcagtg tctgcggccc caggacagaa ggtcaccatc 60

tcctgctctg gaagcagctc cgacattggg aataattatg tatcgtggta ccagcagctc 120

cggaacag cccccaaact cctcatttat gacaataata agcgaccctc agggattcct 180

gaccgattct ctggctccaa gtctggcacg tcagccaccc tgggcatcac cggactccag 240

actggggacg aggccgatta ttactgcgga acatgggata gcagcctgag tgcttgggtg 300

ttcggcggag ggaccaagct gaccgtccta 330

<210> 28

<211> 126

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 28

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Ser Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Asn Gly Leu Glu Trp Val

35 40 45

Ala Phe Met Ser Tyr Asp Gly Ser Asn Lys Lys Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 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 Asp Arg Gly Ile Ser Ala Gly Gly Asn Tyr Tyr Tyr Tyr Gly

100 105 110

Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 29

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 29

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Tyr Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Ile Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro

85 90 95

Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys

100 105

<210> 30

<211> 128

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 30

Glu Val Gln Leu Val Gln Thr Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Thr Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gly Ile Ile Pro Ile Phe Asp Thr Gly Asn Ser Ala Gln Ser Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Val Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Ser Ser Arg Ile Tyr Asp Ala Asn Arg Gln Ala Val Pro Tyr

100 105 110

Tyr Asp Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 31

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 31

Gln Ala Val Leu Thr Gln Pro Ser Ser Val Ser Thr Pro Pro Gly Gln

1 5 10 15

Met Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asp Ile Gly Asn Asn

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln

65 70 75 80

Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu

85 90 95

Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 32

<211> 128

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 32

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Thr Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gly Ile Ile Pro Ile Phe Asp Thr Gly Asn Ser Ala Gln Ser Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Val Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Ser Ser Arg Ile Tyr Asp Phe Thr Ser Gly Leu Ala Pro Tyr

100 105 110

Tyr Asp Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 33

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 33

Gln Ala Val Leu Thr Gln Pro Ser Ser Val Ser Thr Pro Pro Gly Gln

1 5 10 15

Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Asp Asn Asn Lys Arg Pro Pro Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln

65 70 75 80

Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu

85 90 95

Ser Thr Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 34

<211> 128

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 34

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Thr Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gly Ile Ile Pro Ile Phe Asp Ala Gly Asn Ser Ala Gln Ser Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala His

65 70 75 80

Met Glu Val Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Ser Ser Arg Ile Tyr Asp His His Ile Gln Lys Gly Gly Tyr

100 105 110

Tyr Asp Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 35

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 35

Gln Ala Val Leu Thr Gln Pro Ser Ser Val Ser Thr Pro Pro Gly Gln

1 5 10 15

Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln

65 70 75 80

Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu

85 90 95

Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 36

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 36

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Thr Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gly Ile Ile Pro Ile Phe Asp Thr Gly Asn Ser Ala Gln Ser Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Val Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Ser Ser Arg Ile Tyr Asp Tyr His Thr Ile Ala Tyr Tyr Asp

100 105 110

<210> 37

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 37

Gln Ala Val Leu Thr Gln Pro Ser Ser Val Ser Thr Pro Pro Gly Gln

1 5 10 15

Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln

65 70 75 80

Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu

85 90 95

Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 38

<211> 128

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 38

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Thr Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gly Ile Ile Pro Ile Phe Asp Thr Gly Asn Ser Ala Gln Ser Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Lys Val Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Ser Ser Arg Ile Tyr Asp Tyr Ile Pro Gly Met Arg Pro Tyr

100 105 110

Tyr Asp Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 39

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 39

Gln Ala Val Leu Thr Gln Pro Ser Ser Val Ser Thr Pro Pro Gly Gln

1 5 10 15

Lys Val Thr Ile Ser Cys Ser Gly Asn Ser Ser Asn Ile Gly Asn Asn

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Arg Ser Gly Thr Leu Ala Thr Leu Gly Ile Thr Gly Leu Gln

65 70 75 80

Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu

85 90 95

Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 40

<211> 128

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 40

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Thr Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gly Ile Ile Pro Ile Phe Asp Thr Gly Asn Ser Ala Gln Ser Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Val Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Ser Ser Arg Ile Tyr Asp Phe Asn Ser Ser Leu Ile Ala Tyr

100 105 110

Tyr Asp Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 41

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 41

Gln Ala Val Leu Thr Gln Pro Ser Ser Val Ser Thr Pro Pro Gly Gln

1 5 10 15

Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln

65 70 75 80

Thr Gly Asp Glu Thr Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu

85 90 95

Ser Ala Trp Val Phe Ser Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 42

<211> 128

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 42

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Thr Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Thr Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gly Ile Ile Pro Ile Phe Asp Thr Gly Asn Ser Ala Gln Ser Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Val Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Ser Ser Arg Ile Tyr Asp Leu Asn Pro Ser Leu Thr Ala Tyr

100 105 110

Tyr Asp Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 43

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 43

Gln Ala Val Leu Thr Gln Pro Ser Ser Val Ser Thr Pro Pro Gly Gln

1 5 10 15

Lys Val Thr Ile Ser Cys Ser Gly Thr Ser Ser Asn Ile Gly Asn Asn

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln

65 70 75 80

Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu

85 90 95

Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 44

<211> 128

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 44

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Thr Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Thr Gly Asn Ser Ala Gln Ser Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Val Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Ser Ser Arg Ile Tyr Asp Phe Glu Pro Ser Leu Ile Tyr Tyr

100 105 110

Tyr Asp Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 45

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 45

Gln Ala Val Leu Thr Gln Pro Ser Ser Val Ser Thr Pro Pro Gly Gln

1 5 10 15

Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln

65 70 75 80

Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu

85 90 95

Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 46

<211> 128

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 46

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Thr Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gly Ile Ile Pro Ile Phe Asp Thr Gly Asn Ser Ala Gln Ser Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Val Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Ser Ser Arg Ile Tyr Asp Leu Asn Pro Ser Leu Thr Ala Tyr

100 105 110

Tyr Asp Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 47

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 47

Gln Ala Val Leu Thr Gln Pro Ser Ser Val Ser Thr Pro Pro Gly Gln

1 5 10 15

Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asp Ile Gly Asn Asn

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Asp Asn Asn Lys Arg Pro Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln

65 70 75 80

Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu

85 90 95

Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 48

<211> 128

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 48

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Thr Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gly Ile Ile Pro Ile Phe Asp Thr Gly Asn Ser Ala Gln Ser Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Val Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Ser Ser Arg Ile Tyr Asp Leu Asn Pro Ser Leu Thr Ala Tyr

100 105 110

Tyr Asp Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 49

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 49

Gln Ala Val Leu Thr Gln Pro Ser Ser Val Ser Thr Pro Pro Gly Gln

1 5 10 15

Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln

65 70 75 80

Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu

85 90 95

Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 50

<211> 128

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 50

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Thr Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gly Ile Ile Pro Ile Phe Asp Thr Gly Asn Ser Ala Gln Ser Phe

50 55 60

Gln Gly Arg Val Ala Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Val Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Ser Ser Arg Ile Tyr Asp Leu Asn Pro Ser Leu Thr Ala Tyr

100 105 110

Tyr Asp Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 51

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 51

Gln Ala Val Leu Thr Gln Pro Ser Ser Val Ser Thr Pro Pro Gly Gln

1 5 10 15

Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln

65 70 75 80

Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Gly Leu

85 90 95

Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 52

<211> 128

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 52

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Thr Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gly Ile Ile Pro Ile Phe Asp Thr Ser Asn Ser Ala Gln Ser Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Val Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Ser Ser Arg Ile Tyr Asp Phe Arg Thr Leu Tyr Ser Thr Tyr

100 105 110

Tyr Asp Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 53

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 53

Gln Ala Val Leu Thr Gln Pro Ser Ser Val Ser Thr Pro Pro Gly Gln

1 5 10 15

Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Ser Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Ala Gly Leu Gln

65 70 75 80

Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu

85 90 95

Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 54

<211> 128

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 54

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Thr Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gly Ile Ile Pro Ile Phe Asp Thr Gly Asn Ser Ala Gln Ser Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Val Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Ser Ser Arg Ile Tyr Asp Leu Asn Pro Ser Leu Thr Ala Tyr

100 105 110

Tyr Asp Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 55

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 55

Gln Ala Val Leu Thr Gln Pro Ser Ser Val Ser Thr Pro Pro Gly Gln

1 5 10 15

Glu Val Thr Ile Ser Cys Ser Gly Ser Ser Thr Asn Ile Gly Asn Asn

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln

65 70 75 80

Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu

85 90 95

Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 56

<211> 128

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 56

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Arg Ile Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Thr Tyr

20 25 30

Gly Val Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gly Ile Ile Pro Ile Phe Asp Thr Gly Asn Ser Ala Gln Ser Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Val Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Ser Ser Arg Ile Tyr Asp Leu Asn Pro Ser Leu Thr Ala Tyr

100 105 110

Tyr Asp Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 57

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 57

Gln Ala Val Leu Thr Gln Pro Ser Ser Val Ser Thr Pro Pro Gly Gln

1 5 10 15

Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln

65 70 75 80

Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu

85 90 95

Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 58

<211> 126

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 58

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ala Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gly Ile Ile Pro Ile Phe Asn Thr Gly Asn Ser Ala Gln Ser Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Val Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Ser Ser Arg Ile Tyr Asp Leu Asn Pro Ser Leu Thr Ala Tyr

100 105 110

Tyr Asp Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val

115 120 125

<210> 59

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 59

Gln Ala Val Leu Thr Gln Pro Ser Ser Val Ser Thr Pro Pro Gly Gln

1 5 10 15

Lys Val Thr Val Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln

65 70 75 80

Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu

85 90 95

Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 60

<211> 128

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 60

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Thr Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gly Ile Ile Pro Ile Phe Asp Thr Gly Asn Ser Ala Gln Ser Phe

50 55 60

Gln Asp Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Val Gly Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Ser Ser Arg Ile Tyr Asp Leu Asn Pro Ser Leu Thr Ala Tyr

100 105 110

Tyr Asp Met Asp Val Trp Gly His Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 61

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 61

Gln Ala Val Leu Thr Gln Pro Ser Ser Val Ser Thr Pro Pro Gly Gln

1 5 10 15

Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln

65 70 75 80

Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu

85 90 95

Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 62

<211> 128

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 62

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Thr Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Val Trp Ile

35 40 45

Gly Gly Ile Ile Pro Ile Phe Asp Thr Gly Asn Ser Ala Gln Ser Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Val Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Ser Ser Arg Ile Tyr Asp Met Ile Ser Ser Leu Gln Pro Tyr

100 105 110

Tyr Asp Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 63

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 63

Gln Ala Val Leu Thr Gln Pro Ser Ser Val Ser Thr Pro Pro Gly Gln

1 5 10 15

Lys Ala Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln

65 70 75 80

Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu

85 90 95

Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 64

<211> 128

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 64

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Thr Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gly Ile Ile Pro Ile Phe Asp Thr Gly Asn Ser Ala Gln Ser Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Val Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Ser Ser Arg Ile Tyr Asp Phe His Leu Ala Asn Lys Gly Tyr

100 105 110

Tyr Asp Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 65

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 65

Gln Ala Val Leu Thr Gln Pro Ser Ser Val Ser Thr Pro Pro Gly Gln

1 5 10 15

Lys Ala Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Asp Ile Thr Gly Leu Gln

65 70 75 80

Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu

85 90 95

Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 66

<211> 128

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 66

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Thr Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Thr Gly Asn Ser Ala Gln Ser Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Val Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Ser Ser Arg Ile Tyr Asp His His Asn His Val Gly Gly Tyr

100 105 110

Tyr Asp Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 67

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 67

Gln Ala Val Leu Thr Gln Pro Ser Ser Val Ser Thr Pro Pro Gly Gln

1 5 10 15

Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

20 25 30

Tyr Ala Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln

65 70 75 80

Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Gly Ser Leu

85 90 95

Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 68

<211> 128

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 68

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Ala Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Thr Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gly Ile Ile Pro Ile Phe Asp Thr Gly Asn Ser Ala Gln Ser Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Val Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Ser Ser Arg Ile Tyr Asp Leu Asn Pro Ser Leu Thr Ala Tyr

100 105 110

Tyr Asp Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 69

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 69

Gln Ala Val Leu Thr Gln Pro Ser Ser Val Ser Thr Pro Pro Gly Gln

1 5 10 15

Lys Val Thr Ile Ser Cys Ser Gly Ser Asn Ser Asn Ile Gly Asn Asn

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln

65 70 75 80

Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu

85 90 95

Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val

100 105

<210> 70

<211> 128

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 70

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Thr Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gly Ile Ile Pro Ile Phe Asp Thr Gly Asn Ser Ala Gln Ser Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Val Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Ser Ser Arg Ile Tyr Asp Leu Asn Pro Ser Leu Thr Ala Tyr

100 105 110

Tyr Asp Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 71

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 71

Gln Ala Val Leu Thr Gln Pro Ser Ser Val Ser Thr Pro Pro Gly Gln

1 5 10 15

Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Glu Asn Asn

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln

65 70 75 80

Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu

85 90 95

Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 72

<211> 128

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 72

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Thr Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Gly Ile Ile Pro Ile Phe Asp Thr Gly Asn Ser Ala Gln Ser Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Val Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Ser Ser Arg Ile Tyr Asp Ala Thr Thr Gly Leu Thr Pro Tyr

100 105 110

Tyr Asp Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 73

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 73

Gln Ala Val Leu Thr Gln Pro Ser Ser Val Ser Thr Pro Pro Gly Gln

1 5 10 15

Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Arg

65 70 75 80

Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu

85 90 95

Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 74

<211> 128

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 74

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Arg Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Tyr Gly Gly Thr Phe Ser Thr Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Val

35 40 45

Gly Gly Ile Ile Pro Ile Phe Asp Thr Arg Asn Ser Ala Gln Ser Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Val Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Ser Ser Arg Ile Tyr Asp Met Val Ser Thr Leu Ile Pro Tyr

100 105 110

Tyr Asp Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 75

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 75

Gln Ala Val Leu Thr Gln Pro Ser Ser Val Ser Thr Pro Pro Gly Gln

1 5 10 15

Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln

65 70 75 80

Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu

85 90 95

Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 76

<211> 126

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 76

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Arg Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Thr Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gly Ile Ile Pro Ile Phe Asp Thr Gly Asn Ser Ala Gln Ser Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Val Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Ser Ser Arg Ile Tyr Asp Ala His Leu Gln Ala Tyr Tyr Asp

100 105 110

Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 77

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 77

Gln Ala Val Leu Thr Gln Pro Ser Ser Val Ser Thr Pro Pro Gly Gln

1 5 10 15

Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Asp Asn Asn Lys Arg Pro Pro Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln

65 70 75 80

Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Arg Asp Ser Ser Leu

85 90 95

Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 78

<211> 128

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 78

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Ala Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Thr Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Ile

35 40 45

Gly Gly Ile Ile Pro Ile Phe Asp Thr Gly Asn Ser Ala Gln Ser Phe

50 55 60

Gln Gly Arg Val Ala Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Val Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Ser Ser Arg Ile Tyr Asp Ala His Leu Asn His His Gly Tyr

100 105 110

Tyr Asp Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 79

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 79

Gln Ala Val Leu Thr Gln Pro Ser Ser Val Ser Thr Pro Pro Gly Gln

1 5 10 15

Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln

65 70 75 80

Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu

85 90 95

Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 80

<211> 128

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 80

Glu Val Gln Leu Val Gln Ser Gly Ala Val Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Thr Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gly Ile Ile Pro Ile Phe Asp Thr Gly Ser Ser Ala Gln Ser Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Val Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Ser Ser Arg Ile Tyr Asp Leu Asn Pro Ser Leu Thr Ala Tyr

100 105 110

Tyr Asp Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 81

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 81

Gln Ala Val Leu Thr Gln Pro Ser Ser Val Ser Thr Pro Pro Gly Gln

1 5 10 15

Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Thr Gly Asn Asn

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Leu Ser Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln

65 70 75 80

Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu

85 90 95

Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 82

<211> 128

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 82

Glu Met Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Thr Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gly Ile Ile Pro Ile Phe Asp Thr Gly Asn Ser Ala Gln Ser Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Val Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Ser Ser Arg Ile Tyr Asp Phe Asn Ser Ala Leu Ile Ser Tyr

100 105 110

Tyr Asp Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 83

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 83

Gln Ala Val Leu Thr Gln Pro Ser Ser Val Ser Thr Pro Pro Gly Gln

1 5 10 15

Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln

65 70 75 80

Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu

85 90 95

Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val

100 105

<210> 84

<211> 125

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 84

Gln Met Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Thr Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ala Tyr His

20 25 30

Tyr Leu His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Thr Thr Asn Tyr Ala Gln Arg Phe

50 55 60

Gln Asp Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Ala Asp Tyr Val Trp Gly Ser Tyr Arg Pro Asp Trp Tyr Phe

100 105 110

Asp Leu Trp Gly Arg Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 85

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 85

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn

20 25 30

Thr Val Asn Trp Tyr Gln Arg Leu Pro Gly Ala Ala Pro Gln Leu Leu

35 40 45

Ile Tyr Asn Asn Asp Gln Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Gly Ser Leu Val Ile Ser Gly Leu Gln

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ser Trp Asp Asp Ser Leu

85 90 95

Asn Gly Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 86

<211> 125

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 86

Gln Met Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Thr Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly His Thr Phe Ala Tyr His

20 25 30

Tyr Leu His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Thr Thr Asn Tyr Ala Gln Arg Phe

50 55 60

Gln Asp Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Ala Asp Tyr Ala Trp Glu Ser Tyr Gln Pro Pro Gln Ile Asn

100 105 110

Gly Val Trp Gly Arg Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 87

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 87

Gln Ser Val Leu Thr Gln Pro Ser Val Ser Ala Ala Pro Gly Gln

1 5 10 15

Lys Val Thr Ile Thr Cys Ser Gly Ser Thr Ser Asn Ile Gly Asn Asn

20 25 30

Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu Met

35 40 45

Ile Tyr Asp Val Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Asn Ser Ala Ser Leu Asp Ile Ser Gly Leu Gln

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu

85 90 95

Ser Glu Phe Phe Phe Gly Thr Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 88

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 88

Phe Gly Ala Phe Thr

15

<210> 89

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 89

Gly Ile Ile Pro Ile Phe Gly Leu Thr Asn Leu Ala Gln Asn Phe Gln

1 5 10 15

gly

<210> 90

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 90

Ser Ser Arg Ile Tyr Asp Leu Asn Pro Ser Leu Thr Ala Tyr Tyr Asp

1 5 10 15

Met Asp Val

<210> 91

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 91

Ser Gly Ser Ser Ser Asp Ile Gly Asn Asn Tyr Val Ser

1 5 10

<210> 92

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 92

Asp Asn Asn Lys Arg Pro Ser

15

<210> 93

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 93

Gly Thr Trp Asp Ser Ser Leu Ser Ala Trp Val

1 5 10

<210> 94

<211> 128

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 94

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Thr Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Cys Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Ile Phe Asp Thr Gly Asn Ser Ala Gln Ser Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Ser Arg Ile Tyr Asp Leu Asn Pro Ser Leu Thr Ala Tyr

100 105 110

Tyr Asp Met Asp Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 95

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 95

Gln Ser Val Leu Thr Gln Pro Ser Val Ser Ala Ala Pro Gly Gln

1 5 10 15

Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln

65 70 75 80

Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu

85 90 95

Ser Ala Trp Val Phe Gly Cys Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 96

<211> 125

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 96

Gln Met Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Thr Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Pro Tyr His

20 25 30

Tyr Leu His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Thr Thr Asn Tyr Ala Gln Arg Phe

50 55 60

Gln Asp Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Phe Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Ala Asp Tyr Val Trp Glu Ser Tyr His Pro Ala Thr Ser Leu

100 105 110

Ser Leu Trp Gly Arg Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 97

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polypeptide"

<400> 97

Gln Ser Val Leu Thr Gln Pro Ser Val Ser Ala Ala Pro Gly Gln

1 5 10 15

Lys Val Thr Ile Ser Cys Pro Gly Ser Thr Ser Asn Ile Gly Asn Asn

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Arg Pro Gly Lys Ala Pro Lys Leu Met

35 40 45

Ile Tyr Asp Val Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Asn Ser Ala Ser Leu Asp Ile Ser Glu Leu Gln

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu

85 90 95

Ser Glu Phe Leu Phe Gly Thr Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 98

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

peptide"

<400> 98

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 99

<211> 2205

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Artificial sequence description: synthetic

polynucleotide"

<400> 99

ctgcccgccc aggtggcctt taccccttat gctcctgagc ccggctctac ctgccggctg 60

agagagtact acgaccagac cgcccagatg tgctgctcca agtgctctcc tggccagcac 120

gccaaggtgt tctgcaccaa gacctccgat accgtgtgcg actcctgcga ggactccacc 180

tacacccagc tgtggaactg ggtgcccgag tgcctgtcct gcggctccag atgttcctcc 240

gaccaggtgg aaacccaggc ctgcaccaga gagcagaacc ggatctgcac ctgtcggcct 300

ggctggtact gcgccctgtc taagcaggaa ggctgcagac tgtgcgcccc tctgcggaag 360

tgtagacctg gctttggcgt ggccagaccc ggcaccgaga catctgatgt cgtgtgcaag 420

ccttgcgccc ctggcacctt ctccaacacc acctcctcca ccgacatctg ccggcctcac 480

cagatctgca acgtggtggc catccctggc aacgcctcta tggacgccgt gtgcacctct 540

acctccccca ccagaagtat ggcccctggc gctgtgcatc tgccccagcc tgtgtctacc 600

agatcccagc acacccagcc cacccctgag ccttctaccg ccccttctac cagcttcctg 660

ctgcctatgg gccctagccc tcctgctgag ggatctacag gcgacgagcc caagtcctgc 720

gacaagaccc acacctgtcc cccttgtcct gcccctgaac tgctgggcgg accttccgtg 780

ttcctgttcc ccccaaagcc caaggacacc ctgatgatca gccggacccc tgaagtgacc 840

tgcgtggtgg tggatgtgtc ccacgaggat cccgaagtga agttcaattg gtacgtggac 841

ggcgtggaag tgcacaacgc caagaccaag cccagagagg aacagtacaa ctccacctac 960

1020

tgcaaggtgt ccaacaaggc cctgcctgcc cccatcgaaa agaccatctc caaggccaag 1080

ggccagcccc gggaacccca ggtgtacaca ctgcccccta gccgggaaga gatgaccaag 1140

aaccaggtgt ccctgacctg tctcgtgaag ggcttctacc cctccgatat cgccgtggaa 1200

tgggagtcca acggccagcc tgagaacaac tacaagacca ccccccctgt gctggactcc 1260

gacggctcat tcttcctgta ctccaagctg acagtggaca agtcccggtg gcagcaggggc 1320

aacgtgttct cctgctccgt gatgcacgag gccctgcaca accactacac ccagaagtcc 1380

ctgtccctga gccctggaaa aggcggcgga ggatctggcg gaggcggatc tcaggtgcag 1440

ctggtgcagt ctggcgctga agtgaagaaa cccggctcct ccgtgaaggt gtcctgcaag 1500

gcttctggcg gcaccttctc tacctacggc atctcctggg tgcgacaggc ccctggccag 1560

tgcctggaat ggatgggcgg catcatcccc atcttcgaca ccggcaactc cgccgagc 1620

ttccagggca gagtgaccat caccgccgac gagtctacct ccaccgccta catggaactg 1680

tcctccctgc ggagcgagga caccgccgtg tactactgcg cccggtcctc tcggatctac 1740

gacctgaacc cttccctgac cgcctactac gacatggacg tgtggggcca gggcacaatg 1800

gtcaccgtgt catctggtgg tggcggctct ggtggcggag gaagtggggg agggggttct 1860

ggggggggag gatctcagtc tgtgctgacc cagcctcctt ccgtgtctgc tgccccaggc 1920

cagaaagtga caatctcctg cagcggctcc agctccaaca tcggcaacaa ctacgtgtcc 1980

tggtatcagc agctgcccgg caccgctccc aaactgctga tctacgataa caacaagcgg 2040

ccctccggca tccccgcag attctccggc tctaagtccg gcacctctgc caccctgggc 2100

atcaccggac tgcagacagg cgacgaggcc gactactact gtggcacctg ggactcctcc 2160

ctgtccgctt gggtgttcgg ctgcggcacc aaactgactg tgctg 2205

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Claims (17)

1. A binding molecule comprising an NGF antagonist domain (nerve growth factor) and a TNFα antagonist domain (tumor necrosis factor α), wherein the NGF antagonist is an anti-NGF antibody or an antigen-binding fragment thereof, and wherein the anti-NGF antibody or fragment thereof contains a severe variable domain. chain (VH domain) containing a set of hypervariable regions (CDR) HCDR1, HCDR2, HCDR3, and a light chain variable domain (VL domain) containing a set of CDRs LCDR1, LCDR2 and LCDR3, and HCDR1 has the amino acid sequence of SEQ ID NO: 4 , HCDR2 has the amino acid sequence of SEQ ID NO: 5, HCDR3 has the amino acid sequence of any of SEQ ID NO: 6, 11, or 12, LCDR1 has the amino acid sequence of SEQ ID NO: 8, LCDR2 has the amino acid sequence of SEQ ID NO: 9, and LCDR3 has the amino acid sequence the sequence of SEQ ID NO: 10. 2. The binding molecule according to claim 1, wherein the TNFα antagonist inhibits the binding of TNFα to the TNF receptor (TNFR) on the cell surface, thereby blocking TNFα activity. 3. The binding molecule of claim 2, wherein the TNFα antagonist comprises an anti-TNFα antibody or an antigen-binding fragment thereof. 4. The binding molecule of claim 2, wherein the TNFα antagonist comprises a soluble TNFα binding fragment of TNFR. 5. The binding molecule according to claim 4, wherein the TNFR is TNFR-2. 6. The binding molecule according to claim 1, wherein the anti-NGF antibody or fragment thereof contains a VH domain having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical amino acid sequence of SEQ ID NO: 3 or 94, and a VL domain having an amino acid sequence at least 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 7 or 95. 7. The binding molecule of claim 1, wherein the anti-NGF antibody or fragment thereof is a full-length H2L2 antibody, Fab fragment, Fab' fragment, F(ab)2 fragment, or single chain Fv (scFv) fragment. 8. The binding molecule of claim 1, wherein the anti-NGF antibody or fragment thereof is an anti-NGF antibody scFv fragment that contains, N-terminally to C-terminally, a VH domain containing an amino acid sequence at least identical to at least 80%, 85%, 90%, 95%, 97%, 99%, or 100% of the amino acid sequence of SEQ ID NO: 3, a 15 amino acid linker sequence (GGGGS)3 (SEQ ID NO: 15), and A VL domain containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 7. 9. The binding molecule of claim 1, wherein the anti-NGF antibody, or fragment thereof, is an anti-NGF scFv fragment that contains, N-terminally to C-terminally, a VH domain containing an amino acid sequence at least identical to at least 80%, 85%, 90%, 95%, 97%, 99% or 100% of the amino acid sequence of SEQ ID NO: 94, a 20 amino acid linker sequence (GGGGS)4 (SEQ ID NO: 19), and A VL domain containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 95. 10. Selected polynucleotide encoding a binding molecule according to any one of paragraphs. 1-9. 11. An expression vector containing a polynucleotide according to claim 10 operably linked to a promoter. 12. A host cell comprising the polynucleotide of claim 10 or the vector of claim 11 that produces a binding molecule capable of binding NGF and TNFα. 13. The method of obtaining a binding molecule according to any one of paragraphs. 1-9, including culturing the host cell according to claim 12 under conditions that stimulate the expression of the binding molecule, and extracting the binding molecule. 14. Analgesic composition containing a binding molecule according to any one of paragraphs. 1-9 and a pharmaceutically acceptable carrier. 15. A set containing a binding molecule according to any one of paragraphs. 1-9, the polynucleotide of claim 10, the vector of claim 11, or the composition of claim 14, wherein said kit further comprises one or more containers, and wherein the binding molecule present in the kit or encoded by the polynucleotide or vector is intended to be used as analgesic agent. 16. The use of a binding molecule in the manufacture of a medicament for preventing, alleviating, reducing or eliminating pain in a subject, where the binding molecule is a binding molecule according to any one of paragraphs. 1-5. 17. The use of claim 16 wherein the pain is acute pain, transient pain, persistent nociceptive pain, or persistent or chronic neuropathic pain.

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