Compounds and methods for treating pain

Abstract

The present invention provides compositions and methods for controlling pain. In particular, the present invention provides methods for controlling pain comprising co-administration of an NGF antagonist and a TNFα antagonist. The NGF antagonist and TNFα antagonist can be separate molecules or part of a multifunctional polypeptide, such as a multispecific binding molecule comprising an NGF antagonist domain and a TNFα antagonist domain. The present invention also provides multifunctional polypeptides, such as multispecific binding molecules, comprising an NGF antagonist, and a TNFα antagonist domain. The methods provide improved pain control. As provided herein, administration of an NGF antagonist and a TNFα antagonist can control pain in a subject more effectively than administration of an equivalent amount of either the NGF antagonist or the TNFα antagonist alone.

Description

[0001] Related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 61 / 934,828, filed February 2, 2014. All teachings of the application cited above are incorporated herein by reference.

[0003] sequence list

[0004] This application contains a sequence list, which has been electronically submitted in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on January 29, 2015, named 110421-0054-WO1_SL.txt, and is 134,721 bytes in size. Technical Background

[0005] Pain is one of the most common symptoms prompting medical attention, and it is the primary symptom for half of all patients seeking medical care. Despite the existence and widespread use of numerous pain medications, complete elimination of pain, especially chronic pain, remains unfulfilled. Consequently, the societal burden remains high. Multiple studies estimate that pain results in 50 million lost workdays and $61.2 billion in lost productivity annually. For those suffering from chronic pain, only about half have access to available prescription treatments to manage their pain. Furthermore, the total market for prescription pain medications is approximately $25 billion annually. As these figures suggest, a significant demand for safe and effective new analgesics persists.

[0006] Therapeutic agents that reduce tissue levels of secreted nerve growth factor (NGF or β-NGF) or inhibit its effects have the potential to become such new analgesics. NGF plays a well-known key role in the development of the nervous system; however, NGF is also a recognized target of pain because it induces pain in animals and humans. Specifically, in adults, NGF promotes the health and survival of a subset of central and peripheral neurons (Huang & Reichardt, Ann. Rev. Neurosci. 24:677-736 (2001)). NGF also helps in the modulation of the functional properties of these neurons and exerts tonic control on the sensitivity or excitability of pain receptors (called nociceptors) (Priestley et al., Can. J. Physiol. Pharmacol. 80:495-505 (2002); Bennett, Neuroscientist 7:13-17 (2001)). Nociceptors sense various noxious stimuli that induce pain and transmit them to the central nervous system (nociception). NGF receptors are located on nociceptors. NGF expression increases in injured or inflamed tissues and is upregulated in a person in a state of pain. Therefore, due to the role of NGF in nociception, NGF binders that lower NGF levels are practically useful for analgesic treatment.

[0007] Subcutaneous injection of NGF itself induces pain in humans and animals. Injected NGF causes rapid thermal hyperalgesia, followed by delayed thermal hyperalgesia and allodynia (Petty et al., Ann. Neurol. 36:244-46 (1994); McArthur et al., Neurology 54:1080-88 (2000)). Endogenously secreted NGF is similarly pro-nociceptive. Tissue injury-induced NGF release and its subsequent peripheral effects play a major role in the induction of thermal hyperalgesia through a process of "peripheral sensitization" (Mendell & Arvanian, Brain Res. Rev. 40:230-39 (2002)). Tissue injury promotes the release of pro-nociceptive and pro-inflammatory cytokines, which in turn induce the release of NGF from keratinocytes and fibroblasts. This released NGF acts directly on nociceptors, inducing pain or a nociceptive state within minutes of nociceptive invasion. Therefore, NGF also indirectly induces and maintains nociceptive / painful states through feedforward release. It induces mast cell degranulation, releases pronociceptive agents such as histamine and serotonin, and, more importantly, releases more NGF. Furthermore, it stimulates sympathetic nerve endings to release pronociceptive neurotransmitters such as norepinephrine (Ma & Woolf, Neuroreport. 8:807-10 (1997)).

[0008] Tissue levels of NGF were 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)). In rats, NGF enhanced the capsaicin response in the DRG (dorsal root ganglion). Elevated NGF levels were recorded in patients with rheumatoid arthritis (Aloe & Tuveri, Clin. Exp. Rheumatol. 15:433-38 (1997)) or cystitis (Lowe et al., Br. J. Urol. 79:572-77 (1997)). In rodents, peripheral nerve injury increases NGF mRNA expression in macrophages, fibroblasts, and Schwann cells (Heumann et al., J. Cell Biol. 104:1623-31 (1987)). Following nerve injury, overexpression of NGF in transgenic mice resulted in enhanced neuropathic pain behavior compared to wild-type mice (Ramer et al., Pain, Supp. 6:S111-20 (1998)). Elevated NGF levels, both hourly and after 15 days, played a role in promoting central sensitization (enhanced synaptic neurotransmission in the nociceptive pathway of the spinal cord). Central sensitization leads to persistent and chronic hyperalgesia and atypical pain. This process is thought to involve the internalization of NGF into a complex with its high-affinity receptor, tyrosine receptor kinase A (trkA). These complexes enhance the secretion of nociceptive neuropeptides (such as substance P or calcitonin gene-related peptide (CGRP)), activation of protein kinase C (PKC), and activation of N-methyl-D-aspartate (NMDA) receptors in the dorsal horn of the spinal cord through retrograde transport to nociceptive cell bodies (Sah et al., Nat. Rev. Drug Disc. 2:460-72 (2003))—all processes that promote sensitization of the nociceptive pathway. NGF also plays a role in the upregulation and redistribution of voltage-dependent and ligand-gated ion channels, including sodium channel subtypes and capsaicin receptors, transient receptor potential cation channel subfamily V member 1 (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)).Altered activity and / or expression of neurotransmitters, receptors, and cation channels are the basis for increased sensitivity and excitability of nociceptors associated with neuropathic pain.

[0009] NGF-induced nociception / pain is mediated by the high-affinity NGF receptor trkA (tyrosine receptor kinase A) (Sah, et al., Nat. Rev. Drug Disc. 2:460-72 (2003)). Approximately 40-45% of the nociceptor cell bodies in the DRG express trkA. These are cell bodies of small-diameter fibers or C-fibers that express secreted pronociceptive peptides, substance P, and CGRP. These fibers terminate in layers I or II of the dorsal horn, where they transmit nociceptive stimuli sensed by peripheral nociceptors to the central nervous system. Mutations or deletions in the trkA gene in humans (Indo, Clin. Auton. Res. 12 (Supp 1): I20-I32 (2002)) and in trkA knockout mice (de Castro et al., Eur. J. Neurosci. 10:146-52 (1998)) produce a phenotype characterized by analgesia. Notably, trkA expression is upregulated in animals with arthritis (Pozza et al., J. Rheumatol. 27:1121-27 (2000)) or cystitis pain (Qiao & Vizzard, J. Comp. Neurol. 454:200-11 (2002)) models or in animals with inflammatory pain (induced by injection of CFA or carrageenan into the paw) (Cho et al., Brain Res. 716:197-201 (1996)).

[0010] NGF also binds to the p75 neurotrophic protein receptor (p75NTR). The function of p75NTR depends on its cellular environment and the presence of other receptors, which are believed to function as co-receptors or co-receptors. The interaction between trkA and p75NTR leads to the formation of a high-affinity binding site for NGF. The importance of such receptor interactions in NGF-mediated pain signaling is not yet clear, but recent studies have suggested that p75NTR may be involved in cellular processes (Zhang & Nicol, Neurosci. Lett. 366:187-92 (2004)). However, although p75NTR knockout mice exhibit an elevated threshold for noxious stimuli, they still retain responsiveness to the hyperalgesic effects of NGF, indicating that the trkA receptor alone is insufficient to mediate these effects (Bergmann et al., Neurosci. Lett. 255:87-90 (1998)).

[0011] NGF blockade produces a step-change efficacy against NSAIDs in chronic nociceptive pain (e.g., osteoarthritis (OA)) and chronic lower back pain. Several therapeutic antibody candidates targeting NGF are in various preclinical and clinical development stages. Such antibodies include, for example, tanezumab (PF-4383119; Pfizer), a humanized antibody in IgG2 form; SAR164877 / REGN475 (Sanofi-Aventis / Regeneron Pharmaceuticals), a human antibody in IgG4 form; AMG403 (Amgen / Johnson & Johnson), a human antibody in IgG2 form; and PG110 (PanGenetics / Abbott), a humanized antibody in IgG4 form. Other therapeutic antibody candidates are disclosed in WO 2006 / 077441, which relates to NGF antibodies and methods of treating diseases or conditions in which NGF plays a role with said disclosed antibodies. MEDI-578 is a human antibody in the form of IgG4. Despite the development of these candidates, there remains a need to provide analgesia for a wider range of pain conditions via NGF binding agents, which offer potent efficacy and an improved safety profile.

[0012] Tumor necrosis factor-α (TNFα), also known as cachecticin, is a pleiotropic cytokine with a wide range of biological activities, including cytotoxicity, immune cell proliferation, inflammatory responses, tumorigenesis, and viral replication (Kim et al., J. Mol. Biol. 374, 1374 (2007)). TNFα is initially produced as a transmembrane protein (tmTNFα), which is subsequently cleaved by metalloproteinases into a soluble form (sTNFα) (Wallis, Lancet Infect. Dis. 8 (10): 601 (2008)). TNFα (~17 kDa) exists as a rigid homotrimeric molecule, which binds to TNF receptor 1 or TNF receptor 2 on the cell surface, inducing receptor oligomerization and signal transduction.

[0013] Inflammatory cytokines, and more specifically TNFα, are known to play a role in the production of hyperalgesia (Leung, L. and Cahill, CM., J. Neuroinflammation 7:27 (2010)). Some preliminary data have suggested that TNFα inhibitors may be useful in controlling neuropathic pain. See, for example, Sommer C, et al., J. Peripher. Nerv. Syst. 6:67-72 (2001), Cohen et al., A&A Feb 2013, 116, 2, 455-462, Genevay et al., Ann Rheum Dis 2004, 63, 1120-1123. Clinical studies have tested TNFα inhibitors as monotherapy for neuropathic pain, but the results remain uncertain. See Leung and Cahill (2010).

[0014] Although candidates targeting NGF and TNFα are being developed for the treatment of pain, there is still a need for formulations that are more effective than current standard of care to provide analgesia for a wider range of pain conditions. This invention provides a combination therapy targeting both NGF and TNFα, which has the potential to improve efficacy and reduce both the dosage and frequency of administration to patients suffering from pain. Invention Overview

[0016] This invention provides a method for controlling pain in a subject, comprising administering an effective amount of a nerve growth factor (NGF) antagonist and a tumor necrosis factor-α (TNFα) antagonist, or a binding molecule comprising an NGF antagonist domain and a TNFα antagonist domain, to a subject in need. In some embodiments, the administration is more effective in controlling pain in the subject than administering an equivalent amount of the NGF antagonist or TNFα antagonist alone. In some embodiments, the method comprises co-administering a TNFα antagonist and an NGF antagonist. In some embodiments, the TNFα antagonist and the NGF antagonist are administered sequentially or simultaneously.

[0017] In some embodiments, the method is sufficient to prevent, reduce, alleviate, or eliminate pain in the subject. In some embodiments, the pain is acute pain, short-term pain, persistent or chronic nociceptive pain, or persistent or chronic neuropathic pain. In some embodiments, the method is more effective in controlling pain in the subject than administering an equal amount of an NGF antagonist or TNFα antagonist alone at at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0018] In some embodiments, the TNFα antagonist portion of the binding molecule binds to a polypeptide comprising the amino acid sequence of SEQ ID NO:2. In some embodiments, the NGF antagonist binds to a polypeptide comprising the amino acid sequence of SEQ ID NO:1.

[0019] In some embodiments, the NGF antagonist portion of the binding molecule is an anti-NGF antibody or its antigen-binding fragment. In some embodiments, the anti-NGF antibody or its fragment inhibits the binding of NGF to TrkA, p75NRT, or both TrkA and p75NRT. In some embodiments, the binding molecule preferentially blocks the binding of NGF to TrkA relative to the binding of NGF to p75NRT. In some embodiments, the anti-NGF antibody or its fragment binds to human NGF with an affinity of about 0.25-0.44 nM. In some embodiments, the anti-NGF antibody or its fragment binds to the same epitope as MEDI-578. In some embodiments, the anti-NGF antibody or its fragment competitively inhibits the binding of MEDI-578 to human NGF.

[0020] In some embodiments, the anti-NGF antibody or a fragment thereof comprises an antibody VH domain and an antibody VL domain, wherein the antibody VH domain comprises a set of CDRs, namely HCDR1, HCDR2, and HCDR3, and the antibody VL domain comprises a set of CDRs, namely LCDR1, LCDR2, and LCDR3, wherein HCDR1 has the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:4 has an amino acid sequence with more than two amino acid substitutions, HCDR2 has the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:5 has an amino acid sequence with more than two amino acid substitutions, HCDR3 has the amino acid sequence of SEQ ID NO:6, SEQ ID NO:6 has an amino acid sequence with more than two amino acid substitutions, SSRIYDFNSALISYYDMDV (SEQ ID NO:11), or SSRIYDMISSLQPYYDMDV (SEQ ID NO:12), LCDR1 has the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:8 has an amino acid sequence with more than two amino acid substitutions, and LCDR2 has the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:8. NO:9 has an amino acid sequence with one or more amino acid substitutions, and the LCDR3 has the amino acid sequence of SEQ ID NO:10 or SEQ ID NO:10 has an amino acid sequence with one or more amino acid substitutions. In some embodiments, the anti-NGF antibody or fragment thereof comprises a VH having the amino acid sequence of SEQ ID NO:3. In some embodiments, the anti-NGF antibody or fragment thereof comprises a VL having the amino acid sequence of SEQ ID NO:7. In some embodiments, the anti-NGF antibody or fragment thereof comprises a VH having at least 80%, 85%, 90%, 95%, or 99% of the same amino acid sequence as the amino acid sequence of SEQ ID NO:3. In some embodiments, the anti-NGF antibody or fragment thereof comprises a VL having at least 80%, 85%, 90%, 95%, or 99% of the same amino acid sequence as the amino acid sequence of SEQ ID NO:7. In some embodiments, the anti-NGF antibody or fragment thereof comprises a VH having the amino acid sequence of SEQ ID NO:94. In some embodiments, the anti-NGF antibody or fragment thereof comprises a VL having the amino acid sequence of SEQ ID NO:95. In some embodiments, the anti-NGF antibody or a fragment thereof comprises VH, said VH having at least 80%, 85%, 90%, 95%, or 99% of the same amino acid sequence as the amino acid sequence of SEQ ID NO:94.In some embodiments, the anti-NGF antibody or a fragment thereof comprises a VL 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.

[0021] In some embodiments, the anti-NGF antibody or a fragment thereof is a complete H2L2 antibody, 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 a fragment thereof is humanized, chimeric, primatized, or fully human. In some embodiments, the NGF antagonist is an anti-NGF scFv fragment. In some embodiments, the scFv is SS-stabilized. In some embodiments, the anti-NGF scFv fragment comprises, from the N-terminus to the C-terminus, the following: VH comprising the amino acid sequence of SEQ ID NO:3, a 15-amino acid linker sequence (GGGGS)3 (SEQ ID NO:15), and VL comprising the amino acid sequence of SEQ ID NO:7. In some embodiments, the anti-NGF scFv fragment comprises, from the N-terminus to the C-terminus, the following: VH containing the amino acid sequence of SEQ ID NO:94, a 20-amino acid linker sequence (GGGGS)4 (SEQ ID NO:19), and VL containing the amino acid sequence of SEQ ID NO:95.

[0022] In some aspects, the method includes administering a TNFα antagonist that inhibits the binding of TNFα to the 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 its antigen-binding fragment comprises an antibody VH domain and an antibody VL domain, the antibody VH domain comprising a set of CDRs, namely HCDR1, HCDR2, and HCDR3, and the antibody VL domain comprising a set of CDRs, namely LCDR1, LCDR2, and LCDR3, wherein the CDRs are identical to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of infliximab or HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of adalimumab.

[0023] In some embodiments, the binding molecule comprises a complete anti-TNFα antibody and an anti-NGF scFv fused to the C-terminus of the heavy chain of the anti-TNFα antibody. Such a binding molecule may comprise a light chain and a heavy chain, the light chain comprising the amino acid sequence of SEQ ID NO:20 and the heavy chain comprising the amino acid sequence of SEQ ID NO:22.

[0024] In some embodiments, the TNFα antagonist comprises a soluble TNFα-binding fragment of TNFR. In some embodiments, the TNFR is TNFR-2 or a soluble fragment thereof. In other embodiments, the TNFR is TNFR-1 or a soluble fragment thereof. In some embodiments, the soluble fragment of TNFR-1 is a 55kD fragment. In other embodiments, the soluble fragment of TNFR-2 is a 75kD fragment. In some embodiments, the TNFR fragment is fused to an immunoglobulin Fc domain. 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.

[0025] In some embodiments, the binding molecule comprises a fusion protein containing an NGF antagonist fused to a TNFα antagonist via a linker. In some embodiments, the binding molecule is a homodimer of the fusion protein.

[0026] In some embodiments, the NGF antagonist is an anti-NGF scFv domain and the TNFα antagonist is a soluble, TNFR-2 TNFα-binding fragment fused to the immunoglobulin Fc domain at its carboxyl terminus. In some embodiments, the scFv is fused to the carboxyl terminus of the immunoglobulin Fc domain via a linker.

[0027] In some embodiments, the binding molecule comprises a homodimer of a fusion polypeptide comprising, from the N-terminus to the C-terminus: a 75 kD fragment of TNFR-2 binding to TNFα, a human IgG1 Fc domain, a 10-amino acid linker sequence (GGGGS)2, a VH comprising the amino acid sequence of SEQ ID NO:3, a 15-amino acid linker sequence (GGGGS)3 (SEQ ID NO:15), and a VL comprising the amino acid sequence of SEQ ID NO:7. In some embodiments, the binding molecule comprises a homodimer of a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:14. In some embodiments, the binding molecule comprises a homodimer of a fusion polypeptide comprising at least 80%, 85%, 90%, 95%, or 99% of the same amino acid sequence as the amino acid sequence of SEQ ID NO:14.

[0028] In some embodiments, the binding molecule comprises a homodimer of a fusion polypeptide comprising, from the N-terminus to the C-terminus: a 75 kD fragment of TNFR-2 binding to TNFα, a human IgG1 Fc domain, a 10-amino acid linker sequence (GGGGS)2, a VH comprising the amino acid sequence of SEQ ID NO:94, a 20-amino acid linker sequence (GGGGS)4 (SEQ ID NO:19), and a VL comprising the amino acid sequence of SEQ ID NO:95. In some embodiments, the binding molecule comprises a homodimer of a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:17. In some embodiments, the binding molecule comprises a homodimer of a fusion polypeptide comprising at least 80%, 85%, 90%, 95%, or 99% of the same amino acid sequence as the amino acid sequence of SEQ ID NO:17.

[0029] In some embodiments, the binding molecule comprises a homodimer of a fusion polypeptide, the fusion polypeptide comprising, from the N-terminus to the C-terminus, the following: a 75kD fragment of TNFR-2 that binds to TNFα, a human IgG1 Fc domain, an adapter sequence, and an anti-NGF scFv domain.

[0030] This disclosure also provides methods for inhibiting p38 phosphorylation in cells, wherein the methods include contacting cells with any polypeptide described herein (e.g., any binding molecule containing the NGF antagonist domain and TNFα antagonist domain described herein). This disclosure also provides methods for inhibiting ERK phosphorylation in cells, wherein the methods include contacting cells with any polypeptide described herein (e.g., any binding molecule containing the NGF antagonist domain and TNFα antagonist domain described herein). In some embodiments, the cells are neuronal cells. In other embodiments, the cells are peripheral neuronal cells. In still other embodiments, the cells are central neuronal cells. In some embodiments, the cells are in a mammal. In some embodiments, the mammal is a human. In some embodiments, the cells are in a cell culture.

[0031] The present invention also provides polynucleotide sequences encoding the binding molecules described herein, vectors containing such polynucleotide sequences, and host cells containing such polynucleotides or vectors.

[0032] The present invention also provides a method for generating the binding molecules described herein.

[0033] The present invention also provides compositions, pharmaceutical compositions, and kits comprising the binding molecules described herein.

[0034] Brief description of attached figures / charts

[0035] Figure 1: TNFR2-Fc fusion protein (Figure A), and an exemplary multispecific binding molecule TNFR2-Fc_VH#4 (Figure B) containing a TNFR2-Fc domain fused to an anti-NGF scFv domain.

[0036] Figure 2A The results of SEC-HPLC analysis of aggregate, monomer, and protein fragmentation levels in a batch of purified TNFR2-Fc_VH#4 are shown.

[0037] Figure 2B SDS-PAGE analysis of purified TNFR2-Fc_VH#4 and purified TNFR2-Fc protein under reducing and non-reducing conditions is shown. Gel loading order: 1. TNFR2-Fc_VH#4, 2. TNFR2-Fc_VL-VH (TNFR2-Fc fused to anti-NGF scFv with reverse variable domain gene orientation), 3. TNFR2-Fc unrelated scFv 1, 4. TNFR2-Fc, 5. TNFR2-Fc unrelated scFv 2.

[0038] Figure 3A The purity of TNFR2-Fc_VH#4 after purification using protein A column is shown. Figure 3B The purity of TNFR2-Fc_VH#4 after the second purification step on an SP agarose column is shown.

[0039] Figure 4 Stability analysis of TNFR2-Fc_VH#4 performed using differential scanning calorimetry is shown.

[0040] Figure 5 shows the binding of TNFR2-Fc_VH#4 to TNFα and NGF (both alone and together), as determined by ELISA. Figure 5A The binding to NGF was shown. Figure 5B It showed binding to TNFα, while Figure 5C Simultaneous binding to TNFα and NGF was demonstrated.

[0041] Figure 6The sensorgrams for surface plasmon resonance binding assays of TNFR2-Fc_VH#4 are shown. Parallel antigen binding of the TNFR2-Fc_VH#4 multispecific antibody was performed using a BIAcore 2000. Simultaneous antigen binding was assessed by sequentially binding TNFα and NGF to TNFR2-Fc_VH#4 bound to the sensor surface. The first portion of the sensorgram shows the binding of a saturated amount of TNFα to the multispecific antibody, and the second portion shows the binding when a second antigen is applied, either again as TNFα (showing a saturated surface) or as an equimolar mixture of TNFα and NGF. An increase in resonance units corresponds to the binding of NGF to the multispecific molecule and, therefore, to simultaneous antigen engagement. Assays were also performed using antigens added in reverse order to determine these data.

[0042] Figure 7 shows the inhibition of NGF-mediated TF-1 cell proliferation. A. NGF-mediated proliferation in the absence of added NGF antagonists. B. Inhibition of human NGF response by TNFR2-Fc_VH#4. C. Inhibition of mouse NGF response by TNFR2-Fc_VH#4. NGF activity is typically expressed as RLU – relative luminescent units, while the percentage of NGF-mediated proliferation is calculated as the percentage response against NGF ligands alone using the following formula: 100*(well RLU – background RLU) / (total RLU – background RLU), where background RLU = mean of media controls and total RLU = mean of ligand-only controls. D. Inhibition of human NGF response by TNFR2-Fc_VarB and ndimab VarB. E. Inhibition of mouse NGF response by TNFR2-Fc_VarB and ndimab VarB.

[0043] Figure 8 shows the inhibition of TNFα-induced caspase 3 activity in U937 cells. A. TNFα-induced caspase 3 activity in U937 cells without the addition of a TNFα antagonist. B. Inhibition of TNFα-induced caspase 3 activity in U937 cells, shown as a percentage of the response under the condition without the addition of an antagonist. TNF activity is typically expressed as RLU – relative luminescent units, while the % of TNF-mediated caspase 3 release is expressed as a percentage response to the TNF ligand alone, as described above. Figure 7C The formula described in [the document] was used for calculation. C. Similar results were shown for related molecules TNFR2-Fc_varB and ndimabVarB.

[0044] Figure 9This study demonstrated the efficacy of etanercept and MEDI-578 in treating mechanopathic hyperalgesia induced by local sciatic nerve ligation. Results were presented as ipsilateral / contralateral ratios. N = 9–10 per group. Data were analyzed using a two-factor ANOVA with time and treatment as dependent factors. Statistical significance was subsequently obtained using Boniferroni's post-hoc test. Compared to the Op+CAT-251 control group, ***p < 0.001.

[0045] Figure 10A The efficacy of TNFR2-Fc_VH#4 on mechanosensitive hyperalgesia induced by local sciatic nerve ligation was demonstrated. Results were presented as ipsilateral / contralateral ratios. N=10 per group. Data were analyzed using a two-factor ANOVA with time and treatment as correlation factors. Statistical significance was subsequently obtained using the Boniferroni post-hoc test. Compared with a bispecific isotype control, ***p<0.001. Figure 10B Similar results were shown using the related molecule TNFR2-Fc_varB.

[0046] Figure 11 The study demonstrated the effect of co-administration of MEDI-578 and etanercept on pain reduction in a model of mechanical hypersensitivity arthritis. N = 9–10 per group. Data were analyzed using a two-way ANOVA analysis. Statistical significance was subsequently obtained using the Boniferroni post-hoc test. Compared with CAT-251, *P>0.05; ***P<0.001.

[0047] Figure 12 The study demonstrated the pain-reducing effect of TNFR2-Fc_VH#4 in a model of mechanosensitive arthritis. N = 9–10 per group. Data were analyzed using a two-way ANOVA. Statistical significance was subsequently determined using the Boniferroni post-hoc test. Compared with a bispecific isotype control, ***P < 0.001.

[0048] Figure 13 The effects of five different doses of TNFR2-Fc_varB on CFA-induced hyperalgesia were demonstrated in a rat model.

[0049] Figure 14 : A heatmap showing the HTRF ratio from the phosphoric acid-p38 reaction.

[0050] Figure 15 : Dose-response curves, which show the effects of TNFα, NGF, or a combination of TNFα and NGF on p38 phosphorylation.

[0051] Figure 16 : A heatmap showing the HTRF ratio from the phosphoric acid-ERK reaction.

[0052] Figure 17 : Dose-response curves, which show the effects of TNFα, NGF, or a combination of TNFα and NGF on ERK phosphorylation. Invention Details

[0054] definition

[0055] It should be noted that the terms “a” or “an” refer to one or more of the same thing. Similarly, “a” (or “an”), “one or more”, and “at least one” are used interchangeably here.

[0056] Furthermore, the term "and / or" as used herein should be understood to specifically disclose each of two particular features or components together or together with the other. Therefore, when the term "and / or" is used in phrases such as "A and / or B," it is intended to include "A and B," "A or B," "A (alone)," and "B (alone)." Similarly, when the term "and / or" is used in phrases such as "A, B, and / or C," it is intended to cover each of the following aspects: 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; A (alone); B (alone); and C (alone).

[0057] It should be understood that wherever an aspect is described herein with the language “comprising”, similar aspects described with the terms “composed of” and / or “substantially composed of” are also provided.

[0058] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art related to this invention. For example, the Concise Dictionary of Biomedical and Molecular Biology, Juo, Pei-Show, 2nd Edition, 2002, CRC Press; the Dictionary of Cell and Molecular Biology, 3rd Edition, 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, 2000 Revised Edition, Oxford University Press, provide a general dictionary for those skilled in the art of the invention.

[0059] Units, prefixes, and symbols are represented in their form as accepted by the International System of Units (SI). Numerical ranges include the numbers defining the range. Unless otherwise stated, amino acid sequences are written from left to right, from amino to carboxyl. The headings provided herein are not intended to limit the various aspects of the invention, but are incorporated herein by reference as a whole. Therefore, the terms defined immediately following this paragraph are defined more fully by reference to the entire specification.

[0060] As used herein, the term "binding molecule" in its broadest sense refers to a molecule that specifically binds to an antigenic determinant (e.g., an antigen). Non-limiting examples of binding molecules include antibodies or fragments thereof, soluble receptor fusion proteins or fragments thereof, and non-immunoglobulin backbones or fragments thereof, each retaining antigen-specific binding. Exemplary non-immunoglobulin backbones include Tn3 (Koide et al., J Mol Biol 2012 Jan 13; 415(2):393-405), DARPin (Boersma & Pluckthun, Curr Opin Biotechnol. 2011 22(6):849-57), and Anticalin (Gebauer & Skerra, Methods Enzymol. 2012; 503:157-88). Exemplary soluble receptor fusion proteins and antibodies are provided below. In some embodiments, the binding molecule may be engineered to comprise a combination of such antibodies or fragments thereof, soluble receptor fusion proteins or fragments thereof, and non-immunoglobulin-based backbones or fragments thereof.

[0061] The term “binding domain” is used herein to refer to any part of a binding molecule or a portion of a binding molecule that recognizes an antigen. Except as expressly referred to as a full-sized binding molecule such as a naturally occurring antibody, the term “binding molecule” is not limited to encompassing full-sized antibodies or other non-antibody binding molecules, as well as antigen-binding fragments, variants, analogs, or derivatives of such binding molecules, such as naturally occurring antibody or immunoglobulin molecules or engineered binding molecules or fragments that bind antigens in a manner similar to that of full-sized binding molecules.

[0062] In some embodiments, the present invention provides multispecific binding molecules, such as bispecific, trispecific, tetraspecific, etc., or antigen-binding fragments, variants, or derivatives thereof. As used herein, multispecific binding molecules may include one or more antibody-binding domains, one or more non-antibody-binding domains, or combinations thereof.

[0063] As used herein, the term “nerve growth factor” (“NGF”), also literally β-nerve growth factor, refers to a secreted protein that plays a role in the growth and survival of a variety of neurons. Human NGF is presented under Genbank accession number NP_002497.2 and is presented herein under SEQ ID NO:1. As used herein, the term NGF is not limited to human NGF and includes all species orthologs of human NGF. The term “NGF” encompasses the pro-form, pro-NGF, full-length NGF, and any form of NGF produced by intracellular processes. The term also encompasses variants of naturally occurring NGF, such as splice variants, allelic variants, and isotypes. NGF binds to two receptors: the p75 neurotrophic protein receptor (p75(NTR)) and TrkA, a transmembrane tyrosine kinase. NGF is a well-established target for pain and is known to mediate sensitization of nociceptors.

[0064] Several formulations are being tested as antagonists of NGF activity. One such anti-NGF agent is trkA-Fc, which acts as a decoy or scavenger to bind to and thereby inactivate endogenous NGF. TrkA-Fc is a fusion protein consisting of the NGF-binding region of trkA linked to a constant domain fragment (Fc) of an IgG antibody. TrkA-Fc induces hypoalgesia in untreated animals, reduces nociceptor responses, and decreases sprouting in unmyelinated pain-sensing neurons (Bennett, DL et al. (1998) Eur J Neurosci, 10:1282-91).

[0065] NGF-mediated pain is particularly well-suited for safe and effective treatment with the binding molecules described herein because peripheral NGF levels increase in response to noxious stimuli and the antibodies have low blood-brain barrier penetration. Some anti-NGF antibodies and their antigen-binding fragments that can be used in the treatments and compositions described herein are available in the literature, see, for example, PCT Publications Nos. WO02 / 096458 and WO04 / 032870.

[0066] The term “MEDI-578” refers to an antibody that specifically binds to NGF, the subject of International Application No. PCT / GB2006 / 000238 and U.S. Patent Application Publication No. 2008 / 0107658A1, both of which are incorporated herein by reference in their entirety. The heavy and light chain sequences of MEDI-578 are shown in SEQ ID NOs:3 and 7, respectively.

[0067] The term NGF-NG refers to an antibody that specifically binds to NGF. The heavy and light chain sequences of NGF-NG are shown in SEQ ID NOs:24 and 26, respectively.

[0068] The term “tumor necrosis factor α” (“TNFα”), also literally as cachexia, APC1 protein; tumor necrosis factor; TNF; or tumor necrosis factor ligand superfamily 2, as used herein, refers to a specific TNFα protein and not a superfamily of TNF ligands. Human TNFα is presented under Genbank accession number NP_000585.2 and is presented herein under SEQ ID NO:2. As used herein, the term TNFα is not limited to human TNF and includes all species orthologs of human TNFα. The term “TNFα” encompasses the precursor form of TNFα, pro-TNFα, full-length TNFα, and any form of TNFα produced by intracellular processes. The term also encompasses naturally occurring and non-natural variants of TNFα, such as splice variants, allelic variants, and isotypes. TNFα binds to two receptors, TNFR1 (type 1 TNF receptor; CD120a; p55 / 60) and TNFR2 (type 2 TNF receptor; CD120b; p75 / 80). TNFα acts as a pro-inflammatory cytokine, for example, in neuroinflammation. For instance, TNFα is thought to be functionally involved in the development of neuropathic pain (Leung, L. and Cahill, CM., J. Neuroinflammation 7:27 (2010)).

[0069] Numerous TNFα antagonists are known in the art, and many are commercially available therapeutic agents. Commercially available TNF-α antagonists that can be used in the treatments and compositions provided herein include etanercept (…). Amgen / Pfizer), infliximab (e.g.) Centocor), pegylated certolizumab pegol (e.g.) UCB), golimumab (e.g., SIMPONI) TM Centocor and adalimumab (e.g.) Abbott).

[0070] "Separated" binding molecules, peptides, antibodies, polynucleotides, vectors, host cells, or compositions refer to binding molecules, peptides, antibodies, polynucleotides, vectors, host cells, or compositions existing in a non-natural form. Separated binding molecules, peptides, antibodies, polynucleotides, vectors, host cells, or compositions include those that have been altered, adapted, combined, rearranged, engineered, or otherwise manipulated to the point that they are no longer in their natural form. In some respects, the separated binding molecules, peptides, antibodies, polynucleotides, vectors, host cells, or compositions are "recombinant."

[0071] As used herein, the terms “multifunctional peptide” and “bifunctional peptide” refer to non-naturally occurring binding molecules designed to target two or more antigens. Multifunctional peptides described herein are typically genetically engineered fusion proteins designed to deliver two distinct desired biological functions into a single binding molecule. For example, a multifunctional peptide can be a multifunctional binding molecule. An exemplary multifunctional peptide described herein is a multifunctional binding molecule comprising an NGF antagonist domain (e.g., a peptide domain that blocks, reduces, or inhibits the function of one or more natural NGFs) and a TNFα antagonist domain (e.g., a peptide domain that blocks, reduces, or inhibits the function of one or more natural TNFαs).

[0072] This article presents a group of multifunctional peptides that are multispecific binding molecules, such as binding molecules containing one or more antibody-binding domains (e.g., "multispecific antibodies"), one or more non-antibody-binding domains (e.g., decoy receptors), or combinations thereof. For example, a multispecific binding molecule containing one or more antibody-binding domains, one or more non-antibody-binding domains, or combinations thereof is a molecule having binding domains capable of specifically recognizing and binding to at least two different epitopes. These different epitopes may be located within the same molecule (e.g., the same NGF) or on different molecules, thereby enabling the multispecific binding molecule to specifically recognize and bind to NGF and another molecule containing an epitope, such as TNFα, thus enabling the multispecific binding molecule to specifically recognize NGF and TNFα.

[0073] Techniques for fabricating multispecific binding molecules (e.g., multispecific binding molecules containing one or more antibody-binding domains, one or more non-antibody-binding domains, or combinations thereof) are available in the art (Dimasi, N., et al., 2009, JMol 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 US Patent 5,731,168). Antibodies with more than two valencies are also included. For example, trispecific antibodies can be prepared (Tutt et al., J. Immunol. 147:60 (1991)).

[0074] The term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination thereof, via at least one antigen-binding site within the variable region of an immunoglobulin molecule. As used herein, the term "antibody" encompasses 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, and tetraspecific antibodies, etc., derived from intact antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins, and any other modified immunoglobulin molecule containing an antigen-binding site, provided that the antibody exhibits the desired biological activity. Antibodies can belong to any of the five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), which are identified based on their heavy chain constant domains as α, δ, ε, γ, and μ, respectively. Different classes of immunoglobulins have different and well-known subunit structures and three-dimensional conformations.

[0075] In some embodiments, a “blocking” binding molecule, such as a blocking antibody, or an “antagonist” binding molecule, such as an antagonist antibody or fusion protein, is a binding molecule that inhibits or reduces the biological activity of the antigen (such as NGF or TNF) it binds to. In some aspects, blocking antibodies or antagonist binding molecules substantially or completely inhibit the biological activity of the antigen. For example, the biological activity may be reduced by 0.01%, 0.1%, 0.5%, 1%, 5%, 10%, 20%, 30%, 50%, 70%, 80%, 90%, 95%, or even 100%. As used herein, “antagonist” and “antagonist domain” include peptides or other molecules that bind to their targets (e.g., TNFα or NGF), thereby blocking or inhibiting the interaction between the target and the receptor. Thus, NGF and / or TNFα antagonists include molecules that block or inhibit the interaction of NGF with trkA or p75 neurotrophic protein, or the interaction of TNFα with TNFR-1 or TNFR-2. NGF and / or TNFα antagonists also include molecules that reduce p38 phosphorylation and / or ERK phosphorylation. Exemplary antagonists include, but are not limited to, antibodies or their antigen-binding fragments, and target-specific, soluble, non-signal-transmitting receptor peptides (“decoy receptors”, or their ligand-binding fragments).

[0076] The term "antibody fragment" refers to a portion of a complete antibody and to the variable region of the antigenic determinant of a complete 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 document are also provided herein.

[0077] "Monoclonal antibody" refers to a homogeneous group of antibodies involved in the highly specific recognition and binding to a single antigenic determinant or epitope. This contrasts with polyclonal antibodies, which typically contain different antibodies targeting different antigenic determinants. The term "monoclonal antibody" encompasses complete and full-length monoclonal antibodies, as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single-chain (scFv) mutants, fusion proteins containing antibody portions, and any other modified immunoglobulin molecules containing antigen recognition sites. Furthermore, "monoclonal antibody" refers to antibodies produced in any number of ways, including but not limited to hybridomas, phage selection, recombinant expression, and transgenic animals.

[0078] The term "humanized antibody" refers to a form of non-human (e.g., mouse) antibody that is a specific immunoglobulin chain, a chimeric immunoglobulin, or a fragment thereof containing a minimal non-human (e.g., mouse) sequence. Typically, humanized antibodies are human immunoglobulins in which residues from the complementarity-determining region (CDR) are replaced by residues from the CDR of a non-human species (e.g., mouse, rat, rabbit, or hamster) with desired specificity, affinity, and capability (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, the Fv framework region (FR or FW) residues of human immunoglobulins are replaced by corresponding residues from antibodies of a non-human species with desired specificity, affinity, and capability. The humanized antibody can also be modified by substituting additional residues, either in the Fv frame region and / or within the replaced non-human residues, to improve and optimize antibody specificity, affinity, and / or potency. Generally, humanized antibodies contain at least one, and typically two or three, variable domains containing all or substantially all of the CDR regions corresponding to non-human immunoglobulins, but all or substantially all of the FR regions are those shared sequences of human immunoglobulins. Humanized antibodies may also contain at least a portion of immunoglobulin constant regions or domains (Fc), typically constant regions or domains of human immunoglobulins. Methods for generating humanized antibodies are described in U.S. Patents 5,225,539 or 5,639,641.

[0079] The “variable region” of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. The variable regions of both the heavy and light chains are each composed of four framework regions (FRs or FWs) connected by three complementarity-determining regions (CDRs) (also called hypervariable regions). The CDRs in each chain cluster very close together via FRs and promote the formation of the antigen-binding site of the antibody with CDRs from the other chain. At least two techniques are used to determine CDRs: (1) methods based on cross-species sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda Md.)); and (2) methods based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al., (1997) J. Molec. Biol. 273: 927-948). In addition, a combination of these two methods is sometimes used to determine CDRs.

[0080] When referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain), the Kabat numbering system is usually used (e.g., Kabat et al., Sequences of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0081] The amino acid position numbering in Kabat refers to the numbering system of the heavy chain variable domain or light chain variable domain used in the compilation of antibodies, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991). Using this numbering system, the actual linear amino acid sequence can contain fewer or more amino acids corresponding to a shortening or insertion of the FR or CDR of the variable domain. For example, the heavy chain variable domain can include an amino acid insertion after residue 52 of H2 (according to residue 52a in Kabat) and an insertion residue after residue 82 of the heavy chain FR (e.g., according to residues 82a, 82b, and 82c in Kabat). The Kabat number of a given antibody residue can be determined by comparison with the sequence homology region of the antibody using the “standard” Kabat numbering sequence. Instead, Chothia refers to the position of the structural loop (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). When numbered using the Kabat numbering convention, the end of the Chothia CDR-H1 loop varies between H32 and H34, depending on the loop length (this is because the Kabat numbering strategy places the insertion at H35A and H35B; if neither 35A nor 35B exists, the loop ends at 32; if only 35A exists, the loop ends at 33; if both 35A and 35B exist, the loop ends at 34). The AbM hypervariable region represents a compromise between the Kabat CDR and Chothia structural loops and is used by Oxford Molecular's AbM antibody modeling software. A comparison is provided in Table 1 below.

[0082] Table 1: Comparison of Antibody Numbering Systems

[0083]

[0084] The term "human antibody" means a natural human antibody or an antibody having an amino acid sequence corresponding to that of a natural human antibody, made using any technique known in the art. The definition of a human antibody includes full-length or complete antibodies, fragments thereof, and / or antibodies containing at least one human heavy chain and / or light chain polypeptide, such as antibodies containing mouse light chain and human heavy chain polypeptides.

[0085] The term "chimeric antibody" refers to an antibody in which the amino acid sequence of an immunoglobulin molecule originates from two or more species. Typically, the variable regions of both the light and heavy chains correspond to the variable regions of antibodies derived from a mammalian species (e.g., mouse, rat, rabbit, etc.) with desired specificity, affinity, and capability, while the constant regions are homologous to sequences derived from antibodies from another species (typically human) to avoid inducing an immune response in that species. Multispecific binding molecules, such as those comprising one or more antibody-binding domains, one or more non-antibody-binding domains, or combinations thereof, such as the multispecific binding molecules of TNFα antagonists and / or NGF antagonists described herein, may contain antibody constant regions (e.g., Fc regions), wherein at least a portion of one or more constant regions is deleted or modified to provide desired biochemical properties, such as improved tumor localization or reduced serum half-life compared to antibodies with substantially similar immunogenicity and containing native or unaltered constant regions. The modified constant regions described herein may include variations or modifications to one or more of the three heavy chain constant regions (CH1, CH2, or CH3) and / or to the light chain constant region (CL). In some aspects, one or more constant regions may be partially or completely deleted. In some aspects, the entire CH2 region may be deleted (ΔCH2 construct). 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.

[0086] The terms "epitope" or "antigenic determinant" are used interchangeably here and refer to the portion of an antigen that can be recognized and specifically bound by a particular antibody. When the antigen is a polypeptide, epitopes can form from both continuous and non-continuous amino acids juxtaposed through the ternary folding of the protein. Epitopes formed from continuous amino acids are generally preserved during protein denaturation, while epitopes formed through ternary folding are generally lost during protein denaturation. Epitopes typically contain at least three, more commonly at least five, or eight to ten amino acids in a distinctive spatial conformation. Epitopes, as described herein, do not need to be limited to the specific amino acids that form the epitope. In some respects, epitopes can be identified by a set of antigen-specific antibodies, by examining binding to peptide subunits of the polypeptide antigen, or by examining binding competition to the antigen.

[0087] The terms "subject," "individual," "animal," "patient," or "mammal" refer to any subject who requires diagnosis, prognosis, or treatment, especially mammalian subjects. Mammal subjects include humans, domestic animals, farm animals, sports animals, and zoo animals, including, for example, humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, and so on.

[0088] The terms "composition" and "pharmaceutical composition" refer to preparations that are in a form that allows the active ingredient to be biologically effective and do not contain any additional components that would have unacceptable toxicity to the subject to which they are administered. Such compositions may be sterile.

[0089] As used herein, the terms “effective amount” and “therapeutic effective amount” mean an amount of one or more therapeutic compositions that is effective in controlling pain in a subject. The term “pain control” and its grammatically equivalent expressions are used herein to describe any beneficial or desirable effect in a patient requiring pain control. For example, an effective amount of one or more therapeutic compositions described herein may, for example, prevent pain, maintain a tolerable level of pain, reduce pain, decrease pain, minimize pain, or eliminate pain in a subject.

[0090] The term “administration” as used herein means administering one or more of the therapeutic compositions described herein to a subject, such as a bifunctional peptide 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 separate therapeutic compositions, one comprising an NGF antagonist and the other comprising a TNFα antagonist. The term “co-administration” means administering two or more therapeutic compositions to a subject, such as one comprising an NGF antagonist and the other comprising a TNFα antagonist. As used herein, co-administration includes, but does not necessarily require, the simultaneous administration of the two or more therapeutic compositions to a subject. The two or more therapeutic compositions may be administered sequentially to the subject, for example, at intervals of 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, or 5 or more hours. The order and timing of co-administration as described herein may be fixed or may vary based on the judgment of a medical professional.

[0091] The terms "polynucleotide" and "nucleic acid" refer to polynucleotides composed of covalently linked nucleotide residues. Polynucleotides can be DNA, cDNA, RNA, single-stranded or double-stranded, vectors, plasmids, bacteriophages, or viruses.

[0092] The term "vector" refers to a construct that can deliver and express one or more target genes or sequences in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, granules or phage vectors, DNA or RNA expression vectors linked to a cationic condensing agent, DNA or RNA expression vectors encapsulated in liposomes, and some eukaryotic cells such as producer cells.

[0093] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably to refer to amino acid polymers of any length. These polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The term also covers amino acid polymers that are naturally modified or modified by intervention; for example, disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other operation or modification, such as conjugation with a labeled component. Also covered within this definition are, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids) and other modifications known in the art.

[0094] "Conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by another amino acid having a similar side chain. Families of amino acid residues with similar side chains are 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), nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, replacing phenylalanine with tyrosine is a conservative substitution. In some respects, the conservative substitutions in the sequences of the peptides and antibodies provided herein do not eliminate the binding or other functional activities of the peptides containing the amino acid sequences. Methods for identifying conserved substitutions of nucleotides and amino acids 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)).

[0095] Binding molecules containing NGF antagonist domain and TNFα antagonist domain

[0096] This invention provides a bifunctional peptide comprising an NGF antagonist domain and a TNFα antagonist domain. In some aspects, administration of an effective amount of the bifunctional peptide provided herein can more effectively control pain in subjects in need than using an equivalent amount of the NGF antagonist or TNFα antagonist alone. The bifunctional peptide provided herein can comprise the NGF antagonist domain and the TNFα antagonist domain in any order, structure, or conformation. Any suitable NGF antagonist or TNFα antagonist may be part of the bifunctional peptide provided herein. Exemplary NGF antagonists and TNFα antagonists are described in other parts of this document.

[0097] In some respects, an NGF antagonist is a non-antibody molecule or its binding domain that inhibits NGF activity, such as a soluble, TrkA NGF-binding fragment. In other respects, an NGF antagonist is an anti-NGF antibody or its antigen-binding fragment. A suitable anti-NGF antagonist (e.g., an antagonist antibody) can inhibit the binding of NGF to TrkA, to p75NRT, or both TrkA and p75NRT. In some respects, anti-NGF antagonists, such as antagonist antibodies or fragments thereof used in bifunctional molecules (e.g., multispecific binding molecules) provided herein, preferentially block the binding of NGF to TrkA relative to the binding of NGF to p75NRT.

[0098] Exemplary antibodies or fragments thereof used in the bifunctional polypeptides (e.g., multispecific binding molecules) described herein are available from U.S. Patent Application Publication No. 2008 / 0107658, which is incorporated herein by reference in its entirety. In some aspects, anti-NGF antibodies or fragments thereof bind to the same epitope as anti-NGF antibody MEDI-578, competitively inhibit NGF against MEDI-578, or bind to NGF with a greater affinity than MEDI-578. In some embodiments, anti-NGF antibodies or fragments thereof bind to human NGF and / or rat NGF with an affinity of 1, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, or 0.2 nM or less. For example, anti-NGF antibodies or fragments thereof can bind human NGF with an affinity of about 0.2–0.8, 0.2–0.7, 0.2–0.6 (0.2–0.6), 0.2–0.5 and / or 0.25–0.44 nM, and rat NGF with an affinity of about 0.2–0.9, 0.2–0.8 and / or 0.25–0.70 nM.

[0099] In some respects, the anti-NGF antibody or a fragment thereof is MEDI-578. MEDI-578 is disclosed as clone 1252A5 in U.S. Patent Application Publication No. 2008 / 0107658. In other respects, anti-NGF antibodies or fragments thereof include tanizumab (RN-624), a humanized anti-NGF monoclonal antibody (Pfizer; described in Kivitz et al., (2013) PAIN, 154, 9, 1603-161); fulranumab, a fully human anti-NGF monoclonal antibody (Amgen; described in Sanga et al., PAIN, Vol. 154, No. 10, October 2013, pp. 1910–1919); REGN475 / SAR164877, a fully human anti-NGF monoclonal antibody (Regeneron / Sanafi-Aventis); and ABT-110 (PG110), a humanized anti-NGF monoclonal antibody (Abbott Laboratories). Anti-NGF antibodies or fragments thereof contained in bifunctional peptides (such as the multispecific binding molecules provided herein) can be, for example, humanized, chimeric, primate-derived, or fully human.

[0100] In some aspects, anti-NGF antibodies or fragments thereof comprise antibody VH domains containing HCDR1, HCDR2, and HCDR3 domains of MEDI-578, variants of the MEDI-578 heavy chain CDRs having up to 1, 2, 3, 4, 5, or more amino acid substitutions (e.g., conserved amino acid substitutions). For example, an anti-NGF antibody or fragment thereof may comprise HCDR1 having the exact amino acid sequence of SEQ ID NO:4, or HCDR1 having the amino acid sequence of SEQ ID NO:4 substituted with one or more (e.g., 1, 2, 3, 4, 5, or more) amino acids. Similarly, an anti-NGF antibody or fragment thereof may comprise HCDR2 having the exact amino acid sequence of SEQ ID NO:5, or HCDR2 having the amino acid sequence of SEQ ID NO:5 substituted with one or more (e.g., 1, 2, 3, 4, 5, or more) amino acids. Similarly, anti-NGF antibodies or fragments thereof may comprise HCDR3 having the exact amino acid sequence of SEQ ID NO:6, or HCDR3 having an amino acid sequence of SEQ ID NO:6 substituted with one or more (e.g., 1, 2, 3, 4, 5, or more) amino acids. In some aspects, said HCDR3 may comprise the amino acid sequence SSRIBNSALISYYDMDV (SEQ ID NO:11) or SSRIBMISSLQPYYDMDV (SEQ ID NO:12).

[0101] In some aspects, the anti-NGF antibody or a fragment thereof comprises an antibody VL domain comprising the LCDR1, LCDR2, and LCDR3 domains of MEDI-578, a variant of the MEDI-578 light chain CDR having up to 1, 2, 3, 4, 5, or more amino acid substitutions (e.g., conserved amino acid substitutions). In some aspects, the anti-NGF antibody or a fragment thereof may comprise an LCDR1 having the exact amino acid sequence of SEQ ID NO:8, or an LCDR1 having an amino acid sequence of SEQ ID NO:8 substituted with one or more (e.g., 1, 2, 3, 4, 5, or more) amino acids. Similarly, the anti-NGF antibody or a fragment thereof may comprise an LCDR2 having the exact amino acid sequence of SEQ ID NO:9, or an LCDR2 having an amino acid sequence of SEQ ID NO:9 substituted with one or more (e.g., 1, 2, 3, 4, 5, or more) amino acids. Similarly, anti-NGF antibodies or fragments thereof may comprise LCDR3 having the exact amino acid sequence of SEQ ID NO:10, or LCDR3 having the amino acid sequence of SEQ ID NO:10 substituted with one or more (e.g., 1, 2, 3, 4, 5, or more) amino acids.

[0102] In some aspects, an anti-NGF antibody or a fragment thereof comprises an antibody VH domain, said antibody VH domain comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the VH amino acid sequence identical to the amino acid sequence of SEQ ID NO:3. In some aspects, an anti-NGF antibody or a fragment thereof comprises an antibody VH domain, said antibody VH domain comprising the VH amino acid sequence of SEQ ID NO:3.

[0103] In some aspects, an anti-NGF antibody or a fragment thereof comprises an antibody VL domain, said antibody VL domain comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the VL amino acid sequence identical to the amino acid sequence of SEQ ID NO:7. In some aspects, an anti-NGF antibody or a fragment thereof comprises an antibody VL domain, said antibody VL domain comprising the VL amino acid sequence of SEQ ID NO:7.

[0104] In some aspects, an anti-NGF antibody or a fragment thereof comprises an antibody VH domain, said antibody VH domain comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the VH amino acid sequence identical to the amino acid sequence of SEQ ID NO:94. In some aspects, an anti-NGF antibody or a fragment thereof comprises an antibody VH domain, said antibody VH domain comprising the VH amino acid sequence of SEQ ID NO:94.

[0105] In some aspects, an anti-NGF antibody or a fragment thereof comprises an antibody VL domain, said antibody VL domain comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the VL amino acid sequence identical to the amino acid sequence of SEQ ID NO:95. In some aspects, an anti-NGF antibody or a fragment thereof comprises an antibody VL domain, said antibody VL domain comprising the VL amino acid sequence of SEQ ID NO:95.

[0106] In some aspects, anti-NGF antibodies or fragments thereof comprise antibody VH domains comprising HCDR1, HCDR2, and HCDR3 domains of any one 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, and variants thereof having up to 1, 2, 3, 4, 5, or more amino acid substitutions (e.g., conserved amino acid substitutions).

[0107] In some aspects, anti-NGF antibodies or fragments thereof comprise antibody VL domains comprising LCDR1, LCDR2, and LCDR3 domains of any one 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, and variants thereof having up to 1, 2, 3, 4, 5, or more amino acid substitutions (e.g., conserved amino acid substitutions).

[0108] In some aspects, the anti-NGF antibody or a fragment thereof comprises an antibody VH domain, said antibody VH domain comprising at least 0%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the same VH amino acid sequence as any one 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 some aspects, the anti-NGF antibody or a fragment thereof comprises an antibody VH domain, said antibody VH domain comprising the VH amino acid sequence of any one 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.

[0109] In some aspects, the anti-NGF antibody or a fragment thereof comprises an antibody VL domain, said antibody VL domain comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the same VL amino acid sequence as any one 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 some aspects, the anti-NGF antibody or a fragment thereof comprises an antibody VL domain, said antibody VL domain comprising the VL amino acid sequence of any one 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.

[0110] In some aspects, anti-NGF antibodies or fragments thereof comprise antibody VH domains containing HCDR1, HCDR2, and HCDR3 domains of NGF-NG, variants of the NGF-NG heavy chain CDRs having up to 1, 2, 3, 4, 5, or more amino acid substitutions (e.g., conserved amino acid substitutions). For example, an anti-NGF antibody or fragment thereof may comprise HCDR1 having the exact amino acid sequence of SEQ ID NO:88, or HCDR1 having the amino acid sequence of SEQ ID NO:88 substituted with one or more (e.g., 1, 2, 3, 4, 5, or more) amino acids. Similarly, an anti-NGF antibody or fragment thereof may comprise HCDR2 having the exact amino acid sequence of SEQ ID NO:89, or HCDR2 having the amino acid sequence of SEQ ID NO:89 substituted with one or more (e.g., 1, 2, 3, 4, 5, or more) amino acids. Similarly, anti-NGF antibodies or fragments thereof may contain HCDR3 having the exact amino acid sequence of SEQ ID NO:90, or HCDR3 having the amino acid sequence of SEQ ID NO:90 substituted with one or more (e.g., 1, 2, 3, 4, 5, or more) amino acids.

[0111] In some aspects, an anti-NGF antibody or a fragment thereof comprises an antibody VL domain comprising LCDR1, LCDR2, and LCDR3 domains of NGF-NG, having a variant of the NGF-NG light chain CDR with up to 1, 2, 3, 4, 5, or more amino acid substitutions (e.g., conserved amino acid substitutions). In some aspects, an anti-NGF antibody or a fragment thereof may comprise an LCDR1 having the exact amino acid sequence of SEQ ID NO: 91, or an LCDR1 having the amino acid sequence of SEQ ID NO: 91 substituted with one or more (e.g., 1, 2, 3, 4, 5, or more) amino acids. Similarly, an anti-NGF antibody or a fragment thereof may comprise an LCDR2 having the exact amino acid sequence of SEQ ID NO: 92, or an LCDR2 having the amino acid sequence of SEQ ID NO: 92 substituted with one or more (e.g., 1, 2, 3, 4, 5, or more) amino acids. Similarly, anti-NGF antibodies or fragments thereof may comprise LCDR3 having the exact amino acid sequence of SEQ ID NO:93, or LCDR3 having an amino acid sequence of SEQ ID NO:93 substituted with one or more (e.g., 1, 2, 3, 4, 5, or more) amino acids.

[0112] In some aspects, an anti-NGF antibody or a fragment thereof comprises an antibody VH domain, said antibody VH domain comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the VH amino acid sequence identical to the amino acid sequence of SEQ ID NO:24. In some aspects, an anti-NGF antibody or a fragment thereof comprises an antibody VH domain, said antibody VH domain comprising the VH amino acid sequence of SEQ ID NO:24.

[0113] In some aspects, the anti-NGF antibody or a fragment thereof comprises an antibody VL domain, said antibody VL domain containing at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the VL amino acid sequence identical to the amino acid sequence of SEQ ID NO:26. In some aspects, the anti-NGF antibody or a fragment thereof comprises an antibody VL domain, said antibody VH domain containing the VL amino acid sequence of SEQ ID NO:26.

[0114] Multifunctional peptides, such as the multispecific binding molecule provided by this invention, may comprise a complete anti-NGF antibody, i.e., an antibody in the form of H2L2 comprising two complete heavy chains and two complete light chains. When the anti-NGF antibody is a complete antibody, 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 the N-terminus or C-terminus of one or more light chains of the anti-NGF antibody. Alternatively, the multifunctional peptide (i.e., the multispecific binding molecule provided by this invention) may comprise an antigen-binding fragment of the anti-NGF antibody. In some aspects, the anti-NGF antibody fragment may comprise any portion of the constant domain of the antibody or may comprise only the variable domain. Exemplary anti-NGF antibody fragments provided for inclusion in bifunctional peptides (e.g., multispecific binding molecules) include, but are not limited to, Fab fragments, Fab' fragments, F(ab)2 fragments, or single-chain Fv (scFv) fragments.

[0115] In some of the exemplary compositions provided herein, the anti-NGF antibody is an scFv fragment (e.g., the scFv fragment of MEDI-578) or an NGF-binding variant thereof. Anti-NGF scFv peptides may contain VH and VL domains in any order, either N-VH-VL-C or N-VL-VH-C. ScFv molecules are typically engineered to link the VH and VL domains via flexible linkers. 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 understood by those skilled in the art, scFv antibody fragments may have reduced stability relative to the variable domains present in standard Fab constructions. In some respects, scFv can be structurally stabilized by introducing stabilizing mutations or by introducing interchain disulfide bonds (e.g., SS-stabilized). However, stabilizing mutations and / or introduced interchain disulfide bonds are not necessary and are not present in some respects. scFv peptides can be stabilized using many methods recognized in the art.

[0116] Linkers can be used to connect domains / regions of the bifunctional peptides provided herein. Linkers can be used to connect NGF antagonist domains and TNFα antagonist domains of bifunctional molecules, and can also be used to interconnect variable heavy and light chains of scFv. Exemplary, non-limiting examples of linkers are peptide chains containing at least four residues. Multiple portions of such linkers can be flexible, hydrophilic, and have very few or no secondary structures of their own (linker portions or flexible linker portions). Linkers of at least four amino acids can be used to connect adjacent domains and / or regions after the bifunctional peptide molecule is assembled. Longer linkers can also be used. Thus, linkers can be 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, or 50 residues. Linkers can also be, for example, about 100-175 residues. When multiple parts of a bifunctional polypeptide molecule are interconnected using multiple linkers, the linkers can be the same or different (e.g., the same or different lengths and / or amino acid sequences).

[0117] Linkers in bifunctional peptide molecules facilitate the formation of desired structures. Linkers may contain (Gly-Ser) nThe linker contains residues (where n is at least 1, 2, and up to, for example, 3, 4, 5, 6, 10, 20, 50, 100, or more integers) and has some Glu or Lys residues dispersed throughout to increase solubility. Alternatively, some linkers do not contain any serine residues, for example, at the site of O-linking glycosylation of the linker. In some aspects, the linker may contain cysteine ​​residues, for example, if the dimerization of the linker is used to introduce the domain of the bifunctional polypeptide into its properly folded conformation. In some aspects, the bifunctional polypeptide may contain at least 1, 2, 3, 4, or more polypeptide linkers that link the domain of the polypeptide.

[0118] In some aspects, the peptide linker may comprise 1-50 residues, 1-25 residues, 25-50 residues, or 30-50 residues. In some aspects, the peptide linker may comprise a portion of an Fc module. For example, in some aspects, the peptide linker may comprise a portion of an immunoglobulin hinge domain of an IgG1, IgG2, IgG3, and / or IgG4 antibody or a variant thereof.

[0119] In some aspects, peptide linkers may comprise or consist of gly-ser linkers. As used herein, the term "gly-ser linker" refers to a peptide composed of glycine and serine residues. Exemplary gly-ser linkers comprise an amino acid sequence of the formula (Gly4Ser)n, where n is an integer from at least 1, 2 up to 3, 4, 5, 6, 10, 20, 50, 100 or more. In some aspects, peptide linkers may comprise at least a portion of a hinge domain (e.g., derived from IgG1, IgG2, IgG3, or IgG4 molecules) and a sequence of gly-ser amino acid residues (e.g., gly-ser linkers such as (Gly4Ser)n).

[0120] When a multifunctional polypeptide (e.g., a multispecific binding molecule) contains scFv, a flexible linker can connect the heavy and light chains of the scFv. This flexible linker typically does not contain a hinge portion, but is a gly-ser linker or other flexible linker. The length and amino acid sequence of the flexible linker that connects the domains of the scFv can be easily selected and optimized.

[0121] In some aspects, the multifunctional polypeptide (e.g., a multispecific binding molecule) may comprise an anti-NGF scFv fragment comprising, from the N-terminus to the C-terminus: VH, a 15-amino acid linker sequence (GGGGS)3, and VL. In some embodiments, the linker connecting VH and VL to scFv is a 20-amino acid linker sequence (GGGGS)4. In some aspects, the VH comprises the amino acid sequence of SEQ ID NO:3. In some aspects, the VL comprises the amino acid sequence of SEQ ID NO:7. In some embodiments, the VH comprises the amino acid sequence of any one of SEQ ID NOs: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, the VL comprises the amino acid sequence of any one 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 some aspects, the VH domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one 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. In some aspects, the VL domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: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.

[0122] In other respects, the stability of the polypeptide can be improved by adding interchain disulfide bonds between the VH and VL domains, which is done by modifying certain residues in the VH and VL domains 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, glycine residues at positions 100, 101, or 102 in the VL domain can be modified with cysteine ​​residues, and glycine residues at position 44 in the VH domain can be modified with cysteine ​​residues.

[0123] The multifunctional peptides (e.g., multispecific binding molecules) described herein may include a TNFα antagonist domain. In some respects, the TNFα antagonist domain can inhibit the binding of TNFα to the TNF receptor (TNFR) on the cell surface, thereby blocking TNF activity.

[0124] In some respects, the TNFα antagonist domain of the multifunctional peptide provided herein is an anti-TNFα antibody or an antigen-binding fragment thereof. In some respects, the anti-TNFα antibody is an antigen-binding fragment of infliximab, adalimumab, pegylated sertozumab, golimumab, or any of these antibodies.

[0125] In some aspects, an anti-TNFα antibody or a fragment thereof binds to the same epitope as any of the following, competitively inhibits any of the following, or binds to TNFα with, for example, a greater affinity for any of the following: an anti-TNFα antibody infliximab, adalimumab, pegylated cerutuzumab, or golimumab, or an antigen-binding fragment of any of these antibodies. In some aspects, the anti-TNFα antibody is an antigen-binding fragment of infliximab, adalimumab, pegylated cerutuzumab, or golimumab, or an antigen-binding fragment of any of these antibodies. The structure or sequence of these anti-TNFα antibodies can be readily obtained by those skilled in the art, and they can be incorporated into the multifunctional peptides (e.g., multispecific binding molecules) described herein without extensive experimental work. The anti-TNFα antibody or fragment thereof contained in the multifunctional peptide can be, for example, humanized, chimeric, primate-derived, or fully human.

[0126] In some respects, anti-TNFα antibodies or fragments thereof comprise antibody VH domains comprising HCDR1, HCDR2, and HCDR3 domains of infliximab, adalimumab, pegylated tertuzumab, or golimumab, or variants of the heavy chain CDR of infliximab, adalimumab, pegylated tertuzumab, or golimumab having up to 1, 2, 3, 4, 5, or more amino acid substitutions (e.g., conserved amino acid substitutions).

[0127] In some respects, anti-TNFα antibodies or fragments thereof comprise antibody VL domains comprising LCDR1, LCDR2, and LCDR3 domains of infliximab, adalimumab, pegylated tertuzumab, or golimumab, or variants of the light chain CDR of infliximab, adalimumab, pegylated tertuzumab, or golimumab having up to 1, 2, 3, 4, 5, or more amino acid substitutions (e.g., conserved amino acid substitutions).

[0128] In some aspects, the anti-TNFα antibody or a fragment thereof comprises an antibody VH domain, said antibody VH domain comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical VH amino acid sequences to those of infliximab, adalimumab, pegylated tertuzumab, or golimumab. In some aspects, the anti-TNFα antibody or a fragment thereof comprises an antibody VH domain, said antibody VH domain comprising the VH amino acid sequence of infliximab, adalimumab, pegylated tertuzumab, or golimumab.

[0129] In some aspects, the anti-TNFα antibody or a fragment thereof comprises an antibody VL domain, said antibody VL domain comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical VL amino acid sequences of infliximab, adalimumab, pegylated tertuzumab, or golimumab. In some aspects, the anti-TNFα antibody or a fragment thereof comprises an antibody VL domain, said antibody VL domain comprising the VL amino acid sequence of infliximab, adalimumab, pegylated tertuzumab, or golimumab.

[0130] In some aspects, the anti-TNFα antibody or a fragment thereof comprises an antibody VH domain, said antibody VH domain comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the same VH amino acid sequence as the amino acid sequence of SEQ ID NO:28. In some aspects, said anti-TNFα antibody or a fragment thereof comprises an antibody VH domain having the amino acid sequence of SEQ ID NO:28.

[0131] In some aspects, the anti-TNFα antibody or a fragment thereof comprises an antibody VL domain, said antibody VL domain comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the VL amino acid sequence identical to the amino acid sequence of SEQ ID NO:29. In some aspects, the anti-TNFα antibody or a fragment thereof comprises an antibody VL domain having the amino acid sequence of SEQ ID NO:29.

[0132] The multifunctional polypeptide (e.g., a multispecific binding molecule) provided by this invention can comprise a complete anti-TNFα antibody, i.e., an antibody in the form of H2L2 comprising two complete heavy chains and two complete light chains. When the anti-TNFα antibody is a complete antibody, one or more NGF antagonist domains can be fused to the N-terminus or C-terminus of one or more heavy chains of the anti-TNFα antibody or the N-terminus or C-terminus of one or more light chains of the anti-TNFα antibody. Alternatively, the multifunctional polypeptide (e.g., a multispecific binding molecule) provided by this invention can comprise an antigen-binding fragment of an anti-TNFα antibody. In some aspects, the anti-TNFα antibody fragment can comprise any portion of the constant domain of the antibody or may comprise only the variable domain. Exemplary anti-TNFα antibody fragments for inclusion within the multifunctional polypeptide include, but are not limited to, Fab fragments, Fab' fragments, F(ab)2 fragments, or single-chain Fv (scFv) fragments.

[0133] In some aspects, the multifunctional molecule is ndimab varB, which is a molecule comprising a complete anti-TNFα antibody (i.e., an antibody in the form of H2L2 comprising two complete heavy chains and two complete light chains) and MEDI-578scFv fused to the C-terminus of the heavy chain of the anti-TNFα antibody. Ndimab varB comprises a light chain and a heavy chain, the light chain comprising the amino acid sequence of SEQ ID NO:20, and the heavy chain comprising the amino acid sequence of SEQ ID NO:22. In some aspects, the bifunctional molecule comprises a light chain and a heavy chain, the light chain comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence of SEQ ID NO:20, and the heavy chain comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence of SEQ ID NO:22.

[0134] In some respects, anti-TNFα antibodies are scFv fragments, such as those derived from infliximab, adalimumab, pegylated sertozumab, or golimumab, or their TNFα-binding variants. Anti-TNFα scFv peptides can contain VH and VL domains in any order, whether N-VH-VL-C or N-VL-VH-C. ScFv molecules are often engineered to link VH and VL via flexible linkers and can assume several different structures, as described above. Anti-TNFα scFv peptides can be stabilized, also as described above.

[0135] In some aspects, the TNFα antagonist is a soluble fragment of a TNF receptor (e.g., TNFR-1 or TNFR-2) that binds to TNFα, or a variant thereof, or a soluble fragment thereof. In some aspects, the soluble fragment of TNFR-1 is a 55 kD fragment. In some embodiments, the soluble fragment of TNFR-2 is a 75 kD fragment. In some aspects, the TNF receptor fragment is fused to a heterologous polypeptide, such as an immunoglobulin Fc fragment, such as the IgG1 Fc domain. In some aspects, the TNFα antagonist comprises the amino acids shown in SEQ ID NO:13, or a TNFα-binding fragment thereof. The TNFR-2 portion comprises amino acids 1 to 235 of SEQ ID NO:13. In some aspects, variants of the soluble fragment of TNFR-2 that binds to TNFα comprise at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences to amino acids 1 to 235 of SEQ ID NO:13. In some aspects, variants of the TNFR-2 TNFα-binding soluble fragment comprise amino acids 1 to 235 of SEQ ID NO:13, except for insertions, substitutions, or deletions of, for example, 1, 2, 3, 4, 5, 10, 20, 20, 40, or 50 amino acids. The IgG1 Fc portion comprises amino acids 236 to 467 of SEQ ID NO:13. In some aspects, the TNFR-2 TNFα-binding soluble fragment may be fused to the Fc portion of any human or non-human antibody, or to any other protein or non-protein substance providing stability, such as albumin or polyethylene glycol. In some aspects, the TNFR-2 TNFα-binding soluble fragment comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to amino acids 236 to 467 of SEQ ID NO:13. In some aspects, variants of the TNFR-2 soluble fragment binding to TNFα comprise amino acids 236 to 467 of SEQ ID NO:13, except for insertions, substitutions, or deletions of, for example, 1, 2, 3, 4, 5, 10, 20, 20, 40, or 50 amino acids. In some aspects, variants of the TNFR-2 soluble fragment binding to TNFα comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:13. In some aspects, variants of the TNFR-2 soluble fragment binding to TNFα comprise SEQ ID NO:13, except for insertions, substitutions, or deletions of, for example, 1, 2, 3, 4, 5, 10, 20, 20, 40, or 50 amino acids.

[0136] In some respects, the soluble fragment of TNFR-2 that binds to TNFα is a single-chain fusion protein. In other respects, the soluble fragment of TNFR-2 that binds to TNFα is a dimer of two fusion proteins linked by, for example, a disulfide bond between two Fc domains.

[0137] The multifunctional peptides (e.g., multispecific binding molecules) provided herein can have a variety of different structures and conformations. In one aspect, the multifunctional peptides provided herein comprise fusion proteins, wherein the NGF antagonist domain, as described above, is fused to the TNFα antagonist domain, as described above, via a flexible linker. Examples of linkers have been described elsewhere in this document. In some aspects, the multifunctional peptides comprise homodimers of fusion proteins.

[0138] In one exemplary aspect, a multifunctional polypeptide is provided, wherein the NGF antagonist is an anti-NGF scFv domain derived from MEDI-578 and the TNFα antagonist is a soluble, TNFα-binding fragment of TNFR-2, said fragment being fused at its carboxyl terminus to an immunoglobulin Fc domain. In some aspects, the anti-NGF scFv may be fused to the carboxyl terminus of the immunoglobulin Fc domain via a linker. In some aspects, the monomer of such a multifunctional polypeptide forms a homodimer, wherein each subunit, from the N-terminus to the C-terminus, comprises: a 75 kD TNFR-2 TNFα-binding fragment, a human IgG1 Fc domain, a 10-amino acid linker (GGGGS)2 (SEQ ID NO:98), an anti-NGF VH comprising the amino acid sequence of SEQ ID NO:3, a 15-amino acid linker (GGGGS)3 (SEQ ID NO:15), and an anti-NGF VL comprising the amino acid sequence of SEQ ID NO:7. In one aspect, the multifunctional polypeptide is TNFR2-Fc_VH#4, comprising a homodimer of a fusion polypeptide containing the amino acid sequence of SEQ ID NO:14. In some aspects, the multifunctional polypeptide comprises a homodimer of a fusion polypeptide containing at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:14.

[0139] In another exemplary aspect, the multifunctional polypeptide comprises, from its N-terminus to its C-terminus: a 75 kD fragment of TNFR-2 binding to TNFα, a human IgG1 Fc domain, a 10-amino acid linker (GGGGS)2 (SEQ ID NO: 98), an anti-NGF VH comprising 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 VL comprising the amino acid sequence of SEQ ID NO: 95. In some aspects, the multifunctional polypeptide is TNFR2-Fc_varB comprising a homodimer of a fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 17. In some aspects, the multifunctional polypeptide comprises a homodimer of a fusion polypeptide comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO: 17.

[0140] polypeptide

[0141] The peptides containing both an NGF antagonist domain and a TNFα antagonist domain (e.g., multifunctional peptides) provided herein, or individual peptides containing either an NGF antagonist domain or a TNFα antagonist domain, may be recombinant peptides, naturally derived peptides, or synthetic peptides. It will be understood in the art that some amino acid sequences can be varied without significantly affecting the structure or function of the protein. Therefore, the present invention also provides variations of the peptides described herein that have substantially the same activity or contain both an NGF antagonist domain and a TNFα antagonist domain. Such mutants include deletions, insertions, inversions, duplications, and type substitutions.

[0142] The peptides and analogues can be further modified to include additional chemical modules that are not part of the conventional protein moiety. These derived modules can improve solubility, biological half-life, or absorption of the protein. The modules can also reduce or eliminate any undesirable side effects of the protein, etc. A review of these modules can be found in REMINGTON'S PHARMACEUTICAL SCIENCES, 20th edition, Mack Publishing Co., Easton, PA (2000).

[0143] In some respects, the multifunctional peptides presented herein may contain non-antibody NGF or TNFα binding domains. Various methods are known in the art for identifying and producing non-antibody peptides that bind to protein targets with high affinity. 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 reference in its entirety. In some respects, phage display technology can be used to identify / produce suitable multifunctional peptides. In some respects, the multifunctional peptides (e.g., multispecific binding molecules) provided herein may contain a protein backbone selected from the group consisting of: protein A, lipocalin, fribronectin domain, ankyrin consensus repeat domain, and thioredoxin.

[0144] Polynucleotides, vectors, and host cells

[0145] This invention provides nucleic acid molecules comprising polynucleotides encoding multifunctional polypeptides comprising an NGF antagonist domain and a TNFα antagonist domain. This invention also provides nucleic acid molecules comprising polynucleotides encoding separate polypeptides comprising an NGF antagonist and a TNFα antagonist, respectively. In some aspects, such polynucleotides encode peptide domains that specifically bind to NGF or fragments thereof and also bind to TNFα or fragments thereof. For example, this invention provides polynucleotides encoding polypeptide domains comprising anti-NGF antibodies or antigen-binding fragments thereof, and polypeptide domains comprising soluble, TNFα-binding moieties of TNFα antagonists (such as anti-TNFα antibodies or antigen-binding fragments thereof) or TNF receptors (e.g., TNFR2). Polynucleotides may be in RNA or DNA form. DNA includes cDNA, genomic DNA, and synthetic DNA; and may be double-stranded or single-stranded, and if single-stranded, may be a coding strand or a non-coding (antisense) strand.

[0146] In some embodiments, the isolated polynucleotide encoding the multifunctional polypeptide described herein comprises the nucleotide sequence of SEQ ID NO:16, 18 or 99 or a fragment thereof, or a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:16, 18 or 99 or a fragment thereof.

[0147] The isolated polypeptides described herein can be produced by any suitable method known in the art. Such methods range from direct protein synthesis to constructing DNA sequences encoding the isolated polypeptide sequences and expressing these sequences in suitable transformation hosts. In some aspects, DNA sequences are constructed using recombinant techniques by isolating or synthesizing DNA sequences that encode multifunctional polypeptides containing both an NGF antagonist domain and a TNFα antagonist domain, or single polypeptides each containing both an NGF antagonist domain and a TNFα antagonist domain. Therefore, the present invention provides isolated polynucleotides encoding bifunctional polypeptides containing both an NGF antagonist domain and a TNFα antagonist domain as detailed above. Isolated polynucleotides encoding single polypeptides each containing both an NGF antagonist domain and a TNFα antagonist domain are also provided.

[0148] In some respects, DNA sequences encoding a target multifunctional polypeptide (e.g., a multispecific binding molecule) or individual polypeptides containing NGF antagonist and TNFα antagonist domains, respectively, can be constructed chemically using an oligonucleotide synthesis instrument. Such oligonucleotides can be designed based on the amino acid sequence of the desired multifunctional polypeptide, and the selection of these codons is advantageous in the host cell from which the target recombinant protein is produced. Isolated polypeptide sequences encoding the target multifunctional polypeptide can be synthesized using standard methods. For example, a back-translated gene can be constructed using the complete amino acid sequence. Furthermore, DNA oligomers containing nucleotide sequences encoding a specific multifunctional polypeptide or individual polypeptide can be synthesized. For example, small oligonucleotides encoding portions of the desired polypeptide can be synthesized and then ligated. Individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.

[0149] In some respects, the polynucleotides provided herein can contain the coding sequence of a mature polypeptide fused into the same reading frame as the marker sequence, thereby allowing, for example, purification of the encoded polypeptide. For example, the marker sequence could be a 6-histidine tag provided by the pQE-9 vector to provide purification of the mature polypeptide fused to the marker (for bacterial hosts), or the marker sequence could be a hemagglutinin (HA) tag derived from influenza hemagglutinin protein (when using a mammalian host (e.g., COS-7 cells)).

[0150] The polynucleotides described in this article also contain variations in coding regions, non-coding regions, or both. In some respects, polynucleotide variants contain variations that produce silent substitutions, additions, or deletions without altering the activity of the encoded polypeptide. In other respects, nucleotide variants arise through silent substitutions caused by genetic code degeneracy. Polynucleotide variants can arise for a variety of reasons, such as to optimize codon expression for a specific host (changing codons in human mRNA to codons preferred by bacterial hosts such as E. coli).

[0151] Vectors and cells containing the polynucleotides described herein are also provided. Once assembled (by synthesis, directed mutagenesis, or other methods), a polynucleotide sequence encoding a specific target isolated polypeptide can be inserted into the expression vector and operatively ligated to an expression control sequence suitable for protein expression in the desired host. This invention provides such vectors. Nucleotide sequencing, restriction enzyme mapping, and expression of the bioactive polypeptide in a suitable host confirm appropriate assembly. As is well known in the art, in order to obtain high expression levels of a transfected gene in a host, the gene must be operatively ligated to transcriptional and translational expression control sequences that function in the selected expression host.

[0152] In some respects, expression vectors exist that can be used to amplify and express DNA encoding a multifunctional polypeptide (e.g., a multispecific binding molecule) or a single polypeptide, wherein the multifunctional polypeptide comprises an NGF antagonist domain and a TNFα antagonist domain, and the single polypeptide comprises an NGF antagonist domain and a TNFα antagonist domain, respectively. Recombinant expression vectors are reproducible DNA constructs having a synthetic or cDNA-derived DNA fragment encoding a multifunctional polypeptide or a single polypeptide comprising an NGF antagonist domain and a TNFα antagonist domain, respectively, operatively linked to a suitable transcriptional or translational regulatory element from a mammalian, microbial, viral, or insect gene. Transcriptional units typically comprise an assembly of: (1) one or more genetic elements that play a regulatory role in gene expression, such as a transcription promoter or enhancer; (2) a structural or coding sequence transcribed into mRNA and translated into a protein; and (3) appropriate transcriptional and translational initiation and termination sequences, as detailed below. Such regulatory elements may include operator sequences to control transcription. The ability to replicate in the host is typically conferred by the origin of replication, and selection genes that facilitate the recognition of transformants can be additionally integrated. DNA regions can be operatively linked when they are functionally interconnected. For example, DNA for a signal peptide (secretion guide) can be operatively linked to DNA of a polypeptide if it is expressed as a precursor involved in the secretion of that polypeptide; a promoter can be operatively linked to a coding sequence if it controls the transcription of that sequence; or a ribosome-binding site can be operatively linked to a coding sequence if its location allows translation. Structural elements intended for use in yeast expression systems include guide sequences that allow the translated protein to be secreted extracellularly by the host cell. Alternatively, when a recombinant protein is expressed without a guide or transport sequence, it may contain an N-terminal methionine residue. This residue can then optionally be cleaved from the expressed recombinant protein to provide the final product.

[0153] The choice of expression control sequences and expression vectors depends on the choice of host. Many expression host / vector combinations are available. Expression vectors useful for eukaryotic hosts include, for example, vectors containing expression control sequences from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Expression vectors useful for bacterial hosts include known bacterial plasmids, such as those from *E. coli*, including pCR1, pBR322, pMB9, and their derivatives, as well as plasmids with a broader host range, such as M13, and filamentous single-stranded DNA bacteriophages.

[0154] This invention also provides host cells comprising polynucleotides encoding the polypeptides described herein. Suitable host cells for expressing the polypeptides described herein include prokaryotes, yeast, insects, or higher eukaryotic cells under the control of a suitable promoter. Prokaryotes include Gram-negative or Gram-positive organisms, such as *Escherichia coli* or bacilli. Higher eukaryotic cells include established mammalian-derived cell lines as described below. Cell-free translation systems may also be used. Cloning and expression vectors suitable for use with bacterial, fungal, yeast, and mammalian cell hosts are described in Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, NY, 1985), the relevant disclosure of which is incorporated herein by reference. Other methods for protein production (including antibody production) can be found, for example, in U.S. Patent Publication No. 2008 / 0187954, U.S. Patents Nos. 6,413,746 and 6,660,501, and International Patent Publication No. WO04009823, each of which is incorporated herein by reference in its entirety.

[0155] Recombinant proteins can also be advantageously expressed using a variety of mammalian or insect cell culture systems. Expression of recombinant proteins in mammalian cells is possible because these proteins are typically correctly folded, appropriately modified, and fully functional. Examples of suitable mammalian host cell lines include HEK-293 and HEK-293T, the COS-7 line of monkey kidney cells (described in Gluzman (Cell 23:175, 1981)), and other cell lines, including, for example, L cells, C127, 3T3, Chinese hamster ovary cells (CHO), HeLa, and BHK cell lines. Mammalian expression vectors can contain non-transcriptional elements, such as a replication origin linked to the gene to be expressed, suitable promoters and enhancers, and other 5' or 3' flanking non-transcriptional sequences, as well as 5' or 3' non-translational sequences, such as essential ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, and transcription termination sequences. Baculovirus systems for producing heterologous proteins in insect cells were reviewed in Luckow and Summers, Bio / Technology 6:47 (1988).

[0156] This invention also provides methods for producing multifunctional peptides as described herein, or methods for producing individual peptides comprising either an NGF antagonist or a TNFα antagonist. The methods involve culturing host cells as described above under conditions that promote the expression of the multifunctional peptide or the individual peptide, and then recovering the multifunctional peptide or the individual peptide.

[0157] For long-term, high-yield recombinant protein production, stable expression is suitable. For example, cell lines stably expressing multifunctional peptides can be engineered. Host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and selection markers, instead of using expression vectors containing viral origins of replication. After introducing the foreign DNA, engineered bacteria can be allowed to grow in enrichment media for 1-2 days before being transferred to selective media. Selection markers in the recombinant plasmid confer selective resistance and allow cells to stably integrate the plasmid into their chromosomes and grow to form colonies (foci), which can then be cloned and expanded into cell lines. This method can be used to engineer cell lines expressing multifunctional peptides.

[0158] In some embodiments, the multifunctional polypeptides provided herein are expressed in cell lines as transient expression of the multifunctional polypeptide. Transient transfection is a process in which the nucleic acid introduced into the cell does not integrate into the cell's genomic or chromosomal DNA but remains as an extrachromosomal element in the cell, such as an episome. The transcription of the episome's nucleic acid is unaffected and produces a protein encoded by the episome's nucleic acid.

[0159] Cell lines (stable or transiently transfected) are maintained in cell culture media and in conditions known in the art that lead to peptide expression and production. In some embodiments, mammalian cell culture media are based on commercially available culture medium formulations, including, for example, DMEM or Ham's F12. In some embodiments, cell culture media are modified to support cell growth and increased expression of biological proteins. As used herein, the terms “cell culture medium,” “culture medium,” and “culture medium formulation” mean a nutrient solution used for the maintenance, growth, propagation, or expansion of cells in an artificial or in vitro environment outside of a multicellular organism or tissue. Cell culture media can be optimized for specific cell culture applications, including, but not limited to, cell culture growth media (formulated to promote cell growth) or cell culture production media (formulated to promote recombinant protein production). The terms nutrient, component, and part are used interchangeably to refer to the components that make up the cell culture medium.

[0160] In various implementations, a fed-batch method is used to maintain cell lines. As used herein, "fed-batch method" means a method by which additional nutrients are supplied to fed-batch cell cultures after initial incubation with a base medium. For example, a fed-batch method may include adding supplemental culture medium over a given period of time according to a defined feeding protocol. Thus, "fed-batch cell culture" means a cell culture in which cells (typically mammalian cells) and culture medium are initially supplied to a culture vessel and additional culture nutrients are continuously or discretely incrementally fed to the culture during culture, with or without periodic cell and / or product harvesting prior to culture termination.

[0161] In some embodiments, the cell culture medium comprises a base medium and at least one hydrolysate, such as a soybean-based hydrolysate, a yeast-based hydrolysate, or a combination of both types of hydrolysates, which produces a modified base medium. Additional nutrients may sometimes contain only the base medium, such as a concentrated base medium, or may contain only the hydrolysate, such as a concentrated hydrolysate. Suitable substrate media include, but are not limited to, Dulbecco's Modified Eagle's Medium (DMEM), DME / F12, Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, .alpha.-Minimum Essential Medium (.alpha.-MEM), Glasgow's Minimal Essential Medium (G-MEM), PF CHO (see, for example, CHO protein-free medium (Sigma) or EX-CELL.TM.325PF CHO serum-free medium for CHO cells protein-free (SAFC Bioscience), and Iscove's Modified Dulbecco's Medium. Other examples of substrate media that can be used in the techniques described herein include BME substrate medium (Gibco-Invitrogen; Dulbecco's Modified Eagle's Medium (DMEM, powder) (Gibco-Invitrogen (#31600)).

[0162] In some embodiments, the substrate culture medium may be serum-free (meaning the culture medium does not contain serum (e.g., fetal bovine serum (FBS), horse serum, goat serum, or any other animal-derived serum known to those skilled in the art)) or animal protein-free, or a culture medium with a defined chemical composition.

[0163] The basal culture medium can be modified to remove some non-nutritive components found in standard basal culture media, such as various inorganic or organic buffers, surfactants, and sodium chloride. Removing these components from the basal culture medium allows for an increase in the concentration of remaining nutrients and may improve overall cell growth and protein expression. Furthermore, the omitted components can be added back to the cell culture medium containing the modified basal culture medium, depending on the requirements of the cell culture conditions. In some embodiments, the cell culture medium contains a modified basal culture medium and at least one of the following nutrients: an iron source, recombinant growth factors; buffers; surfactants; osmotic regulators; an energy source; and non-animal hydrolysates. Additionally, the modified basal cell culture medium may optionally contain amino acids, vitamins, or a combination of both. In some embodiments, the modified medium also contains glutamine, such as L-glutamine and / or methotrexate.

[0164] In some embodiments, large-scale protein production is carried out via bioreactor processes using fed-batch, perfusion, or continuous fed bioreactor methods known in the art. Large-scale bioreactors have a volume of at least 50 L, sometimes exceeding 500 L or 1,000 to 100,000 L. These bioreactors use agitated impellers to distribute oxygen and nutrients. Small-scale bioreactors generally refer to cell culture in a volume not exceeding about 100 L, and the solvent range can be from about 1 L to about 100 L. Alternatively, single-use bioreactors (SUBs) can be used for both large-scale and small-scale cultures.

[0165] Temperature, pH, agitation, aeration, and inoculation density can vary depending on the host cells used and the recombinant protein to be expressed. For example, recombinant protein cultures can be maintained at temperatures between 30 and 45 degrees Celsius. The pH of the culture medium can be monitored during the culture process to maintain it at an optimal level, which may be in the range of 6.0 to 8.0 for some host cells. Impeller-driven mixing can be used for agitation in such culture methods. The impeller rotation speed can be approximately 50 to 200 cm / sec tip speed, but other known airlift or other mixing / aeration systems can also be used, depending on the type of host cells being cultured. Adequate aeration is provided to maintain a dissolved oxygen concentration of approximately 20% to 80% air saturation in the culture, again depending on the chosen host cells. Alternatively, the bioreactor can inject gas or oxygen directly into the culture medium. Other oxygen supply methods exist, including bubble-free aeration systems using hollow fiber membrane aeration devices.

[0166] Protein purification

[0167] Proteins produced by the host transformed as described above can be purified using any suitable method. Such standard methods include chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or any other standard technique used for protein purification. Affinity tags (such as hexahistidine, maltose-binding domains, influenza coat sequences, and glutathione S-transferases) can be attached to the protein to allow for easy purification by passing it through a suitable affinity column. The isolated protein can also be physically characterized using techniques such as proteolysis, nuclear magnetic resonance, and X-ray crystallography.

[0168] For example, the supernatant from a system that secretes recombinant proteins into a culture medium can be concentrated using a commercially available protein concentrator, such as an Amicon or Millipore Pellicon ultrafiltration unit. After the concentration step, the concentrate is coated onto a suitable purification matrix. Alternatively, anion exchange resins, which are matrix or substrate materials with pendant diethylaminoethyl (DEAE) groups, can be used. The matrix can be acrylamide, agarose, dextran, cellulose, or other types commonly used in protein purification. Suitable cation exchangers include a variety of insoluble matrices, including sulfopropyl or carboxymethyl. Finally, the NGF binder can be further purified using one or more reversed-phase high-performance liquid chromatography (RP-HPLC) steps employing a hydrophobic RP-HPLC medium, such as a silica gel with pendant methyl or other aliphatic groups. Some or all of the aforementioned purification steps can also be used in various combinations to provide homogeneous recombinant proteins.

[0169] Recombinant proteins produced in bacterial cultures can be isolated, for example, by first extracting from the cell precipitate, followed by one or more concentration, salting out, aqueous ion exchange, or size exclusion chromatography steps. High-performance liquid chromatography (HPLC) can be used for the final purification step. The microbial cells used in recombinant protein expression can be disrupted by any convenient method, including repeated freeze-thaw cycles, mechanical disruption, or the use of cell lysis agents.

[0170] Methods known in the art for purifying recombinant peptides include, for example, those described in U.S. Patent Publications Nos. 2008 / 0312425, 2008 / 0177048, and 2009 / 0187005, each of which is incorporated herein by reference in its entirety.

[0171] Method of use and pharmaceutical composition

[0172] This invention provides a method for controlling or treating pain in a subject, comprising administering a therapeutically effective amount of a multifunctional polypeptide (e.g., a multispecific binding molecule) of TNFα and NGF antagonists, as provided herein, or comprising co-administering a TNFα antagonist and an NGF antagonist. In some aspects, the subject is a human being.

[0173] The present invention also provides pharmaceutical compositions comprising multifunctional polypeptides (e.g., multispecific binding molecules) containing TNFα and NGF antagonists as provided herein, or pharmaceutical compositions comprising combinations of TNFα and NGF antagonists as provided herein. In some aspects, the pharmaceutical compositions further comprise a pharmaceutically acceptable carrier. These pharmaceutical compositions are useful for treating pain (e.g., neuropathic pain and inflammatory pain (e.g., osteoarthritis or rheumatoid arthritis)).

[0174] The multifunctional peptides and compositions containing NGF antagonists and TNFα antagonists described herein can be used for a variety of applications, including but not limited to controlling or treating pain, such as neuropathic pain. The methods used can be in vitro, ex vivo, or in vivo.

[0175] In some respects, diseases, conditions, or illnesses treated with NGF binders (such as antibodies or peptides) are associated with pain. In some respects, the pain is associated with chronic nociceptive pain, chronic lower back pain, neuropathic pain, cancer pain, herpes zoster neuralgia (PHN) pain, or visceral pain conditions.

[0176] The present invention provides a method for controlling pain in a subject, comprising administering an effective amount of a nerve growth factor (NGF) antagonist and a tumor necrosis factor (TNFα) antagonist to a subject requiring pain control, wherein the administration controls pain in the subject more effectively than administering an equal amount of the NGF antagonist or TNFα antagonist alone.

[0177] In some respects, the administration is the co-administration of an NGF antagonist and a TNFα antagonist as a combination therapy. As discussed elsewhere in this document, multiple individual components may be administered simultaneously or sequentially. The individual NGF antagonist or TNFα antagonist may be any NGF or TNFα antagonist provided herein, such as a soluble TrkA receptor fragment that binds to NGF, an anti-NGF antibody or its antigen-binding fragment thereof, a soluble TNFα-binding fragment of a TNF receptor (e.g., TNFR-2), or an anti-TNFα antibody or its fragment (e.g., infliximab, adalimumab, pegylated sertuzumab, golimumab, or an antigen-binding fragment of any of these antibodies).

[0178] Co-administration can include multiple doses of various antagonists required to control pain. In some respects, co-administration can include various components at lower doses or less frequent doses than those routinely administered as individual treatments, thereby providing greater safety, convenience, and cost-effectiveness.

[0179] In some respects, the methods for controlling pain provided herein include administering a multifunctional peptide (e.g., a multispecific binding molecule) comprising an NGF antagonist domain and a TNFα antagonist domain. Exemplary multifunctional peptides used in this method are described in detail herein. Based on this disclosure, other multifunctional peptides that can be used in this method will be apparent to those skilled in the art.

[0180] "More effective" at controlling pain than the components administered alone" means that combination therapy is more effective at controlling pain than the individual administration of an equivalent amount of an NGF antagonist or TNFα antagonist. In some respects, and as further detailed below, the methods for controlling pain provided herein can provide synergistic efficacy; for example, the combined effect of administering the NGF antagonist and TNFα antagonist may be greater than the additive effect, or the combined administration of the two may be effective when neither NGF antagonist nor TNFα antagonist is effective alone. In some respects, the combination can allow for dose savings; for example, when administered together, the effective dose of each component may be lower than the effective dose of either component administered alone.

[0181] In some respects, the pain control methods provided herein are more effective in controlling pain in subjects than the administration of equal amounts of NGF antagonists or TNFα antagonists alone, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In some respects, the dose of individual NGF antagonists or TNFα antagonists co-administered to the subject, or the relative dose of NGF antagonists or TNFα antagonists provided after administration of the bifunctional peptides provided herein, may be, for example, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% lower than the dose required to administer the components alone.

[0182] The effectiveness of pain control can be measured by questioning patients to assess the quality and intensity of their pain experience on different scales. The verbal pain scale uses words to describe a range from no pain, mild pain, moderate pain, and severe pain, each assigned a score from 0 to 3. Alternatively, patients can be asked to rate their pain on a numerical pain scale from 0 (no pain) to 10 (maximum possible pain). On the visual analog scale (VAS), vertical or horizontal lines have words describing pain from no pain to maximum possible pain, and patients are asked to mark these lines at points representing their current pain level. The McGill Pain Index allows patients to describe both the quality and intensity of their pain by selecting the best words to describe it from a short candidate list, such as thud, burning, pinching. Other pain scales can be used for adults who experience pain with difficulty, using VAS or numerical scales such as FACES, or for non-verbal patients, such as behavioral rating scales. The functional activity scale (FIS) score assesses how much pain interferes with a patient's experience, and is assessed by asking them to perform a task related to the area of ​​pain. Improvements in pain scores using these types of scales can potentially indicate improvements in analgesic efficacy.

[0183] According to the methods for controlling pain provided herein, the administration is sufficient to control pain in subjects requiring pain control, for example, by co-administering an NGF antagonist and a TNFα antagonist, or by administering a multifunctional polypeptide (e.g., a multispecific binding molecule) containing both an NGF antagonist domain and a TNFα antagonist domain, to prevent, reduce, alleviate, or eliminate pain in the subject. In some aspects, the pain can be acute pain, short-term pain, persistent or chronic nociceptive pain, or persistent or chronic neuropathic pain.

[0184] In some respects, formulations for storage and use have been prepared by combining a multifunctional polypeptide (e.g., a multispecific binding molecule) of TNFα and NGF antagonists as described herein, or a combination of TNFα and NGF antagonists as described herein, with a pharmaceutically acceptable carrier (e.g., a loading agent, an excipient) (Remington, The Science and Practice of Pharmacy, 20th edition, Mack Publishing, 2000). Suitable pharmaceutically acceptable carriers include, but are not limited to, non-toxic buffers such as phosphoric acid, citric acid, and other organic acids; salts such as sodium chloride; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzyl chloride; phenol, butyl or benzyl alcohol; alkyl p-hydroxybenzoates). Parabens, such as methyl or propyl parabens; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol; low molecular weight peptides (e.g., 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 dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and nonionic surfactants such as Tween or polyethylene glycol (PEG).

[0185] The multifunctional polypeptides of the present invention can be formulated as liquids, semi-solids, or solids, depending on the physicochemical properties of the molecules and delivery pathways. Formulations may contain excipients, or combinations of excipients, such as sugars, amino acids, and surfactants. Liquid formulations may contain a variety of polypeptide concentrations and pH values. Solid formulations can be produced, for example, by freeze-drying, spray-drying, or drying using supercritical fluid technology. In some embodiments, any formulation described herein is a freeze-dried formulation.

[0186] In one specific embodiment, the multifunctional polypeptide of the present 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.

[0187] The pharmaceutical compositions described herein can be administered in any quantity and method for local or systemic treatment. Administration can be local (e.g., applied to mucous membranes, including vaginal and rectal delivery) such as transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, or powders; pulmonary (e.g., by inhalation or blowing of powders or aerosols, including via nebulizers; intratracheal, intranasal, epidermal, and transdermal); oral; or parenteral, including intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; or intracranial (e.g., intrathecal or intraventricular) administration.

[0188] The TNFα and NGF antagonist multifunctional peptides or the combination of TNFα antagonists and NGF antagonists provided in this article can be further combined with a second (or third) compound having anti-nociceptive properties in drug formulations or dosing regimens as combination therapy.

[0189] For treatment of pain, the appropriate dosage of the TNFα and NGF antagonist multifunctional peptides (e.g., multispecific binding molecules) or combinations of TNFα and NGF antagonists described herein depends on the type of pain to be treated, the severity and etiology of the pain, the responsiveness of the pain, whether the multifunctional peptide or peptide combination is administered for therapeutic or preventative purposes, prior treatment, the patient's clinical history, etc., all of which are determined by the treating physician. The multifunctional peptide or peptide combination can be administered once or continuously for several days to several months within a series of treatments to maintain effective pain control. The optimal dosing regimen can be calculated from measurements of drug accumulation in the patient's body and varies according to the relative potency of each antibody or peptide. The administering physician can easily determine the optimal dosage, method of administration, and repetition rate.

[0190] The administration of multifunctional peptides (e.g., multispecific binding molecules) or peptide combination therapies described herein provides a “synergistic effect” and is demonstrated to be “synergistic,” meaning that the effect achieved when the active ingredients are used together is greater than the sum of the effects produced by using the compounds separately. The synergistic effect is achieved when the active ingredients: (1) are administered as a single multifunctional fusion peptide; (2) are co-formulated and administered or delivered simultaneously in unit dose formulations of the combination; (3) are delivered as separate formulations in alternating or parallel order; or (4) according to some other protocol. When delivered in alternating therapy, the synergistic effect is achieved when the compounds are administered or delivered sequentially (e.g., via different injections in separate syringes). Generally, during alternating therapy, the effective doses of each active ingredient are administered sequentially, while in combination therapy, the effective doses of two or more active ingredients are administered together.

[0191] pain

[0192] In its broadest usage, "pain" refers to an experiential phenomenon that is highly dependent on the subjectivity of the individual experiencing it and is influenced by that individual's psychological state, including environmental and cultural context. "Physical" pain is usually associated with a third-party-perceived stimulus that causes actual or potential tissue damage. In this sense, according to the International Association for the Study of Pain (IASP), pain can be considered "a sensory and emotional experience associated with or described in accordance with actual or potential tissue damage." However, some instances of pain lack a perceivable trigger. For example, psychogenic pain includes the exacerbation of pre-existing physical pain in individuals with mental illness and no evidence of any perceivable trigger, caused by psychological factors or a syndrome of pain that is sometimes persistently perceived.

[0193] Types of pain

[0194] Pain includes nociceptive pain, neurogenic pain, breakthrough pain, anomalous pain, hyperalgesia, dysesthesia, paresthesia, hyperpathia, phantom limb pain, psychogenic pain, analgesia, neuralgia, and neuritis. Other classifications include malignant pain, angina pectoris, and / or idiopathic pain, complex regional pain syndrome I, and complex regional pain syndrome II. Types and symptoms of pain are not necessarily mutually exclusive. These terms are intended as defined by the IASP.

[0195] Nociceptive pain is initiated by specialized sensory nociceptors in the peripheral nerves in response to noxious stimuli, which encode the stimuli as action potentials. Nociceptors, typically located on Aδ and (Polymodal) C fibers, are free nerve endings terminating under the skin, in tendons, joints, and in body organs. Dorsal root ganglion (DRG) neurons provide the site of communication between the peripheral nervous system and the spinal cord. Signals are processed through the spinal cord to the brainstem and thalamus and ultimately to the cerebral cortex, where they typically (but not always) trigger the sensation of pain. Nociceptive pain can be triggered by a variety of chemical, thermal, biological (e.g., inflammatory), or mechanical events that have the potential to stimulate or damage body tissues, typically exceeding a certain minimum threshold intensity required to induce nociceptive activity in the nociceptors.

[0196] Neuropathic pain is typically caused by abnormal activity in the peripheral or central nervous system, resulting in 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 usually the result of tissue damage and the resulting inflammatory processes. Neuropathic pain can persist sufficiently long after any observable tissue injury has seemingly healed (e.g., months or years later).

[0197] In cases of neuropathic pain, sensory processing from the affected area can manifest as abnormal and harmless stimuli (e.g., heat, contact / pressure) that typically do not cause pain (i.e., abnormal pain), or noxious stimuli that respond to normal painful stimuli can induce an exaggerated perception of pain (i.e., hyperalgesia). Furthermore, sensations similar to electric tingling or shock, or "pins and needles" (i.e., paresthesia) and / or sensations with unpleasant characteristics (i.e., hypoesthesia) may be induced by normal stimuli. Breakthrough pain is an exacerbation of pre-existing chronic pain. Hyperalgesia is a pain syndrome caused by an abnormal painful response to stimuli. These stimuli are, in most cases, repetitive and have an elevated pain threshold, which can be considered the minimum painful experience a patient can recognize as pain.

[0198] Examples of neuropathic pain include tactile paresthesia (e.g., pain induced after nerve injury), neuralgia (e.g., postherpetic neuralgia, or trigeminal neuralgia), reflex sympathetic dystrophy / burning pain (neurotrauma), components of cancer pain (e.g., pain caused by cancer itself or related conditions such as inflammation, or pain caused by treatments such as chemotherapy, surgery, or radiation therapy), phantom limb pain, entrapment neuropathy (e.g., carpal tunnel syndrome), and neuropathy such as peripheral neuropathy (e.g., caused by diabetes, HIV, chronic alcohol use, exposure to other toxins (including many chemotherapy treatments), vitamin deficiencies, and a wide variety of other medical conditions). Neuropathic pain includes pain induced by the expression of neuropathological operations following nerve injury caused by a variety of factors, such as surgery, trauma, shingles, diabetic neuropathy, amputation of the leg or arm, cancer, etc. Medical conditions associated with neuropathic pain include traumatic nerve injury, stroke, multiple sclerosis, syringomyelia, spinal cord injury, and cancer.

[0199] The stimuli that trigger pain often elicit an inflammatory response, which itself contributes to the pain experience. In some cases, pain manifests as a complex mixture of nociceptive and neuropathic factors. For example, chronic pain often includes inflammatory nociceptive pain or neuropathic pain, or a mixture of both. Initial neurological disturbances or damage may trigger the release of inflammatory mediators and subsequent neuroinflammatory reactions. For instance, migraines can represent a mixture of neuropathic and nociceptive pain. Similarly, pelvic pain may be secondary pain originating from nociceptive input to the muscles, but the abnormal muscle activity may be caused by a neurological condition.

[0200] Kits containing TNFα and NGF antagonists

[0201] This disclosure provides kits comprising a multifunctional peptide (e.g., a multispecific binding molecule) containing TNFα and NGF antagonists as described herein, or a combination of TNFα antagonists and NGF antagonists as described herein, which can be used to perform the methods described herein. In some aspects, the kit contains at least one multifunctional fusion peptide in one or more containers, the multifunctional fusion peptide comprising a TNFα antagonist and an NGF antagonist, such as a peptide containing the amino acid sequence of SEQ ID NO: 14 or 17, or the kit contains a combination of an NGF antagonist (e.g., MEDI-578) and a TNFα antagonist (e.g., an anti-TNFα antibody such as infliximab or adalimumab, or a soluble fragment of the TNF receptor that binds to TNFα, such as TNFR2-Fc). Those skilled in the art will readily recognize that the TNFα and NGF antagonists disclosed herein can be readily integrated into one of the established kit formats, which are well known in the art. Example

[0202] The present invention is generally described herein and will be more readily understood by referring to the following embodiments, which are included only for illustrating some aspects and embodiments of the invention and are not intended to limit the invention.

[0203] Example 1. Construction and characterization of a multispecific binding molecule against NGF scFv / TNFR2-Fc

[0204] A multifunctional molecule (specifically a multispecific binding molecule) containing an anti-NGF antibody domain and a TNFR2-Fc domain was generated below. An anti-NGF antibody scFv fragment was fused to the C-terminus of a TNFR2-Fc fusion protein (SEQ ID NO:13) via the heavy chain CH3 domain, in the form of Bs3Ab as described in Dimasi, N., et al., J Mol Biol. 393:672-92 (2009) and PCT Publication No. WO 2013 / 070565. The structure is illustrated in Figure 1. DNA constructs encoding the TNFR2-Fc polypeptide and the multispecific binding molecule were synthesized using GeneArt (Invitrogen). For the multispecific binding molecule, an anti-NGF scFv containing the VH (SEQ ID NO:3) and VL (SEQ ID NO:7) domains of MEDI-578 linked together by a 15-amino acid linker sequence (GGGGS)3 (SEQ ID NO:15) was constructed. The N-terminus of scFv was fused to the C-terminus of SEQ ID NO:13 via a 10-amino acid linker sequence (GGGGS)2. This multispecific binding molecule is referred to herein as TNFR2-Fc_VH#4. The DNA construct encoding the multispecific binding molecule was engineered to contain a stop codon and an EcoRI restriction site at the 3' end for cloning into the Bs3Ab expression vector. The DNA sequence encoding TNFR2-Fc_VH#4 is shown as SEQ ID NO:16 and its amino acid sequence is SEQ ID NO:14.

[0205] 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 MEDI-578scFv portion of the multispecific binding molecule. This was accomplished by introducing a G->C mutation at amino acid 44 in the VH domain (SEQ ID NO:94) and amino acid 102 in the VL domain (SEQ ID NO:95). This clone is named TNFR2-Fc_varB. The amino acid sequence of TNFR2-Fc_varB is represented as SEQ ID NO:17. The DNA sequence encoding TNFR2-Fc_varB is represented as SEQ ID NO:18. The codon-optimized DNA sequence encoding TNFR2-Fc_varB is represented as SEQ ID NO:99. The difference between TNFR2-Fc_varB and TNFR2-Fc_VH#4 lies in the replacement of the 15-amino acid linker sequence (GGGGS)3 connecting VH and VL in the scFv region with a 20-amino acid linker sequence (GGGGS)4 (SEQ ID NO:19). Differential scanning fluorescence (DSF) was used to measure the thermal stability (Tm) of TNFR2-Fc_VH#4 and TNFR2-Fc_varB. This method measures the incorporation of the fluorescent dye Sypro Orange (Invitrogen), which binds to the hydrophobic surface revealed during protein domain unfolding upon exposure to elevated temperatures. In the DSF assay, the Tm of TNFR2-Fc_VH#4 was 62 °C, while the Tm of TNFR2-Fc_varB was 66 °C. Therefore, the addition of interchain disulfide bonds to the MEDI-578 scFv region of the multispecific molecule increased the molecule's thermal stability by 4 °C.

[0206] TNFR2-Fc protein and TNFR2-Fc_VH#4 were transiently expressed in suspension CHO cells using polyethyleneimine (PEI) (Polysciences) as the transfection reagent. Cells were maintained in CD-CHO medium (Life Technologies). 1 ml of HiTrap MabSelect SuRe was added. TM Affinity chromatography was used to purify the culture harvest from small-scale transfection according to the manufacturer's specifications (GE Healthcare), followed by buffer exchange in 1% sucrose, 100 mM NaCl, 25 mM L-arginine hydrochloride, and 25 mM sodium phosphate (pH 6.3). The purity of the recombinant protein was analyzed under reducing conditions by SDS-PAGE and analytical size exclusion chromatography (see Methods below), and the concentration was determined by reading the absorbance at 280 nm using a theoretically determined extinction coefficient.

[0207] Small-scale transient expression of the TNFR2-Fc fusion protein and the TNF-NGF multispecific construct TNFR2-Fc_VH#4, and protein A-column purification yielded 36.6 and 79.9 mg L, respectively. -1 Yield / output ratio.

[0208] Large batches of TNFR2-Fc_VH#4 were generated as described below. Crude culture harvests from large-scale transfection (up to 6 L) were filtered using deep filtration and loaded onto a 1.6 x 20 cm Protein A agarose column (GE Healthcare) pre-equilibrated with buffer A (phosphate-buffered saline, pH 7.2). The column was then washed with buffer A and eluted in a discontinuous gradient of buffer B (50 mM sodium acetate, pH < 4.0). Further purification was achieved by loading the product onto a 1.6 x 20 cm Poros HS 50 column (Applied Biosystems) pre-equilibrated with buffer C (50 mM sodium acetate buffer, pH < 5.5), followed by washing with buffer C and elution in a linear gradient of 0 to 1 M NaCl in 50 mM sodium acetate buffer at pH < 5.5. The resulting eluents were analyzed by size exclusion HPLC. Protein concentration was determined using the A280 spectroscopy method with a Beckman DU520 spectrophotometer and a calculated extinction coefficient of 1.36.

[0209] Characterization method of TNFR2-Fc_VH#4

[0210] Western blot analysis was performed using standard experimental protocols. Xcell SureLock was used. TM The Invitrogen system transferred proteins onto a polyvinylidene fluoride membrane (Life Technologies) according to the manufacturer's instructions. The membrane was blocked with 3% (w / v) skim milk powder in phosphate-buffered saline (PBS) at room temperature for 1 hour. Western blotting was then performed using a standard experimental protocol with an HRP-conjugated anti-human IgG Fc-specific antibody (Sigma).

[0211] Size exclusion HPLC was performed using a Gilson HPLC system (isocratic pump-307, UV / Vis-151 detector, liquid manipulator-215, and injection module-819), a Phenomenex BioSep-SEC-S3000 (300 x 7.8 mm) column, with D-PBS (life Technologies) as the mobile phase, at a flow rate of 1 ml / min. 25 μL of sample was injected onto the column, and protein separation was monitored at A280 nm.

[0212] Enzymatic deglycosylation of small-scale purified TNFR2-Fc_VH#4 was performed using the EDGLY kit (Sigma Aldrich) according to the manufacturer's specifications. The protein was deglycosylated under both denaturing and native conditions. For denatured proteins, 30 μg of protein was deglycosylated at 37°C for 3 h using PNGase F, O-glycosidase, and α-(2→3,6,8,9)-neuraminidase, β-N-acetylglucosaminease, and β-(1→4)-galactosidase. Under untreated conditions, 35 μg of protein was deglycosylated at 37°C for 3 days using the same enzymes. The deglycosylated protein was analyzed by Coomassie Brilliant Blue stained SDS-PAGE and by Western blotting using standard assay protocols.

[0213] N-terminal amino acid sequencing of TNFR2-Fc_VH#4 was performed as described below. Approximately 2 μg of TNFR2-Fc_VH#4 was processed on an SDS-PAGE gel using standard experimental protocols. Xcell SureLock was used. TM The Invitrogen system was used to transfer the protein onto a PVDF membrane according to the manufacturer's instructions. The membrane was stained with 0.1% (w / v) Amino Black for approximately 15 minutes on an orbital oscillation platform, followed by washing with dH2O to reduce background staining on the PVDF membrane. The membrane was air-dried and then N-terminal sequencing was performed. The target band was excised and analyzed using an Applied Biosystems 494HT sequencer (Applied Biosystems, San Francisco, CA, USA) with online hydantoin sequencing. Sequence confirmation of the N-terminus of the multispecific binding molecule was performed using an Applied Biosystems 140A micro HPLC system.

[0214] Characterization results

[0215] An overview of purified TNFR2-Fc_VH#4 and TNFR2-Fc was provided by SEC-HPLC at the aggregate, monomer, and protein fragmentation levels. Figure 2A and 2BThe main peak containing monomers comprised approximately 90% of the total protein, along with the remaining approximately 10% of protein masses with lower column retention times, indicating the presence of higher-order species or aggregates. However, the monomer peak from SEC-HPLC exhibited two prominent shoulders, indicating that the protein within the peak was not a single species. SDS-PAGE analysis with Coomassie Brilliant Blue staining revealed two distinct bands (approximately 100 and 75 kDa) for TNFR2-Fc_VH#4 under reducing conditions, and similarly, two distinct bands (approximately 70 and 45 kDa) were observed for the TNFR2-Fc fusion protein. Figure 2B Under non-reducing conditions, TNFR2-Fc_VH#4 exhibited three main bands (between 150 and 250 kD), while the TNFR2-Fc fusion protein had one main band and one secondary band (located at approximately 150 and 120 kD, respectively). Since the molecular weight difference between the two bands under reducing conditions was approximately equal to the size of the scFv fragment (~26.5 kD), further analysis was performed to determine the form of multispecific binding molecule being generated. Mass spectrometry analysis under untreated conditions confirmed the SDS-PAGE data, showing three molecular weights at approximately 125, 152, and 176 kD in the purified TNFR2-Fc_VH#4 preparation for both separately purified protein preparations (Figure 2C).

[0216] If the band pattern observed by SDS-PAGE is due to differential glycosylation of TNFR2-Fc_VH#4, it resolves back to a single band upon deglycosylation. However, the band pattern is maintained under both reducing and non-reducing conditions regardless of whether TNFR2-Fc_VH#4 is deglycosylated as a native or denatured protein (data not shown). Western blot staining of both glycosylated and deglycosylated TNFR2-Fc_VH#4 with polyclonal anti-human IgG Fc-specific antibody showed that both the full-length expected band and the lower molecular weight band were reactive to the anti-Fc-specific antibody (data not shown).

[0217] The truncated product was definitively identified by N-terminal amino acid sequencing of the protein. This revealed that the first eight amino acids at the N-terminus of the stage protein were SMAPGAVH, corresponding to amino acids 176 to 183 of the TNFR2-Fc_VH#4 sequence (SEQ ID NO: 14). This represents a 175-amino acid truncation at the N-terminus of TNFR2-Fc_VH#4, leaving only the 42 amino acids of the TNFR2 domain. This allowed for precise interpretation of a large amount of data from SDS-PAGE, mass spectrometry, and SEC-HPLC analyses. Three possible combinations of TNFR2-Fc_VH#4 dimers were found in the purified protein preparation: (1) a full-length homodimer, (2) a heterodimer of both the full-length and truncated species, and (3) a homodimer of the truncated species. To accurately measure the bioactivity of both in vivo and in vitro, the full-length homodimer preparation was generated using a two-step column chromatography process. In the first step, after protein A purification, the product contains 80.5% monomer ( Figure 3A The monomer percentage was 97.8% after the second column purification step (SP agarose). Figure 3B The overall yield / output ratio for the process was 7.3%.

[0218] Example 2. Thermal stability analysis by differential scanning calorimetry (DSC)

[0219] An automated MicroCal VP capillary DSC (GE Healthcare, USA) was used for calorimetry. Protein samples were tested at 1 mg / mL in 25 mM histidine / histidine-HCl buffer (pH 6.0). The protein samples and buffer were linearly heated from 25°C to 100°C at a rate of 95°C per hour. Thermal transitions were determined by subtracting the reference buffer from the protein samples using Origin 7 software.

[0220] Thermochromatogram of TNFR2-Fc_VH#4 Figure 4The study showed three distinct unfolding transitions with denaturation temperatures (Tm) of 64, 67, and 84 °C. We infer that a Tm of 64 °C corresponds to the denaturation of both the TNFR2 domain and the anti-NGF scFv domain, while Tm values ​​of 67 °C and 84 °C are typical denaturation temperatures for the IgG1 CH2 and CH3 domains, respectively (e.g., Dimasi, N., et al., J Mol Biol. 393:672-92 (2009), and PCT Publication No. WO 2013 / 070565). Unwilling to be bound by theory, scFvs typically have lower denaturation temperatures than other antibody domains, and their unfolding is characterized by a single transition event (Roberge et al., 2006; Jung et al., 1999; Tischenko et al., 1998).

[0221] Example 3. Confirmation of antigen binding to TNFR2-Fc_VH#4

[0222] A. Single or double antigen binding via ELISA

[0223] Dilute the Nunc Maxisorp well with 50 μl of PBS (pH 7.4) to a final volume of 5 μg / ml. -1 TNFα (R&D Systems) was coated overnight at 4°C. On the following day, the coated solution was removed, and the wells were blocked at room temperature for 1 hour with 150 μl of blocking buffer [3% skim milk-PBS]. The wells were rinsed three times in PBS, and then 50 μl of the prepared TNFR2-Fc_VH#4 dilution series was added. After 1 hour at room temperature, the wells were washed three times in PBS-Tween 20 (0.1% v / v; PBS-T). Then, 50 μL of biotinylated NGF was added to the wells, and the wells were incubated at room temperature for another hour. The wells were then washed as described above, and 50 μl of streptavidin-HRP (1:100) was added. After 1 hour at room temperature, the wells were washed with PBS-T, and 50 μl of 3,3′,5,5′-tetramethylbenzidine matrix was added to the wells, allowing for color development. The reaction was terminated by adding 1M H2SO4, and the absorbance was measured at 450 nm using a microtiter plate reader. The data were analyzed using Prism 5 software (GraphPad, San Diego, CA). For single antigen binding ELISA, wells were coated with TNFα or NGF-biotin as described above, and antibody binding was detected using an anti-human IgG Fc-specific HRP-conjugated antibody (1:5000), followed by color development as described above.

[0224] The ELISA results are shown in Figure 5. TNFR2-Fc_VH#4 was designed to bind both TNFα and NGF antigens. Single antigen binding was performed by first immobilizing one antigen onto a 96-well microtiter plate and then adding serial dilutions of TNFR2-Fc_VH#4. Specific binding was detected using a horseradish peroxidase (HRP)-conjugated anti-IgG Fc-specific antibody. For the dual antigen binding ELISA, the first antigen TNFα was immobilized onto the ELISA plate, followed by serial dilutions of TNFR2-Fc_VH#4, and then a biotinylated second antigen, i.e., a fixed concentration of NGF, was added. Specific binding was then detected using HRP-conjugated streptavidin. TNFR2-Fc_VH#4 binds to both TNFα and NGF in the single antigen binding ELISA. Figure 5A And B). In dual-antigen binding ELISA, TNFR2-Fc_VH#4 binds to both TNFα and NGF (and B). Figure 5C ).

[0225] B. Antigen binding via surface plasmon resonance

[0226] Meanwhile, antigen binding assays were performed using a BIAcore 2000 instrument (GE Healthcare), as described in Dimasi, N., et al., J Mol Biol. 393:672-92 (2009). In short, approximately 1500 resonance units of TNFR2-Fc_VH#4 were immobilized at 100 nM using a CM5 sensor chip. The sensor chip surface was then used for the simultaneous binding of TNFα and NGF. The antigen was prepared in HBS-EP buffer (10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, 0.005% P2O). Binding measurements were performed at a flow rate of 30 μl / min. To determine the simultaneous binding of the multispecific antibody to TNFα and NGF, 1 μM TNFα (molecular weight 17.5 kDa) was injected onto the sensor chip surface, followed by a mixture of TNFα and NGF (molecular weight 13.5 kDa), both 1 μM, at the end of the injection. TNFα was included in the mixture with NGF to prevent signal loss due to TNFα dissociation during the NGF binding phase. As a control, a similar binding procedure was performed, with only TNFα added at the final injection; this time, the resonance unit did not increase further, indicating that TNFα was bound at saturation levels. Similar binding and control experiments were performed where the injection order of TNFα and NGF was reversed.

[0227] Simultaneous antigen binding of TNFR2-Fc_VH#4 was characterized by surface plasmon resonance. Binding events were qualitatively analyzed in a sequential manner. TNFR2-Fc_VH#4 was covalently immobilized onto the sensor chip surface using amine coupling chemistry. Subsequently, a first antigen was injected to provide saturation-level binding to TNFR2-Fc_VH#4, followed by the injection of a second antigen as an equimolar co-incorporation with antigen 1. The binding sensing plot clearly showed that TNFR2-Fc_VH#4 simultaneously binds to TNFα and NGF (…). Figure 6 Regardless of the order of antigen injection, simultaneous binding to both antigens occurs.

[0228] Example 4. Inhibition of NGF-induced TF-1 cell proliferation

[0229] TF-1 cells (ECACC product number: 93022307) were loaded at a rate of 1.5 x 10⁻⁶. 4 Cells were seeded in 50 μl of serum-free medium in 96-well Corning Costar plates and incubated at 37°C with 5% CO2 for 18 hours. Recombinant human (Sigma) or mouse NGF (R&D Systems) was pre-incubated at 37°C in Greiner 96-well round-bottom plates for 30 minutes with a dilution of TNFR2-Fc_VH#4, MEDI-578IgG1™ YTE (a non-bound IgG1™ YTE isotype control of MEDI-578), or a non-bound bispecific isotype control R347Bs3Ab. Then, 50 μl of each sample was added to the cell plates and incubated at 37°C for 48 hours. After the incubation period, 100 μl of cell titrease was added. The assay buffer (Promega) was used, and the plates were incubated at 37°C with 5% CO2 for 10 minutes. The luminescence was then measured using a standard luminescence protocol. Standard NGF-induced TF-1 proliferation in the absence of antibody was observed... Figure 7A middle.

[0230] The functional activity of TNFR2-Fc_VH#4 was determined using NGF-induced TF-1 proliferation. TNFR2-Fc_VH#4 completely inhibited NGF-induced proliferation in both humans and mice (respectively...). Figure 7B and 7C ). Figure 7B : Use the corresponding EC 80 TF-1 cells were stimulated with a concentration of recombinant human NGF. Cells were incubated with serially diluted ligand and antibody solutions for 48 hours, followed by cell titration. Cell proliferation was quantified by culturing in Promega buffer for 10 minutes. Figure 7C : Use the corresponding EC 80TF-1 cells were stimulated with a concentration of recombinant mouse NGF. Cells were incubated with serially diluted ligands and antibodies for 48 hours, followed by cell titration. Cell proliferation was quantified by incubating in assay buffer (Promega) for 10 minutes. These data demonstrate that the NGF-inhibitory moiety of TNFR2-Fc_VH#4 is biologically active and inhibits NGF-induced proliferation with efficacy similar to IgG1™ against MEDI-578. Similar data were observed for TNFR2-Fc_varB and another TNF-NGF multispecific binding molecule, ndimab var B. Figure 7D and 7E ndimab varB comprises a complete anti-TNFα antibody, namely an antibody comprising two complete heavy chains and two complete light chains in the form of H2L2, and MEDI-578scFv fused to the C-terminus of the heavy chain of the anti-TNFα antibody. The light chain of ndimab varB is described in SEQ ID NO:20 and the heavy chain of ndimab varB is described in SEQ ID NO:22.

[0231] Example 5. Inhibition of TNFα-induced apoptosis in U937 cells

[0232] U937 cells (ECACC product number: 85011440) were used at 8x10 5 The cell / well concentration was placed in 50 μl of culture medium in a blackwalled 96-well Corning Costar agar plate. The medium was prepared using the appropriate EC value. 80 U937 cells were stimulated with a concentration of recombinant human TNFα. Cells were incubated with serially diluted ligand and antibody solutions for 2 hours, followed by quantification of caspase 3 activity by incubation with caspase 3 assay reaction buffer for 2 hours. Cells were pre-incubated at 37°C for 30 minutes with TNFR2-Fc_VH#4, a non-binding bispecific isotype control, R347Bs3Ab, and etanercept. Then, 50 μl of recombinant human TNFα (R&D Systems) was added to obtain a final assay concentration of 20 ng / ml, followed by incubation at 37°C for 2 hours. After incubation, 50 μl of caspase 3 assay reaction buffer (0.2% w / v CHAPS, 0.5% v / v Igepal CA-630, 200 mM NaCl, 50 mM HEPES, 20 μM DEVD-R110 substrate (Invitrogen)) was added, and cells were incubated at 37°C for 2.5 hours. Fluorescence was measured by excitation at 475 nm and emission at 512 nm. Caspase activity lacking TNFα antagonists was shown... Figure 8A .

[0233] The functional activity of TNFR2-Fc_VH#4 was determined by measuring TNFα-induced caspase-3 activity in U937 cells. TNFR2-Fc_VH#4 completely inhibited TNFα-induced caspase-3 activity, just as etanercept did. Figure 8B This clearly demonstrates that the TNFα inhibitory portion of TNFR2-Fc_VH#4 is biologically active and possesses similar potency to etanercept. Similar data were also observed for TNFR2-Fc_varB and ndimab varB (see [link to relevant data]). Figure 8C ).

[0234] Example 6. In vivo assay

[0235] All in vivo procedures were conducted in accordance with the UK Home Office Animal (Scientific Procedures) Act (1986) and approved by the local ethics committee. Female C57Bl / 6 mice (Charles River, UK) were used throughout the process. Mice were housed in groups of 5-6 in individually ventilated cages (IVCs) with free access to food and water during a 12-hour light / dark cycle (lights on from 07:00 to 19:00). The housing and handling areas were maintained at 24°C with constant background noise via a regular radio broadcast. For identification purposes, all mice underwent transponder insertion under anesthesia (3% isoflurane in oxygen) for at least 5 days prior to the start of each study.

[0236] A. Seltzer model of neuropathic pain

[0237] Mechanohyperalgesia was determined using an analgysemeter (Randall LO, Selitto JJ, Arch Int Pharmacodyn Ther. 111:409-19 (1957)) (Ugo Basile). Increasing forces were applied alternately to the dorsal surface of each hind paw until a withdrawal response was observed. At this point, the force was stopped, and the weight was recorded in grams. Data were expressed as withdrawal thresholds for the ipsilateral and contralateral paws, in grams. After establishing baseline readings, mice were divided into two groups with approximately equal ipsilateral / contralateral ratios and underwent surgery. Mice were anesthetized with 3% isoflurane. Subsequently, the left sciatic nerve was exposed approximately 1 cm via a blunt dissection through an incision at the mid-thigh level. A suture (10 / 0 Virgin Silk: Ethicon) was then passed through the third dorsal nerve and secured. The wound was closed with glue, and mice were allowed to recover for at least 7 days before starting testing. Mice undergoing sham surgery underwent the same experimental protocol, but the wound was glued after nerve exposure and allowed to recover. Hyperalgesia in mice was tested on days 7 and 10 post-surgery. Following testing on day 10, the sham-operated mice were further subdivided into groups receiving CAT251 IgG1 isotype control (0.03 mg / kg subcutaneously), etanercept (0.01 mg / kg subcutaneously), MEDI-578 (0.03 mg / kg subcutaneously), or a combination of etanercept (0.01 mg / kg subcutaneously) and MEDI-578 (0.03 mg / kg subcutaneously). All sham-operated mice received CAT251 (0.03 mg / kg subcutaneously). Mechanical hyperalgesia was measured at hour 4 and on days 1, 2, 3, 4, and 7 post-administration.

[0238] In a mechanical hyperalgesia model, co-administration of etanercept and MEDI-578 showed a significant reduction in the ipsilateral / contralateral ratio on day 10 post-surgery compared to the sham-operated control group. Figure 9 Single administration of etanercept (0.01 mg / kg subcutaneously) or MEDI-578 (0.03 mg / kg subcutaneously) did not significantly reverse this hyperalgesia. Co-administration of etanercept (0.01 mg / kg subcutaneously) together with MEDI-578 (0.03 mg / kg subcutaneously) significantly reversed mechanical hyperalgesia at 4 hours post-administration, and the effect lasted until 7 days post-administration.

[0239] In the second study, the efficacy of TNFR2-Fc_VH#4 was evaluated. After establishing mechanotropic hyperalgesia, mice were administered R347Bs3Ab isotype control (0.03 mg / kg subcutaneously), etanercept (0.01 mg / kg subcutaneously), MEDI-578 (0.03 mg / kg subcutaneously), or TNFR2-Fc_VH#4 (0.01 mg / kg or 0.03 mg / kg subcutaneously) on day 13 post-surgery. Sham-operated animals received the R347Bs3Ab isotype control (0.03 mg / kg subcutaneously). Mechanotropic hyperalgesia in mice was tested at 4 hours post-administration and on days 1, 2, 4, and 7 post-administration, as described above.

[0240] Administration of TNFR2-Fc_VH#4 on day 10 post-surgery produced a significant reduction in the ipsilateral / contralateral ratio compared to the sham-operated control group. Figure 10A Administration of etanercept (0.01 mg / kg subcutaneously) or MEDI-578 (0.03 mg / kg subcutaneously) did not significantly reverse this mechanoalgesia. However, administration of TNFR2-Fc_VH#4 (0.01 and 0.03 mg / kg subcutaneously) produced a significant reversal of mechanoalgesia 4 hours after administration, and this effect lasted until up to 6 days after administration. No effect was observed after administration of the R347 control Bs3Ab. Similar data were observed when TNFR2-Fc_varB was administered (see [link to relevant data]). Figure 10B These data suggest that TNFR2-Fc_VH#4 can significantly reverse pain at very low doses, while the same amount of MEDI-578 or etanercept alone has shown no effect or minimal effect.

[0241] B. Chronic joint pain model

[0242] Mechanohypersensitivity was determined in a mouse bipedal incapacitance tester (Linton Instrumentation). Mice were placed in the device with their hind paws resting on separate sensors, and body weight distribution was calculated over a 4-second time interval. Data are expressed in grams as the ratio of ipsilateral to contralateral weight-bearing.

[0243] After establishing baseline readings, mice were divided into two groups with approximately equal ipsilateral / contralateral ratios. Intra-articular injection was performed using the following technique: animals were anesthetized with 3% isoflurane in oxygen, and the left knee was shaved and cleaned. Each mouse's knee was injected with 10 μl of Freund's complete adjuvant (FCA) (10 mg / ml) or a medium (light mineral oil) using a 25-gauge needle mounted on a 100 μl Hamilton syringe. The injection was directed into the synovial space of the knee joint. Mice were allowed to recover as described above, and changes in mechanosensitiveness were retested at 7 and 10 days post-injection. Following testing on day 10, FCA-treated mice were further randomized to their respective groups, and on day 13, mice were administered etanercept (0.01 mg / kg intraperitoneally) or the medium, followed by a single dose of MEDI-578 (0.03 mg / kg intravenously) or a CAT251 isotype control (0.03 mg / kg intravenously). As described above, the mechanical hypersensitivity of mice was tested 4 hours after administration and on days 1, 2, 4 and 7 after administration.

[0244] The effects of co-administration of etanercept and MEDI-578 were evaluated using an intra-articular FCA model of inflammatory pain. Intra-articular FCA induced mechanosensitive hypersensitivity, which was shown to be a significant reduction in the ipsilateral / contralateral ratio at days 7 and 10 compared to the mediator control. Figure 11 No decrease in the ipsilateral / contralateral ratio compared to baseline was observed in the sham surgery group. Administration of etanercept (0.01 mg / kg intraperitoneal) + CAT251 (0.03 mg / kg intravenous) or PBS (10 ml / kg intraperitoneal) + MEDI-578 (0.03 mg / kg intravenous) caused slight reversal of FCA-induced mechanosensitivity at 4 hours or on days 1, 2, 4, and 7 post-administration, but this did not reach statistical significance. However, administration of etanercept (0.01 mg / kg intraperitoneal) + MEDI-578 (0.03 mg / kg intravenous) caused significant reversal of FCA-induced mechanosensitivity at all test times post-administration.

[0245] In the second study, the efficacy of TNFR2-Fc_VH#4 was evaluated. After establishing FCA-induced mechanosensitivity, mice were administered the following agents on day 13 post-FCA: R347Bs3Ab isotype control (0.01 mg / kg subcutaneously), etanercept (0.01 mg / kg subcutaneously), MEDI-578 (0.01 mg / kg subcutaneously), or TNFR2-Fc_VH#4 (0.003 mg / kg or 0.01 mg / kg subcutaneously). Mechanosensitivity in mice was retested at 4 hours post-administration and on days 1, 2, 4, and 7 post-administration, as described above.

[0246] The efficacy of TNFR2-Fc_VH#4 (“bispecific”) was compared with that of etanercept and MEDI-578 alone. Figure 12 In this study, etanercept (0.01 mg / kg subcutaneously) and MEDI-578 (0.01 mg / kg subcutaneously) did not significantly reverse FCA-induced mechanosensitivity at any time point after administration. However, administration of TNFR2-Fc_VH#4 significantly reversed FCA-induced mechanosensitivity. Higher doses of TNFR2-Fc_VH#4 (0.01 mg / kg subcutaneously) showed significant activity over the duration of the study, while lower doses (0.003 mg / kg subcutaneously) reached significance on day 1 after administration and remained at a similar level to the higher dose over the duration of the study.

[0247] C. Establishment of an FCA-induced model of mechanosensitive hypersensitivity in rats

[0248] Intraplantar injection of Freund's complete adjuvant (FCA) elicits an inflammatory response, inducing hypersensitivity and edema, and mimicking some aspects of clinical inflammatory pain. These effects can be investigated using weight-bearing apparatus. The potential anti-hyperalgesic properties of TNFR2-Fc_VH#4 were assessed using weight-bearing methods to investigate FCA-induced hypersensitivity. In untreated rats, body weight was evenly distributed between the two hind paws. However, when the injected (left) hind paw was inflamed and / or painful, its weight redistributed, resulting in less weight on the affected paw (reduced weight-bearing on the injured paw). Weight-bearing on each hind limb was measured using a rat bipedal balance analgesia meter (Linton Instruments, UK). Rats were placed in the bipedal balance analgesia meter with their hind paws on separate sensors, and the average force exerted by both hind limbs was recorded over 4 seconds.

[0249] For this study, untreated rats ( Male Sprague Dawley rats (Harlan, UK, 198-258g) were acclimatized to the operating room in their living cages with free access to food and water. Acclimation to a bipedal balance analgesia device was conducted over several days. Baseline weight-bearing measurements were obtained before injury induction. Inflammatory hypersensitivity was induced by a plantar injection of FCA (available from Sigma, 100 μl of a 1 mg / ml solution) into the left hind paw. Weight-bearing measurements before treatment were used to assess hypersensitivity 23 hours after FCA.

[0250] Animals were then graded using a Latin square design based on the load-bearing FCA window and randomized to the treatment group. Twenty-four hours after FCA injection, animals were treated with intravenous TNFR2-Fc_VH#4 (“bispecific”) at doses of 0.003, 0.01, 0.03, 0.3, and 3 mg / kg, intravenous negative control antibody NIP228 (producing antibodies to bind to the hapten nitrophenol) at doses of 3 mg / kg, or oral mediator (1% methylcellulose) at doses of 2 ml / kg, or oral indomethacin at doses of 10 mg / kg.

[0251] Weight-bearing capacity was assessed 4 or 24 hours after antibody / drug treatment. Data were analyzed by comparing the treated group with the media control group at each time point. Statistical analysis included repeated measures ANOVA followed by a planned comparison test using InVivoStat (invivostat.co.uk), with p < 0.05 considered significant. Results are shown in Figure 13 Significant reversal of hypersensitivity to indomethacin (10 mg / kg) was observed at 4 and 24 hours. TNFR2-Fc_VH#4 administered at 0.3 and 3 mg / kg showed significant reversal of hypersensitivity at 4 and 24 hours, and TNFR2-Fc_VH#4 administered at 0.003 and 0.03 mg / kg also showed significant reversal of hypersensitivity, but only at 24 hours. The isotype control NIP228 showed no significant effect on FCA response at any time point.

[0252] Example 7. Phosphorylation of TNFα and NGF at p38

[0253] Literature indicates that p38 phosphorylation plays an important role in the progression of neuropathic pain. For example, treatment with p38 inhibitors has been shown to prevent the progression of neuropathic pain symptoms in models of preservative nerve injury (Wen YR et al., Anesthesiology 2007, 107:312-321) and sciatic neuritis models (Milligan ED et al., J Neurosci 2003, 23:1026-1040). In this study, the effects of TNFα, NGF, and combinations of TNFα and NGF on p38 phosphorylation were investigated in cell culture assays. Briefly, Neuroscreen-1 cells (a subclone of PC-12 rat neuroendocrine cells) were incubated with increased amounts of TNFα, NGF, or combinations of TNFα and NGF. After a 20-minute incubation period, phosphorylated p38 was quantified using homogenized time-resolved fluorescence (HTRF) assays (Cisbio).

[0254] HTRF assay: After stimulation with TNFα, NGF, or a combination of TNFα and NGF, cell supernatant was rapidly removed and cells were lysed in lysis buffer. In a sandwich assay, phosphate-p38 MAPK (Thr180 / Tyr182) was detected in lysis buffer using two different specific antibodies: an anti-phosphate-p38 antibody conjugated to europium cryptate (donor fluorophore) and an anti-p38 (total) antibody conjugated to d2 (recipient fluorophore). The antibodies were incubated with cell lysis buffer, and the HTRF ratio was calculated from fluorescence measurements at 665 nm and 620 nm, obtained using an EnVision Multilabel Plate Reader (Perkin Elmer).

[0255] Data are expressed as HTRF ratios, calculated as the ratio between emission at 665 nm and emission at 620 nm. A heatmap showing the HTRF ratios from the phosphoric acid-p38 reaction is shown in [image / image / description]. Figure 14 The dose-response curves showing the effects of TNFα, NGF, or a combination of TNFα and NGF are shown in [the figure]. Figure 15 In the middle. For example Figure 15 As shown, the combined effect of higher concentrations of TNFα and NGF on phosphate-p38 is greater than the predicted total amount of phosphate-p38 signaling induced by either single factor. These data suggest that TNFα and NGF can work together to induce p38 phosphorylation, and that both pathways may be involved in the molecular signaling that leads to pain.

[0256] Example 8. ERK phosphorylation by TNFα and NGF

[0257] Like p38, ERK is also activated during the progression of neuropathic pain (Zhuang ZY et al., Pain 2005, 114:149-159). In this study, the effects of TNFα, NGF, and combinations of TNFα and NGF on ERK phosphorylation were investigated in cell culture assays. Briefly, Neuroscreen-1 cells (a subclone of PC-12 rat neuroendocrine cells) were incubated with increased amounts of TNFα, NGF, or combinations of TNFα and NGF. After a 20-minute incubation period, phospho-ERK was quantified using homogenized time-resolved fluorescence (HTRF) assays (Cisbio).

[0258] HTRF assay: Following stimulation, cell supernatant was rapidly removed and cells were lysed in lysis buffer. In a sandwich assay, phospho-ERK MAPK (Thr202 / Tyr204) was detected in lysis buffer using two different specific antibodies: an anti-phospho-ERK antibody conjugated to a europium cryptoid (donor fluorophore) and an anti-ERK (total) antibody conjugated to d2 (recipient fluorophore). The antibodies were incubated with the cell lysis buffer, and the HTRF ratio was calculated from fluorescence measurements at 665 nm and 620 nm, obtained using an EnVision Multilabel Plate Reader (Perkin Elmer).

[0259] The data is expressed as an HTRF ratio, calculated as the ratio between emission at 665 nm and emission at 620 nm. A heatmap showing the HTRF ratio from the phosphoric acid-ERK reaction is shown in [image / image / description]. Figure 16 The dose-response curves showing the effects of TNFα, NGF, or a combination of TNFα and NGF are shown in [the figure]. Figure 17 In the middle. For example Figure 17 As shown, low levels of TNFα alone do not induce phospho-ERK, but higher levels enhance NGF-induced phospho-ERK. These data suggest that TNFα and NGF can work together to induce ERK phosphorylation, and that both pathways may be involved in the molecular signaling that leads to pain.

[0260] sequence list

[0261] SEQ ID NO:1 NP_002497.2|β-Nerve Growth Factor Precursor [Human (Homo sapiens)]

[0262]

[0263] SEQ ID NO:2 NP_000585.2|Tumor Necrosis Factor [Human]

[0264]

[0265] SEQ ID NO:3 MEDI-578VH(1256A5VH)

[0266]

[0267] SEQ ID NO:4 MEDI-578VHCDR1

[0268] 1 TYGIS

[0269] SEQ ID NO:5 MEDI-578VHCDR2

[0270] 1 GIIPIFDTGN SAQSFQG

[0271] SEQ ID NO:6 MEDI-578VHCDR3

[0272] 1 SSRIYDLNPS LTAYYDMDV

[0273] SEQ ID NO:7 MEDI-578VL(1256A5VL)

[0274]

[0275] SEQ ID NO:8 MEDI-578VLCDR1

[0276] 1 SGSSSNIGNN YVS

[0277] SEQ ID NO:9 MEDI-578VLCDR2

[0278] 1 DNNKRPS

[0279] SEQ ID NO:10 MEDI-578VLCDR3

[0280] 1 GTWDSSLSAW V

[0281] SEQ ID NO:11

[0282] 1 SSRIYDFNSA LISYYDMDV

[0283] SEQ ID NO:12

[0284] 1 SSRIYDMISS LQPYYDMDV

[0285] SEQ ID NO:13 Soluble TNFR2 amino acid sequence

[0286]

[0287]

[0288] SEQ ID NO:14 TNFR2-Fc_VH#4-amino acid sequence

[0289]

[0290] SEQ ID NO:15 (Gly4Ser)3 15aa connector sequence

[0291] 1 GGGGSGGGGS GGGGS

[0292] SEQ ID NO:16 TNFR2-Fc_VH#4 – Nucleotide Sequence

[0293]

[0294]

[0295] SEQ ID NO:17–TNFR2-Fc_varB-amino acid sequence

[0296]

[0297]

[0298] SEQ ID NO:18-TNFR2-Fc_varB-nucleotide sequence

[0299]

[0300]

[0301] SEQ ID NO:19-(Gly4Ser)4 20aa Connector Sequence

[0302] 1 GGGGSGGGGS GGGGSGGGGS

[0303] SEQ ID NO:20 - ndimab varB-L chain amino acid sequence

[0304]

[0305] SEQ ID NO:21-ndimab varB-L chain nucleotide sequence

[0306]

[0307]

[0308] SEQ ID NO:22-ndimab varB-H chain amino acid sequence

[0309]

[0310] SEQ ID NO:23-ndimab varB-H chain nucleotide sequence

[0311]

[0312]

[0313]

[0314] SEQ ID NO:24–NGF-NG VH amino acid sequence

[0315] QVQLVQSGAEVKKPGSSVKVSCKASGGTFWFGAFTWVRQAPGQGLEWMGGIIPIFGLTNLAQNFQGRVTITADESTSTVYMELSSLRSEDTAVYYCARSSRIYDLNPSLTAYYDMDVWGQGTMVTVSS

[0316] SEQ ID NO:25–NGF-NG VH nucleotide sequence

[0317]

[0318] SEQ ID NO:26–NGF-NG VL amino acid sequence

[0319] QSVLTQPPSVSAAPGQKVTISCSGSSSDIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTVL

[0320] SEQ ID NO:27–NGF-NG VL nucleotide sequence

[0321]

[0322] SEQ ID NO:28–ndimab VH amino acid sequence

[0323]

[0324]

[0325] SEQ ID NO:29–ndimab VL amino acid sequence

[0326]

[0327] SEQ ID NO:30–1126F1VH amino acid sequence

[0328] EVQLVQTGAEVKKPGSSVKVSCKASGGTFSTYGISWVRQAPGQGLEWIGGIIPIFDTGNSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDANRQAVPYYDMDVWGQGTMVTVSS

[0329] SEQ ID NO: 31 - Amino acid sequence of 1126F1 VL

[0330] QAVLTQPSSVSTPPGQMVTISCSGSSSDIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTVL

[0331] SEQ ID NO: 32 - Amino acid sequence of 1126G5 VH

[0332] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGISWVRQAPGQGLEWIGGIIPIFDTGNSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDFTSGLAPYYDMDVWGQGTMVTVSS

[0333] SEQ ID NO: 33 - Amino acid sequence of 1126G5 VL

[0334] QAVLTQPSSVSTPPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPPGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSTWVFGGGTKLTVL

[0335] SEQ ID NO: 34 - Amino acid sequence of 1126H5 VH

[0336] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGISWVRQAPGQGLEWIGGIIPIFDAGNSAQSFQGRVTITADESTSTAHMEVSSLRSEDTAVYYCASSSRIYDHHIQKGGYYDMDVWGQGTMVTVSS

[0337] SEQ ID NO: 35 - Amino acid sequence of 1126H5 VL

[0338] QAVLTQPSSVSTPPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTVL

[0339] SEQ ID NO: 36 – 1127D9VH Amino Acid Sequence

[0340] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGISWVRQAPGQGLEWIGGIIPIFDTGNSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDYHTIAYYD

[0341] SEQ ID NO: 37 – 1127D9VL Amino Acid Sequence

[0342] QAVLTQPSSVSTPPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTVL

[0343] SEQ ID NO: 38 – 1127F9VH Amino Acid Sequence

[0344] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGISWVRQAPGQGLEWIGGIIPIFDTGNSAQSFQGRVTITADESTSTAYMKVSSLRSDDTAVYYCASSSRIYDYIPGMRPYYDMDVWGQGTMVTVSS

[0345] SEQ ID NO: 39 – 1127F9VL Amino Acid Sequence

[0346] QAVLTQPSSVSTPPGQKVTISCSGNSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSRSGTLATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTVL

[0347] SEQ ID NO: 40 – 1131D7VH Amino Acid Sequence

[0348] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGISWVRQAPGQGLEWIGGIIPIFDTGNSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDFNSSLIAYYDMDVWGQGTMVTVSS

[0349] SEQ ID NO: 41 – 1131D7 VL Amino Acid Sequence

[0350] QAVLTQPSSVSTPPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDETDYYCGTWDSSLSAWVFSGGTKLTVL

[0351] SEQ ID NO: 42 – 1131H2 VH Amino Acid Sequence

[0352] EVQLVQSGAEVKKPGSTVKVSCKASGGTFSTYGISWVRQAPGQGLEWIGGIIPIFDTGNSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDLNPSLTAYYDMDVWGQGTMVTVSS

[0353] SEQ ID NO: 43 – 1131H2 VL Amino Acid Sequence

[0354] QAVLTQPSSVSTPPGQKVTISCSGTSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTVL

[0355] SEQ ID NO: 44 – 132A9 VH Amino Acid Sequence

[0356] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGISWVRQAPGQGLEWIGGIIPIFGTGNSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDFEPSLIYYYDMDVWGQGTMVTVSS

[0357] SEQ ID NO: 45 – 132A9 VL Amino Acid Sequence

[0358] QAVLTQPSSVSTPPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTVL

[0359] SEQ ID NO: 46 – 1132H9VH Amino Acid Sequence

[0360] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGISWVRQAPGQGLEWIGGIIPIFDTGNSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDLNPSLTAYYDMDVWGQGTMVTVSS

[0361] SEQ ID NO: 47 – 1132H9VL Amino Acid Sequence

[0362] QAVLTQPSSVSTPPGQKVTISCSGSSSDIGNNYVSWYQQLPGTAPKLLIYDNNKRPTGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTVL

[0363] SEQ ID NO: 48 – 1133C11VH Amino Acid Sequence

[0364] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGISWVRQAPGQGLEWIGGIIPIFDTGNSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDLNPSLTAYYDMDVWGQGTMVTVSS

[0365] SEQ ID NO: 49 – 1133C11VL Amino Acid Sequence

[0366] QAVLTQPSSVSTPPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTVL

[0367] SEQ ID NO: 50 – 1134D9VH Amino Acid Sequence

[0368] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGISWVRQAPGQGLEWIGGIIPIFDTGNSAQSFQGRVAITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDLNPSLTAYYDMDVWGQGTMVTVSS

[0369] SEQ ID NO: 51 – 1134D9 VL Amino Acid Sequence

[0370] QAVLTQPSSVSTPPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSGLSAWVFGGGTKLTVL

[0371] SEQ ID NO: 52 – 1145D1 VH Amino Acid Sequence

[0372] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGISWVRQAPGQGLEWIGGIIPIFDTSNSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDFRTLYSTYYDMDVWGQGTMVTVSS

[0373] SEQ ID NO: 53 – 1145D1 VL Amino Acid Sequence

[0374] QAVLTQPSSVSTPPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGISDRFSGSKSGTSATLGIAGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTVL

[0375] SEQ ID NO: 54 – 1146D7 VH Amino Acid Sequence

[0376] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGISWVRQAPGQGLEWIGGIIPIFDTGNSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDLNPSLTAYYDMDVWGQGTMVTVSS

[0377] SEQ ID NO: 55 – 1146D7 VL Amino Acid Sequence

[0378] QAVLTQPSSVSTPPGQEVTISCSGSSTNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTVL

[0379] SEQ ID NO: 56–1147D2VH Amino acid sequence

[0380] EVQLVQSGAEVKKPGSSVRISCKASGGTFSTYGVSWVRQAPGQGLEWIGGIIPIFDTGNSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDLNPSLTAYYDMDVWGQGTMVTVSS

[0381] SEQ ID NO: 57–1147D2VL Amino acid sequence

[0382] QAVLTQPSSVSTPPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGVPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTVL

[0383] SEQ ID NO: 58–1147G9VH Amino acid sequence

[0384] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSAYGISWVRQAPGQGLEWIGGIIPIFNTGNSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDLNPSLTAYYDMDVWGQGTMVTV

[0385] SEQ ID NO: 59–1147G9VL Amino acid sequence

[0386] QAVLTQPSSVSTPPGQKVTVSCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTVL

[0387] SEQ ID NO: 60–1150F1VH Amino acid sequence

[0388] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGISWVRQAPGQGLEWIGGIIPIFDTGNSAQSFQDRVTITADESTSTAYMEVGSLRSDDTAVYYCASSSRIYDLNPSLTAYYDMDVWGHGTMVTVSS

[0389] SEQ ID NO: 61–1150F1VL Amino Acid Sequence

[0390] QAVLTQPSSVSTPPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTVL

[0391] SEQ ID NO: 62–1152H5VH Amino Acid Sequence

[0392] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGISWVRQAPGQGLVWIGGIIPIFDTGNSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDMISSLQPYYDMDVWGQGTMVTVSS

[0393] SEQ ID NO: 63–1152H5VL Amino Acid Sequence

[0394] QAVLTQPSSVSTPPGQKATISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTVL

[0395] SEQ ID NO: 64–1155H1VH Amino Acid Sequence

[0396] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGISWVRQAPGQGLEWIGGIIPIFDTGNSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDFHLANKGYYDMDVWGQGTMVTVSS

[0397] SEQ ID NO: 65–1155H1VL Amino Acid Sequence

[0398] QAVLTQPSSVSTPPGQKATISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLDITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTVL

[0399] Amino acid sequence of SEQ ID NO: 66–1158A1VH

[0400] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGISWVRQAPGQGLEWIGGIIPIFGTGNSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDHHNHVGGYYDMDVWGQGTMVTVSS

[0401] Amino acid sequence of SEQ ID NO: 67–1158A1VL

[0402] QAVLTQPSSVSTPPGQKVTISCSGSSSNIGNNYASWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDGSLSAWVFGGGTKLTVL

[0403] Amino acid sequence of SEQ ID NO: 68–1160E3VH

[0404] EVQLVQSGAEVKKPGSSAKVSCKASGGTFSTYGISWVRQAPGQGLEWIGGIIPIFDTGNSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDLNPSLTAYYDMDVWGQGTMVTVSS

[0405] Amino acid sequence of SEQ ID NO: 69–1160E3VL

[0406] QAVLTQPSSVSTPPGQKVTISCSGSNSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTV

[0407] Amino acid sequence of SEQ ID NO: 70–1165D4VH

[0408] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGISWVRQAPGQGLEWIGGIIPIFDTGNSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDLNPSLTAYYDMDVWGQGTMVTVSS

[0409] SEQ ID NO: 71 – 1165D4VL Amino Acid Sequence

[0410] QAVLTQPSSVSTPPGQKVTISCSGSSSNIENNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTVL

[0411] SEQ ID NO: 72 – 1175H8VH Amino Acid Sequence

[0412] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGISWVRQAPGQRLEWIGGIIPIFDTGNSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDATTGLTPYYDMDVWGQGTMVTVSS

[0413] SEQ ID NO: 73 – 1175H8VL Amino Acid Sequence

[0414] QAVLTQPSSVSTPPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLRTGDEADYYCGTWDSSLSAWVFGGGTKLTVL

[0415] SEQ ID NO: 74 – 1211G10VH Amino Acid Sequence

[0416] EVQLVQSGAEVRKPGSSVKVSCKAYGGTFSTYGISWVRQAPGQGLEWVGGIIPIFDTRNSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDMVSTLIPYYDMDVWGQGTMVTVSS

[0417] SEQ ID NO: 75 – 1211G10VL Amino Acid Sequence

[0418] QAVLTQPSSVSTPPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTVL

[0419] SEQ ID NO: 76 – 1214A1VH Amino Acid Sequence

[0420] EVQLVQSGAEVKKPGSSVRVSCKASGGTFSTYGISWVRQAPGQGLEWIGGIIPIFDTGNSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDAHLQAYYDMDVWGQGTMVTVSS

[0421] SEQ ID NO: 77 – 1214A1VL Amino Acid Sequence

[0422] QAVLTQPSSVSTPPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPPGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTRDSSLSAWVFGGGTKLTVL

[0423] SEQ ID NO: 78 – 1214D10VH Amino Acid Sequence

[0424] EVQLVQSGAEAKKPGSSVKVSCKASGGTFSTYGISWVRQAPGRGLEWIGGIIPIFDTGNSAQSFQGRVAITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDAHLNHHGYYDMDVWGQGTMVTVSS

[0425] SEQ ID NO: 79 – 1214D10VL Amino Acid Sequence

[0426] QAVLTQPSSVSTPPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQAGDEADYYCGTWDSSLSAWVFGGGTKLTVL

[0427] SEQ ID NO: 80 – 1218H5VH Amino Acid Sequence

[0428] EVQLVQSGAVVKKPGSSVKVSCKASGGTFSTYGISWVRQAPGQGLEWIGGIIPIFDTGSSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDLNPSLTAYYDMDVWGQGTMVTVSS

[0429] Amino Acid Sequence of 1218H5VL, SEQ ID NO: 81

[0430] QAVLTQPSSVSTPPGQKVTISCSGSSSNTGNNYVSWYQQLSGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTVL

[0431] Amino Acid Sequence of 1230H7VH, SEQ ID NO: 82

[0432] EMQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGISWVRQAPGQGLEWIGGIIPIFDTGNSAQSFQGRVTITADESTSTAYMEVSSLRSDDTAVYYCASSSRIYDFNSALISYYDMDVWGQGTMVTVSS

[0433] Amino Acid Sequence of 1230H7VL, SEQ ID NO: 83

[0434] QAVLTQPSSVSTPPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLTV

[0435] Amino Acid Sequence of 1083H4VH, SEQ ID NO: 84

[0436] QMQLVQSGAEVKKTGSSVKVSCKASGYTFAYHYLHWVRQAPGQGLEWMGGIIPIFGTTNYAQRFQDRVTITADESTSTAYMELSSLRSEDTAVYYCASADYVWGSYRPDWYFDLWGRGTMVTVSS

[0437] Amino Acid Sequence of 1083H4VL, SEQ ID NO: 85

[0438] QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQRLPGAAPQLLIYNNDQRPSGIPDRFSGSKSGTSGSLVISGLQSEDEADYYCASWDDSLNGRVFGGGTKLTVL

[0439] Amino Acid Sequence of 1227H8 VH of SEQ ID NO: 86

[0440] QMQLVQSGAEVKKTGSSVKVSCKASGHTFAYHYLHWVRQAPGQGLEWMGGIIPIFGTTNYAQRFQDRVTITADESTSTAYMELSSLRSEDTAVYYCASADYAWESYQPPQINGVWGRGTMVTVSS

[0441] Amino Acid Sequence of 1227H8 VL of SEQ ID NO: 87

[0442] QSVLTQPPSVSAAPGQKVTITCSGSTSNIGNNYVSWYQQHPGKAPKLMIYDVSKRPSGVPDRFSGSKSGNSASLDISGLQSEDEADYYCAAWDDSLSEFFFGTGTKLTVL

[0443] HCDR1 of NGF-NG of SEQ ID NO: 88

[0444] FGAFT

[0445] HCDR2 of NGF-NG of SEQ ID NO: 89

[0446] GIIPIFGLTNLAQNFQG

[0447] HCDR3 of NGF-NG of SEQ ID NO: 90

[0448] SSRIYDLNPSLTAYYDMDV

[0449] LCDR1 of NGF-NG of SEQ ID NO: 91

[0450] SGSSSDIGNNYVS

[0451] LCDR2 of NGF-NG of SEQ ID NO: 92

[0452] DNNKRPS

[0453] LCDR3 of NGF-NG of SEQ ID NO: 93

[0454] GTWDSSLSAWV

[0455] SEQ ID NO:94–MEDI-578VH amino acid sequence, with G->C

[0456]

[0457] The amino acid sequence SEQ ID NO:95–MEDI-578VL has G->C

[0458]

[0459] SEQ ID NO:96–1230D8VH amino acid sequence

[0460] QMQLVQSGAEVKKTGSSVKVSCKASGYTFPYHYLHWVRQAPGQGLEWMGGIIPIFGTTNYAQRFQDRVTITADESTSTAYMEFSSLRSEDTAVYYCASADYVWESYHPATSLSLWGRGTMVTVSS

[0461] SEQ ID NO:97–1230D8VL amino acid sequence

[0462] QSVLTQPPSVSAAPGQKVTISCPGSTSNIGNNYVSWYQQRPGKAPKLMIYDVSKRPSGVPDRFSGSKSGNSASLDISELQSEDEADYYCAAWDDSLSEFLFGTGTKLTVL

[0463] SEQ ID NO:98

[0464] GGGGSGGGGS

[0465] SEQ ID NO:99–TNFR2-Fc_varB–Codon-optimized nucleotide sequence

[0466]

[0467]

[0468]

[0469] ***

[0470] The scope of this invention is not limited to the specific aspects described, which are meant as a single description of an individual aspect of the invention, and any functionally equivalent combination or method is within the scope of this invention. In fact, various modifications to the invention added to what is shown and described herein will be apparent to those skilled in the art based on the description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

[0471] All publications and patent applications mentioned in this specification are incorporated herein by reference in such a manner as to specifically and individually describe each individual publication or patent application by reference.

Claims

1. A binding molecule capable of binding NGF and TNFα, wherein the binding molecule is: (a) A homodimer of a fusion polypeptide, said fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 14; or (b) Homodimer of a fusion polypeptide, the fusion polypeptide consisting of the amino acid sequence of SEQ ID NO:

17.

2. The binding molecule according to claim 1, wherein the binding molecule is a homodimer of a fusion polypeptide, the fusion polypeptide consisting of the amino acid sequence of SEQ ID NO:

17.

3. The binding molecule according to claim 1, wherein the binding molecule is a homodimer of a fusion polypeptide, the fusion polypeptide consisting of the amino acid sequence of SEQ ID NO: 14.

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