Anti-trka Antibodies, Derivatives And Uses Thereof

Abstract

The present invention relates to an antibody, recombinant or synthetic antigen-binding fragments thereof able to recognise and bind an epitope comprised in the TrkA amino acid sequence, medical uses thereof and a pharmaceutical composition comprising at least one of the above antibody, recombinant or synthetic antigen-binding fragments thereof.

Description

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This patent application is a continuation of U.S. application Ser. No. 13/894,489, filed on May 15, 2013, that in turn claims benefit of priority from European Patent Application No. 12171752.4, filed Jun. 13, 2012, the contents of all of which are incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to anti-TrkA antibodies and to derivatives thereof which are able to recognise and bind an amino acid sequence comprised in the Nerve Growth Factor NGF-binding site of the high affinity tyrosine kinase receptor of NGF (TrkA), thus acting as NGF antagonists and preventing the functional activation of TrkA by NGF. The antibodies of the invention are useful in the treatment of conditions associated with the expression and/or activity of TrkA, including pain states.

BACKGROUND OF THE INVENTION

Monoclonal Antibodies.

[0003] Monoclonal antibodies (Mabs) represent the fastest-growing market segment within the pharmaceutical industry. Despite a number of drawbacks, they are particularly appreciated among biotherapeutics, thanks to their unique features, including extremely high target specificity, favourable pharmacokinetics (long half-life) as well as faster development and higher success rate, as compared to small molecules. Soluble ligands and membrane-bound receptors involved in pain signalling represent ideal targets for the Mab, making it possible to obtain anti-pain biologics with higher specificity and/or different mechanism of action (MoA) as compared to currently available analgesics.

[0004] The huge body of evidence suggesting that the NGF pathway provides new molecular targets for pain therapy has spurred the development of neutralizing antibodies against NGF and its receptors. In general, antibodies in the whole immunoglobulin format (IgG) are big molecules that do not go through the blood brain barrier when they are systemically administered. As for antibodies targeting the NGF pathway this means they are likely to be effective in periphery (as it is desirable for many pain conditions) but not in central nervous system (CNS). This is a clear advantage, since NGF is known to exert neurotrophic and neuroprotective effects on specific TrkA-expressing cell populations in the CNS, like basal forebrain cholinergic neurons). From a theoretical point of view anti-NGF neutralizing antibodies represent the most efficacious tool to reduce NGF bioactivity, especially if both receptors are thought to be involved; moreover when the neutralizing agent is an antibody in the whole Immunoglobulin format (IgG), it is usually safer to block a ligand instead of a membrane-bound receptor, which would increase the risk of complement-dependent cytolysis (CDC) and antigen-dependent cell cytolysis (ADCC) for the receptor bearing cell. On the other hand, targeting NGF in Peripheral nervous System (PNS) might indirectly affect the concentration of the same factor in CNS, altering the balance between PNS and CNS pools (peripheral sink effect), with possible deleterious effects on NGF-responsive neurons in the CNS; moreover, in certain cases (e.g., the massive release of NGF in a short time, in inflammation) it is much easier to target and block the receptor than the NGF ligand. The neutralisation of either receptor would therefore avoid the drawbacks associated with NGF targeting as well as the side effects depending on signalling through the other receptor.

[0005] Various anti-TrkA antibodies have also been generated. One such antibody is the monoclonal called 5C3 as disclosed in WO97/21732. The antibody interacts within the juxtamembrane/IgG2 domain of TrkA receptor. However, this antibody was found to be a TrkA agonist and therefore not useful for inhibiting TrkA-triggered activities and consequently having a therapeutic effect, as reducing pain. As a matter of fact, when binding to TrkA, this antibody does not prevent the functional activation of the receptor, since, on the contrary, it induces the receptor functional activation upon its binding. Moreover it was not raised against specific loops in the TrkAd5 domain that are known to be essential for NGF binding.

[0006] An anti-TrkA monoclonal antibody referred to as MNAC13 is disclosed in WO00/73344. This antibody and its derivatives are said to prevent the functional activation of TrkA in a range of biological systems. In particular, the MNAC13 antibody is able to reduce pain in relevant animal models (Ugolini et al., 2007. Proc Natl Acad Sci USA. 104: 2985-2990). However, structural evidence (Covaceuszach et al., 2005. Proteins. 58: 717-727) demonstrates that the MNAC13 antibody does not interact with the NGF binding site on the TrkA receptor (TrkAd5 domain), whereas it binds to a more N-terminal portion of the TrkA extracellular domain (ECD) (i.e. the TrkAd4 domain).

[0007] WO06/131952 discloses medical uses of the MNAC13 antibody in treating chronic pain.

[0008] WO05/061540 discloses a method of antibody humanization in which structural data from crystallographic studies are employed to conduct the first design steps of the humanization process. Anti-TrkA antibodies, such as MNAC13, as disclosed in WO00/73344, are described as examples of mouse antibodies humanized using the method. Several different humanized variants of MNAC13 are disclosed in WO09/098238.

[0009] A human antibody and derivatives thereof that acts as a powerful NGF activity antagonist by recognizing and binding specific loops in the TrkAd5 domain essential for NGF binding has not been provided yet. Moreover there is the need to obtain antibodies with a defined binding specificity to fine regulating the activity thereof.

Chronic Pain as a Therapeutic Area of Largely Unmet Need.

[0010] Persistent pain represents a major health problem. It can show different levels of severity and is associated to different pathologies, such as back injury, migraine headaches, arthritis, herpes zoster, diabetic neuropathy, temporomandibular joint syndrome, and cancer. Mild pain is presently treated with acetaminophen, aspirin, and NSAIDs. The NSAIDs inhibit COX and thereby reduce prostaglandin synthesis. However, they are associated with gastrointestinal toxicity, and although COX-2-selective inhibitors have significantly reduced adverse gastric effects, there is still a raised risk of cardiovascular disease (Zeilhofer, 2007. Biochem Pharmacol. 73: 165-174). Moderate pain can be controlled using corticosteroidal drugs such as cortisol and prednisone, inhibiting phospholipase A2 (Flower and Blackwell, 1979. Nature. 278: 456-459). Nonetheless, corticosteroids display remarkable adverse effects including weight gain, insomnia, and immune system weakening. Severe pain may be treated with strong opioids such as morphine and fentanyl. However, long-term use of opiates is limited by several serious drawbacks, including development of tolerance and physical dependence (Przewlocki and Przewlocka, 2001. Eur J Pharmacol. 429: 79-91).

[0011] As current pain therapies are often poorly effective or have undesirable side effects, an urgent need therefore exists to develop more specifically efficacious drugs directed against new molecular targets, with particular emphasis for the therapeutic area of chronic pain.

Molecular Mechanisms Underlying Pain.

[0012] Chronic pain may be of either nociceptive or neuropathic origin. In some cases, complex pain syndromes are produced by a combination of both, as it is the case for many types of oncologic pain. Nociceptive pain is induced by noxious mechanical, chemical, or thermal stimuli acting through pain specific receptors, mainly expressed on the peripheral endings of sensory neurons. Activation of nociceptive Ad-fibers (small diameter, rapidly-conducting, and myelinated) and C-fibers (small diameter, slower-conducting, and unmeylinated) results in pain perception. Tonic or chronic nociceptive pain may arise from sustained inflammatory disorders, resulting in hyperalgesia (increased sensitivity to painful stimuli) and/or allodynia (lowering of the threshold beyond which a stimulus is perceived as painful). Neuropathic pain may be induced by neural lesion or dysfunction, sometimes implying central neuroplasticity.

[0013] Following injury, inflammatory mediators are released from both damaged tissues and activated immune cells. Proinflammatory cytokines such as tumor necrosis factor-a (TNFa) and interleukin-1 (IL-1) are secreted by neutrophils and activated cells of the monocyte/macrophage lineage. These cytokines may stimulate the release of Nerve growth factor (NGF) both from structural sources (fibroblasts, keratinocytes, and Schwann cells) and from inflammatory cell types (lymphocytes, macrophages, and mast cells). Moreover, mast cell degranulation releases other proinflammatory substances, which make up the so-called inflammatory soup: histamine, cytokines, prostaglandins, bradykinin, serotonin (5-hydroxytryptamine [5-HT]), adenosine triphosphate (ATP) and H+. Upon binding to their own specific receptors on nociceptive neurons, they all contribute to pain signalling (Pezet and McMahon, 2006. Annu Rev Neurosci. 29: 507-538).

TrkA Receptor Signalling in Persistent Pain.

[0014] Nerve growth factor (NGF) is a multi-functional molecule that exerts its biological functions in a variety of neural and non-neural cells (Levi-Montalcini, 1987. Science. 237: 1154-1162), by means of two types of receptors: the TrkA tyrosine kinase receptor and the p75 neurotrophin receptor (p75NTR), belonging to the molecular family of Tumor Necrosis Factor receptors. TrkA mediates the survival and neurite outgrowth-promoting effects of NGF during development. Each NGF dimer binds two TrkA monomers, resulting in dimerization and trans-autophosphorylation of specific tyrosine residues. These phosphotyrosine residues form docking sites for several adaptor proteins coupling the receptor to intracellular signalling pathways including the mitogen activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K) and PLC pathways (Kaplan and Miller, 2000. Curr Opin Neurobiol. 10: 381-391); (Huang and Reichardt, 2003. Annu Rev Biochem. 72: 609-642). It is known that the exogenous administration of NGF induces pain both in animals and in humans (Mantyh et al., Anesthesiology. 115: 189-204) furthermore, the first line of evidence associating NGF signalling and pain comes from genetic studies. For example, congenital insensitivity to pain with anhydrosis is an autosomal recessive disorder characterized by the absence/abnormal development of several subsets of sensory and sympathetic neurons, which makes affected individuals unresponsive to pain and unable to sweat (anhydrosis). Null mutations in the gene encoding TrkA (NTRK1) have been recognized to be responsible for this disorder (Indo, 2001. Hum Mutat. 18: 462-471; Indo et al., 2001. Hum Mutat. 18: 308-318; Indo et al., 1996. Nat Genet. 13: 485-488).

[0015] NGF plays a key role in pain transduction mechanisms in adult nervous system. Peripheral nociceptors strongly express the TrkA and p75NTR receptors and are developmentally and functionally dependent on NGF. NGF is a peripherally produced mediator of several persistent pain states, notably those associated with inflammation, also thanks to its dual action on inflammatory mast cells that are recruited by NGF to the injured or painful site, and are induced by NGF to release inflammatory mediators. NGF is released by mast cells, fibroblasts and other cell types present in peripheral sites where inflammation is taking place. In particular, mast cells seem to play a key role (Woolf et al., 1996. J Neurosci. 16: 2716-2723). In fact, they produce NGF and display functional TrkA receptors on their surface in the same time, which makes them capable of responding to NGF itself, (Horigome et al., 1993. J Biol Chem. 268: 14881-14887). Thus the NGF-TrkA system appears to mediate mast cell activation through an autocrine loop, allowing local amplification of the activation process.

[0016] Therefore, prior art still fails to disclose an anti-TrkA molecule that specifically recognises and binds an epitope in the TrkAd5 domain, comprising the sequence from aa 294 to to aa 299 of TrkA amino acid sequence of SEQ ID NO: 65, and more effectively acts as NGF antagonists specifically preventing the functional activation of TrkA by NGF.

SUMMARY OF THE INVENTION

[0017] An object of the invention is an antibody, recombinant or synthetic antigen-binding fragments thereof which is able to recognise and bind a sequence having at least 80% identity to an epitope comprising the sequence from amino acid residue 294 to amino acid residue 299 of the human TrkA amino acid sequence (UniProt P04629133-796) of SEQ ID NO: 65 (i.e., the sequence VEMHHW, SEQ ID NO: 66).

[0018] The antibody, recombinant or synthetic antigen-binding fragments thereof according to the invention is preferably a NGF antagonist.

[0019] The antibody, recombinant or synthetic antigen-binding fragments thereof of the invention is able to specifically antagonize the activity of NGF triggered by TrkA, by preventing the functional activation of TrkA by NGF (also known as neutralizing antibodies).

[0020] In the present invention an antibody refers to: [0021] a) a monoclonal, a polyclonal or a chimeric, or a humanized, or a deimmunized, or an affinity matured antibody, or a fully human antibody; and [0022] b) a recombinant or synthetic antigen-binding fragments thereof, as well as molecules comprising such antigen-binding fragments, including engineered antibody fragments, antibody derivatives, bispecific antibodies and other multispecific molecules.

[0023] In a preferred embodiment, said epitope consists of the sequence from amino acid residue 294 to amino acid residue 299 of the human TrkA amino acid sequence of SEQ ID NO: 65 (i.e., the sequence VEMHHW, SEQ ID NO: 66).

[0024] In another embodiment of the invention, the above sequence having at least 80% identity to an epitope is the sequence VEQHHW (SEQ ID NO: 67).

[0025] The antibody, recombinant or synthetic antigen-binding fragments thereof according to the invention preferably comprises at least one heavy chain complementary determining region (CDRH3) amino acid sequence having at least 80% identity to an amino acid sequence selected from the group consisting of: SEQ ID NOs: 64, 43, 46, 49, 52, 55, 58 and 61.

[0026] The antibody, recombinant or synthetic antigen-binding fragments thereof according to the present invention preferably comprises at least one heavy chain complementary determining region (CDRH1) amino acid sequence having at least 80% identity to an amino acid sequence selected from the group consisting of: SEQ ID NOs: 62, 41, 44, 47, 50, 53, 56 and 59.

[0027] The antibody, recombinant or synthetic antigen-binding fragments thereof according to the present invention preferably comprises at least one heavy chain complementary determining region (CDRH2) amino acid sequence having at least 80% identity to an amino acid sequence selected from the group consisting of: SEQ ID NOs: 63, 42, 45, 48, 51, 54, 57 and 60.

[0028] The antibody, recombinant or synthetic antigen-binding fragments thereof of the invention preferably comprises a heavy chain complementary determining regions (CDRH1) amino acid sequence having at least 80% identity to an amino acid sequence selected from the group consisting of: SEQ ID NOs: 41, 44, 47, 50, 53, 56, 59 and 62 and a heavy chain complementary determining regions (CDRH2) amino acid sequence having at least 80% identity to an amino acid sequence selected from the group consisting of: SEQ ID NOs: 42, 45, 48, 51, 54, 57, 60 and 63 and a heavy chain complementary determining regions (CDRH3) amino acid sequence having at least 80% identity to an amino acid sequence selected from the group consisting of: SEQ ID NOs: 43, 46, 49, 52, 55, 58, 61 and 64.

[0029] In a preferred embodiment, the antibody, recombinant or synthetic antigen-binding fragments thereof comprises a CDRH1 amino acid sequence having at least 80% identity to SEQ ID NO: 62, a CDRH2 amino acid sequence having at least 80% identity to SEQ ID NO: 63 and a CDRH3 amino acid sequence having at least 80% identity to SEQ ID NO: 64.

[0030] The antibody, recombinant or synthetic antigen-binding fragments thereof according to the present invention preferably comprises one light chain complementary determining region (CDRL3) amino acid sequence having at least 80% identity to an amino acid sequence selected from the group consisting of: SEQ ID NOs: 40, 19, 22, 25, 28, 31, 34 and 37.

[0031] The antibody, recombinant or synthetic antigen-binding fragments thereof according to the present invention preferably comprises at least one light chain complementary determining region (CDRL1) amino acid sequence having at least 80% identity to an amino acid sequence selected from the group consisting of: SEQ ID NOs: 38, 17, 20, 23, 26, 29, 32 and 35.

[0032] The antibody, recombinant or synthetic antigen-binding fragments thereof according to the present invention preferably comprises one light chain complementary determining region (CDRL2) amino acid sequence having at least 80% identity to an amino acid sequence selected from the group consisting of: SEQ ID NOs: 39, 18, 21, 24, 27, 30, 33 and 36.

[0033] The antibody, recombinant or synthetic antigen-binding fragments thereof of the invention preferably comprises a light chain complementary determining regions (CDRL1) amino acid sequence having at least 80% identity to an amino acid sequence selected from the group consisting of: SEQ ID NOs: 17, 20, 23, 26, 29, 32, 35 and 38 and a light chain complementary determining regions (CDRL2) amino acid sequence having at least 80% identity to an amino acid sequence selected from the group consisting of: SEQ ID NOs: 18, 21, 24, 27, 30, 33, 36 and 39 and a light chain complementary determining regions (CDRL3) amino acid sequence having at least 80% identity to an amino acid sequence selected from the group consisting of: SEQ ID NOs: 19, 22, 25, 28, 31, 34, 37 and 40.

[0034] In a preferred embodiment of the invention, the antibody, recombinant or synthetic antigen-binding fragments thereof comprises a CDRL1 amino acid sequence having at least 80% identity to SEQ ID NO: 38, a CDRL2 amino acid sequence having at least 80% identity to SEQ ID NO: 39 and a CDRL3 amino acid sequence having at least 80% identity to SEQ ID NO: 40.

[0035] The antibody, recombinant or synthetic antigen-binding fragments thereof according to the present invention preferably comprises a heavy chain variable region comprising SEQ ID NOs: 15, 1, 3, 5, 7, 9, 11 or 13.

[0036] The antibody, recombinant or synthetic antigen-binding fragments thereof according to the present invention preferably comprises a light chain variable region comprising SEQ ID NOs: 16, 2, 4, 6, 8, 10, 12 or 14.

[0037] According to one aspect of the invention, the antibody, recombinant or synthetic antigen-binding fragments thereof according to the present invention comprises: [0038] a) a variable heavy chain comprising a sequence selected from any of CRB0022VH (SEQ ID NO: 1), CRB0036VH (SEQ ID NO: 3), CRB0069VH (SEQ ID NO: 5), CRB0072VH (SEQ ID NO: 7), CRB0082VH (SEQ ID NO: 9), CRB0084VH (SEQ ID NO: 11), CRB0088VH (SEQ ID NO: 13) or CRB0089VH (SEQ ID NO: 15) or variants thereof or [0039] b) a variable light chain comprising a sequence selected from any of CRB0022VK (SEQ ID NO: 2), CRB0036VK (SEQ ID NO: 4), CRB0069VK (SEQ ID NO: 6), CRB0072VK (SEQ ID NO: 8), CRB0082VL (SEQ ID NO: 10), CRB0084VL (SEQ ID NO: 12), CRB0088VL (SEQ ID NO: 14) or CRB0089VL (SEQ ID NO: 16) or variants thereof.

[0040] More preferably, the antibody comprises both a variable heavy chain as described in a) above and variable light chain as described in b), i.e. it comprises one of the following 64 combinations of light and heavy chains:

CRB0022VH_CRB0022VK; CRB0022VH_CRB0036VK; CRB0022VH_CRB0069VK; CRB0022VH_CRB0072VK; CRB0022VH_CRB0082VL; CRB0022VH_CRB0084VL; CRB0022VH_CRB0088VL; CRB0022VH_CRB0089VL; CRB0036VH_CRB0022VK; CRB0036VH_CRB0036VK; CRB0036VH_CRB0069VK; CRB0036VH_CRB0072VK; CRB0036VH_CRB0082VL; CRB0036VH_CRB0084VL; CRB0036VH_CRB0088VL; CRB0036VH_CRB0089VL; CRB0069VH_CRB0022VK; CRB0069VH_CRB0036VK; CRB0069VH_CRB0069VK; CRB0069VH_CRB0072VK; CRB0069VH_CRB0082VL; CRB0069VH_CRB0084VL; CRB0069VH_CRB0088VL; CRB0069VH_CRB0089VL; CRB0072VH_CRB0022VK; CRB0072VH_CRB0036VK; CRB0072VH_CRB0069VK; CRB0072VH_CRB0072VK; CRB0072VH_CRB0082VL; CRB0072VH_CRB0084VL; CRB0072VH_CRB0088VL; CRB0072VH_CRB0089VL; CRB0082VH_CRB0022VK; CRB0082VH_CRB0036VK; CRB0082VH_CRB0069VK; CRB0082VH_CRB0072VK; CRB0082VH_CRB0082VL; CRB0082VH_CRB0084VL; CRB0082VH_CRB0088VL; CRB0082VH_CRB0089VL; CRB0084VH_CRB0022VK; CRB0084VH_CRB0036VK; CRB0084VH_CRB0069VK; CRB0084VH_CRB0072VK; CRB0084VH_CRB0082VL; CRB0084VH_CRB0084VL; CRB0084VH_CRB0088VL; CRB0084VH_CRB0089VL; CRB0088VH_CRB0022VK; CRB0088VH_CRB0036VK; CRB0088VH_CRB0084VL; CRB0088VH_CRB0088VL; CRB0088VH_CRB0089VL; CRB0089VH_CRB0022VK; CRB0089VH_CRB0036VK; CRB0089VH_CRB0069VK; CRB0089VH_CRB0072VK; CRB0089VH_CRB0082VL; CRB0089VH_CRB0084VL; CRB0089VH_CRB0088VL; and CRB0089VH_CRB0089VL.

[0041] Particularly preferred anti-TrkA antibodies which fall within the scope of the present invention comprises a plurality of hypervariable regions (CDR), at least one of which, or at least two of which, or at least three of which, or at least four of which, or at least five of which, or six of which is/are selected from the group consisting of: [0042] CDRH1 regions, comprising sequences selected from the group consisting of SEQ ID NOs: 41, 44, 47, 50, 53, 56, 59, 62; [0043] CDRH2 regions, comprising sequences selected from the group consisting of SEQ ID NOs: 42, 45, 48, 51, 54, 57, 60, 63; [0044] CDRH3 regions, comprising sequences selected from the group consisting of SEQ ID NOs: 43, 46, 49, 52, 55, 58, 61, 64; [0045] CDRL1 regions, comprising sequences selected from the group consisting of SEQ. ID NOs: 17, 20, 23, 26, 29, 32, 35, 38; [0046] CDRL2 regions, comprising sequences selected from the group consisting of SEQ. ID NOs: 18, 21, 24, 27, 30, 33, 36, 39; and [0047] CDRL3 regions, comprising sequences selected from the group consisting of SEQ. ID NOs: 19, 22, 25, 28, 31, 34, 37, 40.

[0048] Other particularly preferred anti-TrkA antibodies which fall within the scope of the present invention are disclosed herein below:

[0049] an anti-TrkA antibody which comprises a heavy chain variable region comprising CDRH1, CDRH2 and CDRH3, and/or a light chain variable region comprising CDRL1, CDRL2, and CDRL3 wherein both CDRHs and CDRLs amino acid sequence are combined as described in the table below:

TABLE-US-00001 CDRH1 CDRH2 CDRH3 CDRL1 CDRL2 CDRL3 SEQ ID NO: 41 42 43 17 18 19 SEQ ID NO: 44 45 46 20 21 22 SEQ ID NO: 47 48 49 23 24 25 SEQ ID NO: 50 51 52 26 27 28 SEQ ID NO: 53 54 55 29 30 31 SEQ ID NO: 56 57 58 32 33 34 SEQ ID NO: 59 60 61 35 36 37 SEQ ID NO: 62 63 64 38 39 40 SEQ ID NO: 41 45 46 20 21 22 SEQ ID NO: 41 48 49 23 24 25 SEQ ID NO: 41 51 52 26 27 28 SEQ ID NO: 41 54 55 29 30 31 SEQ ID NO: 41 57 58 32 33 34 SEQ ID NO: 41 60 61 35 36 37 SEQ ID NO: 41 63 64 38 39 40 SEQ ID NO: 44 42 43 17 18 19 SEQ ID NO: 44 48 49 23 24 25 SEQ ID NO: 44 51 52 26 27 28 SEQ ID NO: 44 54 55 29 30 31 SEQ ID NO: 44 57 58 32 33 34 SEQ ID NO: 44 60 61 35 36 37 SEQ ID NO: 44 63 64 38 39 40 SEQ ID NO: 47 42 43 17 18 19 SEQ ID NO: 47 45 46 20 21 22 SEQ ID NO: 47 51 52 26 27 28 SEQ ID NO: 47 54 55 29 30 31 SEQ ID NO: 47 57 58 32 33 34 SEQ ID NO: 47 60 61 35 36 37 SEQ ID NO: 47 63 64 38 39 40 SEQ ID NO: 50 42 43 17 18 19 SEQ ID NO: 50 45 46 20 21 22 SEQ ID NO: 50 48 49 23 24 25 SEQ ID NO: 50 54 55 29 30 31 SEQ ID NO: 50 57 58 32 33 34 SEQ ID NO: 50 60 61 35 36 37 SEQ ID NO: 50 63 64 38 39 40 SEQ ID NO: 53 42 43 17 18 19 SEQ ID NO: 53 45 46 20 21 22 SEQ ID NO: 53 48 49 23 24 25 SEQ ID NO: 53 51 52 26 27 28 SEQ ID NO: 53 57 58 32 33 34 SEQ ID NO: 53 60 61 35 36 37 SEQ ID NO: 53 63 64 38 39 40 SEQ ID NO: 56 42 43 17 18 19 SEQ ID NO: 56 45 46 20 21 22 SEQ ID NO: 56 48 49 23 24 25 SEQ ID NO: 56 51 52 26 27 28 SEQ ID NO: 56 54 55 29 30 31 SEQ ID NO: 56 60 61 35 36 37 SEQ ID NO: 56 63 64 38 39 40 SEQ ID NO: 59 42 43 17 18 19 SEQ ID NO: 59 45 46 20 21 22 SEQ ID NO: 59 48 49 23 24 25 SEQ ID NO: 59 51 52 26 27 28 SEQ ID NO: 59 54 55 29 30 31 SEQ ID NO: 59 57 58 32 33 34 SEQ ID NO: 59 63 64 38 39 40 SEQ ID NO: 62 42 43 17 18 19 SEQ ID NO: 62 45 46 20 21 22 SEQ ID NO: 62 48 49 23 24 25 SEQ ID NO: 62 51 52 26 27 28 SEQ ID NO: 62 54 55 29 30 31 SEQ ID NO: 62 57 58 32 33 34 SEQ ID NO: 62 60 61 35 36 37 SEQ ID NO: 44 42 46 20 21 22 SEQ ID NO: 47 42 49 23 24 25 SEQ ID NO: 50 42 52 26 27 28 SEQ ID NO: 53 42 55 29 30 31 SEQ ID NO: 56 42 58 32 33 34 SEQ ID NO: 59 42 61 35 36 37 SEQ ID NO: 62 42 64 38 39 40 SEQ ID NO: 41 45 43 17 18 19 SEQ ID NO: 47 45 49 23 24 25 SEQ ID NO: 50 45 52 26 27 28 SEQ ID NO: 53 45 55 29 30 31 SEQ ID NO: 56 45 58 32 33 34 SEQ ID NO: 59 45 61 35 36 37 SEQ ID NO: 62 45 64 38 39 40 SEQ ID NO: 41 48 43 17 18 19 SEQ ID NO: 44 48 46 20 21 22 SEQ ID NO: 50 48 52 26 27 28 SEQ ID NO: 53 48 55 29 30 31 SEQ ID NO: 56 48 58 32 33 34 SEQ ID NO: 59 48 61 35 36 37 SEQ ID NO: 62 48 64 38 39 40 SEQ ID NO: 41 51 43 17 18 19 SEQ ID NO: 44 51 46 20 21 22 SEQ ID NO: 47 51 49 23 24 25 SEQ ID NO: 53 51 55 29 30 31 SEQ ID NO: 56 51 58 32 33 34 SEQ ID NO: 59 51 61 35 36 37 SEQ ID NO: 62 51 64 38 39 40 SEQ ID NO: 41 54 43 17 18 19 SEQ ID NO: 44 54 46 20 21 22 SEQ ID NO: 47 54 49 23 24 25 SEQ ID NO: 50 54 52 26 27 28 SEQ ID NO: 56 54 58 32 33 34 SEQ ID NO: 59 54 61 35 36 37 SEQ ID NO: 62 54 64 38 39 40 SEQ ID NO: 41 57 43 17 18 19 SEQ ID NO: 44 57 46 20 21 22 SEQ ID NO: 47 57 49 23 24 25 SEQ ID NO: 50 57 52 26 27 28 SEQ ID NO: 53 57 55 29 30 31 SEQ ID NO: 59 57 61 35 36 37 SEQ ID NO: 62 57 64 38 39 40 SEQ ID NO: 41 60 43 17 18 19 SEQ ID NO: 44 60 46 20 21 22 SEQ ID NO: 47 60 49 23 24 25 SEQ ID NO: 50 60 52 26 27 28 SEQ ID NO: 53 60 55 29 30 31 SEQ ID NO: 56 60 58 32 33 34 SEQ ID NO: 62 60 64 38 39 40 SEQ ID NO: 41 63 43 17 18 19 SEQ ID NO: 44 63 46 20 21 22 SEQ ID NO: 47 63 49 23 24 25 SEQ ID NO: 50 63 52 26 27 28 SEQ ID NO: 53 63 55 29 30 31 SEQ ID NO: 56 63 58 32 33 34 SEQ ID NO: 59 63 61 35 36 37 SEQ ID NO: 44 45 43 20 21 22 SEQ ID NO: 47 48 43 23 24 25 SEQ ID NO: 50 51 43 26 27 28 SEQ ID NO: 53 54 43 29 30 31 SEQ ID NO: 56 57 43 32 33 34 SEQ ID NO: 59 60 43 35 36 37 SEQ ID NO: 62 63 43 38 39 40 SEQ ID NO: 41 42 46 17 18 19 SEQ ID NO: 47 48 46 23 24 25 SEQ ID NO: 50 51 46 26 27 28 SEQ ID NO: 53 54 46 29 30 31 SEQ ID NO: 56 57 46 32 33 34 SEQ ID NO: 59 60 46 35 36 37 SEQ ID NO: 62 63 46 38 39 40 SEQ ID NO: 41 42 49 17 18 19 SEQ ID NO: 44 45 49 20 21 22 SEQ ID NO: 50 51 49 26 27 28 SEQ ID NO: 53 54 49 29 30 31 SEQ ID NO: 56 57 49 32 33 34 SEQ ID NO: 59 60 49 35 36 37 SEQ ID NO: 62 63 49 38 39 40 SEQ ID NO: 41 42 52 17 18 19 SEQ ID NO: 44 45 52 20 21 22 SEQ ID NO: 47 48 52 23 24 25 SEQ ID NO: 53 54 52 29 30 31 SEQ ID NO: 56 57 52 32 33 34 SEQ ID NO: 59 60 52 35 36 37 SEQ ID NO: 62 63 52 38 39 40 SEQ ID NO: 41 42 55 17 18 19 SEQ ID NO: 44 45 55 20 21 22 SEQ ID NO: 47 48 55 23 24 25 SEQ ID NO: 50 51 55 26 27 28 SEQ ID NO: 56 57 55 32 33 34 SEQ ID NO: 59 60 55 35 36 37 SEQ ID NO: 62 63 55 38 39 40 SEQ ID NO: 41 42 58 17 18 19 SEQ ID NO: 44 45 58 20 21 22 SEQ ID NO: 47 48 58 23 24 25 SEQ ID NO: 50 51 58 26 27 28 SEQ ID NO: 53 54 58 29 30 31 SEQ ID NO: 59 60 58 35 36 37 SEQ ID NO: 62 63 58 38 39 40 SEQ ID NO: 41 42 61 17 18 19 SEQ ID NO: 44 45 61 20 21 22 SEQ ID NO: 47 48 61 23 24 25 SEQ ID NO: 50 51 61 26 27 28 SEQ ID NO: 53 54 61 29 30 31 SEQ ID NO: 56 57 61 32 33 34 SEQ ID NO: 62 63 61 38 39 40 SEQ ID NO: 41 42 64 17 18 19 SEQ ID NO: 44 45 64 20 21 22 SEQ ID NO: 47 48 64 23 24 25 SEQ ID NO: 50 51 64 26 27 28 SEQ ID NO: 53 54 64 29 30 31 SEQ ID NO: 56 57 64 32 33 34 SEQ ID NO: 59 60 64 35 36 37 SEQ ID NO: 44 45 46 17 21 22 SEQ ID NO: 47 48 49 17 24 25 SEQ ID NO: 50 51 52 17 27 28 SEQ ID NO: 53 54 55 17 30 31 SEQ ID NO: 56 57 58 17 33 34 SEQ ID NO: 59 60 61 17 36 37 SEQ ID NO: 62 63 64 17 39 40 SEQ ID NO: 41 42 43 20 18 19 SEQ ID NO: 47 48 49 20 24 25 SEQ ID NO: 50 51 52 20 27 28 SEQ ID NO: 53 54 55 20 30 31 SEQ ID NO: 56 57 58 20 33 34 SEQ ID NO: 59 60 61 20 36 37 SEQ ID NO: 62 63 64 20 39 40 SEQ ID NO: 41 42 43 23 18 19 SEQ ID NO: 44 45 46 23 21 22 SEQ ID NO: 50 51 52 23 27 28 SEQ ID NO: 53 54 55 23 30 31 SEQ ID NO: 56 57 58 23 33 34 SEQ ID NO: 59 60 61 23 36 37 SEQ ID NO: 62 63 64 23 39 40 SEQ ID NO: 41 42 43 26 18 19 SEQ ID NO: 44 45 46 26 21 22 SEQ ID NO: 47 48 49 26 24 25 SEQ ID NO: 53 54 55 26 30 31 SEQ ID NO: 56 57 58 26 33 34 SEQ ID NO: 59 60 61 26 36 37 SEQ ID NO: 62 63 64 26 39 40 SEQ ID NO: 41 42 43 29 18 19 SEQ ID NO: 44 45 46 29 21 22 SEQ ID NO: 47 48 49 29 24 25 SEQ ID NO: 50 51 52 29 27 28 SEQ ID NO: 56 57 58 29 33 34 SEQ ID NO: 59 60 61 29 36 37 SEQ ID NO: 62 63 64 29 39 40 SEQ ID NO: 41 42 43 32 18 19 SEQ ID NO: 44 45 46 32 21 22 SEQ ID NO: 47 48 49 32 24 25 SEQ ID NO: 50 51 52 32 27 28 SEQ ID NO: 53 54 55 32 30 31 SEQ ID NO: 59 60 61 32 36 37 SEQ ID NO: 62 63 64 32 39 40 SEQ ID NO: 41 42 43 35 18 19 SEQ ID NO: 44 45 46 35 21 22 SEQ ID NO: 47 48 49 35 24 25 SEQ ID NO: 50 51 52 35 27 28 SEQ ID NO: 53 54 55 35 30 31 SEQ ID NO: 56 57 58 35 33 34 SEQ ID NO: 62 63 64 35 39 40 SEQ ID NO: 41 42 43 38 18 19 SEQ ID NO: 44 45 46 38 21 22 SEQ ID NO: 47 48 49 38 24 25 SEQ ID NO: 50 51 52 38 27 28 SEQ ID NO: 53 54 55 38 30 31 SEQ ID NO: 56 57 58 38 33 34 SEQ ID NO: 59 60 61 38 36 37 SEQ ID NO: 44 45 46 20 18 22 SEQ ID NO: 47 48 49 23 18 25 SEQ ID NO: 50 51 52 26 18 28 SEQ ID NO: 53 54 55 29 18 31 SEQ ID NO: 56 57 58 32 18 34 SEQ ID NO: 59 60 61 35 18 37 SEQ ID NO: 62 63 64 38 18 40 SEQ ID NO: 41 42 43 17 21 19 SEQ ID NO: 47 48 49 23 21 25 SEQ ID NO: 50 51 52 26 21 28 SEQ ID NO: 53 54 55 29 21 31 SEQ ID NO: 56 57 58 32 21 34 SEQ ID NO: 59 60 61 35 21 37 SEQ ID NO: 62 63 64 38 21 40 SEQ ID NO: 41 42 43 17 24 19

SEQ ID NO: 44 45 46 20 24 22 SEQ ID NO: 50 51 52 26 24 28 SEQ ID NO: 53 54 55 29 24 31 SEQ ID NO: 56 57 58 32 24 34 SEQ ID NO: 59 60 61 35 24 37 SEQ ID NO: 62 63 64 38 24 40 SEQ ID NO: 41 42 43 17 27 19 SEQ ID NO: 44 45 46 20 27 22 SEQ ID NO: 47 48 49 23 27 25 SEQ ID NO: 53 54 55 29 27 31 SEQ ID NO: 56 57 58 32 27 34 SEQ ID NO: 59 60 61 35 27 37 SEQ ID NO: 62 63 64 38 27 40 SEQ ID NO: 41 42 43 17 30 19 SEQ ID NO: 44 45 46 20 30 22 SEQ ID NO: 47 48 49 23 30 25 SEQ ID NO: 50 51 52 26 30 28 SEQ ID NO: 56 57 58 32 30 34 SEQ ID NO: 59 60 61 35 30 37 SEQ ID NO: 62 63 64 38 30 40 SEQ ID NO: 41 42 43 17 33 19 SEQ ID NO: 44 45 46 20 33 22 SEQ ID NO: 47 48 49 23 33 25 SEQ ID NO: 50 51 52 26 33 28 SEQ ID NO: 53 54 55 29 33 31 SEQ ID NO: 59 60 61 35 33 37 SEQ ID NO: 62 63 64 38 33 40 SEQ ID NO: 41 42 43 17 36 19 SEQ ID NO: 44 45 46 20 36 22 SEQ ID NO: 47 48 49 23 36 25 SEQ ID NO: 50 51 52 26 36 28 SEQ ID NO: 53 54 55 29 36 31 SEQ ID NO: 56 57 58 32 36 34 SEQ ID NO: 62 63 64 38 36 40 SEQ ID NO: 41 42 43 17 39 19 SEQ ID NO: 44 45 46 20 39 22 SEQ ID NO: 47 48 49 23 39 25 SEQ ID NO: 50 51 52 26 39 28 SEQ ID NO: 53 54 55 29 39 31 SEQ ID NO: 56 57 58 32 39 34 SEQ ID NO: 59 60 61 35 39 37 SEQ ID NO: 44 45 46 20 21 19 SEQ ID NO: 47 48 49 23 24 19 SEQ ID NO: 50 51 52 26 27 19 SEQ ID NO: 53 54 55 29 30 19 SEQ ID NO: 56 57 58 32 33 19 SEQ ID NO: 59 60 61 35 36 19 SEQ ID NO: 62 63 64 38 39 19 SEQ ID NO: 41 42 43 17 18 22 SEQ ID NO: 47 48 49 23 24 22 SEQ ID NO: 50 51 52 26 27 22 SEQ ID NO: 53 54 55 29 30 22 SEQ ID NO: 56 57 58 32 33 22 SEQ ID NO: 59 60 61 35 36 22 SEQ ID NO: 62 63 64 38 39 22 SEQ ID NO: 41 42 43 17 18 25 SEQ ID NO: 44 45 46 20 21 25 SEQ ID NO: 50 51 52 26 27 25 SEQ ID NO: 53 54 55 29 30 25 SEQ ID NO: 56 57 58 32 33 25 SEQ ID NO: 59 60 61 35 36 25 SEQ ID NO: 62 63 64 38 39 25 SEQ ID NO: 41 42 43 17 18 28 SEQ ID NO: 44 45 46 20 21 28 SEQ ID NO: 47 48 49 23 24 28 SEQ ID NO: 53 54 55 29 30 28 SEQ ID NO: 56 57 58 32 33 28 SEQ ID NO: 59 60 61 35 36 28 SEQ ID NO: 62 63 64 38 39 28 SEQ ID NO: 41 42 43 17 18 31 SEQ ID NO: 44 45 46 20 21 31 SEQ ID NO: 47 48 49 23 24 31 SEQ ID NO: 50 51 52 26 27 31 SEQ ID NO: 56 57 58 32 33 31 SEQ ID NO: 59 60 61 35 36 31 SEQ ID NO: 62 63 64 38 39 31 SEQ ID NO: 41 42 43 17 18 34 SEQ ID NO: 44 45 46 20 21 34 SEQ ID NO: 47 48 49 23 24 34 SEQ ID NO: 50 51 52 26 27 34 SEQ ID NO: 53 54 55 29 30 34 SEQ ID NO: 59 60 61 35 36 34 SEQ ID NO: 62 63 64 38 39 34 SEQ ID NO: 41 42 43 17 18 37 SEQ ID NO: 44 45 46 20 21 37 SEQ ID NO: 47 48 49 23 24 37 SEQ ID NO: 50 51 52 26 27 37 SEQ ID NO: 53 54 55 29 30 37 SEQ ID NO: 56 57 58 32 33 37 SEQ ID NO: 62 63 64 38 39 37 SEQ ID NO: 41 42 43 17 18 40 SEQ ID NO: 44 45 46 20 21 40 SEQ ID NO: 47 48 49 23 24 40 SEQ ID NO: 50 51 52 26 27 40 SEQ ID NO: 53 54 55 29 30 40 SEQ ID NO: 56 57 58 32 33 40 SEQ ID NO: 59 60 61 35 36 40

[0050] The antibody, recombinant or synthetic antigen-binding fragments thereof according to the present invention preferably comprises a heavy chain variable region comprising SEQ ID NO: 15 and a light chain variable region comprising SEQ ID NO: 16.

[0051] In the present invention "at least 80% identity" means that the identity may be at least 80% or at least 85% or 90% or 95% or 100% sequence identity to referred sequences.

[0052] A derivative of said antibody is also object of the invention; wherein the derivative is capable of binding VEMHHW epitope on TrkA receptor.

[0053] The anti-TrkA antibodies of the present invention may comprise any suitable framework variable domain sequence, provided that the binding activity to TrkA is substantially retained. For example, the anti-TrkA antibodies of the invention comprise a human subgroup III heavy chain framework consensus sequence. For example, the framework consensus sequence comprises the heavy chain variable domain sequence of claim 1 of U.S. Pat. No. 7,608,453 B2 which is incorporated herein by reference:

TABLE-US-00002 (SEQ ID NO: 102) QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVSV ISGDGSNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGD YWGQGTLVTVSS.

[0054] The antibody, recombinant or synthetic antigen-binding fragments thereof according to the invention is preferably a monoclonal antibody or a chimeric or a humanized, or a deimmunized or an affinity matured antibody or a fully human antibody.

[0055] Another object of the invention is also a nucleic acid molecule encoding the antibody, recombinant or synthetic antigen-binding fragments thereof as above defined, preferably said nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NOs: 90 (CRB0089_IgG4 derived VH--CH1-H--CH2-CH3) and 91 (CRB0089_VLCL).

[0056] Other objects of the invention are an expression vector encoding the antibody recombinant or synthetic antigen-binding fragments thereof of the invention, a host cell comprising said nucleic acid which preferably produces the antibody, recombinant or synthetic antigen-binding fragments thereof of the invention.

[0057] A further object of the invention is a method of producing the antibody, recombinant or synthetic antigen-binding fragments thereof of the invention comprising culturing the above cell that produces the antibody as described above and recovering the antibody from the cell culture.

[0058] The antibodies are useful for therapeutic applications in humans. Typically, the antibodies are fully human or chimeric or humanized to minimize the risk for immune responses against the antibodies when administered to a patient. As described herein, other antigen-binding molecules such as, e.g., antigen-binding antibody fragments, antibody derivatives, and multispecific molecules, can be designed or derived from such antibodies.

[0059] Antibody-binding fragments of such antibodies, as well as molecules comprising such antigen-binding fragments, including engineered antibody fragments, antibody derivatives, bispecific antibodies and other multispecific molecules, are also objects of the invention.

[0060] It is a further object of the invention, the antibody, recombinant or synthetic antigen-binding fragments thereof according to the invention for use as a medicament, preferably, for use in the treatment of pain, cancer, neuronal disorders, inflammation-related diseases, diabetes.

[0061] It is another object of the invention a pharmaceutical composition comprising at least one antibody, recombinant or synthetic antigen-binding fragments thereof as described above and pharmaceutically acceptable excipients.

[0062] Pharmaceutical compositions comprising the antibody and/or a fragment and/or a recombinant derivative and/or a conjugate thereof in admixture with at least one pharmaceutically acceptable excipient and/or vehicle are included in the scope of the present invention.

[0063] In a preferred embodiment, the composition according to the invention is for use in parenteral administration.

[0064] A further object of the invention is the use of the antibody, recombinant or synthetic antigen-binding fragments thereof according to the invention for inhibiting TrkA.

[0065] It is another object of the invention a method of reducing and/or inhibiting TrkA comprising administering an effective amount of the antibody, recombinant or synthetic antigen-binding fragments thereof as described above.

[0066] In the present invention mutants of the disclosed CDRs may be generated by mutating one or more amino acids in the sequence of the CDRs. It is known that a single amino acid substitution appropriately positioned in a CDR can be sufficient to improve the affinity. Researchers have used site directed mutagenesis to increase affinity of some immunoglobulin products by about 10 fold. This method of increasing or decreasing (i.e., modulating) affinity of antibodies by mutating CDRs is common knowledge (see, e.g., Paul, W. E., 1993). Thus, the substitution, deletion, or addition of amino acids to the CDRs of the invention to increase or decrease (i.e., modulate) binding affinity or specificity is also within the scope of this invention.

[0067] For sake of brevity, the preferred antibody according to the present invention is identified with the name CRB0089_IgG4 (comprising SEQ ID NO: 15 and SEQ ID NO: 16). While the present invention focuses on such antibody, as an exemplification of the present invention, one of ordinary skill in the art will appreciate that, once given the present disclosure, other similar antibodies, and antibody fragments thereof, as well as antibody fragments of these similar antibodies may be produced and used within the scope of the present invention. Such similar antibodies may be produced by a reasonable amount of experimentation by those skilled in the art.

[0068] Still preferably, the antibody is a scFv, Fv fragment, a Fab fragment, a F(ab)2 fragment, a multimeric antibody, a peptide or a proteolytic fragment containing the epitope binding region. Preferably the scFv fragment comprises a sequence selected from the group of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16.

[0069] It is a further object of the present invention a nucleic acid encoding the antibody or functional derivatives thereof of the invention, or hybridizing with the above nucleic acid, or consisting of a degenerated sequence thereof.

[0070] The process for the preparation of the monoclonal antibody is within the skills of the man skilled in the art and comprises cultivating host cell and isolating the antibody according to standard procedures.

[0071] As far as the industrial aspects of the present invention are concerned, the antibody herein disclosed shall be suitably formulated in pharmaceutical compositions as normally done in this technical field.

[0072] The antibodies of the present invention may comprises at least one CDRH as defined above that contains one or more amino acid substitutions, deletions or insertions of no more than 4 amino acids, preferably of no more than 2 amino acids. The antibodies of the present invention may further comprises at least one CDRL as defined above that contains one or more amino acid substitutions, deletions or insertions of no more than 4 amino acids, preferably of no more than 2 amino acids.

[0073] In some aspects, the invention comprises a method for treating or preventing pain, cancer, neuronal disorders, inflammation-related diseases, diabetes, the method comprising administering to a subject in need thereof an effective amount of at least one antibody, recombinant or synthetic antigen-binding fragments thereof of the invention simultaneously or sequentially with an agent that specifically blocks said disease.

[0074] The antibody, recombinant or synthetic antigen-binding fragments thereof of the invention are neutralizing antibody (i.e. an antibody that reduces or abolishes the biological activity of the related antigen) that binds to TrkA and reduces the likelihood that TrkA binds to NGF.

[0075] Preferably, the antibody, recombinant or synthetic antigen-binding fragments thereof of the invention binds to TrkA at a location that overlaps with a location at which NGF binds to TrkA.

[0076] The invention provides formulations comprising a therapeutically effective amount of an antibody as disclosed herein, a buffer maintaining the pH in the range from about 4.5 to about 7.5, and, optionally, a surfactant.

[0077] The formulations are typically for an antibody as disclosed herein, recombinant or synthetic antigen-binding fragments thereof of the invention as active principle concentration from about 0.1 mg/ml to about 100 mg/ml. In certain embodiments, the antibody, recombinant or synthetic antigen-binding fragments thereof concentration is from about 0.1 mg/ml to about 100 mg/ml. For the purposes herein, a "pharmaceutical composition" is one that is adapted and suitable for administration to a mammal, especially a human. Thus, the composition can be used to treat a disease or disorder in the mammal. Moreover, the antibody in the composition has been subjected to one or more purification or isolation steps, such that contaminant(s) that might interfere with its therapeutic use have been separated therefrom. Generally, the pharmaceutical composition comprises the therapeutic protein and a pharmaceutically acceptable carrier or diluent. The composition is usually sterile and may be lyophilized. Pharmaceutical preparations are described in more detail below.

[0078] Therapeutic formulations of the antibody/antibodies can be prepared by mixing the antibody having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed., 1980), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and may include buffers, antioxidants, preservatives, peptides, proteins, hydrophilic polymers, chelating agents such as EDTA, sugars, salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN.RTM., PLURONICS.RTM. or polyethylene glycol (PEG).

[0079] The active ingredients may also be entrapped in microcapsule prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (16th edition, Osol, A. Ed., 1980). The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0080] In another embodiment, for the prevention or treatment of disease, the appropriate dosage of anti-TrkA antibody/antibodies of the present invention, will depend on the type of disease to be treated, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 .mu.g/kg to 15 mg/kg of antibody or fragment thereof is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. The progress of this therapy is easily monitored by conventional techniques and assays.

[0081] The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

[0082] An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.

[0083] Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0084] An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of the antibody to its antigen in a competition assay by 50% or more. An exemplary competition assay is provided herein.

[0085] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.

[0086] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called [alpha], [delta], [epsilon], [gamma], and [mu], respectively.

[0087] The term "Fc region" herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

[0088] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0089] The terms "full length antibody," "intact antibody," and "whole antibody" are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.

[0090] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0091] A "human antibody" is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

[0092] A "human consensus framework" is a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3.

[0093] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0094] A "deimmunized" antibody is an antibody with reduced immunogenicity based on disruption of HLA binding, an underlying requirement for T cell stimulation.

[0095] The term "hypervariable region" or "HVR," as used herein refers to each of the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops ("hypervariable loops"). Generally, native four-chain antibodies comprise six HVRs; three in the VH (HI, H2, H3), and three in the VL (LI, L2, L3). HVRs generally comprise amino acid residues from the hypervariable loops and/or from the "complementarity determining regions" (CDRs), the latter being of highest sequence variability and/or involved in antigen recognition. Exemplary hypervariable loops occur at amino acid residues 26-32 (LI), 50-52 (L2), 91-96 (L3), 26-32 (HI), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917, 1987). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acid residues 24-34 of LI, 50-56 of L2, 89-97 of L3, 31-35B of HI, 50-65 of H2, and 95-102 of H3 (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991). With the exception of CDR1 in VH, CDRs generally comprise the amino acid residues that form the hypervariable loops. CDRs also comprise "specificity determining residues," or "SDRs," which are residues that contact antigen. SDRs are contained within regions of the CDRs called abbreviated-CDRs, or a-CDRs. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) occur at amino acid residues 31-34 of LI, 50-55 of L2, 89-96 of L3, 31-35B of HI, 50-58 of H2, and 95-102 of H3 (See Almagro and Fransson, Front. Biosci. 13: 1619-1633, 2008). Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al.

[0096] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.

[0097] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0098] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs, See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91, 2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively (See, e.g., Portolano et al., J. Immunol. 150:880-887, 1993; Clarkson et al., Nature 352:624-628, 1991).

[0099] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors".

[0100] In another aspect, the antibody or derivatives thereof comprises a heavy chain variable domain (VH) sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group of: SEQ ID NOs: 15, 1, 3, 5, 7, 9, 11 and 13.

[0101] In another aspect, the antibody or derivatives thereof comprises a light chain variable domain (VK or VL) sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group of: SEQ ID NOs: 16, 2, 4, 6, 8, 10, 12 and 14.

[0102] In certain embodiments, the VH sequence or VK/VL sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NOs: 15, 1, 3, 5, 7, 9, 11 and 13 and SEQ ID NOs: 16, 2, 4, 6, 8, 10, 12 and 14, respectively, contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-TrkA antibody comprising that sequence retains the ability to bind to the TrkAd5 domain. In certain embodiments, a total of 1 to 4 amino acids have been substituted, inserted and/or deleted in the sequence of the CDRH3 such as in SEQ ID NO: 49. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs).

[0103] Preferably, the antibody of the invention is an ScFv antibody CRB0022, CRB0036, CRB0069, CRB0072, CRB0082, CRB0084, CRB0088, CRB0089 (SEQ ID NOs: 94, 95, 96, 97, 98, 99, 101 and 100, respectively). The respective IgG4 are also part of the invention.

[0104] In certain embodiments, the antibody, recombinant or synthetic antigen-binding fragments thereof of the invention has a dissociation constant (Kd) of <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM or less, e.g. from 10.sup.-8 M to 10.sup.-13 M, e.g., from 10.sup.-9 M to 10.sup.-13 M.

[0105] In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA) performed with the Fab version of an antibody of interest and its antigen as described by the following assay. Solution binding affinity of Fabs for antigen is measured by equilibrating Fab with a minimal concentration of (I)-labeled antigen in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol. 293:865-881(1999)).

[0106] Recombinant and/or biotechnological derivatives as well as fragments of any of the above-disclosed anti-TrkA antibodies also fall within the scope of the invention, provided that the binding activity to TrkA is substantially retained.

[0107] Within the scope of the present invention are also anti-TrkA antibodies that compete with any of the above-disclosed anti-TrkA antibodies for binding to TrkA.

An anti-TrkA antibody falling within the scope of the present invention is preferably a monoclonal antibody, which is for example produced by recombinant techniques. As an alternative, it is a polyclonal antibody.

[0108] A chimeric antibody is for example an antibody comprising antigen binding sequences from a non-human donor grafted to a heterologous non-human, human, or humanized sequence (e.g., framework and/or constant domain sequences). In one embodiment, the non-human donor is a mouse. In a further embodiment, an antigen binding sequence is synthetic, e.g., obtained by mutagenesis (e.g., phage display or SPLINT screening, etc.). In a particular embodiment, a chimeric antibody of the invention has murine V regions and a human C region. In one embodiment, the murine light chain V region is fused to a human kappa light chain. In another embodiment, the murine heavy chain V region is fused to a human IgG1 or a IgG4 C region.

[0109] In some embodiments, the antibodies of the invention are of the IgG class (e.g., IgG1 or IgG4) and comprise at least one mutation in E233, L234, G236, D265, D270, N297, E318, K320, K322, A327, A330, P331, and/or P329 (numbering according to the EU index). In some embodiments, the antibodies comprise the mutations L234A/L235A or D265A/N297A.

[0110] Antibodies of the IgG4 isotype are shown to be dynamic molecules, undergoing Fab arm exchange in vivo and in vitro. The ability to engage in Fab arm exchange appears to be an inherent feature of IgG4 that involves the third constant domain in addition to the hinge region and that only requires a reducing environment to be activated. In some embodiments, the antibodies of the invention are characterized by S228P mutation in the IgG4 core-hinge that was demonstrated to be involved in the reduction in IgG4 half antibody formation.

[0111] The antibodies of the invention bind (such as specifically bind) TrkA and in some embodiments, they modulate (e.g., inhibit) one or more aspects of TrkA signaling (such as TrkA phosphorylation) and/or neutralization of any biologically relevant TrkA and/or TrkA ligand biological pathway and/or treatment or prevention of a disorder associated with TrkA activation (such as increased TrkA expression and/or activity).

[0112] The inventors have also found that an isolated full length IgG anti-TrkA antibody of the present invention generally binds human TrkA with a Kd in a nM range or stronger. As is well-established in the art, binding affinity of a ligand to its receptor can be determined using any of a variety of assays, and expressed in terms of a variety of quantitative values. Accordingly, the binding affinity is expressed as Kd values and reflects intrinsic binding affinity (e.g., with minimized avidity effects). Generally and preferably, binding affinity is measured in vitro, whether in a cell-free or cell-associated setting. Any of a number of assays known in the art, including those described herein, can be used to obtain binding affinity measurements, including, for example, Biacore and ELISA.

[0113] In some embodiments, the anti-TrkA antibody of the present invention specifically binds to a polypeptide consisting of or consisting essentially of a TrkA (e.g., a human or mouse TrkA), preferably with a Kd of 1.times.10.sup.-8 M or stronger.

[0114] The anti-TrkA antibody of the present invention, as well as the derivatives and fragments thereof, which are capable of recognising and binding the NGF-binding site of TrkA, are advantageously effective in a number of applications, including those discussed herein below.

[0115] In a further aspect of the invention there is provided a compound of the invention for the treatment of a disorder for which a TrkA inhibitor is indicated.

In a further aspect of the invention there is provided use of a compound of the invention for the preparation of a medicament for the treatment of a disorder for which a TrkA inhibitor is indicated.

[0116] In a further aspect of the invention there is provided a method of treating a disorder in an animal (preferably a mammal, more preferably a human) for which a TrkA inhibitor is indicated, comprising administering to said animal a therapeutically effective amount of a compound of the invention.

[0117] Disorder for which a TrkA inhibitor is indicated to include pain, particularly neuropathic, nociceptive and inflammatory pain.

[0118] The antibody, recombinant or synthetic antigen-binding fragments thereof according to the present invention are effective in the treatment of both chronic pain and acute pain. The treatment of chronic pain is preferred. The pain may for example be associated with any of the following: pancreatitis, kidney stones, IBD, Crohn's disease, post surgical adhesions, gall bladder stones, headaches, dysmenorrhea, musculoskeletal pain, sprains, visceral pain, ovarian cysts, prostatitis (in particular the chronic abatteric variant), cystitis, interstitial cystitis, post-operative pain, pain due to vertebral fracture associated with osteoporosis, migraine, trigeminal neuralgia, pain from burns and/or wounds, pain associated with trauma, neuropathic pain, pain associated with musculoskeletal diseases, rheumatoid arthritis, ankylosing spondylitis, periarticular pathologies, HIV infection. Osteoarthritis, endometriosis, uterine leiomyomas, oncological pain and, in particular, pain from bone metastases are examples of pathological conditions in which associated pain is reduced by treatment with the anti-TrkA antibodies and derivatives of the present invention.

[0119] The antibody, recombinant or synthetic antigen-binding fragments thereof according to the present invention may also be employed to treat cancer. Several tumor types express TrkA. TrkA activation by NGF might underlie tumor growth (e.g. of prostate and pancreatic cancers). TrkA activation by NGF also facilitates the growth and infiltration of nerve fibers into tumor masses. By preventing TrkA activation, it is also possible to significantly reduce the formation of neuromas. Furthermore, the anti-trkA antibodies of the present invention and their derivatives could be coupled to a cytotoxic agent and employed to target cancer cells expressing TrkA.

[0120] The antibody, recombinant or synthetic antigen-binding fragments thereof according to the present invention may also be employed in the treatment of various neuronal disorders. It is known that NGF can be used in the treatment of Alzheimer's disease (Cattaneo et al., 2008. J Alzheimers Dis. 15:255-283). Unfortunately, it also induces hyperalgesia, due to its action on peripheral TrkA receptors. The antibody, recombinant or synthetic antigen-binding fragments thereof according to the present invention may therefore be used in combination with NGF-based treatments to reduce undesired NGF-evoked hyperalgesia.

[0121] The antibody, recombinant or synthetic antigen-binding fragments thereof according to the present invention may also be employed in the treatment of inflammation-related diseases. NGF is released by mast cells, fibroblasts and other cell types in the peripheral sites where inflammatory processes occur. In particular, mast cells appear to play a fundamental role. They produce NGF and at the same time express functional TrkA receptors at their surface. The NGF/TrkA system appears to mediate mastocyte activation through an autocrine positive feedback mechanism which allows local amplification of the algogenic inflammatory signal. Examples of inflammatory disorders that may be treated include inflammatory forms of the urinary tract and of the pelvic region, osteoarthritis, rheumatoid arthritis, asthma.

BRIEF DESCRIPTION OF THE DRAWINGS

[0122] The invention will be described now by non-limiting examples referring to the following figures.

[0123] FIG. 1. shows the NGF bind TrkA extracellular domain (ECD); the loop chosen for the selection of SPLINT libraries on human TrkA ECD comprises sequence VEMHHW.

[0124] FIG. 2. ELISA reactivity of isolated anti-TrkA scFvs versus human TrkA immunoadhesin (hTrkA-Fc) and an unrelated scFv (unr) as negative control. hTrkA-Fc antigen was coated at 2 mg/mL. Anti-TrkA scFvs were used at 50 and/or 5 mg/mL. The mean absorbance at 450 nm of the experiments performed in duplicate wells is shown with SD indicated by the bars.

[0125] FIG. 3. Specificity ELISA of isolated anti-TrkA scFvs versus human TrkA immunoadhesin (hTrkA-Fc), human TrkB immunoadhesins (hTrkB-Fc), human TrkC immunoadhesins (hTrkC-Fc) and human p75 immunoadhesins (hp75-Fc). Human antigens were coated at 2 .mu.g/mL. Anti-TrkA scFvs were used at 50 .mu.g/mL (upper panel) and/or 5 .mu.g/mL (lower panel). The mean absorbance at 450 nm of the experiments performed in duplicate wells is shown with SD indicated by the bars.

[0126] FIG. 4. MTT assay of isolated anti-TrkA scFvs. Cell proliferation in TF1 was measured following incubation with NGF and the indicated anti-TrkA scFvs. The MTT assay is a colorimetric assay for measuring the activity of enzymes that reduce MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole) to formazan dyes, giving a purple color. It is apply in this experiment to assess the proliferation of TF1 cells. Cells were challenged with various concentrations of scFv (20 ng/mL; 200 ng/mL; 2,000 ng/mL and 20,000 ng/mL). % of inhibition of TF1 proliferation was calculated using 10 ng/ml as 100% standard. All anti-TrkA scFvs inhibit NGF-mediated proliferation in a dose-dependent manner.

[0127] FIG. 5. FACS analysis performed with Bioanalyzer 2100. A cell fluid chip-based fluorescent cytometric assay that runs on Bioanalyzer 2100 (Agilent) for fast characterization of small population cell phenotypes was assessed. The assay determines the expression of specific cell surface markers. In this experiment, the specificity of scFvs on TF-1 cell line expressing human TrkA receptor was assessed. Six samples can be analyzed on each chip in one automated process. Results were in good agreement with conventional flow cytometry in quantisation.

[0128] FIG. 6. Evaluation of analgesic activity in the formalin test in the mouse. The licking activity was recorded continuously for 1 hour, calculated in blocks of consecutive 5-minutes periods and analyzed as the early (0-5 min) and the late (15-35 min) phases of the formalin test. Each mouse was subcutaneous injected into the dorsal surface of the right hind paw one hour before the test with 20 .mu.l (40 .mu.g) anti-TrkA scFv or PBS as control groups. The two phases characterizing the formalin test were separately analyzed by unpaired Student's t test (for two groups).

[0129] FIG. 7. Western blot analysis of human TrkA, TrkB, TrkC and p75NTR immunoadhesins.

[0130] FIG. 8. NGF biological assay performed with 3T3TrkA cells. Western blot results showing that CRB0022, CRB0036, CRB0069, CRB0072, CRB0082, CRB0084, CRB0088 and CRB0089 lead a differential reduction of phospho-TrkA levels after NGF stimulation in mouse 3T3 cells overexpressing TrkA.

[0131] FIG. 9. Specificity ELISA of anti-TrkA CRB0089_IgG4 mAb versus human TrkA immunoadhesin (hTrkA-Fc) and human TrkB immunoadhesins (hTrkB-Fc), Human antigens were coated at 2 .mu.g/mL. CRB0089_IgG4 was used at different concentration (20 .mu.g/mL-0.02 .mu.g/mL). The mean absorbance at 450 nm of the experiments performed in duplicate wells is shown with SD indicated by the bars. CRB0089_IgG4 binds hTrkA-Fc in a dose-dependent manner. A minimal binding activity was shown also for hTrkB-Fc but only at higher doses.

[0132] FIG. 10. FACS analysis performed with Bioanalyzer 2100. A cell fluid chip-based fluorescent cytometric assay that runs on Bioanalyzer 2100 (Agilent) for fast characterization of small population cell phenotypes was assessed. In this experiment, the specificity of anti-TrkA CRB0089_IgG4 on TF-1 cell line expressing human TrkA receptor was assessed. Results were in good agreement with conventional flow cytometry in quantisation.

[0133] FIG. 11. Dose response curve with IC50 determination. MTT potency assay was used for the determination of potency of CRB0089_IgG4. Cell proliferation of TF1 cells was measured following incubation with NGF and the indicated concentration of CRB0089_IgG4. CRB0089_IgG4 inhibited NGF-mediated proliferation in a dose-dependent manner. Half-maximal inhibition (IC50) by CRB0089_IgG4 was calculated around 40 nM.

[0134] FIG. 12. Formalin-induced licking behavior in mice: effect of increasing doses of CRB0089_IgG4 injected locally in the right dorsal paw. Each mouse was subcutaneous injected with CRB0089_IgG4 at the indicated doses (5-20 .mu.g of mAb) into the dorsal surface of the right hind paw eighteen hours before formalin injection (1% formalin). The licking activity was recorded continuously for 1 hour, calculated in blocks of consecutive 5-minutes periods and analyzed as the early (0-5 min) and the late (15-35 min) phases of the formalin test. The two phases characterizing the formalin test were separately analyzed by unpaired Student's t test (for two groups).

[0135] FIG. 13. Formalin-induced licking behavior in mice: effect of increasing doses of CRB0089_IgG4 subcutaneously injected. Each mouse was subcutaneously injected (systemically) with 300 .mu.l CRB0089_IgG4 or PBS as control group, 18 hours before the test. As internal control of the experiment, a group of mice (n=8) was treated locally in the right dorsal paw with 20 .mu.g (1 .mu.g/.mu.l) CRB0089_IgG4 as in the previous experiment shown in FIG. 11.

[0136] FIG. 14. CFA-induced inflammatory pain in rats: effect of increasing subcutaneous doses of CRB0089_IgG4. Male Wistar rats were injected into the right hind footpad with 300 .mu.g of Mycobacterium tuberculosis in 100 .mu.L of liquid paraffin (Complete Freund's Adjuvant; CFA). Seventy-two hours later, CRB0089_IgG4 (5 or 20 .mu.g) was administered subcutaneously and 18 h after the administration, the response to noxious mechanical stimulation was assessed by measuring PWT with an analgesimeter of the Randall-Selitto type.

[0137] FIG. 15. CFA-induced inflammatory pain in rats: effect of increasing subcutaneous doses of CRB0089_IgG4 in the controlateral paw 18 h after injection. The response to noxious mechanical stimulation mechanical threshold was measured also in the controlateral non-injured hind paw. A slight but significant hypoalgesic-like effect was recognized after treatment with 20 .mu.g/rat dose of CRB0089_IgG4.

[0138] FIG. 16. CFA-induced paw edema in rats: effect of increasing subcutaneous doses of CRB0089_IgG4 in the ipsilateral paw 18 h after injection. The paw edema induced by the CFA injury in the right hind paw was evaluated. As for the hyperalgesic test, CRB0089_IgG4 was administered 72 h after the CFA injection. Eighteen hours after CRB0089_IgG4 administration the paw volume was measured by means of a Plethysmometer (UgoBasile, Italy).

[0139] FIG. 17. CCI-induced neuropathic pain in rats: effect of a single injection of 20 .mu.g/rat CRB0089_IgG4 on mechanical hyperalgesia. Experiments were performed on male Wistar rats. The response to noxious mechanical stimulation was assessed by measuring PWT with an analgesimeter.

[0140] FIG. 18. Acidic saline-induced pain in rats: effect of a single injection of 20 .mu.g/rat CRB0089_IgG4 on mechanical allodynia. Ipsilateral and contralateral paw withdrawal thresholds in response to mechanical stimuli were measured on Days 0 (baseline--0 d), 5 (5 d), 6 (6 d), 9 (9 d), and 12 (12 d). Nociceptive thresholds, expressed in grams (g), were measured with a Dynamic Plantar Aesthesiometer by applying increasing pressure to the right and left hind paw until the rat withdrew the paw.

DETAILED DESCRIPTION OF THE INVENTION

Examples

Materials and Methods

[0141] SPLINT Library from Human Lymphocytes

[0142] The development of therapeutic antibodies for use in the treatment of human diseases has long been a goal for many researchers in the antibody field. One way to obtain these antibodies is through SPLINT libraries constructed from human lymphocytes. SPLINT technology express human scFv (single chain antibody fragment) libraries cloned in pMV1 vector, a vector derived from pLinker220 vector (Visintin et al., 2004. J Immunol Methods. 290:135-153.), as fusion to the VP16 activation domain. The variable regions are linked with a small peptide linker (SGGSTSGSGKPGSGEGSSGT, SEQ ID NO: 93). pMV1 contains LEU2 gene that permits maintenance of the plasmid and selection on media lacking leucine in yeast strain L40 and the bla gene that permits the selection of plasmid in E. coli.

[0143] For construction of human SPLINT libraries the peripheral blood donations from one hundred, non-immunized donors were used. Approximately 2-20 ml of blood samples from each donor were collected. B-lymphocytes were isolated from peripheral blood by using Ficoll plaque reagent (Amersham, USA). Briefly, the diluted blood sample (1:1 of blood per PBS) was carefully layered on top of the Ficoll plaque reagent, and then the two phase solution was centrifuged at 400.times.g for 30 minutes. B-lymphocytes were collected from the interface between the two phases. Total RNA was extracted from B-lymphocytes by RNeasy Mini Kit (Qiagen) according to manufacturer's instruction. Total RNA was prepared from the B lymphocytes and pooled together before being used for the isolation of mRNA. mRNA was prepared using Oligotex mRNA mini kit (Qiagen) according to manufacturer's instruction. ThermoScript.TM. RT-PCR System (Invitrogen) was used for cDNA synthesis reactions according to manufacturer's instruction.Oligo (dT)20 were used to synthesize cDNA of V-genes repertoire. In order to reduce amplification bias, we performed 62 independent PCR reactions to amplify V gene segments, using all possible combinations within a primer set (data not shown). The primer sequences, which in theory encompass the entire repertoire of human antibody genes, were obtained from IMGT/GENE-DB (Giudicelli et al., 2005. Stud Health Technol Inform. 116:3-8.), and modified according to previously published protocols (Sblattero and Bradbury, 1998. Immunotechnology. 3:271-278; Marks et al., 1991. Eur J Immunol. 21:985-991; Orlandi et al., 1992. Biotechnology. 24:527-531). In this method, the individual rearranged heavy- and light-chain variable regions are amplified separately and are linked through a series of overlap polymerase chain reaction (PCR) steps to give the final scFv products that are used for cloning (Visintin et al., 2004. J Immunol Methods. 290:135-153).

[0144] The PCR reactions included seven VH forward primers paired with four VH reverse primers which generated a total of twenty-eight reactions; whereas four V.kappa. forward primers paired with four reverse primers generated a total of sixteen reactions; and nine V.lamda. forward primers paired with two V.lamda. reverse primers generated a total of eighteen reactions. The PCRs led to the representation in the repertoire of variable regions derived from all conceivable framework assemblies. All primers contained either BssHII or NheI restriction sites or linker sequence. The final pull-through PCR could be done with two primers (PTfw&PTrv). After the final scFv gene repertoires had been sequentially digested with BssHII and NheI, they were ligated directly into pre-digested and dephosphorylated pMV1 vector. From one ligation reaction and thirty electroporations for the library, we were able to obtain the final huSPLINT_09 library consisting of .about.10.sup.8 different scFv molecules with 0.04% of clones from no-insert ligation.

Preparation of TrkA Dimer Bait.

[0145] pMICBD1 (Visintin et al., 2004. J Immunol Methods. 290:135-153) vector was used to clone I27 titin modified to expose two loop of human TrkA (SEQ ID NO 68; SEQ: ID NO: 69). The bait was constructed by assembly PCR. The oligodeoxynucleotides for the first step of assembly PCR (Table V upper panel; SEQ ID NOs: 80, 81, 82, 83, 84, 85, 86 and 87) were diluted to 0.125 .mu.g/.mu.L with double distilled water, while the oligodeoxynucleotides for the second PCR step (Table V lower panel; SEQ ID NOs: 88 and 89) were diluted to 0.25 .mu.g/.mu.L. For the first PCR reaction, 4 .mu.L of each oligo, 4 .mu.L of 5 mM dNTPs, 10 .mu.L of 10.times.PFU buffer (Promega), 1.5 .mu.L of PFU DNA polymerase (Promega, 3 U/.mu.L), and 68.5 .mu.L of double distilled water were combined. This mixture was then subjected to 8 cycles of amplification at 94.degree. C. (1.5 min), 54.degree. C. (2 min), and 72.degree. C. (3 min). During the first cycle, the 94.degree. C. step was performed for 7 min. After the last cycle completed, an additional 5 min 72.degree. C. elongation step was performed. For the second PCR reaction, 1 .mu.L of the crude mixture from the first PCR reaction was mixed with 4 .mu.L of each primer, 4 .mu.L of 5 mM dNTPs, 10 .mu.L of 10.times.PFU buffer (Promega), 1.5 .mu.L of PFU DNA polymerase (Promega, 3 U/.mu.L), and 75.5 .mu.L of double distilled water. This mixture was then subjected to 25 cycles of amplification. Each cycle consisted of a 30 second 94.degree. C. step, a 2 min 54.degree. C. step, and a 1.5 min 72.degree. C. step. Prior to the first cycle, a 5 min 94.degree. C. step was used. A 5 min 72.degree. C. elongation step was included following the final cycle. The PCR mixtures were analyzed by 1.5% agarose gel electrophoresis and then purified by PCR purification kit (Qiagen). The purified cDNA was then first digested with restriction enzymes EcoRI-BamHI. The digested fragment was subsequently run by gel electrophoresis and the isolated DNA was subjected to gel extraction kit (Qiagen). The purified EcoRI-BamHI fragment was subsequently cloned in pMICBD1 vector. The final construct was checked by sequence analysis and western blot analysis and X-gal assay as previously described (Visintin and Cattaneo, 2001. Antibody Engineering. 1:790)(Visintin et al., 2002. J Mol Biol. 317:73-83.).

Selection of Anti-TrkAscFv from SPLINT Library.

[0146] L40 expressing bait TrkA dimer bait was transformed with 250 .mu.g of human SPLINT library according to the protocol described below:

[0147] Day 1: inoculation of L40 containing the bait in 5 ml of YC-UW overnight;

[0148] Day 2: inoculation of 100 ml of YC-UW with an aliquot of the overnight culture in order to have a dilution that allows the algorithmic growth phase to be reached the next day; and

[0149] Day 3: the overnight culture is transferred in 1 l of heated YPAD to obtain a culture with an OD 600=0.3; the yeast is grown at 30.degree. C. for 3 h, the cells are centrifuged at 1500 rpm for 5 min at room temperature; the yeast pellet is washed with 500 ml of 1.times.TE and then centrifuged at 1500 rpm for 5 min at room temperature; the pellet is resuspended in 20 ml of 1.times.LiAC, 0.5.times.TE and transferred to a fresh flask; 250 .mu.g of human SPLINT library and 1 ml of denatured salmon sperm are added; 140 ml of 1.times.LiAc, 40% PEG 3350, 1.times.TE; the product is mixed and incubated in a low-speed agitator for 30 min at 30.degree. C.; 17.6 ml of DMSO are added and mixed. Thermal shock is performed for 10 min at 42.degree. C. while agitating occasionally. The product is quickly cooled by adding 400 ml of YPA. The yeast is pelleted by centrifugation and washed once with 500 ml of YPA. After centrifugation, the pellet is resuspended in 1 l of YPAD preheated to 30.degree. C. The product is incubated for 1 h at 30.degree. C.; 1 ml of the culture is isolated and the pellet obtained by centrifugation of this ml is resuspended in 1 ml of YC-UWL. Dilutions of 1:10, 1:100, and 1:1000 are seeded on YC-UWL plates to calculate the efficiency of the transformation. The pellet obtained from the remaining culture is washed twice with YC-WHULK. The final pellet is resuspended in 10 ml of YC-WHULK. The aliquots are plated on YC-WHULK plates and after 3-4 days the colonies that have grown are analyzed to determine the interaction.

[0150] Thousand colonies grown on YC-WHULK and testing blue on beta-Gal assay were analyzed by PCR-fingerprinting analysis using the BstNI restriction enzyme. The analysis of digestion patterns and scFv sequences isolated from the HIS+lacZ+ colonies permit the isolation of 189 different scFv which 61 that recognize the bait in a secondary screening (specificity screening) (see Table I).

Expression, Refolding and Purification of scFv from E. coli Inclusion Bodies.

[0151] cDNA encoding isolated scFvs were cloned into pETM-13 vector (http://www.embl.de/ExternalInfo/protein_unit/draft_frames_save/flowchart- /clo_vector/frame_our_Ec_vectors.html) for expression and induction of inclusion bodies into the cell cytoplasm of E. coli. Appropriate strain must me employed to maximize expression levels: standard choice is BL21(DE3) strain (Novagen). If the codon usage is very different Rosetta 2 (DE3) (Novagen) can be employed. Single colonies carrying the expression plasmid were cultured O/N at 37.degree. C. Next day diluted overnight growths were grown to OD.sub.600>0.75 and IPTG to 1.5 mM final concentration was added. After 4-5 hours incubation cell were collected and centrifuged at 6000 RPM for 15 minutes. Pellet were frozen at -80.degree. C. Pellet were resuspended in 20 mL of Lysis Buffer (50 mM Tris pH 8, 0.5 mM EDTA, 10 mg/mL DNase, 20 mg/mL Lysozyme). The lysates were incubated by shaking for 1 hour. Then the lysated were subsequently sonicated three times for 45 seconds and keep in ice for 1 minute after each sonication. The lysates were centrifuged at 6000 RPM. After centrifugation the supernatant were discarded. Pellets were then resuspended and vortex in 20 mL wash buffer (10 mM Tris pH 8, 1 mM EDTA, 1% Triton X100). After centrifugation at 10000 RPM for 10 minutes the pellets were subsequently resuspended in 20 mL wash buffer 2 (10 mM Tris pH 8, 1 mM EDTA, 1M NaCl). After centrifugation at 10000 RPM for 10 minutes the pellets were subsequently resuspended in 20 mL wash buffer 3 (10 mM Tris pH 8, 1 mM EDTA). After centrifugation at 10000 RPM for 10 minutes the pellets were subsequently resuspended and vortex in 5 mL/g of solubilization buffer (6M Guanidinium, 100 mM Tris pH 8, 1 mM EDTA, 100 mM DTT). The solubilized samples were incubated shaking for 2 hours at room temperature. After centrifugation at 10000 RPM for 10 minutes the supernatant were pH lowered to 3-4 by adding acetic acid. The samples were dialyzed against 250 mL of 6M Guanidinium pH 3.5 (4 hours at RT or 12 hours at 4.degree. C.), three times changing buffer. After the inclusion bodies have been solubilized by high concentrations of denaturing agents, refolding is then accomplished by the controlled removal of excess denaturant. This was allowed to occur in the presence of a suitable redox system and of other folding promotion agents according to the model of "pulse renaturation". Dilution of the solubilized protein (35 mg/L) directly stirring into the renaturation buffer (0.5M Arginine, 100 mM Tris pH 8.5, 5 mM EDTA, 375 mM L-glutathione oxidized (freshly added) was performed every 50 minutes (incubation at 4.degree. C. in the meantime) till all the solubilized sample has been added. After incubation at 4.degree. C. overnight, the samples were dialyzed against 5 L of IEXA buffer (according to the pI of scFv and thus to ionic exchange protocol subsequently employed) 4 hours at RT or overnight at 4.degree. C., with two buffer changes. Centrifuge at 10000 RPM for 10 min and filter were necessary before ion exchange chromatography. The purified proteins were aliquoted and stored at -80.degree. C. after quantification and analysis by Bioanalyzer 2100 (Agilent).

Preparation of TrkA Immunoadhesins.

[0152] Soluble human and murine TrkA, TrkB, TrkC and p75NTR receptors were engineered as immunoadhesins (Chamow and Ashkenazi, 1996. Trends Biotechnol. 14:52-60) by linking the extracellular domain of the receptors to the Fc portion (immunoglobulin heavy chain constant region) of IgG2a camel antibody (Camelus dromedarius) (SEQ ID NOs: 70, 71, 72, 73 and 74). The DNA sequences coding for the immunoadhesins were cloned into pCDNA3 vector (Invitrogen) for expression in mammalian cell lines and the proteins were purified by Protein A-Sepharose chromatography from culture medium. The purified proteins were aliquoted and stored at -80.degree. C. after quantification and analysis by Bioanalyzer 2100 (Agilent).

[0153] The purified proteins were also subjected to western blot analysis using an anti-Camel antibody (Bethyl) as primary antibody (1:4000) and an anti-rabbit-HRP secondary antibody diluted 1:2000 (DAKO). ECL reagents (Amersham) was used for the detection of the protein according to the manufacturing instruction (FIG. 7).

Specificity ELISA.

[0154] Microtitre plate wells were coated with 50 .mu.l of 2 .mu.g/mL human TrkA immunoadhesin, 10 .mu.g/ml bovine serum albumin (BSA) or PBS (the uncoated well). After preblock of the microtitre plates, 50 .mu.l of soluble scFv from each selected clones (at 50-5 .mu.g/mL concentration) was added to a well coated with hTrkA, BSA or an uncoated well. HRP activity was visualized using TMB (Sigma). The results of eight isolated clones are shown in FIG. 7.

Crossreactivity ELISA.

[0155] Microtitre plate wells were coated with 50 .mu.l of either 2 .mu.g/ml human TrkA immunoadhesin, 2 .mu.g/ml mouse TrkA immunoadhesin, 10 .mu.g/ml bovine serum albumin (BSA) or PBS (the uncoated well). After preblock of the microtitre plates, 50 .mu.l of soluble scFv from each isolated clone was added to a well coated with either hTrkA, mTrkA, BSA or an uncoated well. As above, HRP activity was visualized using TMB (Sigma). Clones were considered to be cross-reacting with human and mouse TrkA if the ELISA signal generated in the mTrkA coated well was at least 0.5-fold less than the signal on hTrkA (data not shown).

Specificity determination by BIACore X-100.TM..

[0156] The antibodies were also shown to be specific for hTrkA and mTrkA by relative binding to the BIACore sensor chips coated with the appropriate antigen. Antibodies were immobilized by ammine coupling to Biosensor CM5 sensorchips (Pharmacia) according to the manufacturers instructions. Anti-TrkA scFv proteins were diluted at 20-50 .mu.g/ml in suitable pre-concentration buffer (at least 2 pH unit below the pI of the scFv in order to get a net positive charge), chosen among the Acetate buffers. The scFvs were immobilized at 100 RU to get a low density immobilization. As binder, the recombinant protein consisting of D4-D5 domain of human and murine TrkA receptor (hTrkA Ig1,2 and mTrkA Ig1,2 respectively) were injected over the immobilized scFv with a contact time of 60 seconds and with a dissociation time of 400 seconds and the assay workflow placed 5 serial dilutions of the TrkA Ig1,2 (starting in the micromolar range and diluting 1:2 each time). The regeneration condition were mild (contact time 30 seconds, 10 mM Glycine pH2). On the basis of the resulting sensorgrams, the concentrations of TrkA Ig1,2 were adjusted to optimize, contact time, dissociation time and regeneration. Data were analysed by Bioevaluation Software (see results on Table II and Table III). The quality of the data fitting were checked by the value of Chi.sup.2 and of the U-value.

NGF Biological Assay with TF1 Cells.

[0157] This is a quantitative assay to measure the functional effect of anti-NGF/TrkA antibodies on the interaction between human NGF and the human TrkA receptor in vitro. TF-1 cells, a human hematopoietic cell line which expresses the native human TrkA receptor but not the p75 NGF-receptor, proliferate in response to exogenous human NGF (Chevalier et al., Blood 1994, vol. 83:1479-85). The TF-1 proliferation assay as described by Chevalier et al., formed the basis for a potency assay to measure the effects of NGF/TrkA neutralising antibodies on the NGF-mediated proliferation of TF1 cells. Before testing with a MTT cell proliferation assay kit (ATCC), TF1 cells are cultured for 1 week in RPMI-1640 containing 10% FBS with 2 ng/ml GM-CSF. Cells for testing are centrifuged (1000 rpm, 5 min), washed (RPMI-1640), centrifuged again and resuspended in RPMI-1640+10% FBS to a concentration of 300,000-400,000 cells/ml. They are then replated on 96-well microplates (15,000-20,000 cells per well in 50 ml) and TrkA neutralising antibodies are added soon after seeding. After 60 min (pre-incubation with TrkA neutralising antibodies), TF1 are exposed to 10 ng/mL NGF in RPMI-1640 containing 10% FBS (50 ml of 2.times.NGF is added per well, each well has a final volume of 100 ml). Control wells are included, either containing medium alone, or containing TF1 cells in the absence of NGF ("cellular blank"). Each treatment is performed in triplicate. After a 40 h incubation period, at 37.degree. C., 5% CO.sub.2, 10 .mu.l of MTT reagent (MTT cell proliferation assay kit) is added for 4 h incubation at 37.degree. C. Thereafter, wells are incubated with Detergent Reagent (MTT cell proliferation assay kit: 100 .mu.l per well; gently mixing, no pipetting) for overnight (O/N) incubation at room temperature in the dark. Absorbance is recorded at 570 nm. 100% inhibition is set as the value of inhibition corresponding to the average O.D. value observed for cells cultured without NGF, in the absence of antibody. 0% inhibition is set as the value of inhibition corresponding to the average O.D. value observed for cells exposed to 10 ng/ml NGF, in the absence of anti-TrkA antibodies.

FACS Analysis of Anti-TrkAscFvs.

[0158] Fluorescence activated cell sorter (FACS) is a powerful tool to measure and analyze cell surface molecules of single cells which flow in fluid stream through a beam of light to detect the fluorescences of the cells. FACS was applied to determine the binding profiling of the various scFv onto TF-1 receptor TrkA. FACS Tests are performed only on population with cell viability>95%. Method is an adaptation of protocols reported on the Application Note "Detection of antibody-stained cell surface and intracellular protein targets with Agilent 2100 Bioanalyzer".

[0159] 10.sup.6 cells/sample=tot: 6.times.10.sup.6 cells are first centrifuged at 350 g for 5 min at room temperature (RT) and wash with 12 ml of Staining Buffer (SB) at RT. The pellet was resuspended in 3 ml of SB at 4.degree. C. and aliquot 0.5 ml of suspension in 1.5 ml conical tubes. Cells were centrifuged again at 350 g for 5 min at 4.degree. C. and the pellet was resuspended in 1 ml HBSS at 4.degree. C. 125 .mu.l 16% p-formaldehyde (final conc 1.7%) was added into each tube and then the sample was incubated for 10 min at 4.degree. C. on tube rotator. After 10 minutes, 75 .mu.l of 10% detergent solution-Tween 20 (final conc 0.6%) was added and the samples were then incubated for 10 min at 4.degree. C. on tube rotator. Samples were then washed once with FACS Buffer (0.8 ml/sample) at 4.degree. C. and then centrifuged at 350 g for 5 min at 4.degree. C. Cells were resuspended with 80 .mu.l/sample of FACS Buffer. 20 .mu.l of a 5.times. primary antibody solution were added (final concentration of scFvs is 5 .mu.g/ml). After incubation for 1 hour at 4.degree. C. on tube rotator samples were washed with FACS Buffer (0.5 ml/sample) at 4.degree. C. and then centrifuged 5 min at 350 g at 4.degree. C. Cells were resuspended with 80 .mu.l/sample of FACS Buffer. 20 .mu.l of a 5.times. secondary antibody solution were added. Final concentration of mouse anti-V5 antibody is 0.20 .mu.g/ml. The samples were incubated for 1 hour at 4.degree. C. on tube rotator. After incubation for 1 hour at 4.degree. C. on tube rotator samples were washed with FACS Buffer (0.5 ml/sample) at 4.degree. C. and then centrifuged 5 min at 350 g at 4.degree. C. Cells were resuspended with 80 .mu.l/sample of FACS Buffer. 20 .mu.l of a 5.times. tertiary antibody solution were added. Final concentration of anti-mouse IgG-Cy5 is 4 .mu.g/ml (1:250 diluted). The samples were incubated for 1 hour at 4.degree. C. on tube rotator. After incubation for 1 hour at 4.degree. C. on tube rotator samples were washed with FACS Buffer (0.5 ml/sample) at 4.degree. C. and then centrifuged 5 min at 350 g at 4.degree. C. Samples were then resuspended in 300 .mu.l of 3 .mu.M SYTO16 diluted in FACS Buffer and incubated for 30 min at 37.degree. C. (no mix). After incubation samples were washed with FACS Buffer (0.5 ml/sample) at 4.degree. C. and then centrifuged 5 min at 350 g at 4.degree. C. Cells were then counted in order to obtain a cell suspension of about 2 million cells/ml diluting the pellet with Cell Buffer (Agilent Cell Kit) (100-50 .mu.l depending on the cell pellet). Cells were loaded into the chip as reported in the Agilent Cell Assay Guide. Samples were analysed by the 2100 Expert Software Assay "Antibody Staining Series II". Gates are chosen according to results obtained from negative controls (unrelated primary antibody).

FACS Analysis of Anti-TrkA mAbs.

[0160] FACS Tests are performed only on population with cell viability>95%. Method is an adaptation of protocols reported on the Application Note "Detection of antibody-stained cell surface and intracellular protein targets with Agilent 2100 Bioanalyzer".

[0161] 10.sup.6 cells/sample=tot: 6.times.10.sup.6 cells are first centrifuged at 350 g for 5 min at room temperature (RT) and wash with 12 ml of Dye Loading Buffer at RT. Cell pellets were resuspended in 2 ml of Dye Loading Buffer. 1 .mu.l of CALCEIN AM was added then 330 .mu.l/sample are aliquoted in eppendorf and incubated for 30 min at 37.degree. C. in termoblock under dim light. After incubation the samples were washed with FACS Buffer (0.5 ml/sample) at 4.degree. C. and then centrifuged 5 min at 350 g at RT. Cells were resuspended in 100 .mu.l/sample of Antibody Solution (con:0.2-20 .mu.g/ml in FACS Buffer) and then incubated for 1 hour at 4.degree. C. on rotating wheel. Proper controls are included (FACS Buffer). After incubation the samples were washed with FACS Buffer (0.5 ml/sample) at 4.degree. C. and then centrifuged 5 min at 350 g at RT. Cells were resuspended in 100 .mu.l/sample of Antibody Solution in FACS buffer 4 .mu.g/ml (1:500) and incubated for 30 minutes at 4.degree. C. on rotating wheel. After incubation the samples were washed with FACS Buffer (0.5 ml/sample) at 4.degree. C. and then centrifuged 5 min at 350 g at RT. Cells were resuspended in 100 .mu.l/sample of Cell Buffer (Component of Agilent Cell Reagents) and proceeded with the loading of the chip as reported in the Agilent Cell Assay Kit Guide).

NGF Biological Assay with 3T3TrkA Cells.

[0162] This assay is described in (Ugolini et al., 2007. Proc Natl Acad Sci USA. 104:2985-2990). 3T3-TrkA cells are cultured in DMEM (+10% FBS+1.times.GlutaMAX+100 units/ml penicillin and 0.1 mg/ml streptomycin) and can be used for the test from 3 days up to 2 months following seeding. The day before the test, cells are seeded in a 6 multi-well plate (2 ml of a suspension containing 5.times.10.sup.5 cells per well). The day after, growth medium is removed and adherent cells are washed with PBS (+Ca/Mg) before being incubated with serum-free medium supplemented with 0.05% BSA for 1 h at 37.degree. C. in CO.sub.2 incubator. At this step, antibodies or other compounds neutralizing NGF/TrkA are added to the corresponding wells at the dilution/s to be evaluated, so that they are present in the medium for 1 h before testing. At the end of such pre-incubation step, 100 ng/ml of NGF are added to each well (except for negative control) for 10 min at 37.degree. C. in CO.sub.2 incubator. After PBS (+Ca/Mg) wash, cells are scraped on ice in 250 ml of cold RIPA buffer supplemented with phosphatases and proteases inhibitors and insoluble material is removed by 5 min 10000 g centrifugation (4.degree. C.). Extracts are separated on SDS polyacrylamide 10% gels and transferred to nitrocellulose using standard protocols. After blocking 1 h at RT with PBS (+5% non fat dry milk) with gentle agitation, filter are incubated O/N at 4.degree. C. with either anti-phospho TrkA antibody (1:1000) or anti-TrkA antibody (1:1000), followed by the corresponding HRP-conjugated secondary antibody (anti-rabbit/anti-goat 1:1000) for 1 h at RT. After 3 washes in PBS containing 0.1% Tween20 and 3 washes in PBS at RT (gentle agitation), the HRP conjugates are detected by ECL.

Reformatting of Anti-TrkA scFvs to Entire IgG Antibodies

[0163] Anti-TrkA CRB0089 scFv was reformatted to entire IgG antibodies. The cDNA encoding the light and heavy chain (human IgG.sub.4) were generated by GENEART (Germany) with suitable restriction sites for subcloning. Sequences were optimized for mammalian expression (CHO--S cell line) (SEQ ID NOs: 90 for heavy chain and 91 for light chain). After synthesis of both chains, the cDNAs were sub-cloned in expression plasmids (pcDNA3.1 derivates containing an extended CMV promoter for expression of the gene of interest) using HindIII and XhoI as cloning sites. For each antibody chain, two expression plasmids were generated: one plasmid containing the cDNA encoding the light chain, one containing the cDNA encoding the heavy chain. The expression plasmid containing the correct inserts were verified by restriction analysis and DNA sequence analysis of the insert.

Production of Recombinant CRB0089_IgG4 Antibody from Transfected Cells

[0164] Anti-TrkA antibody was produced from transfected cells. CHO--S cells were transfected with plasmids encoding CRB0089 heavy and light chains. Conditioned media from transfected cells were recovered by removing cells and debris. Clarified conditioned media were loaded onto protein A-sepharose column. Non-specific bindings were removed by extensively binding buffer washes (20 mM sodium phosphate pH 7.0). Bound antibody proteins on the protein A column were recovered by acidic antibody elution from protein A (0.1 M glycine-HCl pH 3.0). Eluted proteins were immediately neutralized with 1M Tris-HCl pH 9.0 (100 mL per mL eluted fractions). Pooled eluted fractions were dialyzed against PBS. Aggregated antibody proteins were removed by size exclusion chromatography.

Formalin-Induced Licking Behavior in Mice.

[0165] The experiment was performed in two days sessions, between 9 and 16 hours, 8 animals/groups of treatment. On the day before the experiment, 16 mice were weighed and allocated 4 per cage; 20 .mu.l of formalin solution (1% in saline) were subcutaneously injected into the plantar surface of the right hind paw using an Hamilton micro-syringe equipped with a 26-gauge needle; four animals at a time were placed in a transparent plexiglass box (11.times.12.times.12 cm), allowed to move freely, and the observation period started. A mirror was placed behind the boxes to allow an unimpeded view of the animals hind paws. The licking activity, i.e. the total amount of time the animal spent licking the injected paw, was taken as index of pain. The licking activity was recorded continuously for 1 hour, calculated in blocks of consecutive 5-minutes periods and analyzed as the early (0-5 min) and the late (15-35 min) phases of the formalin test. Each mouse was subcutaneously injected into the dorsal surface of the right hind paw using an Hamilton micro-syringe (26-gauge needle) with 20 .mu.l anti-TrkA (5-20 mg/paw) mAb or PBS as control group, 18 hours before the test.

[0166] Alternatively, mice were subcutaneously injected (systemically) with 300 .mu.l CRB0089_IgG4 (5-20 mg) or PBS as control group, 18 hours before the test.

CFA-Induced Inflammatory Pain in Rats.

[0167] Male Wistar rats were injected into the right hind footpad with 300 .mu.g of Mycobacterium tuberculosis in 100 .mu.L of liquid paraffin (Complete Freund's Adjuvant; CFA). Seventy-two hours later, CRB0089_IgG4 (5 or 20 .mu.g) was administered subcutaneously and 18 h after the administration, the response to noxious mechanical stimulation was assessed by measuring paw withdrawal threshold (PWT) with an analgesimeter of the Randall-Selitto type. Animals were gently restrained, and steadily increasing pressure was applied to the dorsal surface of both the ipsilateral (CFA-treated) and the controlateral paw via a dome-shaped plastic tip.

[0168] To evaluate the paw edema induced by the CFA injury, CRB0089_IgG4 was administered in the right hind paw 72 hours later the CFA injection. Eighteen hours after CRB0089_IgG4 administration, the paw volume was measured by means of a Plethysmometer (UgoBasile, Italy).

Chronic Constriction Injury (CCI)-Induced Neuropathic Pain in Rats.

[0169] Experiments were performed on male Wistar rats (Charles River) weighing 225-250 g at the time of surgery. A minimum of 7 days was allowed for acclimatization before the beginning of the experiments. On each test day, the rats were brought into the experimental room 2 hours prior to the session in order to habituate them to the environment. The experiments were performed by a single experimenter. The CCI was carried out as described previously by Bennet and Xie (Bennett and Xie, 1988. Pain. 33:87-107). Rats were anesthetized with sodium pentobarbital (50 mg/kg i.p.). The common right sciatic nerve was exposed at mid-thigh level, proximal to the sciatic trifurcation. Four chronic gut ligatures (4/0 silk) with about 1 mm spacing were loosely tied around the nerve, so that the vascular supply was not compromised. The overlying muscle was closed in layers with 4/0 synthetic absorbable surgical suture. The skin was closed by application of acrylic glue. In sham animals, an identical dissection was performed, except that the sciatic nerve was not ligated. The tests were conducted on animals at least 1 week after surgery. The response to noxious mechanical stimulation was assessed by measuring PWT with an analgesimeter. Animals were gently restrained, and steadily increasing pressure was applied to the dorsal surface of the ipsilateral (CFA-treated) paw via a dome-shaped plastic tip. The latency to paw withdrawal was determined before surgery, after surgery and at a selected time after test compound or vehicle injection.

A Non-Inflammatory Model of Chronic Muscle Pain in Rats: Bilateral Allodynia Induced by Unilateral Injection of Acidic Saline in the Gastrocnemius Muscle.

[0170] The acidic saline animal model of pain is thought to mimic human chronic pain syndromes such as fibromyalgia. Repeated intramuscular injections of acidic saline is a model of non-inflammatory pain characterized by bilateral long-lasting allodynia of the paw which is believed to be centrally mediated.

[0171] Male Wistar rats were brought to the behavioral testing room 1 h before the test. The right gastrocnemius muscle was injected with 150 .mu.L of preservative-free sterile saline (pH=4). Five days later (5 d), the same gastrocnemius muscle was re-injected. As a control for the injection procedure, a separate group of animals were injected with sterile saline. Ipsilateral and contralateral paw withdrawal thresholds in response to mechanical stimuli were measured on Days 0 (baseline--0 d), 5 (5 d), 6 (6 d), 9 (9 d), and 12 (12 d). Nociceptive thresholds, expressed in grams (g), were measured with a Dynamic Plantar Aesthesiometer by applying increasing pressure to the right and left hind paw until the rat withdrew the paw. A maximal cut-off of 50 g was used to prevent tissue damage. The threshold was tested three times for each paw and the mean value was calculated. On Day 5, 6 h after the second saline injection, CRB0089_IgG4 was administered subcutaneously at a dose of 20 .mu.g/rat. A saline subcutaneous injection was used as vehicle control. Mechanical withdrawal thresholds of both hind paws were measured 18 h (6 d), 90 h (9 d), and 162 h (12 d) after CRB0089_IgG4 injection. Two injections of acidic saline into the gastrocnemius muscle produced bilateral decreases in the mechanical withdrawal threshold of the paw 24 h after the second injection.

Results

Selection of Specific Anti-VEMHHW Epitope of Human TrkAscFvs Using SPLINT Technology.

[0172] To select specific anti-VEMHHW epitope of human TrkA receptor by SPLINT technology, the VEMHHW peptide (SEQ ID NO: 66) was engineered to be part of the two loops of the immunoglobulin like (Ig-like) domain of 127 Titin protein (SEQ ID NOs: 68, 69). Ig-like domain is a common structural unit across many protein families that are functionally unrelated (Wright et al., 2004. Protein Eng Des Sel. 17:443-453. Epub 2004 June 2018). The beta-sandwich fold provides a very robust structural scaffold upon which it is possible to insert long peptides without altering either the structure or folding of the domain. VEMHHW peptide was inserted between A76-N77 and E27-D29 of 127 Ig-like domain of titin protein (SEQ ID NO: 92). The recombinant 2.times.VEMHHW-I27 protein was subsequently cloned at the 3' of LexA and used to challenge a mouse SPLINT (mSPLINT) and a human SPLINT libraries (huSPLINT_09) (Visintin et al., 2004. J Immunol Methods. 290:135-153).

[0173] From the selection procedure using hSPLINT_09 a total of 189 colonies able to grow in the absence of histidine and showing activation of .beta.-Galactosidase were obtained. The scFv-VP16 plasmids were isolated and sorted by their restriction patterns and sequences. The specificity of scFvs with different DNA fingerprints were re-analyzed using yeast strains expressing LexA-2.times.VEMHHW-127 and LexA-127, as non-relevant antigen. 61 different anti-VEMHHW scFvs were thus identified. Analysis of the V region nucleotide sequences of the selected anti-VEMHHW scFvs revealed that they were derived from germline V region genes. The amino acid sequence of V regions of the isolated anti-VEMHHW scFvs are in the group of sequences consisting of SEW NO: 1, 2, 3, 4 from the selection of mSPLINT and SEQ ID NOs: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16 from the selection of hSPLINT_09.

Expression and Refolding of Anti-TrkA in the Cytoplasm of E. coli

[0174] To identify potential anti-TrkA in vivo binders cDNA expressing anti-VEMHHW scFv were cloned into E. coli pETM-13 expression vector. The proteins were well expressed in the cytoplasm and mostly retained in inclusion bodies (TB). scFv fragments can be refolded by dialysis after solubilization of IB (Umetsu et al., 2003. J Biol Chem. 278:8979-8987. Epub 2003 January 8977). We performed the technique of refolding by dilution (Patil et al., 2008. J Biotechnol. 134:218-221. Epub 2008 January 2018). The refolding condition of scFv was optimized for each sample. Refolded scFv were subsequently quantified by Bioanalyzer 2100 (Agilent) and tested by ELISA and Biacore analysis.

Binding Specificity and Cross-Reactivity of Anti-TrkA to Human and murineTrkA

[0175] The purified anti-TrkA scFv were first analysed by FACS analysis for binding to TrkA receptor. TrkA binding analysis by flow cytometry on 3T3-TrkA and TF1 expressing TrkA receptor was performed with the panel of isolated anti-TrkA scFvs. All the anti-TrkA scFvs were able to recognize the TrkA receptor under physiological condition. Following identification of TrkA expressing cells by anti-TrkA scFv, we analyzed the panel of scFv for specificity and crossreactivity with mouse TrkA by ELISA assay. In order to use the extracellular domain (ECD) of Trk receptor we have engineered a panel of human Trk receptors (TrkA, TrkB, TrkC and p75NTR) as immunoadhesin proteins. These recombinant proteins were constructed to have the ECD domain of the Trk receptors linked to the Fc portion of a IgG2a camel antibody (Camelus dromedaries) (SEQ ID NOs: 70, 71, 72, 73 and 74). The recombinant protein were expressed in mammalian cell line (CHO--S cell line) and purified by protein A column. After purification the receptor chimera were analysed by SDS-PAGE western blot analysis under reducing condition (FIG. 7). The purified immunoadhesins were used as ligand in ELISA. As shown in FIG. 2, all the scFv were able to recognize at the concentration of 50 .mu.g/mL and 5 .mu.g/mL the TrkA immunoadhesins. The same scFvs were tested against the Trk family immunoadhesins. The anti-TrkA seems to be specific for TrkA only, even if a slight crossreactivity was shown for some scFv (CRB0036 and CRB0069) on TrkB and TrkC immunoadhesins (FIG. 3).

[0176] Like other receptor tyrosine kinases, TrkA undergoes dimerization and activation upon ligand binding. In the absence of NGF, some domains of the receptor, perhaps the same ones responsible for ligand binding, impede its spontaneous dimerization at the cell surface (Arevalo et al., 2000. Mol Cell Biol. 20:5908-5916). It was demonstrated that a recombinant deleted protein of TrkA receptor, Ig-1,2 which express both Ig-1 and Ig-2 domains of the extracellular domain (ECD) of the receptor TrkA was able to dimerize also in the absence of NGF (Arevalo et al., 2000. Mol Cell Biol. 20:5908-5916.). We have used two recombinant proteins engineered to express both Ig-likes domains and able to dimerize in the absence of NGF for Biacore analysis.

[0177] We have carried out surface plasmon resonance (SPR) analyses to determine the binding kinetics of a panel of isolated anti-TrkA scFvs (SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16). The scFvs were immobilized on a CM5 chip followed by injections at various concentration of human and murine Ig 1,2 TrkA (SEQ ID NO: 78) and mouse Ig1,2 TrkA (SEQ ID NO: 79). As shown in Table II and III, all the scFvs were able to recognize both human and mouse Ig 1,2 recombinant TrkA proteins in the range of nanomolar affinity.

Measurement of Antagonism of NGF Activity of Anti-TrkA scFvs.

[0178] In order to evaluate the potency of anti-TrkA scFvs, the TF1 cell proliferation assay (MTT cell proliferation kit, ATCC) was used (concentration/response study). Final average OD values for triplicate measurements were calculated by subtraction of the average values for the cellular blank. Maximal inhibition was set corresponding to the average OD value observed for cells cultured without NGF, in the absence of test antibody. Zero inhibition was set corresponding to the average OD value observed for cells exposed to 10 ng/ml NGF in the absence of test scFv. As shown in FIG. 5, all the isolated anti-TrkA scFv were able to inhibit TF1 cell proliferation mediated by human NGF in a range between 20-60%.

[0179] To test whether the anti-TrkA scFvs were also able to inhibit NGF triggered phosphorylation on Tyr residues under physiological condition in 3T3TrkA cell line, we treated the cell line with NGF and 100 mg/mL anti-TrkA for 1 hour. As shown in FIG. 8 all the scFv tested were able to inhibit the phosphorylation of TrkA at Tyr 490. As a positive control, a strong antagonist of NGF that totally inhibit the phosphorylation of TrkA was used (anti-NGF).

[0180] To test whether the anti-TrkA scFvs were able to inhibit inflammatory pain induced by subcutaneous injection of formalin in mouse model, 20 mg of each purified anti-TrkA scFv was injected subcutaneously in the mouse hind paw. The injection resulted in a biphasic licking response: the first phase represents a basic pain response to direct stimulation of the nerve endings, the second phase represents a tonic pain response to subsequent inflammation. The effect of different anti-TrkA scFv is summarized in FIG. 6. All anti-TrkA scFv were able to inhibit the second phase of formalin induced pain with a percent of analgesic effect in a range between 27-55%.

mAb CRB0089_IgG4: Biochemical Characterization of the Reformatted Immunoglobulin

[0181] A complete IgG4 immunoglobulin was assembled by amplifying the individual V-regions of isolated anti-TrkA CRB0089 into a vector enabling the transfer of V-regions from scFv to full length immunoglobulin for mammalian expression.

[0182] Whole IgG4 was produced and purified from transfected CHO cell line. The anti-TrkA CRB0089_IgG4 was tested for binding in ELISA. As shown in FIG. 9, the antibody was able to bind in a dose specific way TrkA receptor. A weak interaction with TrkB receptor was also detected even if the specificity of this binding must be confirmed by other specific binding assay.

[0183] Furthermore, the kinetic analysis of CRB0089_IgG4 to TrkA Ig1,2 on Biacore X-100 was performed. As shown in Table IV, the kinetics rates of association and dissociation and affinity constants were calculated both for human and murine TrkA receptor. mAb CRB0089_IgG4 cross reacts with similar affinity both human and murine receptor thus indicated that murine models of pain can be used for further preclinical development of the antibody.

[0184] Mab CRB0089_IgG4 was also tested to bind TF1 expressing TrkA receptor by FACS analysis. The antibody was able to bind to TrkA receptor in a dose dependent manner. At higher concentration (20 mg/mL) the antibody display 99% gated events on more than 2800 cells tested. The antibody strongly bind to TrkA receptor even at lower concentrations (FIG. 10) as compared with an unrelated antibody (unr-IgG4) used as negative control.

Measurement of Anti-TrkA Antagonism Activity of CRB0089_IgG4.

[0185] In order to evaluate the potency of anti-TrkA CRB0089_IgG4, a TF1 cell proliferation assay was employed (concentration response study). The inhibitory potency of anti-TrkA CRB0089_IgG4 antibody was quantified as IC.sub.50 values (i.e., the concentration of antibody required to reduce the NGF-mediated proliferative response by 50%) using Sigma Plot software. Inhibition curves were plotted individually in order to obtain discrete IC.sub.50 values for each test antibody in each experiment. Measures of cell proliferation were normalized with respect to maximum OD values obtained within that assay, in the absence of added test antibody. Normalized responses were then plotted against test antibody concentration on a log scale, and IC.sub.50 values were derived using the Sigma Plot nonlinear curve fitting function "log (inhibitor) vs response-variable slope".

[0186] As shown in FIG. 11, CRB0089_IgG4 was able to inhibit NGF-mediated proliferative response in a dose dependent manner with an IC.sub.50 of approximately 43 nM.

In Vivo Activity of CRB0089_IgG4 in Inhibiting NGF-Mediated Pain

[0187] A series of in vivo experiments were conducted to assess the activity of CRB0089_IgG4 in rodent models of inflammatory/neurogenic and neuropathic pain.

Two "classical" screening models were initially used to evaluate the analgesic properties of CRB0089_IgG4: a) the formalin-induced licking behavior in the mouse, b) the complete Freund's adjuvant (CFA)-induced mechanical hyperalgesia in the rat.

[0188] a) The experiment was performed in two days sessions with 8 animals/groups of treatment. CRB0089_IgG4, injected into the dorsal surface of the paw, showed to be active at both used dose (5-20 .mu.g/paw) to inhibit both the early and the late phase of the formalin-induced behavior in mice (FIG. 12). In another experiment, each mouse was subcutaneously injected with 300 .mu.l CRB0089_IgG4 or PBS as control group, 18 hours before the test. As internal control of the experiment, a group of mice (n=8) was treated locally in the right dorsal paw with 20 .mu.g (1 .mu.g/.mu.l) CRB0089_IgG4 as in the previous experiment.

[0189] CRB0089_IgG4, injected s.c. in the range 5-20 .mu.g, showed to inhibit the late phase of the formalin-induced behavior in mice at both the used doses (FIG. 13). No treatments induced significant changes related to the early phase of the formalin-induced behavior in mice.

[0190] b) Male Wistar rats were injected into the right hind footpad with Mycobacterium tuberculosis in liquid paraffin (Complete Freund's Adjuvant; CFA). Seventy-two hours later, CRB0089_IgG4 (5 or 20 .mu.g) was administered subcutaneously and 18 h after the administration, the response to noxious mechanical stimulation was assessed by measuring PWT with an analgesimeter of the Randall-Selitto type. Animals were gently restrained, and steadily increasing pressure was applied to the dorsal surface of both the ipsilateral (CFA-treated) and the controlateral paw via a dome-shaped plastic tip.

[0191] In this experimental model of inflammatory pain, CRB0089_IgG4 dose-dependently reduced mechanical hyperalgesia in the CFA-injured hind paw 18 h after the antibody injection (ED.sub.50=.about.5 .mu.g/rat, ED.sub.100=.about.20 .mu.g/rat; FIG. 14). When the mechanical threshold was measured in the controlateral non-injured hind paw, a slight but significant hypoalgesic-like effect was recognized after treatment with 20 .mu.g/rat dose of CRB0089_IgG4 (FIG. 15).

[0192] In this experiment it was also evaluated the paw edema induced by the CFA injury in the right hind paw. As for the hyperalgesic test previous described, we administered CRB0089_IgG4 72 h later the CFA injection. Eighteen hours after CRB0089_IgG4 administration the paw volume was measured by means of a Plethysmometer. As shown in FIG. 16, both concentrations of CRB0089_IgG4 did not reduced paw edema in treated rats.

[0193] To test neuropathic pain, two different animal models were used: c) chronic constriction injury (CCI)-induced neuropathic pain in rats and d) a non-inflammatory model of chronic muscle pain in rats-bilateral allodynia induced by unilateral injection of acidic saline in the gastrocnemius muscle.

[0194] c) Experiments were performed on male Wistar rats (Charles River) weighing 225-250 g at the time of surgery. The CCI was carried out as described previously (Bennett and Xie, 1988. Pain. 33:87-107). The tests were conducted on animals at least 1 week after surgery. The response to noxious mechanical stimulation was assessed by measuring PWT with an analgesimeter. Animals were gently restrained, and steadily increasing pressure was applied to the dorsal surface of the ipsilateral (CFA-treated) paw via a dome-shaped plastic tip. The latency to paw withdrawal was determined before surgery, after surgery and at a selected time after test compound or vehicle injection. As shown in FIG. 17, CRB0089_IgG4 at the concentration of 20 mg/rat induced 100% reversion of hyperalgesia after 18 h post injection.

[0195] d) The acidic saline animal model of pain is thought to mimic human chronic pain syndromes such as fibromyalgia. Repeated intramuscular injections of acidic saline is a model of non-inflammatory pain characterized by bilateral long-lasting allodynia of the paw which is believed to be centrally mediated. Ipsilateral and contralateral paw withdrawal thresholds in response to mechanical stimuli were measured on Days 0 (baseline--0 d), 5 (5 d), 6 (6 d), 9 (9 d), and 12 (12 d). Nociceptive thresholds, expressed in grams (g), were measured with a Dynamic Plantar Aesthesiometer by applying increasing pressure to the right and left hind paw until the rat withdrew the paw. CRB0089_IgG4 was administered subcutaneously at a dose of 20 .mu.g/rat 6 h after the second acidic saline injection. Mechanical withdrawal thresholds of both hind paws were measured 18 h (6 d), 90 h (9 d), and 162 h (12 d) after CRB0089_IgG4 injection. Two injections of acidic saline into the gastrocnemius muscle produced bilateral decreases in the mechanical withdrawal threshold of the paw 24 h after the second injection. CRB0089_IgG4 (20 .mu.g/rat), injected subcutaneously 18 h before the measure of mechanical allodynia (6 d), increased the withdrawal threshold of both hind paws. The anti-nociceptive effects of a single injection of 20 .mu.g/rat CRB0089_IgG4 lasted almost 90 h before returning to basal allodynic parameters. As shown in FIG. 18 CRB0089_IgG4 at the concentration of 20 .mu.g/rat induced 100% reversion of hyperalgesia after 90 h of injection in the ipsilateral paw. Moreover, at the same concentration and at the same time, CRB0089_IgG4 induced 60% reversion hyperalgesia also in the controlateral paw.

Tables

TABLE-US-00003 [0196] TABLE I Results of anti-TrkA SPLINT screening: N. .noteq.clones N. positive clones BAIT (1 screening) (II screening) TrkA_loopA 189 61

TABLE-US-00004 TABLE II Biacore analysis of anti-TrkA scFvs vs human TrkA: Human Ig1, 2 TrkA scFv ka (1/Ms) kd (1/s) KD (M) CRB0022 9192 2.94E-05 3.2 nM CRB0036 2731 3.75E-05 13.7 nM CRB0069 6266 3.57E-05 5.7 nM CRB0072 3675 7.48E-06 2.04 nM CRB0082 2090 8.79.E-05 42.1 nM CRB0084 9602 1.63E-02 1.70 .mu.M CRB0088 2755 3.2E-06 1.16 nM CRB0089 4598 7.76E-05 16.9 nM

TABLE-US-00005 TABLE III Biacore analysis of anti-TrkA scFvs vs mouse TrkA: Mouse Ig1, 2 TrkA scFv ka (1/Ms) kd (1/s) KD (M) CRB0022 9426 1.09E-05 1.16 nM CRB0036 6440 7.07E-06 1.10 nM CRB0069 6120 1.02E-05 1.67 nM CRB0072 2907 4.93E-05 .sup. 17 nM CRB0082 2630 344.E-04 131 nM CRB0084 1.07E+04 2.33E-05 2.18 nM CRB0088 3413 1.31E-04 38.5 nM CRB0089 2247 3.19E-05 14.2 nM

TABLE-US-00006 TABLE IV Biacore analysis of CRB0089_IgG4: mAb ka (1/Ms) kd (1/s) KD (M) Human Ig1, 2 TrkA CRB0089_IgG4 6424 1.03E-04 1.61E-08 Mouse Ig1, 2 TrkA CRB0089_IgG4 4950 1.36E-04 2.76E-08

TABLE-US-00007 TABLE V TrkA_bait PCR assembly primers: SEQ ID NO I PCR-assembly 80 5'-CATCATGAATTCCTAATAGAAGTGGAAAAGCCTCTGTACG GAGTAGAGGTG-3' 81 5'-CACAGAAAGTTCAATTTCAAAGTGGGCTGTTTCACCAACA AACACCTCTACTCCGTACAGAGG-3' 82 5'-CCACTTTGAAATTGAACTTTCTGTGGAGATGCACCACTGG GTTCACGGCCAGTGGAAGCTG-3' 83 5'-CAATGATTTCACAGTCAGGGGAAGCTGTCAAAGGCTGTCC TTTCAGCTTCCACTGGCCGT-3' 84 5'-CTTCCCCTGACTGTGAAATCATTGAGGATGGAAAGAAGCA TATTCTGATCCTTCATAACTGTCAGC-3' 85 5'-TCTCCACCTGGAAGGAAACCTCTCCTGTCATACCCAGCTG ACAGTTATGAAGGATCAGAATAT-3' 86 5'-GGTTTCCTTCCAGGTGGAGATGCACCACTGGAAATCTGCA GCCAATCTGAAAGTGAAAGAATT-3' 87 5'-TAATACGACTCACTATAGTCGACGGATCCTTACAATTCTT TCACTTTCAGATTGGCTG-3' II PCR-assembly 88 5'-CATCATGAATTCCTAATAGAAGTGGAAAAG-3' 89 5'-TAATACGACTCACTATAGTCGACGG-3'

Sequence CWU 1

1

1021120PRTArtificial SequenceCRB0022VH 1Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Glu Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gly Ile Asp Pro Ala Asn Gly Asn Thr Ile Tyr Val Pro Lys Phe 50 55 60 Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Asp Phe Asp Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 2106PRTArtificial SequenceCRB0022VK 2Asp Ile Val Leu Thr Gln Ser Pro Ala Leu Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Arg Ser Ser Pro Lys Pro Trp Ile Tyr 35 40 45 Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Tyr Thr 85 90 95 Phe Gly Gly Arg Thr Arg Leu Glu Ile Lys 100 105 3116PRTArtificial SequenceCRB0036VH 3Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Leu Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Asn Tyr 20 25 30 Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Leu Pro Gly Ser Gly Ser Ala His Tyr Ser Glu Lys Phe 50 55 60 Lys Asp Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Thr Arg Ser Pro Pro Asn Pro Gly Tyr Trp Gly Gln Gly Thr Ser Val 100 105 110 Thr Val Ser Ser 115 4112PRTArtificial SequenceCRB0036VK 4Asp Ile Val Met Thr Gln Ala Ala Pro Ser Val Pro Val Thr Pro Gly 1 5 10 15 Glu Ser Val Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30 Ser Gly Ile Thr Ser Leu Tyr Trp Tyr Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Gln Leu Pro Ile Tyr Arg Met Ser Ser Leu Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Val Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His 85 90 95 Leu Glu Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 5123PRTArtificial SequenceCRB0069VH 5Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25 30 Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45 Arg Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55 60 Glu Ser Val Lys Ser Arg Ile Ile Ile Asn Ser Gly Thr Ser Lys Asn 65 70 75 80 Gln Phe Ser Leu His Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85 90 95 Tyr Tyr Cys Gly Arg Ala Lys Asp Gly Trp Tyr Asp Ala Phe Asp Ile 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 6110PRTArtificial SequenceCRB0069VK 6Gln Pro Gly Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Val Gly Ala Gly 20 25 30 Tyr Thr Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Thr Leu 35 40 45 Leu Ile Tyr Asp Asn Asn Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Arg Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu 65 70 75 80 Arg Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Arg Ser 85 90 95 Arg Val Tyr Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu 100 105 110 7127PRTArtificial SequenceCRB0072VH 7Glu Val Gln Leu Val Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Ser 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ile Asn Tyr 20 25 30 Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser Ile Trp Tyr Glu Gly Ser Asn Glu Lys Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Tyr Thr Leu His 65 70 75 80 Leu Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Asp Tyr Tyr Cys 85 90 95 Val Arg Asp Arg Gly Thr Gly Asp Tyr Arg Asn Ser Arg Phe Tyr Tyr 100 105 110 Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 8107PRTArtificial SequenceCRB0072VK 8Asp Ile Gln Met Thr Gln Ser Pro Ile Ser Leu Ser Ala Thr Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Arg Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Val Phe Thr Leu Thr Ile Ser Gly Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Cys Cys Gln Gln Ser His Ser Thr Ala Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 9117PRTArtificial SequenceCRB0082VH 9Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Phe 20 25 30 Thr Val Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45 Arg Leu Gly Arg Thr Tyr Arg Arg Ser Lys Asp Tyr Ala Glu Tyr Met 50 55 60 Arg Ser Arg Leu Thr Ile Asn Ala Asp Thr Ser Lys Asn Gln Leu Ser 65 70 75 80 Leu Gln Leu Asp Ser Val Thr Pro Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gln Asn Ser Ala Phe Asp Leu Trp Gly Gln Arg Thr Met 100 105 110 Val Thr Val Ser Ser 115 10108PRTArtificial SequenceCRB0082VL 10Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Thr 1 5 10 15 Thr Ala Arg Leu Ser Cys Glu Ala Thr Lys Ile Gly Ser Gln Arg Leu 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Met Val Ile Ser 35 40 45 Phe Asp Ser Asp Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60 Arg Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ser Ser Gly Tyr His 85 90 95 Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 11120PRTArtificial SequenceCRB0084VH 11Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Arg Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Phe Ser Asp Ser 20 25 30 Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Asn Asn His Arg Gly Ser Thr Asn Tyr Ser Pro Thr Leu Arg 50 55 60 Ser Arg Leu Ser Ile Ser Ile Asp Ser Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Val Pro Tyr Arg Leu Arg Ser Arg Ile Phe Asp Val Trp Gly Gln 100 105 110 Gly Thr Met Val Thr Val Ser Ser 115 120 12108PRTArtificial SequenceCRB0084VL 12Gln Pro Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Arg Gln 1 5 10 15 Thr Ala Thr Ile Thr Cys Gly Gly Asn Tyr Ile Gly Ser Lys Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Asp Pro Val Leu Val Val Asn 35 40 45 Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Ala Asn Thr Ala Thr Leu Thr Ile Ser Gly Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Thr Glu Pro 85 90 95 Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 13121PRTArtificial SequenceCRB0088VH 13Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp His 20 25 30 Tyr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Asn Lys Ala Asn Ser Tyr Thr Thr Glu Tyr Ala Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Lys Thr 65 70 75 80 Leu Tyr Leu Gln Met Thr Ser Leu Lys Thr Asp Asp Thr Ala Leu Tyr 85 90 95 Tyr Cys Ala Arg Asp Leu Gly Ala Ser Asp Ala Phe Asp Leu Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 14111PRTArtificial SequenceCRB0088VL 14Gln Pro Val Leu Thr Gln Ser Pro Pro Ala Ser Ala Ser Leu Gly Ala 1 5 10 15 Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gly His Ser Ser Tyr Ala 20 25 30 Ile Ala Trp His Gln Gln Gln Pro Glu Lys Gly Pro Arg Tyr Leu Met 35 40 45 Lys Val Asn Ser Asp Gly Ser His Asn Lys Gly Asp Gly Ile Pro Asp 50 55 60 Arg Phe Ser Gly Ser Arg Ser Gly Ala Glu Arg Tyr Leu Thr Ile Ser 65 70 75 80 Ser Leu His Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Trp Gly 85 90 95 Ala Gly Gly Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 15122PRTArtificial SequenceCRB0089VH 15Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25 30 Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45 Trp Leu Gly Arg Thr Phe His Arg Ser Arg Trp Tyr Asn Glu Tyr Pro 50 55 60 Val Ser Val Arg Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Asn Asn 65 70 75 80 Gln Phe Thr Leu Gln Leu Asn Ser Val Thr Pro Asp Asp Thr Ala Val 85 90 95 Tyr Tyr Cys Ala Arg Ala Pro Val Ala Gly Leu Thr Phe Asp Ile Trp 100 105 110 Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 16105PRTArtificial SequenceCRB0089VL 16Gln Thr Val Val Thr Gln Glu Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Ile Ile Gly Ala Gly 20 25 30 Tyr Glu Val His Trp Tyr Gln His Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Asp Ser Ile Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Ser Ala Ile Phe 85 90 95 Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 1710PRTArtificial SequenceCDRL1_22 17Ser Ala Ser Ser Ser Val Ser Tyr Met Tyr 1 5 10 187PRTArtificial SequenceCDRL2_22 18Leu Thr Ser Asn Leu Ala Ser 1 5 199PRTArtificial SequenceCDRL3_22 19Gln Gln Trp Ser Ser Asn Pro Tyr Thr 1 5 2016PRTArtificial SequenceCDRL1_36 20Arg Ser Ser Lys Ser Leu Leu His Ser Ser Gly Ile Thr Ser Leu Tyr 1 5 10 15 217PRTArtificial SequenceCDRL2_36 21Arg Met Ser Ser Leu Ala Ser 1 5 229PRTArtificial SequenceCDRL3_36 22Met Gln His Leu Glu Tyr Pro Leu Thr 1 5 2314PRTArtificial SequenceCDRL1_69 23Thr Gly Ser Ser Ser Asn Val Gly Ala Gly Tyr Thr Val His 1 5 10 247PRTArtificial SequenceCDRL2_69 24Asp Asn Asn Asn Arg Pro Ser 1 5 2510PRTArtificial SequenceCDRL3_69 25Gln Ser Tyr Asp Arg Ser Arg Val Tyr Val 1 5 10 2611PRTArtificial SequenceCDRL1_72 26Arg Ala Ser Gln Ser Ile Gly Arg Tyr Leu Asn 1 5 10 277PRTArtificial SequenceCDRL2_72 27Gly Ala Ser Ser Leu Glu Ser 1 5 289PRTArtificial SequenceCDRL3_72 28Gln Gln Ser His Ser Thr Ala Trp Thr 1 5 2911PRTArtificial SequenceCDRL1_82 29Glu Ala Thr Lys Ile Gly Ser Gln Arg Leu His 1 5 10 307PRTArtificial SequenceCDRL2_82 30Phe Asp Ser Asp Arg Pro Ser 1 5 3111PRTArtificial SequenceCDRL3_82 31Asn Ser Arg Asp Ser Ser Gly Tyr His Leu Val 1 5 10 3211PRTArtificial SequenceCDRL1_84 32Gly Gly Asn Tyr Ile Gly Ser Lys Ser Val His 1 5 10 337PRTArtificial SequenceCDRL2_84 33Asp Asp Ser Asp Arg Pro Ser 1 5 3411PRTArtificial SequenceCDRL3_84 34Gln Val Trp Asp Ser Ser Thr Glu Pro Val Val 1 5 10 3512PRTArtificial SequenceCDRL1_88 35Thr Leu Ser Ser Gly His Ser Ser Tyr Ala Ile Ala 1 5 10 3611PRTArtificial SequenceCDRL2_88 36Val Asn Ser Asp Gly Ser His Asn Lys Gly Asp 1 5 10 379PRTArtificial SequenceCDRL3_88 37Gln Thr Trp Gly Ala Gly Gly Val Val 1 5 3814PRTArtificial SequenceCDRL1_89 38Thr Gly Ser Ser Ser Ile Ile Gly Ala Gly Tyr Glu Val His 1 5 10 397PRTArtificial SequenceCDRL2_89 39Asp Ser Ile Asn Arg Pro Ser 1 5 405PRTArtificial SequenceCDRL3_89 40Gln Ser Ser Ala Ile 1 5 415PRTArtificial SequenceCDRH1_22 41Asp Thr Tyr Met His 1 5 4217PRTArtificial SequenceCDRH2_22 42Gly Ile Asp Pro Ala Asn Gly Asn Thr Ile Tyr Val Pro Lys Phe Gln 1 5

10 15 Gly 4311PRTArtificial SequenceCDRH3_22 43Gly Gly Tyr Asp Phe Asp Tyr Ala Met Asp Tyr 1 5 10 445PRTArtificial SequenceCDRH1_36 44Asn Tyr Trp Ile Glu 1 5 4517PRTArtificial SequenceCDRH2_36 45Glu Ile Leu Pro Gly Ser Gly Ser Ala His Tyr Ser Glu Lys Phe Lys 1 5 10 15 Asp 467PRTArtificial SequenceCDRH3_36 46Ser Pro Pro Asn Pro Gly Tyr 1 5 477PRTArtificial SequenceCDRH1_69 47Ser Asn Ser Ala Ala Trp Asn 1 5 4818PRTArtificial SequenceCDRH2_69 48Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Glu Ser Val 1 5 10 15 Lys Ser 4911PRTArtificial SequenceCDRH3_69 49Ala Lys Asp Gly Trp Tyr Asp Ala Phe Asp Ile 1 5 10 505PRTArtificial SequenceCDRH1_72 50Asn Tyr Gly Val His 1 5 5117PRTArtificial SequenceCDRH2_72 51Ser Ile Trp Tyr Glu Gly Ser Asn Glu Lys Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 5218PRTArtificial SequenceCDRH3_72 52Asp Arg Gly Thr Gly Asp Tyr Arg Asn Ser Arg Phe Tyr Tyr Gly Met 1 5 10 15 Asp Val 537PRTArtificial SequenceCDRH1_82 53Ser Phe Thr Val Ala Trp Asn 1 5 5415PRTArtificial SequenceCDRH2_82 54Arg Thr Tyr Arg Arg Ser Lys Asp Tyr Ala Glu Tyr Met Arg Ser 1 5 10 15 558PRTArtificial SequenceCDRH3_82 55Gly Gln Asn Ser Ala Phe Asp Leu 1 5 565PRTArtificial SequenceCDRH1_84 56Asp Ser Tyr Trp Thr 1 5 5716PRTArtificial SequenceCDRH2_84 57Glu Asn Asn His Arg Gly Ser Thr Asn Tyr Ser Pro Thr Leu Arg Ser 1 5 10 15 5812PRTArtificial SequenceCDRH3_84 58Val Pro Tyr Arg Leu Arg Ser Arg Ile Phe Asp Val 1 5 10 595PRTArtificial SequenceCDRH1_88 59Asp His Tyr Met Asp 1 5 6019PRTArtificial SequenceCDRH2_88 60Arg Ile Arg Asn Lys Ala Asn Ser Tyr Thr Thr Glu Tyr Ala Ala Ser 1 5 10 15 Val Lys Gly 6110PRTArtificial SequenceCDRH3_88 61Asp Leu Gly Ala Ser Asp Ala Phe Asp Leu 1 5 10 627PRTArtificial SequenceCDRH1_89 62Ser Asn Ser Ala Ala Trp Asn 1 5 6318PRTArtificial SequenceCDRH2_89 63Arg Thr Phe His Arg Ser Arg Trp Tyr Asn Glu Tyr Pro Val Ser Val 1 5 10 15 Arg Ser 6410PRTArtificial SequenceCDRH3_89 64Ala Pro Val Ala Gly Leu Thr Phe Asp Ile 1 5 10 65796PRTHomo sapiens 65Met Leu Arg Gly Gly Arg Arg Gly Gln Leu Gly Trp His Ser Trp Ala 1 5 10 15 Ala Gly Pro Gly Ser Leu Leu Ala Trp Leu Ile Leu Ala Ser Ala Gly 20 25 30 Ala Ala Pro Cys Pro Asp Ala Cys Cys Pro His Gly Ser Ser Gly Leu 35 40 45 Arg Cys Thr Arg Asp Gly Ala Leu Asp Ser Leu His His Leu Pro Gly 50 55 60 Ala Glu Asn Leu Thr Glu Leu Tyr Ile Glu Asn Gln Gln His Leu Gln 65 70 75 80 His Leu Glu Leu Arg Asp Leu Arg Gly Leu Gly Glu Leu Arg Asn Leu 85 90 95 Thr Ile Val Lys Ser Gly Leu Arg Phe Val Ala Pro Asp Ala Phe His 100 105 110 Phe Thr Pro Arg Leu Ser Arg Leu Asn Leu Ser Phe Asn Ala Leu Glu 115 120 125 Ser Leu Ser Trp Lys Thr Val Gln Gly Leu Ser Leu Gln Glu Leu Val 130 135 140 Leu Ser Gly Asn Pro Leu His Cys Ser Cys Ala Leu Arg Trp Leu Gln 145 150 155 160 Arg Trp Glu Glu Glu Gly Leu Gly Gly Val Pro Glu Gln Lys Leu Gln 165 170 175 Cys His Gly Gln Gly Pro Leu Ala His Met Pro Asn Ala Ser Cys Gly 180 185 190 Val Pro Thr Leu Lys Val Gln Val Pro Asn Ala Ser Val Asp Val Gly 195 200 205 Asp Asp Val Leu Leu Arg Cys Gln Val Glu Gly Arg Gly Leu Glu Gln 210 215 220 Ala Gly Trp Ile Leu Thr Glu Leu Glu Gln Ser Ala Thr Val Met Lys 225 230 235 240 Ser Gly Gly Leu Pro Ser Leu Gly Leu Thr Leu Ala Asn Val Thr Ser 245 250 255 Asp Leu Asn Arg Lys Asn Val Thr Cys Trp Ala Glu Asn Asp Val Gly 260 265 270 Arg Ala Glu Val Ser Val Gln Val Asn Val Ser Phe Pro Ala Ser Val 275 280 285 Gln Leu His Thr Ala Val Glu Met His His Trp Cys Ile Pro Phe Ser 290 295 300 Val Asp Gly Gln Pro Ala Pro Ser Leu Arg Trp Leu Phe Asn Gly Ser 305 310 315 320 Val Leu Asn Glu Thr Ser Phe Ile Phe Thr Glu Phe Leu Glu Pro Ala 325 330 335 Ala Asn Glu Thr Val Arg His Gly Cys Leu Arg Leu Asn Gln Pro Thr 340 345 350 His Val Asn Asn Gly Asn Tyr Thr Leu Leu Ala Ala Asn Pro Phe Gly 355 360 365 Gln Ala Ser Ala Ser Ile Met Ala Ala Phe Met Asp Asn Pro Phe Glu 370 375 380 Phe Asn Pro Glu Asp Pro Ile Pro Val Ser Phe Ser Pro Val Asp Thr 385 390 395 400 Asn Ser Thr Ser Gly Asp Pro Val Glu Lys Lys Asp Glu Thr Pro Phe 405 410 415 Gly Val Ser Val Ala Val Gly Leu Ala Val Phe Ala Cys Leu Phe Leu 420 425 430 Ser Thr Leu Leu Leu Val Leu Asn Lys Cys Gly Arg Arg Asn Lys Phe 435 440 445 Gly Ile Asn Arg Pro Ala Val Leu Ala Pro Glu Asp Gly Leu Ala Met 450 455 460 Ser Leu His Phe Met Thr Leu Gly Gly Ser Ser Leu Ser Pro Thr Glu 465 470 475 480 Gly Lys Gly Ser Gly Leu Gln Gly His Ile Ile Glu Asn Pro Gln Tyr 485 490 495 Phe Ser Asp Ala Cys Val His His Ile Lys Arg Arg Asp Ile Val Leu 500 505 510 Lys Trp Glu Leu Gly Glu Gly Ala Phe Gly Lys Val Phe Leu Ala Glu 515 520 525 Cys His Asn Leu Leu Pro Glu Gln Asp Lys Met Leu Val Ala Val Lys 530 535 540 Ala Leu Lys Glu Ala Ser Glu Ser Ala Arg Gln Asp Phe Gln Arg Glu 545 550 555 560 Ala Glu Leu Leu Thr Met Leu Gln His Gln His Ile Val Arg Phe Phe 565 570 575 Gly Val Cys Thr Glu Gly Arg Pro Leu Leu Met Val Phe Glu Tyr Met 580 585 590 Arg His Gly Asp Leu Asn Arg Phe Leu Arg Ser His Gly Pro Asp Ala 595 600 605 Lys Leu Leu Ala Gly Gly Glu Asp Val Ala Pro Gly Pro Leu Gly Leu 610 615 620 Gly Gln Leu Leu Ala Val Ala Ser Gln Val Ala Ala Gly Met Val Tyr 625 630 635 640 Leu Ala Gly Leu His Phe Val His Arg Asp Leu Ala Thr Arg Asn Cys 645 650 655 Leu Val Gly Gln Gly Leu Val Val Lys Ile Gly Asp Phe Gly Met Ser 660 665 670 Arg Asp Ile Tyr Ser Thr Asp Tyr Tyr Arg Val Gly Gly Arg Thr Met 675 680 685 Leu Pro Ile Arg Trp Met Pro Pro Glu Ser Ile Leu Tyr Arg Lys Phe 690 695 700 Thr Thr Glu Ser Asp Val Trp Ser Phe Gly Val Val Leu Trp Glu Ile 705 710 715 720 Phe Thr Tyr Gly Lys Gln Pro Trp Tyr Gln Leu Ser Asn Thr Glu Ala 725 730 735 Ile Asp Cys Ile Thr Gln Gly Arg Glu Leu Glu Arg Pro Arg Ala Cys 740 745 750 Pro Pro Glu Val Tyr Ala Ile Met Arg Gly Cys Trp Gln Arg Glu Pro 755 760 765 Gln Gln Arg His Ser Ile Lys Asp Val His Ala Arg Leu Gln Ala Leu 770 775 780 Ala Gln Ala Pro Pro Val Tyr Leu Asp Val Leu Gly 785 790 795 666PRTHomo sapiens 66Val Glu Met His His Trp 1 5 676PRTMus musculus 67Val Glu Gln His His Trp 1 5 68285DNAHomo sapiens 68ctaatagaag tggaaaagcc tctgtacgga gtagaggtgt ttgttggtga aacagcccac 60tttgaaattg aactttctgt ggagatgcac cactgggttc acggccagtg gaagctgaaa 120ggacagcctt tgacagcttc ccctgactgt gaaatcattg aggatggaaa gaagcatatt 180ctgatccttc ataactgtca gctgggtatg acaggagagg tttccttcca ggtggagatg 240caccactgga aatctgcagc caatctgaaa gtgaaagaat tgtaa 2856994PRTHomo sapiens 69Leu Ile Glu Val Glu Lys Pro Leu Tyr Gly Val Glu Val Phe Val Gly 1 5 10 15 Glu Thr Ala His Phe Glu Ile Glu Leu Ser Val Glu Met His His Trp 20 25 30 Val His Gly Gln Trp Lys Leu Lys Gly Gln Pro Leu Thr Ala Ser Pro 35 40 45 Asp Cys Glu Ile Ile Glu Asp Gly Lys Lys His Ile Leu Ile Leu His 50 55 60 Asn Cys Gln Leu Gly Met Thr Gly Glu Val Ser Phe Gln Val Glu Met 65 70 75 80 His His Trp Lys Ser Ala Ala Asn Leu Lys Val Lys Glu Leu 85 90 70667PRTArtificial SequenceHuman TrkA-CamelFc 70Met Leu Arg Gly Gly Arg Arg Gly Gln Leu Gly Trp His Ser Trp Ala 1 5 10 15 Ala Gly Pro Gly Ser Leu Leu Ala Trp Leu Ile Leu Ala Ser Ala Gly 20 25 30 Ala Ala Pro Cys Pro Asp Ala Cys Cys Pro His Gly Ser Ser Gly Leu 35 40 45 Arg Cys Thr Arg Asp Gly Ala Leu Asp Ser Leu His His Leu Pro Gly 50 55 60 Ala Glu Asn Leu Thr Glu Leu Tyr Ile Glu Asn Gln Gln His Leu Gln 65 70 75 80 His Leu Glu Leu Arg Asp Leu Arg Gly Leu Gly Glu Leu Arg Asn Leu 85 90 95 Thr Ile Val Lys Ser Gly Leu Arg Phe Val Ala Pro Asp Ala Phe His 100 105 110 Phe Thr Pro Arg Leu Ser Arg Leu Asn Leu Ser Phe Asn Ala Leu Glu 115 120 125 Ser Leu Ser Trp Lys Thr Val Gln Gly Leu Ser Leu Gln Glu Leu Val 130 135 140 Leu Ser Gly Asn Pro Leu His Cys Ser Cys Ala Leu Arg Trp Leu Gln 145 150 155 160 Arg Trp Glu Glu Glu Gly Leu Gly Gly Val Pro Glu Gln Lys Leu Gln 165 170 175 Cys His Gly Gln Gly Pro Leu Ala His Met Pro Asn Ala Ser Cys Gly 180 185 190 Val Pro Thr Leu Lys Val Gln Val Pro Asn Ala Ser Val Asp Val Gly 195 200 205 Asp Asp Val Leu Leu Arg Cys Gln Val Glu Gly Arg Gly Leu Glu Gln 210 215 220 Ala Gly Trp Ile Leu Thr Glu Leu Glu Gln Ser Ala Thr Val Met Lys 225 230 235 240 Ser Gly Gly Leu Pro Ser Leu Gly Leu Thr Leu Ala Asn Val Thr Ser 245 250 255 Asp Leu Asn Arg Lys Asn Val Thr Cys Trp Ala Glu Asn Asp Val Gly 260 265 270 Arg Ala Glu Val Ser Val Gln Val Asn Val Ser Phe Pro Ala Ser Val 275 280 285 Gln Leu His Thr Ala Val Glu Met His His Trp Cys Ile Pro Phe Ser 290 295 300 Val Asp Gly Gln Pro Ala Pro Ser Leu Arg Trp Leu Phe Asn Gly Ser 305 310 315 320 Val Leu Asn Glu Thr Ser Phe Ile Phe Thr Glu Phe Leu Glu Pro Ala 325 330 335 Ala Asn Glu Thr Val Arg His Gly Cys Leu Arg Leu Asn Gln Pro Thr 340 345 350 His Val Asn Asn Gly Asn Tyr Thr Leu Leu Ala Ala Asn Pro Phe Gly 355 360 365 Gln Ala Ser Ala Ser Ile Met Ala Ala Phe Met Asp Asn Pro Phe Glu 370 375 380 Phe Asn Pro Glu Asp Pro Ile Pro Val Ser Phe Ser Pro Val Asp Thr 385 390 395 400 Asn Ser Thr Ser Gly Asp Pro Val Glu Lys Ala Ala Ala Asp Arg Lys 405 410 415 Ile Pro Gln Pro Gln Pro Lys Pro Gln Pro Gln Pro Gln Pro Gln Pro 420 425 430 Lys Pro Gln Pro Lys Pro Glu Pro Glu Cys Thr Cys Pro Lys Cys Pro 435 440 445 Ala Pro Glu Leu Pro Gly Gly Pro Ser Val Phe Val Phe Pro Pro Lys 450 455 460 Pro Lys Asp Val Leu Ser Ile Ser Gly Arg Pro Glu Val Thr Cys Val 465 470 475 480 Val Val Asp Val Gly Lys Lys Asp Pro Glu Val Asn Phe Asn Trp Tyr 485 490 495 Val Asp Gly Val Glu Val Arg Thr Ala Asn Thr Lys Pro Lys Glu Glu 500 505 510 Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Ile Gln His 515 520 525 Gln Asp Trp Leu Thr Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys 530 535 540 Ala Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Ala Lys Gly Gln 545 550 555 560 Thr Arg Glu Pro Gln Val Tyr Thr Leu Ala Pro His Arg Glu Glu Leu 565 570 575 Ala Lys Asp Thr Val Ser Val Thr Cys Leu Val Lys Gly Phe Tyr Pro 580 585 590 Pro Asp Ile Asn Val Glu Trp Gln Arg Asn Arg Gln Pro Glu Ser Glu 595 600 605 Gly Ala Tyr Ala Thr Thr Leu Pro Gln Leu Asp Asn Asp Gly Thr Tyr 610 615 620 Phe Leu Tyr Ser Lys Leu Ser Val Gly Lys Asn Thr Trp Gln Arg Gly 625 630 635 640 Glu Thr Phe Thr Cys Val Val Met His Glu Ala Leu His Asn His Tyr 645 650 655 Thr Gln Lys Ser Ile Thr Gln Ser Ser Gly Lys 660 665 71687PRTArtificial Sequencehuman TrkB-CamelFc 71Met Ser Ser Trp Ile Arg Trp His Gly Pro Ala Met Ala Arg Leu Trp 1 5 10 15 Gly Phe Cys Trp Leu Val Val Gly Phe Trp Arg Ala Ala Phe Ala Cys 20 25 30 Pro Thr Ser Cys Lys Cys Ser Ala Ser Arg Ile Trp Cys Ser Asp Pro 35 40 45 Ser Pro Gly Ile Val Ala Phe Pro Arg Leu Glu Pro Asn Ser Val Asp 50 55 60 Pro Glu Asn Ile Thr Glu Ile Phe Ile Ala Asn Gln Lys Arg Leu Glu 65 70 75 80 Ile Ile Asn Glu Asp Asp Val Glu Ala Tyr Val Gly Leu Arg Asn Leu 85 90 95 Thr Ile Val Asp Ser Gly Leu Lys Phe Val Ala His Lys Ala Phe Leu 100 105 110 Lys Asn Ser Asn Leu Gln His Ile Asn Phe Thr Arg Asn Lys Leu Thr 115 120 125 Ser Leu Ser Arg Lys His Phe Arg His Leu Asp Leu Ser Glu Leu Ile 130 135 140 Leu Val Gly Asn Pro Phe Thr Cys Ser Cys Asp Ile Met Trp Ile Lys 145 150 155 160 Thr Leu Gln Glu Ala Lys Ser Ser Pro Asp Thr Gln Asp Leu Tyr Cys 165 170 175 Leu Asn Glu Ser Ser Lys Asn Ile Pro Leu Ala Asn Leu Gln Ile Pro 180 185 190 Asn Cys Gly Leu Pro Ser Ala Asn Leu Ala Ala Pro Asn Leu Thr Val 195 200 205 Glu Glu Gly Lys Ser Ile Thr Leu Ser Cys Ser Val Ala Gly Asp Pro 210 215 220 Val Pro Asn Met Tyr Trp Asp Val Gly Asn Leu Val Ser Lys His Met 225 230 235 240 Asn Glu Thr Ser His Thr Gln Gly Ser Leu Arg Ile Thr Asn Ile Ser 245 250 255 Ser Asp Asp Ser Gly Lys Gln Ile Ser Cys Val Ala Glu Asn Leu Val 260 265 270 Gly Glu Asp Gln Asp Ser Val Asn Leu Thr Val His Phe Ala Pro Thr 275 280 285 Ile Thr Phe Leu Glu Ser Pro Thr Ser Asp His His Trp Cys Ile Pro 290 295 300

Phe Thr Val Lys Gly Asn Pro Lys Pro Ala Leu Gln Trp Phe Tyr Asn 305 310 315 320 Gly Ala Ile Leu Asn Glu Ser Lys Tyr Ile Cys Thr Lys Ile His Val 325 330 335 Thr Asn His Thr Glu Tyr His Gly Cys Leu Gln Leu Asp Asn Pro Thr 340 345 350 His Met Asn Asn Gly Asp Tyr Thr Leu Ile Ala Lys Asn Glu Tyr Gly 355 360 365 Lys Asp Glu Lys Gln Ile Ser Ala His Phe Met Gly Trp Pro Gly Ile 370 375 380 Asp Asp Gly Ala Asn Pro Asn Tyr Pro Asp Val Ile Tyr Glu Asp Tyr 385 390 395 400 Gly Thr Ala Ala Asn Asp Ile Gly Asp Thr Thr Asn Arg Ser Asn Glu 405 410 415 Ile Pro Ser Thr Asp Val Thr Asp Lys Thr Gly Arg Glu His Ala Ala 420 425 430 Ala Asp Arg Lys Ile Pro Gln Pro Gln Pro Lys Pro Gln Pro Gln Pro 435 440 445 Gln Pro Gln Pro Lys Pro Gln Pro Lys Pro Glu Pro Glu Cys Thr Cys 450 455 460 Pro Lys Cys Pro Ala Pro Glu Leu Pro Gly Gly Pro Ser Val Phe Val 465 470 475 480 Phe Pro Pro Lys Pro Lys Asp Val Leu Ser Ile Ser Gly Arg Pro Glu 485 490 495 Val Thr Cys Val Val Val Asp Val Gly Lys Lys Asp Pro Glu Val Asn 500 505 510 Phe Asn Trp Tyr Val Asp Gly Val Glu Val Arg Thr Ala Asn Thr Lys 515 520 525 Pro Lys Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu 530 535 540 Thr Ile Gln His Gln Asp Trp Leu Thr Gly Lys Glu Phe Lys Cys Lys 545 550 555 560 Val Asn Asn Lys Ala Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys 565 570 575 Ala Lys Gly Gln Thr Arg Glu Pro Gln Val Tyr Thr Leu Ala Pro His 580 585 590 Arg Glu Glu Leu Ala Lys Asp Thr Val Ser Val Thr Cys Leu Val Lys 595 600 605 Gly Phe Tyr Pro Pro Asp Ile Asn Val Glu Trp Gln Arg Asn Arg Gln 610 615 620 Pro Glu Ser Glu Gly Ala Tyr Ala Thr Thr Leu Pro Gln Leu Asp Asn 625 630 635 640 Asp Gly Thr Tyr Phe Leu Tyr Ser Lys Leu Ser Val Gly Lys Asn Thr 645 650 655 Trp Gln Arg Gly Glu Thr Phe Thr Cys Val Val Met His Glu Ala Leu 660 665 670 His Asn His Tyr Thr Gln Lys Ser Ile Thr Gln Ser Ser Gly Lys 675 680 685 72686PRTArtificial Sequencehuman TrkC-CamelFc 72Met Asp Val Ser Leu Cys Pro Ala Lys Cys Ser Phe Trp Arg Ile Phe 1 5 10 15 Leu Leu Gly Ser Val Trp Leu Asp Tyr Val Gly Ser Val Leu Ala Cys 20 25 30 Pro Ala Asn Cys Val Cys Ser Lys Thr Glu Ile Asn Cys Arg Arg Pro 35 40 45 Asp Asp Gly Asn Leu Phe Pro Leu Leu Glu Gly Gln Asp Ser Gly Asn 50 55 60 Ser Asn Gly Asn Ala Ser Ile Asn Ile Thr Asp Ile Ser Arg Asn Ile 65 70 75 80 Thr Ser Ile His Ile Glu Asn Trp Arg Ser Leu His Thr Leu Asn Ala 85 90 95 Val Asp Met Glu Leu Tyr Thr Gly Leu Gln Lys Leu Thr Ile Lys Asn 100 105 110 Ser Gly Leu Arg Ser Ile Gln Pro Arg Ala Phe Ala Lys Asn Pro His 115 120 125 Leu Arg Tyr Ile Asn Leu Ser Ser Asn Arg Leu Thr Thr Leu Ser Trp 130 135 140 Gln Leu Phe Gln Thr Leu Ser Leu Arg Glu Leu Gln Leu Glu Gln Asn 145 150 155 160 Phe Phe Asn Cys Ser Cys Asp Ile Arg Trp Met Gln Leu Trp Gln Glu 165 170 175 Gln Gly Glu Ala Lys Leu Asn Ser Gln Asn Leu Tyr Cys Ile Asn Ala 180 185 190 Asp Gly Ser Gln Leu Pro Leu Phe Arg Met Asn Ile Ser Gln Cys Asp 195 200 205 Leu Pro Glu Ile Ser Val Ser His Val Asn Leu Thr Val Arg Glu Gly 210 215 220 Asp Asn Ala Val Ile Thr Cys Asn Gly Ser Gly Ser Pro Leu Pro Asp 225 230 235 240 Val Asp Trp Ile Val Thr Gly Leu Gln Ser Ile Asn Thr His Gln Thr 245 250 255 Asn Leu Asn Trp Thr Asn Val His Ala Ile Asn Leu Thr Leu Val Asn 260 265 270 Val Thr Ser Glu Asp Asn Gly Phe Thr Leu Thr Cys Ile Ala Glu Asn 275 280 285 Val Val Gly Met Ser Asn Ala Ser Val Ala Leu Thr Val Tyr Tyr Pro 290 295 300 Pro Arg Val Val Ser Leu Glu Glu Pro Glu Leu Arg Leu Glu His Cys 305 310 315 320 Ile Glu Phe Val Val Arg Gly Asn Pro Pro Pro Thr Leu His Trp Leu 325 330 335 His Asn Gly Gln Pro Leu Arg Glu Ser Lys Ile Ile His Val Glu Tyr 340 345 350 Tyr Gln Glu Gly Glu Ile Ser Glu Gly Cys Leu Leu Phe Asn Lys Pro 355 360 365 Thr His Tyr Asn Asn Gly Asn Tyr Thr Leu Ile Ala Lys Asn Pro Leu 370 375 380 Gly Thr Ala Asn Gln Thr Ile Asn Gly His Phe Leu Lys Glu Pro Phe 385 390 395 400 Pro Glu Ser Thr Asp Asn Phe Ile Leu Phe Asp Glu Val Ser Pro Thr 405 410 415 Pro Pro Ile Thr Val Thr His Lys Pro Glu Glu Asp Thr Ala Ala Ala 420 425 430 Asp Arg Lys Ile Pro Gln Pro Gln Pro Lys Pro Gln Pro Gln Pro Gln 435 440 445 Pro Gln Pro Lys Pro Gln Pro Lys Pro Glu Pro Glu Cys Thr Cys Pro 450 455 460 Lys Cys Pro Ala Pro Glu Leu Pro Gly Gly Pro Ser Val Phe Val Phe 465 470 475 480 Pro Pro Lys Pro Lys Asp Val Leu Ser Ile Ser Gly Arg Pro Glu Val 485 490 495 Thr Cys Val Val Val Asp Val Gly Lys Lys Asp Pro Glu Val Asn Phe 500 505 510 Asn Trp Tyr Val Asp Gly Val Glu Val Arg Thr Ala Asn Thr Lys Pro 515 520 525 Lys Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 530 535 540 Ile Gln His Gln Asp Trp Leu Thr Gly Lys Glu Phe Lys Cys Lys Val 545 550 555 560 Asn Asn Lys Ala Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Ala 565 570 575 Lys Gly Gln Thr Arg Glu Pro Gln Val Tyr Thr Leu Ala Pro His Arg 580 585 590 Glu Glu Leu Ala Lys Asp Thr Val Ser Val Thr Cys Leu Val Lys Gly 595 600 605 Phe Tyr Pro Pro Asp Ile Asn Val Glu Trp Gln Arg Asn Arg Gln Pro 610 615 620 Glu Ser Glu Gly Ala Tyr Ala Thr Thr Leu Pro Gln Leu Asp Asn Asp 625 630 635 640 Gly Thr Tyr Phe Leu Tyr Ser Lys Leu Ser Val Gly Lys Asn Thr Trp 645 650 655 Gln Arg Gly Glu Thr Phe Thr Cys Val Val Met His Glu Ala Leu His 660 665 670 Asn His Tyr Thr Gln Lys Ser Ile Thr Gln Ser Ser Gly Lys 675 680 685 73506PRTArtificial Sequencehuman p75NTR-CamelFc 73Met Gly Ala Gly Ala Thr Gly Arg Ala Met Asp Gly Pro Arg Leu Leu 1 5 10 15 Leu Leu Leu Leu Leu Gly Val Ser Leu Gly Gly Ala Lys Glu Ala Cys 20 25 30 Pro Thr Gly Leu Tyr Thr His Ser Gly Glu Cys Cys Lys Ala Cys Asn 35 40 45 Leu Gly Glu Gly Val Ala Gln Pro Cys Gly Ala Asn Gln Thr Val Cys 50 55 60 Glu Pro Cys Leu Asp Ser Val Thr Phe Ser Asp Val Val Ser Ala Thr 65 70 75 80 Glu Pro Cys Lys Pro Cys Thr Glu Cys Val Gly Leu Gln Ser Met Ser 85 90 95 Ala Pro Cys Val Glu Ala Asp Asp Ala Val Cys Arg Cys Ala Tyr Gly 100 105 110 Tyr Tyr Gln Asp Glu Thr Thr Gly Arg Cys Glu Ala Cys Arg Val Cys 115 120 125 Glu Ala Gly Ser Gly Leu Val Phe Ser Cys Gln Asp Lys Gln Asn Thr 130 135 140 Val Cys Glu Glu Cys Pro Asp Gly Thr Tyr Ser Asp Glu Ala Asn His 145 150 155 160 Val Asp Pro Cys Leu Pro Cys Thr Val Cys Glu Asp Thr Glu Arg Gln 165 170 175 Leu Arg Glu Cys Thr Arg Trp Ala Asp Ala Glu Cys Glu Glu Ile Pro 180 185 190 Gly Arg Trp Ile Thr Arg Ser Thr Pro Pro Glu Gly Ser Asp Ser Thr 195 200 205 Ala Pro Ser Thr Gln Glu Pro Glu Ala Pro Pro Glu Gln Asp Leu Ile 210 215 220 Ala Ser Thr Val Ala Gly Val Val Thr Thr Val Met Gly Ser Ser Gln 225 230 235 240 Pro Val Val Thr Arg Gly Thr Thr Asp Ala Ala Ala Asp Arg Lys Ile 245 250 255 Pro Gln Pro Gln Pro Lys Pro Gln Pro Gln Pro Gln Pro Gln Pro Lys 260 265 270 Pro Gln Pro Lys Pro Glu Pro Glu Cys Thr Cys Pro Lys Cys Pro Ala 275 280 285 Pro Glu Leu Pro Gly Gly Pro Ser Val Phe Val Phe Pro Pro Lys Pro 290 295 300 Lys Asp Val Leu Ser Ile Ser Gly Arg Pro Glu Val Thr Cys Val Val 305 310 315 320 Val Asp Val Gly Lys Lys Asp Pro Glu Val Asn Phe Asn Trp Tyr Val 325 330 335 Asp Gly Val Glu Val Arg Thr Ala Asn Thr Lys Pro Lys Glu Glu Gln 340 345 350 Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Ile Gln His Gln 355 360 365 Asp Trp Leu Thr Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Ala 370 375 380 Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Ala Lys Gly Gln Thr 385 390 395 400 Arg Glu Pro Gln Val Tyr Thr Leu Ala Pro His Arg Glu Glu Leu Ala 405 410 415 Lys Asp Thr Val Ser Val Thr Cys Leu Val Lys Gly Phe Tyr Pro Pro 420 425 430 Asp Ile Asn Val Glu Trp Gln Arg Asn Arg Gln Pro Glu Ser Glu Gly 435 440 445 Ala Tyr Ala Thr Thr Leu Pro Gln Leu Asp Asn Asp Gly Thr Tyr Phe 450 455 460 Leu Tyr Ser Lys Leu Ser Val Gly Lys Asn Thr Trp Gln Arg Gly Glu 465 470 475 480 Thr Phe Thr Cys Val Val Met His Glu Ala Leu His Asn His Tyr Thr 485 490 495 Gln Lys Ser Ile Thr Gln Ser Ser Gly Lys 500 505 74670PRTArtificial Sequencemouse TrkA-CamelFc 74Met Leu Arg Gly Gln Arg Leu Gly Gln Leu Gly Trp His Arg Pro Ala 1 5 10 15 Ala Gly Leu Gly Ser Leu Met Thr Ser Leu Met Leu Ala Cys Ala Ser 20 25 30 Ala Ala Ser Cys Arg Glu Val Cys Cys Pro Val Gly Pro Ser Gly Leu 35 40 45 Arg Cys Thr Arg Ala Gly Ser Leu Asp Thr Leu Arg Gly Leu Arg Gly 50 55 60 Ala Gly Asn Leu Thr Glu Leu Tyr Val Glu Asn Gln Gln His Leu Gln 65 70 75 80 Arg Leu Glu Phe Glu Asp Leu Gln Gly Leu Gly Glu Leu Arg Ser Leu 85 90 95 Thr Ile Val Lys Ser Gly Leu Arg Phe Val Ala Pro Asp Ala Phe Arg 100 105 110 Phe Thr Pro Arg Leu Ser His Leu Asn Leu Ser Ser Asn Ala Leu Glu 115 120 125 Ser Leu Ser Trp Lys Thr Val Gln Gly Leu Ser Leu Gln Asp Leu Thr 130 135 140 Leu Ser Gly Asn Pro Leu His Cys Ser Cys Ala Leu Phe Trp Leu Gln 145 150 155 160 Arg Trp Glu Gln Glu Gly Leu Cys Gly Val His Thr Gln Thr Leu His 165 170 175 Asp Ser Gly Pro Gly Asp Gln Phe Leu Pro Leu Gly His Asn Thr Ser 180 185 190 Cys Gly Val Pro Thr Val Lys Ile Gln Met Pro Asn Asp Ser Val Glu 195 200 205 Val Gly Asp Asp Val Phe Leu Gln Cys Gln Val Glu Gly Leu Ala Leu 210 215 220 Gln Gln Ala Asp Trp Ile Leu Thr Glu Leu Glu Gly Ala Ala Thr Val 225 230 235 240 Lys Lys Phe Gly Asp Leu Pro Ser Leu Gly Leu Ile Leu Val Asn Val 245 250 255 Thr Ser Asp Leu Asn Lys Lys Asn Val Thr Cys Trp Ala Glu Asn Asp 260 265 270 Val Gly Arg Ala Glu Val Ser Val Gln Val Ser Val Ser Phe Pro Ala 275 280 285 Ser Val His Leu Gly Leu Ala Val Glu Gln His His Trp Cys Ile Pro 290 295 300 Phe Ser Val Asp Gly Gln Pro Ala Pro Ser Leu Arg Trp Leu Phe Asn 305 310 315 320 Gly Ser Val Leu Asn Glu Thr Ser Phe Ile Phe Thr Gln Phe Leu Glu 325 330 335 Ser Ala Leu Thr Asn Glu Thr Met Arg His Gly Cys Leu Arg Leu Asn 340 345 350 Gln Pro Thr His Val Asn Asn Gly Asn Tyr Thr Leu Leu Ala Ala Asn 355 360 365 Pro Tyr Gly Gln Ala Ala Ala Ser Val Met Ala Ala Phe Met Asp Asn 370 375 380 Pro Phe Glu Phe Asn Pro Glu Asp Pro Ile Pro Val Ser Phe Ser Pro 385 390 395 400 Val Asp Gly Asn Ser Thr Ser Arg Asp Pro Val Glu Lys Ala Ala Ala 405 410 415 Asp Arg Lys Ile Pro Gln Pro Gln Pro Lys Pro Gln Pro Gln Pro Gln 420 425 430 Pro Gln Pro Lys Pro Gln Pro Lys Pro Glu Pro Glu Cys Thr Cys Pro 435 440 445 Lys Cys Pro Ala Pro Glu Leu Pro Gly Gly Pro Ser Val Phe Val Phe 450 455 460 Pro Pro Lys Pro Lys Asp Val Leu Ser Ile Ser Gly Arg Pro Glu Val 465 470 475 480 Thr Cys Val Val Val Asp Val Gly Lys Lys Asp Pro Glu Val Asn Phe 485 490 495 Asn Trp Tyr Val Asp Gly Val Glu Val Arg Thr Ala Asn Thr Lys Pro 500 505 510 Lys Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 515 520 525 Ile Gln His Gln Asp Trp Leu Thr Gly Lys Glu Phe Lys Cys Lys Val 530 535 540 Asn Asn Lys Ala Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Ala 545 550 555 560 Lys Gly Gln Thr Arg Glu Pro Gln Val Tyr Thr Leu Ala Pro His Arg 565 570 575 Glu Glu Leu Ala Lys Asp Thr Val Ser Val Thr Cys Leu Val Lys Gly 580 585 590 Phe Tyr Pro Pro Asp Ile Asn Val Glu Trp Gln Arg Asn Arg Gln Pro 595 600 605 Glu Ser Glu Gly Ala Tyr Ala Thr Thr Leu Pro Gln Leu Asp Asn Asp 610 615 620 Gly Thr Tyr Phe Leu Tyr Ser Lys Leu Ser Val Gly Lys Asn Thr Trp 625 630 635 640 Gln Arg Gly Glu Thr Phe Thr Cys Val Val Met His Glu Ala Leu His 645 650 655 Asn His Tyr Thr Gln Lys Ser Ile Thr Gln Ser Ser Gly Lys 660 665 670 75799PRTMus musculus 75Met Leu Arg Gly Gln Arg Leu Gly Gln Leu Gly Trp His Arg Pro Ala 1 5 10 15 Ala Gly Leu Gly Ser Leu Met Thr Ser Leu Met Leu Ala Cys Ala Ser 20 25 30 Ala Ala Ser Cys Arg Glu Val Cys Cys Pro

Val Gly Pro Ser Gly Leu 35 40 45 Arg Cys Thr Arg Ala Gly Ser Leu Asp Thr Leu Arg Gly Leu Arg Gly 50 55 60 Ala Gly Asn Leu Thr Glu Leu Tyr Val Glu Asn Gln Gln His Leu Gln 65 70 75 80 Arg Leu Glu Phe Glu Asp Leu Gln Gly Leu Gly Glu Leu Arg Ser Leu 85 90 95 Thr Ile Val Lys Ser Gly Leu Arg Phe Val Ala Pro Asp Ala Phe Arg 100 105 110 Phe Thr Pro Arg Leu Ser His Leu Asn Leu Ser Ser Asn Ala Leu Glu 115 120 125 Ser Leu Ser Trp Lys Thr Val Gln Gly Leu Ser Leu Gln Asp Leu Thr 130 135 140 Leu Ser Gly Asn Pro Leu His Cys Ser Cys Ala Leu Phe Trp Leu Gln 145 150 155 160 Arg Trp Glu Gln Glu Gly Leu Cys Gly Val His Thr Gln Thr Leu His 165 170 175 Asp Ser Gly Pro Gly Asp Gln Phe Leu Pro Leu Gly His Asn Thr Ser 180 185 190 Cys Gly Val Pro Thr Val Lys Ile Gln Met Pro Asn Asp Ser Val Glu 195 200 205 Val Gly Asp Asp Val Phe Leu Gln Cys Gln Val Glu Gly Leu Ala Leu 210 215 220 Gln Gln Ala Asp Trp Ile Leu Thr Glu Leu Glu Gly Ala Ala Thr Val 225 230 235 240 Lys Lys Phe Gly Asp Leu Pro Ser Leu Gly Leu Ile Leu Val Asn Val 245 250 255 Thr Ser Asp Leu Asn Lys Lys Asn Val Thr Cys Trp Ala Glu Asn Asp 260 265 270 Val Gly Arg Ala Glu Val Ser Val Gln Val Ser Val Ser Phe Pro Ala 275 280 285 Ser Val His Leu Gly Leu Ala Val Glu Gln His His Trp Cys Ile Pro 290 295 300 Phe Ser Val Asp Gly Gln Pro Ala Pro Ser Leu Arg Trp Leu Phe Asn 305 310 315 320 Gly Ser Val Leu Asn Glu Thr Ser Phe Ile Phe Thr Gln Phe Leu Glu 325 330 335 Ser Ala Leu Thr Asn Glu Thr Met Arg His Gly Cys Leu Arg Leu Asn 340 345 350 Gln Pro Thr His Val Asn Asn Gly Asn Tyr Thr Leu Leu Ala Ala Asn 355 360 365 Pro Tyr Gly Gln Ala Ala Ala Ser Val Met Ala Ala Phe Met Asp Asn 370 375 380 Pro Phe Glu Phe Asn Pro Glu Asp Pro Ile Pro Val Ser Phe Ser Pro 385 390 395 400 Val Asp Gly Asn Ser Thr Ser Arg Asp Pro Val Glu Lys Lys Asp Glu 405 410 415 Thr Pro Phe Gly Val Ser Val Ala Val Gly Leu Ala Val Ser Ala Ala 420 425 430 Leu Phe Leu Ser Ala Leu Leu Leu Val Leu Asn Lys Cys Gly Gln Arg 435 440 445 Ser Lys Phe Gly Ile Asn Arg Pro Ala Val Leu Ala Pro Glu Asp Gly 450 455 460 Leu Ala Met Ser Leu His Phe Met Thr Leu Gly Gly Ser Ser Leu Ser 465 470 475 480 Pro Thr Glu Gly Lys Gly Ser Gly Leu Gln Gly His Ile Met Glu Asn 485 490 495 Pro Gln Tyr Phe Ser Asp Thr Cys Val His His Ile Lys Arg Gln Asp 500 505 510 Ile Ile Leu Lys Trp Glu Leu Gly Glu Gly Ala Phe Gly Lys Val Phe 515 520 525 Leu Ala Glu Cys Tyr Asn Leu Leu Asn Asp Gln Asp Lys Met Leu Val 530 535 540 Ala Val Lys Ala Leu Lys Glu Ala Ser Glu Asn Ala Arg Gln Asp Phe 545 550 555 560 Gln Arg Glu Ala Glu Leu Leu Thr Met Leu Gln His Gln His Ile Val 565 570 575 Arg Phe Phe Gly Val Cys Thr Glu Gly Gly Pro Leu Leu Met Val Phe 580 585 590 Glu Tyr Met Arg His Gly Asp Leu Asn Arg Phe Leu Arg Ser His Gly 595 600 605 Pro Asp Ala Lys Leu Leu Ala Gly Gly Glu Asp Val Ala Pro Gly Pro 610 615 620 Leu Gly Leu Gly Gln Leu Leu Ala Val Ala Ser Gln Val Ala Ala Gly 625 630 635 640 Met Val Tyr Leu Ala Ser Leu His Phe Val His Arg Asp Leu Ala Thr 645 650 655 Arg Asn Cys Leu Val Gly Gln Gly Leu Val Val Lys Ile Gly Asp Phe 660 665 670 Gly Met Ser Arg Asp Ile Tyr Ser Thr Asp Tyr Tyr Arg Val Gly Gly 675 680 685 Arg Thr Met Leu Pro Ile Arg Trp Met Pro Pro Glu Ser Ile Leu Tyr 690 695 700 Arg Lys Phe Ser Thr Glu Ser Asp Val Trp Ser Phe Gly Val Val Leu 705 710 715 720 Trp Glu Ile Phe Thr Tyr Gly Lys Gln Pro Trp Tyr Gln Leu Ser Asn 725 730 735 Thr Glu Ala Ile Glu Cys Ile Thr Gln Gly Arg Glu Leu Glu Arg Pro 740 745 750 Arg Ala Cys Pro Pro Asp Val Tyr Ala Ile Met Arg Gly Cys Trp Gln 755 760 765 Arg Glu Pro Gln Gln Arg Leu Ser Met Lys Asp Val His Ala Arg Leu 770 775 780 Gln Ala Leu Ala Gln Ala Pro Pro Ser Tyr Leu Asp Val Leu Gly 785 790 795 76799PRTRattus rattus 76Met Leu Arg Gly Gln Arg His Gly Gln Leu Gly Trp His Arg Pro Ala 1 5 10 15 Ala Gly Leu Gly Gly Leu Val Thr Ser Leu Met Leu Ala Cys Ala Cys 20 25 30 Ala Ala Ser Cys Arg Glu Thr Cys Cys Pro Val Gly Pro Ser Gly Leu 35 40 45 Arg Cys Thr Arg Ala Gly Thr Leu Asn Thr Leu Arg Gly Leu Arg Gly 50 55 60 Ala Gly Asn Leu Thr Glu Leu Tyr Val Glu Asn Gln Arg Asp Leu Gln 65 70 75 80 Arg Leu Glu Phe Glu Asp Leu Gln Gly Leu Gly Glu Leu Arg Ser Leu 85 90 95 Thr Ile Val Lys Ser Gly Leu Arg Phe Val Ala Pro Asp Ala Phe His 100 105 110 Phe Thr Pro Arg Leu Ser His Leu Asn Leu Ser Ser Asn Ala Leu Glu 115 120 125 Ser Leu Ser Trp Lys Thr Val Gln Gly Leu Ser Leu Gln Asp Leu Thr 130 135 140 Leu Ser Gly Asn Pro Leu His Cys Ser Cys Ala Leu Leu Trp Leu Gln 145 150 155 160 Arg Trp Glu Gln Glu Asp Leu Cys Gly Val Tyr Thr Gln Lys Leu Gln 165 170 175 Gly Ser Gly Ser Gly Asp Gln Phe Leu Pro Leu Gly His Asn Asn Ser 180 185 190 Cys Gly Val Pro Ser Val Lys Ile Gln Met Pro Asn Asp Ser Val Glu 195 200 205 Val Gly Asp Asp Val Phe Leu Gln Cys Gln Val Glu Gly Gln Ala Leu 210 215 220 Gln Gln Ala Asp Trp Ile Leu Thr Glu Leu Glu Gly Thr Ala Thr Met 225 230 235 240 Lys Lys Ser Gly Asp Leu Pro Ser Leu Gly Leu Thr Leu Val Asn Val 245 250 255 Thr Ser Asp Leu Asn Lys Lys Asn Val Thr Cys Trp Ala Glu Asn Asp 260 265 270 Val Gly Arg Ala Glu Val Ser Val Gln Val Ser Val Ser Phe Pro Ala 275 280 285 Ser Val His Leu Gly Lys Ala Val Glu Gln His His Trp Cys Ile Pro 290 295 300 Phe Ser Val Asp Gly Gln Pro Ala Pro Ser Leu Arg Trp Phe Phe Asn 305 310 315 320 Gly Ser Val Leu Asn Glu Thr Ser Phe Ile Phe Thr Gln Phe Leu Glu 325 330 335 Ser Ala Leu Thr Asn Glu Thr Met Arg His Gly Cys Leu Arg Leu Asn 340 345 350 Gln Pro Thr His Val Asn Asn Gly Asn Tyr Thr Leu Leu Ala Ala Asn 355 360 365 Pro Tyr Gly Gln Ala Ala Ala Ser Ile Met Ala Ala Phe Met Asp Asn 370 375 380 Pro Phe Glu Phe Asn Pro Glu Asp Pro Ile Pro Val Ser Phe Ser Pro 385 390 395 400 Val Asp Thr Asn Ser Thr Ser Arg Asp Pro Val Glu Lys Lys Asp Glu 405 410 415 Thr Pro Phe Gly Val Ser Val Ala Val Gly Leu Ala Val Ser Ala Ala 420 425 430 Leu Phe Leu Ser Ala Leu Leu Leu Val Leu Asn Lys Cys Gly Gln Arg 435 440 445 Ser Lys Phe Gly Ile Asn Arg Pro Ala Val Leu Ala Pro Glu Asp Gly 450 455 460 Leu Ala Met Ser Leu His Phe Met Thr Leu Gly Gly Ser Ser Leu Ser 465 470 475 480 Pro Thr Glu Gly Lys Gly Ser Gly Leu Gln Gly His Ile Met Glu Asn 485 490 495 Pro Gln Tyr Phe Ser Asp Thr Cys Val His His Ile Lys Arg Gln Asp 500 505 510 Ile Ile Leu Lys Trp Glu Leu Gly Glu Gly Ala Phe Gly Lys Val Phe 515 520 525 Leu Ala Glu Cys Tyr Asn Leu Leu Asn Asp Gln Asp Lys Met Leu Val 530 535 540 Ala Val Lys Ala Leu Lys Glu Thr Ser Glu Asn Ala Arg Gln Asp Phe 545 550 555 560 His Arg Glu Ala Glu Leu Leu Thr Met Leu Gln His Gln His Ile Val 565 570 575 Arg Phe Phe Gly Val Cys Thr Glu Gly Gly Pro Leu Leu Met Val Phe 580 585 590 Glu Tyr Met Arg His Gly Asp Leu Asn Arg Phe Leu Arg Ser His Gly 595 600 605 Pro Asp Ala Lys Leu Leu Ala Gly Gly Glu Asp Val Ala Pro Gly Pro 610 615 620 Leu Gly Leu Gly Gln Leu Leu Ala Val Ala Ser Gln Val Ala Ala Gly 625 630 635 640 Met Val Tyr Leu Ala Ser Leu His Phe Val His Arg Asp Leu Ala Thr 645 650 655 Arg Asn Cys Leu Val Gly Gln Gly Leu Val Val Lys Ile Gly Asp Phe 660 665 670 Gly Met Ser Arg Asp Ile Tyr Ser Thr Asp Tyr Tyr Arg Val Gly Gly 675 680 685 Arg Thr Met Leu Pro Ile Arg Trp Met Pro Pro Glu Ser Ile Leu Tyr 690 695 700 Arg Lys Phe Ser Thr Glu Ser Asp Val Trp Ser Phe Gly Val Val Leu 705 710 715 720 Trp Glu Ile Phe Thr Tyr Gly Lys Gln Pro Trp Tyr Gln Leu Ser Asn 725 730 735 Thr Glu Ala Ile Glu Cys Ile Thr Gln Gly Arg Glu Leu Glu Arg Pro 740 745 750 Arg Ala Cys Pro Pro Asp Val Tyr Ala Ile Met Arg Gly Cys Trp Gln 755 760 765 Arg Glu Pro Gln Gln Arg Leu Ser Met Lys Asp Val His Ala Arg Leu 770 775 780 Gln Ala Leu Ala Gln Ala Pro Pro Ser Tyr Leu Asp Val Leu Gly 785 790 795 77830PRTMacaca mulatta 77Met Ser Ala Glu Ala Trp Gln Gln Gln Leu Arg Ala His Arg Arg Leu 1 5 10 15 Pro Arg Arg Ser Lys Ala Gly Ala Ala Ala Met Leu Arg Gly Gly Arg 20 25 30 Arg Gly Gln Leu Gly Trp His Ser Trp Ala Ala Gly Pro Gly Ser Leu 35 40 45 Leu Ala Trp Leu Met Leu Ala Ser Ala Gly Ala Ser Pro Cys Pro Asp 50 55 60 Ala Cys Cys Pro His Gly Ser Ser Gly Leu Arg Cys Thr Arg Asp Gly 65 70 75 80 Ala Leu Asp Ser Leu His His Leu Pro Gly Ala Glu Asn Leu Thr Glu 85 90 95 Leu Val Gln Gly Ser Trp Ala Ala Gly Phe Arg Glu Ile Glu Gly Arg 100 105 110 Ile Ser Gly His Gln Gly Lys Gly Gln Ala Ser Pro His Pro Leu Pro 115 120 125 Cys Tyr Glu Glu Ala Thr Pro Ala Phe Leu Leu Glu Leu Gly Ile Glu 130 135 140 Leu Ala Gly Ile Gly Gly Arg Cys Tyr Ile Glu Asn Gln Gln His Leu 145 150 155 160 Gln His Leu Glu Leu Arg Asp Leu Arg Gly Leu Gly Glu Leu Arg Asn 165 170 175 Leu Val Leu Ser Gly Asn Pro Leu His Cys Ser Cys Ala Leu Arg Trp 180 185 190 Leu Gln Arg Trp Glu Glu Glu Gly Leu Gly Gly Val His Glu Gln Lys 195 200 205 Leu Gln Cys His Gly Gln Gly Pro Leu Ala His Met Pro Asn Ala Ser 210 215 220 Cys Gly Val Pro Met Leu Lys Val Gln Val Pro Asn Ala Ser Val Asp 225 230 235 240 Val Gly Asp Asp Val Leu Leu Trp Cys Gln Val Glu Gly Arg Gly Leu 245 250 255 Glu Gln Ala Gly Trp Ile Leu Thr Glu Leu Glu Gln Ser Ala Thr Val 260 265 270 Met Lys Ser Gly Ala Leu Pro Ser Leu Gly Leu Thr Leu Ala Asn Val 275 280 285 Thr Ser Asp Leu Asn Arg Lys Asn Val Thr Cys Trp Ala Glu Asn Asp 290 295 300 Val Gly Arg Ala Glu Leu Ser Val Gln Val Asn Val Ser Phe Pro Ala 305 310 315 320 Ser Val Gln Leu His Thr Ala Val Glu Met His His Trp Cys Ile Pro 325 330 335 Phe Ser Val Asp Gly Gln Pro Ala Pro Ser Leu Arg Trp Leu Phe Asn 340 345 350 Gly Ser Val Leu Asn Glu Thr Ser Phe Ile Phe Thr Glu Phe Leu Glu 355 360 365 Pro Ala Ala Asn Glu Thr Val Arg His Gly Cys Leu Arg Leu Asn Gln 370 375 380 Pro Thr His Val Asn Asn Gly Asn Tyr Thr Leu Leu Ala Ala Asn Pro 385 390 395 400 Phe Gly Gln Ala Ser Ala Ser Ile Met Ala Ala Phe Met Asp Asn Pro 405 410 415 Phe Glu Phe Asn Pro Glu Asp Pro Ile Pro Val Ser Phe Ser Pro Val 420 425 430 Asp Thr Asn Ser Thr Ser Gly Asp Pro Val Glu Lys Lys Asp Glu Thr 435 440 445 Pro Phe Gly Val Ser Val Ala Val Gly Leu Ala Val Phe Ala Cys Leu 450 455 460 Phe Leu Ser Met Leu Leu Leu Val Leu Asn Lys Cys Gly Arg Arg Asn 465 470 475 480 Lys Phe Gly Ile Asn Arg Pro Ala Val Leu Ala Pro Glu Asp Gly Leu 485 490 495 Ala Met Ser Leu His Phe Met Thr Leu Gly Gly Ser Ser Leu Ser Pro 500 505 510 Thr Glu Gly Lys Gly Ser Gly Leu Gln Gly His Ile Ile Glu Asn Pro 515 520 525 Gln Tyr Phe Ser Asp Ala Cys Val His His Ile Lys Arg Arg Asp Ile 530 535 540 Val Leu Lys Trp Glu Leu Gly Glu Gly Ala Phe Gly Lys Val Phe Leu 545 550 555 560 Ala Glu Cys His Asn Leu Leu Pro Glu Gln Asp Lys Met Leu Val Ala 565 570 575 Val Lys Ala Leu Lys Glu Val Ser Glu Ser Ala Arg Gln Asp Phe Gln 580 585 590 Arg Glu Ala Glu Leu Leu Thr Met Leu Gln His Gln His Ile Val Arg 595 600 605 Phe Phe Gly Val Cys Thr Glu Gly Arg Pro Leu Leu Met Val Phe Glu 610 615 620 Tyr Met Arg His Gly Asp Leu Asn Arg Phe Leu Arg Ser His Gly Pro 625 630 635 640 Asp Ala Lys Leu Leu Ala Gly Gly Glu Asp Val Ala Pro Gly Pro Leu 645 650 655 Gly Leu Gly Gln Leu Leu Ala Val Ala Ser Gln Val Ala Ala Gly Met 660 665 670 Val Tyr Leu Ala Gly Leu His Phe Val His Arg Asp Leu Ala Thr Arg 675 680 685 Asn Cys Leu Val Gly Gln Gly Leu Val Val Lys Ile Gly Asp Phe Gly 690 695 700 Met Ser Arg Asp Ile Tyr Ser Thr Asp Tyr Tyr Arg Val Gly Gly Arg 705 710 715 720 Thr Met Leu Pro Ile Arg Trp Met Pro Pro Glu Ser Ile Leu Tyr Arg 725 730 735 Lys Phe Thr Thr Glu Ser Asp Val

Trp Ser Phe Gly Val Val Leu Trp 740 745 750 Glu Ile Phe Thr Tyr Gly Lys Gln Pro Trp Tyr Gln Leu Ser Asn Thr 755 760 765 Glu Ala Ile Asp Cys Ile Thr Gln Gly Arg Glu Leu Glu Arg Pro Arg 770 775 780 Ala Cys Pro Pro Glu Val Tyr Ala Ile Met Arg Gly Cys Trp Gln Arg 785 790 795 800 Glu Pro Gln Gln Arg His Ser Ile Lys Asp Val His Ala Arg Leu Gln 805 810 815 Ala Leu Ala Gln Ala Pro Pro Val Tyr Leu Asp Val Leu Gly 820 825 830 78216PRTArtificial Sequencehuman Ig1,2 TrkA 78Met Ala Cys Gly Val Pro Thr Leu Lys Val Gln Val Pro Asn Ala Ser 1 5 10 15 Val Asp Val Gly Asp Asp Val Leu Leu Arg Cys Gln Val Glu Gly Arg 20 25 30 Gly Leu Glu Gln Ala Gly Trp Ile Leu Thr Glu Leu Glu Gln Ser Ala 35 40 45 Thr Val Met Lys Ser Gly Gly Leu Pro Ser Leu Gly Leu Thr Leu Ala 50 55 60 Asn Val Thr Ser Asp Leu Asn Arg Lys Asn Leu Thr Cys Trp Ala Glu 65 70 75 80 Asn Asp Val Gly Arg Ala Glu Val Ser Val Gln Val Asn Val Ser Phe 85 90 95 Pro Ala Ser Val Gln Leu His Thr Ala Val Glu Met His His Trp Cys 100 105 110 Ile Pro Phe Ser Val Asp Gly Gln Pro Ala Pro Ser Leu Arg Trp Leu 115 120 125 Phe Asn Gly Ser Val Leu Asn Glu Thr Ser Phe Ile Phe Thr Glu Phe 130 135 140 Leu Glu Pro Ala Ala Asn Glu Thr Val Arg His Gly Cys Leu Arg Leu 145 150 155 160 Asn Gln Pro Thr His Val Asn Asn Gly Asn Tyr Thr Leu Leu Ala Ala 165 170 175 Asn Pro Phe Gly Gln Gly Ser Ala Ser Ile Met Ala Ala Phe Met Asp 180 185 190 Asn Pro Phe Glu Phe Asn Pro Glu Asp Pro Ile Pro Asp Asn Asn Thr 195 200 205 His Leu Glu Thr Arg Trp Arg Arg 210 215 79223PRTArtificial Sequencemouse Ig1,2 TrkA 79Met Ala Cys Gly Val Pro Thr Val Lys Ile Gln Met Pro Asn Asp Ser 1 5 10 15 Val Glu Val Gly Asp Asp Val Phe Leu Gln Cys Gln Val Glu Gly Leu 20 25 30 Ala Leu Gln Gln Ala Asp Trp Ile Leu Thr Glu Leu Glu Gly Ala Ala 35 40 45 Thr Val Lys Lys Phe Gly Asp Leu Pro Ser Leu Gly Leu Ile Leu Val 50 55 60 Asn Val Thr Ser Asp Leu Asn Lys Lys Asn Val Thr Cys Trp Ala Glu 65 70 75 80 Asn Asp Val Gly Arg Ala Glu Val Ser Val Gln Val Ser Val Ser Phe 85 90 95 Pro Ala Ser Val His Leu Gly Leu Ala Val Glu Gln His His Trp Cys 100 105 110 Ile Pro Phe Ser Val Asp Gly Gln Pro Ala Pro Ser Leu Arg Trp Leu 115 120 125 Phe Asn Gly Ser Val Leu Asn Glu Thr Ser Phe Ile Phe Thr Gln Phe 130 135 140 Leu Glu Ser Ala Leu Thr Asn Glu Thr Met Arg His Gly Cys Leu Arg 145 150 155 160 Leu Asn Gln Pro Thr His Val Asn Asn Gly Asn Tyr Thr Leu Leu Ala 165 170 175 Ala Asn Pro Tyr Gly Gln Ala Ala Ala Ser Val Met Ala Ala Phe Met 180 185 190 Asp Asn Pro Phe Glu Phe Asn Pro Glu Asp Pro Ile Pro Val Ser Phe 195 200 205 Ser Pro Val Asp Gly Asn Ser Thr Ser Arg Asp Pro Val Glu Lys 210 215 220 8051DNAArtificial Sequencesynthetic oligonucleotide 80catcatgaat tcctaataga agtggaaaag cctctgtacg gagtagaggt g 518163DNAArtificial Sequencesynthetic oligonucleotide 81cacagaaagt tcaatttcaa agtgggctgt ttcaccaaca aacacctcta ctccgtacag 60agg 638261DNAArtificial Sequencesynthetic oligonucleotide 82ccactttgaa attgaacttt ctgtggagat gcaccactgg gttcacggcc agtggaagct 60g 618360DNAArtificial Sequencesynthetic oligonucleotide 83caatgatttc acagtcaggg gaagctgtca aaggctgtcc tttcagcttc cactggccgt 608466DNAArtificial Sequencesynthetic oligonucleotide 84cttcccctga ctgtgaaatc attgaggatg gaaagaagca tattctgatc cttcataact 60gtcagc 668563DNAArtificial Sequencesynthetic oligonucleotide 85tctccacctg gaaggaaacc tctcctgtca tacccagctg acagttatga aggatcagaa 60tat 638663DNAArtificial Sequencesynthetic oligonucleotide 86ggtttccttc caggtggaga tgcaccactg gaaatctgca gccaatctga aagtgaaaga 60att 638758DNAArtificial Sequencesynthetic oligonucleotide 87taatacgact cactatagtc gacggatcct tacaattctt tcactttcag attggctg 588830DNAArtificial Sequencesynthetic oligonucleotide 88catcatgaat tcctaataga agtggaaaag 308925DNAArtificial Sequencesynthetic oligonucleotide 89taatacgact cactatagtc gacgg 25901410DNAArtificial SequenceCRB0089 IgG4 derived VH-CH1-H-CH2-CH3 90atggaatggt ccggcgtgtt catgttcctg ctgtccgtga ccgctggcgt gcactcccag 60gtgcagctgc aggaatccgg ccctggcctg gtgaaaccct cccagaccct gtccctgacc 120tgcgccatct ccggcgactc cgtgtcctcc aactccgccg cctggaactg gatccggcag 180tccccttctc ggggcctgga atggctgggc agaaccttcc atcggtccag atggtacaac 240gagtaccccg tgtccgtgcg gtcccggatc accatcaacc ccgacacctc caacaaccag 300ttcaccctgc agctgaactc cgtgaccccc gacgacaccg ccgtgtacta ctgcgccaga 360gcccctgtgg ccggcctgac cttcgatatc tggggccagg gcacaatggt gacagtgtcc 420tccgcctcca ccaagggccc ctctgtgttc cctctggccc cttgctcccg gtccacctct 480gagtctaccg ccgctctggg ctgtctggtg aaagactact tccccgagcc cgtgaccgtg 540tcctggaata gtggcgccct gacctccggc gtgcacacct ttccagccgt gctgcagtcc 600tccggcctgt actccctgtc ctccgtggtg accgtgccct cctccagcct gggcaccaag 660acctacacct gtaacgtgga ccacaagccc tccaacacca aggtggacaa gcgggtggaa 720tctaagtacg gccctccctg cccaccctgt cctgctccag agtttctggg cggaccctcc 780gtgttcctgt tccccccaaa gcccaaggac accctgatga tctcccggac ccccgaagtg 840acctgcgtgg tggtggacgt gtcccaggaa gatcccgagg tccagttcaa ttggtacgtg 900gacggcgtgg aagtgcacaa cgccaagacc aagcccagag aggaacagtt caactccacc 960taccgggtgg tgtctgtgct gaccgtgctg caccaggact ggctgaacgg caaagagtac 1020aagtgcaagg tctccaacaa gggcctgccc tccagcatcg aaaagaccat ctccaaggcc 1080aagggccagc cccgcgagcc tcaggtgtac acactgcccc ctagccaaga agagatgacc 1140aagaaccagg tgtccctgac atgcctggtg aagggcttct acccctccga tatcgccgtg 1200gaatgggagt ccaacggcca gcccgagaac aactacaaga ccaccccccc tgtgctggac 1260tccgacggct ccttcttcct gtactctcgg ctgaccgtgg acaagtcccg gtggcaggaa 1320ggcaacgtct tctcctgctc cgtgatgcac gaggccctgc acaaccacta cacccagaag 1380tctctgagcc tgtccctggg caagtgatga 141091696DNAArtificial SequenceCRB0089_VLCL 91atggaatggt ccggcgtgtt catgttcctg ctgtccgtga ccgctggcgt gcactcccag 60accgtggtga cacaggaacc ctccgtctct ggcgcccctg gccagagagt gaccatctcc 120tgcaccggct cctccagcat catcggcgct ggctacgagg tgcactggta tcagcatctg 180cccggcaccg cccccaagct gctgatctac gactccatca accggccctc cggcgtgccc 240gaccggttct ctggctccaa gtccggcacc tccgcctccc tggctatcac cggactgcag 300gccgaggacg aggccgacta ctactgccag tcctccgcca tcttcggcgg aggcaccaag 360ctgaccgtgc tgggccagcc caaggccaac cccaccgtga ccctgttccc cccatcctcc 420gaggaactgc aggccaacaa ggccaccctg gtctgcctga tctccgactt ctaccctggc 480gccgtgaccg tggcctggaa ggctgacggc tctcctgtga aggccggcgt ggaaaccacc 540aagccctcca agcagtccaa caacaaatac gccgcctcct cctacctgtc cctgaccccc 600gagcagtgga agtcccaccg gtcctacagc tgccaggtca cacacgaggg ctccaccgtg 660gaaaagaccg tggctcctac cgagtgctcc tgatga 6969289PRTArtificial SequenceI27 Ig-like domain of titin protein 92Leu Ile Glu Val Glu Lys Pro Leu Tyr Gly Val Glu Val Phe Val Gly 1 5 10 15 Glu Thr Ala His Phe Glu Ile Glu Leu Ser Glu Pro Asp Val His Gly 20 25 30 Gln Trp Lys Leu Lys Gly Gln Pro Leu Thr Ala Ser Pro Asp Cys Glu 35 40 45 Ile Ile Glu Asp Gly Lys Lys His Ile Leu Ile Leu His Asn Cys Gln 50 55 60 Leu Gly Met Thr Gly Glu Val Ser Phe Gln Ala Ala Asn Ala Lys Ser 65 70 75 80 Ala Ala Asn Leu Lys Val Lys Glu Leu 85 9320PRTArtificial Sequencepeptide linker 93Ser Gly Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly 1 5 10 15 Ser Ser Gly Thr 20 94247PRTArtificial SequenceCRB0022 scFv 94Asp Ile Val Leu Thr Gln Ser Pro Ala Leu Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Arg Ser Ser Pro Lys Pro Trp Ile Tyr 35 40 45 Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Tyr Thr 85 90 95 Phe Gly Gly Arg Thr Arg Leu Glu Ile Lys Pro Ser Gly Gly Ser Thr 100 105 110 Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Asp Ser Ser Gly Ala Gln 115 120 125 Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala Ser 130 135 140 Val Glu Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr 145 150 155 160 Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly 165 170 175 Gly Ile Asp Pro Ala Asn Gly Asn Thr Ile Tyr Val Pro Lys Phe Gln 180 185 190 Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr Leu 195 200 205 Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala 210 215 220 Arg Gly Gly Tyr Asp Phe Asp Tyr Ala Met Asp Tyr Trp Gly Gln Gly 225 230 235 240 Thr Ser Val Thr Val Ser Ser 245 95249PRTArtificial SequenceCRB0036 scFv 95Asp Ile Val Met Thr Gln Ala Ala Pro Ser Val Pro Val Thr Pro Gly 1 5 10 15 Glu Ser Val Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30 Ser Gly Ile Thr Ser Leu Tyr Trp Tyr Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Gln Leu Pro Ile Tyr Arg Met Ser Ser Leu Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Val Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His 85 90 95 Leu Glu Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 Arg Ser Gly Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu 115 120 125 Gly Ser Ser Gly Thr Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu 130 135 140 Leu Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr 145 150 155 160 Thr Phe Ser Asn Tyr Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His 165 170 175 Gly Leu Glu Trp Ile Gly Glu Ile Leu Pro Gly Ser Gly Ser Ala His 180 185 190 Tyr Ser Glu Lys Phe Lys Asp Lys Ala Thr Phe Thr Ala Asp Thr Ser 195 200 205 Ser Asn Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser 210 215 220 Ala Val Tyr Phe Cys Thr Arg Ser Pro Pro Asn Pro Gly Tyr Trp Gly 225 230 235 240 Gln Gly Thr Ser Val Thr Val Ser Ser 245 96253PRTArtificial SequenceCRB0069 scFv 96Gln Pro Gly Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Val Gly Ala Gly 20 25 30 Tyr Thr Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Thr Leu 35 40 45 Leu Ile Tyr Asp Asn Asn Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Arg Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu 65 70 75 80 Arg Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Arg Ser 85 90 95 Arg Val Tyr Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu Ser Gly 100 105 110 Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Ser 115 120 125 Gly Thr Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro 130 135 140 Ser Gln Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser 145 150 155 160 Ser Asn Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly 165 170 175 Leu Glu Arg Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp 180 185 190 Tyr Ala Glu Ser Val Lys Ser Arg Ile Ile Ile Asn Ser Gly Thr Ser 195 200 205 Lys Asn Gln Phe Ser Leu His Leu Asn Ser Val Thr Pro Glu Asp Thr 210 215 220 Ala Val Tyr Tyr Cys Gly Arg Ala Lys Asp Gly Trp Tyr Asp Ala Phe 225 230 235 240 Asp Ile Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 245 250 97254PRTArtificial SequenceCRB0072 scFv 97Asp Ile Gln Met Thr Gln Ser Pro Ile Ser Leu Ser Ala Thr Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Arg Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Val Phe Thr Leu Thr Ile Ser Gly Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Cys Cys Gln Gln Ser His Ser Thr Ala Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Ser Gly Gly Ser Thr 100 105 110 Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Ser Gly Thr Glu 115 120 125 Val Gln Leu Val Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Ser Ser 130 135 140 Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ile Asn Tyr Gly 145 150 155 160 Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Val Ala 165 170 175 Ser Ile Trp Tyr Glu Gly Ser Asn Glu Lys Tyr Ala Asp Ser Val Lys 180 185 190 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Tyr Thr Leu His Leu 195 200 205 Gln Leu Asn Ser Leu Arg Ala Glu Asp Thr Ala Asp Tyr Tyr Cys Val 210 215 220 Arg Asp Arg Gly Thr Gly Asp Tyr Arg Asn Ser Arg Phe Tyr Tyr Gly 225 230 235 240 Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 245 250 98245PRTArtificial SequenceCRB0082 scFv 98Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Thr 1 5 10 15 Thr Ala Arg Leu Ser Cys Glu Ala Thr Lys Ile Gly Ser Gln Arg Leu 20 25 30 His Trp Tyr

Gln Gln Lys Pro Gly Gln Ala Pro Val Met Val Ile Ser 35 40 45 Phe Asp Ser Asp Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60 Arg Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ser Ser Gly Tyr His 85 90 95 Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser Gly Gly Ser 100 105 110 Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Ser Gly Thr 115 120 125 Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 130 135 140 Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Phe 145 150 155 160 Thr Val Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 165 170 175 Arg Leu Gly Arg Thr Tyr Arg Arg Ser Lys Asp Tyr Ala Glu Tyr Met 180 185 190 Arg Ser Arg Leu Thr Ile Asn Ala Asp Thr Ser Lys Asn Gln Leu Ser 195 200 205 Leu Gln Leu Asp Ser Val Thr Pro Asp Asp Thr Ala Val Tyr Tyr Cys 210 215 220 Ala Arg Gly Gln Asn Ser Ala Phe Asp Leu Trp Gly Gln Arg Thr Met 225 230 235 240 Val Thr Val Ser Ser 245 99248PRTArtificial SequenceCRB0084 scFv 99Gln Pro Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Arg Gln 1 5 10 15 Thr Ala Thr Ile Thr Cys Gly Gly Asn Tyr Ile Gly Ser Lys Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Asp Pro Val Leu Val Val Asn 35 40 45 Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Ala Asn Thr Ala Thr Leu Thr Ile Ser Gly Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Thr Glu Pro 85 90 95 Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser Gly Gly Ser 100 105 110 Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Ser Gly Thr 115 120 125 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Arg Pro Ser Glu 130 135 140 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Phe Ser Asp Ser 145 150 155 160 Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Ile 165 170 175 Gly Glu Asn Asn His Arg Gly Ser Thr Asn Tyr Ser Pro Thr Leu Arg 180 185 190 Ser Arg Leu Ser Ile Ser Ile Asp Ser Ser Lys Asn Gln Phe Ser Leu 195 200 205 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 210 215 220 Arg Val Pro Tyr Arg Leu Arg Ser Arg Ile Phe Asp Val Trp Gly Gln 225 230 235 240 Gly Thr Met Val Thr Val Ser Ser 245 100247PRTArtificial SequenceCRB0089 scFv 100Gln Thr Val Val Thr Gln Glu Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Ile Ile Gly Ala Gly 20 25 30 Tyr Glu Val His Trp Tyr Gln His Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Asp Ser Ile Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Ser Ala Ile Phe 85 90 95 Gly Gly Gly Thr Lys Leu Thr Val Leu Ser Gly Gly Ser Thr Ser Gly 100 105 110 Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Ser Gly Thr Gln Val Gln 115 120 125 Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser 130 135 140 Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn Ser Ala Ala 145 150 155 160 Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu Trp Leu Gly 165 170 175 Arg Thr Phe His Arg Ser Arg Trp Tyr Asn Glu Tyr Pro Val Ser Val 180 185 190 Arg Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Asn Asn Gln Phe Thr 195 200 205 Leu Gln Leu Asn Ser Val Thr Pro Asp Asp Thr Ala Val Tyr Tyr Cys 210 215 220 Ala Arg Ala Pro Val Ala Gly Leu Thr Phe Asp Ile Trp Gly Gln Gly 225 230 235 240 Thr Met Val Thr Val Ser Ser 245 101252PRTArtificial SequenceCRB0088 scFv 101Gln Pro Val Leu Thr Gln Ser Pro Pro Ala Ser Ala Ser Leu Gly Ala 1 5 10 15 Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gly His Ser Ser Tyr Ala 20 25 30 Ile Ala Trp His Gln Gln Gln Pro Glu Lys Gly Pro Arg Tyr Leu Met 35 40 45 Lys Val Asn Ser Asp Gly Ser His Asn Lys Gly Asp Gly Ile Pro Asp 50 55 60 Arg Phe Ser Gly Ser Arg Ser Gly Ala Glu Arg Tyr Leu Thr Ile Ser 65 70 75 80 Ser Leu His Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Thr Trp Gly 85 90 95 Ala Gly Gly Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser 100 105 110 Gly Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser 115 120 125 Ser Gly Thr Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln 130 135 140 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 145 150 155 160 Ser Asp His Tyr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 165 170 175 Glu Trp Val Gly Arg Ile Arg Asn Lys Ala Asn Ser Tyr Thr Thr Glu 180 185 190 Tyr Ala Ala Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser 195 200 205 Lys Lys Thr Leu Tyr Leu Gln Met Thr Ser Leu Lys Thr Asp Asp Thr 210 215 220 Ala Leu Tyr Tyr Cys Ala Arg Asp Leu Gly Ala Ser Asp Ala Phe Asp 225 230 235 240 Leu Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 245 250 102112PRTArtificial SequenceConsensus 102Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Ser Gly Asp Gly Ser Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105 110

Claims

1. An antibody, recombinant or synthetic antigen-binding fragments thereof which is able to recognise and bind a sequence having at least 80% identity to an epitope comprising the sequence from amino acid residue 294 to amino acid residue 299 of the human TrkA amino acid sequence of SEQ ID NO: 65.

2. The antibody, recombinant or synthetic antigen-binding fragments thereof according to claim 1, which is an NGF antagonist.

3. The antibody, recombinant or synthetic antigen-binding fragments thereof according to claim 1, wherein said epitope consists of the sequence from amino acid residue 294 to amino acid residue 299 of the human TrkA amino acid sequence of SEQ ID NO: 65.

4. The antibody, recombinant or synthetic antigen-binding fragments thereof according to claim 1, comprising at least one heavy chain complementary determining region (CDRH3) amino acid sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 64, 43, 46, 49, 52, 55, 58 and 61.

5. The antibody, recombinant or synthetic antigen-binding fragments thereof according to claim 1, comprising at least one heavy chain complementary determining region (CDRH1) amino acid sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 62, 41, 44, 47, 50, 53, 56 and 59.

6. The antibody, recombinant or synthetic antigen-binding fragments thereof according to claim 1, comprising at least one heavy chain complementary determining region (CDRH2) amino acid sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 63, 42, 45, 48, 51, 54, 57 and 60.

7. The antibody, recombinant or synthetic antigen-binding fragments thereof according to claim 6, comprising a CDRH1 amino acid sequence having at least 80% identity to SEQ ID NO: 62, a CDRH2 amino acid sequence having at least 80% identity to SEQ ID NO: 63 and a CDRH3 amino acid sequence having at least 80% identity to SEQ ID NO: 64.

8. The antibody, recombinant or synthetic antigen-binding fragments thereof according to claim 1, comprising at least one light chain complementary determining region (CDRL3) amino acid sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 40, 19, 22, 25, 28, 31, 34 and 37.

9. The antibody, recombinant or synthetic antigen-binding fragments thereof according to claim 1, comprising at least one light chain complementary determining region (CDRL1) amino acid sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 38, 17, 20, 23, 26, 29, 32 and 35.

10. The antibody, recombinant or synthetic antigen-binding fragments thereof according to claim 1, comprising at least one light chain complementary determining region (CDRL2) amino acid sequence having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 39, 18, 21, 24, 27, 30, 33 and 36.

11. The antibody, recombinant or synthetic antigen-binding fragments thereof according to claim 10, comprising a CDRL1 amino acid sequence having at least 80% identity to SEQ ID NO: 38, a CDRL2 amino acid sequence having at least 80% identity to SEQ ID NO: 39 and a CDRL3 amino acid sequence having at least 80% identity to SEQ ID NO: 40.

12. The antibody, recombinant or synthetic antigen-binding fragments thereof according to claim 1, comprising a heavy chain variable region comprising SEQ ID NOs: 15, 1, 3, 5, 7, 9, 11 or 13.

13. The antibody, recombinant or synthetic antigen-binding fragments thereof according to claim 1, comprising a light chain variable region comprising SEQ ID NOs: 16, 2, 4, 6, 8, 10, 12 or 14.

14. A method for treating pain, cancer, neuronal disorders, inflammation-related diseases, or diabetes, in a subject in need thereof comprising administering an effective amount of at least one of an antibody, recombinant or synthetic antigen-binding fragments thereof, according to claim 1.

15. A pharmaceutical composition comprising at least one antibody, recombinant or synthetic antigen-binding fragments thereof according to claim 1, and pharmaceutically acceptable excipients.

16. A method of inhibiting TrkA in a subject in need thereof, comprising administering an effective amount of at least one of an antibody, recombinant or synthetic antigen-binding fragments thereof, according to claim 1.

17. The antibody according to claim 1.