Fusion proteins comprising extracellular domains of ROR1 and ROR2 to inhibit ROR2 signalling and activate cartilage development

Abstract

The present invention relates to receptor tyrosine kinase-like orphan receptor 2 (ROR2) inhibitors which are fused to IgG2-Fc domains.

Description

[0001] TREATMENT

[0002] FIELD OF THE INVENTION

[0003] FUSION PROTEINS COMPRISING EXTRACELLULAR DOMAINS OF ROR1 AND ROR2 TO INHIBIT ROR2 SIGNALLING AND ACTIVATE CARTILAGE DEVELOPMENT

[0004] BACKGROUND OF THE INVENTION Osteoarthritis (OA) is a joint disease characterized by breakdown of the articular cartilage, the tissue that covers the ends of the long bones allowing frictionless joint motion and bone changes including thickening of the bone supporting the cartilage and excessive bone formation at the margins of the joint (osteophytes). OA is usually caused by pathological, chronic mechanical injury, for instance excessive body weightjoint malalignment, or joint instability, such as lesions to ligaments and menisci.

[0005] OA is one of the leading causes of disability, affecting 528 million people globally (WHO, 2022). There are no disease-modifying treatments for osteoarthritis. Current treatment consists of non-steroidal anti-inflammatory drugs, and eventually joint replacement (NICE, 2014). Autologous chondrocyte implantation and microfracture are used to treat other cartilage disorders / problems. Autologous chondrocyte implantation is indicated only for localized, relatively small lesions, but it is not feasible in osteoarthritic joints because surrounding healthy cartilage is required for containing the implanted chondrocytes and for suturing. Furthermore, it is very costly and therefore not realistic for large / public healthcare providers. Similarly, microfracture is only indicated for small cartilage defects.

[0006] The present inventors previously investigated the role of ROR-family of receptor tyrosine kinases (RTKs) in cartilage formation and the treatment of OA. In particular, the present inventors previously found that receptor tyrosine kinase-like orphan receptor 2 (ROR2) is upregulated in cartilage following injury and that silencing of R0R2 using siRNA and shRNA approaches was useful for treating OA in mice and humans (see WO 2019 / 097247). There are, however, certain disadvantages associated with siRNA and shRNA treatments, particularly around delivery and therapeutic persistence owing to both extracellular and intracellular challenges. For example, in the serum, siRNAs may be degraded by circulating endonucleases and may also be rapidly cleared via the renal system. Consequently, siRNAs have half-life in the range of minutes to hours. siRNAs also suffer from poor cell penetration owing to their hydrophilic and their negative charge. Unproductive endosomal processing can also reduce the efficacy of siRNA-based approaches.

[0007] Owing to these drawbacks, the inventors previously found that it was necessary to carry out intra-articular injections of anti-ROR2 siRNAs in mice every five days. The need for frequent injections would limit the utility of this approach in humans as such injections would need to be carried out by a physician, are painful and carry the risk of infection. Thus, there remains a need for new OA treatments based on the inhibition of R0R2.

[0008] SUMMARY OF THE INVENTION

[0009] The present invention is based, at least in part, on the finding that the extracellular domains of R0R1 and R0R2 are able to inhibit R0R2 signalling and activate cartilage development (chondrogenesis). Furthermore, the inventors have surprisingly found that fusion proteins comprising the extracellular domain (ECD) of R0R2 are able to stimulate cartilage development (chondrogenesis). Such fusion proteins may have greater half-life and / or efficacy than sh / siRNA-inhibitors of R0R2 signalling. A preferred fusion protein comprises the ECD of R0R2 fused to a fragment crystallizable (Fc) region of a human immunoglobulin, such as IgG, A, M or E, but preferably of human IgG. IgGl, IgG4 and IgG2 are particularly preferred, with IgG2 most preferred. IgGl has “effector function”, in that IgGl -tagged constructs tend to kill cells that they bind to, whereas IgG2 and IgG4 have reduced or no effector function, which is preferred in the context of stimulating chondrogenesis. It was observed that a fusion protein comprising an IgG2 Fc domain fused to the N-terminus of the R0R2 ECD performed particularly well. To tag a protein of interest with an IgG Fc domain, tagging is usually performed at the C-terminus of the protein of interest. This is because Fc is the C-terminal part of an IgG molecule. Therefore, it is surprising that the N-terminus IgG2-Fc tagged R0R2 ECD was found to be the most effective at stimulating chondrogenesis. The inventors have also suprisingly found that untagged R0R2 ECD and R0R1 ECD both inhibited R0R2 signalling and stimulated cartilage development (chondrogenesis). Further, the inventors have found that untagged and IgG2-Fc tagged R0R2 ECD are able to inhibit canonical WNT signalling. In contrast to R0R2 silencing (e.g. using an siRNA-based or shRNA-based approach), which only suppresses YAP-TEAD signalling but has the potential to de-repress canonical WNT signalling, it has been unexpectly found that R0R2-ECD advantageously suppresses both YAP and canonical WNT signalling, thereby blocking the two major pathogenic signalling pathways in osteoarthritis.

[0010] Thus, in one aspect, the present invention provides a receptor tyrosine kinase-like orphan receptor 2 (R0R2) inhibitor which is a fusion protein comprising or consisting of a receptor tyrosine kinase-like orphan receptor extracellular domain (ROR-ECD) and an immunoglobulin G2 fragment crystallisable domain (IgG2-Fc).

[0011] In one embodiment, the ROR-ECD is an R0R2-ECD. In one embodiment, the R0R2- ECD comprises or consists of the amino acid sequence of SEQ ID NO: 1 or a functional variant or fragment thereof having at least about 80% identity thereto. In one embodiment, the ROR2-ECD is encoded by the nucleotide sequence of SEQ ID NO: 2 or a functional variant thereof having at least about 80% identity thereto. In one embodiment, the ROR2 inhibitor comprising an ROR2-ECD is capable of inhibiting canonical WNT signalling.

[0012] In one embodiment, the ROR-ECD is an ROR1-ECD. In one embodiment, the ROR1-ECD comprises or consists of the amino acid sequence of SEQ ID NO: 3 or a functional variant or fragment thereof having at least about 80% identity thereto. In one embodiment, the ROR1-ECD is encoded by the nucleotide sequence of SEQ ID NO: 4 or a functional variant thereof having at least about 80% identity thereto.

[0013] In one embodiment, the IgG2-Fc domain comprises or consists of the amino acid sequence of SEQ ID NO: 7 or a functional variant or fragment thereof having at least about 80% identity thereto. In one embodiment, the IgG2-Fc domain is encoded by the nucleotide sequence of SEQ ID NO: 8 or a functional variant thereof having at least about 80% sequence identity thereto.

[0014] In one embodiment, the IgG2-Fc domain is N-terminal to the ROR-ECD. In one embodiment, the ROR2 inhibitor has the structure: Nter-IgG2-Fc-ROR2-ECD-Cter. In one embodiment, the fusion protein comprises or consists of the amino acid sequence of SEQ ID NO: 15 or a functional variant or fragment thereof having at least about 80% identity thereto. In one embodiment, the fusion protein is encoded by the nucleotide sequence of SEQ ID NO: 16 or a functional variant thereof having at least about 80% identity thereto. In one embodiment, the IgG2-Fc domain is C-terminal to the ROR-ECD. In one embodiment, the ROR2 inhibitor has the structure: Nter-ROR2-ECD-IgG2-Fc-Cter. In one embodiment, the fusion protein comprises or consists of the amino acid sequence of SEQ ID NO: 11 or a functional variant or fragment thereof having at least about 80% identity thereto. In one embodiment, the fusion protein is encoded by the nucleotide sequence of SEQ ID NO: 12 or a functional variant thereof having at least about 80% identity thereto. In one embodiment, the ROR2 inhibitor is capable of stimulating chondrogenesis. In one embodiment, the ROR2 inhibitor is capable of upregulating the expression of Aggrecan. In one embodiment, the ROR2 inhibitor is capable of upregulating the expression of growth / differentiation factor 5 (GDF5). In one embodiment, the ROR2 inhibitor is capable of inhibiting yes-associated protein (YAP) signalling. In one embodiment, the ROR2 inhibitor is capable of inhibiting the expression of cysteine-rich angiogenic inducer 61 (CYR61).

[0015] In one aspect, the present invention provides an isolated nucleic acid encoding an ROR2 inhibitor of the invention. In one embodiment, the isolated nucleic acid comprises or consists of the nucleotide sequence of SEQ ID NO: 16 or a functional variant thereof of having at least 80% sequence identity thereto. In one embodiment, the isolated nucleic acid comprises or consists of the nucleotide sequence of SEQ ID NO: 12 or a functional variant thereof having at least 80% sequence identity thereto.

[0016] In one aspect, the present invention provides a vector comprising the nucleic acid of the invention. In one embodiment, the vector is viral vector. In one embodiment, the vector is selected from the group consisting of a lentiviral vector, a retroviral vector, and adeno- associated viral (AAV) vector. In one embodiment, the vector comprises or consists of the nucleotide sequence of SEQ ID NO: 18 or a functional variant thereof of having at least 80% sequence identity thereto. In one embodiment, the vector comprises or consists of the nucleotide sequence of SEQ ID NO: 22 or a functional variant thereof of having at least 80% sequence identity thereto.

[0017] In one aspect, the present invention provides an isolated cell comprising the nucleic acid of the invention. In one aspect, the present invention provides an isolated cell comprising the vector of the invention.

[0018] In one aspect, the present invention provides a pharmaceutical composition comprising the R0R2 inhibitor of the invention, the nucleic acid of the invention, the vector of the invention or the cell of the invention, together with a pharmaceutically acceptable carrier or excipient.

[0019] In one aspect, the invention provides the R0R2 inhibitor of the invention, the nucleic acid of the invention, the vector of the invention, the cell of the invention or the pharmaceutical composition of the invention for use as a medicament.

[0020] In one aspect, the invention provides a method of treating osteoarthritis comprising administering to an individual in need thereof an R0R2 inhibitor of the invention, a nucleic acid of the invention, a vector of the invention, a cell of the invention, or a pharmaceutical composition of the invention.

[0021] In one aspect, the invention provides a method of treating osteoarthritis-associated pain comprising administering to an individual in need thereof an R0R2 inhibitor of the invention, a nucleic acid of the invention, a vector of the invention, a cell of the invention, or a pharmaceutical composition of the invention.

[0022] In one aspect, the invention provides a method of stimulating cartilage production comprising administering to an individual in need thereof an R0R2 inhibitor of the invention, a nucleic acid of the invention, a vector of the invention, a cell of the invention, or a pharmaceutical composition of the invention.

[0023] BRIEF DESCRIPTION OF THE FIGURES

[0024] Figure 1 shows the results of Alcian blue staining to assess cartilage- specific extracellular matrix production in C3H10T1 / 2 cells transfected with plasmids encoding the following constructs: (A) C-terminal IgGl-Fc R0R2-ECD; (B) N-terminal IgGl-Fc R0R2-ECD; (C) C-terminal IgG2-Fc R0R2-ECD; and (D) N-terminal IgG2-Fc R0R2-ECD. Figure 2 shows the results of Alcian blue staining indicating cartilage formation following three days exposure of C3H10T1 / 2 cells with C-terminal IgG2-Fc tagged R0R2-ECD, BMP2, or IgG2-Fc alone.

[0025] Figure 3 shows the results of HOPFlash reporter assays to assess the capacity of R0R2 ECD and R0R1 ECD to suppress YAP signalling in a dose-dependent manner. Panel (A) shows the raw firefly luciferase activity results while panel (B) shows the results normalised for renilla luciferase activity.

[0026] Figure 4 shows the results of HOPFlash reporter assays to assess the capacity of IgGl- R0R2-ECD fusions to suppress YAP signalling in a dose-dependent manner.

[0027] Figure 5 shows the results of RT-PCR assays to assess the expression of Aggrecan (A), Cyr61 (B) and Gdf5 (C) in C3H10T1 / 2 cells were transfected with plasmids encoding for R0R1-ECD, R0R2-ECD, and empty plasmid used as negative control. Data were normalised relative beta-actin.

[0028] Figure 6 shows the results of a HEK293 TOPFlash reporter assay to assess the capacity of fusion proteins to suppress WNT signalling.

[0029] Figure 7 shows the results of Alcian blue staining to assess the capacity of N-terminally IgG2 tagged R0R2-ECD-Fc to increase the amount of cartilage-specific glycosaminoglycan rich extracellular matrix in C3H10T1 / 2 cells cultured in micromass.

[0030] Figure 8 shows the results of HOPFlash reporter assays to assess the capacity of N- terminally IgG2 tagged R0R2-ECD-Fc to suppress TEAD transcriptional activity.

[0031] Figure 9 shows the results of TOPFlash reporter assays to assess the capacity of R0R2 over-expression to suppress WNT signalling.

[0032] Figure 10 shows the results of an in vitro experiment to assess the effects of the re- introduction of R0R2 expression on chondrogenesis in C3H10T1 / 2 cells in which ROR2 expression was knocked out using CRISPR technology. The effects of ROR2 expression on cartilage formation were assessed using Alcian blue staining. Figure 11 shows the results of an in vivo experiment to assess the effects of tamoxifen- inducible R0R2 expression in a murine instability-induced model of osteoarthritis (meniscal / ligamentous injury; MLI). At the age of approximately 10 weeks, male WT, heterozygous and KO mice were subjected to meniscal / ligamentous injury (MLI) surgery. Starting at 4 weeks after MLI, tamoxifen was inj ected for 5 consecutive days in order to knock out ROR2 expression in the cartilage. Following euthanasia at 8 weeks after MLI surgery, Osteoarthritis Research Society International (OARSI) scores were assessed in the femur and tibia.

[0033] Figure 12 shows the results of TEAD reporter assays to assess the capacity of C-terminal and N-terminal IgGl-Fc tagged ROR2-ECD fusions to suppress TEAD transcriptional activity in a dose-dependent manner.

[0034] DETAILED DESCRIPTION OF THE INVENTION

[0035] Unless otherwise defined herein, technical and scientific terms used in the present description have the meanings that are commonly understood by those of ordinary skill in the art. For purposes of interpreting this specification, the following description of terms will apply and whenever appropriate, terms used in the singular (“a”, “an”, and “the”) will also include the plural and vice versa unless the content clearly dictates otherwise. Thus, for example, reference to “a protein” includes “proteins”, and the like. In the event that any description of a term set forth conflicts with any document incorporated herein by reference, the description of the term set forth below shall control.

[0036] The term "comprises" (comprise, comprising) should be understood to have its normal meaning in the art, i.e. that the stated feature or group of features is included, but that the term does not exclude any other stated feature or group of features from also being present. The term “ consists of should also be understood to have its normal meaning in the art, i.e. that the stated feature or group of features is included, to the exclusion of further features. For every embodiment in which “comprises" or “ comprising' is used, the present invention provides a further embodiment in which “ consists of’ or “ consisting of is used. Thus, every disclosure of “ comprises" should be considered to be a disclosure of "consists of . In instances where the terms "comprising ' and “comprises” are used, also provided is something “consisting essentially of or “consisting of what is set out. The terms “patient" and “ subjecf are used interchangeably and typically refer to a human.

[0037] ROR2

[0038] ROR-family receptor tyrosine kinases form a small subfamily of receptor tyrosine kinases (RTKs), characterized by a conserved, unique domain architecture. ROR RTKs are evolutionary conserved throughout the animal kingdom and act as alternative receptors and co-receptors for Wnt (also referred to as WNT herein) ligands.

[0039] The Ror2 gene encodes Receptor Tyrosine Kinase Like Orphan Receptor (R0R2), tyrosine kinase and type I transmembrane protein, which is also known as Tyrosine-Protein Kinase Transmembrane Receptor ROR2, Neurotrophic Tyrosine Kinase Receptor-Related 2 (NTRKR2).

[0040] ROR2 proteins are integral membrane protein comprising an extracellular portion, a transmembrane domain and an intracellular portion. The terms “extracellular portion of an ROR2 protein" and “extracellular domain of an ROR2 protein" may be used interchangeably herein. The extracellular portion / domain of an ROR2 protein may also be referred to as the “ECD" of an R0R2 protein. Typically, the extracellular portion of ROR2 comprises, from the N-terminus: a signal peptide, an immunoglobulin (Ig) domain, a cysteine rich domain (CRD) and a Kringle domain. C-terminal to the Kringle domain is a transmembrane domain followed by the intracellular portion. The intracellular portion of ROR2 typically comprises, from the N-terminus: a tyrosine kinase domain, a first serine / threonine (Ser / Thr) rich domain, a proline rich domain and a second Ser / Thr rich domain. Each of the Ig domain, CRD and the Kringle domain of the extracellular portion are likely to mediate protein-protein interactions, for example interactions with activating ligands of ROR2, such as Wnt family members. In particular, the CRD is known to bind activating ligands of the Wnt family.

[0041] An example human ROR2 protein has the amino acid sequence:

[0042] MARGSALPRRPLLCIPAVWAAAALLLSVSRTSGEVEVLDPNDPLGPLDGQDGPIPTLKGY FLNFLEPVNNITIVQGQTAILHCKVAGNPPPNVRWLKNDAPWQEPRRI I IRKTEYGSRL RIQDLDTTDTGYYQCVATNGMKTITATGVLFVRLGPTHSPNHNFQDDYHEDGFCQPYRGI ACARFIGNRTIYVDSLQMQGEIENRITAAFTMIGTSTHLSDQCSQFAIPSFCHFVFPLCD ARSRAPKPRELCRDECEVLESDLCRQEYTIARSNPLILMRLQLPKCEALPMPESPDAANC MRIGIPAERLGRYHQCYNGSGMDYRGTASTTKSGHQCQPWALQHPHSHHLSSTDFPELGG GHAYCRNPGGQMEGPWCFTQNKNVRMELCDVPSCSPRDSSKMGILYILVPS IAIPLVIAC LFFLVCMCRNKQKASASTPQRRQLMASPSQDMEMPLINQHKQAKLKEISLSAVREMEELG EDRFGKVYKGHLFGPAPGEQTQAVAIKTLKDKAEGPLREEFRHEAMLRARLQHPNWCLL GWTKDQPLSMI FSYCSHGDLHEFLVMRSPHSDVGSTDDDRTVKSALEPPDFVHLVAQIA AGMEYLSSHHWHKDLATRNVLVYDKLNVKISDLGLFREVYAADYYKLLGNSLLPIRWMA PEAIMYGKES IDSDIWSYGWLWEVFSYGLQPYCGYSNQDWEMIRNRQVLPCPDDCPAW VYALMIECWNEFPSRRPRFKDIHSRLRAWGNLSNYNSSAQTSGASNTTQTSSLSTSPVSN VSNARYVGPKQKAPPFPQPQFIPMKGQIRPMVPPPQLYIPVNGYQPVPAYGAYLPNFYPV QIPMQMAPQQVPPQMVPKPSSHHSGSGSTSTGYVTTAPSNTSMADRAALLSEGADDTQNA PEDGAQSTVQEAEEEEEGSVPETELLGDCDTLQVDEAQVQLEA

[0043] (SEQ ID NO: 23)

[0044] Within the amino acid sequence of SEQ ID NO: 23, the following amino acid residues correspond to the domains of R0R2 as shown in Table 1 below.

[0045] An example human cDNA encoding R0R2 has the nucleotide sequence: ggacgcatcgtagaaaggggtggtggcgcccgaccccgcgccccggcccgaagctctgagg gcttcccggcccccactgcctgcggcatggcccggggctcggcgctcccgcggcggccgct gctgtgcatcccggccgtctgggcggccgccgcgcttctgctctcagtgtcccggacttca ggtgaagtggaggttctggatccgaacgaccctttaggaccccttgatgggcaggacggcc cgattccaactctgaaaggttactttctgaattttctggagccagtaaacaatatcaccat tgtccaaggccagacggcaattctgcactgcaaggtggcaggaaacccaccccctaacgtg cggtggctaaagaatgatgccccggtggtgcaggagccgcggcggatcatcatccggaaga cagaatatggttcacgactgcgaatccaggacctggacacgacagacactggctactacca gtgcgtggccaccaacgggatgaagaccattaccgccactggcgtcctgtttgtgcggctg ggtccaacgcacagcccaaatcataactttcaggatgattaccacgaggatgggttctgcc agccttaccggggaattgcctgtgcacgcttcattggcaaccggaccatttatgtggactc gcttcagatgcagggggagattgaaaaccgaatcacagcggcctt caeca tgatcggcacg tctacgcacctgtcggaccagtgctcacagttcgccatcccatccttctgccacttcgtgt ttcctctgtgcgacgcgcgctcccgggcacccaagccgcgtgagctgtgccgcgacgagtg cgaggtgctggagagcgacctgtgccgccaggagtacaccatcgcccgctccaacccgctc atcctcatgcggcttcagctgcccaagtgtgaggcgctgcccatgcctgagagccccgacg ctgccaactgcatgcgcattggcatcccagccgagaggctgggccgctaccatcagtgcta taacggctcaggcatggattacagaggaacggcaagcaccaccaagtcaggccaccagtgc cagccgtgggccctgcagcacccccacagccaccacctgtccagcacagacttccctgagc ttggaggggggcacgcctactgccggaaccccggaggccagatggagggcccctggtgctt tacgcagaataaaaacgtacgcatggaactgtgtgacgtaccctcgtgtagtccccgagac agcagcaagatggggattctgtacatcttggtccccagcatcgcaattccactggtcatcg cttgccttttcttcttggtttgcatgtgccggaataagcagaaggcatctgcgtccacacc gcagcggcgacagctgatggcctcgcccagccaagacatggaaatgcccctcattaaccag cacaaacaggccaaactcaaagagatcagcctgtctgcggtgaggttcatggaggagctgg gagaggaccggtttgggaaagtctacaaaggtcacctgttcggccctgccccgggggagca gacccaggctgtggccatcaaaacgctgaaggacaaagcggaggggcccctgcgggaggag ttccggcatgaggctatgctgcgagcacggctgcaacaccccaacgtcgtctgcctgctgg gcgtggtgaccaaggaccagcccct gagcat gat cttcagct act gttcgcacggcgacct ccacgaattcctggtcatgcgctcgccgcactcggacgtgggcagcaccgatgatgaccgc acggtgaagtccgccctggagccccccgacttcgtgcaccttgtggcacagatcgcggcgg ggatggagtacctatccagccaccacgtggttcacaaggacctggccacccgcaatgtgct agtgtacgacaagctgaacgtgaagatctcagacttgggcctcttccgagaggtgtatgcc gccgattactacaagctgctggggaactcgctgctgcctatccgctggatggccccagagg ccatcatgtacggcaagttctccatcgactcagacatctggtcctacggtgtggtcctgtg ggaggtcttcagctacggcctgcagccctactgcgggtattccaaccaggatgtggtggag atgatccggaaccggcaggtgctgccttgccccgatgactgtcccgcctgggtgtatgccc tcatgatcgagtgctggaacgagttccccagccggcggccccgcttcaaggacatccacag ccggctccgagcctggggcaacctttccaactacaacagctcggcgcagacctcgggggcc agcaacaccacgcagaccagctccctgagcaccagcccagtgagcaatgtgagcaacgccc gctacgtggggcccaagcagaaggccccgcccttcccacagccccagttcatccccatgaa gggccagatcagacccatggtgcccccgccgcagctctacatccccgtcaacggctaccag ccggtgccggcctatggggcctacctgcccaacttctacccggtgcagatcccaatgcaga tggccccgcagcaggtgcctcctcagatggtccccaagcccagctcacaccacagtggcag tggctccaccagcacaggctacgtcaccacggccccctccaacacatccatggcagacagg gcagccctgctctcagagggcgctgatgacacacagaacgccccagaagatggggcccaga gcaccgtgcaggaagcagaggaggaggaggaaggctctgtcccagagactgagctgctggg ggactgtgacactctgcaggtggacgaggcccaagtccagctggaagcttgagtggcacca gggcccagggttcggggatagaagccccgccgagaccccacagggacctcagtcacctttg agaagacaccatactcagcaatcacaagagcccgccggccagtgggcttgtttgcagactg ggtgaggtggagccctgctcctctctgtcctctgacacagctgccctgcctaggagcaccc aagccaggcagggggtctggcagcacggcgtcctggggagcaggacacatggtcatcccca gggctgtatacattgattctggtggtagactggtagtgagcagcaaatgcctttcaagaaa ataggtggcagcttcactccatgtcatatatggagtgaatatttcaaaacgttgggaataa gggcctgcaaaaggca

[0046] (SEQ ID NO: 24)

[0047] The extracellular portion of an example human R0R2 protein has the amino acid sequence:

[0048] MARGSALPRRPLLCIPAVWAAAALLLSVSRTSGEVEVLDPNDPLGPLDGQDGPIPTLKGYF LNFLEPVNNITIVQGQTAILHCKVAGNPPPNVRWLKNDAPWQEPRRI I IRKTEYGSRLRI QDLDTTDTGYYQCVATNGMKTITATGVLFVRLGPTHSPNHNFQDDYHEDGFCQPYRGIACA RFIGNRTIYVDSLQMQGEIENRITAAFTMIGTSTHLSDQCSQFAIPSFCHFVFPLCDARSR APKPRELCRDECEVLESDLCRQEYTIARSNPLILMRLQLPKCEALPMPESPDAANCMRIGI PAERLGRYHQCYNGSGMDYRGTASTTKSGHQCQPWALQHPHSHHLSSTDFPELGGGHAYCR NPGGQMEGPWCFTQNKNVRMELCDVPSCSPRDSSKMGILY

[0049] (SEQ ID NO: 25) The R0R2 extracellular portion of SEQ ID NO: 25 may be encoded by the nucleotide sequence:

[0050] ATGGCCCGGGGCTCGGCGCTCCCGCGGCGGCCGCTGCTGTGCATCCCGGCCGTCTGGGCGG

[0051] CCGCCGCGCTTCTGCTCTCAGTGTCCCGGACTTCAGGTGAAGTGGAGGTTCTGGATCCGAA

[0052] CGACCCTTTAGGACCCCTTGATGGGCAGGACGGCCCGATTCCAACTCTGAAAGGTTACTTT

[0053] CTGAATTTTCTGGAGCCAGTAAACAATATCACCATTGTCCAAGGCCAGACGGCAATTCTGC

[0054] ACTGCAAGGTGGCAGGAAACCCACCCCCTAACGTGCGGTGGCTAAAGAATGATGCCCCGGT

[0055] GGTGCAGGAGCCGCGGCGGATCATCATCCGGAAGACAGAATATGGTTCACGACTGCGAATC

[0056] CAGGACCTGGACACGACAGACACTGGCTACTACCAGTGCGTGGCCACCAACGGGATGAAGA

[0057] CCATTACCGCCACTGGCGTCCTGTTTGTGCGGCTGGGTCCAACGCACAGCCCAAATCATAA

[0058] CTTTCAGGATGATTACCACGAGGATGGGTTCTGCCAGCCTTACCGGGGAATTGCCTGTGCA

[0059] CGCTTCATTGGCAACCGGACCATTTATGTGGACTCGCTTCAGATGCAGGGGGAGATTGAAA

[0060] ACCGAATCACAGCGGCCTTCACCATGATCGGCACGTCTACGCACCTGTCGGACCAGTGCTC

[0061] ACAGTTCGCCATCCCATCCTTCTGCCACTTCGTGTTTCCTCTGTGCGACGCGCGCTCCCGG GCACCCAAGCCGCGTGAGCTGTGCCGCGACGAGTGCGAGGTGCTGGAGAGCGACCTGTGCC GCCAGGAGTACACCATCGCCCGCTCCAACCCGCTCATCCTCATGCGGCTTCAGCTGCCCAA GTGTGAGGCGCTGCCCATGCCTGAGAGCCCCGACGCTGCCAACTGCATGCGCATTGGCATC CCAGCCGAGAGGCTGGGCCGCTACCATCAGTGCTATAACGGCTCAGGCATGGATTACAGAG GAACGGCAAGCACCACCAAGTCAGGCCACCAGTGCCAGCCGTGGGCCCTGCAGCACCCCCA CAGCCACCACCTGTCCAGCACAGACTTCCCTGAGCTTGGAGGGGGGCACGCCTACTGCCGG AACCCCGGAGGCCAGATGGAGGGCCCCTGGTGCTTTACGCAGAATAAAAACGTACGCATGG AACTGTGTGACGTACCCTCGTGTAGTCCCCGAGACAGCAGCAAGATGGGGATTCTGTAC

[0062] (SEQ ID NO: 26)

[0063] The CRD of an example human R0R2 protein has the amino acid sequence:

[0064] CQPYRGIACARFIGNRT IYVDSLQMQGE IENRI TAAFTMIGTSTHLSDQCSQFAI PS FCHF

[0065] VFPLCDARSRAPKPRELCRDECEVLESDLCRQEYT IARSNPLILMRLQLPKCEALPMPESP DAAN

[0066] (SEQ ID NO: 27)

[0067] The CRD domain of SEQ ID NO: 27 may be encoded by the nucleotide sequence:

[0068] CTGCCAGCCTTACCGGGGAATTGCCTGTGCACGCTTCATTGGCAACCGGACCATTTATGTG

[0069] GACTCGCTTCAGATGCAGGGGGAGATTGAAAACCGAATCACAGCGGCCTTCACCATGATCG

[0070] GCACGTCTACGCACCTGTCGGACCAGTGCTCACAGTTCGCCATCCCATCCTTCTGCCACTT

[0071] CGTGTTTCCTCTGTGCGACGCGCGCTCCCGGGCACCCAAGCCGCGTGAGCTGTGCCGCGAC

[0072] GAGTGCGAGGTGCTGGAGAGCGACCTGTGCCGCCAGGAGTACACCATCGCCCGCTCCAACC

[0073] CGCTCATCCTCATGCGGCTTCAGCTGCCCAAGTGTGAGGCGCTGCCCATGCCTGAGAGCCC CGACGCTGCCAAC

[0074] (SEQ ID NO: 28)

[0075] An example mouse R0R2 protein has the amino acid sequence of:

[0076] MARGWVRPSRVPLCARAVWTAAALLLWTPWTAGEVEDSEAIDTLGQPDGPDSPLPTLKGY FLNFLEPVNNI T IVQGQTAILHCKVAGNPPPNVRWLKNDAPWQEPRRVI IRKTEYGSRL RIQDLDTTDTGYYQCVATNGLKT I TATGVLYVRLGPTHSPNHNFQDDDQEDGFCQPYRGI

[0077] ACARFIGNRT IYVDSLQMQGE IENRI TAAFTMIGTSTQLSDQCSQFAI PS FCHFVFPLCD ARSRAPKPRELCRDECEVLENDLCRQEYTIARSNPLILMRLQLPKCEALPMPESPDAANC MRIGIPAERLGRYHQCYNGSGADYRGMASTTKSGHQCQPWALQHPHSHRLSSTEFPELGG GHAYCRNPGGQVEGPWCFTQNKNVRVELCDVPPCSPRDGSKMGILYILVPS IAIPLVIAC L FFLVCMCRNKQKASAS T PQRRQLMAS PS QDMEMPL I S QHKQAKLKE I S LS TVRFMEELG EDRFGKVYKGHLFGPAPGEPTQAVAIKTLKDKAEGPLREEFRQEAMLRARLQHPNIVCLL GWTKDQPLSMI FSYCSHGDLHEFLVMRSPHSDVGSTDDDRTVKSALEPPDFVHWAQIA AGMEFLSSHHWHKDLATRNVLVYDKLNVRISDLGLFREVYSADYYKLMGNSLLPIRWMS PEAVMYGKFS IDSDIWSYGWLWEVFSYGLQPYCGYSNQDWEMIRSRQVLPCPDDCPAW VYALMIECWNEFPSRRPRFKDIHSRLRSWGNLSNYNSSAQTSGASNTTQTSSLSTSPVSN VSNARYMAPKQKAQPFPQPQFIPMKGQIRPLVPPAQLYIPVNGYQPVPAYGAYLPNFYPV QIPMQMAPQQVPPQMVPKPSSHHSGSGSTSTGYVTTAPSNTSVADRAALLSEGTEDAQNI AEDVAQSPVQEAEEEEEGSVPETELLGDNDTLQVTEAAHVQLEA

[0078] (SEQ ID NO: 29)

[0079] An example mouse cDNA encoding R0R2 has the nucleotide sequence: atggctcggggctgggtgcggccgagccgtgtgcctctgtgcgcccgggccgtctggacg gctgcggcgctcctgctctggacaccctggacggcaggtgaagtggaagattcggaggca atcgacaccttgggacaacctgatggaccggacagcccacttcccactctgaaaggctac tttctgaattttctggagccagtcaacaatatcaccattgttcagggccagacggcaatc ctgcactgcaaggtggcgggaaacccacctcccaatgtgcggtggctgaagaatgatgcc ccggttgtgcaagagccacgaagggtcgtcatccggaagacagaatacggctcccggctg cggatccaagacctggacacaacagacacaggctactaccagtgtgtggctaccaacggg ctgaagaccatcactgccactggggttctatatgtgcggctcggtccgacgcacagcccg aaccacaattttcaggatgacgatcaggaagatggcttctgccagccgtaccgagggatc gcttgtgcgcgcttcattgggaaccggactatttatgtggactccctccagatgcagggg gaga ttgaaaaccgaatcacagctgcctt caeca tgatcggcacctccacgcaactgtca gaccagtgttcacagtttgccatcccatccttctgccacttcgtcttccctctgtgcgac gcatgctcccgggcgcccaagcctcgcgaactgtgccgggatgaatgtgaggtgctggag aacgacctgtgccgccaggagtacaccatcgcccgctccaacccgctcatcctcatgcgg ctccagctgcccaagtgcgaagcgctgcccatgcccgagagcccggatgctgcgaactgc atgcgcatcgggatccccgcggagaggctgggtcgctaccaccagtgctacaacggctcc ggcgccgattacagggggatggccagtaccaccaagtcaggccaccagtgtcagccttgg gctctgcagcacccccacagccatcgcctatccagcacggaattccctgagctgggagga ggccatgcctactgccggaaccccgggggccagatggaaggcccgtggtgctttacgcag aataaaaacgtacgcgtggaactgtgtgacgtacccccgtgtagtccccgatatggcagc aagatggggattctgtacatcctggtccccagcattgctatccccctggtcatcgcttgc ctgttcttcctcgtctgcatgtgccgcaacaaacagaaggcttcggcctccaccccacag cgccggcagctgatggcctctcccagccaggacatggagatgccactcatcagccagcac aaacaggccaaactcaaagagatcagcttgtccacagtgaggttcatggaggagctcggg gaggaccggtttggcaaggtctacaaaggccacctgtt egggee tgccccaggagaacca acccaggccgtggccatcaagacgctgaaagacaaggctgaggggcccctgcgggaggag ttccggcaagaggcgatgctccgggcccgactgcagcaccccaacatcgtctgtc tecta ggcgtcgtgaccaaggaccaaccctt gagcat gat cttcagct act gttcccatggcgac cttcatgaattcctggtcatgcgctcgccgcactccgatgtgggcagcaccgatgacgac cgcacagtgaagtcagccctggagcccccggacttcgtgcacgtggtggcgcagatcgct gcggggatggagttcctgtccagccaccacgtgtgccataaggacctggccacacgcaat gtgctggtgtacgacaagctgaacgtgaggatctcagacttgggcctcttccgtgaggta tactccgcagattactacaaactcatgggcaattcactgctgcccatccgctggatgtcc cccgaggccgtcatgtatggaaagttctccatcgactctgacatctggtcctacggtgtg gtcctctgggaggtctttagctacggcctgcagccctactgtgggtactccaaccaggac gtggtggagatgatccggagccggcaggtgctgccctgcccggatgactgccccgcctgg gtctatgccctcatgattgaatgctggaatgagttcccaagccggaggccccgctttaag gacatccacagccggctccggtcctggggcaacctatccaactataatagttccgcgcag acctcaggagccagcaacaccacacagaccagctccctgagcaccagccccgtaagcaat gtgagcaatgcccgctatatggcccccaagcagaaggcccagcccttcccacagcctcag ttcatccccatgaagggtcagatcagacccttggtgccccccgcacagctgtacatcccg gtgaacggctatcagccggtaccggcatacggggcctacctgcccaacttctacccagtc cagatccccatgcagatggccccacagcaggtgccccctcagatggtccccaagccgagc tcacaccacagtggcagcggctccaccagcactggctacgtcaccacggcgccctccaat acatctgtggcggacagggcggccctactctctgagggcaccgaggatgtacagaacatc gcggaagacgtggcccagagccctgtgcaggaagcagaggaggaggaggaggggtctgtc cctgagactgaactcctgggagacaatgacacgctccaggtgaccgaggcggctcatgtc cagcttgaagcctga

[0080] (SEQ ID NO: 30)

[0081] The ROR2 receptor has been shown to bind members of the Wnt family of secreted extracellular glycoproteins involved in a variety of signalling pathways, including Wnt5a, Wnt3a, Wntl, Wntl 1, Wnt4 and Wntl6. Wnt5a, Wnt3a, Wntl, Wntl 1, Wnt4 and Wntl6 can activate ROR-2 dependent signalling (Gao et al., (2011) Dev. Cell 20: 163-176).Wnt5a has been shown to induce ROR2 homo-dimerization and tyrosine phosphorylation in U2OS human osteoblastic cells. Furthermore, in the presence of Wnt5a increased phosphorylation of the R0R2 substrate 14-3-3beta scaffold protein was observed, indicating that Wnt5a binding causes activation of the R0R2 signalling cascade (Liu et al. 2008). The Wnt5a / ROR2 signalling pathway inhibits canonical Wnt-P-catenin signalling (mediated by the receptor Frizzled).

[0082] R0R2 targets the Ctgf gene which encodes connective tissue growth factor. R0R2 also targets the Yap, Taz, and Wht5a genes which encode YAP (yes-associated protein), TAZ and WNT5A proteins respectively (Park et al. (2015) Cell 162: 780-794). Monomeric ROR2 may also interact with other proteins to form a receptor complex capable of downstream signalling. For example, on activation two R0R2 molecules may interact to form a homodimer. Alternatively, ROR2 may form a heterodimer with ROR1. ROR2 may also interact (e.g. bind to) other ROR2 co-receptors, including but not limited to frizzled receptors or scaffolding proteins (e.g. syndecans). These interactions may be necessary for downstream signalling by ROR2 and therefore ROR2 signalling may be inhibited by disrupting these interactions. R0R2 expression is also positively correlated with increased expression of YAP1 and of YAP1 regulated genes including cysteine-rich angiogenic inducer 61 (Cyr61\ Thus, R0R2 activation / inhibition may be evaluated by a number of different methods, such as measuring (i) R0R2 induced inhibition of Wnt-P-catenin signalling, (ii) R0R2 tyrosine phosphorylation, (iii) phosphorylation of R0R2 downstream targets, such as 14-3- 3beta scaffold protein, (iv) expression of R0R2 target genes, such as Ctgf or (v) the capacity of R0R2 to form a receptor complex (e.g. whether R0R2 can interact with other members of the receptor complex). A person skilled in the art, would be able to select appropriate techniques for measuring each of (i) to (v). R0R2 activation / inhibition may also be evaluated by measuring the expression of genes such as Aggrecan, and Cyr61.

[0083] ROR1

[0084] The Rorl gene encodes Receptor Tyrosine Kinase Like Orphan Receptor (R0R1), tyrosine kinase and type I transmembrane protein, which is also known as Tyrosine-Protein Kinase Transmembrane Receptor R0R1, Neurotrophic Tyrosine Kinase Receptor-Related 1 (NTRKR1).

[0085] R0R1 proteins are integral membrane protein comprising an extracellular portion, a transmembrane domain and an intracellular portion. The terms “extracellular portion of an R0R1 protein" and “extracellular domain of an R0R1 protein" may be used interchangeably herein. The extracellular portion / domain of an R0R1 protein may also be referred to as the “ECD" of an R0R1 protein.

[0086] Typically, the extracellular portion of R0R1 comprises, from the N-terminus: a signal peptide, an immunoglobulin (Ig) domain, a cysteine rich domain (CRD) (also known as a Frizzled domain) and a Kringle domain. C-terminal to the Kringle domain is a transmembrane domain followed by the intracellular portion. The intracellular portion of R0R1 typically comprises, from the N-terminus: a tyrosine kinase domain, a first serine / threonine (Ser / Thr) rich domain, a proline rich domain and a second Ser / Thr rich domain. Each of the Ig domain, CRD and the Kringle domain of the extracellular portion are likely to mediate protein-protein interactions, for example interactions with activating ligands of R0R1, such as Wnt family members. In particular, the CRD is known to bind activating ligands of the Wnt family. The R0R2 and R0R1 proteins / fragments of the invention are referred to herein collectively as “ROR proteins”. Accordingly, the term “ROR ECD” used herein is intended to encompass extracellular domains from both R0R2 and R0R1 proteins.

[0087] ROR2 inhibitors

[0088] The present invention relates to R0R2 inhibitors and therapeutic uses thereof in treating and / or preventing cartilage loss and / or treating and / or preventing osteoarthritis, treating and / or preventing osteoarthritis associated pain, promoting cartilage repair, and / or preventing cartilage degradation.

[0089] An R0R2 inhibitor may partially or fully inhibit (e.g. reduce, prevent, abolish, or ameliorate) at least one function or activity of R0R2. For example, as defined herein, an R0R2 inhibitor of the invention inhibits at least one activity or function of R0R2 that results in, for example, stimulation of chondrocyte differentiation (which may be associated with increased expression of chondrocytic differentiation markers e.g. C0L2A1, AGGRECAN, ERG, GDF5), decreased expression of Coll Al and / or ColX, decreased expression of collagen degrading enzymes ADAMTS-4 and ADAMTS-5, increased accumulation of GAG (for example reduced degradation of GAG and / or increased expression of GAG), and / or reduction in OA associated pain. Accordingly, in one aspect, an R0R2 inhibitor of the invention is capable of stimulating chondrogenesis. Inhibition of R0R2 may also result in decreased transcription of an endogenous Ctgf gene and / or decreased expression of an endogenous CTGF protein. Inhibition of R0R2 may also result in increased transcription of an endogenous AGGRECAN gene and an endogenous growth / differentiation factor 5 gene (GDF5). Accordingly, in one aspect, an R0R2 inhibitor of the invention is capable of upregulating the expression of Aggrecan and / or growth / differentiation factor 5 (GDF5). Inhibition of R0R2 may also result in decreased transcription of an endogenous cysteine-rich angiogenic inducer 61 gene (Cyr61f Accordingly, in one aspect, an R0R2 inhibitor of the invention is capable of inhibiting the expression of cysteine-rich angiogenic inducer 61 (CYR61).

[0090] An R0R2 inhibitor of the invention may act via any mechanism that reduces at least one function or activity of R0R2. By way of example, an R0R2 inhibitor of the invention may inhibit a function or activity of R0R2 by: (a) reducing or preventing the interaction between an R0R2 protein and one or more activating ligands such as Wnt family members (e.g. Wnt5a, Wnt3a, Wntl, Wntl 1, Wnt4 and Wntl 6);

[0091] (b) reducing or preventing the interaction between an R0R2 protein and a member of an R0R2 receptor complex, for example by reducing or preventing the interaction between a first R0R2 protein and a second R0R2 protein, and / or reducing or preventing the interaction between an R0R2 protein and an R0R1 protein, a frizzled receptor protein and / or a scaffold protein (e.g. a syndecan);

[0092] (c) reducing or preventing the interaction between R0R2 interaction with downstream targets, such as 14-3-3beta scaffold protein, and / or

[0093] (d) reducing or preventing R0R2’s ability to phosphorylate a downstream target protein (e.g. 14-3-3beta).

[0094] An R0R2 inhibitor of the invention may reduce or prevent the interaction between an endogenous R0R2 and its activating ligands by competing by binding to one or more activating ligands of R0R2, such as a Wnt family member (e.g. Wnt5a, Wnt3a, Wntl, Wntl 1, Wnt4 and / or Wntl 6).

[0095] An R0R2 inhibitor of the invention may reduce or prevent yes-associated protein (YAP) signalling. Preferably, the R0R2 inhibitor of the invention inhibits YAP signalling.

[0096] The canonical WNT pathway is usually highly conserved and activated via the binding of extracellular WNT ligands to membrane receptors. Once activated, the canonical WNT pathway induces the stability of P-catenin and transfers it to the nucleus, ultimately facilitating the expression of genes involved in cell proliferation, survival, differentiation, and migration. In the absence of WNT ligands, the transmembrane receptors FZD and LRP5 / 6 are located on the plasma membrane separately. In the cytoplasm, a “destruction complex” comprising adenomatous polyposis coli (APC), AXIN, casein kinase 1 (CK1) and glycogen synthase kinase 3 protein (GSK3 protein) captures P-catenin by phosphorylating CK1 and GSK3, thus activating the process of P-catenin degradation, thereby permitting GROUCHOU, which binds to TCF / LEF, to inhibit the transcription of target genes. When WNT ligands are recognized by FZD and LRP5 / 6, the “destruction complex” is recruited to the cell membrane by interacting with FZD, which loses the ability to degrade P-catenin. The induced P-catenin translocates to the nucleus and activates the transcription of target genes by interacting with TCF / LEF. R0R2 activity inhibits canonical WNT signalling (Gao et al., Dev Cell. 2011 Feb 15;20(2):163-76; Park et al., Cell. 2015 Aug 13; 162(4): 780-94; Thorup et al., 2020) at least in part by competing with the WNT co-receptors LRP5 and LRP6 for the binding to FZD receptors (Gao et al., 2011). Therefore, without wishing to be bound by theory, it would be expected that complete suppression of ROR2 activity would result in activation of canonical WNT signalling. This would not be desirable given that canonical WNT signalling is pathogenic in osteoarthritis (Corr et al., Nat Clin Pract Rheumatol. 2008 Oct;4(10):550-6). However, contrary to this expectation, the inventors have unexpectedly found that ROR2 inhibitors which comprise or consist of an ROR2-ECD are capable of inhibiting canonical WNT signalling (see Examples 7, 12 and 13). Thus, where the ROR2 inhibitor of the invention comprises or consists of an ROR2-ECD, the R0R2 inhibitor may be capable of inhibiting canonical WNT signalling.

[0097] In one embodiment, the ROR2 inhibitor is a soluble ROR2 protein. The terms “ soluble ROR2 protein" and “ soluble fragment of an ROR2 protein" are used interchangeably herein. The soluble ROR2 protein may comprise all or a portion of one or more extracellular domains of a full-length ROR2 protein, such as an ROR2 protein having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 1 or an ROR2 protein encoded by a polynucleotide having at having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 2.

[0098] The soluble ROR2 protein may comprise all or a portion of one or more extracellular domains of an ROR2 protein having the sequence of SEQ ID NO: 1. The soluble ROR2 protein may comprise all or a portion of one or more extracellular domains of an ROR2 protein encoded by a polynucleotide having the sequence of SEQ ID NO: 2. Preferably, the ROR2 inhibitor of the invention comprises or consists of the amino acid sequence of SEQ ID NO: 1 or a functional variant or fragment thereof having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, or at least about 99.9% sequence identity thereto. Preferably, the R0R2 inhibitor of the invention is encoded by the nucleotide sequence of SEQ ID NO: 2 or a functional variant thereof having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, or at least about 99.9% sequence identity thereto.

[0099] As discussed above, the extracellular portion / domain of R0R2 comprises, from the N- terminus: a signal peptide, an immunoglobulin (Ig) domain, a cysteine rich domain (CRD) and a Kringle domain. The extracellular portion / domain of human ROR2 protein may have amino acid sequence of SEQ ID NO: 1. The soluble ROR2 protein may comprise a polypeptide having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 1. The soluble ROR2 protein may comprise a polypeptide having the sequence of SEQ ID NO: 1.

[0100] The soluble ROR2 protein may comprise a polypeptide encoded by a polynucleotide having the sequence of SEQ ID NO: 2 or a polynucleotide having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 2.

[0101] The soluble ROR2 protein may comprise all or a portion of the CRD domain of ROR2. As discussed above, an example CRD domain of a human ROR2 protein has the sequence of SEQ ID NO 27. An example CRD domain of a human ROR2 protein is encoded by a polynucleotide having the sequence of SEQ ID NO: 28. The soluble ROR2 protein may comprise a polypeptide having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 27. The soluble protein may comprise a polypeptide having the sequence of SEQ ID NO: 27. The soluble R0R2 protein may comprise a polypeptide encoded by a polynucleotide having the sequence of SEQ ID NO: 28 or a polynucleotide having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 28.

[0102] The soluble ROR2 protein may comprise a fragment of an extracellular portion / domain of an ROR2 protein. The fragment of the extracellular portion / domain may be a functional fragment capable of partially or fully inhibiting (e.g. reducing, preventing, abolishing, or ameliorating) at least one function or activity of ROR2. For example, the fragment of the extracellular portion / domain of an ROR2 protein may be capable of:

[0103] (i) reducing or preventing the interaction between an ROR2 protein and one or more activating ligands such as Wnt family members (e.g. Wnt5a, Wnt3a, Wntl, Wntl 1, Wnt4 and Wntl 6);

[0104] (ii) reducing or preventing the interaction between an ROR2 protein and a member of an ROR2 receptor complex, for example by reducing or preventing the interaction between a first ROR2 protein and a second ROR2 protein, and / or reducing or preventing the interaction between an ROR2 protein and an ROR1 protein, a frizzled receptor protein and / or a scaffold protein (e.g. a syndecan);

[0105] (iii) reducing or preventing the interaction between ROR2 interaction with downstream targets, such as 14-3-3beta scaffold protein, and / or

[0106] (iv) reducing or preventing ROR2’ s ability to phosphorylate a downstream target protein (e.g. 14-3-3beta).

[0107] The fragment of the extracellular portion / domain an ROR2 protein may be capable of reducing or preventing the interaction between an endogenous ROR2 and its activating ligands by competing by binding to one or more activating ligands of ROR2, such as a Wnt family member (e.g. Wnt5a, Wnt3a, Wntl, Wntl l, Wnt4 and / or Wntl6).

[0108] The soluble ROR2 protein may comprise a fragment of a polypeptide having the sequence of SEQ ID NO: 25 or a polypeptide having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 25. The soluble R0R2 protein may comprise a fragment of a polypeptide encoded by a polynucleotide having the sequence of SEQ ID NO: 26 or a polynucleotide having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% sequence identity to SEQ ID NO: 26.

[0109] The fragment of the extracellular portion / domain of an ROR2 protein may be at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250, 275, 300, 325, 350, 375, or 400 amino acids in length. The fragment of the extracellular portion / domain of an ROR2 protein may be about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250, 275, 300, 325, 350, 375, or 400 amino acids in length. The fragment of the extracellular portion / domain of an ROR2 protein may be from 10 to 400, from 20 to 375, from 30 to 350, from 40 to 325, from 50 to 300, from 60 to 275, from 70 to 250, from 90 to 225, from 100 to 200, from 120 to 180, from 140 to 160 amino acids in length.

[0110] The fragment of the extracellular portion / domain of an ROR2 protein may comprise at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250, 275, 300, 325, 350, 375, or 400 contiguous amino acids of SEQ ID NO: 1 or a polypeptide having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 1. The fragment of the extracellular portion / domain of an ROR2 protein may comprise about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250, 275, 300, 325, 350, 375, or 400 contiguous amino acids of SEQ ID NO: 1 or a polypeptide having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 1. The fragment of the extracellular portion / domain of an ROR2 protein may comprise from 10 to 400, from 20 to 375, from 30 to 350, from 40 to 325, from 50 to 300, from 60 to 275, from 70 to 250, from 90 to 225, from 100 to 200, from 120 to 180, from 140 to 160 contiguous amino acids of SEQ ID NO: 1 or a polypeptide having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 1.

[0111] The fragment of the extracellular portion / domain of an ROR2 protein may comprise at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250, 275, 300, 325, 350, 375, or 400 contiguous amino acids of a polypeptide encoded by a polynucleotide having the sequence of SEQ ID NO: 2 or a polynucleotide having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 2. The fragment of the extracellular portion / domain of an ROR2 protein may comprise about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250, 275, 300, 325, 350, 375, or 400 contiguous amino acids of a polypeptide encoded by a polynucleotide having the sequence of SEQ ID NO: 2 or a polynucleotide having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 2. The fragment of the extracellular portion / domain of an ROR2 protein may comprise from 10 to 400, from 20 to 375, from 30 to 350, from 40 to 325, from 50 to 300, from 60 to 275, from 70 to 250, from 90 to 225, from 100 to 200, from 120 to 180, from 140 to 160 contiguous amino acids of a polypeptide encoded by a polynucleotide having the sequence of SEQ ID NO: 2 or a polynucleotide having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 2.

[0112] In one embodiment, the ROR2 inhibitor is a soluble ROR1 protein. The terms “ soluble ROR1 protein” and “ soluble fragment of an ROR1 protein” are used interchangeably herein. The soluble ROR1 protein may comprise all or a portion of one or more extracellular domains of a full-length ROR1 protein, such as an ROR1 protein having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 3 or an R0R1 protein encoded by a polynucleotide having at having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 4.

[0113] The soluble ROR1 protein may comprise all or a portion of one or more extracellular domains of an ROR2 protein having the sequence of SEQ ID NO: 3. The soluble ROR1 protein may comprise all or a portion of one or more extracellular domains of an ROR1 protein encoded by a polynucleotide having the sequence of SEQ ID NO: 4. In one aspect, the ROR2 inhibitor of the invention comprises or consists of the amino acid sequence of SEQ ID NO: 3 or a functional variant or fragment thereof having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, or at least about 99.9% sequence identity thereto. In one aspect, the ROR2 inhibitor of the invention is encoded by the nucleotide sequence of SEQ ID NO: 4 or a functional variant thereof having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, or at least about 99.9% sequence identity thereto.

[0114] As discussed above, the extracellular portion / domain of ROR1 comprises, from the N- terminus: a signal peptide, an immunoglobulin (Ig) domain, a cysteine rich domain (CRD) and a Kringle domain. The extracellular portion / domain of human ROR1 protein may have amino acid sequence of SEQ ID NO: 3. The soluble ROR1 protein may comprise a polypeptide having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 3. The soluble ROR1 protein may comprise a polypeptide having the sequence of SEQ ID NO: 3. The soluble R0R1 protein may comprise a polypeptide encoded by a polynucleotide having the sequence of SEQ ID NO: 4 or a polynucleotide having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 4.

[0115] The soluble ROR1 protein may comprise all or a portion of the CRD domain of ROR1. An example CRD domain of a human ROR1 protein has the sequence of SEQ ID NO: 39. An example CRD domain of a human ROR1 protein is encoded by a polynucleotide having the sequence of SEQ ID NO: 40. The soluble ROR1 protein may comprise a polypeptide having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 39. The soluble protein may comprise a polypeptide having the sequence of SEQ ID NO: 39. The soluble ROR2 protein may comprise a polypeptide encoded by a polynucleotide having the sequence of SEQ ID NO: 40 or a polynucleotide having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 40.

[0116] The soluble ROR1 protein may comprise a fragment of an extracellular portion / domain of an ROR1 protein. The fragment of the extracellular portion / domain may be a functional fragment capable of partially or fully inhibiting (e.g. reducing, preventing, abolishing, or ameliorating) at least one function or activity of ROR2. For example, the fragment of the extracellular portion / domain of an ROR1 protein may be capable of:

[0117] (i) reducing or preventing the interaction between an ROR2 protein and one or more activating ligands such as Wnt family members (e.g. Wnt5a, Wnt3a, Wntl, Wntl 1, Wnt4 and Wntl 6);

[0118] (ii) reducing or preventing the interaction between an ROR2 protein and a member of an ROR2 receptor complex, for example by reducing or preventing the interaction between a first ROR2 protein and a second ROR2 protein, and / or reducing or preventing the interaction between an ROR2 protein and an ROR1 protein, a frizzled receptor protein and / or a scaffold protein (e.g. a syndecan); (iii) reducing or preventing the interaction between R0R2 interaction with downstream targets, such as 14-3-3beta scaffold protein, and / or

[0119] (iv) reducing or preventing R0R2’s ability to phosphorylate a downstream target protein (e.g. 14-3-3beta).

[0120] The fragment of the extracellular portion / domain an R0R1 protein may be capable of reducing or preventing the interaction between an endogenous R0R2 and its activating ligands by competing by binding to one or more activating ligands of R0R2, such as a Wnt family member (e.g. Wnt5a, Wnt3a, Wntl, Wntl l, Wnt4 and / or Wntl6).

[0121] The fragment of the extracellular portion / domain of an R0R1 protein may be at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250, 275, 300, 325, 350, 375, or 400 amino acids in length. The fragment of the extracellular portion / domain of an ROR1 protein may be about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250, 275, 300, 325, 350, 375, or 400 amino acids in length. The fragment of the extracellular portion / domain of an ROR1 protein may be from 10 to 400, from 20 to 375, from 30 to 350, from 40 to 325, from 50 to 300, from 60 to 275, from 70 to 250, from 90 to 225, from 100 to 200, from 120 to 180, from 140 to 160 amino acids in length.

[0122] The fragment of the extracellular portion / domain of an ROR1 protein may comprise at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250, 275, 300, 325, 350, 375, or 400 contiguous amino acids of SEQ ID NO: 3 or a polypeptide having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 3. The fragment of the extracellular portion / domain of an ROR1 protein may comprise about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250, 275, 300, 325, 350, 375, or 400 contiguous amino acids of SEQ ID NO: 3 or a polypeptide having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 3. The fragment of the extracellular portion / domain of an ROR1 protein may comprise from 10 to 400, from 20 to 375, from 30 to 350, from 40 to 325, from 50 to 300, from 60 to

[0123] 275, from 70 to 250, from 90 to 225, from 100 to 200, from 120 to 180, from 140 to 160 contiguous amino acids of SEQ ID NO: 3 or a polypeptide having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 3.

[0124] The fragment of the extracellular portion / domain of an ROR1 protein may comprise at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250, 275, 300, 325, 350, 375, or 400 contiguous amino acids of a polypeptide encoded by a polynucleotide having the sequence of SEQ ID NO: 4 or a polynucleotide having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 4. The fragment of the extracellular portion / domain of an ROR1 protein may comprise about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250, 275, 300, 325, 350, 375, or 400 contiguous amino acids of a polypeptide encoded by a polynucleotide having the sequence of SEQ ID NO: 4 or a polynucleotide having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 4. The fragment of the extracellular portion / domain of an ROR1 protein may comprise from 10 to 400, from 20 to 375, from 30 to 350, from 40 to 325, from 50 to 300, from 60 to 275, from 70 to 250, from 90 to 225, from 100 to 200, from 120 to 180, from 140 to 160 contiguous amino acids of a polypeptide encoded by a polynucleotide having the sequence of SEQ ID NO: 4 or a polynucleotide having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 4.

[0125] The ROR2 inhibitors of the invention may usefully comprise other domains, linkers, cleavage tags, and / or affinity tags in addition to an extracellular portion / domain of an ROR protein. The ROR2 inhibitors may be tagged one or more fragment crystallizable (Fc) regions of a human immunoglobulin, such as IgG, A, M or E, but preferably of human IgG. According to the invention, the soluble ROR-ECD is fused to an immunoglobulin G2 fragment crystallisable domain (IgG2-Fc). In one aspect, the IgG2-Fc domain comprises all or a portion of one or more IgG2-Fc domains, such as an IgG2-Fc domain having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, or at least about 99.9% sequence identity to SEQ ID NO: 7 or an IgG2-Fc domain encoded by a polynucleotide having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, or at least about 99.9% sequence identity to SEQ ID NO: 8.

[0126] The IgG2-Fc domain may comprise or consist of the amino acid sequence of SEQ ID NO: 7. The IgG2-Fc domain may comprise a functional variant or fragment of SEQ ID NO: 7, having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, or at least about 99.9% sequence identity thereto. The IgG2- Fc domain may be encoded by a polynucleotide having the sequence of SEQ ID NO: 8. The IgG2-Fc domain may be encoded by a functional variant of SEQ ID NO: 8, having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, or at least about 99.9% sequence identity thereto.

[0127] The IgG2-Fc domain may be fused to the N-terminal to the soluble ROR2 or ROR1 protein. Preferably, the IgG2-Fc domain is N-terminal to the ROR2-ECD. Such an ROR2 inhibitor has the following structure: Nterminal-IgG2-Fc-ROR2-ECD-Cterminal. The IgG2-Fc domain may be N-terminal to the ROR1-ECD. Such an ROR2 inhibitor has the following structure Nterminal-IgG2-Fc-RORl-ECD-Cterminal. Thus, the present invention provides an R0R2 inhibitor comprising the polypeptide of SEQ ID NO: 15. The present invention also provides an R0R2 inhibitor comprising a polypeptide having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 15. The present invention also provides an ROR2 inhibitor comprising a polypeptide encoded by SEQ ID NO: 16. The present invention also provides an ROR2 inhibitor comprising a polypeptide encoded by a polynucleotide having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 16.

[0128] The IgG2-Fc domain may be fused to the C-terminal to the soluble ROR2 or ROR1 protein. Preferably, the IgG2-Fc domain is C-terminal to the ROR2-ECD. Such an ROR2 inhibitor has the following structure: Nterminal-ROR2-ECD- IgG2-Fc-Cterminal. The IgG2-Fc domain may be C-terminal to the ROR1-ECD. Such an ROR2 inhibitor has the following structure Nterminal- ROR1-ECD- IgG2-Fc-Cterminal. Thus, the present invention provides an ROR2 inhibitor comprising the polypeptide of SEQ ID NO: 11. The present invention also provides an ROR2 inhibitor comprising a polypeptide having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 11. The present invention also provides an ROR2 inhibitor comprising a polypeptide encoded by SEQ ID NO: 12. The present invention also provides an ROR2 inhibitor comprising a polypeptide encoded by a polynucleotide having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 12.

[0129] In one aspect, the ROR2 inhibitors of the invention at tagged with at least one, at least two, at least three, at least four, or at least five IgG2-Fc domains disclosed herein. Additionally or alternatively, the ROR2 inhibitors of the invention are tagged with human serum albumin. Additionally or alternatively, the ROR2 inhibitors of the invention are tagged with a IgG4-Fc domain. The ROR2 inhibitors of the invention may be tagged with at least one, at least two, at least three, at least four, or at least five IgG4-Fc domains. Other examples of suitable tags which may be fused to the R0R2 inhibitors of the invention can be found in Czajkowsky et al., EMBO Mol Med. 2012;4(10): 1015-1028.

[0130] In one aspect, the R0R2 inhibitor may further comprise a suitable membrane anchor capable of targeting the R0R2 inhibitor to cells. Any suitable membrane anchor known in the art may be used in the R0R2 inhibitors of the present invention. The person skilled in the art is able to select a suitable membrane anchor. An example membrane anchor that can be used in the R0R2 inhibitors of the present invention is a GPI anchor. A GPI anchor typically comprises a core of phosphoethanolamine (PE)-3Man-GlcN- phosphatidylinositol (Pl)-glycerol linked to alkyl, alkyl fatty acids or ceramide chains, which are able to interact hybrophobically with a lipid bilayer e.g. a cell membrane. The membrane anchor may be connected to the C-terminus of the extracellular domain of the R0R2 inhibitor.

[0131] The membrane anchor may be connected to the extracellular portion / domain of the R0R2 inhibitor by a linker. Accordingly, where the R0R2 protein comprises a GPI anchor, said GPI anchor may be connected to the C-terminus of the extracellular portion / domain of the R0R2 inhibitor by a linker, which may be referred to as a GPI linker. An example GPI linker may have the following amino acid sequence SSSTTTTTTTTLLLLLLLLLLLLLL (SEQ ID NO: 31) The GPI linker may be encoded by the following nucleotide sequence: AGCAGCAGCACCACCACCACCACCACCACCACCCTGCTGCTGCTGCTGCTGCT GCTGCTGCTGCTGCTGCTGCTG (SEQ ID NO: 32). SEQ ID NO: 32 may be followed by a stop codon e.g. TGA, TAA or TAG (for example in a vector).

[0132] The R0R2 inhibitor may further comprise an affinity tag. The inclusion of an affinity tag allows the R0R2 inhibitor to be readily isolated and / or purified. Any suitable affinity tag know in the art may be used in the R0R2 inhibitor of the invention. The person skilled in the art is able to select a suitable affinity tag. The affinity tag may be selected from the group consisting of a Myc / His tag, a Myc tag, a His tag, a glutathione S-transferase (GST) tag, a maltose binding protein (MBP) tag, a Strep tag II, a FLAG tag, an alkaline phosphatase tag, a bacteriophage T7 epitope tag (T7-tag), a calmodulin binding peptide (CBP) tag, a galactose binding protein (GBP) tag, a human influenza hemagglutinin (HA) tag, and combinations thereof. The affinity tag may be C-terminal to the extracellular portion / domain, IgG2-Fc domain or the membrane anchor. In one embodiment, the R0R2 inhibitor further comprises a cleavage tag arranged to permit the affinity tag to be cleaved from the R0R2 inhibitor. In this way, R0R2 inhibitor may be captured via the affinity tag and then recovered by cleaving the cleavage tag. The cleavage tag may be C-terminal to the extracellular portion / domain or the membrane anchor and N-terminal to the affinity tag. The cleavage tag is selected from the group consisting of a enterokinase cleavage tag, a tobacco etch virus protease cleavage site, a thrombin cleavage tag, a factor Xa (FXa) cleavage tag, a human rhinovirus (HRV) 3C Protease (‘PreScission’) cleavage tag. In one embodiment, the cleavage tag is an enterokinase cleavage tag. For example, an enterokinase cleavage tag having the amino acid sequence KDDDD (SEQ ID NO: 33). The enterokinase cleavage tag may be encoded by the nucleotide sequence AAAGATGATGATGAT (SEQ ID NO: 34). Alternatively, the enterokinase cleavage tag may be encoded by the nucleotide sequence: AGCAGCAGCACCACCACCACCACCACCACCACCCTGCTGCTGCTGCTGCTGCTGCTGCTGC

[0133] TGCTGCTGCTGCT GAAAGAT GAT GAT GAT GAACAAAAAC T CAT C T CAGAAGAGGAT C T GAA TATGCATACCGGTCATCATCACCATCACCATTGA (SEQ ID NO: 35).

[0134] In one embodiment, the cleavage tag is a tobacco etch virus protease cleavage tag. For example, a tobacco etch virus protease cleavage tag having the amino acid sequence GQFYLNE (SEQ ID NO: 36). The tobacco etch virus protease cleavage tag may be encoded by the nucleotide sequence GGCCAGTTTTATCTGAACGAA (SEQ ID NO: 37). Alternatively, the tobacco etch virus protease cleavage tag may be encoded by the nucleotide sequence: AGCAGCAGCACCACCACCACCACCACCACCACCCTGCTGCTGCTGCTGCTGCTGCTGCTGC T GC T GC T GC T GC T GGGCCAGT T T TAT C T GAACGAAGAACAAAAAC T CAT C T CAGAAGAGGA T C T GAAT AT G C AT AC C G G T C AT C AT C AC CAT C AC C AT T GA (SEQ ID NO: 38).

[0135] In any polypeptide R0R2 inhibitor described herein, the polypeptide sequence may be modified by at least one, at least two, at least three, at least four, or at least five additions, deletions or substitutions, provided that a polypeptide having the modified sequence exhibits the same or increased R0R2 inhibition, as compared to a polypeptide having the unmodified sequence. By “the same” it is to be understood that the polypeptide of the modified sequence does not exhibit significantly reduced R0R2 inhibition as compared to polypeptide of the unmodified sequence. Any comparison of R0R2 inhibition between sequences is to be conducted using the same assay. Unless otherwise specified, modifications to an amino acid sequence are preferably conservative amino acid substitutions. Conservative substitutions replace amino acids with other amino acids of similar chemical structure, similar chemical properties or similar side-chain volume. The amino acids introduced may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality or charge to the amino acids they replace. Alternatively, the conservative substitution may introduce another amino acid that is aromatic or aliphatic in the place of a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well-known in the art and may be selected in accordance with the properties of the 20 main amino acids as defined in Table Al below. Where amino acids have similar polarity, this can be determined by reference to the hydropathy scale for amino acid side chains in Table A2.

[0136] Table Al - Chemical properties of amino acids

[0137] Table A2 - Hydropathy scale Side Chain Hydropathy

[0138] IIe 4.5 Vai 4.2 Leu 3.8 Phe 2.8 Cys 2.5 Met 1.9 Ala 1.8 Gly -0.4 Thr -0.7 Ser -0.8 Tip -0.9 Tyr -1.3 Pro -1.6 His -3.2 Glu -3.5 Gin -3.5 Asp -3.5 Asn -3.5 Lys -3.9 Arg -4.5 For the purpose of this invention, in order to determine the percent identity of two sequences (such as two polypeptide sequences), the sequences are aligned for optimal comparison purposes (e.g. gaps can be introduced in a first sequence for optimal alignment with a second sequence). The amino acids at each position are then compared. When a position in the first sequence is occupied by the same amino acid as the corresponding position in the second sequence, then the amino acids are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions in the reference sequence x 100).

[0139] Typically the sequence comparison is carried out over the length of the reference sequence. For example, if the user wished to determine whether a given (“test”) sequence is less than 80% identical to SEQ ID NO: 1, SEQ ID NO: 1 would be the reference sequence. To assess whether a sequence is less 80% identical to SEQ ID NO: 1 (an example of a reference sequence), the skilled person would carry out an alignment over the length of SEQ ID NO: 1, and identify how many positions in the test sequence were identical to those of SEQ ID NO: 1. If less than 80% of the positions are identical, the test sequence is less than 80% identical to SEQ ID NO: 1. If the sequence is shorter than SEQ ID NO: 1, the gaps or missing positions should be considered to be non-identical positions.

[0140] The skilled person is aware of different computer programs that are available to determine the homology or identity between two sequences. For instance, a comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In an embodiment, the percent identity between two amino acid or nucleic acid sequences is determined using the Needleman and Wunsch (1970) algorithm which has been incorporated into the GAP program in the Accelrys GCG software package (available at http: / / www.accelrys.com / products / gcg / ), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0141] Nucleic acids

[0142] The present invention also provides isolated nucleic acids encoding a R0R2 inhibitor of the invention. The terms “ nucleic acid', “oligonucleotide”, “polynucleotide” and “nucleic acid molecule" are used interchangeably herein and refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.

[0143] Non-limiting examples of polynucleotides of the present invention include a gene, a gene fragment, messenger RNA (mRNA), cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide of the invention may be provided in isolated or substantially isolated form. By substantially isolated, it is meant that there may be substantial, but not total, isolation of the polypeptide from any surrounding medium. The polynucleotides may be mixed with carriers or diluents which will not interfere with their intended use and still be regarded as substantially isolated. A nucleic acid sequence which “encodes” a selected polypeptide is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences, for example in an expression vector. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. For the purposes of the invention, such nucleic acid sequences can include, but are not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic sequences from viral or prokaryotic DNA or RNA, and even synthetic DNA sequences. A transcription termination sequence may be located 3' to the coding sequence.

[0144] Polynucleotides can be synthesised according to methods well known in the art, as described by way of example in Sambrook et al (1989, Molecular Cloning - a laboratory manual; Cold Spring Harbor Press). The nucleic acid molecules of the present invention may be provided in the form of an expression cassette which includes control sequences operably linked to the inserted sequence, thus allowing for expression of the polypeptide of the invention in vivo. These expression cassettes, in turn, are typically provided within vectors (e.g., plasmids or recombinant viral vectors). Such an expression cassette may be administered directly to a host subject. Alternatively, a vector comprising a polynucleotide of the invention may be administered to a host subject. Preferably the polynucleotide is prepared and / or administered using a genetic vector. A suitable vector may be any vector which is capable of carrying a sufficient amount of genetic information, and allowing expression of a R0R2 inhibitor of the invention. The present invention provides an isolated polynucleotide having the sequence of SEQ ID NO: 16 or an isolated polynucleotide having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 16. The present invention also provides an isolated polynucleotide having the sequence of SEQ ID NO: 12 or an isolated polynucleotide having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 12.

[0145] Vectors

[0146] The present invention also provides a vector comprising a nucleotide sequence encoding a ROR2 inhibitor of the invention. As used herein, the term “vector” refers to a molecule or construct suitable for delivering a nucleotide sequence encoding a ROR2 inhibitor of the invention to a target cell. A suitable vector may be any vector which is capable of carrying a sufficient amount of genetic information, and allowing expression of a ROR2 inhibitor of the invention.

[0147] Viral vectors

[0148] In one embodiment the vector is viral vector, optionally a viral vector selected from the group consisting of a lentiviral vector, a retroviral vector, and adeno-associated viral (AAV) vector. In one embodiment, the AAV vector is a self-complementary AAV (scAAV) vector. The viral vectors of the present invention may be derived or derivable from any suitable virus.

[0149] In one embodiment, the viral vector is a recombinant viral particle. A recombinant viral particle is capable of transducing a target cell with the nucleotide sequence encoding a ROR2 inhibitor of the invention. For a retroviral particle, once within the cell the RNA genome from the vector particle is reverse transcribed into DNA and integrated into the genome of the target cell. The present invention provides a vector having the sequence of SEQ ID NO: 18 or a vector having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 18. The present invention also provides a vector having the sequence of SEQ ID NO: 22 or a vector having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 22.

[0150] Isolated cells

[0151] The present invention also provides an isolated cell comprising an ROR2 inhibitor, a nucleic acid, and / or a vector of the present invention. The present invention also relates to the therapeutic uses of such isolated cells in treating and / or preventing cartilage loss and / or treating and / or preventing osteoarthritis, treating and / or preventing osteoarthritis associated pain, promoting cartilage repair, and / or preventing cartilage degradation.

[0152] Also described herein is the use of inhibitors of R0R2 in methods of enhancing the capacity of autologous cell preparations to produce cartilage in vivo.

[0153] Thus, in one aspect, the present invention provides a method for enhancing the capacity of a cell to produce cartilage. In one embodiment, the method is an in vitro method. In one embodiment, the method is an ex -vivo. In one embodiment, the in vitro method enhances the capacity of cells to produce cartilage following administration to patient.

[0154] Cells of the present invention or cells that may be used in the methods of the present invention may be selected from the group consisting of: differentiated cells, nondifferentiated cells, for example stem cells, such as pluripotent stem cells, multipotent stem cells, induced pluripotent stem cells, mesenchymal stem cells (MSCs). In one embodiment, the cells of the invention are MSCs. The MSCs may be condensing MSCs. MSCs can be stimulated to differentiate into condensing stem cells by contacting the MSCs with one or more growth or differentiation factors, such as one or more factors selected from the group consisting of bone morphogenetic protein (BMP)-5a, BMP-5b, fibroblast growth factor (FGF)-4, FGF-8, FGF-10, FGF basic, N-cadherin, Neural cell adhesion molecule (NCAM), Perlecan, transforming growth factor (TGF)-beta 1, TGF-beta 2 and Veriscan.

[0155] In one embodiment, the cells of the invention are chondrocytes. Chondrocytes are cells found in cartilage, which produce and maintain the cartilaginous matrix. Condensing MSC can be stimulated to differentiate into chondrocytes by contacting the condensing MSC with one or more growth or differentiation factors, such as a one or more factors selected from the group consisting of BMP -2, BMP-4, BMP-5 BMP-7, growth differentiation factor (GDF)-5 / BMP-14, FGF basic, insulin-like growth factor 1 (IGF-1), TGF-beta 1 and TGF- beta 2.

[0156] Pharmaceutical compositions

[0157] The present invention also provides a pharmaceutical composition comprising an R0R2 inhibitor of the invention, a nucleic acid of the invention, a vector of the invention, or an isolated cell of the invention. The pharmaceutical composition will also typically comprise a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition may be formulated to help it be compatible with its intended route of administration.

[0158] Examples of routes of administration which may be employed in the invention, and which in some cases include parenteral, e.g., intra-articular, intravenous, intradermal, subcutaneous, intraperitoneal, intramuscular, oral (e.g., inhalation), transdermal (topical), transmucosal, administration may be employed. The pharmaceutical compositions, R0R2 inhibitors, nucleic acids, vectors, or isolated cells of the invention may be, for instance, delivered by such routes. The pharmaceutical compositions of the invention may be formulated to aid compatibility with any of the preceding routes of administration. A preferred route for delivery for the pharmaceutical compositions, R0R2 inhibitors, nucleic acids, vectors, or isolated cells of the invention is via intra-articular administration. In one embodiment, the intra-articular administration is via intra-articular injection or infusion (also known as joint injection or infusion). It is predicted that the fusion proteins of the present invention will be suitable for weekly or bi-weekly systemic delivery or for intraarticular delivery every 3-6 months in patients. This is more convenient for patients because the delivery is less frequent than the injections required for delivering anti-ROR2 siRNA. In one embodiment, the pharmaceutical compositions, R0R2 inhibitors, nucleic acids, vectors, or isolated cells of the invention are delivered intra-articularly by a slow release implant. Where a patient is suffering from cartilage loss or a related disorder, such as osteoarthritis, of a knee joint, the slow release implant may be implanted into the infrapatellar fat pad of the knee of the patient. In one embodiment, the slow release implant is configured to control the release rate of the R0R2 inhibitor, the nucleic acid, the vectors, or the isolated cells of the invention are delivered intra-articularly. The slow release implant may comprise a carrier portion to encapsulate the R0R2 inhibitor, the nucleic acid, the vectors, or the isolated cells of the invention. In one embodiment, carrier portion comprises atelocollagen.

[0159] Pharmaceutical compositions of the invention, including in particular solutions or suspensions used for parenteral, intradermal, subcutaneous or intra-articular application, may, for example, include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. Possible excipients may in some instances be selected from water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and polyvinylpyrrolidone. The pH of the composition may be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The composition, for instance a composition for parenteral preparation, may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. In a preferred instance, a composition of the invention has a physiological pH.

[0160] Pharmaceutical compositions suitable for injectable use, for example suitable for use in intra-articular injection, include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For suitable carriers may include physiological saline, bacteriostatic water, Cremophor EL(TM) (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). The compositions will typically be sterile. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The carrier may be suitable for parenteral, e.g. intra-articular, intravenous, intramuscular or subcutaneous administration (e.g., by injection or infusion). The carrier may be particularly suitable for intra-articular administration. Depending on the route of administration, an anti-ROR2 antibody of the invention may be coated in a material to protect the antibody from the action of acids and other natural conditions that may inactivate or denature the antibody.

[0161] Preferred pharmaceutically acceptable carriers comprise aqueous carriers or diluents. Examples of suitable aqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, buffered water and saline. Examples of other carriers include ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.

[0162] A pharmaceutical composition of the invention also may include a pharmaceutically acceptable anti-oxidant. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

[0163] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the active agent (e.g. antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active agent into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active agent plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0164] Pharmaceutical compositions of the invention may comprise one or more one or more R0R2 inhibitors, nucleic acids, vectors, or isolated cells of the invention. Pharmaceutical compositions of the invention may comprise additional active ingredients as well as one or more R0R2 inhibitors, nucleic acids, vectors, or isolated cells of the invention. They may also comprise additional therapeutic or prophylactic agents. The additional therapeutic agents or prophylactic agents may be useful for treating or preventing osteoarthritis and / or promoting cartilage repair and / or treating or preventing osteoarthritis associated pain.

[0165] Pharmaceutical compositions comprising nucleic acids of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters of the nucleic acid. In certain instances, a composition of the invention may include more than one nucleic acid of the invention. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents may also be employed. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts. A prodrug may, for instance, include the incorporation of additional nucleosides at one or both ends of an oligomeric compound which are cleaved by endogenous nucleases within the body, to form the active nucleic acid.

[0166] Administration may be, for instance, by inhalation. Systemic administration may be, for instance, by transmucosal or transdermal means. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the R0R2 inhibitors may be, for instance, formulated into a transdermal patches or plasters, ointments, salves, gels, or creams. The R0R2 inhibitors may be, for instance, prepared in the form of suppositories or retention enemas. In some instances, the R0R2 inhibitors may be formulated with carriers that protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.

[0167] In one embodiment, the pharmaceutical compositions may be formulated in unit dosage forms. In some embodiments the compositions may be formulated in ampoules. The pharmaceutical compositions may be included in a container, pack, or dispenser together with instructions for administration.

[0168] The present invention also provides a kit comprising one or more R0R2 inhibitors, nucleic acids, vectors, or isolated cells of the invention and optionally instructions for administration to a subject for treating osteoarthritis and / or promoting cartilage repair and / or treating osteoarthritis associated pain, preferably such a kit has the one or more R0R2 inhibitors, nucleic acids, vectors, or isolated cells of the invention provided in the form of a pharmaceutical composition of the invention. The kit may also include means for administering the one or more R0R2 inhibitors, nucleic acids, vectors, or isolated cells of the invention or pharmaceutical composition, for instance a syringe or other appropriate delivery device. The kit may comprise any of the means of delivery discussed herein. In one instance, the kit also comprises lipofectin and in particular a nucleic acid of the invention formulated with lipofectin. The kit may comprise an R0R2 inhibitor, a nucleic acid, a vector, of the invention together with a slow release implant, for example a slow release implant, which comprises a carrier portion comprising atelocollagen.

[0169] The dosage of the R0R2 inhibitors, nucleic acids, vectors, or isolated cells of the invention to be administered will depend upon the particular method or route of delivery being carried out, and when it is being administered to a subject, the nature of disease, the condition of the subject, the particular formulation, and the route of administration. Examples of intracellular concentrations for the nucleic acids of the invention include those in the range from about 0.005 to 50 pM, or more preferably 0.02 to 5 pM. For administration to a subject such as a human, a daily dosage ranging from about 0.001 to 50 mg / kg, preferably 0.01 to 10 mg / kg, and more preferably from 0.1 to 5 mg / kg may be employed. The skilled person and particularly an appropriate physician will be able to identify an appropriate dosage, for instance taking factors such as age, sex, weight and so on into account.

[0170] Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single dose may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0171] Administration may be in single or multiple doses. Multiple doses may be administered via the same or different routes and to the same or different locations. Alternatively, doses can be via a sustained release formulation, in which case less frequent administration is required. Dosage and frequency may vary depending on the half-life of the one or more R0R2 inhibitors, nucleic acids, vectors, or isolated cells of the invention in the subject and the duration of treatment desired.

[0172] Methods of treatment

[0173] The present invention also provides a method of treating and / or preventing cartilage loss, treating and / or preventing osteoarthritis, treating and / or preventing osteoarthritis associated pain, promoting cartilage repair, or preventing cartilage degradation, the method comprising administering to a subject in need thereof one or more R0R2 inhibitors, nucleic acids, vectors, or isolated cells of the invention.

[0174] In addition, the present invention provides one or more R0R2 inhibitors, nucleic acids, vectors or isolated cells of the invention, for use in treating and / or preventing cartilage loss, treating and / or preventing osteoarthritis, treating and / or preventing osteoarthritis associated pain, promoting cartilage repair, or preventing cartilage degradation in a subject.

[0175] In addition, the present invention provides one of more R0R2 inhibitors, nucleic acids, vectors or isolated cells of the invention, for use in the manufacture of a medicament for treating and / or preventing cartilage loss, treating and / or preventing osteoarthritis, treating and / or preventing osteoarthritis associated pain, promoting cartilage repair, or preventing cartilage degradation in a subject.

[0176] In some embodiments, the subject may be a human subject. The subject may be a female subject. In some embodiments, the subject may be a non-human subject. The subject may have been previously diagnosed as having cartilage loss, osteoarthritis, osteoarthritis associated pain and / or cartilage degradation. The subject may be at risk for developing cartilage loss, osteoarthritis, osteoarthritis associated pain and / or cartilage degradation, for example, the subject may have been previously determined to carry genetic markers which indicate that the subject has a higher risk of developing cartilage loss, osteoarthritis, osteoarthritis associated pain and / or cartilage degradation. The subject may be suspected of having OA. The subject may be undergoing treatment for cartilage loss, osteoarthritis, osteoarthritis associated pain and / or cartilage degradation. For example, the subject may be undergoing treatment with one or more R0R2 inhibitors, such as an R0R2 inhibitor of the present invention. The subject may have sustained an injury or trauma or have undergone a surgical intervention putting them at a higher risk of developing cartilage loss, osteoarthritis, osteoarthritis associated pain and / or cartilage degradation. For example, the subject may have previously undergone surgery to treat an anterior cruciate ligament (ACL) injury or an injury, such as a tear, to a knee meniscus. The subject may be an elderly subject. For example, the subject may be at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80 at least 85, at least 90, or at least 95 years in age.

[0177] The subject may be unresponsive to other methods or composition for treating and / or preventing cartilage loss, osteoarthritis, osteoarthritis associated pain and / or cartilage degradation. For example, the subject may be unresponsive to treatment with a NS AID or an opioid analgesic.

[0178] In one embodiment, the method treatment of the invention ameliorates OA associated pain. Pain is typically measured and monitored using self-administered questionnaires such as the WOMAC, IKDC, Lysholm, KOOS, Oxford Knee score and similar validated scoring systems (Collins et al. (2015) Arthritis Care Res (Hoboken). 2011 Nov; 63(0 11): S208- S228.). Such scoring systems assess both pain and function with several sub-scores. More pain-focussed scoring systems can also be used to separate different components of pain such as pain at weight bearing from allodynia or central sensitization (for instance

[0179] PainDETECT and S-LANSS; Moreton et al. (2015) Arthritis Care & Research Volume 67, Issue 4, pages 519-528, April 2015). Functional tests such as Timed ‘Up & Go’ or ‘Stair climbing test’ as described in Baert I, et al. Knee Surgery, Sports Traumatology, Arthroscopy, 2014.

[0180] The present invention is further illustrated by the following examples which should not be construed as further limiting.

[0181] EXAMPLES

[0182] Example 1 - Materials and methods

[0183] The materials and methods that were employed when performing the experiments described in the subsequent Examples are in accordance with those which are standard in the art. It would be within the technical capability of the skilled person to perform the experiments described in the Examples using such standard materials and methods.

[0184] Micromass Culture and Alcian Blue Staining

[0185] Cells were resuspended at a density of 1 x 107cells / mL in complete medium, and micromass cultures were obtained by pipetting 15 pL drops of cell suspension into each well of a 24 well plate. The cells were allowed to attach for 3h and then ImL of complete medium was added (with stimulation if indicated). Micromasses were cultured for 3 days. Micromasses were harvested for RT-PCR gene expression analysis or fixed and wholemount stained with Alcian blue. Alcian blue extraction and quantification was performed as previously described. Images were acquired at room temperature with Leica microscope.

[0186] Example 2

[0187] Aim

[0188] IgGl-Fc is the most common Fc tag used for the generation of biologies. However, IgGl Fc domains “effector function”, which means they can fix complement and damage the cells that they are bound to. This is advantageous in situations where is beneficial to block pathogenic cells (such as immune cells in autoimmune conditions), but may not be desirable in the present circumstances in which it is desirable to support chondrocytes to preserve cartilage integrity. Therefore, constructs comprising IgGl-Fc were compared with constructs comprising IgG2-Fc which has lesser or no effector function. In addition, since the presence of an Fc tag can cause steric hinderance C- and N-terminal tagging was compared.

[0189] Methods

[0190] Four constructs were therefore generated and compared for their capacity to support chondrogenesis in C3H10T1 / 2 cells cultured in micromass. In more detail, subconfluent C3H10T1 / 2 cells were transfected with plasmids encoding the following constructs: (a) C- terminal IgGl-Fc tagged R0R2-ECD; (b) C-terminal IgG2-Fc tagged R0R2-ECD; (c) N- terminal IgGl-Fc tagged R0R2-ECD; or (d) N-terminal IgG2-Fc tagged R0R2-ECD. For cells transfected with (a) and (c), a construct encoding IgGl-Fc was used as a negative control. As a negative control for cells transfected with (b) and (d), a construct encoding IgG2-Fc was used as a negative control. Three days later the cells were plated in micromass (150,000 cell per micromass at a concentration of 20 x 106cells / ml. The following day the micromasses were fixed and stained overnight with 1% Alcian Blue pH=0.2 and imaged. Staining density was assessed and compared using Imaged software. Alcian Blue staining measures the amount of cartilage-specific highly sulphated glycosaminoglycans in the extracellular matrix so is an indicator of chondrogenesis (cartilage development).

[0191] Results

[0192] As shown in Figure 1A and Figure IB, the IgGl-Fc tagged ROR2-ECD did not result in any improved chondrogenesis, either at the C-terminal or the N-terminal or ROR2-ECD. In fact, N-terminal IgGl-Fc tagged ROR2-ECD was found to be anti-chondrogenic. However, the N-terminal IgG2-Fc tagged ROR2-ECD shows significantly increased chondrogenesis compared to the negative control (Figure ID).

[0193] Example 3

[0194] Aim

[0195] The aim of this experiment was to investigate the dose-response of C-terminally tag IgG2- Fc tagging were chondrogenic. Accordingly, the chondrogenic activity of C-terminal IgG2-Fc tagging of ROR2-ECD was compared to the IgG2-Fc alone as control at concentrations similar to those at which Etanercept (a biological containing an IgGl-Fc tag) is active in a dose-response curve. Method

[0196] C3H10T1 / 2 cells were plated in micromass and exposed to increasing concentration of C- terminal IgG2-Fc tagged R0R2-ECD, BMP2 or IgG2-Fc alone for 3 days. At the end of the experiment, the micromasses were stained with Alcian Blue at a pH of 0.2 and densitometric analysis was used to compare glycosaminoglycan content.

[0197] Results

[0198] High doses of C-terminal IgG2 -Fc-tagged ROR2-ECD were able to induce chondrogenesis, but not with the same efficiency as N-terminal IgG2 -Fc-tagged ROR2- ECD (Figure 2). For comparison, Etanercept (MW-150 kDalton), at a dose of 25 mg twice weekly, and with a distribution volume of 12L, is expected to reach a concentration of 3 mg / L (Korth-Bradley et al. Ann Pharmacother 2000 Feb;34(2): 161-4; PMID: 10676822 https: / / pubmed.ncbi.nlm.nih.gov / 10676822 / ) which, considering the molecular of Etanercept is around 150 kDa, equates to a concentration of about 20 nM.

[0199] Example 4

[0200] Aim

[0201] The previously described experiments suggest that the N-terminal IgG2-Fc tag construct is the most efficacious, but this does not exclude the possibility that, e.g. if dose-response curves are bell-shaped, other configurations may perform better. To investigate this possibility of a bell-shaped dose-response curve for the IgG2-Fc tagged constructed, the activation of the TEAD reporter assay was used. This assay measures the activation of YAP signalling as a readout because suppression of YAP signalling is required for the chondrogenic function of R0R2 blockade (Thorup et al. Science Translational Medicine 2020, 12(561)).

[0202] Methods

[0203] Subconfluent C3H10T1 / 2 cells were transfected with plasmids encoding for firefly luciferase under the transcriptional control of a TEAD-responsive promoter and renilla luciferase under the control of a ubiquitous promoter to control for transfection efficiency (HOPFlash reporter assay). The cells were transferred to serum-free medium, treated with recombinant, untagged ROR1-ECD, ROR2-ECD, WNT5A, Verteporfin or DMSO (negative control for Verteporfin) or PBS (negative control for recombinant proteins) for 24 hours and then harvested. Luciferase substrate was added for luminometry measurements.

[0204] Results

[0205] The results show readings of firefly luciferase activity with (Figure 3B) or without (Figure 3A) normalization for renilla luciferase activity. As expected, WNT5A induced activation of the HOPFlash reporter. TEAD is essential in mediating YAP-dependent gene expression. Since luciferase expression is under the control of a TEAD-responsive promoter, luciferase activity is indicative of YAP signaling. The results indicate that both R0R1-ECD and R0R2-ECD can suppress YAP signaling in a dose-dependent manner.

[0206] Example 5

[0207] Aim

[0208] The aim of these experiments was to further validate the finding that IgGl-Fc tagging results in an inactive R0R2-ECD molecule.

[0209] Methods

[0210] Subconfluent C3H10T1 / 2 cells were transfected with plasmids encoding for C-terminal (MHH) or N-terminal (WZM) IgGl-Fc tagged R0R2-ECD, or IgGl-Fc as control as indicated, plus plasmids encoding firefly luciferase under the transcriptional control of a TEAD-responsive promoter and renilla luciferase under the control of a ubiquitous promoter to control for transfection efficiency (HOPFlash reporter assay). The cells were transferred to serum-free medium for 24 hours and then harvested. Luciferase substrate was added for luminometry measurements.

[0211] Results

[0212] The results show readings of firefly luciferase activity with normalization for renilla luciferase activity (Figure 4). The results show that neither C-terminal nor N-terminal IgGl-Fc tagged R0R2-ECD cause increased luciferase expression. TEAD is essential in mediating YAP-dependent gene expression. Since luciferase expression is under the control of a TEAD-responsive promoter, luciferase activity is indicative of YAP signaling. Accordingly, the results indicate that neither C- nor N-terminal IgGl -tagged constructs are able to inhibit YAP signalling. Methods

[0213] A dose-response curve was generated with recombinant R0R2-ECD tagged at either terminus with IgGl-Fc spanning concentrations between 22.2 fM and 22.2 nM. To ensure that the osmotic pressure did not vary from sample to sample, isolated IgGl-Fc was used as a diluent so that each sample had a constant molar concentration of R0R2-ECD-Fc + isolated IgGl-Fc. For instance, the sample comprising no R0R2-ECD-Fc contained 22.2 nM of IgGl-Fc. In more detail, subconfluent C3H10T1 / 2 cells were transfected with plasmids encoding for firefly luciferase under the transcriptional control of a TEAD- responsive promoter and renilla luciferase under the control of a ubiquitous promoter to control for transfection efficiency (HOPFlash reporter assay). The cells were transferred to serum -free medium for 24 hours and treated for 24 hours with N- or C-terminal IgGl-Fc tagged R0R2-ECD at the molarity indicated. Isolated IgGl-Fc was used as control and as a diluent so that the total molarity of R0R2-ECD-Fc and free IgGl-Fc would stay constant at 22.2 nM. Luciferase activity was measured after 24 hours.

[0214] Results

[0215] The results show readings of firefly luciferase activity with normalization for renilla luciferase activity (Figure 13). The results show that tagging R0R2-ECD with IgGl-Fc at either terminus results in failure to induce increased luciferase expression.

[0216] Example 6

[0217] Aim

[0218] The aim of this experiment was to compare the capacity of the R0R1 and R0R2 ECDs to inhibit R0R2 signalling and activate chondrogenesis. To this end, the capacity of both ECD was evaluated using untagged constructs.

[0219] Methods

[0220] C3H10T1 / 2 cells were transfected in monolayer with plasmids encoding for untagged R0R1-ECD, untagged R0R2-ECD and empty plasmid used as negative control. The cells were then plated in micromass at 150,000 cells per micromass and 5 days later were used for RNA extraction and RT-PCR. Data are expressed as ratio to the housekeeping gene P actin.

[0221] Results As shown in Figure 5, both R0R1-ECD and R0R2-ECD increase expression of Aggrecan (Figure 5A) and Gdf5 (Figure 5C) and decrease expression of Cyr61 (Figure 5B). Aggrecan is a marker of cartilage formation, Gdf5 is a marker of stable articular cartilage formation, and Cyr61 is a reporter of R0R2 activity. The results therefore indicate that both untagged R0R1-ECD and R0R2-ECD inhibit R0R2 signalling and activate chondrogenesis.

[0222] Example 7

[0223] Aim

[0224] R0R2 activity inhibits canonical WNT signalling (Gao et al., 2011; Park et al., 2015; Thorup et al., 2020) at least in part by competing with the WNT co-receptors LRP5 and LRP6 for the binding to FZD receptors (Gao et al., 2011). Therefore, without wishing to be bound by theory, it would be expected that suppression of R0R2 activity would result in activation of canonical WNT signalling. This would not be desirable given that canonical WNT signalling is pathogenic in osteoarthritis (Corr et al., 2008). Accordingly, the inventors investigated the effects of ROR1 ECD and ROR2 ECD on canonical WNT signalling.

[0225] Methods

[0226] To perform a TOPFlash assay, HEK293 cells were transfected with a plasmid encoding for ROR2, a plasmid encoding for ROR1-ECD or ROR2-ECD or an empty plasmid (denoted EV) . The empty plasmid was used as a control so that the total amount of DNA would be constant in all transfections. The cells were then treated with vehicle or WNT3 A as indicated. The fusion proteins, ROR2-ECD and ROR1-ECD, used here do not comprise an Fc tag.

[0227] Results

[0228] The results show that unexpectedly ROR2-ECD, but not ROR1-ECD, suppresses canonical WNT signalling (Figure 6). As expected, WNT3A upregulates canonical WNT signalling. As previously reported, ROR2 blocks the capacity of WNT3A to activate canonical WNT signalling, thereby showing that the signalling intracellular portion of ROR2 is not required for the canonical WNT inhibitory activity and that such activity relies most likely on the capacity of ROR2-ECD to compete with the canonical WNT co-receptors LRP5 and LRP6 to bind FZD receptors. Example 8

[0229] Aim

[0230] The aim of this experiment was to confirm the capacity of N-terminally tagged R0R2- ECD-IgG2-Fc to increase the amount of cartilage-specific glycosaminoglycan rich extracellular matrix in C3H10T1 / 2 cells cultured in micromass.

[0231] Methods

[0232] C3H10T1 / 2 cells were plated in micromass and treated with either 100 ng / mL WNT5A alone or 100 ng / mL WNT5A and 40 nM N-terminal IgG2 tagged ROR2-ECD-Fc. Three days later, the cartilage-specific highly sulphated glycosaminoglycan content in the extracellular matrix was quantified by staining with Alcian Blue at pH = 0.2.

[0233] Results

[0234] As shown in Figure 7, treatment with N-terminal IgG2 tagged ROR2-ECD-Fc increased cartilage-specific extracellular matrix production. This experiment confirms the capacity of N-terminal IgG2 tagged ROR2-ECD-Fc to increase the amount of cartilage-specific glycosaminoglycan rich extracellular matrix in C3H10T1 / 2 cells cultured in micromass. The results indicate that N-terminal tagging of ROR2-ECD with IgG2-Fc preserves the biological activity of the ROR2-ECD.

[0235] Example 9

[0236] Aim

[0237] The aim of this experiment was to confirm the capacity of N-terminally tagged ROR2- ECD-IgG2-Fc to supress TEAD transcriptional activity.

[0238] Methods

[0239] C28 / I2 human chondrocytes were transfected with plasmids encoding for firefly luciferase under the transcriptional control of a TEAD-responsive promoter and renilla luciferase under the control of a ubiquitous promoter to control for transfection efficiency and plated in monolayer (HOPFlash reporter assay). The cells were treated with WNT5A and N-terminally tagged ROR2-ECD-IgG2-Fc and luciferase activity was assessed 24h later.

[0240] Results

[0241] The results show that N-terminally tagged ROR2-ECD-IgG2-Fc efficiently blocks TEAD transcriptional activity both in the presence and the absence of WNT5A (Figure 8).

[0242] Example 10

[0243] Aim

[0244] The aim of this experiment was to investigate the effect of R0R2 overexpression on canonical WNT signalling using a TOPFlash reporter assay.

[0245] Methods

[0246] C3H10T1 / 2 cells were transfected with plasmids encoding firefly luciferase under the control of TCF / LEF responsive elements (TOPFlash reporter assay) and renilla luciferase under the transcriptional control of a ubiquitous promoter for control of transfection efficiency, ROR2 plasmid or empty vector (EV) and treated with recombinant WNT3 A or WNT5A. The reporter assay was assessed the following day.

[0247] Results

[0248] The results show that overexpression of ROR2 inhibits the capacity of WNT3A to activate canonical WNT signalling (Figure 9; compare positions 2 and 6 on the axis of the plot). Therefore, ROR2 signalling inhibits canonical WNT signalling.

[0249] Without wishing to be bound by theory, the inventors hypothesize that, in a cell in which canonical WNT signalling is active, for instance by binding of WNT3A to its receptors FZD and LRP6, a R0R2 ligand such as WNT5A will bind ROR2, which will outcompete LRP6 for FZD binding. This results in activation of YAP signalling (Thorup et al., 2020) and suppression of canonical WNT signalling (Gao et al., 2011; Thorup et al., 2020) because LRP6 is removed from FZD.

[0250] Example 11 Aim

[0251] Without wishing to be bound by theory, it was hypothesised that, when the R0R2 ligand WNT5A is present, it will bind with high affinity to R0R2, which will compete with the canonical WNT receptors LRP5 and LRP6. Therefore, YAP, which is downstream of R0R2, will enter the nucleus and beta catenin, downstream of canonical WNT signalling, will be degraded. Since both YAP and beta catenin signalling inhibit cartilage formation, without wishing to be bound by theory, it is believed that there is a “Goldilocks zone” where some degree of R0R2 inhibition is good, because it represses YAP signalling, whereas too great a degree of R0R2 inhibition it is not beneficial because it de-represses beta catenin signalling. For example, when R0R2 expression is silenced (e.g. with an siRNA or shRNA-based approach), there is a potential risk that the level of canonical WNT signalling will fall outside the “Goldilocks zone” and consequently beta catenin will be disinhibited because LRP5 and LRP6 are not competed by R0R2. In this scenario, beta catenin signalling may cause cartilage degradation. This experiment aims to test this hypothesis in vitro.

[0252] Methods

[0253] C3H10T1 / 2 cells were made in which R0R2 expression was knocked out using CRISPR technology. Expression of R0R2 was then re-introduced to R0R2 KO cells at increasing concentrations using plasmid transfection. Chondrogenesis was measured by the intensity of Alcian blue staining.

[0254] Results

[0255] As shown in Figure 10, the re-introduction of small amounts of R0R2 expression in the ROR2 KO cells enhanced chondrogenesis. However, the re-introduction of higher amounts of ROR2 expression in ROR2 KO cells was found to suppresses chondrogenesis, likely because of activation of YAP signalling. Thus, levels of ROR2 expression outside of the “Goldilocks zone” were found to be deleterious for chondrogenesis. Without wishing to be bound by theory, it is hypothesized that this may be due to the fact that completely removing ROR2, which competes with the WNT receptors LRP5 and LRP6 for binding to FZD receptors, thereby de-represses WNT signalling. The same effects would not be expected with ROR2 ECD which suppress both canonical WNT signalling and YAP signalling, as demonstrated above. Example 12

[0256] Aim

[0257] The aim of this experiment was to investigate the effect of R0R2 expression levels in vivo in a mouse model for cartilage degeneration associated with osteoarthritis.

[0258] Methods

[0259] Conditional inducible, cartilage-specific R0R2 knockout mice were generated by crossing mice harbouring LoxP sites flanking exon 3 and 4 of the R0R2 gene (JAX 018354 - B6; 129S4-Ror2tml.lMeg / J) with transgenic mice expressing tamoxifen-inducible creERT2 inserted into 3'UTR of the Aggrecan promoter (JAX 019148 - B6.Cg-

[0260] Acantml (cre / ERT2)Crm / J) which are both in the C57BL / 6 background. At the age of approximately 10 weeks, the male mice were subjected to meniscal / ligamentous injury (MLI) surgery. Starting at 4 weeks after MLI, tamoxifen was injected for 5 consecutive days. All mice were then euthanized at 8 weeks after MLI surgery. The Osteoarthritis Research Society International (OARSI) score was assessed in the femur and tibia of WT, heterozygous, and KO male mice.

[0261] Results

[0262] As shown in Figure 11, homozygous ROR2 KO mice developed as much cartilage breakdown as wild type mice. However, heterozygous mice, harbouring only one copy of the ROR2 gene, were protected from osteoarthritis. These results indicate that partial, but not total absence of ROR2 protects from cartilage breakdown in this instability-induced model of osteoarthritis.

[0263] SEQUENCE LISTING

[0264] ROR2-ECD (SEQ ID NO: 1):

[0265] EVEVLDPNDPLGPLDGQDGPI PTLKGYFLNFLEPVNNITIVQGQTAILHCKVAGNPPPNVRWLKNDAPVVQEP RRI I IRKTEYGSRLRIQDLDTTDTGYYQCVATNGMKTITATGVLFVRLGPTHSPNHNFQDDYHEDGFCQPYRG IACARFIGNRTIYVDSLQMQGEIENRITAAFTMIGTSTHLSDQCSQFAI PSFCHFVFPLCDARSRTPKPRELC RDECEVLESDLCRQEYTIARSNPLILMRLQLPKCEALPMPESPDAANCMRIGI PAERLGRYHQCYNGSGMDYR GTASTTKSGHQCQPWALQHPHSHHLSSTDFPELGGGHAYCRNPGGQMEGPWCFTQNKNVRMELCDVPSCSPRD SSKMG

[0266] ROR2-ECD (SEQ ID NO: 2):

[0267] GAGGTGGAGGTGCTGGACCCAAACGACCCACTGGGCCCCCTGGACGGGCAGGACGGCCCCATTCCTACCCTGA AGGGCTACTTTCTCAACTTTCTCGAGCCCGTGAATAACATCACAATCGTCCAGGGCCAGACAGCTATTCTGCA CTGTAAGGTGGCCGGGAACCCTCCCCCTAACGTGCGTTGGCTGAAGAATGATGCCCCCGTGGTGCAGGAACCT CGGCGGATCATCATCAGAAAGACTGAATACGGCTCTCGCCTGCGGATTCAGGATCTGGACACCACCGACACAG GCTATTACCAGTGTGTGGCCACCAATGGGATGAAGACCATCACTGCTACCGGCGTGCTGTTCGTGCGCCTGGG C C CT AC C CAT AGT C CT AAC CAT AAT T T C CAGGAC GACT AC CAC GAGGAT GGAT T T T GC CAGC C CT ACAGGGGC ATTGCCTGCGCCCGCTTCATCGGCAACCGGACAATCTACGTTGACAGCCTGCAGATGCAGGGCGAGATCGAGA ACAGGAT CACT GC C GC CT T TAG CAT GAT T GGCACAAGT AC C CAC CT GT CAGAT CAGT GCT C C CAGT T T GC CAT CCCATCCTTCTGCCACTTCGTGTTCCCACTGTGTGACGCCAGGTCCCGGACTCCAAAACCCCGCGAGCTGTGT AGAGAC GAGT GC GAGGT GCT GGAGAGC GAC CT T T GCAGACAGGAGT ACAC CAT TGC CAGGT C CAAC C C C CT GA T C CT GAT GAGGCT GCAGCT GC C CAAGT GC GAAGC C CT GC C CAT GC C C GAGAGT C CAGAC GC C GC CAAT T GCAT GAGAAT C GGAAT C C CT GC C GAGAGGCT GGGCAGGT AC CAC CAGT GT T ACAAC GGCT C C GGGAT GGACT AT AGG GGCAC C GC CT C CAC CACAAAGAGC GGC CAC CAGT GT CAGC CAT GGGC C CT GCAGCAC C C C CACAGC CAC CAC C TGAGCTCCACCGATTTTCCTGAGCTGGGGGGGGGCCACGCCTACTGCCGCAACCCTGGCGGCCAGATGGAGGG GCCATGGTGCTTCACACAGAATAAGAATGTGCGCATGGAGCTGTGTGATGTGCCCTCCTGCTCACCTAGGGAC AGCAGCAAGAT GGGG

[0268] R0R1-ECD (SEQ ID NO: 3):

[0269] QETELSVSAELVPTSSWNI SSELNKDSYLTLDE PMNNITTSLGQTAELHCKVSGNPPPTIRWFKNDAPVVQE P RRLS FRSTIYGSRLRIRNLDTTDTGYFQCVATNGKEVVSSTGVLFVKFGPPPTAS PGYSDEYEEDGFCQPYRG IACARFI GNRTVYMESLHMQGE IENQITAAFTMI GTSSHLSDKCSQFAI PSLCHYAFPYCDETSSVPKPRDLC RDECE I LENVLCQTEYI FARSNPMI LMRLKLPNCEDLPQPES PEAANCIRI GI PMADPINKNHKCYNSTGVDY RGTVSVTKSGRQCQPWNSQYPHTHTFTALRFPELNGGHSYCRNPGNQKEAPWCFTLDENFKSDLCDI PACDSK DSKEKNKME

[0270] ROR1-ECD (SEQ ID NO: 4):

[0271] CAAGAAACAGAGCT GT CAGT CAGT GCT GAATTAGT GCCTACCT CAT CAT GGAACAT CT CAAGT GAACT CAACA AAGATTCTTACCTGACCCTCGATGAACCAATGAATAACATCACCACGTCTCTGGGCCAGACAGCAGAACTGCA CTGCAAAGTCTCTGGGAATCCACCTCCCACCATCCGCTGGTTCAAAAATGATGCTCCTGTGGTCCAGGAGCCC CGGAGGCTCTCCTTTCGGTCCACCATCTATGGCTCTCGGCTGCGGATTAGAAACCTCGACACCACAGACACAG GCTACTTCCAGTGCGTGGCAACAAACGGCAAGGAGGTGGTTTCTTCCACTGGAGTCTTGTTTGTCAAGTTTGG CCCCCCTCC CACT GCAAGT C CAGGAT ACT CAGAT GAGT AT GAAGAAGAT GGAT T CT GT CAGC CAT ACAGAGGG AT T GCAT GT GCAAGAT T TAT T GGCAAC C GCAC C GT CT AT AT GGAGT CT T T GCACAT GCAAGGGGAAAT AGAAA ATCAGATCACAGCTGCCTTCACTATGATTGGCACTTCCAGTCACTTATCTGATAAGTGTTCTCAGTTCGCCAT TCCTTCCCTGTGCCACTATGCCTTCCCGTACTGCGATGAAACTTCATCCGTCCCAAAGCCCCGTGACTTGTGT C GC GAT GAAT GT GAAAT C CT GGAGAAT GT C CT GT GT CAAACAGAGT ACAT T T T T GCAAGAT CAAAT C C CAT GA TTCTGATGAGGCTGAAACTGCCAAACTGTGAAGATCTCCCCCAGCCAGAGAGCCCAGAAGCTGCGAACTGTAT C C GGAT T GGAAT T C C CAT GGCAGAT C CT AT AAAT AAAAAT CACAAGT GT T AT AACAGCACAGGT GT GGACT AC C GGGGGAC C GT CAGT GT GAC CAAAT CAGGGC GC CAGT GC CAGC CAT GGAAT T C C CAGT AT C C C CACACACACA CTTTCACCGCCCTTCGTTTCCCAGAGCTGAATGGAGGCCATTCCTACTGCCGCAACCCAGGGAATCAAAAGGA AGCTCCCTGGTGCTTCACCTTGGATGAAAACTTTAAGTCTGATCTGTGTGACATCCCAGCGTGCGATTCAAAG GAT T C CAAGGAGAAGAAT AAAAT GGAA

[0272] IgGl Fc (SEQ ID NO: 5):

[0273] E PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPRE PQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLS PGK

[0274] IgGl Fc (SEQ ID NO: 6):

[0275] GAGCCAAAGAGCTGCGACAAGACCCACACCTGCCCACCCTGCCCAGCCCCTGAACTGCTGGGCGGCCCCAGCG TGTTCCTGTTTCCGCCCAAGCCAAAGGACACCCTGATGATCTCTAGAACACCAGAAGTGACTTGTGTGGTGGT GGAT GT GT C C CAC GAAGAT C CT GAGGT GAAAT T CAACT GGT AC GT GGAT GGT GT GGAAGT GCACAAT GC CAAG ACAAAGCCCCGGGAGGAGCAGTACAATTCTACTTACAGAGTGGTCTCAGTGCTGACCGTGCTGCACCAGGACT GGCT GAAC GGCAAGGAGT AT AAGT GCAAAGT GAGT AAT AAGGC CCTGCCCGCCCC CAT C GAGAAAAC CAT CT C T AAGGC CAAGGGGCAGC CT AGAGAAC CT CAGGT GT ACAC CCTGCCCCC CAGCAGAGAGGAGAT GAC CAAGAAT CAGGTGTCACTGACCTGTCTGGTTAAGGGCTTCTACCCTTCCGATATTGCCGTGGAGTGGGAAAGCAATGGCC AGCCCGAGAACAACTACAAGACTACCCCTCCCGTGCTGGACTCTGATGGAAGCTTTTTTCTGTACAGCAAGCT GAC C GT GGACAAGT C CAGGT GGCAGCAGGGCAAC GT GT T CT C CT GCAGC GT GAT GCAC GAGGC C CT GCACAAT CAC T AC AC AC AGAAGT CCCTGTCCCTGAGCC C AGGC AAG

[0276] IgG2 Fc (SEQ ID NO: 7):

[0277] ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVQFNWYVDGMEVHNAKTKPR EEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTI SKTKGQPRE PQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLS PGK IgG2 Fc (SEQ ID NO: 8):

[0278] GAGAGAAAGTGTTGCGTGGAGTGCCCCCCTTGCCCCGCCCCTCCAGTGGCCGGCCCCAGCGTGTTTCTGTTCC C C C CT AAGC C CAAGGACAC C CT GAT GAT CAGCAGGAC C C C C GAGGT GACT T GC GT GGT GGT GGAC GT CT C C CA T GAGGAT C C C GAGGT GCAGT T T AAT T GGT AC GT T GAT GGCAT GGAGGT GCAT AAC GC CAAGAC CAAAC CAC GG GAGGAGCAGTTCAACAGCACATTTCGCGTGGTCTCTGTGCTGACCGTCGTGCACCAGGACTGGCTGAACGGAA AAGAGT AT AAGT GCAAGGT GT C CAACAAGGGC CT GC CAGC C C CT AT C GAAAAGACT AT CT C CAAGAC CAAGGG GCAGC CAC GC GAGC C C CAGGT GT ACACT CT C C CAC C CAGC C GGGAGGAGAT GACAAAGAAC CAGGT GT CT CT G ACCTGTCTGGTGAAGGGGTTCTACCCTAGCGATATCGCCGTGGAGTGGGAGTCTAACGGCCAGCCCGAGAACA ACTACAAGACAACCCCTCCCATGCTGGACTCTGACGGCTCCTTCTTTCTGTATTCTAAGCTCACCGTGGACAA GAGT AGAT GGCAGCAGGGCAAC GT GT T T T CT T GCAGC GT GAT GCAC GAGGC C CT GCACAAT CACT ACACT CAG AAGAGCCTGAGCCTGTCCCCCGGCAAG

[0279] C-terminal IgGl-Fc ROR2-ECD (SEQ ID NO: 9); residues corresponding to the IgGl-Fc domain are underlined:

[0280] EVEVLDPNDPLGPLDGQDGPI PTLKGYFLNFLE PVNNITIVQGQTAI LHCKVAGNPPPNVRWLKNDAPVVQE P RRI I IRKTEYGSRLRIQDLDTTDTGYYQCVATNGMKTITATGVLFVRLGPTHS PNHNFQDDYHEDGFCQPYRG IACARFI GNRTIYVDSLQMQGE IENRITAAFTMI GTSTHLSDQCSQFAI PS FCHFVFPLCDARSRTPKPRELC RDECEVLESDLCRQEYTIARSNPLI LMRLQLPKCEALPMPES PDAANCMRI GI PAERLGRYHQCYNGSGMDYR GTASTTKSGHQCQPWALQHPHSHHLSSTDFPELGGGHAYCRNPGGQMEGPWCFTQNKNVRMELCDVPSCS PRD S S KMGI LYE PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPRE PQVYTLPP SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLS PGK

[0281] C-terminal IgGl-Fc R0R2-ECD (SEQ ID NO: 10):

[0282] GAGGTGGAGGTGCTGGATCCCAACGATCCTCTGGGCCCACTGGACGGCCAGGATGGCCCTATCCCTACACTGA AAGGGTACTTCCTGAACTTCCTGGAGCCAGTGAATAACATTACCATCGTGCAGGGCCAGACCGCCATCCTGCA CT GCAAGGT GGCAGGAAAC C CT C C C C CAAAC GT GAGGT GGCT GAAGAAC GAC GC C C CAGT C GT GCAGGAAC CT C GCAGAAT CAT CAT C C GGAAGAC C GAGT AC GGCAGCAGACT GAGAAT C CAGGAC CT GGACACAACAGACAC C G GCTACTACCAGTGTGTGGCCACTAACGGCATGAAAACCATCACTGCCACAGGCGTGCTGTTCGTGCGGCTGGG GCCCACTCACAGCCCTAACCACAATTTCCAGGACGATTACCACGAAGACGGCTTCTGCCAGCCCTATCGCGGT AT T GC CT GC GC C C GGT T T AT C GGAAACAGGACAAT T T AC GT GGACT C C CT GCAGAT GCAGGGC GAGAT C GAGA AT C GAAT CAC CGCCGCCTT CAC CAT GAT C GGCAC CAGCACACAC CT GAGC GAC CAGT GCAGT CAGT T T GC CAT CCCCTCCTTTTGCCACTTTGTGTTCCCCCTGTGCGACGCCCGCTCCAGAACACCAAAACCCCGCGAACTGTGT AGAGAC GAGT GT GAGGT GCT GGAGT C C GAC CT GT GCAGACAGGAGT AT AC CAT C GC C C GAAGT AAT C C C CT GA TCCTGATGCGGCTGCAGCTGCCCAAGTGCGAGGCCCTGCCAATGCCCGAGTCCCCCGACGCCGCCAATTGTAT GAGAAT C GGGAT C C C C GC C GAGAGACT GGGC C GCT AC CAT CAGT GCT AT AAT GGCAGC GGAAT GGAT TAG C GG GGCACTGCCTCTACCACCAAGTCCGGGCACCAGTGCCAGCCCTGGGCCCTGCAACACCCCCACAGTCATCATC TGAGCTCTACCGATTTCCCCGAACTGGGCGGGGGACACGCTTACTGTCGAAACCCCGGGGGCCAGATGGAAGG CCCCTGGTGCTT CAC C CAGAACAAGAAT GT GAGAAT GGAGGT GT GC GAC GT GC CAT CT T GCAGC C C CAGGGAC T C CAGCAAGAT GGGGAT T CT GT AT GAGC CAAAAT CT T GC GACAAGACACACAC CT GT CCCCCCTGTCCCGCCC CTGAGCTCCTGGGCGGCCCTAGCGTGTTCTTGTTCCCTCCCAAGCCCAAGGACACCCTGATGATCAGCCGGAC T C C C GAGGT CAC CT GC GT GGT C GT GGAC GT GT C C CAT GAGGAT C C C GAGGT CAAAT T T AACT GGT AT GT GGAT GGC GT GGAAGT GCAT AAC GCAAAGAC CAAAC CAAGGGAGGAGCAGT ACAACT C CAC CT ACAGGGT GGT GAGC G TGCTCACCGTGCTGCACCAGGACTGGCTGAACGGAAAGGAATATAAGTGCAAAGTGTCAAACAAAGCCCTGCC C GC C C CAAT C GAGAAGAC CAT CT C CAAGGCAAAAGGT CAGC C CAGGGAGC CACAGGT GT ACACACT GC CT C CA AGCAGGGAGGAGAT GACAAAAAAC CAGGT GT CACT GACAT GC CT GGT GAAGGGCT T T TAG C CAT CT GAT AT C G C C GT GGAGT GGGAGT CT AAC GGC CAGC C C GAGAAT AAT T ACAAAAC CAC CCCTCCCGT GCT GGAT T C C GAT GG CAGCTTCTTTCTCTACTCCAAGCTGACCGTCGACAAAAGTCGGTGGCAGCAGGGTAACGTGTTTAGTTGCAGC GT GAT GCAT GAGGCACT GCACAAT CACTATACCCAGAAGT CACT GT CACT GT CACCT GGCAAG

[0283] C-terminal IgG2-Fc ROR2-ECD (SEQ ID NO: 11); residues corresponding to the IgG2-Fc domain are underlined:

[0284] EVEVLDPNDPLGPLDGQDGPI PTLKGYFLNFLE PVNNITIVQGQTAI LHCKVAGNPPPNVRWLKNDAPVVQE P RRI I IRKTEYGSRLRIQDLDTTDTGYYQCVATNGMKTITATGVLFVRLGPTHS PNHNFQDDYHEDGFCQPYRG IACARFI GNRTIYVDSLQMQGE IENRITAAFTMI GTSTHLSDQCSQFAI PS FCHFVFPLCDARSRTPKPRELC RDECEVLESDLCRQEYTIARSNPLI LMRLQLPKCEALPMPES PDAANCMRI GI PAERLGRYHQCYNGSGMDYR GTASTTKSGHQCQPWALQHPHSHHLSSTDFPELGGGHAYCRNPGGQMEGPWCFTQNKNVRMELCDVPSCS PRD S S KMGI LYERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVQFNWYVDGMEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTI SKTKGQPRE PQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHE

[0285] ALHNHYTQKSLSLS PGK

[0286] C-terminal IgG2-Fc R0R2-ECD (SEQ ID NO: 12):

[0287] GAGGTGGAGGTGCTGGACCCAAACGACCCACTGGGCCCCCTGGACGGGCAGGACGGCCCCATTCCTACCCTGA AGGGCTACTTTCTCAACTTTCTCGAGCCCGTGAATAACATCACAATCGTCCAGGGCCAGACAGCTATTCTGCA CTGTAAGGTGGCCGGGAACCCTCCCCCTAACGTGCGTTGGCTGAAGAATGATGCCCCCGTGGTGCAGGAACCT CGGCGGATCATCATCAGAAAGACTGAATACGGCTCTCGCCTGCGGATTCAGGATCTGGACACCACCGACACAG GCTATTACCAGTGTGTGGCCACCAATGGGATGAAGACCATCACTGCTACCGGCGTGCTGTTCGTGCGCCTGGG C C CT AC C CAT AGT C CT AAC CAT AAT T T C CAGGAC GACT AC CAC GAGGAT GGAT T T T GC CAGC C CT ACAGGGGC ATTGCCTGCGCCCGCTTCATCGGCAACCGGACAATCTACGTTGACAGCCTGCAGATGCAGGGCGAGATCGAGA ACAGGAT CACT GC C GC CT T TAG CAT GAT T GGCACAAGT AC C CAC CT GT CAGAT CAGT GCT C C CAGT T T GC CAT CCCATCCTTCTGCCACTTCGTGTTCCCACTGTGTGACGCCAGGTCCCGGACTCCAAAACCCCGCGAGCTGTGT AGAGAC GAGT GC GAGGT GCT GGAGAGC GAC CT T T GCAGACAGGAGT ACAC CAT TGC CAGGT C CAAC C C C CT GA T C CT GAT GAGGCT GCAGCT GC C CAAGT GC GAAGC C CT GC C CAT GC C C GAGAGT C CAGAC GC C GC CAAT T GCAT GAGAAT C GGAAT C C CT GC C GAGAGGCT GGGCAGGT AC CAC CAGT GT T ACAAC GGCT C C GGGAT GGACT AT AGG GGCAC C GC CT C CAC CACAAAGAGC GGC CAC CAGT GT CAGC CAT GGGC C CT GCAGCAC C C C CACAGC CAC CAC C TGAGCTCCACCGATTTTCCTGAGCTGGGGGGGGGCCACGCCTACTGCCGCAACCCTGGCGGCCAGATGGAGGG GCCATGGTGCTTCACACAGAATAAGAATGTGCGCATGGAGCTGTGTGATGTGCCCTCCTGCTCACCTAGGGAC AGCAGCAAGAT GGGGAT C CT GT AC GAGC GCAAGT GCT GC GT GGAGT GC C C C C CAT GC C CAGC CCCTCCCGTGG C C GGC C C CT C C GT GT T C CT GT T T C CT C CAAAGC CAAAGGACAC C CT GAT GAT CAGCAGGACAC C C GAGGT GAC CT GC GT GGT GGT GGAC GT GAGT CAT GAGGAT C C C GAGGT GCAGT T CAACT GGT AC GT GGAT GGCAT GGAGGT C CAT AAC GC CAAGACAAAGC C C C GGGAAGAGCAGT T CAACT C CACAT T T AGAGT GGT CT C C GT C CT GAC C GT GG T GCAT CAGGACT GGCT GAAC GGGAAAGAAT AT AAAT GT AAGGT GT C CAACAAGGGC CT GC C C GC C C CT AT T GA AAAGAC CAT T T CT AAGACAAAAGGACAGC C C C GC GAGC C C CAGGT GT AT AC CCTGCCCC CAAGC C GC GAGGAA ATGACAAAAAATCAGGTGTCACTGACCTGCCTGGTGAAGGGATTTTACCCTAGCGATATCGCTGTCGAGTGGG AATCTAACGGCCAGCCAGAGAACAACTACAAGACCACCCCCCCCATGCTGGATAGCGACGGCTCTTTCTTTCT GTACTCCAAGCTGACAGTTGACAAGTCTCGCTGGCAGCAGGGCAATGTGTTCAGCTGCAGCGTGATGCATGAG GCCCTGCATAATCACTATACACAGAAGAGTCTCAGCCTGTCCCCTGGCAAG

[0288] N-terminal IgGl-Fc ROR2-ECD (SEQ ID NO: 13); residues corresponding to the IgGl-Fc domain are underlined:

[0289] E PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPRE PQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLS PGKEVEVLDPNDPLGPLDGQDGPI PTLKGYFLNFLE PVNNITIVQGQTAI LHCKVAGNPPPNV RWLKNDAPVVQE PRRI I IRKTEYGSRLRIQDLDTTDTGYYQCVATNGMKTITATGVLFVRLGPTHS PNHNFQD DYHEDGFCQPYRGIACARFI GNRTIYVDSLQMQGE IENRITAAFTMI GTSTHLSDQCSQFAI PS FCHFVFPLC DARSRTPKPRELCRDECEVLESDLCRQEYTIARSNPLI LMRLQLPKCEALPMPES PDAANCMRI GI PAERLGR YHQCYNGSGMDYRGTASTTKSGHQCQPWALQHPHSHHLSSTDFPELGGGHAYCRNPGGQMEGPWCFTQNKNVR MELCDVPSCS PRDSSKMGI LY

[0290] N-terminal IgGl-Fc R0R2-ECD (SEQ ID NO: 14):

[0291] GAGCCAAAGAGCTGCGACAAGACCCACACCTGCCCACCCTGCCCAGCCCCTGAACTGCTGGGCGGCCCCAGCG TGTTCCTGTTTCCGCCCAAGCCAAAGGACACCCTGATGATCTCTAGAACACCAGAAGTGACTTGTGTGGTGGT GGAT GT GT C C CAC GAAGAT C CT GAGGT GAAAT T CAACT GGT AC GT GGAT GGT GT GGAAGT GCACAAT GC CAAG ACAAAGCCCCGGGAGGAGCAGTACAATTCTACTTACAGAGTGGTCTCAGTGCTGACCGTGCTGCACCAGGACT GGCT GAAC GGCAAGGAGT AT AAGT GCAAAGT GAGT AAT AAGGC CCTGCCCGCCCC CAT C GAGAAAAC CAT CT C T AAGGC CAAGGGGCAGC CT AGAGAAC CT CAGGT GT ACAC CCTGCCCCC CAGCAGAGAGGAGAT GAC CAAGAAT CAGGTGTCACTGACCTGTCTGGTTAAGGGCTTCTACCCTTCCGATATTGCCGTGGAGTGGGAAAGCAATGGCC AGCCCGAGAACAACTACAAGACTACCCCTCCCGTGCTGGACTCTGATGGAAGCTTTTTTCTGTACAGCAAGCT GAC C GT GGACAAGT C CAGGT GGCAGCAGGGCAAC GT GT T CT C CT GCAGC GT GAT GCAC GAGGC C CT GCACAAT CACTACACACAGAAGTCCCTGTCCCTGAGCCCAGGCAAGGAGGTGGAGGTGCTGGACCCAAACGACCCACTGG GCCCCCTGGACGGGCAGGACGGCCCCATTCCTACCCTGAAGGGCTACTTTCTCAACTTTCTCGAGCCCGTGAA TAACATCACAATCGTCCAGGGCCAGACAGCTATTCTGCACTGTAAGGTGGCCGGGAACCCTCCCCCTAACGTG C GT T GGCT GAAGAAT GAT GC C C C C GT GGT GCAGGAAC CT C GGC GGAT CAT CAT CAGAAAGACT GAAT AC GGCT CTCGCCTGC GGAT T CAGGAT CT GGACAC CAC C GACACAGGCT AT TAG CAGT GT GT GGC CAC CAAT GGGAT GAA GACCATCACTGCTACCGGCGTGCTGTTCGTGCGCCTGGGCCCTACCCATAGTCCTAACCATAATTTCCAGGAC GACT AC CAC GAGGAT GGAT T T T GC CAGC C CT ACAGGGGCAT TGCCTGCGCCCGCTT CAT C GGCAAC C GGACAA T CT AC GT T GACAGC CT GCAGAT GCAGGGC GAGAT C GAGAACAGGAT CACT GC C GC CT T TAG CAT GAT T GGCAC AAGTACCCACCTGTCAGATCAGTGCTCCCAGTTTGCCATCCCATCCTTCTGCCACTTCGTGTTCCCACTGTGT GAC GC CAGGT C C C GGACT C CAAAAC C C C GC GAGCT GT GT AGAGAC GAGT GC GAGGT GCT GGAGAGC GAG CT T T GCAGACAGGAGT ACAC CAT TGC CAGGT C CAAC C C C CT GAT C CT GAT GAGGCT GCAGCT GC C CAAGT GC GAAGC C CT GC C CAT GC C C GAGAGT C CAGAC GC C GC CAAT T GCAT GAGAAT C GGAAT C C CT GC C GAGAGGCT GGGCAGG TAG CAC CAGT GT T ACAAC GGCT C C GGGAT GGACT AT AGGGGCAC C GC CT C CAC CACAAAGAGC GGC CAC CAGT GTCAGCCATGGGCCCTGCAGCACCCCCACAGCCACCACCTGAGCTCCACCGATTTTCCTGAGCTGGGGGGGGG C CAC GC CT ACT GC C GCAAC CCTGGCGGC CAGAT GGAGGGGC CAT GGT GCT T CACACAGAAT AAGAAT GT GC GC ATGGAGCTGTGTGATGTGCCCTCCTGCTCACCTAGGGACAGCAGCAAGATGGGGATCCTGTAC

[0292] N-terminal IgG2-Fc R0R2-ECD (SEQ ID NO: 15); residues corresponding to IgG2-Fc are underlined:

[0293] ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVQFNWYVDGMEVHNAKTKPR EEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTI SKTKGQPRE PQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLS PGKEVEVLDPNDPLGPLDGQDGPI PTLKGYFLNFLE PVNNITIVQGQTAI LHCKVAGNPPPNVRWLK NDAPVVQE PRRI I IRKTEYGSRLRIQDLDTTDTGYYQCVATNGMKTITATGVLFVRLGPTHS PNHNFQDDYHE DGFCQPYRGIACARFI GNRTIYVDSLQMQGE IENRITAAFTMI GTSTHLSDQCSQFAI PS FCHFVFPLCDARS RTPKPRELCRDECEVLESDLCRQEYTIARSNPLI LMRLQLPKCEALPMPES PDAANCMRI GI PAERLGRYHQC YNGSGMDYRGTASTTKSGHQCQPWALQHPHSHHLSSTDFPELGGGHAYCRNPGGQMEGPWCFTQNKNVRMELC DVPSCS PRDSSKMGI LY

[0294] N-terminal IgG2-Fc R0R2-ECD (SEQ ID NO: 16):

[0295] GAGAGAAAGTGTTGCGTGGAGTGCCCCCCTTGCCCCGCCCCTCCAGTGGCCGGCCCCAGCGTGTTTCTGTTCC C C C CT AAGC C CAAGGACAC C CT GAT GAT CAGCAGGAC C C C C GAGGT GACT T GC GT GGT GGT GGAC GT CT C C CA T GAGGAT C C C GAGGT GCAGT T T AAT T GGT AC GT T GAT GGCAT GGAGGT GCAT AAC GC CAAGAC CAAAC CAC GG GAGGAGCAGTTCAACAGCACATTTCGCGTGGTCTCTGTGCTGACCGTCGTGCACCAGGACTGGCTGAACGGAA AAGAGT AT AAGT GCAAGGT GT C CAACAAGGGC CT GC CAGC C C CT AT C GAAAAGACT AT CT C CAAGAC CAAGGG GCAGC CAC GC GAGC C C CAGGT GT ACACT CT C C CAC C CAGC C GGGAGGAGAT GACAAAGAAC CAGGT GT CT CT G ACCTGTCTGGTGAAGGGGTTCTACCCTAGCGATATCGCCGTGGAGTGGGAGTCTAACGGCCAGCCCGAGAACA ACTACAAGACAACCCCTCCCATGCTGGACTCTGACGGCTCCTTCTTTCTGTATTCTAAGCTCACCGTGGACAA GAGT AGAT GGCAGCAGGGCAAC GT GT T T T CT T GCAGC GT GAT GCAC GAGGC C CT GCACAAT CACT ACACT CAG AAGAGCCTGAGCCTGTCCCCCGGCAAGGAGGTGGAGGTGCTGGACCCAAACGACCCACTGGGCCCCCTGGACG GGCAGGACGGCCCCATTCCTACCCTGAAGGGCTACTTTCTCAACTTTCTCGAGCCCGTGAATAACATCACAAT CGTCCAGGGCCAGACAGCTATTCTGCACTGTAAGGTGGCCGGGAACCCTCCCCCTAACGTGCGTTGGCTGAAG AATGATGCCCCCGTGGTGCAGGAACCTCGGCGGATCATCATCAGAAAGACTGAATACGGCTCTCGCCTGCGGA T T CAGGAT CT GGACAC CAC C GACACAGGCT AT TAG CAGT GT GT GGC CAC CAAT GGGAT GAAGAC CAT CACT GC TACCGGCGTGCTGTTCGTGCGCCTGGGCCCTACCCATAGTCCTAACCATAATTTCCAGGACGACTACCACGAG GAT GGAT T T T GC CAGC C CT ACAGGGGCAT TGCCTGCGCCCGCTT CAT C GGCAAC C GGACAAT CT AC GT T GACA GC CT GCAGAT GCAGGGC GAGAT C GAGAACAGGAT CACT GC C GC CT T TAG CAT GAT T GGCACAAGT AC C CAC CT GTCAGATCAGTGCTCCCAGTTTGCCATCCCATCCTTCTGCCACTTCGTGTTCCCACTGTGTGACGCCAGGTCC C GGACT C CAAAAC C C C GC GAGCT GT GT AGAGAC GAGT GC GAGGT GCT GGAGAGC GAC CT T T GCAGACAGGAGT ACACCATTGCCAGGTCCAACCCCCTGATCCTGATGAGGCTGCAGCTGCCCAAGTGCGAAGCCCTGCCCATGCC C GAGAGT C CAGAC GC C GC CAAT T GCAT GAGAAT C GGAAT C C CT GC C GAGAGGCT GGGCAGGT AC CAC CAGT GT T ACAAC GGCT C C GGGAT GGACT AT AGGGGCAC C GC CT C CAC CACAAAGAGC GGC CAC CAGT GT CAGC CAT GGG CCCTGCAGCACCCCCACAGCCACCACCTGAGCTCCACCGATTTTCCTGAGCTGGGGGGGGGCCACGCCTACTG C C GCAAC CCTGGCGGC CAGAT GGAGGGGC CAT GGT GCT T CACACAGAAT AAGAAT GT GC GCAT GGAGCT GT GT GATGTGCCCTCCTGCTCACCTAGGGACAGCAGCAAGATGGGGATCCTGTAC

[0296] Plasmid VB2101280-1127wzm encoding N-terminal IgGl-Fc R0R2-ECD (SEQ ID NO: 17): aatgtagtcttatgcaatactcttgtagtcttgcaacatggtaacgatgagttagcaaca tgccttacaaggagagaaaaagcaccgtgcatgccgattggtggaagtaaggtggtacga tcgtgccttattaggaaggcaacagacgggtctgacatggattggacgaaccactgaatt gccgcattgcagagatattgtatttaagtgcctagctcgatacataaacgggtctctctg gttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcc tcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctgg taactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccg aacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggctt gctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttg actagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggaga attagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaatta aaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgtta gaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacagga tcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaagg atagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagt aagaccaccgcacagcaagcggccgctgatcttcagacctggaggaggagatatgaggga caattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagc acccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagc tttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgct gacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgag ggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctcca ggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttgggg ttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataa atctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaa ttacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatga acaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaa ttggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaat agtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtt tcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaagaagg tggagagagagacagagacagatccattcgattagtgaacggatctcgacggtatcgcta gcttttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacata atagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaatttt actagtgattatcggatcaactttgtatagaaaagttgtagttattaatagtaatcaatt acggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaat ggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgtt cccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaa actgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtc aatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcct acttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcag tacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccatt gacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaac aactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagc agagctggtttagtgaaccgtcagatccaagtttgtacaaaaaagcaggctgccaccatg gagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtgac gagccaaagagctgcgacaagacccacacctgcccaccctgcccagcccctgaactgctg ggcggccccagcgtgttcctgtttccgcccaagccaaaggacaccctgatgatctctaga acaccagaagtgacttgtgtggtggtggatgtgtcccacgaagatcctgaggtgaaattc aactggtacgtggatggtgtggaagtgcacaatgccaagacaaagccccgggaggagcag tacaattctacttacagagtggtctcagtgctgaccgtgctgcaccaggactggctgaac ggcaaggagtataagtgcaaagtgagtaataaggccctgcccgcccccatcgagaaaacc atctctaaggccaaggggcagcctagagaacctcaggtgtacaccctgccccccagcaga gaggagatgaccaagaatcaggtgtcactgacctgtctggttaagggcttctacccttcc gatattgccgtggagtgggaaagcaatggccagcccgagaacaactacaagactacccct cccgtgctggactctgatggaagcttttttctgtacagcaagctgaccgtggacaagtcc aggtggcagcagggcaacgtgttctcctgcagcgtgatgcacgaggccctgcacaatcac tacacacagaagtccctgtccctgagcccaggcaaggaggtggaggtgctggacccaaac gacccactgggccccctggacgggcaggacggccccattcctaccctgaagggctacttt ctcaactttctcgagcccgtgaataacatcacaatcgtccagggccagacagctattctg cactgtaaggtggccgggaaccctccccctaacgtgcgttggctgaagaatgatgccccc gtggtgcaggaacctcggcggatcatcatcagaaagactgaatacggctctcgcctgcgg attcaggatctggacaccaccgacacaggctattaccagtgtgtggccaccaatgggatg aagaccatcactgctaccggcgtgctgttcgtgcgcctgggccctacccatagtcctaac cataatttccaggacgactaccacgaggatggattttgccagccctacaggggcattgcc tgcgcccgcttcatcggcaaccggacaatctacgttgacagcctgcagatgcagggcgag atcgagaacaggatcactgccgcctttaccatgattggcacaagtacccacctgtcagat cagtgctcccagtttgccatcccatccttctgccacttcgtgttcccactgtgtgacgcc aggtcccggactccaaaaccccgcgagctgtgtagagacgagtgcgaggtgctggagagc gacctttgcagacaggagtacaccattgccaggtccaaccccctgatcctgatgaggctg cagctgcccaagtgcgaagccctgcccatgcccgagagtccagacgccgccaattgcatg agaatcggaatccctgccgagaggctgggcaggtaccaccagtgttacaacggctccggg atggactataggggcaccgcctccaccacaaagagcggccaccagtgtcagccatgggcc ctgcagcacccccacagccaccacctgagctccaccgattttcctgagctgggggggggc cacgcctactgccgcaaccctggcggccagatggaggggccatggtgcttcacacagaat aagaatgtgcgcatggagctgtgtgatgtgccctcctgctcacctagggacagcagcaag atggggatcctgtactgaacccagctttcttgtacaaagtggtgataatcgaattccgat aatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgct ccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgt atggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttg tggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccact ggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccct attgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctg ttgggcactgacaattccgtggtgttgtcggggaagctgacgtcctttccatggctgctc gcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctc aatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtctt cgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcatcgggaattc ccgcggttcgctttaagaccaatgacttacaaggcagctgtagatcttagccacttttta aaagaaaaggggggactggaagggctaattcactcccaacgaagacaagatctgcttttt gcttgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaacta gggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcc cgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaa atctctagcagtagtagttcatgtcatcttattattcagtatttataacttgcaaagaaa tgaatatcagagagtgagaggaacttgtttattgcagcttataatggttacaaataaagc aatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttg tccaaactcatcaatgtatcttatcatgtctggctctagctatcccgcccctaactccgc ccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattt tttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgag gaggcttttttggaggcctagggacgtacccaattcgccctatagtgagtcgtattacgc gcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaact taatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcac cgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcgg cgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgc cctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttcc ccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacct cgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagac ggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaac tggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgat ttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaa aatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatt tgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataa atgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgccctt attcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaa gtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaac agcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcactttt aaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggt cgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcat cttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataac actgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttg cacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagcc ataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaa ctattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggag gcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgct gataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagat ggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaa cgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagac caagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatc taggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttc cactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctg cgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccg gatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagatacca aatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccg cctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcg tgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctga acggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatac ctacagcgtgagctatgagaaagcgccacgcttcccgaagagagaaaggcggacaggtat ccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcc tggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtga tgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttc ctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtg gataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgag cgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctcccc gcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggc agtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacac tttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacagga aacagctatgaccatgattacgccaagcgcgcaattaaccctcactaaagggaacaaaag ctggagctgcaagctt

[0297] Plasmid VB210128-1119hbp encoding N-terminal IgG2-Fc ROR2-ECD (SEQ ID NO:

[0298] 18): aatgtagtcttatgcaatactcttgtagtcttgcaacatggtaacgatgagttagcaaca tgccttacaaggagagaaaaagcaccgtgcatgccgattggtggaagtaaggtggtacga tcgtgccttattaggaaggcaacagacgggtctgacatggattggacgaaccactgaatt gccgcattgcagagatattgtatttaagtgcctagctcgatacataaacgggtctctctg gttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcc tcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctgg taactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccg aacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggctt gctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttg actagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggaga attagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaatta aaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgtta gaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacagga tcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaagg atagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagt aagaccaccgcacagcaagcggccgctgatcttcagacctggaggaggagatatgaggga caattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagc acccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagc tttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgct gacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgag ggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctcca ggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttgggg ttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataa atctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaa ttacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatga acaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaa ttggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaat agtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtt tcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaagaagg tggagagagagacagagacagatccattcgattagtgaacggatctcgacggtatcgcta gcttttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacata atagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaatttt actagtgattatcggatcaactttgtatagaaaagttgtagttattaatagtaatcaatt acggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaat ggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgtt cccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaa actgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtc aatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcct acttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcag tacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccatt gacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaac aactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagc agagctggtttagtgaaccgtcagatccaagtttgtacaaaaaagcaggctgccaccatg gagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtgac gagagaaagtgttgcgtggagtgccccccttgccccgcccctccagtggccggccccagc gtgtttctgttcccccctaagcccaaggacaccctgatgatcagcaggacccccgaggtg acttgcgtggtggtggacgtctcccatgaggatcccgaggtgcagtttaattggtacgtt gatggcatggaggtgcataacgccaagaccaaaccacgggaggagcagttcaacagcaca tttcgcgtggtctctgtgctgaccgtcgtgcaccaggactggctgaacggaaaagagtat aagtgcaaggtgtccaacaagggcctgccagcccctatcgaaaagactatctccaagacc aaggggcagccacgcgagccccaggtgtacactctcccacccagccgggaggagatgaca aagaaccaggtgtctctgacctgtctggtgaaggggttctaccctagcgatatcgccgtg gagtgggagtctaacggccagcccgagaacaactacaagacaacccctcccatgctggac tctgacggctccttctttctgtattctaagctcaccgtggacaagagtagatggcagcag ggcaacgtgttttcttgcagcgtgatgcacgaggccctgcacaatcactacactcagaag agcctgagcctgtcccccggcaaggaggtggaggtgctggacccaaacgacccactgggc cccctggacgggcaggacggccccattcctaccctgaagggctactttctcaactttctc gagcccgtgaataacatcacaatcgtccagggccagacagctattctgcactgtaaggtg gccgggaaccctccccctaacgtgcgttggctgaagaatgatgcccccgtggtgcaggaa cctcggcggatcatcatcagaaagactgaatacggctctcgcctgcggattcaggatctg gacaccaccgacacaggctattaccagtgtgtggccaccaatgggatgaagaccatcact gctaccggcgtgctgttcgtgcgcctgggccctacccatagtcctaaccataatttccag gacgactaccacgaggatggattttgccagccctacaggggcattgcctgcgcccgcttc atcggcaaccggacaatctacgttgacagcctgcagatgcagggcgagatcgagaacagg atcactgccgcctttaccatgattggcacaagtacccacctgtcagatcagtgctcccag tttgccatcccatccttctgccacttcgtgttcccactgtgtgacgccaggtcccggact ccaaaaccccgcgagctgtgtagagacgagtgcgaggtgctggagagcgacctttgcaga caggagtacaccattgccaggtccaaccccctgatcctgatgaggctgcagctgcccaag tgcgaagccctgcccatgcccgagagtccagacgccgccaattgcatgagaatcggaatc cctgccgagaggctgggcaggtaccaccagtgttacaacggctccgggatggactatagg ggcaccgcctccaccacaaagagcggccaccagtgtcagccatgggccctgcagcacccc cacagccaccacctgagctccaccgattttcctgagctgggggggggccacgcctactgc cgcaaccctggcggccagatggaggggccatggtgcttcacacagaataagaatgtgcgc atggagctgtgtgatgtgccctcctgctcacctagggacagcagcaagatggggatcctg tactgaacccagctttcttgtacaaagtggtgataatcgaattccgataatcaacctctg gattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgcta tgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcatt ttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtc aggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcatt gccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcg gaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgac aattccgtggtgttgtcggggaagctgacgtcctttccatggctgctcgcctgtgttgcc acctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggac cttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccct cagacgagtcggatctccctttgggccgcctccccgcatcgggaattcccgcggttcgct ttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaagggg ggactggaagggctaattcactcccaacgaagacaagatctgctttttgcttgtactggg tctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactg cttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgt gactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagt agtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagag agtgagaggaacttgtttattgcagcttataatggttacaaataaagcaatagcatcaca aatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatc aatgtatcttatcatgtctggctctagctatcccgcccctaactccgcccatcccgcccc taactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatg cagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttg gaggcctagggacgtacccaattcgccctatagtgagtcgtattacgcgcgctcactggc cgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgc agcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttc ccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgc ggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgc tcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctct aaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaa acttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccc tttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacact caaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattg gttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgct tacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttc taaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataa tattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattccctttttt gcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgct gaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatc cttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgcta tgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacac tattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggc atgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaac ttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggg gatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgac gagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggc gaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagtt gcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctgga gccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcc cgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacag atcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactca tatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatc ctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtca gaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgc tgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagcta ccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttctt ctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctc gctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccggg ttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcg tgcacacagcccagcttggagcgaacgacctacaccgaact gaga tacctacagcgt gag ctatgagaaagcgccacgcttcccgaagagagaaaggcggacaggtatccggtaagcggc agggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttat agtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggg gggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgc tggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtatt accgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtca gtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccg attcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaac gcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccg gctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgac catgattacgccaagcgcgcaattaaccctcactaaagggaacaaaagctggagctgcaa gctt

[0299] Plasmid VB21011 l-1095bmq encoding IgK signal peptide - IgGl-Fc (SEQ ID NO: 19): aatgtagtcttatgcaatactcttgtagtcttgcaacatggtaacgatgagttagcaaca tgccttacaaggagagaaaaagcaccgtgcatgccgattggtggaagtaaggtggtacga tcgtgccttattaggaaggcaacagacgggtctgacatggattggacgaaccactgaatt gccgcattgcagagatattgtatttaagtgcctagctcgatacataaacgggtctctctg gttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcc tcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctgg taactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccg aacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggctt gctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttg actagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggaga attagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaatta aaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgtta gaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacagga tcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaagg atagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagt aagaccaccgcacagcaagcggccgctgatcttcagacctggaggaggagatatgaggga caattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagc acccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagc tttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgct gacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgag ggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctcca ggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttgggg ttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataa atctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaa ttacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatga acaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaa ttggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaat agtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtt tcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaagaagg tggagagagagacagagacagatccattcgattagtgaacggatctcgacggtatcgcta gcttttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacata atagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaatttt actagtgattatcggatcaactttgtatagaaaagttgtagttattaatagtaatcaatt acggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaat ggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgtt cccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaa actgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtc aatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcct acttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcag tacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccatt gacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaac aactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagc agagctggtttagtgaaccgtcagatccaagtttgtacaaaaaagcaggctgccaccatg gagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtgac gagccaaagagctgcgacaagacccacacctgcccaccctgcccagcccctgaactgctg ggcggccccagcgtgttcctgtttccgcccaagccaaaggacaccctgatgatctctaga acaccagaagtgacttgtgtggtggtggatgtgtcccacgaagatcctgaggtgaaattc aactggtacgtggatggtgtggaagtgcacaatgccaagacaaagccccgggaggagcag tacaattctacttacagagtggtctcagtgctgaccgtgctgcaccaggactggctgaac ggcaaggagtataagtgcaaagtgagtaataaggccctgcccgcccccatcgagaaaacc atctctaaggccaaggggcagcctagagaacctcaggtgtacaccctgccccccagcaga gaggagatgaccaagaatcaggtgtcactgacctgtctggttaagggcttctacccttcc gatattgccgtggagtgggaaagcaatggccagcccgagaacaactacaagactacccct cccgtgctggactctgatggaagcttttttctgtacagcaagctgaccgtggacaagtcc aggtggcagcagggcaacgtgttctcctgcagcgtgatgcacgaggccctgcacaatcac tacacacagaagtccctgtccctgagcccaggcaagtgaacccagctttcttgtacaaag tggtgataatcgaattccgataatcaacctctggattacaaaatttgtgaaagattgact ggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttg tatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttg ctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtg tttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccggg actttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgc tgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaagctg acgtcctttccatggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttc tgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggct ctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggcc gcctccccgcatcgggaattcccgcggttcgctttaagaccaatgacttacaaggcagct gtagatcttagccactttttaaaagaaaaggggggactggaagggctaattcactcccaa cgaagacaagatctgctttttgcttgtactgggtctctctggttagaccagatctgagcc tgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttga gtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcaga cccttttagtcagtgtggaaaatctctagcagtagtagttcatgtcatcttattattcag tatttataacttgcaaagaaatgaatatcagagagtgagaggaacttgtttattgcagct tataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttca ctgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggctctag ctatcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctc cgccccatggctgactaattttttttatttatgcagaggccgaggccgcctcggcctctg agctattccagaagtagtgaggaggcttttttggaggcctagggacgtacccaattcgcc ctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactggga aaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcg taatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcga atgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgt gaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttct cgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccg atttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtag tgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaa tagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttga tttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaa atttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcgggga aatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctc atgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtatt caacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgct cacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggt tacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgt tttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgac gccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtac tcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgct gccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccg aaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgg gaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagca atggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaa caattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggccctt ccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatc attgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacgggg agtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgatt aagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaactt catttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatc ccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatct tcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgcta ccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggc ttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccac ttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggct gctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggat aaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacg acctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaa gagagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagg gagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctga cttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagc aacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcct gcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgct cgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgccca atacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacagg tttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcat taggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagc ggataacaatttcacacaggaaacagctatgaccatgattacgccaagcgcgcaattaac cctcactaaagggaacaaaagctggagctgcaagctt

[0300] Plasmid VB21011 l-1096fvp encoding IgK signal peptide - IgG2-Fc (SEQ ID NO: 20): aatgtagtcttatgcaatactcttgtagtcttgcaacatggtaacgatgagttagcaaca tgccttacaaggagagaaaaagcaccgtgcatgccgattggtggaagtaaggtggtacga tcgtgccttattaggaaggcaacagacgggtctgacatggattggacgaaccactgaatt gccgcattgcagagatattgtatttaagtgcctagctcgatacataaacgggtctctctg gttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcc tcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctgg taactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccg aacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggctt gctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttg actagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggaga attagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaatta aaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgtta gaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacagga tcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaagg atagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagt aagaccaccgcacagcaagcggccgctgatcttcagacctggaggaggagatatgaggga caattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagc acccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagc tttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgct gacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgag ggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctcca ggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttgggg ttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataa atctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaa ttacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatga acaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaa ttggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaat agtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtt tcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaagaagg tggagagagagacagagacagatccattcgattagtgaacggatctcgacggtatcgcta gcttttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacata atagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaatttt actagtgattatcggatcaactttgtatagaaaagttgtagttattaatagtaatcaatt acggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaat ggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgtt cccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaa actgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtc aatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcct acttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcag tacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccatt gacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaac aactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagc agagctggtttagtgaaccgtcagatccaagtttgtacaaaaaagcaggctgccaccatg gagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtgac gagagaaagtgttgcgtggagtgccccccttgccccgcccctccagtggccggccccagc gtgtttctgttcccccctaagcccaaggacaccctgatgatcagcaggacccccgaggtg acttgcgtggtggtggacgtctcccatgaggatcccgaggtgcagtttaattggtacgtt gatggcatggaggtgcataacgccaagaccaaaccacgggaggagcagttcaacagcaca tttcgcgtggtctctgtgctgaccgtcgtgcaccaggactggctgaacggaaaagagtat aagtgcaaggtgtccaacaagggcctgccagcccctatcgaaaagactatctccaagacc aaggggcagccacgcgagccccaggtgtacactctcccacccagccgggaggagatgaca aagaaccaggtgtctctgacctgtctggtgaaggggttctaccctagcgatatcgccgtg gagtgggagtctaacggccagcccgagaacaactacaagacaacccctcccatgctggac tctgacggctccttctttctgtattctaagctcaccgtggacaagagtagatggcagcag ggcaacgtgttttcttgcagcgtgatgcacgaggccctgcacaatcactacactcagaag agcctgagcctgtcccccggcaagtgaacccagctttcttgtacaaagtggtgataatcg aattccgataatcaacctctggattacaaaatttgtgaaagattgactggtattcttaac tatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctatt gcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttat gaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgca acccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttc cccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggg gctcggctgttgggcactgacaattccgtggtgttgtcggggaagctgacgtcctttcca tggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtccct tcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctctt ccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcat cgggaattcccgcggttcgctttaagaccaatgacttacaaggcagctgtagatcttagc cactttttaaaagaaaaggggggactggaagggctaattcactcccaacgaagacaagat ctgctttttgcttgtactgggtctctctggttagaccagatctgagcctgggagctctct ggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagta gtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtca gtgtggaaaatctctagcagtagtagttcatgtcatcttattattcagtatttataactt gcaaagaaatgaatatcagagagtgagaggaacttgtttattgcagcttataatggttac aaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagt tgtggtttgtccaaactcatcaatgtatcttatcatgtctggctctagctatcccgcccc taactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggct gactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccaga agtagtgaggaggcttttttggaggcctagggacgtacccaattcgccctatagtgagtc gtattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgt tacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaaga ggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgcc ctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacact tgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgc cggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgcttt acggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgcc ctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactctt gttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggat tttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaa ttttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcgga acccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataa ccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgt gtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacg ctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactg gatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatg agcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagag caactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcaca gaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatg agtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaacc gcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctg aatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacg ttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagac tggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctgg tttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactg gggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaact atggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaa ctgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaattt aaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgag ttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcct ttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtt tgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcg cagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactct gtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggc gataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcgg tcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaa ctgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagagagaaaggcg gacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccaggg ggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcga tttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggccttt ttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccct gattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccga acgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccg cctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactgg aaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccag gctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaattt cacacaggaaacagctatgaccatgattacgccaagcgcgcaattaaccctcactaaagg gaacaaaagctggagctgcaagctt

[0301] Plasmid VB201230-1093mhh encoding C-terminal IgGl-Fc R0R2-ECD (SEQ ID NO:

[0302] 21): aatgtagtcttatgcaatactcttgtagtcttgcaacatggtaacgatgagttagcaaca tgccttacaaggagagaaaaagcaccgtgcatgccgattggtggaagtaaggtggtacga tcgtgccttattaggaaggcaacagacgggtctgacatggattggacgaaccactgaatt gccgcattgcagagatattgtatttaagtgcctagctcgatacataaacgggtctctctg gttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcc tcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctgg taactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccg aacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggctt gctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttg actagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggaga attagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaatta aaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgtta gaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacagga tcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaagg atagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagt aagaccaccgcacagcaagcggccgctgatcttcagacctggaggaggagatatgaggga caattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagc acccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagc tttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgct gacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgag ggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctcca ggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttgggg ttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataa atctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaa ttacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatga acaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaa ttggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaat agtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtt tcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaagaagg tggagagagagacagagacagatccattcgattagtgaacggatctcgacggtatcgcta gcttttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacata atagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaatttt actagtgattatcggatcaactttgtatagaaaagttgtagttattaatagtaatcaatt acggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaat ggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgtt cccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaa actgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtc aatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcct acttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcag tacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccatt gacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaac aactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagc agagctggtttagtgaaccgtcagatccaagtttgtacaaaaaagcaggctgccaccatg gagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtgac gaggtggaggtgctggatcccaacgatcctctgggcccactggacggccaggatggccct atccctacactgaaagggtacttcctgaacttcctggagccagtgaataacattaccatc gtgcagggccagaccgccatcctgcactgcaaggtggcaggaaaccctcccccaaacgtg aggtggctgaagaacgacgccccagtcgtgcaggaacctcgcagaatcatcatccggaag accgagtacggcagcagactgagaatccaggacctggacacaacagacaccggctactac cagtgtgtggccactaacggcatgaaaaccatcactgccacaggcgtgctgttcgtgcgg ctggggcccactcacagccctaaccacaatttccaggacgattaccacgaagacggcttc tgccagccctatcgcggtattgcctgcgcccggtttatcggaaacaggacaatttacgtg gac t ccc tgcagatgcagggcgagatcgagaatcgaatcaccgccgcctt caeca t gate ggcaccagcacacacctgagcgaccagtgcagtcagtttgccatcccctccttttgccac tttgtgttccccctgtgcgacgcccgctccagaacaccaaaaccccgcgaactgtgtaga gacgagtgtgaggtgctggagtccgacctgtgcagacaggagtataccatcgcccgaagt aatcccctgatcctgatgcggctgcagctgcccaagtgcgaggccctgccaatgcccgag tcccccgacgccgccaattgtatgagaatcgggatccccgccgagagactgggccgctac catcagtgctataatggcagcggaatggattaccggggcactgcctctaccaccaagtcc gggcaccagtgccagccctgggccctgcaacacccccacagtcatcatctgagctctacc gatttccccgaactgggcgggggacacgcttactgtcgaaaccccgggggccagatggaa ggcccctggtgcttcacccagaacaagaatgtcagaatggagctgtgcgacgtgccatct tgcagccccagggactccagcaagatggggattctgtatgagccaaaatcttgcgacaag acacacacctgtcccccctgtcccgcccctgagctcctgggcggccctagcgtgttcttg ttccctcccaagcccaaggacaccctgatgatcagccggactcccgaggtcacctgcgtg gtcgtggacgtgtcccatgaggatcccgaggtcaaatttaactggtatgtggatggcgtg gaagtgcataacgcaaagaccaaaccaagggaggagcagtacaactccacctacagggtg gtgagcgtgctcaccgtgctgcaccaggactggctgaacggaaaggaatataagtgcaaa gtgtcaaacaaagccctgcccgccccaatcgagaagaccatctccaaggcaaaaggtcag cccagggagccacaggtgtacacactgcctccaagcagggaggagatgacaaaaaaccag gtgtcactgacatgcctggtgaagggcttttacccatctgatatcgccgtggagtgggag tctaacggccagcccgagaataattacaaaaccacccctcccgtgctggattccgatggc agcttctttctctactccaagctgaccgtcgacaaaagtcggtggcagcagggtaacgtg tttagttgcagcgtgatgcatgaggcactgcacaatcactatacccagaagtcactgtca ctgtcacctggcaagtgaacccagctttcttgtacaaagtggtgataatcgaattccgat aatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgct ccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgt atggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttg tggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccact ggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccct attgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctg ttgggcactgacaattccgtggtgttgtcggggaagctgacgtcctttccatggctgctc gcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctc aatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtctt cgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcatcgggaattc ccgcggttcgctttaagaccaatgacttacaaggcagctgtagatcttagccacttttta aaagaaaaggggggactggaagggctaattcactcccaacgaagacaagatctgcttttt gcttgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaacta gggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcc cgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaa atctctagcagtagtagttcatgtcatcttattattcagtatttataacttgcaaagaaa tgaatatcagagagtgagaggaacttgtttattgcagcttataatggttacaaataaagc aatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttg tccaaactcatcaatgtatcttatcatgtctggctctagctatcccgcccctaactccgc ccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattt tttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgag gaggcttttttggaggcctagggacgtacccaattcgccctatagtgagtcgtattacgc gcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaact taatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcac cgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcgg cgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgc cctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttcc ccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacct cgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagac ggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaac tggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgat ttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaa aatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatt tgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataa atgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgccctt attcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaa gtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaac agcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcactttt aaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggt cgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcat cttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataac actgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttg cacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagcc ataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaa ctattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggag gcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgct gataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagat ggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaa cgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagac caagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatc taggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttc cactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctg cgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccg gatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagatacca aatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccg cctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcg tgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctga acggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatac ctacagcgtgagctatgagaaagcgccacgcttcccgaagagagaaaggcggacaggtat ccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcc tggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtga tgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttc ctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtg gataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgag cgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctcccc gcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggc agtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacac tttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacagga aacagctatgaccatgattacgccaagcgcgcaattaaccctcactaaagggaacaaaag ctggagctgcaagctt

[0303] Plasmid VB201230-1095jgq encoding C-terminal IgG2-Fc R0R2-ECD (SEQ ID NO: 22): aatgtagtcttatgcaatactcttgtagtcttgcaacatggtaacgatgagttagcaaca tgccttacaaggagagaaaaagcaccgtgcatgccgattggtggaagtaaggtggtacga tcgtgccttattaggaaggcaacagacgggtctgacatggattggacgaaccactgaatt gccgcattgcagagatattgtatttaagtgcctagctcgatacataaacgggtctctctg gttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcc tcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctgg taactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccg aacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggctt gctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttg actagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggaga attagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaatta aaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgtta gaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacagga tcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaagg atagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagt aagaccaccgcacagcaagcggccgctgatcttcagacctggaggaggagatatgaggga caattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagc acccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagc tttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgct gacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgag ggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctcca ggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttgggg ttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataa atctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaa ttacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatga acaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaa ttggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaat agtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtt tcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaagaagg tggagagagagacagagacagatccattcgattagtgaacggatctcgacggtatcgcta gcttttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacata atagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaatttt actagtgattatcggatcaactttgtatagaaaagttgtagttattaatagtaatcaatt acggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaat ggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgtt cccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaa actgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtc aatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcct acttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcag tacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccatt gacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaac aactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagc agagctggtttagtgaaccgtcagatccaagtttgtacaaaaaagcaggctgccaccatg gagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtgac gaggtggaggtgctggacccaaacgacccactgggccccctggacgggcaggacggcccc attcctaccctgaagggctactttctcaactttctcgagcccgtgaataacatcacaatc gtccagggccagacagctattctgcactgtaaggtggccgggaaccctccccctaacgtg cgttggctgaagaatgatgcccccgtggtgcaggaacctcggcggatcatcatcagaaag actgaatacggctctcgcctgcggattcaggatctggacaccaccgacacaggctattac cagtgtgtggccaccaatgggatgaagaccatcactgctaccggcgtgctgttcgtgcgc ctgggccctacccatagtcctaaccataatttccaggacgactaccacgaggatggattt tgccagccctacaggggcattgcctgcgcccgcttcatcggcaaccggacaatctacgtt gacagcctgcagatgcagggcgagatcgagaacaggatcactgccgcctttaccatgatt ggcacaagtacccacctgtcagatcagtgctcccagtttgccatcccatccttctgccac ttcgtgttcccactgtgtgacgccaggtcccggactccaaaaccccgcgagctgtgtaga gacgagtgcgaggtgctggagagcgacctttgcagacaggagtacaccattgccaggtcc aaccccctgatcctgatgaggctgcagctgcccaagtgcgaagccctgcccatgcccgag agtccagacgccgccaattgcatgagaatcggaatccctgccgagaggctgggcaggtac caccagtgttacaacggctccgggatggactataggggcaccgcctccaccacaaagagc ggccaccagtgtcagccatgggccctgcagcacccccacagccaccacctgagctccacc gattttcctgagctgggggggggccacgcctactgccgcaaccctggcggccagatggag gggccatggtgcttcacacagaataagaatgtgcgcatggagctgtgtgatgtgccctcc tgctcacctagggacagcagcaagatggggatcctgtacgagcgcaagtgctgcgtggag tgccccccatgcccagcccctcccgtggccggcccctccgtgttcctgtttcctccaaag ccaaaggacaccctgatgatcagcaggacacccgaggtgacctgcgtggtggtggacgtg agtcatgaggatcccgaggtgcagttcaactggtacgtggatggcatggaggtccataac gccaagacaaagccccgggaagagcagttcaactccacatttagagtggtctccgtcctg accgtggtgcatcaggactggctgaacgggaaagaatataaatgtaaggtgtccaacaag ggcctgcccgcccctattgaaaagaccatttctaagacaaaaggacagccccgcgagccc caggtgtataccctgcccccaagccgcgaggaaatgacaaaaaatcaggtgtcactgacc tgcctggtgaagggattttaccctagcgatatcgctgtcgagtgggaatctaacggccag ccagagaacaactacaagaccaccccccccatgctggatagcgacggctctttctttctg tactccaagctgacagttgacaagtctcgctggcagcagggcaatgtgttcagctgcagc gtgatgcatgaggccctgcataatcactatacacagaagagtctcagcctgtcccctggc aagtgaacccagctttcttgtacaaagtggtgataatcgaattccgataatcaacctctg gattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgcta tgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcatt ttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtc aggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcatt gccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcg gaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgac aattccgtggtgttgtcggggaagctgacgtcctttccatggctgctcgcctgtgttgcc acctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggac cttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccct cagacgagtcggatctccctttgggccgcctccccgcatcgggaattcccgcggttcgct ttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaagggg ggactggaagggctaattcactcccaacgaagacaagatctgctttttgcttgtactggg tctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactg cttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgt gactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagt agtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagag agtgagaggaacttgtttattgcagcttataatggttacaaataaagcaatagcatcaca aatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatc aatgtatcttatcatgtctggctctagctatcccgcccctaactccgcccatcccgcccc taactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatg cagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttg gaggcctagggacgtacccaattcgccctatagtgagtcgtattacgcgcgctcactggc cgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgc agcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttc ccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgc ggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgc tcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctct aaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaa acttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccc tttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacact caaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattg gttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgct tacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttc taaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataa tattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattccctttttt gcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgct gaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatc cttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgcta tgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacac tattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggc atgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaac ttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggg gatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgac gagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggc gaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagtt gcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctgga gccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcc cgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacag atcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactca tatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatc ctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtca gaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgc tgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagcta ccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttctt ctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctc gctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccggg ttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcg tgcacacagcccagcttggagcgaacgacctacaccgaact gaga tacctacagcgt gag ctatgagaaagcgccacgcttcccgaagagagaaaggcggacaggtatccggtaagcggc agggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttat agtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggg gggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgc tggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtatt accgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtca gtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccg attcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaac gcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccg gctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgac catgattacgccaagcgcgcaattaaccctcactaaagggaacaaaagctggagctgcaa gctt

[0304] Amino acid sequence of example human R0R2 amino acid sequence (SEQ ID NO: 23): MARGSALPRRPLLCI PAVWAAAALLLSVSRTSGEVEVLDPNDPLGPLDGQDGPI PTLKGYFLNFLEPVNNITI VQGQTAILHCKVAGNPPPNVRWLKNDAPVVQEPRRI I IRKTEYGSRLRIQDLDTTDTGYYQCVATNGMKTITA TGVLFVRLGPTHSPNHNFQDDYHEDGFCQPYRGIACARFIGNRTIYVDSLQMQGEIENRITAAFTMIGTSTHL SDQCSQFAI PSFCHFVFPLCDARSRAPKPRELCRDECEVLESDLCRQEYTIARSNPLILMRLQLPKCEALPMP ESPDAANCMRIGI PAERLGRYHQCYNGSGMDYRGTASTTKSGHQCQPWALQHPHSHHLSSTDFPELGGGHAYC RNPGGQMEGPWCFTQNKNVRMELCDVPSCSPRDSSKMGILYILVPSIAI PLVIACLFFLVCMCRNKQKASAST PQRRQLMASPSQDMEMPLINQHKQAKLKEISLSAVRFMEELGEDRFGKVYKGHLFGPAPGEQTQAVAIKTLKD KAEGPLREEFRHEAMLRARLQHPNVVCLLGVVTKDQPLSMI FSYCSHGDLHEFLVMRSPHSDVGSTDDDRTVK SALEPPDFVHLVAQIAAGMEYLSSHHVVHKDLATRNVLVYDKLNVKISDLGLFREVYAADYYKLLGNSLLPIR WMAPEAIMYGKFSIDSDIWSYGVVLWEVFSYGLQPYCGYSNQDVVEMIRNRQVLPCPDDCPAWVYALMIECWN EFPSRRPRFKDIHSRLRAWGNLSNYNSSAQTSGASNTTQTSSLSTSPVSNVSNARYVGPKQKAPPFPQPQFI P MKGQIRPMVPPPQLYI PVNGYQPVPAYGAYLPNFYPVQI PMQMAPQQVPPQMVPKPSSHHSGSGSTSTGYVTT APSNTSMADRAALLSEGADDTQNAPEDGAQSTVQEAEEEEEGSVPETELLGDCDTLQVDEAQVQLEA

[0305] Nucleotide sequence of example human cDNA encoding example human R0R2 (SEQ ID NO: 24): ggacgcatcgtagaaaggggtggtggcgcccgaccccgcgccccggcccgaagctctgagggcttcccggccc ccactgcctgcggcatggcccggggctcggcgctcccgcggcggccgctgctgtgcatcccggccgtctgggc ggccgccgcgcttctgctctcagtgtcccggacttcaggtgaagtggaggttctggatccgaacgacccttta ggaccccttgatgggcaggacggcccgattccaactctgaaaggttactttctgaattttctggagccagtaa acaatatcaccattgtccaaggccagacggcaattctgcactgcaaggtggcaggaaacccaccccctaacgt gcggtggctaaagaatgatgccccggtggtgcaggagccgcggcggatcatcatccggaagacagaatatggt tcacgactgcgaatccaggacctggacacgacagacactggctactaccagtgcgtggccaccaacgggatga agaccattaccgccactggcgtcctgtttgtgcggctgggtccaacgcacagcccaaatcataactttcagga tgattaccacgaggatgggttctgccagccttaccggggaattgcctgtgcacgcttcattggcaaccggacc atttatgtggactcgcttcagatgcagggggagattgaaaaccgaatcacagcggccttcaccatgatcggca cgtctacgcacctgtcggaccagtgctcacagttcgccatcccatccttctgccacttcgtgtttcctctgtg cgacgcgcgctcccgggcacccaagccgcgtgagctgtgccgcgacgagtgcgaggtgctggagagcgacctg tgccgccaggagtacaccatcgcccgctccaacccgctcatcctcatgcggcttcagctgcccaagtgtgagg cgctgcccatgcctgagagccccgacgctgccaactgcatgcgcattggcatcccagccgagaggctgggccg ctaccatcagtgctataacggctcaggcatggattacagaggaacggcaagcaccaccaagtcaggccaccag tgccagccgtgggccctgcagcacccccacagccaccacctgtccagcacagacttccctgagcttggagggg ggcacgcctactgccggaaccccggaggccagatggagggcccctggtgctttacgcagaataaaaacgtacg catggaactgtgtgacgtaccctcgtgtagtccccgagacagcagcaagatggggattctgtacatcttggtc cccagcatcgcaattccactggtcatcgcttgccttttcttcttggtttgcatgtgccggaataagcagaagg catctgcgtccacaccgcagcggcgacagctgatggcctcgcccagccaagacatggaaatgcccctcattaa ccagcacaaacaggccaaactcaaagagatcagcctgtctgcggtgaggttcatggaggagctgggagaggac cggtttgggaaagtctacaaaggtcacctgttcggccctgccccgggggagcagacccaggctgtggccatca aaacgctgaaggacaaagcggaggggcccctgcgggaggagttccggcatgaggctatgctgcgagcacggct gcaacaccccaacgtcgtctgcctgctgggcgtggtgaccaaggaccagcccctgagcatgatcttcagctac tgttcgcacggcgacctccacgaattcctggtcatgcgctcgccgcactcggacgtgggcagcaccgatgatg accgcacggtgaagtccgccctggagccccccgacttcgtgcaccttgtggcacagatcgcggcggggatgga gtacctatccagccaccacgtggttcacaaggacctggccacccgcaatgtgctagtgtacgacaagctgaac gtgaagatctcagacttgggcctcttccgagaggtgtatgccgccgattactacaagctgctggggaactcgc tgctgcctatccgctggatggccccagaggccatcatgtacggcaagttctccatcgactcagacatctggtc ctacggtgtggtcctgtgggaggtcttcagctacggcctgcagccctactgcgggtattccaaccaggatgtg gtggagatgatccggaaccggcaggtgctgccttgccccgatgactgtcccgcctgggtgtatgccctcatga tcgagtgctggaacgagttccccagccggcggccccgcttcaaggacatccacagccggctccgagcctgggg caacctttccaactacaacagctcggcgcagacctcgggggccagcaacaccacgcagaccagctccctgagc accagcccagtgagcaatgtgagcaacgcccgctacgtggggcccaagcagaaggccccgcccttcccacagc cccagttcatccccatgaagggccagatcagacccatggtgcccccgccgcagctctacatccccgtcaacgg ctaccagccggtgccggcctatggggcctacctgcccaacttctacccggtgcagatcccaatgcagatggcc ccgcagcaggtgcctcctcagatggtccccaagcccagctcacaccacagtggcagtggctccaccagcacag gctacgtcaccacggccccctccaacacatccatggcagacagggcagccctgctctcagagggcgctgatga cacacagaacgccccagaagatggggcccagagcaccgtgcaggaagcagaggaggaggaggaaggctctgtc ccagagactgagctgctgggggactgtgacactctgcaggtggacgaggcccaagtccagctggaagcttgag tggcaccagggcccagggttcggggatagaagccccgccgagaccccacagggacctcagtcacctttgagaa gacaccatactcagcaatcacaagagcccgccggccagtgggcttgtttgcagactgggtgaggtggagccct gctcctctctgtcctctgacacagctgccctgcctaggagcacccaagccaggcagggggtctggcagcacgg cgtcctggggagcaggacacatggtcatccccagggctgtatacattgattctggtggtagactggtagtgag cagcaaatgcctttcaagaaaataggtggcagcttcactccatgtcatatatggagtgaatatttcaaaacgt tgggaataagggcctgcaaaaggca

[0306] Amino acid sequence of extracellular portion of example human R0R2 protein (SEQ ID NO: 25):

[0307] MARGSALPRRPLLCI PAVWAAAALLLSVSRTSGEVEVLDPNDPLGPLDGQDGPI PTLKGYFLNFLE PVNNITI VQGQTAI LHCKVAGNPPPNVRWLKNDAPVVQE PRRI I IRKTEYGSRLRIQDLDTTDTGYYQCVATNGMKTITA TGVLFVRLGPTHS PNHNFQDDYHEDGFCQPYRGIACARFI GNRTIYVDSLQMQGE IENRITAAFTMI GTSTHL SDQCSQFAI PS FCHFVFPLCDARSRAPKPRELCRDECEVLESDLCRQEYTIARSNPLI LMRLQLPKCEALPMP ES PDAANCMRI GI PAERLGRYHQCYNGSGMDYRGTASTTKSGHQCQPWALQHPHSHHLSSTDFPELGGGHAYC RNPGGQMEGPWCFTQNKNVRMELCDVPSCS PRDSSKMGI LY

[0308] Nucleotide sequence encoding extracellular portion of example human R0R2 protein (SEQ ID NO: 26):

[0309] ATGGCCCGGGGCTCGGCGCTCCCGCGGCGGCCGCTGCTGTGCATCCCGGCCGTCTGGGCGGCCGCCGCGCTTC TGCTCTCAGTGTCCCGGACTTCAGGTGAAGTGGAGGTTCTGGATCCGAACGACCCTTTAGGACCCCTTGATGG GCAGGACGGCCCGATTCCAACTCTGAAAGGTTACTTTCTGAATTTTCTGGAGCCAGTAAACAATATCACCATT GT C CAAGGC CAGAC GGCAAT T CT GCACT GCAAGGT GGCAGGAAAC C CAC C C C CT AAC GT GC GGT GGCT AAAGA AT GAT GCCCCGGTGGT GCAGGAGC C GC GGC GGAT CAT CAT C C GGAAGACAGAAT AT GGT T CAC GACT GC GAAT C CAGGAC CT GGACAC GACAGACACT GGCT ACT AC CAGT GC GT GGC CAC CAAC GGGAT GAAGAC CAT TAG C GC C ACTGGCGTCCTGTTTGTGCGGCTGGGTC CAAC GCACAGC C CAAAT CAT AACT T T CAGGAT GAT TAG CAC GAGG ATGGGTTCTGCCAGCCTTACCGGGGAATTGCCTGTGCACGCTTCATTGGCAACCGGACCATTTATGTGGACTC GGT T CAGAT GCAGGGGGAGAT T GAAAAC C GAAT CACAGC GGC CT T CAC CAT GAT C GGCAC GT CT AC GCAC CT G TCGGACCAGTGCTCACAGTTCGCCATCCCATCCTTCTGCCACTTCGTGTTTCCTCTGTGCGACGCGCGCTCCC GGGCAC C CAAGC C GC GT GAGCT GT GC C GC GAG GAGT GC GAGGT GGT GGAGAGC GAG CT GT GC C GC CAGGAGT A CACCATCGCCCGCTCCAACCCGCTCATCCTCATGCGGCTTCAGCTGCCCAAGTGTGAGGCGCTGCCCATGCCT GAGAGCCCCGACGCTGCCAACTGCATGCGCATTGGCATCCCAGCCGAGAGGCTGGGCCGCTACCATCAGTGCT AT AAC GGCT CAGGCAT GGAT T ACAGAGGAAC GGCAAGCAC CAC CAAGT CAGGC CAC CAGT GC CAGC C GT GGGC CCTGCAGCACCCCCACAGCCACCACCTGTCCAGCACAGACTTCCCTGAGCTTGGAGGGGGGCACGCCTACTGC C GGAAC C C C GGAGGC CAGAT GGAGGGC CCCTGGTGCTT TAG GCAGAAT AAAAAC GT AC GGAT GGAACT GT GT G AC GT AC C CT C GT GT AGT C C C C GAGACAGCAGCAAGAT GGGGAT T CT GT AC

[0310] Amino acid sequence of CRD of example human R0R2 protein (SEQ ID NO: 27):

[0311] CQPYRGIACARFI GNRTIYVDSLQMQGE IENRITAAFTMI GTSTHLSDQCSQFAI PS FCHFVFPLCDARSRAP KPRELCRDECEVLESDLCRQEYTIARSNPLI LMRLQLPKCEALPMPES PDAAN

[0312] Nucleotide sequence encoding CRD of example human R0R2 protein (SEQ ID NO: 28):

[0313] CTGCCAGCCTTACCGGGGAATTGCCTGTGCACGCTTCATTGGCAACCGGACCATTTATGTGGACTCGCTTCAG AT GCAGGGGGAGAT T GAAAAC C GAAT CACAGC GGC CT T CAC CAT GAT C GGCAC GT CT AC GCAC CT GT C GGAC C AGTGCTCACAGTTCGCCATCCCATCCTTCTGCCACTTCGTGTTTCCTCTGTGCGACGCGCGCTCCCGGGCACC CAAGC C GC GT GAGCT GT GC C GC GAC GAGT GC GAGGT GCT GGAGAGC GAC CT GT GC C GC CAGGAGT ACAC CAT C GCCCGCTCCAACCCGCTCATCCTCATGCGGCTTCAGCTGCCCAAGTGTGAGGCGCTGCCCATGCCTGAGAGCC CCGACGCTGCCAAC

[0314] Example mouse R0R2 protein (SEQ ID NO: 29):

[0315] MARGWVRPSRVPLCARAVWTAAALLLWTPWTAGEVEDSEAIDTLGQPDGPDS PLPTLKGY FLNFLEPVNNITIVQGQTAILHCKVAGNPPPNVRWLKNDAPVVQEPRRVI IRKTEYGSRL RIQDLDTTDTGYYQCVATNGLKTITATGVLYVRLGPTHSPNHNFQDDDQEDGFCQPYRGI ACARFIGNRTIYVDSLQMQGEIENRITAAFTMIGTSTQLSDQCSQFAI PSFCHFVFPLCD ARSRAPKPRELCRDECEVLENDLCRQEYTIARSNPLILMRLQLPKCEALPMPESPDAANC MRIGI PAERLGRYHQCYNGSGADYRGMASTTKSGHQCQPWALQHPHSHRLSSTEFPELGG GHAYCRNPGGQVEGPWCFTQNKNVRVELCDVPPCSPRDGSKMGILYILVPSIAI PLVIAC LFFLVCMCRNKQKASASTPQRRQLMASPSQDMEMPLISQHKQAKLKEISLSTVRFMEELG EDRFGKVYKGHLFGPAPGEPTQAVAIKTLKDKAEGPLREEFRQEAMLRARLQHPNIVCLL GVVTKDQPLSMI FSYCSHGDLHEFLVMRSPHSDVGSTDDDRTVKSALEPPDFVHVVAQIA AGMEFLSSHHVVHKDLATRNVLVYDKLNVRISDLGLFREVYSADYYKLMGNSLLPIRWMS PEAVMYGKFSIDSDIWSYGVVLWEVFSYGLQPYCGYSNQDVVEMIRSRQVLPCPDDCPAW VYALMIECWNEFPSRRPRFKDIHSRLRSWGNLSNYNSSAQTSGASNTTQTSSLSTSPVSN VSNARYMAPKQKAQPFPQPQFI PMKGQIRPLVPPAQLYI PVNGYQPVPAYGAYLPNFYPV QI PMQMAPQQVPPQMVPKPSSHHSGSGSTSTGYVTTAPSNTSVADRAALLSEGTEDAQNI AEDVAQSPVQEAEEEEEGSVPETELLGDNDTLQVTEAAHVQLEA

[0316] Nucleotide sequence of example mouse cDNA encoding example mouse R0R2 protein (SEQ ID NO: 30): atggctcggggctgggtgcggccgagccgtgtgcctctgtgcgcccgggccgtctggacg gctgcggcgctcctgctctggacaccctggacggcaggtgaagtggaagattcggaggca atcgacaccttgggacaacctgatggaccggacagcccacttcccactctgaaaggctac tttctgaattttctggagccagtcaacaatatcaccattgttcagggccagacggcaatc ctgcactgcaaggtggcgggaaacccacctcccaatgtgcggtggctgaagaatgatgcc ccggttgtgcaagagccacgaagggtcgtcatccggaagacagaatacggctcccggctg cggatccaagacctggacacaacagacacaggctactaccagtgtgtggctaccaacggg ctgaagaccatcactgccactggggttctatatgtgcggctcggtccgacgcacagcccg aaccacaattttcaggatgacgatcaggaagatggcttctgccagccgtaccgagggatc gcttgtgcgcgcttcattgggaaccggactatttatgtggactccctccagatgcagggg gaga ttgaaaaccgaatcacagctgcctt caeca tgatcggcacctccacgcaactgtca gaccagtgttcacagtttgccatcccatccttctgccacttcgtcttccctctgtgcgac gcatgctcccgggcgcccaagcctcgcgaactgtgccgggatgaatgtgaggtgctggag aacgacctgtgccgccaggagtacaccatcgcccgctccaacccgctcatcctcatgcgg ctccagctgcccaagtgcgaagcgctgcccatgcccgagagcccggatgctgcgaactgc atgcgcatcgggatccccgcggagaggctgggtcgctaccaccagtgctacaacggctcc ggcgccgattacagggggatggccagtaccaccaagtcaggccaccagtgtcagccttgg gctctgcagcacccccacagccatcgcctatccagcacggaattccctgagctgggagga ggccatgcctactgccggaaccccgggggccagatggaaggcccgtggtgctttacgcag aataaaaacgtacgcgtggaactgtgtgacgtacccccgtgtagtccccgatatggcagc aagatggggattctgtacatcctggtccccagcattgctatccccctggtcatcgcttgc ctgttcttcctcgtctgcatgtgccgcaacaaacagaaggcttcggcctccaccccacag cgccggcagctgatggcctctcccagccaggacatggagatgccactcatcagccagcac aaacaggccaaactcaaagagatcagcttgtccacagtgaggttcatggaggagctcggg gaggaccggtttggcaaggtctacaaaggccacctgtt egggee tgccccaggagaacca acccaggccgtggccatcaagacgctgaaagacaaggctgaggggcccctgcgggaggag ttccggcaagaggcgatgctccgggcccgactgcagcaccccaacatcgtctgtc tecta ggcgtcgtgaccaaggaccaacccttgagcatgatcttcagctactgttcccatggcgac cttcatgaattcctggtcatgcgctcgccgcactccgatgtgggcagcaccgatgacgac cgcacagtgaagtcagccctggagcccccggacttcgtgcacgtggtggcgcagatcgct gcggggatggagttcctgtccagccaccacgtgtgccataaggacctggccacacgcaat gtgctggtgtacgacaagctgaacgtgaggatctcagacttgggcctcttccgtgaggta tactccgcagattactacaaactcatgggcaattcactgctgcccatccgctggatgtcc cccgaggccgtcatgtatggaaagttctccatcgactctgacatctggtcctacggtgtg gtcctctgggaggtctttagctacggcctgcagccctactgtgggtactccaaccaggac gtggtggagatgatccggagccggcaggtgctgccctgcccggatgactgccccgcctgg gtctatgccctcatgattgaatgctggaatgagttcccaagccggaggccccgctttaag gacatccacagccggctccggtcctggggcaacctatccaactataatagttccgcgcag acctcaggagccagcaacaccacacagaccagctccctgagcaccagccccgtaagcaat gtgagcaatgcccgctatatggcccccaagcagaaggcccagcccttcccacagcctcag ttcatccccatgaagggtcagatcagacccttggtgccccccgcacagctgtacatcccg gtgaacggctatcagccggtaccggcatacggggcctacctgcccaacttctacccagtc cagatccccatgcagatggccccacagcaggtgccccctcagatggtccccaagccgagc tcacaccacagtggcagcggctccaccagcactggctacgtcaccacggcgccctccaat acatctgtggcggacagggcggccctactctctgagggcaccgaggatgtacagaacatc gcggaagacgtggcccagagccctgtgcaggaagcagaggaggaggaggaggggtctgtc cctgagactgaactcctgggagacaatgacacgctccaggtgaccgaggcggctcatgtc cagcttgaagcctga

[0317] Amino acid sequence of GPI linker (SEQ ID NO: 31):

[0318] SSSTTTTTTTTLLLLLLLLLLLLLL

[0319] Nucleotide sequence encoding GPI linker (SEQ ID NO: 32):

[0320] AGCAGCAGCACCACCACCACCACCACCACCACCCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGC TG

[0321] Amino acid sequence of enterokinase cleavage tag (SEQ ID NO: 33):

[0322] KDDDD

[0323] Nucleotide sequence encoding enterokinase cleavage tag (SEQ ID NO: 34):

[0324] AAAGAT GAT GAT GAT

[0325] Alternative nucleotide sequence encoding enterokinase cleavage tag (SEQ ID NO: 35):

[0326] AGCAGCAGCACCACCACCACCACCACCACCACCCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGC T GAAAGAT GAT GAT GAT GAACAAAAACT CAT CT CAGAAGAGGAT CT GAATAT GCATACCGGT CAT CAT CACCA TCACCATTGA

[0327] Amino acid sequence of tobacco etch virus protease cleavage tag (SEQ ID NO: 36):

[0328] GQFYLNE

[0329] Nucleotide sequence encoding tobacco etch virus protease cleavage tag (SEQ ID NO: 37):

[0330] GGC CAGT T T T AT CT GAAC GAA

[0331] Alternative nucleotide sequence encoding tobacco etch virus protease cleavage tag (SEQ

[0332] ID NO: 38):

[0333] AGCAGCAGCACCACCACCACCACCACCACCACCCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGC T GGGC CAGT T T TAT CT GAAC GAAGAACAAAAACT CAT CT CAGAAGAGGAT CT GAATAT GCAT AC C GGT CAT CA T C AC CAT C AC CAT T GA

[0334] Amino acid sequence of CRD of R0R1 (SEQ ID NO: 39):

[0335] CQPYRGIACARFI GNRTVYMESLHMQGE IENQITAAFTMI GTSSHLSDKCSQFAI PSLCHYAFPYCDETSSVP KPRDLCRDECE I LENVLCQTEYI FARSNPMI LMRLKLPNCEDLPQPES PEAANC

[0336] Nucleotide sequence of CRD of R0R1 (SEQ ID NO: 40):

[0337] T GT CAGC CAT ACAGAGGGAT T GCAT GT GCAAGAT T TAT T GGCAAC C GCAC C GT CT AT AT GGAGT CT T T GCACA TGCAAGGGGAAATAGAAAATCAGATCACAGCTGCCTTCACTATGATTGGCACTTCCAGTCACTTATCTGATAA GTGTTCTCAGTTCGCCATTCCTTCCCTGTGCCACTATGCCTTCCCGTACTGCGATGAAACTTCATCCGTCCCA AAGCCCCGTGACTTGTGTCGC GAT GAAT GT GAAAT C C T GGAGAAT GTCCTGTGT C AAAC AGAGT AC AT T T T T G CAAGATCAAATCCCATGATTCTGATGAGGCTGAAACTGCCAAACTGTGAAGATCTCCCCCAGCCAGAGAGCCC AGAAGCT GCGAACT GT

[0338] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described aspects of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology, cell biology, immunology or related fields are intended to be within the scope of the following claims.

Claims

CLAIMS1. A receptor tyrosine kinase-like orphan receptor 2 (R0R2) inhibitor which is a fusion protein comprising or consisting of a receptor tyrosine kinase-like orphan receptor extracellular domain (ROR-ECD) and an immunoglobulin G2 fragment crystallisable domain (IgG2-Fc).

2. The ROR2 inhibitor of claim 1, wherein the ROR-ECD is an ROR2-ECD.

3. The ROR2 inhibitor of claim 2, wherein:(a) (i) the ROR2-ECD comprises or consists of the amino acid sequence of SEQ ID NO: 1 or a functional variant or fragment thereof having at least about 80% identity thereto; and / or(ii) the ROR2-ECD is encoded by the nucleotide sequence of SEQ ID NO: 2 or a functional variant thereof having at least about 80% identity thereto; and / or(b) the ROR2 inhibitor is capable of inhibiting canonical WNT signalling.

4. The ROR2 inhibitor of claim 1, wherein the ROR-ECD is an ROR1-ECD.

5. The ROR2 inhibitor of claim 4, wherein:(i) the ROR1-ECD comprises or consists of the amino acid sequence of SEQ ID NO: 3 or a functional variant or fragment thereof having at least about 80% identity thereto; and / or(ii) the ROR1-ECD is encoded by the nucleotide sequence of SEQ ID NO: 4 or a functional variant thereof having at least about 80% identity thereto.

6. The ROR2 inhibitor of any one of the preceding claims, wherein:(i) the IgG2-Fc domain comprises or consists of the amino acid sequence of SEQ ID NO: 7 or a functional variant or fragment thereof having at least about 80% identity thereto; and / or(ii) the IgG2-Fc domain is encoded by the nucleotide sequence of SEQ ID NO: 8 or a functional variant thereof having at least about 80% sequence identity thereto.

7. The R0R2 inhibitor of any one of the preceding claims, wherein the IgG2-Fc domain is N-terminal to the ROR-ECD.

8. The R0R2 inhibitor of claim 7 having the structure: Nter-IgG2-Fc-ROR2-ECD- Cter.

9. The R0R2 inhibitor of claim 8, wherein:(i) the fusion protein comprises or consists of the amino acid sequence of SEQ ID NO: 15 or a functional variant or fragment thereof having at least about 80% identity thereto; and / or(ii) the fusion protein is encoded by the nucleotide sequence of SEQ ID NO: 16 or a functional variant thereof having at least about 80% identity thereto.

10. The ROR2 inhibitor of any one of claims 1-6, wherein the IgG2-Fc domain is C- terminal to the ROR-ECD.

11. The ROR2 inhibitor of claim 10 which has the structure: Nter-ROR2-ECD-IgG2- Fc-Cter.

12. The ROR2 inhibitor of claim 11, wherein:(i) the fusion protein comprises or consists of the amino acid sequence of comprises or consists of the amino acid sequence of SEQ ID NO: 11 or a functional variant thereof having at least about 80% identity thereto; and / or(ii) the fusion protein is encoded by the nucleotide sequence of SEQ ID NO: 12 or a functional variant thereof having at least about 80% identity thereto.

13. The ROR2 inhibitor of any one of the preceding claims which is capable of stimulating chondrogenesis and / or upregulating the expression of Aggrecan and / or growth / differentiation factor 5 (GDF5).

14. The ROR2 inhibitor of any one of the preceding claims which is capable of inhibiting yes-associated protein (YAP) signalling and / or inhibiting the expression of cysteine-rich angiogenic inducer 61 (CYR61).

15. An isolated nucleic acid encoding an R0R2 inhibitor as defined in any one of the preceding claims.

16. The isolated nucleic acid of claim 15, which comprises or consists of the nucleotide sequence of SEQ ID NO: 16 or a functional variant thereof of having at least 80% sequence identity thereto, or SEQ ID NO: 12 or a functional variant thereof having at least 80% sequence identity thereto.

17. A vector comprising the nucleic acid of claim 15 or 16.

18. The vector of claim 17, wherein the vector is a viral vector optionally selected from the group consisting of a lentiviral vector, a retroviral vector, and adeno-associated viral (AAV) vector.

19. The vector of claim 17 or 18, wherein the vector comprises or consists of the nucleotide sequence of SEQ ID NO: 18 or a functional variant thereof of having at least 80% sequence identity thereto, or SEQ ID NO: 22 or a functional variant thereof of having at least 80% sequence identity thereto.

20. An isolated cell comprising the nucleic acid of claim 15 or 16 and / or the vector of any one of claims 17-19.

21. A pharmaceutical composition comprising the ROR2 inhibitor of any one of claims 1-14, the nucleic acid of claim 15, the vector of any one of claims 17-19, and / or the cell of 20, together with together with a pharmaceutically acceptable carrier or excipient.

22. An ROR2 inhibitor as defined in any one of claims 1-14, a nucleic acid as defined in claim 15 or 16, a vector as defined in any one of claims 17-19, a cell as defined in claim 20, or a pharmaceutical composition as defined in claim 21, for use as a medicament.

23. A method of treating osteoarthritis comprising administering to an individual in need thereof an ROR2 inhibitor as defined in any one of claims 1-14, a nucleic acid as defined in claim 15 or 16, a vector as defined in any one of claims 17-19, a cell as defined in claim 20, or a pharmaceutical composition as defined in claim 21.

24. A method of treating osteoarthritis-associated pain comprising administering to an individual in need thereof an R0R2 inhibitor as defined in any one of claims 1-14, a nucleic acid as defined in claim 15 or 16, a vector as defined in any one of claims 17-19, a cell as defined in claim 20, or a pharmaceutical composition as defined in claim 21.

25. A method of stimulating cartilage production comprising administering to an individual in need thereof an R0R2 inhibitor as defined in any one of claims 1-14, a nucleic acid as defined in claim 15 or 16, a vector as defined in any one of claims 17-19, a cell as defined in claim 20, or a pharmaceutical composition as defined in claim 21.

Images