WO2014029752A1

WO2014029752A1 - Anti lrp6 antibodies

Info

Publication number: WO2014029752A1
Application number: PCT/EP2013/067271
Authority: WO
Inventors: Jeremy Griggs; Alan Peter Lewis; Trevor Anthony Kenneth Wattam
Original assignee: Glaxo Group Limited
Priority date: 2012-08-22
Filing date: 2013-08-20
Publication date: 2014-02-27
Also published as: EP2888279A1

Abstract

The present disclosure concerns antigen binding polypeptides and fragments thereof which specifically bind Low-density lipoprotein receptor-related protein 6 (LRP6) particularly human LRP6 (hl_RP6) and which inhibit the binding of Wnt ligands to the LRP6 receptor. Further disclosed are pharmaceutical compositions, screening and medical treatment methods.

Description

ANTI LRP6 ANTIBODIES

Field of the invention

The present invention relates to antigen binding polypeptides that specifically bind Low-density lipoprotein receptor-related protein 6 (LRP6) and in particular human LRP6.

The present invention also concerns methods of treating diseases or disorders with said antigen binding polypeptides, pharmaceutical compositions comprising said antigen binding polypeptides and methods of manufacture. Other aspects of the present invention will be apparent from the description below.

Background of the invention Mammalian wnt signalling is associated with numerous developmental processes and plays a key role in tissue homeostasis. Wnt signalling occurs through a large family of 19 wnt ligands, a family of frizzled receptors and co-receptors including LRP5 and LRP6. Wnts are expressed in a wide variety of tissues. Regulation of wnt signalling is complex and involves a variety of secreted antagonists including SFRP family members, WIF, dickkopf (DKK) family members and sclerostin (SOST).

The wnt ligand co-receptor LRP6 belongs to a subfamily of low-density-lipoprotein receptor related proteins. The extracellular domain of LRP6 is composed of structural motifs in common with other LRP family members: YWTD β-propellers, EGF-like domains, and LDLR type A (LA) domains. The particular arrangement of the extracellular motifs is unique to LRP6 and the closest related LRP family member, LRP5.

LRP6 acts functionally as a Wnt ligand co-receptor with the frizzled family of Wnt receptors. Evidence supports the existence of two independent Wnt ligand binding sites on LRP6, one located within β - propellers 1 and 2 (E1 E2), and one within β -propellers 3 and 4 (E3E4). Different wnt ligands bind preferentially to one site or another, and can be grouped into two classes based on this binding behaviour (e.g. E1 E2-binding wnts (wntl class) 1 , 2, 2b, 6, 8a, 9a, 9b, 10b and E3E4-binding (wnt3 class) wnts 3, 3a).

Abnormal wnt signalling is implicated in a variety of pathologies. For example, constitutive activation of wnt signalling that results from abnormal nuclear accumulation of beta-catenin (e.g. as a result of mutations in the tumour suppressor gene APC) is a common feature of colorectal cancers. Aberrant wnt signalling is also associated with other cancers including breast, gastric, liver, lung and melanoma. Abnormal wnt signalling is also implicated in diseases including, but not limited to cardiac disease, diabetes, fibrosis, neuronal degenerative diseases, non-oncogenic proliferative diseases, obesity, osteoporosis, osteoarthritis, polycystic kidney disease, schizophrenia, and vascular disease.

Abberent wnt signalling may therefore represent a target for therapeutic intervention in a wide variety of diseases.

Brief Description of Figures Figure 1 Identification of mAb of interest using the Hek293 Topflash Assay Ultrafiltered conditioned media from hybridomas expressing putative novel anti-LRP6 mAbs were tested in the Hek293 Topflash Assay for the capacity to modulate Wnt-mediated signalling. Conditioned medium sample "F2" antagonised Wnt 1- and Wnt 3a-mediated signalling in both the "non-potentiated" (A) and the "potentiated" (B) assay formats. The control LRP6-binding protein H027 achieved antagonism of Wnt 1 -mediated signalling and reciprocal potentiation of Wnt 3a-mediated signalling in both assay formats. Conversely, the control LRP6-binding protein k020 antagonised Wnt 3a-mediated signalling with reciprocal potentiation of Wnt 1-mediated signalling.

Figure 2 Characterisation of purified S360103E04 in the Hek293 Topflash Assay Purified monoclonal antibody S360103E04, corresponding to hybridoma sample "F2" was tested in the Hek293 Topflash assay. S360103E04 achieved antagonism of Wnt 1-mediated (A) and Wnt 3a- mediated (B) signalling in a dose-dependent manner.

Figure 3 Characterisation of chimeric antibody DMS10031 The murine antibody S360103E04 was chimerised onto a human lgG1 framework, designated DMS10031 and tested in the Hek293 Topflash assay to confirm retention of Wnt antagonism activity. Treatment with DMS10031 resulted in a dose- dependent antagonism of Wnt 1-mediated (A) and Wnt 3a-mediated (B) signalling.

Figure 4 Simultaneous antagonism of Wnt 1- and Wnt 3a mediated signalling by DMS10031 in the Hek293 Topflash assay The capacity of DMS10031 to antagonise Wnt signalling in the simultaneous presence of Wnt 3a and Wnt 1 was tested in the Hek293 Topflash assay. Cells were co- transfected with Topflash and Wnt 1 expression vectors and recombinant Wnt 3a was added. Treatment with DMS10031 resulted in a dose-dependent antagonism of total Wnt signal in the absence of any evidence of signal potentiation.

Figure 5 Orthologue cross-reactivity of S360103E04 / DMS10031 Binding of DMS10031 to cells transfected to express cynomolgous or murine LRP6 was assessed by flow cytometry. (A) DMS10031 (open) bound to cells expressing cynomolgous LRP6 compared to an isotype control (grey). (B) In contrast, DMS10031 (open) failed to show differential binding to cells expressing murine LRP6 compared to and isotype control (grey). Figure 6 LRP6 specificity of S360103E04 Lack of binding of S360103E04 to recombinant human LRP5 was assessed by ELISA. No binding was observed over the concentration range tested. The integrity of the assay was confirmed by the concentration-dependent binding shown by the control antibody.

Figure 7 LRP6 specificity of S360103E04 Lack of binding of S360103E04 to human LRP5 was assessed by flow cytometry. Hek293 cells were transfected to overexpress human LRP5, LRP6 or were mock transfected. Binding of the LRP5/LRP6-reactive positive control antibody was confirmed for both LRP5- and LRP6-expressing cells and negligible binding to mock-transfected cells was observed. In contrast, S360103E04 bound only to LRP6-transfected cells and no substantive binding to LRP5-expressing or mock-transfected cells was observed.

Figure 8 Unique binding epitope of S360103E04 Competition ELISAs were used to assess whether the unique Wnt-antagonising activities of S360103E04 are the result of binding to a unique epitope on LRP6. S360103E04 binding to recombinant human LRP6 was tested in the presence of absence of a fixed concentration of prototypical anti-LRP6 mAbs and binding proteins (i.e. those achieving reciprocal antagonism and potentiation of alternate Wnt ligand classes). The presence of a molar excess of Arca135 (A), YW31 1.31.57 (B), YW31 1.61.62 (C), YW210.09 (D), H027 or k020 (E) failed to alter the concentration-dependent binding of S360103E04 to LRP6. These data confirm that S360103E04 binds an epitope distinct from the epitope(s) bound by either of the test agents.

Figure 9 Activity of S360103E04, DMS10031 and DMS10064 in the HT1080 Topflash Assay

The HT1080 Topflash assay was used to characterise the capacity of S360103E04, DMS10031 and DMS10064 to modulate signalling mediated by Wnt 1 , Wnt 3a or Wnt7b. Treatment with S360103E04, DMS10031 or DMS10064 achieved a dose-dependent antagonism of wnt3a-mediated signalling (A) but showed no evidence of reciprocal potentiation of wntl-mediated signalling (B). In contrast, YW31 1.31.57 antagonised wnt3a-mediated signalling and potentiated wntl-mediated signalling. Consistent with expectations, YW210.09 antagonised wntl-mediated signalling and potentiated wnt3a-mediated signalling. Of note, neither S360103E04, DMS10031 or DMS10064 substantially modulated Wnt7b-mediated signalling, compared with, for example, YW31 1.31.57, which strongly potentiated Wnt7b-mediated signalling (C).

Figure 10 Biacore analysis of DMS10031 and DMS10064 binding. Representative sensorgrams from which surface plasmon resonance binding kinetics data for DMS10031 (A) and DMS10064 (B) were obtained. Test mAbs were captured on the protein A derivitised sensorchip followed by injection of LRP6 ECD. Association and dissociation times were 240s and 600s, respectively. mAbs were captured on protein A at 2 concentrations and tested with four concentrations of LRP6-ECD at 18nM, 36nM, 71 nM and 143nM. These were double referenced and are represented by traces 1-4, respectively. Both antibody variants showed similar LRP6 binding profiles. Figure 11 Humanisation of S360103E04 / DMS10031 The Hek293 Topflash assay was used to confirm the retention of Wnt modulation activity in the humanised variant DMS10064 compared with the parental murine mAb S360103E04 and the chimeric human lgG1 mAb DMS10031. Treatment with DMS10064 resulted in a dose-dependent antagonism of Wnt 1- (A) and Wnt 3- (B) -mediated signalling in a dose-dependent manner that was comparable to that achieved by S360103E04 and DMS10064.

Summary of the Invention

The present invention provides antigen binding polypeptides which specifically bind to Low-density lipoprotein receptor-related protein 6 (LRP6). In particular the present invention provides antigen binding polypeptides which specifically bind to human Low-density lipoprotein receptor-related protein 6 (LRP6) and which comprise a paired VH/VL wherein the paired VH/VL antagonises or does not potentiate the signalling of one Wnt ligand and does not cause activation or potentiation of signalling of a second Wnt ligand.

In one aspect of the invention as herein described there is provided an antigen binding polypeptide which specifically binds to Low-density lipoprotein receptor-related protein 6 (LRP6), and which comprises a paired VH/VL wherein the paired VH/VL antagonises or does not potentiate signalling mediated by a Wnt 3 class ligand and does not activate or potentiate signalling mediated by a Wnt 1 class ligand.

In another aspect of the invention as herein described there is provided an antigen binding polypeptide which specifically binds to Low-density lipoprotein receptor-related protein 6 (LRP6), and which comprises a paired VH/VL wherein the paired VH/VL antagonises or does not potentiate signalling mediated by a Wnt 1 class ligand and does not activate or potentiate signalling mediated by a Wnt 3 class ligand. In another aspect of the invention as herein described there is provided an antigen binding polypeptide which specifically binds to Low-density lipoprotein receptor-related protein 6 (LRP6), and which comprises a paired VH/VL wherein the paired VH/VL antagonises or does not potentiate signalling by at least 2 Wnt ligands. The antigen binding polypeptides of the present invention are related to, or derived from a murine monoclonal antibody S360103E04 (also referred to as E04). The E04 murine heavy chain variable region amino acid sequence is provided as SEQ ID NO. 7 and the E04 murine light chain variable region amino acid sequence is provided as SEQ ID NO. 8. The heavy chain variable regions (VH) of the present invention may comprise the following CDRs or variants of these CDR's (as defined by Kabat (Kabat et al; Sequences of proteins of Immunological Interest NIH, 1987)): CDRH1 is provided as SEQ ID NO. 1

CDRH2 is provided as SEQ ID NO. 2

CDRH3 is provided as SEQ ID NO. 3

The light chain variable regions (VL) of the present invention may comprise the following CDRs or variants of these CDR's (as defined by Kabat (Kabat et al; Sequences of proteins of Immunological Interest NIH, 1987)):

CDRL1 is provided as SEQ ID NO. 4

CDRL2 is provided as SEQ ID NO. 5

CDRL3 is provided as SEQ ID NO. 6

The invention also provides a polynucleotide sequence encoding a heavy chain variable region of any of the antigen-binding proteins described herein, and a polynucleotide encoding a light chain variable region of any of the antigen-binding proteins described herein.

The invention also provides a polynucleotide sequence encoding a heavy chain of any of the antigen- binding proteins described herein, and a polynucleotide encoding a light chain of any of the antigen- binding proteins described herein.

Such polynucleotides represent the coding sequence which corresponds to the equivalent polypeptide sequences, however it will be understood that such polynucleotide sequences could be cloned into an expression vector along with a start codon, an appropriate signal sequence and a stop codon.

The invention also provides a recombinant transformed or transfected host cell comprising one or more polynucleotides encoding a heavy chain and or a light chain of any of the antigen-binding proteins described herein.

The invention further provides a method for the production of any of the antigen-binding proteins described herein which method comprises the step of culturing a host cell comprising a first and second vector, said first vector comprising a polynucleotide encoding a heavy chain of any of the antigen-binding proteins described herein and said second vector comprising a polynucleotide encoding a light chain of any of the antigen-binding proteins described herein, in a suitable culture media, for example serum- free culture media.

The invention further provides a pharmaceutical composition comprising an antigen-binding protein as described herein and a pharmaceutically acceptable carrier. In a further aspect there is provided method of treatment or prophylaxis of a disease or disorder responsive to inhibiting or blocking LRP6 such as the modulation of the interaction between LRP6 and its Wnt 1 class or Wnt 3 class or Wnt7 class ligands which method comprises the step of

administering to said patient a therapeutically effective amount of the antigen binding polypeptide or fragment thereof the present invention.

In one aspect of the invention there is provided a therapeutic approach to the treatment of inflammatory disorders or diseases inflammatory bowel disease, ulcerative colitis or bone related disorders or diseases such as osteoporosis, osteoarthritis, bone fractures and bone lesions or cancer including but not limited to gastrointestinal cancer, pancreatic cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer (including renal cell carcinoma), prostate cancer small cell lung carcinoma, non small cell lung carcinoma, germ cell or embryonal or teratocarcinoma tumors or vascular related disorders such as but not limited to Norrie disease, osteoporosis- pseudoglioma syndrome (OPPG), familial exudative vitreoretinopathy (FEVR), retinopathy of prematurity (Rap), diabetic retinopathy, age-related macular degeneration, retinopathy of prematurity, Coats' disease and Coats' like reaction, and retinal artery or vein occlusion, and myocardial-related conditions, such as myocardial infarction and ischemic heart disease. In particular it is an object of the present invention to provide antigen binding polypeptides or fragments thereof, especially antibodies that specifically bind LRP6 (e.g. human LRP6) and antagonise the interaction between LRP6 and its ligands such as Wnt 3 class and/or Wnt 1 class and/or Wnt 7 class ligands in the treatment of diseases and disorders responsive to modulation of that interaction.

Antigen-binding proteins including monoclonal antibodies have been identified that can antagonize wnt ligand binding to LRP6. However, all inhibitory monoclonal antibodies and antibody fragments identified to date are capable only of antagonizing either E1 E2 or E3E4 ligands, not both simultaneously. Moreover, bivalent antigen binding proteins capable of antagonising signalling by one wnt ligand class typically cause potentiation of signalling mediated by alternate class ligands. Thus, inhibition of canonical wnt signalling would not be achieved by such agents if ligand(s) from both classes were present.

This finding is of particular significance when considering the utility of anti-LRP6 mAbs and other LRP6 modulatory proteins in the therapeutic modulation of Wnt-mediated signalling. For example, ligands from more than one Wnt ligand family may be associated with disease progression and may be present and contributing to disease progression in vivo. The use of an anti-LRP6 mAb or other modulatory protein exhibiting reciprocal antagonism and potentiation of signalling from different ligand classes may lead to an exacerbation of the disease phenotype resulting from the potentiation component of its effects. In contrast, the use of a dual-antagonist mAb would not be expected to effect Wnt signalling potentiation, irrespective of the class(es) or identity(ies) of Wnt ligands present.

In one aspect of the invention there is provided an antigen binding polypeptide or fragment thereof according to the invention as herein described for use in treating a disease or disorder selected from but not limited to inflammatory disorders or diseases inflammatory bowel disease, ulcerative colitis or bone related disorders or diseases such as osteoporosis, osteoarthritis, bone fractures and bone lesions or cancer including but not limited to gastrointestinal cancer, pancreatic cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer (including renal cell carcinoma), prostate cancer small cell lung carcinoma, non small cell lung carcinoma, germ cell or embryonal or teratocarcinoma tumors or vascular related disorders such as but not limited to Norrie disease, osteoporosis-pseudoglioma syndrome (OPPG), familial exudative vitreoretinopathy (FEVR), retinopathy of prematurity (Rap), diabetic retinopathy, age-related macular degeneration, retinopathy of prematurity, Coats' disease and Coats' like reaction, and retinal artery or vein occlusion, and myocardial-related conditions, such as myocardial infarction and ischemic heart disease.

In one aspect of the invention there is provided the use of antigen binding polypeptide or fragment thereof according to the invention as herein described for the manufacture of a medicament for treating a disease or disorder selected from but not limited to inflammatory disorders or diseases inflammatory bowel disease, ulcerative colitis or bone related disorders or diseases such as osteoporosis, osteoarthritis, bone fractures and bone lesions or cancer including but not limited to gastrointestinal cancer, pancreatic cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer (including renal cell carcinoma), prostate cancer small cell lung carcinoma, non small cell lung carcinoma , germ cell or embryonal or teratocarcinoma tumors or vascular related disorders such as but not limited to Norrie disease, osteoporosis-pseudoglioma syndrome (OPPG), familial exudative vitreoretinopathy (FEVR), retinopathy of prematurity (Rap), diabetic retinopathy, age-related macular degeneration, retinopathy of prematurity, Coats' disease and Coats' like reaction, and retinal artery or vein occlusion, and myocardial-related conditions, such as myocardial infarction and ischemic heart disease.

In another aspect of the present invention there is provided a method of treating a human patient afflicted with an inflammatory disorders or diseases inflammatory bowel disease, ulcerative colitis or bone related disorders or diseases such as osteoporosis, osteoarthritis, bone fractures and bone lesions or cancer including but not limited to gastrointestinal cancer, pancreatic cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer (including renal cell carcinoma), prostate cancer small cell lung carcinoma, non small cell lung carcinoma, germ cell or embryonal or teratocarcinoma tumors or vascular related disorders such as but not limited to Norrie disease, osteoporosis-pseudoglioma syndrome (OPPG), familial exudative vitreoretinopathy (FEVR), retinopathy of prematurity (Rap), diabetic retinopathy, age-related macular degeneration, retinopathy of prematurity, Coats' disease and Coats' like reaction, and retinal artery or vein occlusion, and myocardial-related conditions, such as myocardial infarction and ischemic heart disease.

In one aspect the disease or disorder occurs in mammals, in one specific aspect the disease or disorder occurs in humans.

Detailed Description of the Invention

The present invention provides antigen binding polypeptides which specifically bind to Low-density lipoprotein receptor-related protein 6 (LRP6). In particular the present invention provides antigen binding polypeptides which specifically bind to human Low-density lipoprotein receptor-related protein 6 (LRP6) and which comprise a paired VH/VL wherein the paired VH/VL antagonises or does not potentiate the signalling of one Wnt ligand and does not activate or potentiate signalling of a second Wnt ligand.

In one aspect of the invention as herein described there is provided an antigen binding polypeptide which specifically binds to Low-density lipoprotein receptor-related protein 6 (LRP6), and which comprises a paired VH/VL wherein the paired VH/VL antagonises or does not potentiate signalling mediated by a Wnt 3 class ligand and does not activate or potentiate signalling mediated by a Wnt 1 class ligand signalling.

In another aspect of the invention as herein described there is provided an antigen binding polypeptide which specifically binds to Low-density lipoprotein receptor-related protein 6 (LRP6), and which comprises a paired VH/VL antagonises or does not potentiate signalling mediated by a Wnt 1 class ligand and does not activate or potentiate signalling mediated by a Wnt 3 class ligand signalling.

In one aspect of the invention as herein described there is provided an antigen binding which specifically binds to Low-density lipoprotein receptor-related protein 6 (LRP6), and which comprises a paired VH/VL wherein the paired VH/VL antagonises or does not potentiate signalling mediated by a Wnt 1 class ligand and does not activate or potentiate signalling mediated by a Wnt 7 class ligand signalling. In one aspect of the invention as herein described there is provided an antigen binding which specifically binds to Low-density lipoprotein receptor-related protein 6 (LRP6), and which comprises a paired VH/VL wherein the paired VH/VL antagonises or does not potentiate signalling mediated by a Wnt 7 class ligand and does not activate or potentiate signalling mediated by a Wnt 1 class ligand signalling. In one aspect of the invention as herein described there is provided an antigen binding which specifically binds to Low-density lipoprotein receptor-related protein 6 (LRP6), and which comprises a paired VH/VL wherein the paired VH/VL antagonises or does not potentiate signalling mediated by a Wnt 7 class ligand and does not activate or potentiate signalling mediated by a Wnt 3 class ligand signalling.

In one aspect of the invention as herein described there is provided an antigen binding which specifically binds to Low-density lipoprotein receptor-related protein 6 (LRP6), and which comprises a paired VH/VL wherein the paired VH/VL antagonises or does not potentiate signalling mediated by a Wnt 3 class ligand and does not activate or potentiate signalling mediated by a Wnt 7 class ligand signalling.

In another aspect of the invention as herein described there is provided an antigen binding polypeptide which specifically binds to Low-density lipoprotein receptor-related protein 6 (LRP6), and which comprises a paired VH/VL wherein the paired VH/VL antagonises or does not potentiate signalling of at least 2 Wnt ligands.

In one aspect the Wnt ligands are from the same Wnt ligand class. In a further aspect the Wnt ligands are from different Wnt ligand classes. For example in one aspect one of the Wnt ligands is from Wnt 1 Class and the other is from Wnt 7 Class, or for example one of the Wnt ligands is from Wnt 1 Class and the other is from Wnt 7 Class, or for example one of the Wnt ligands is from Wnt 3 Class and the other is from Wnt 7 Class.

In one aspect the paired VH/VL antagonises all three classes of Wnt ligand. A Wnt 1 class ligand is herein defined as comprising all Wnt ligands except for Wnt3, Wnt 3a, Wnt 4, Wnt7a, Wnt 7b and Wnt 10a

For example a Wnt 1 class ligand is more specifically defined as comprising Wnt 1 and/or Wnt2 and/or Wnt 2b and/or Wnt 6 and/or, Wnt 8a and/or Wnt 9a and/or Wnt 9b and/or Wnt 10b. A Wnt 3 class ligand is herein defined as comprising Wnt 3 and/or Wnt 3a

It has been proposed that an additional binding site exists, to which, for example, wnt4, Wnt7a, Wnt 7b, Wnt 10a can bind and activate signalling. Canonical wnt signaling is activated by a wnt ligand binding to and causing heterodimerisation of a member of the frizzled family and the coreceptor LRP5 or LRP6. This mediates phosphorylation of LRP5 or LRP6 and a sequence of signalling events, including the prevention of phosphorylation and degradation of cytosolic beta-catenin. Wnt-mediated signal transduction ultimately permits the translocation of beta-cetenin into the nucleus which leads to the activation of TCF target genes. A Wnt 7 class ligand is herein defined as comprising Wnt 4 and/or Wnt7a and/or Wnt 7b and/or Wnt 10a

In one aspect of the invention there is provided an antigen binding polypeptide as herein described wherein the antigen binding polypeptide comprises CDRH3 of SEQ ID NO.3 or a variant of SEQ ID NO. 3.

In a further aspect of the invention there is provided an antigen binding polypeptide as herein described wherein the antigen binding polypeptide further comprises one or more of: CDR H1 of SEQ. ID. NO: 1 , CDRH2: SEQ. ID. NO: 2: CDRL1 : SEQ. ID. NO: 4, CDRL2: SEQ. ID. NO: 5 and/or CDRL3: SEQ. ID. NO: 6 and or variants thereof.

In yet a further aspect the antigen binding polypeptide comprises CDR H3 of SEQ. ID. NO: 3:

CDRH2: SEQ. ID. NO: 2: CDR H1 of SEQ. ID. NO:1 : CDRL1 : SEQ. ID. NO: 4: CDRL2: SEQ. ID. NO: 5 and CDRL3: SEQ. ID. NO: 6.

The antigen binding polypeptides of the present invention are derived from the murine antibody having the variable regions as described in SEQ ID NO:7 and SEQ ID NO:8 or non-murine equivalents thereof, such as rat, human, chimeric or humanised variants thereof.

In another aspect the antigen binding polypeptides of the present invention are derived from the humanised antibody having the variable heavy chain sequences as described in SEQ ID NO:13 or SEQ ID NO:93 or SEQ ID NO:95 or SEQ ID NO:97 or SEQ ID NO:99, or SEQ ID NO: 101 , or SEQ ID NO: 103, or SEQ ID NO: 105 and/or the variable light chain sequences as described in SEQ ID NO: 14.

In one aspect of the invention there is provided an antigen binding polypeptide comprising an isolated heavy chain variable domain selected from any on the following: SEQ ID NO: 13 or SEQ ID NO:93 or SEQ ID NO:95 or SEQ ID NO:97 or SEQ ID NO:99, or SEQ ID NO: 101 , or SEQ ID NO:103, or SEQ ID NO: 105.

In another aspect of the invention there is provided an antigen binding polypeptide comprising an isolated light chain variable domain selected from any on the following: SEQ ID NO: 14.

In one aspect the antigen binding polypeptide of the present invention comprises a heavy chain variable region encoded by SEQ. ID. NO: 13 and a light chain variable region encoded by SEQ. ID. NO: 14

In one aspect the antigen binding polypeptide of the present invention comprises a heavy chain variable region encoded by SEQ. ID. NO: 105 and a light chain variable region encoded by SEQ. ID. NO: 14 In one aspect there is provided a polynucleotide encoding an isolated variable heavy chain said polynucleotide comprising SEQ ID NO:90 or SEQ ID NO:94 or SEQ ID NO:96 or SEQ ID NO:98 or SEQ ID NO: 100 or SEQ ID NO: 102, or SEQ ID NO: 104 , or SEQ ID NO: 106.

In one aspect there is provided a polynucleotide encoding an isolated variable light chain said polynucleotide comprising SEQ. ID. NO. 92.

In a further aspect there is provided a polynucleotide encoding an isolated variable heavy chain said polynucleotide comprising SEQ ID NO:90 or SEQ ID NO:94 or SEQ ID NO:96 or SEQ ID NO:98 or SEQ ID NO: 100 or SEQ ID NO: 102, or SEQ ID NO: 104 , or SEQ ID NO: 106 and a polynucleotide encoding an isolated variable light chain said polynucleotide comprising SEQ. ID. NO. 92.

In a further aspect the antigen binding polypeptide may comprise any one of the variable heavy chains as described herein in combination with any one of the light chains as described herein.

In one aspect the antigen binding polypeptide is an antibody or antigen binding fragment thereof comprising one or more CDR's according to the invention described herein, or one or both of the heavy or light chain variable domains according to the invention described herein. In one aspect the antigen binding polypeptide binds primate LRP6. In one such aspect the antigen binding polypeptide additionally binds non-human primate LRP6, for example cynomolgus macaque monkey LRP6.

The antigen binding polypeptides of the present invention may comprise heavy chain variable regions and light chain variable regions of the invention which may be formatted into the structure of a natural antibody or functional fragment or equivalent thereof. An antigen binding polypeptide of the invention may therefore comprise the VH regions of the invention formatted into a full length antibody, a (Fab')2 fragment, a Fab fragment, a bi-specific or biparatopic molecule or equivalent thereof (such as scFV, bi- tri- or tetra-bodies, Tandabs etc.), when paired with an appropriate light chain. The antibody may be an lgG1 , lgG2, lgG3, or lgG4; or IgM; IgA, IgE or IgD or a modified variant thereof. The constant domain of the antibody heavy chain may be selected accordingly. The light chain constant domain may be a kappa or lambda constant domain. The antigen binding polypeptide may also be a chimeric antibody of the type described in WO86/01533 which comprises an antigen binding region and a non- immunoglobulin region.

The constant region is selected according to the functionality required for example, an lgG1 may demonstrate lytic ability through binding to complement and/or will mediate ADCC (antibody dependent cell cytotoxicity).

In another aspect the antigen binding polypeptide is selected from the group consisting of a Fab, Fab', F(ab')2, Fv, diabody, triabody, tetrabody, miniantibody, and a minibody, In one aspect of the present invention the antigen binding polypeptide is a humanised or chimaeric antibody, in a further aspect the antibody is humanised.

In one aspect the antibody is a monoclonal antibody.

In one aspect the antigen binding polypeptide is bi-specific or biparatopic.

In one aspect of the present invention there is provided an antigen binding polypeptide comprising a heavy chain sequence as set forth in SEQ ID NO: 16 or SEQ ID NO: 18 or SEQ ID NO: 19 or SEQ ID NO: 20 or SEQ ID NO: 21 or SEQ ID NO: 22 or SEQ ID NO: 23 or SEQ ID NO: 24.

In one aspect of the present invention there is provided an antigen binding polypeptide comprising a light chain sequence as set forth in SEQ ID NO: 17.

In a further aspect of the invention there is provided an antigen binding polypeptide comprising a heavy chain sequence of SEQ ID NO: 24 and a light chain sequence as set forth in SEQ ID NO: 17

In a further aspect of the invention there is provided an antigen binding polypeptide comprising a heavy chain sequence of SEQ ID NO: 23 and a light chain sequence as set forth in SEQ ID NO: 17 In a further aspect of the invention there is provided an antigen binding polypeptide comprising a heavy chain sequence of SEQ ID NO: 22 and a light chain sequence as set forth in SEQ ID NO: 17

In a further aspect of the invention there is provided an antigen binding polypeptide comprising a heavy chain sequence of SEQ ID NO: 21 and a light chain sequence as set forth in SEQ ID NO: 17

In one aspect there is provided an antigen binding polypeptide which does not compete with an antigen binding polypeptide which comprises the CDR sequences of SEQ ID NO:s 36-41.

In one aspect there is provided an antigen binding polypeptide which does not compete with an antigen binding polypeptide which comprises the CDR sequences of SEQ ID NO:s 47-52.

In one aspect there is provided an antigen binding polypeptide which does not compete with an antigen binding polypeptide which comprises the CDR sequences of SEQ ID NO:s 59-64. In one aspect there is provided an antigen binding polypeptide which does not compete with an antigen binding polypeptide which comprises the CDR sequences of SEQ ID NO:s 71-76.

In one aspect there is provided an antigen binding polypeptide which does not compete with an antigen binding polypeptide which comprises a variable heavy chain sequence selected from SEQ ID NO: 42 or SEQ ID NO:53 or SEQ ID NO:65 or SEQ ID NO: 77. In one aspect there is provided an antigen binding polypeptide which does not compete with an antigen binding polypeptide which comprises a variable heavy chain sequence selected from SEQ ID NO: 42 and a variable light chain sequence selected from SEQ ID NO:43.

In one aspect there is provided an antigen binding polypeptide which does not compete with an antigen binding polypeptide which comprises a variable heavy chain sequence selected from SEQ ID NO: 53 and a variable light chain sequence selected from SEQ ID NO:54. In one aspect there is provided an antigen binding polypeptide which does not compete with an antigen binding polypeptide which comprises a variable heavy chain sequence selected from SEQ ID NO: 65 and a variable light chain sequence selected from SEQ ID NO:66.

In one aspect there is provided an antigen binding polypeptide which does not compete with an antigen binding polypeptide which comprises a variable heavy chain sequence selected from SEQ ID NO: 77 or SEQ ID NO:79 and a variable light chain sequence of SEQ ID NO:81.

In one aspect there is provided an antigen binding polypeptide which competes with an antigen binding polypeptide of the invention as herein described. In one such aspect there is therefore provided an antigen binding polypeptide which competes with an antigen binding polypeptide which comprises the CDR sequences according to SEQ ID NO: s 1-6. In a further aspect there is provided an antigen binding protein which competes with an antigen binding protein which comprises a variable heavy chain sequence selected from SEQ ID NO: 13 or SEQ ID NO:93 or SEQ ID NO:95 or SEQ ID NO:97 or SEQ ID NO:99, or SEQ ID NO:101 , or SEQ ID NO:103, or SEQ ID NO: 105 and the variable light chain sequence of SEQ ID NO: 14.

In one aspect there is provided an antigen binding polypeptide which binds to the same epitope as an antibody which has the variable heavy chain sequence of SEQ ID NO: 13or SEQ ID NO:93 or SEQ ID NO:95 or SEQ ID NO:97 or SEQ ID NO:99, or SEQ ID NO: 101 , or SEQ ID NO: 103, or SEQ ID NO: 105 and the variable light chain sequence of SEQ ID NO: 14.

The "same epitope" can be considered to have been bound if an antigen binding protein binds to the same or overlapping amino acid residues or sterically inhibits the binding of an antigen binding protein of the present invention. The epitope of a mAb is the region of its antigen to which the mAb binds. Two antibodies bind to the same or overlapping epitope if each competitively inhibits (blocks) binding of the other to the antigen. That is, a 1x, 5x, 10x, 20x or 100x excess of one antibody inhibits binding of the other by at least 50% but preferably 75%, 90% or even 99% as measured in a competitive binding assay compared to a control lacking the competing antibody (see, e.g., Junghans et al., Cancer Res. 50: 1495, 1990, which is incorporated herein by reference). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Also the same epitope may include "overlapping epitopes" eg if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. In one aspect the antigen binding polypeptide according to the invention described herein does not block binding of DKK1 and/or SOST to LRP6. For example the antigen binding polypeptide according to the invention described herein does not compete for binding to LRP6 with DKK1 and/or SOST.

In one aspect the antigen binding polypeptide according to the invention described herein blocks binding of DKK1 and/or SOST to LRP6. For example the antigen binding polypeptide according to the invention described herein competes for binding to LRP6 with DKK1 and/or SOST.

In another aspect the antigen binding polypeptide binds to human LRP6 with high affinity for example when measured by Biacore the antigen binding polypeptide binds to human LRP6 with an affinity of 1- 1000nM or 500nM or less or an affinity of 200nM or less or an affinity of 100nM or less or an affinity of 50 nM or less or an affinity of 500pM or less or an affinity of 400pM or less, or 300pM or less. In a further aspect the antigen binding polypeptide binds to human LRP6 when measured by Biacore of between about 50nM and about 200nM or between about 50nM and about 150nM. In one aspect of the present invention the antigen binding polypeptide binds LRP6 with an affinity of less than 100nM. In one such aspect, this is measured by Biacore, for example as set out in Example 8.

In another aspect the antigen binding polypeptide binds to human LRP6 and antagonises or neutralises the cellular signalling mediated by binding of Wnt 3 Class ligands or Wnt 1 Class ligands or Wnt 7 Class ligands. For example antagonises or neutralises the cellular signalling mediated by binding of Wnt 3a and/or Wnt 1 to the LRP6 receptor in a cell neutralisation assay wherein the antigen binding polypeptide has an IC50 in the nanomolar range for example of between about 1 nM and about 500nM, or between about 1 nM and about 100nM, or between about 10pM and 50nM, or between about 1 nM and about 50nM, or between about 1 nM and about 25nM, or between about 1 nM and about 15nM. For example the antigen binding polypeptide has an IC 50 of less than 25nM.

For example antagonises or neutralises the cellular signalling mediated by binding of Wnt 3a and/or Wnt 1 to the LRP6 receptor in a cell neutralisation assay wherein the antigen binding polypeptide has an IC50 in the picomolar range for example of between about 1 pM and about 500pM, or between about 1 pM and about 100pM, or between about 1 pM and 50pM, or between about 1 pM and about 25pM. For example the antigen binding polypeptide has an IC 50 of less than 1 1 pM.

In one such aspect, this is measured by a cellular gene reporter assay, for example as set out in Examples 2 and 7.

The antigen binding polypeptides, for example antibodies of the present invention may be produced by transfection of a host cell with an expression vector comprising the coding sequence for the antigen binding polypeptide of the invention. An expression vector or recombinant plasmid is produced by placing these coding sequences for the antigen binding polypeptide in operative association with conventional regulatory control sequences capable of controlling the replication and expression in, and/or secretion from, a host cell. Regulatory sequences include promoter sequences, e.g., CMV promoter, and signal sequences which can be derived from other known antibodies.

Similarly, a second expression vector can be produced having a DNA sequence which encodes a complementary antigen binding polypeptide light or heavy chain. In certain aspects this second expression vector is identical to the first except insofar as the coding sequences and selectable markers are concerned, so to ensure as far as possible that each polypeptide chain is functionally expressed. Alternatively, the heavy and light chain coding sequences for the antigen binding polypeptide may reside on a single vector.

A selected host cell is co-transfected by conventional techniques with both the first and second vectors (and simply transfected by a single vector) to create the transfected host cell of the invention comprising either the recombinant or synthetic light and heavy chains. The transfected cell is then cultured by conventional techniques to produce the engineered antigen binding polypeptide of the invention. The antigen binding polypeptide which includes the association of both the recombinant heavy chain and/or light chain is screened from culture by appropriate assay, such as ELISA or RIA. Similar conventional techniques may be employed to construct other antigen binding polypeptides.

Suitable vectors for the cloning and subcloning steps employed in the methods and construction of the compositions of this invention may be selected by one of skill in the art. For example, the conventional pUC series of cloning vectors may be used. One vector, pUC19, is commercially available from supply houses, such as Amersham (Buckinghamshire, United Kingdom) or Pharmacia (Uppsala, Sweden). Additionally, any vector which is capable of replicating readily, has an abundance of cloning sites and selectable genes (e.g., antibiotic resistance), and is easily manipulated may be used for cloning. Thus, the selection of the cloning vector is not a limiting factor in this invention.

The expression vectors may also be characterized by genes suitable for amplifying expression of the heterologous DNA sequences, e.g., the mammalian dihydrofolate reductase gene (DHFR). Other vector sequences include a poly A signal sequence, such as from bovine growth hormone (BGH) and the betaglobin promoter sequence (betaglopro). The expression vectors useful herein may be synthesized by techniques well known to those skilled in this art.

The components of such vectors, e.g. replicons, selection genes, enhancers, promoters, signal sequences and the like, may be obtained from commercial or natural sources or synthesized by known procedures for use in directing the expression and/or secretion of the product of the recombinant DNA in a selected host. Other appropriate expression vectors of which numerous types are known in the art for mammalian, bacterial, insect, yeast, and fungal expression may also be selected for this purpose. The present invention also encompasses a cell line transfected with a recombinant plasmid containing the coding sequences of the antigen binding polypeptides of the present invention. Host cells useful for the cloning and other manipulations of these cloning vectors are also conventional. However, cells from various strains of E. Coli may be used for replication of the cloning vectors and other steps in the construction of antigen binding polypeptides of this invention.

Suitable host cells or cell lines for the expression of the antigen binding polypeptides of the invention include mammalian cells such as NSO, Sp2/0, CHO (e.g. DG44), COS, HEK, a fibroblast cell (e.g., 3T3), and myeloma cells, for example it may be expressed in a CHO or a myeloma cell. Human cells may be used, thus enabling the molecule to be modified with human glycosylation patterns.

Alternatively, other eukaryotic cell lines may be employed. The selection of suitable mammalian host cells and methods for transformation, culture, amplification, screening and product production and purification are known in the art. See, e.g., Sambrook et al., cited above. Bacterial cells may prove useful as host cells suitable for the expression of the recombinant Fabs or other aspects of the present invention (see, e.g., Pluckthun, A., Immunol. Rev., 130: 151-188 (1992)). However, due to the tendency of proteins expressed in bacterial cells to be in an unfolded or improperly folded form or in a non-glycosylated form, any recombinant Fab produced in a bacterial cell would have to be screened for retention of antigen binding ability. If the molecule expressed by the bacterial cell was produced in a properly folded form, that bacterial cell would be a desirable host, or in alternative aspects the molecule may express in the bacterial host and then be subsequently refolded. For example, various strains of E. Coli used for expression are well-known as host cells in the field of biotechnology. Various strains of B. Subtilis, Streptomyces, other bacilli and the like may also be employed in this method.

Where desired, strains of yeast cells known to those skilled in the art are also available as host cells, as well as insect cells, e.g. Drosophila and Lepidoptera and viral expression systems. See, e.g. Miller et al., Genetic Engineering, 8:277-298, Plenum Press (1986) and references cited therein.

The general methods by which the vectors may be constructed, the transfection methods required to produce the host cells of the invention, and culture methods necessary to produce the antigen binding polypeptide of the invention from such host cell may all be conventional techniques. Typically, the culture method of the present invention is a serum-free culture method, usually by culturing cells serum-free in suspension. Likewise, once produced, the antigen binding polypeptides of the invention may be purified from the cell culture contents according to standard procedures of the art, including ammonium peroxide precipitation, affinity columns, column chromatography, gel electrophoresis and the like. Such techniques are within the skill of the art and do not limit this invention. For example, preparations of altered antibodies are described in WO 99/58679 and WO 96/16990.

Yet another method of expression of the antigen binding polypeptides may utilize expression in a transgenic animal, such as described in U. S. Patent No. 4,873,316. This relates to an expression system using the animals casein promoter which when transgenically incorporated into a mammal permits the female to produce the desired recombinant protein in its milk.

In a further aspect of the invention there is provided a method of producing an antibody of the invention which method comprises the step of culturing a host cell transformed or transfected with a vector encoding the light and/or heavy chain of the antibody of the invention and recovering the antibody thereby produced.

In accordance with the present invention there is provided a method of producing an anti-LRP6 antibody of the present invention which binds to and neutralises the activity of human LRP6 which method comprises the steps of;

providing a first vector encoding a heavy chain of the antibody;

providing a second vector encoding a light chain of the antibody;

transforming a mammalian host cell (e.g. CHO) with said first and second vectors;

culturing the host cell of step (c) under conditions conducive to the secretion of the antibody from said host cell into said culture media;

recovering the secreted antibody of step (d).

Once expressed by the desired method, the antibody is then examined for in vitro activity by use of an appropriate assay. Presently conventional ELISA assay formats are employed to assess qualitative and quantitative binding of the antibody to BCMA. Additionally, other in vitro assays may also be used to verify neutralizing efficacy prior to subsequent human clinical studies performed to evaluate the persistence of the antibody in the body despite the usual clearance mechanisms.

The dose and duration of treatment relates to the relative duration of the molecules of the present invention in the human circulation, and can be adjusted by one of skill in the art depending upon the condition being treated and the general health of the patient. It is envisaged that repeated dosing (e.g. once a week or once every two weeks or once every 3 weeks) over an extended time period (e.g. four to six months) maybe required to achieve maximal therapeutic efficacy..

In one aspect of the present invention there is provided a recombinant transformed, transfected or transduced host cell comprising at least one expression cassette, for example where the expression cassette comprises a polynucleotide encoding a heavy chain of an antigen binding polypeptide according to the invention described herein and further comprises a polynucleotide encoding a light chain of an antigen binding polypeptide according to the invention described herein or where there are two expression cassettes and the 1 st encodes the light chain and the second encodes the heavy chain. For example in one aspect the first expression cassette comprises a polynucleotide encoding a heavy chain of an antigen binding polypeptide comprising a constant region or antigen binding fragment thereof which is linked to a constant region according to the invention described herein and further comprises a second cassette comprising a polynucleotide encoding a light chain of an antigen binding polypeptide comprising a constant region or antigen binding fragment thereof which is linked to a constant region according to the invention described herein for example the first expression cassette comprises a polynucleotide encoding a heavy chain selected from SEQ. ID. NO:16, or SEQ. ID. NO: 18 or SEQ. ID. NO: 19 or SEQ. ID. NO: 20 or SEQ. ID. NO: 21 or SEQ. ID. NO: 22 or SEQ. ID. NO: 23 or SEQ. ID. NO: 24 and a second expression cassette comprising a polynucleotide encoding a light chain of SEQ. ID. NO: 17.

In another aspect of the invention there is provided a stably transformed host cell comprising a vector comprising one or more expression cassettes encoding a heavy chain and/or a light chain of the antibody comprising a constant region or antigen binding fragment thereof which is linked to a constant region as described herein. For example such host cells may comprise a first vector encoding the heavy chain and a second vector encoding the light chain, for example the first vector comprises a polynucleotide sequence selected from SEQ. ID. NO: 27 or SEQ. ID. NO: 29 or SEQ. ID. NO: 30 or SEQ. ID. NO: 31 or SEQ. ID. NO: 32 or SEQ. ID. NO: 33 or SEQ. ID. NO: 34 or SEQ. ID. NO: 35 and a second vector comprises a polynucleotide sequence of SEQ ID NO: 28. In one such example the first vector encodes a heavy chain selected from SEQ. ID. NO: 32 and a second vector encoding a light chain for example the light chain of SEQ ID NO: 28.

In another aspect of the present invention there is provided a host cell according to the invention described herein wherein the cell is eukaryotic, for example where the cell is mammalian. Examples of such cell lines include CHO or NS0.

In another aspect of the present invention there is provided a method for the production of an antibody comprising a constant region or antigen binding fragment thereof which is linked to a constant region according to the invention described herein which method comprises the step of culturing a host cell in a culture media, for example serum- free culture media.

In another aspect of the present invention there is provided a method according to the invention described herein wherein said antibody is further purified to at least 95% or greater (e.g. 98% or greater) with respect to said antibody containing serum- free culture media. In yet another aspect there is provided a pharmaceutical composition comprising an antigen binding polypeptide and a pharmaceutically acceptable carrier.

In another aspect of the present invention there is provided a kit-of-parts comprising the composition according to the invention described herein described together with instructions for use.

The mode of administration of the therapeutic agent of the invention may be any suitable route which delivers the agent to the host. The antigen binding polypeptides, and pharmaceutical compositions of the invention are particularly useful for parenteral administration, i.e., subcutaneously (s.c), intrathecally, intraperitoneally, intramuscularly (i.m.) or intravenously (i.v.). In one such aspect the antigen binding polypeptides of the present invention are administered intravenously or subcutaneously.

Therapeutic agents of the invention may be prepared as pharmaceutical compositions containing an effective amount of the antigen binding polypeptide of the invention as an active ingredient in a pharmaceutically acceptable carrier. In one aspect the prophylactic agent of the invention is an aqueous suspension or solution containing the antigen binding polypeptide in a form ready for injection. In one aspect the suspension or solution is buffered at physiological pH. In one aspect the compositions for parenteral administration will comprise a solution of the antigen binding polypeptide of the invention or a cocktail thereof dissolved in a pharmaceutically acceptable carrier. In one aspect the carrier is an aqueous carrier. A variety of aqueous carriers may be employed, e.g., 0.9% saline, 0.3% glycine, and the like. These solutions may be made sterile and generally free of particulate matter. These solutions may be sterilized by conventional, well known sterilization techniques (e.g., filtration). The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, etc. The concentration of the antigen binding polypeptide of the invention in such pharmaceutical formulation can vary widely, i.e., from less than about 0.5%, usually at or at least about 1 % to as much as about 15 or 20% by weight and will be selected primarily based on fluid volumes, viscosities, etc., according to the particular mode of administration selected.

Thus, a pharmaceutical composition of the invention for intramuscular injection could be prepared to contain about 1 mL sterile buffered water, and between about 1 ng to about 100 mg, e.g. about 50 ng to about 30 mg or about 5 mg to about 25 mg, of an antigen binding polypeptide, for example an antibody of the invention. Similarly, a pharmaceutical composition of the invention for intravenous infusion could be made up to contain about 250 ml of sterile Ringer's solution, and about 1 to about 30 or 5 mg to about 25 mg of an antigen binding polypeptide of the invention per ml of Ringer's solution. Actual methods for preparing parenterally administrable compositions are well known or will be apparent to those skilled in the art and are described in more detail in, for example, Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pennsylvania. For the preparation of intravenously administrable antigen binding polypeptide formulations of the invention see Lasmar U and Parkins D "The formulation of Biopharmaceutical products", Pharma.

Sci.Tech.today, page 129-137, Vol.3 (3rd April 2000); Wang, W "Instability, stabilisation and formulation of liquid protein pharmaceuticals", Int. J. Pharm 185 (1999) 129-188; Stability of Protein Pharmaceuticals Part A and B ed Ahern T.J., Manning M.C., New York, NY: Plenum Press (1992); Akers.M.J. "Excipient-Drug interactions in Parenteral Formulations", J. Pharm Sci 91 (2002) 2283- 2300; Imamura, K et al "Effects of types of sugar on stabilization of Protein in the dried state", J Pharm Sci 92 (2003) 266-274; Izutsu, Kkojima, S. "Excipient crystalinity and its protein-structure- stabilizing effect during freeze-drying", J Pharm. Pharmacol, 54 (2002) 1033-1039; Johnson, R, "Mannitol-sucrose mixtures-versatile formulations for protein peroxidise19g19n", J. Pharm. Sci, 91 (2002) 914-922; and Ha,E Wang W, Wang Y.j. "Peroxide formation in polysorbate 80 and protein stability", J. Pharm Sci, 91 , 2252-2264,(2002) the entire contents of which are incorporated herein by reference and to which the reader is specifically referred.

In one aspect the therapeutic agent of the invention, when in a pharmaceutical preparation, is present in unit dose forms. The appropriate therapeutically effective dose will be determined readily by those of skill in the art. Suitable doses may be calculated for patients according to their weight, for example suitable doses may be in the range of about 0.1 to about 20mg/kg, for example about 1 to about 20mg/kg, for example about 10 to about 20mg/kg or for example about 1 to about 15mg/kg, for example about 10 to about 15mg/kg. To effectively treat conditions such as Multiple myeloma, SLE or IPT in a human, suitable doses may be within the range of about 0.1 to about 1000 mg, for example about 0.1 to about 500mg, for example about 500mg, for example about 0.1 to about 100mg, or about 0.1 to about 80mg, or about 0.1 to about 60mg, or about 0.1 to about 40mg, or for example about 1 to about 100mg, or about 1 to about 50mg, of an antigen binding polypeptide of this invention, which may be administered parenterally, for example subcutaneously, intravenously or

intramuscularly. Such dose may, if necessary, be repeated at appropriate time intervals selected as appropriate by a physician.

The antigen binding polypeptides described herein can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional immunoglobulins and art-known peroxidise and reconstitution techniques can be employed.

In another aspect there is provided an antigen binding polypeptide according to the invention as herein described for use as a medicament.

In one aspect, the invention relates to a method of treating a human patient with a disease, the method comprising administering an antigen binding protein according to the invention.

The invention also relates to an antigen binding protein as disclosed herein for the treatment of disease in a human.

The invention also relates to use of an antigen binding protein as disclosed herein in the manufacture of a medicament for the treatment of disease, and an antigen binding protein as disclosed herein for use in treatment of disease.

For example in one aspect of the invention there is provided the use of the antigen binding polypeptide as described herein for use in the treatment or prophylaxis of diseases and disorders responsive to antagonising (such as inhibiting or blocking) of the interaction between a Wnt 1 Class ligand and/or a Wnt 3 class and/or a Wnt7 Class ligand and the LRP6 receptor.

In one aspect, the disease to be treated by the antigen binding protein of the invention is an inflammatory disorder or disease such as but not limited to inflammatory bowel disease or ulcerative colitis. In another aspect the bone related disease or disorder such as but not limited to osteoporosis, osteoarthritis, bone fractures or bone lesions.

In another aspect, the cancer related disease or disorder is selected from the group consisting of gastrointestinal cancer, pancreatic cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer (including renal cell carcinoma), prostate cancer, small cell lung carcinoma, non small cell lung carcinoma, germ cell or embryonal or teratocarcinoma tumors. In one aspect of the present invention the disease is Non small cell lung carcinoma.

In one aspect of the present invention the disease is Breast Cancer

In one aspect of the present invention the disease is Prostate Cancer

In one aspect of the present invention the disease is Pancreatic Cancer In one aspect of the present invention the disease is Germ cell teratocarcinoma. Definitions

The term "antigen binding polypeptide" as used herein refers to antibodies and fragments thereof and other protein constructs, such as domains, which are capable of binding to LRP6.

The term "antibody" is used herein in the broadest sense to refer to molecules with an

immunoglobulin-like domain (for example IgG, IgM, IgA, IgD or IgE) and includes monoclonal, recombinant, polyclonal, chimeric, human, humanised, multispecific antibodies, including bispecific antibodies, and heteroconjugate antibodies; a single variable domain (e.g., VH, VHH, VL, domain antibody (dAb™)), antigen binding antibody fragments, Fab, F(ab')2, Fv, disulphide linked Fv, single chain Fv, disulphide-linked scFv, diabodies, TANDABS™, etc. and modified versions of any of the foregoing (for a summary of alternative "antibody" formats see Holliger and Hudson, Nature

Biotechnology, 2005, Vol 23, No. 9, 1 126-1 136). Alternative antibody formats include alternative scaffolds in which the one or more CDRs of any molecules in accordance with the disclosure can be arranged onto a suitable non-immunoglobulin protein scaffold or skeleton, such as an affibody, a SpA scaffold, an LDL receptor class A domain, an avimer (see, e.g., U.S. Patent Application Publication Nos. 2005/0053973, 2005/0089932, 2005/0164301 ) or an EGF domain.

The term "domain" refers to a folded protein structure which retains its tertiary structure independent of the rest of the protein. Generally domains are responsible for discrete functional properties of proteins and in many cases may be added, removed or transferred to other proteins without loss of function of the remainder of the protein and/or of the domain. The term "single variable domain" refers to a folded polypeptide domain comprising sequences characteristic of antibody variable domains. It therefore includes complete antibody variable domains such as VH, VHH and VL and modified antibody variable domains, for example, in which one or more loops have been replaced by sequences which are not characteristic of antibody variable domains, or antibody variable domains which have been truncated or comprise N- or C-terminal extensions, as well as fragments of variable domains which retain at least the binding activity and specificity of the full-length domain. A single variable domain is capable of binding an antigen or epitope independently of a different variable region or domain. A "domain antibody" or "dAb™" may be considered the same as a "single variable domain". A single variable domain may be a human single variable domain, but also includes single variable domains from other species such as rodent (for example, as disclosed in WO 00/29004), nurse shark and Camelid VHH dAbsTM. Camelid VHH are immunoglobulin single variable domains that are derived from species including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies naturally devoid of light chains. Such VHH domains may be humanised according to standard techniques available in the art, and such domains are considered to be "single variable domains". As used herein VH includes camelid VHH domains.

"Antigen binding site" or "paratope" refers to a site on an antigen binding polypeptide, which is capable of specifically binding to an antigen, this may be a single variable domain, or it may be paired VH/VL domains as can be found on a standard antibody. Single-chain Fv (ScFv) domains can also provide antigen-binding sites.

The term "potentiate" as used herein refers to an increase in Wnt-mediated signalling The term "antagonise" as used herein refers to a decrease in the level of signalling. For example in one embodiment the signalling is decreased by at least 10% or by at least 25% or by at least 40% or by at least 50% or by at least 60% or by at least 75% or by

at least 90%. In one aspect there is 100% (complete blocking) of the signal. "Signalling" can be measured by a cellular gene reporter assay, for example as set out in Examples 2 and 7.

The term "multi-specific" antigen binding polypeptide refers to antigen binding polypeptides which comprise at least two different antigen binding sites. Each of these antigen-binding sites will be capable of binding to a different epitope, which may be present on the same antigen or different antigens. The multi-specific antigen binding polypeptide will have specificity for more than one antigen, for example two antigens, or for three antigens, or for four antigens.

Examples of multi-specific antigen binding polypeptides include those that consist of, or consist essentially of, an Fc region of an antibody, or a part thereof, linked at each end, directly or indirectly (for example, via a linker sequence) to a binding domain. Such an antigen binding polypeptide may comprise two binding domains separated by an Fc region, or part thereof. By separated is meant that the binding domains are not directly linked to one another, and may be located at opposite ends (C and N terminus) of an Fc region, or any other scaffold region.

The antigen binding polypeptide may comprise two scaffold regions each bound to two binding domains, for example at the N and C termini of each scaffold region, either directly or indirectly via a linker. Each binding domain may bind to a different antigen.

As used herein, the term mAbdAb refers to a monoclonal antibody linked to a further binding domain, in particular a single variable domain such as a domain antibody. A mAbdAb has at least two antigen binding sites, at least one of which is from a domain antibody, and at least one is from a paired VH/VL domain. mAbdAbs are described in WO2009/068649.

A "dAb™ conjugate" refers to a composition comprising a dAb to which a drug is chemically conjugated by means of a covalent or noncovalent linkage. Preferably, the dAb and the drug are covalently bonded. Such covalent linkage could be through a peptide bond or other means such as via a modified side chain. The noncovalent bonding may be direct (e.g., electrostatic interaction, hydrophobic interaction) or indirect (e.g., through noncovalent binding of complementary binding partners (e.g., biotin and avidin), wherein one partner is covalently bonded to drug and the complementary binding partner is covalently bonded to the dAb™). When complementary binding partners are employed, one of the binding partners can be covalently bonded to the drug directly or through a suitable linker moiety, and the complementary binding partner can be covalently bonded to the dAb™ directly or through a suitable linker moiety.

As used herein, "dAb™ fusion" refers to a fusion protein that comprises a dAb™ and a polypeptide drug (which could be a dAb™ or mAb). The dAb™ and the polypeptide drug are present as discrete parts (moieties) of a single continuous polypeptide chain.

In one aspect, antigen binding polypeptides of the present disclosure show cross-reactivity between human LRP6 and LRP6 from another species, such as cyno LRP6. In an aspect, the antigen binding polypeptides of the invention specifically bind human and cyno LRP6. This is particularly useful, since drug development typically requires testing of lead drug candidates in other tox species before the drug is tested in humans. The provision of a drug that can bind human and cyno species allows one to test results in these system and make side-by-side comparisons of data using the same drug. This avoids the complication of needing to find a drug that works against a cyno LRP6 and a separate drug that works against human LRP6 and also avoids the need to compare results in humans and cyno using non-identical drugs. Cross reactivity between other species used in disease models such as dog, is also envisaged.

Optionally, the binding affinity of the antigen binding polypeptide for at least cyno LRP6 and the binding affinity for human LRP6 differ by no more than a factor of 2, 5, 10, 50 or 100. Affinity is the strength of binding of one molecule, e.g. an antigen binding polypeptide of the invention, to another, e.g. its target antigen, at a single binding site. The binding affinity of an antigen binding polypeptide to its target may be determined by equilibrium methods (e.g. enzyme-linked

immunoabsorbent assay (ELISA) or radioimmunoassay (RIA)), or kinetics (e.g. BIACORE™ analysis). For example, the Biacore™ methods described in Example 8 may be used to measure binding affinity.

Avidity is the sum total of the strength of binding of two molecules to one another at multiple sites, e.g. taking into account the valency of the interaction. In an aspect, the equilibrium dissociation constant (KD) of the antigen binding polypeptide LRP6 interaction is 100 nM or less, 10 nM or less, 2 nM or less or 1 nM or less. Alternatively the KD may be between 5 and 10 nM; or between 1 and 2 nM. The KD may be between 1 pM and 500 pM; or between 500 pM and 1 nM. A skilled person will appreciate that the smaller the KD numerical value, the stronger the binding. The reciprocal of KD (i.e. 1/KD) is the equilibrium association constant (KA) having units M-1. A skilled person will appreciate that the larger the KA numerical value, the stronger the binding.

The dissociation rate constant (kd) or "off-rate" describes the stability of the antigen binding polypeptide-LRP6 complex, i.e. the fraction of complexes that decay per second. For example, a kd of 0.01 s-1 equates to 1 % of the complexes decaying per second.

The term "neutralises" as used throughout the present specification means that the Wnt ligand- mediated cellular signalling resulting from interactions of said ligand(s) with LRP6 is reduced in the presence of an antigen binding polypeptide as described herein in comparison to the Wnt-mediated signalling activity in the absence of the antigen binding polypeptide, in vitro or in vivo. Neutralisation may be due to one or more of blocking Wnt ligands binding to LRP6, preventing Wnt ligands from activatingl_RP6, down regulating LRP6, or affecting effector functionality. For example, the methods described in Examples 1 , 2, 3, 7, 9 may be used to assess the neautralising capability of an LRP6- binding protein. "CDRs" are defined as the complementarity determining region amino acid immunoglobulin heavy and light chains. There are three heavy chain and three light chain CDRs (or CDR regions) in the variable portion of an immunoglobulin. Thus, "CDRs" as used herein refers to all three heavy chain CDRs, all three light chain CDRs, all heavy and light chain CDRs, or at least two CDRs.

Throughout this specification, amino acid residues in variable domain sequences and full length antibody sequences are numbered according to the Kabat numbering convention. Similarly, the terms "CDR", "CDRL1 ", "CDRL2", "CDRL3", "CDRH1 ", "CDRH2", "CDRH3" used in the Examples follow the Kabat numbering convention. For further information, see Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987). It will be apparent to those skilled in the art that there are alternative numbering conventions for amino acid residues in variable domain sequences and full length antibody sequences. There are also alternative numbering conventions for CDR sequences, for example those set out in Chothia et al. (1989) Nature 342: 877-883. The structure and protein folding of the antibody may mean that other residues are considered part of the CDR sequence and would be understood to be so by a skilled person.

Other numbering conventions for CDR sequences available to a skilled person include "AbM" (University of Bath) and "contact" (University College London) methods. The minimum overlapping region using at least two of the Kabat, Chothia, AbM and contact methods can be determined to provide the "minimum binding unit". The minimum binding unit may be a sub-portion of a CDR.

Table 1 below represents one definition using each numbering convention for each CDR or binding unit. The Kabat numbering scheme is used in Table 1 to number the variable domain amino acid sequence. It should be noted that some of the CDR definitions may vary depending on the individual publication used.

Table 1

CDRs or minimum binding units may be modified by at least one amino acid substitution, deletion or addition, wherein the modified antigen binding polypeptide substantially retains the biological characteristics of the unmodified protein, such as binding to LRP6.

It will be appreciated that each of CDR H1 , H2, H3, L1 , L2, L3 may be modified alone or in combination with any other CDR, in any permutation or combination. In one aspect, a CDR is modified by the substitution, deletion or addition of up to 3 amino acids, for example 1 or 2 amino acids, for example 1 amino acid. Typically, the modification is a substitution, particularly a conservative substitution, for example as shown in Table 2 below.

Table 2:

Hydrophobic Met, Ala, Val, Leu, lie

Neutral hydrophilic Cys, Ser, Thr

Acidic Asp, Glu

Basic Asn, Gin, His, Lys, Arg

Residues that influence chain Gly, Pro

orientation

Aromatic Trp, Tyr, Phe

For example, in a variant CDR, the amino acid residues of the minimum binding unit may remain the same, but the flanking residues that comprise the CDR as part of the Kabat or Chothia definition(s) may be substituted with a conservative amino acid residue.

Such antigen binding polypeptides comprising modified CDRs or minimum binding units as described above may be referred to herein as "functional CDR variants" or "functional binding unit variants".

In one aspect of the invention there is provided an antigen binding protein having 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 98% or greater, 99% or greater or 100% identity to any of one the variable heavy chain sequences selected from SEQ ID NO:13 or SEQ ID NO:89 or SEQ ID NO:93 or SEQ ID NO:95 or SEQ ID NO:97 or SEQ ID NO:99, or SEQ ID NO: 101 , or SEQ ID NO:103, or SEQ ID NO:105.

In a further aspect of the invention there is provided an antigen binding protein having 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 98% or greater, 99% or greater or 100% identity in the framework regions to any of one the variable heavy chain sequences selected from SEQ ID NO: 13 or SEQ ID NO:93 or SEQ ID NO:95 or SEQ ID NO:97 or SEQ ID NO:99, or SEQ ID NO: 101 , or SEQ ID NO:103, or SEQ ID NO:105. For example in one aspect the humanised heavy chain variable domain may comprise the CDRs listed in SEQ ID NO: 1-3; within an acceptor antibody framework having 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 98% or greater, 99% or greater or 100% identity in the framework regions to any of one the variable heavy chain sequences selected from SEQ ID NO: 13 or SEQ ID NO:93 or SEQ ID NO:95 or SEQ ID NO:97 or SEQ ID NO:99, or SEQ ID NO: 101 , or SEQ ID NO:103, or SEQ ID NO:105.

In one aspect of the invention there is provided an antigen binding protein having 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 98% or greater, 99% or greater or 100% identity to any of one the variable light chain sequences selected from SEQ ID NO:14.

In a further aspect of the invention there is provided an antigen binding protein having 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 98% or greater, 99% or greater or 100% identity in the framework regions to any of one the variable light chain sequences selected from SEQ ID NO: 14.

For example in one aspect the humanised heavy chain variable domain may comprise the CDRs listed in SEQ ID NO: 4-6; within an acceptor antibody framework having 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 98% or greater, 99% or greater or 100% identity in the framework regions to the variable light chain sequences selected from SEQ ID NO: 14.

In one aspect there is provided an antigen binding polypeptide comprising:

(a) CDRH1 as shown in SEQ ID NO: 1 ; and a CDRH2 as shown in SEQ ID NO:2; CDRH3 as shown in SEQ ID NO:3; CDRL1 as shown in SEQ ID NO:4; CDRL2 as shown in SEQ ID NO:5; CDRL3 as shown in SEQ ID NO:6: or

(b) a VH domain comprising an amino acid sequence at least 90% identical to an amino acid sequence as

shown in any one of SEQ ID NO: 13 or SEQ ID NO:93 or SEQ ID NO:95 or SEQ ID NO:97 or SEQ ID NO:99 or SEQ ID NO: 101 or SEQ ID NO:103 or, SEQ ID NO: 105; and a VL domain comprising an amino acid sequence at least 90% identical to an amino acid sequence as shown in SEQ ID NO:14. wherein the antigen binding protein comprises a paired VH/VL wherein the paired VH/VL antagonises or does not potentiate the signalling of one Wnt ligand and does not activate or potentiate signalling of a second Wnt ligand.

In one aspect there is provided an antigen binding polypeptide comprising:

(b) a VH domain comprising an amino acid sequence at least 95% identical to an amino acid sequence as

The term "epitope" as used herein refers to that portion of the antigen that makes contact with a particular antigen binding site (paratope) on the antigen binding polypeptide. An epitope may be linear or conformational/discontinuous. A conformational or discontinuous epitope comprises amino acid residues that are separated by other sequences, i.e. not in a continuous sequence in the antigen's primary sequence. Although the residues may be from different regions of the peptide chain, they are in close proximity in the three dimensional structure of the antigen. In the case of multimeric antigens, a conformational or discontinuous epitope may include residues from different peptide chains.

Particular residues comprised within an epitope can be determined through computer modelling programs or via three-dimensional structures obtained through methods known in the art, such as X- ray crystallography.

Competition between the antigen binding polypeptide and a reference antibody may be determined by competition ELISA, FMAT or BIAcore. In one aspect, the competition assay is carried out by Biacore. There are several possible reasons for this competition: the two proteins may bind to the same or overlapping epitopes, there may be steric inhibition of binding, or binding of the first protein may induce a conformational change in the antigen that prevents or reduces binding of the second protein. The reduction or inhibition in biological activity may be partial or total. A neutralising antigen binding polypeptide may neutralise the activity of LRP6 by at least 20%, 30% 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% relative to LRP6 activity in the absence of the antigen binding polypeptide.

Neutralisation may be determined or measured using one or more assays known to the skilled person or as described herein.

The CDRs L1 , L2, L3, H1 and H2 tend to structurally exhibit one of a finite number of main chain conformations. The particular canonical structure class of a CDR is defined by both the length of the CDR and by the loop packing, determined by residues located at key positions in both the CDRs and the framework regions (structurally determining residues or SDRs). Martin and Thornton (1996; J Mol Biol 263:800-815) have generated an automatic method to define the "key residue" canonical templates. Cluster analysis is used to define the canonical classes for sets of CDRs, and canonical templates are then identified by analysing buried hydrophobics, hydrogen-bonding residues, and conserved glycines and prolines. The CDRs of antibody sequences can be assigned to canonical classes by comparing the sequences to the key residue templates and scoring each template using identity or similarity matrices. There may be multiple variant CDR canonical positions per CDR, per corresponding CDR, per binding unit, per heavy or light chain variable region, per heavy or light chain, and per antigen binding polypeptide, and therefore any combination of substitution may be present in the antigen binding polypeptide of the invention, provided that the canonical structure of the CDR is maintained such that the antigen binding polypeptide is capable of specifically binding LRP6.

As discussed above, the particular canonical structure class of a CDR is defined by both the length of the CDR and by the loop packing, determined by residues located at key positions in both the CDRs and the framework regions.

"Percent identity" between a query nucleic acid sequence and a subject nucleic acid sequence is the "Identities" value, expressed as a percentage, that is calculated by the BLASTN algorithm when a subject nucleic acid sequence has 100% query coverage with a query nucleic acid sequence after a pair-wise BLASTN alignment is performed. Such pair-wise BLASTN alignments between a query nucleic acid sequence and a subject nucleic acid sequence can be performed by using the default settings of the BLASTN algorithm available on the National Center for Biotechnology Information's website with the filter for low complexity regions turned off. Importantly, a query nucleic acid sequence may be described by a nucleic acid sequence identified in one or more claims herein or elsewhere in this application.

"Percent identity" between a query amino acid sequence and a subject amino acid sequence is the "Identities" value, expressed as a percentage, that is calculated by the BLASTP algorithm when a subject amino acid sequence has 100% query coverage with a query amino acid sequence after a pair-wise BLASTP alignment is performed. Such pair-wise BLASTP alignments between a query amino acid sequence and a subject amino acid sequence can be performed by using the default settings of the BLASTP algorithm available on the National Center for Biotechnology Information's website with the filter for low complexity regions turned off. Importantly, a query amino acid sequence may be described by an amino acid sequence identified in one or more claims herein or elsewhere in this application.

Also disclosed are amino acid sequences comprising amino-terminal deletions and carboxy terminal deletions of the amino acid sequence(s).

The skilled person will appreciate that, upon production of an antigen binding polypeptide such as an antibody, in particular depending on the cell line used and particular amino acid sequence of the antigen binding polypeptide, post-translational modifications may occur. For example, this may include the cleavage of certain leader sequences, the addition of various sugar moieties in various glycosylation and phosphorylation patterns, deamidation, oxidation, disulfide bond scrambling, isomerisation, C-terminal lysine clipping, and N-terminal glutamine cyclisation. The present invention encompasses the use of antigen binding polypeptides which have been subjected to, or have undergone, one or more post-translational modifications. Thus an "antigen binding polypeptide" or "antibody" of the invention includes an "antigen binding polypeptide" or "antibody", respectively, as defined earlier which has undergone a post-translational modification such as described herein. Glycosylation of antibodies at conserved positions in their constant regions is known to have a profound effect on antibody function, particularly effector functioning, see for example, Boyd et al. (1996) Mol. Immunol. 32: 131 1-1318. Glycosylation variants of the antigen binding polypeptides of the invention wherein one or more carbohydrate moiety is added, substituted, deleted or modified are contemplated. Introduction of an asparagine-X-serine or asparagine-X-threonine motif creates a potential site for enzymatic attachment of carbohydrate moieties and may therefore be used to manipulate the glycosylation of an antibody. In Raju et al. (2001 ) Biochemistry 40: 8868-8876 the terminal sialyation of a TNFR-lgG immunoadhesin was increased through a process of

regalactosylation and/or resialylation using beta-1 , 4-galactosyltransferace and/or alpha, 2,3 sialyltransferase. Increasing the terminal sialylation is believed to increase the half-life of the immunoglobulin. Antibodies, in common with most glycoproteins, are typically produced as a mixture of glycoforms. This mixture is particularly apparent when antibodies are produced in eukaryotic, particularly mammalian cells. A variety of methods have been developed to manufacture defined glycoforms, see Zhang et al. (2004) Science 303: 371 : Sears et al. (2001 ) Science 291 : 2344; Wacker et al. (2002) Science 298: 1790; Davis et al. (2002) Chem. Rev. 102: 579; Hang et al. (2001 ) Acc. Chem. Res 34: 727. The antibodies (for example of the IgG isotype, e.g. lgG1 ) as herein described may comprise a defined number (e.g. 7 or less, for example 5 or less, such as two or a single) of glycoform(s).

Deamidation is an enzymatic reaction primarily converting asparagine (N) to iso-aspartic acid and aspartic acid (D) at approximately 3:1 ratio. To a much lesser degree, deamidation can occur with glutamine residues in a similar manner. Deamidation in a CDR results in a change in charge of the molecule, but typically does not result in a change in antigen binding, nor does it impact on PK/PD. Oxidation can occur during production and storage (i.e. in the presence of oxidizing conditions) and results in a covalent modification of a protein, induced either directly by reactive oxygen species or indirectly by reaction with secondary by-products of oxidative stress. Oxidation happens primarily with methionine residues, but occasionally can occur at tryptophan and free cysteine residues.

Disulfide bond scrambling can occur during production and basic storage conditions. Under certain circumstances, disulfide bonds can break or form incorrectly, resulting in unpaired cysteine residues (- SH). These free (unpaired) sulfhydryls (-SH) can promote shuffling.

Isomerization typically occurs during production, purification, and storage (at acidic pH) and usually occurs when aspartic acid is converted to isoaspartic acid through a chemical process.

N-terminal glutamine in the heavy chain and/or light chain is likely to form pyroglutamate (pGlu). Most pGlu formation happens in the production bioreactor, but it can be formed non-enzymatically, depending on pH and temperature of processing and storage conditions. pGlu formation is considered as one of the principal degradation pathways for recombinant mAbs.

C-terminal lysine clipping is an enzymatic reaction catalyzed by carboxypeptidases, and is commonly observed in recombinant mAbs. Variants of this process include removal of lysine from one or both heavy chains. Lysine clipping does not appear to impact bioactivity and has no effect on mAb effector function.

Naturally occurring autoantibodies exist in humans that can bind to proteins. Autoantibodies can thus bind to endogenous proteins (present in naive subjects) as well as to proteins or peptides which are administered to a subject for treatment. Therapeutic protein-binding autoantibodies and antibodies that are newly formed in response to drug treatment are collectively termed anti-drug antibodies (ADAs). Pre-existing antibodies against molecules such as therapeutic proteins and peptides, administered to a subject can affect their efficacy and could result in administration reactions, hypersensitivity, altered clinical response in treated patients and altered bioavailability by sustaining, eliminating or neutralizing the molecule. It could be advantageous to provide molecules for therapy which comprise human immunoglobulin (antibody) single variable domains or dAbs™ which have reduced immunogenicity (i.e. reduced ability to bind to pre-existing ADAs when administered to a subject, in particular a human subject.

Antigen binding polypeptide as described herein may be incorporated into pharmaceutical compositions for use in the treatment of the human diseases described herein. In one aspect, the pharmaceutical composition comprises an antigen binding polypeptide in combination with one or more pharmaceutically acceptable carriers and/or excipients.

Such compositions comprise a pharmaceutically acceptable carrier as known and called for by acceptable pharmaceutical practice, see e.g. Remingtons Pharmaceutical Sciences, 16th edition (1980) Mack Publishing Co.

Pharmaceutical compositions may be administered by injection or continuous infusion (examples include, but are not limited to, intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular and intraportal). In one aspect, the composition is suitable for intravenous administration.

Pharmaceutical compositions may be suitable for topical administration (which includes, but is not limited to, epicutaneous, inhaled, intranasal or ocular administration) or enteral administration (which includes, but is not limited to, oral or rectal administration).

Pharmaceutical compositions may comprise between 1 mg to 10g of antigen binding polypeptide, for example between 5 mg and 1 g of antigen binding polypeptide. Alternatively, the composition may comprise between 5 mg and 500 mg, for example between 5 mg and 50 mg.

Methods for the preparation of such pharmaceutical compositions are well known to those skilled in the art. Other excipients may be added to the composition as appropriate for the mode of administration and the particular protein used.

Effective doses and treatment regimes for administering the antigen binding polypeptide may be dependent on factors such as the age, weight and health status of the patient and disease to be treated. Such factors are within the purview of the attending physician. Guidance in selecting appropriate doses may be found in e.g. Smith et al (1977) Antibodies in human diagnosis and therapy, Raven Press, New York.

The pharmaceutical composition may comprise a kit of parts of the antigen binding polypeptide together with other medicaments, optionally with instructions for use. For convenience, the kit may comprise the reagents in predetermined amounts with instructions for use.

The terms "individual", "subject" and "patient" are used herein interchangeably. In one aspect, the subject is a mammal, such as a primate, for example a cynomolgous macaque or marmoset or monkey. In another aspect, the subject is a human.

The antigen binding polypeptide described herein may also be used in methods of treatment.

Treatment can be therapeutic, prophylactic or preventative. Treatment encompasses alleviation, reduction, or prevention of at least one aspect or symptom of a disease and encompasses prevention or cure of the diseases described herein.

The antigen binding polypeptide described herein is used in an effective amount for therapeutic, prophylactic or preventative treatment. A therapeutically effective amount of the antigen binding polypeptide described herein is an amount effective to ameliorate or reduce one or more symptoms of, or to prevent or cure, the disease. Production Methods

Antigen binding polypeptides may be prepared by any of a number of conventional techniques. For example, antigen binding polypeptides may purified from cells that naturally express them (e.g., an antibody can be purified from a hybridoma that produces it), or produced in recombinant expression systems. See, for example, Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.), Plenum Press, New York (1980); and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988).

Any expression system can be used to make the antigen binding polypeptides of the invention. In general, host cells are transformed with a recombinant expression vector encoding the desired antigen binding polypeptide. Among the host cells that may be employed are prokaryotes (including bacteria), yeast (for example S. Cerevisiae, S. Pombe, P. pastoris, Aspergilus), or higher eukaryotic cells. Prokaryotes include gram negative or gram positive organisms, for example E. coli or Bacilli. Higher eukaryotic cells include insect cells and cell lines of mammalian origin (for example, CHO, Perc6, HEK293, HeLa).

Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985). Methods of cloning are known in the art and are generally described in Sambrook J et al. 2000 Molecular Cloning: A Laboratory Manual (Third Edition).

The cells can be cultured under conditions that promote expression of the antigen binding polypeptide, and the polypeptide recovered by conventional protein purification procedures. The antigen binding polypeptides contemplated for use herein include substantially homogeneous antigen binding polypeptides substantially free of contaminating materials.

The host cell may be an isolated host cell. The host cell is usually not part of a multicellular organism (e.g., plant or animal). The host cell may be a non-human host cell.

The skilled person will appreciate that, upon production of the antigen binding polypeptide , in particular depending on the cell line used and particular amino acid sequence of the antigen binding polypeptide, post-translational modifications may occur. This may include the cleavage of certain leader sequences, the addition of various sugar moieties in various glycosylation patterns, deamidation (for example at an asparagine or glutamine residue), oxidation (for example at a methionine, tryptophan or free cysteine residue), disulfide bond scrambling, isomerisation (for example at an aspartic acid residue), C-terminal lysine clipping (for example from one or both heavy chains), and N-terminal glutamine cyclisation (for example in the heavy and/or light chain). The present invention encompasses the use of antibodies which have been subjected to, or have undergone, one or more post-translational modifications. The modification may occur in a CDR, the variable framework region, or the constant region. The modification may result in a change in charge of the molecule. The modification typically does not result in a change in antigen binding, function, bioactivity, nor does it impact the PK/PD. Increased half-life, or half-life extension, can be useful in in vivo applications of antigen binding polypeptides, especially antibodies and most especially antibody fragments of small size. Such fragments (Fvs, disulphide bonded Fvs, Fabs, scFvs, dAbsTM) are generally rapidly cleared from the body. Antigen binding polypeptides in accordance with the disclosure can be adapted or modified to provide increased serum half-life in vivo and consequently longer persistence, or residence, times of the functional activity of the antigen binding polypeptide in the body. Suitably, such modified molecules have a decreased clearance and increased Mean Residence Time compared to the non- adapted molecule. Increased half-life can improve the pharmacokinetic and pharmacodynamic properties of a therapeutic molecule and can also be important for improved patient compliance. The antigen binding polypeptides of the disclosure can be stabilized in vivo and their half-life increased by binding to molecules which resist degradation and/or clearance or sequestration ("half- life extending moiety" or "half-life extending molecule"). Half-life extension strategies are reviewed, for example, in "Therapeutic Proteins: Strategies to Modulate Their Plasma Half-Lives", Edited by Roland Kontermann, Wiley-Blackwell, 2012, ISBN: 978-3-527-32849-9. Suitable half-life extension strategies include: PEGylation, polysialylation, HESylation, recombinant PEG mimetics, N- glycosylation, O-glycosylation, Fc fusion, engineered Fc, IgG binding, albumin fusion, albumin binding, albumin coupling and nanoparticles.

Antigen binding polypeptides of the disclosure and antagonists comprising these can be formatted to have a larger hydrodynamic size, for example, by attachment of a PEG group, serum albumin, transferrin, transferrin receptor or at least the transferrin-binding portion thereof, an antibody Fc region, or by conjugation to an antibody domain. For example, polypeptides dAbsTM and antagonists may be formatted as a larger antigen-binding fragment of an antibody or as an antibody (e.g., formatted as a Fab, Fab', F(ab)2, F(ab')2, IgG, scFv).

As used herein, "hydrodynamic size" refers to the apparent size of a molecule (e.g., an antigen binding polypeptide) based on the diffusion of the molecule through an aqueous solution. The diffusion or motion of a protein through solution can be processed to derive an apparent size of the protein, where the size is given by the "Stokes radius" or "hydrodynamic radius" of the protein particle. The "hydrodynamic size" of a protein depends on both mass and shape (conformation), such that two proteins having the same molecular mass may have differing hydrodynamic sizes based on the overall conformation and charge of the protein. An increase in hydrodynamic size can give an associated decrease in renal clearance leading to an observed increase in half life (t1/2).

Hydrodynamic size of the antigen binding polypeptides (e.g., domain antibody monomers and multimers) of the disclosure may be determined using methods which are well known in the art. For example, gel filtration chromatography may be used to determine the hydrodynamic size of an antigen binding polypeptide. Suitable gel filtration matrices for determining the hydrodynamic sizes of antigen binding polypeptides, such as cross-linked agarose matrices, are well known and readily available. The size of an antigen binding polypeptide format (e.g., the size of a PEG moiety attached to a domain antibody monomer), can be varied depending on the desired application. For example, where antigen binding polypeptide is intended to leave the circulation and enter into peripheral tissues, it is desirable to keep the hydrodynamic size of the antigen binding polypeptide low to facilitate extravazation from the blood stream. Alternatively, where it is desired to have the antigen binding polypeptide remain in the systemic circulation for a longer period of time the size of the antigen binding polypeptide can be increased, for example by formatting as an Ig like protein.

In one aspect, the half-life extending moiety or molecule is a polyethylene glycol moiety or a PEG mimetic. In one aspect, the antigen binding polypeptide comprises (optionally consists of) a single variable domain of the disclosure linked to a polyethylene glycol moiety (optionally, wherein said moiety has a size of about 20 to about 50 kDa, optionally about 40 kDa linear or branched PEG). Reference is made to WO04081026 for more detail on PEGylation of domain antibodies and binding moieties. In one aspect, the antagonist consists of a domain antibody monomer linked to a PEG, wherein the domain antibody monomer is a single variable domain according to the disclosure.

Suitable PEG mimetics are reviewed, for example in Chapter 4, pages 63-80, "Therapeutic Proteins: Strategies to Modulate Their Plasma Half-Lives" Edited by Roland Kontermann, Wiley-Blackwell, 2012, ISBN: 978-3-527-32849-9.

The interaction between the Fc region of an antibody and various Fc receptors (FcyR) is believed to mediate phagocytosis and half-life/clearance of an antibody or antibody fragment. The neonatal FcRn receptor is believed to be involved in both antibody clearance and the transcytosis across tissues (see Junghans (1997) Immunol. Res 16: 29-57; and Ghetie et al. (2000) Annu. Rev. Immunol. 18: 739-

766). In one aspect, the half-life extending moiety may be an Fc region from an antibody. Such an Fc region may incorporate various modifications depending on the desired property. For example, a salvage receptor binding epitope may be incorporated into the antibody to increase serum half life, see US 5,739,277.

Human lgG1 residues determined to interact directly with human FcRn includes Ile253, Ser254, Lys288, Thr307, Gln31 1 , Asn434 and His435. Accordingly, substitutions at any of the positions described in this section may enable increased serum half-life and/or altered effector properties of the antibodies.

Half-life extension by fusion to the Fc region is reviewed, for example, in Chapter 9, pages 157-188, "Therapeutic Proteins: Strategies to Modulate Their Plasma Half-Lives" Edited by Roland

Kontermann, Wiley-Blackwell, 2012, ISBN: 978-3-527-32849-9.

Typically, a polypeptide that enhances serum half-life in vivo, i.e. a half-life extending molecule, is a polypeptide which occurs naturally in vivo and which resists degradation or removal by endogenous mechanisms which remove unwanted material from the organism (e.g., human). Typically, such molecules are naturally occurring proteins which themselves have a long half-life in vivo.

For example, a polypeptide that enhances serum half-life in vivo can be selected from proteins from the extracellular matrix, proteins found in blood, proteins found at the blood brain barrier or in neural tissue, proteins localized to the kidney, liver, muscle, lung, heart, skin or bone, stress proteins, disease-specific proteins, or proteins involved in Fc transport. Suitable polypeptides are described, for example, in WO2008/096158.

Such an approach can also be used for targeted delivery of an antigen binding polypeptide, e.g. a single variable domain, in accordance with the disclosure to a tissue of interest. In one aspect targeted delivery of a high affinity single variable domain in accordance with the disclosure is provided.

In one aspect, an antigen binding polypeptide, e.g. single variable domain, in accordance with the disclosure can be linked, i.e. conjugated or associated, to serum albumin, fragments and analogues thereof. Half-life extension by fusion to albumin is reviewed, for example in Chapter 12, pages 223- 247, "Therapeutic Proteins: Strategies to Modulate Their Plasma Half-Lives" Edited by Roland Kontermann, Wiley-Blackwell, 2012, ISBN: 978-3-527-32849-9.

Examples of suitable albumin, albumin fragments or albumin variants are described, for example, in WO2005077042 and WO2003076567.

In another aspect, an antigen binding polypeptide in accordance with the disclosure can be linked, i.e. conjugated or associated, to transferrin, fragments and analogues thereof.

In one aspect, half-life extension can be achieved by targeting an antigen or epitope that increases half-live in vivo. The hydrodynamic size of an antigen binding polypeptide and its serum half-life may be increased by conjugating or associating an antigen binding polypeptide of the disclosure to a binding domain that binds a naturally occurring molecule and increases half-live in vivo.

For example, the antigen binding polypeptide in accordance with the invention can be conjugated or linked to an anti-serum albumin or anti-neonatal Fc receptor antibody or antibody fragment, e.g. an anti-SA or anti-neonatal Fc receptor dAbTM, Fab, Fab' or scFv, or to an anti-SA affibody or anti- neonatal Fc receptor Affibody or an anti-SA avimer, or an anti-SA binding domain which comprises a scaffold selected from, but not limited to, the group consisting of CTLA-4, lipocallin, SpA, an affibody, an avimer, GroEI and fibronectin (see WO2008096158 for disclosure of these binding domains). Conjugating refers to a composition comprising antigen binding polypeptides of the invention bonded (covalently or noncovalently) to a binding domain such as a binding domain that binds serum albumin. In another aspect, the binding domain may be a polypeptide domain such as an Albumin Binding Domain (ABD) or a small molecule which binds albumin (reviewed, for example in Chapter 14, pages 269-283 and Chapter 15, pages 285-296, "Therapeutic Proteins: Strategies to Modulate Their Plasma Half-Lives" Edited by Roland Kontermann, Wiley-Blackwell, 2012, ISBN: 978-3-527-32849-9).

In one aspect, there is provided a fusion protein comprising an antigen binding polypeptide in accordance with the invention and an anti-serum albumin or anti-neonatal Fc receptor antibody or antibody fragment.

Where a (one or more) half-life extending moiety (e.g. , albumin, transferrin and fragments and analogues thereof or binding domain) is used to format the antigen binding polypeptide of the disclosure, it can be conjugated using any suitable method. In addition, a half-life extending moiety may be added through genetic fusion, for example by using a single nucleotide construct that encodes a fusion protein, wherein the fusion protein is encoded as a single polypeptide chain with the half-life extending moiety located N- or C-terminally to the antigen binding polypetide. Alternatively, conjugation can be achieved by using a peptide linker between moieties, e.g., a peptide linker as described in WO2003076567 or WO2004003019. In one aspect, a peptide linker is the amino acid sequence AST.

Half-life may also be extended by pharmaceutical formulations such as liposomes or nanoparticles.

"Half-life (t1/2)" refers to the time required for the concentration of the antigen binding polypeptide to reach half of its original value. The serum half-life of proteins can be measured by pharmacokinetic studies according to the method described by Kim et al. (Eur. J. of Immuno. 24: 542, 1994). According to this method, radiolabeled protein is injected intravenously into mice and its plasma concentration is periodically measured as a function of time, for example, at about 3 minutes to about 72 hours after the injection. Other methods for pharmacokinetic analysis and determination of the half-life of a molecule will be familiar to those skilled in the art. Details may be found in Kenneth, A et al: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and in Peters et al, Pharmacokinetic analysis: A Practical Approach (1996). Reference is also made to "Pharmacokinetics", M Gibaldi & D Perron, published by Marcel Dekker, 2nd Rev. ex edition (1982), which describes pharmacokinetic parameters such as t alpha and t beta half lives and area under the curve (AUC), and "Clinical Pharmacokinetics: Concepts and Applications", Rowland and Tozer, Third Edition (1995).

"Clearance (CL)" refers to the volume of plasma irreversibly cleared of a protein per unit time.

Clearance is calculated as the Dose/AUC (AUC : is the Area Under Curve or Area under the plasma drug concentration time curve). Clearance can also be calculated by the rate of drug elimination divided by the plasma concentration of the drug (rate of elimination = CL^*concentration). "Mean Residence Time (MRT)" is the average time that the antigen binding polypeptides reside in the body before being irreversibly eliminated. Calculated as MRT= AUMC/AUC.

"Steady state concentration" (Css) is the concentration reached when the drug elimination rate becomes equal to drug administration rate as a result of continued drug administration. Css fluctuates between peak and trough levels and is measured in microgram/ml. "Mean steady-state trough concentration" refers to the mean of the trough level across the patient population at a given time.

"Comparable mean steady-state trough concentration" refers to mean steady-state trough concentration which is the same or within about 10% to 30% of the stated value. Comparable mean steady-state trough concentration for the antigen binding polypeptides of the invention may be considered to be those mean steady-state trough concentrations that are 0.8 to 1.25 times the mean steady-state trough concentration achieved with an IgG comprising the light chain sequence of SEQ ID No. 17 and the heavy chain sequence of SEQ ID No. 24.

Half lives and AUC can be determined from a curve of serum concentration of drug (for example theantigen binding polypeptide of the present invention) against time. Half life may be determined through compartmental or non-com partmental analysis. The WINNONLIN™ analysis package (available from Pharsight Corp., Mountain View, CA94040, USA) can be used, for example, to model the curve. In one aspect, "half life" refers to the terminal half life.

The curve may be modelled to a 2-compartmental model. The t alpha (t a) half life is the half life of the first phase and the t beta (t β) half life is the half life of the second, in this case, terminal, phase i.e the terminal half life. Alternatively, the curve may be modelled to a 3-compartmental model. Here, the terminal phase is the t gamma (t λ) half-life i.e. terminal half life.

In a first phase (the alpha phase) of either a 2- or 3-compartmental distribution, the antigen binding polypeptide is undergoing mainly distribution in the patient, along with elimination. Thus, in one aspect, the present disclosure provides an antigen binding polypeptide or a composition comprising an antigen binding polypeptide according to the disclosure having a t alpha half life in the range of 15 minutes or more. In one aspect, the lower end of the range is 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 10 hours, 1 1 hours or 12 hours. In addition, or alternatively, an antigen binding polypeptide or composition according to the disclosure will have a t alpha half life in the range of up to and including 12 hours. In one aspect, the upper end of the range is 1 1 , 10, 9, 8, 7, 6 or 5 hours. An example of a suitable range is 1 to 6 hours, 2 to 5 hours or 3 to 4 hours.

In a 2- or 3-compartmental model, the terminal phase is when the antigen binding polypeptide has been distributed and the serum concentration is decreasing as the ligand is cleared from the patient. Terminal half life can also be determined from non-compartmental analysis.

In one aspect, the present disclosure provides an antigen binding polypeptide or a composition comprising an antigen binding polypeptide according to the disclosure having a terminal half-life in the range of about 2.5 hours or more. In one aspect, the lower end of the range is about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 10 hours, about 1 1 hours, or about 12 hours. In addition, or alternatively, an antigen binding polypeptide composition according to the disclosure has a terminal half life in the range of up to and including 21 days. In one aspect, the upper end of the range is about 12 hours, about 24 hours, about 2 days, about 3 days, about 5 days, about 10 days, about 15 days or about 20 days. In one aspect an antigen binding polypeptide or composition according to the disclosure will have a terminal half life in the range about 12 to about 60 hours. In a further aspect, it will be in the range about 12 to about 48 hours. In a further aspect still, it will be in the range about 12 to about 26 hours.

In addition, or alternatively to the above criteria, the present disclosure provides an antigen binding polypeptide or a composition comprising an antigen binding polypeptide according to the disclosure having an AUC value (area under the curve) in the range of about 1 mg/min/ml or more. In one aspect, the lower end of the range is about 5, about 10, about 15, about 20, about 30, about 100, about 200 or about 300 mg/min/ml. In addition, or alternatively, an antigen binding polypeptide or composition according to the disclosure has an AUC in the range of up to about 600 mg/min/ml. In one aspect, the upper end of the range is about 500, about 400, about 300, about 200, about 150, about 100, about 75 or about 50 mg/min/ml. In one aspect an antigen binding polypeptide according to the disclosure will have a AUC in the range selected from the group consisting of the following: about 15 to about 150 mg/min/ml, about 15 to about 100 mg/min/ml, about 15 to about 75 mg/min/ml, and about 15 to about 50mg/min/ml.

The half-life of an antigen binding polypeptide is increased if its concentration and/or functional activity persists, in vivo, for a longer period than a similar antigen binding polypeptide which is not bound to or does not bind a half-life increasing molecule. Thus, for example, an antigen binding polypeptide which comprises a half-life increasing molecule, or a moiety specific for a half life increasing molecule, and a moiety specific for a target molecule is compared with the same antigen binding polypeptide wherein half-life increasing moiety is not present. Thus, for example, an antigen binding polypeptide which comprises a moiety specific for serum albumin and a moiety specific for a target molecule is compared with the same antigen binding polypeptide wherein the moiety with specificity to serum albumin is not present. The antigen binding polypeptide for comparison may lack a moiety specific for serum albumin and/or may comprise a "dummy dAbTM", i.e. a non-binding dAbTM, instead of the moiety specific for serum albumin. Typically, the half-life is increased by 10%, 20%, 30%, 40%, 50% or more. Increases in the range of 2x, 3x, 4x, 5x, 10x, 20x, 30x, 40x, 50x or more of the half-life are possible. Alternatively, or in addition, increases in the range of up to 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 150x of the half life are possible.

For the determination of an increase in the in vivo half-life of the modified IgGs or fragments thereof, the clearance rate in β-phase is calculated and compared with that of the unmodified IgG.

"Volume of distribution" may be an important parameter for some molecules e.g. for a molecule with a short half-life but rapid targeting to a particular tissue.

In another aspect the antigen binding polypeptide consists of, or consists essentially of, an Fc region of an antibody, or a part thereof, linked at each end, directly or indirectly (for example, via a linker sequence) to an epitope binding domain. Such an antigen binding construct may comprise 2 epitope- binding domains separated by an Fc region, or part thereof. By separated is meant that the epitope- binding domains are not directly linked to one another, and in one aspect are located at opposite ends (C and N terminus) of an Fc region, or any other scaffold region.

Antigen binding proteins of the present invention may be linked to epitope-binding domains by the use of linkers. Examples of suitable linkers include amino acid sequences which may be from 1 amino acid to 150 amino acids in length, or from 1 amino acid to 140 amino acids, for example, from 1 amino acid to 130 amino acids, or from 1 to 120 amino acids, or from 1 to 80 amino acids, or from 1 to 50 amino acids, or from 1 to 20 amino acids, or from 1 to 10 amino acids, or from 5 to 18 amino acids. Such sequences may have their own tertiary structure, for example, a linker of the present invention may comprise a single variable domain. The size of a linker in one aspect is equivalent to a single variable domain. Suitable linkers may be of a size from 1 to 100 angstroms, for example may be of a size from 20 to 80 angstroms or for example may be of a size from 20 to 60 angstroms or for example less than 40 angstroms, or less than 20 angstroms, or less than 5 angstroms in length. The long half-life of IgG antibodies is reported to be dependent on its binding to FcRn. Therefore, substitutions that increase the binding affinity of IgG to FcRn at pH 6.0 while maintaining the pH dependence of the interaction by engineering the constant region have been extensively studied (Ghetie et al., Nature Biotech. 15: 637-640, 1997; Hinton et al., JBC 279: 6213-6216, 2004;

Dall'Acqua et al., 10 J Immunol 1 17: 1 129-1 138, 2006). Another means of modifying antigen binding polypeptides of the present invention involves increasing the in-vivo half life of such proteins by modification of the immunoglobulin constant domain or FcRn (Fc receptor neonate) binding domain. In adult mammals, FcRn, also known as the neonatal Fc receptor, plays a key role in maintaining serum antibody levels by acting as a protective receptor that binds and salvages antibodies of the IgG isotype from degradation. IgG molecules are endocytosed by endothelial cells, and if they bind to FcRn, are recycled out into circulation. In contrast, IgG molecules that do not bind to FcRn enter the cells and are targeted to the lysosomal pathway where they are degraded.

The neonatal FcRn receptor is believed to be involved in both antibody clearance and the transcytosis across tissues (see Junghans R.P (1997) Immunol. Res 16. 29-57 and Ghetie et al (2000)

Annu.Rev.lmmunol. 18, 739-766). Human lgG1 residues determined to interact directly with human FcRn includes Ile253, Ser254, Lys288, Thr307, Gln31 1 , Asn434 and His435. Switches at any of these positions described in this section may enable increased serum half-life and/or altered effector properties of antigen binding polypeptides of the invention. Antigen binding polypeptides of the present invention may have amino acid modifications that increase the affinity of the constant domain or fragment thereof for FcRn. Increasing the half-life of therapeutic and diagnostic IgG's and other bioactive molecules has many benefits including reducing the amount and/or frequency of dosing of these molecules. In one aspect there is therefore provided an antigen binding according to the invention provided herein or a fusion protein comprising all or a portion (an FcRn binding portion) of an IgG constant domain having one or more of these amino acid modifications and a non-lgG protein or non-protein molecule conjugated to such a modified IgG constant domain, wherein the presence of the modified IgG constant domain increases the in vivo half life of the antigen binding polypeptide.

PCT Publication No. WO 00/42072 discloses a polypeptide comprising a variant Fc region with altered FcRn binding affinity, which polypeptide comprises an amino acid modification at any one or more of amino acid positions 238, 252, 253, 254, 255, 256, 265, 272, 286, 288, 303, 305, 307, 309, 31 1 , 312, 317, 340, 356, 360, 362, 376, 378, 380, 386,388, 400, 413, 415, 424,433, 434,435, 436, 439, and 447 of the Fc region, wherein the numbering of the residues in the Fc region is that of the EU index (Kabat et al).

PCT Publication No. WO 02/060919 A2 discloses a modified IgG comprising an IgG constant domain comprising one or more amino acid modifications relative to a wild-type IgG constant domain, wherein the modified IgG has an increased half-life compared to the half-life of an IgG having the wild-type IgG constant domain, and wherein the one or more amino acid modifications are at one or more of positions 251 , 253, 255, 285-290, 308-314, 385-389, and 428-435. Shields et al. (2001 , J Biol Chem ; 276:6591-604) used alanine scanning mutagenesis to alter residues in the Fc region of a human lgG1 antibody and then assessed the binding to human FcRn. Positions that effectively abrogated binding to FcRn when changed to alanine include I253, S254, H435, and Y436. Other positions showed a less pronounced reduction in binding as follows: E233- G236, R255, K288, L309, S415, and H433. Several amino acid positions exhibited an improvement in FcRn binding when changed to alanine; notable among these are P238, T256, E272, V305, T307, Q31 1 , D312, K317, D376, E380, E382, S424, and N434. Many other amino acid positions exhibited a slight improvement (D265, N286, V303, K360, Q362, and A378) or no change (S239, K246, K248, D249, M252, E258, T260, S267, H268, S269, D270, K274, N276, Y278, D280, V282, E283, H285, T289, K290, R292, E293, E294, Q295, Y296, N297, S298, R301 , N315, E318, K320, K322, S324, K326, A327, P329, P331 , E333, K334, T335, S337, K338, K340, Q342, R344, E345, Q345, Q347, R356, M358, T359, K360, N361 , Y373, S375, S383, N384, Q386, E388, N389, N390, K392, L398, S400, D401 , K414, R416, Q418, Q419, N421 , V422, E430, T437, K439, S440, S442, S444, and K447) in FcRn binding.

The most pronounced effect was found for combination variants with improved binding to FcRn. At pH 6.0, the E380A/N434A variant showed over 8-fold better binding to FcRn, relative to native lgG1 , compared with 2-fold for E380A and 3.5-fold for N434A. Adding T307A to this effected a 12-fold improvement in binding relative to native lgG1. In one aspect the antigen binding polypeptide of the invention comprises the E380A/N434A mutations and has increased binding to FcRn.

Dall'Acqua et al. (2002, J Immunol. ;169:5171-80) described random mutagenesis and screening of human lgG1 hinge-Fc fragment phage display libraries against mouse FcRn. They disclosed random mutagenesis of positions 251 , 252, 254-256, 308, 309, 31 1 , 312, 314, 385-387, 389, 428, 433, 434, and 436. The major improvements in lgG1 -human FcRn complex stability occur in substituting residues located in a band across the Fc-FcRn interface (M252, S254, T256, H433, N434, and Y436) and to lesser extend substitutions of residues at the periphery like V308, L309, Q31 1 , G385, Q386, P387, and N389. The variant with the highest affinity to human FcRn was obtained by combining the M252Y/S254T/T256E and H433K/N434F/Y436H mutations and exhibited a 57-fold increase in affinity relative to the wild-type lgG1. The in vivo behaviour of such a mutated human lgG1 exhibited a nearly 4-fold increase in serum half-life in cynomolgus monkey as compared to wild-type lgG1.

The present invention therefore provides a variant of an antigen binding polypeptide with optimized binding to FcRn. In a preferred aspect, the said variant of an antigen binding polypeptide comprises at least one amino acid modification in the Fc region of said antigen binding polypeptide, wherein said modification is selected from the group consisting of 226, 227, 228, 230, 231 , 233, 234, 239, 241 , 243, 246, 250, 252, 256, 259, 264, 265, 267, 269, 270, 276, 284, 285, 288, 289, 290, 291 , 292, 294, 297, 298, 299, 301 , 302, 303, 305, 307, 308, 309, 311 , 315, 317, 320, 322, 325, 327, 330, 332, 334, 335, 338, 340, 342, 343, 345, 347, 350, 352, 354, 355, 356, 359, 360, 361 , 362, 369, 370, 371 , 375, 378, 380, 382, 384, 385, 386, 387, 389, 390, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401 403, 404, 408, 41 1 , 412, 414, 415, 416, 418, 419, 420, 421 , 422, 424, 426, 428, 433, 434, 438, 439, 440, 443, 444, 445, 446 and 447 of the Fc region as compared to said parent polypeptide, wherein the numbering of the amino acids in the Fc region is that of the EU index in Kabat., In a further aspect of the invention the modifications are M252Y/S254T/T256E.

Additionally, various publications describe methods for obtaining physiologically active molecules whose half-lives are modified either by introducing an FcRn-binding polypeptide into the molecules (WO 97/43316; U.S. Patent N° 5,869,046; U.S. Patent N° 5,747,035; WO 96/32478; WO 91/14438) or by fusing the molecules with antibodies whose FcRn-binding affinities are preserved but affinities for other Fc receptors have been greatly reduced (WO 99/43713) or fusing with FcRn binding domains of antibodies (WO 00/09560; U.S. Patent N°4, 703,039).

Although substitutions in the constant region are able to significantly improve the functions of therapeutic IgG antibodies, substitutions in the strictly conserved constant region have the risk of immunogenicity in human (Presta, supra, 2008; De Groot and Martin, Clin Immunol 131 : 189-201 , 2009) and substitution in the highly diverse variable region sequence might be less immunogenic. Reports concerned with the variable region include engineering the CDR residues to improve binding affinity to the antigen (Rothe et al., Expert Opin Bioi Ther 6: 177-187,2006; Bostrom et al., Methods Mol Bioi 525: 353-376,2009; Thie et al., Methods Mol Bioi 525: 309-322, 2009) and engineering the CDR and framework residues to improve stability (Worn and Pluckthun, J Mol Bioi 305: 989-1010, 2001 ; Ewert et al., Methods 34: 184-199, 2004) and decrease immunogenicity risk (De Groot and Martin, supra, 2009; Jones et al., Methods Mol Bio 525: 405-423, xiv, 2009). As reported, improved affinity to the antigen can be achieved by affinity maturation using the phage or ribosome display of a randomized library.

Improved stability can be rationally obtained from sequence- and structure-based rational design. Decreased immunogenicity risk (deimmunization) can be accomplished by various humanization methodologies and the removal of T-cell epitopes, which can be predicted using in silico technologies or determined by in vitro assays. Additionally, variable regions have been engineered to lower pi. A longer half life was observed for these antibodies as compared to wild type antibodies despite comparable FcRn binding. Engineering or selecting antibodies with pH dependent antigen binding to modify antibody and/or antigen half life eg lgG2 antibody half life can be shortened if antigen- mediated clearance mechanisms normally degrade the antibody when bound to the antigen. Similarly, the antigen:antibody complex can impact the half-life of the antigen, either extending half-life by protecting the antigen from the typical degradation processes, or shortening the half-life via antibody- mediated degradation. One embodiment relates to antibodies with higher affinity for antigen at pH 7.4 as compared to endosomal pH (i.e., pH 5.5-6.0) such that the KD ratio at pH5.5/ pH 7.4 or at pH 6.0/ pH 7.4 is 2 or more. For example to enhance the pharmacokinetic (PK) and

pharmacodynamic (PD) properties of the antibody, it is possible to engineer pH-sensitive binding to the antibody by introducing histidines into CDR residues.

Additionally, methods of producing an antigen binding polypeptide with a decreased biological half-life are also provided. A variant IgG in which His435 is mutated to alanine results in the selective loss of FcRn binding and a significantly reduced serum half-life (Firan et al. 2001 , International immunology 13:993). U.S. Pat. No. 6,165,745 discloses a method of producing an antigen binding polypeptide with a decreased biological half-life by introducing a mutation into the DNA segment encoding the antigen binding polypeptide. The mutation includes an amino acid substitution at position 253, 310, 31 1 , 433, or 434 of the Fc-hinge domain.

The term "Effector Function" as used herein is meant to refer to one or more of Antibody dependant cell mediated cytotoxic activity (ADCC), Complement-dependant cytotoxic activity (CDC) mediated responses, Fc-mediated phagocytosis or antibody dependant cellular phagocytosis (ADCP) and antibody recycling via the FcRn receptor.

The interaction between the constant region of an antigen binding polypeptide and various Fc receptors (FcR) including FcyRI (CD64), FcyRII (CD32) and FcyRMI (CD16) is believed to mediate the effector functions of the antigen binding polypeptide. Significant biological effects can be a consequence of effector functionality. Usually, the ability to mediate effector function requires binding of the antigen binding polypeptide to an antigen and not all antigen binding polypeptides will mediate every effector function.

Effector function can be measured in a number of ways including for example via binding of the FcyRMI to Natural Killer cells or via FcyRI to monocytes/macrophages to measure for ADCC effector function. For example an antigen binding polypeptide of the present invention can be assessed for ADCC effector function in a Natural Killer cell assay. Examples of such assays can be found in Shields et al, 2001 The Journal of Biological Chemistry, Vol. 276, p6591-6604; Chappel et al, 1993 The Journal of Biological Chemistry, Vol 268, p25124-25131 ; Lazar et al, 2006 PNAS, 103; 4005- 4010.

Examples of assays to determine CDC function include that described in 1995 J Imm Meth 184:29-38. Human lgG1 constant regions containing specific mutations or altered glycosylation on residue Asn297 have also been described to enhance binding to Fc receptors. In some cases these mutations have also been shown to enhance ADCC and CDC (Lazar et al. PNAS 2006, 103; 4005- 4010; Shields et al. J Biol Chem 2001 , 276; 6591-6604; Nechansky et al. Mol Immunol, 2007, 44; 1815-1817).

Examples of mutations known to increase effector function include S239D, I332E, A330L, E333A, K326W, G236A and combinations thereof.

In one aspect of the present invention there is provided an antigen binding polypeptide comprising a chimaeric heavy chain constant region for example an antigen binding polypeptide comprising a chimaeric heavy chain constant region with at least one CH2 domain from lgG3 such that the antigen binding polypeptide has enhanced effector function, for example wherein it has enhanced ADCC or enhanced CDC, or enhanced ADCC and CDC functions,. In one such aspect, the antigen binding polypeptide may comprise one CH2 domain from lgG3 or both CH2 domains may be from lgG3. Also provided is a method of producing an antigen binding polypeptide according to the invention comprising the steps of:

a) culturing a recombinant host cell comprising an expression vector comprising an isolated nucleic acid as described herein wherein the expression vector comprises a nucleic acid sequence encoding an Fc domain having both lgG1 and lgG3 Fc domain amino acid residues; and

b) recovering the antigen binding polypeptide.

Such methods for the production of antigen binding polypeptides can be performed, for example, using the COMPLEGENT™ technology system available from BioWa, Inc. (Princeton, NJ) and Kyowa Hakko Kogyo (now, Kyowa Hakko Kirin Co., Ltd.) Co., Ltd. In which a recombinant host cell comprising an expression vector in which a nucleic acid sequence encoding a chimeric Fc domain having both lgG1 and lgG3 Fc domain amino acid residues is expressed to produce an antigen binding polypeptide having enhanced complement dependent cytotoxicity (CDC) activity that is increased relative to an otherwise identical antigen binding polypeptide lacking such a chimeric Fc domain. Aspects of the COMPLEGENT™ technology system are described in WO2007011041 and US20070148165 each of which are incorporated herein by reference. In an alternative aspect CDC activity may be increased by introducing sequence specific mutations into the Fc region of an IgG chain. Those of ordinary skill in the art will also recognize other appropriate systems.

In an alternative aspect of the present invention, there is provided an antigen binding polypeptide comprising a heavy chain constant region with an altered glycosylation profile such that the antigen binding polypeptide has enhanced effector function. For example, wherein the antigen binding polypeptide has enhanced ADCC or enhanced CDC or wherein it has both enhanced ADCC and CDC effector function. Examples of suitable methodologies to produce antigen binding polypeptides with an altered glycosylation profile are described in WO2003011878, WO2006014679 and EP1229125, all of which can be applied to the antigen binding polypeptides of the present invention.

The present invention also provides a method for the production of an antigen binding polypeptide according to the invention comprising the steps of:

a) culturing a recombinant host cell comprising an expression vector comprising the isolated nucleic acid as described herein, wherein the FUT8 gene encoding alpha-1 ,6-fucosyltransferase has been inactivated in the recombinant host cell; and

b) recovering the antigen binding polypeptide.

Such methods for the production of antigen binding polypeptides can be performed, for example, using the POTELLIGENT™ technology system available from BioWa, Inc. (Princeton, NJ) in which CHOK1SV cells lacking a functional copy of the FUT8 gene produce monoclonal antibodies having enhanced antibody dependent cell mediated cytotoxicity (ADCC) activity that is increased relative to an identical monoclonal antibody produced in a cell with a functional FUT8 gene. Aspects of the POTELLIGENT™ technology system are described in US7214775, US6946292, WO0061739 and WO0231240 all of which are incorporated herein by reference. Those of ordinary skill in the art will also recognize other appropriate systems.

It will be apparent to those skilled in the art that such modifications may not only be used alone but may be used in combination with each other in order to further enhance effector function. Also provided is an immunoconjugate (interchangeably referred to as "antibody-drug conjugates," or "ADCs")comprising an antigen binding polypeptide according to the invention as herein described including, but not limited to, an antibody conjugated to one or more cytotoxic agents, such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).

Immunoconjugates have been used for the local delivery of cytotoxic agents, i.e., drugs that kill or inhibit the growth or proliferation of cells, in the treatment of cancer (Lambert, J. (2005) Curr. Opinion in Pharmacology 5:543-549; Wu et al. (2005) Nature Biotechnology 23(9): 1 137-1 146; Payne, G.

(2003) i 3:207-212; Syrigos and Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drug Deliv. Rev. 26: 151-172; U.S. Pat. No. 4,975,278). Immunoconjugates allow for the targeted delivery of a drug moiety to a tumor, and intracellular accumulation therein, where systemic administration of unconjugated drugs may result in unacceptable levels of toxicity to normal cells as well as the tumor cells sought to be eliminated (Baldwin et al., Lancet (Mar. 15, 1986) pp. 603-05; Thorpe (1985) "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications (A. Pinchera et al., eds) pp. 475-506. Both polyclonal antibodies and monoclonal antibodies have been reported as useful in these strategies (Rowland et al., (1986) Cancer Immunol. Immunother. 21 : 183-87). Drugs used in these methods include daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al., (1986) supra). Toxins used in antibody-toxin conjugates include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as geldanamycin (Mandler et al (2000) J. Nat. Cancer Inst. 92(19): 1573-1581 ; Mandler et al (2000) Bioorganic & Med. Chem. Letters 10: 1025-1028; Mandler et al (2002) Bioconjugate Chem. 13:786-791 ), maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al (1998) Cancer Res.

58:2928; Hinman et al (1993) Cancer Res. 53:3336-3342).

In certain aspects, an immunoconjugate comprises an antigen binding polypeptide, including but not limited to, an antibody and a chemotherapeutic agent or other toxin. Chemotherapeutic agents useful in the generation of immunoconjugates are described herein. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. See, e.g., WO 93/21232 published Oct. 28, 1993. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 212Bi, 1311, 131 In, 90Y, and 186Re. Antigen binding polypeptides of the present invention may also be conjugated to one or more toxins, including, but not limited to, a calicheamicin, maytansinoids, dolastatins, aurostatins, a trichothecene, and CC1065, and the derivatives of these toxins that have toxin activity. Suitable cytotoxic agents include, but are not limited to, an auristatin including dovaline-valine-dolaisoleunine-dolaproine- phenylalanine (MMAF) and monomethyl auristatin E (MMAE) as well as ester forms of MMAE, a DNA minor groove binding agent, a DNA minor groove alkylating agent, an enediyne, a lexitropsin, a duocarmycin, a taxane, including paclitaxel and docetaxel, a puromycin, a dolastatin, a maytansinoid, and a vinca alkaloid. Specific cytotoxic agents include topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, dolastatin-10, echinomycin, combretatstatin, chalicheamicin, maytansine, DM-1 , DM-4, netropsin. Other suitable cytotoxic agents include anti-tubulin agents, such as an auristatin, a vinca alkaloid, a podophyllotoxin, a taxane, a baccatin derivative, a cryptophysin, a maytansinoid, a combretastatin, or a dolastatin. Antitubulin agent include dimethylvaline-valine- dolaisoleuine-dolaproine-phenylalanine-p-phenylened- iamine (AFP), MMAF, MMAE, auristatin E, vincristine, vinblastine, vindesine, vinorelbine, VP-16, camptothecin, paclitaxel, docetaxel, epothilone A, epothilone B, nocodazole, colchicines, colcimid, estramustine, cemadotin, discodermolide, maytansine, DM-1 , DM-4 or eleutherobin.

Antibody drug conjugates can be produced by conjugating the small molecule anti-tubulin agent monomethylauristatin E (MMAE) or monomethylauristatin F (MMAF) to the antibodies. In the case of MMAE the linker consists of a thiol-reactive maleimide, a caproyl spacer, the dipeptide valine- citrulline, and p-aminobenzyloxycarbonyl, a self-immolative fragmenting group. In the case of MMAF a protease-resistant maleimidocaproyl linker is used. The conjugation process leads to heterogeneity in drug-antibody attachment, varying in both the number of drugs bound to each antibody molecule (mole ratio [MR]), and the site of attachment. The most prevalent species is the material with an MR = 4; less prevalent are materials with MR of 0, 2, 6, and 8. The overall average drug-to-antibody MR is approximately 4.

The present invention is now described by way of example only. The appended claims may include a generalisation of one of more of the following examples.

Examples

Example 1 : Immunisations and screening of hybridomas. To generate murine monoclonal antibodies with specificity for LRP6, mice were immunised by a variety of methods. Seven groups of SJL Mice were immunised via intraperitoneal or subcutaneous routes using a RIMMS (Rapid immunisation at multiple sites) or conventional protocol. Typically, mice received 5 intraperitoneal immunisations over 3 months for a conventional protocol and 3 subcutaneous immunisations over 12 days for a RIMMs protocol. The mice were euthanized and immune tissues were removed for use in generating hybridomas.

Mice were immunised with a combination of LRP6 proteins consisting of recombinant LRP6-Fc protein (R&D systems cat 1505-LR) and/or LRP6 extracellular domain (ECD) (GSK, in-house) as free protein and/or conjugated to the carrier protein, purified protein derivative (PPD). These immunogens were suspended in a range of adjuvants, including MPL® + TDM Adjuvant (Sigma, M-6536), AS01 b (GSK Rixensart), AS02a (GSK Rixensart) or AS04c adjuvant (GSK Rixensart).

Cells from disrupted immune tissue were fused with myeloma cells and plated out into semi-solid media containing fluorescently-labelled anti-mouse IgG secondary antibody. The resulting hybridomas were grown for 10 days in a humidified incubator at 37°C 5% C0₂. Hybridoma cell colonies for further characterisation were initially identified based on microscopic observation of concentrated, localised fluorescence signal resulting from secondary antibody binding to secreted murine IgG. Colonies were picked using a ClonePix FL instrument into 96 well tissue culture plates containing cell culture media (JRH 610 medium supplemented with 10% FCS, 1 % penicillin / streptomycin and 1 % glutamax (Invitrogen)) and grown for a further seven days with replenishment of medium at day 5. Hybridoma conditioned medium samples were harvested and tested for anti-LRP6 and anti-LRP5 antibody content by ELISA using LRP6 ECD protein (GSK GRITS, in-house) and LRP5-Fc protein (GSK, in- house), respectively. Samples which achieved preferential binding to LRP6 (LRP6:LRP5 ELISA absorbance ratio >0.6) were selected for further analysis.

Hybridomas of interest were expanded and grown in culture medium containing ultra-low bovine IgG foetal bovine serum (low IgG FBS cell culture medium). After 2 days incubation, conditioned medium was removed and used for further characterisation.

Conditioned medium was subjected to ultrafiltration to generate material compatible with the cell- based Topflash assay. After pre-filtration through 1.2μιη glass fibre filter plates (Millipore), samples were applied to 100 KDa MWCO membranes in 96 well deep well block ultrafiltration units (PALL life sciences) and centrifuged with PBS washing.

Example 2: Identification of monoclonal antibodies of interest in ultrafiltered hybridoma conditioned medium For initial identification of monoclonal antibodies with the desired Wnt-signalling modulation characteristics, ultrafiltered conditioned medium from hybridomas identified as being of potential interest in Example 1 were assessed in the Hek293 Topflash Assay for their capacity to modulate Wnt-mediated signalling. The Topflash assay is a well-characterised assay in which a vector encoding luciferase under the control of Wnt response elements provides a gene reporter readout that reflects cellular Wnt signalling by quantitative determination of luciferase expression. Hek293 cells (ATCC) were routinely maintained in RPMI1640 medium supplemented with 10% foetal calf serum, glutamine and Hepes buffer (Gibco). Cells were lifted using cell dissociation buffer (Gibco), washed, resuspended in growth medium and seeded into white 384 well tissue culture plates to achieve 6,000 cells per well in 25 μΙ volume. Cells were incubated overnight in a humidified incubator at 37°C 5% C0₂ prior co-transfection with the Topflash vector (Millipore) and expression plasmids for either Wnt 1 or Wnt 3a (GSK in-house). Briefly, per well, 0.015 μg Topflash and 0.015 μg Wnt expression plasmids were combined in 1.5 μΙ Optimem (Gibco) to which was added 1.5 μΙ optimum containing 0.074 μΙ 293fectin reagent (Invitrogen). The mixture was incubated for 20 minutes at room temperature prior to addition of 7 μΙ growth medium. The resulting 10 μΙ of transfection mixture was added to cells which were typically incubated for 2-4 hours. This protocol was designated the "non-potentiated" assay format. Ultra-filtered hybridoma conditioned medium samples were added to transfected cells in quadruplicate and incubated overnight. Luciferase expression was determined using the Steady-Glo reagent according to the manufacturer's instructions (Promega) and luminescence was detected using a multifunctional plate-reader (Wallac). Data from Wnt 1- and Wnt 3a-transfected cells were analysed to identify ultrafiltered hybridoma conditioned medium samples containing antibodies capable of modulating signalling mediated by either of the ligand classes represented by Wnt 1 and Wnt 3a.

Antagonism of specific Wnt ligands using bivalent anti-LRP6 antibodies or other protein-binding agents has been reported to achieve reciprocal potentiation of alternative ligand class(es) (Ettenberg ef a/, 2010; Gong ef a/, 2010). For example, bivalent antibodies which antagonise Wnt 1-mediated signalling are expected to potentiate Wnt 3a-mediated signalling. Conversely, bivalent antibodies which antagonise Wnt 3a-mediated signalling are expected to potentiate Wnt 1-mediated signalling. Reference control agents with these characteristics were therefore included in the TOPflash assay. H037 is a bivalent LRP6-binding protein that exhibits Wnt 1 antagonism / Wnt 3a potentiation. K020 is a bivalent LRP6-binding protein that exhibits Wnt 3a antagonism / Wnt 1 potentiation. Testing of hybridoma conditioned medium samples identified a single sample, designated "F2" and corresponding to hybridoma cell line S360103E04, that had the capacity to antagonise Wnt 1- and Wnt 3a-mediated signalling (Figure 1A). This hybridoma resulted from the RIMMs immunisation technique as described in Example 1 which comprised immunisation with free LRP6 ECD and PPD- conjugated LRP6-Fc suspended in RIBI adjuvant.

As an alternative to the "non-potentiated format" assay, the "potentiated format" assay was additionally employed to identify mAbs capable of antagonising potentiated Wnt signalling. Thus, cells that had been co-transfected with Topflash and a Wnt 3a-expressing plasmid were treated with 1 ug/ml of (Wnt 3a-potentiating) H037 in addition to test samples. Test samples that were capable of abrogating the potentiation mediated by H037 would therefore be identified as having Wnt 3a- antagonising potential. Conversely, cells that had been co-transfected with Topflash and a Wnt 1- expressing plasmid were treated with 1 ug/ml of (Wnt 1-potentiating) k020 in addition to test samples. Test samples that were capable of abrogating the potentiation mediated by k020 would be identified as having Wnt 1-antagonising potential. This alternate assay format provided an additional, independent means of identifiying novel monoclonal antibodies of interest. Consistent with the outcome of the "non-potentiated" assay, the hybridoma sample designated F2 and corresponding to hybridoma cell line S360103E04 was identified as having the capacity to antagonise potentiated Wnt 1- and Wnt 3a-mediated signalling (Figure 1 B).

A combined total of approximately 1569 hybridomas expressing anti-LRP6 antibodies were generated from the immunisation strategies described in Example 1 , of which approximately 80% were screened in the Topflash assay.

Purification, isotvpinq & RNA extraction

For generation of purified monoclonal antibodies of interest based on Topflash screening, conditioned medium from hybridoma lines including S360103E04 that were maintained in low IgG medium was harvested and centrifuged to remove cellular debris prior to filtration (0.2μιη). Monoclonal antibodies were purified using standard protein-A affinity chromatography using an Akta Xpress (GE Life Sciences). IgG isotype was determined using an isotyping kit (Pierce) according to the manufacturer's instructions. The antibody produced by cell line S360103E04 was determined to be of the murine lgG1/K isotype and retained the designation S360103E04.

To validate the characteristics of the non-purified S360103E04 described above, purified murine antibody was used in the non-potentiated format of the Topflash assay over a concentration range from 5x10^"05 g/ml - 3x10^"11 g/ml and a dose-dependent antagonism of Wnt 1-mediated (Figure 2A) and Wnt 3a-mediated (Figure 2B) signalling was observed, confirming the unexpected dual- antagonist activities of this antibody.

Example 3: Sequencing and Chimerisation of Antibody S360103E04 Antibody RNA was extracted from hybridomas using a robotic RNA preparation method (Promega) and a Biomek 2000 robot. Total RNA was extracted from the S360103E04 hybridoma cell line (3E04) and the cDNA of the heavy and light variable domains was produced by reverse transcription (AccessQuick RT-PCR system, Promega Cat# A1701 ) using primers specific for the leader sequence and the antibody constant regions according to the pre-determined isotype (lgGI/κ). The cDNA of the variable heavy and light domains was then cloned into a plasmid for sequencing. The 3E04 V_H region amino acid sequence is shown in SEQ ID NO: 7. The 3E04 V_L region amino acid sequence is shown in SEQ ID NO: 8. The Kabat CDR sequences for S360103E04 ("3E04") are shown in SEQ ID NO's : 1-6. The chimeric antibody was constructed by taking variable regions from the S360103E04 murine monoclonal antibody (V_H: SEQ ID NO: 7; V_L: SEQ ID NO: 8) and grafting these on to human lgG1/k wild type constant regions. The original murine signal sequences (as shown in SEQ ID NO: 9 and SEQ ID NO: 10 for the heavy chain and light chain, respectively) were utilised in the construction of these constructs. In brief, the murine variable regions were cloned into pTT mammalian expression vectors (National Research Council Canada, with a modified Multiple Cloning Site (MCS)) after RT- PCR using the InFusion HD Cloning Kit (Clontech Cat# 639650). Primers used in the RT-PCR reaction were designed to have nucleotide extensions overlapping with those of the vector ends, allowing for direct InFusion cloning after the RT-PCR procedure. Clones with the correct V_H and V_L sequences were identified and plasmids prepared using standard molecular biology techniques for expression in HEK293/6E cells. The amino acid sequences of the open reading frame of the chimeric antibody is shown in SEQ ID NO: 1 1 (Heavy chain) and SEQ ID NO: 12 (Light chain). Antibodies were purified from the HEK cell supernatant using immobilised Protein-A columns and quantified by reading the absorbance at 280nm. The human lgG1 chimeric mAb was designated DMS10031.

Characterisation of S360103E04 / DMS10031 - Signalling Modulation

The unpotentiated format Topflash assay was used to confirm that the Wnt signalling antagonising properties of S360103E04 had been retained in the chimeric version DMS10031. As expected, treatment with DMS10031 antagonised Wnt 1- or Wnt 3a-mediated signalling in a dose-dependent manner (Figure 3A, 3B).

To further interrogate the observation that S360103E04 and DMS10031 have the unexpected feature of antagonism of both ligand classes, the capacity of DMS10031 to antagonise Wnt signalling in the simultaneous presence of Wnt 1 and Wnt 3a was tested. The non-potentiated Topflash assay using cotransfection of Topflash and a Wnt 1-expression plasmid was modified to include the addition of 100ng/ml final concentration of recombinant Wnt 3a (R&D systems). Treatment with DMS10031 resulted in a dose-dependent antagonism of luminescence signal with no evidence of potentiation, consistent with its capacity to antagonise both ligand classes as a single agent (Figure 4). Example 4: Characterisation of S360103E04 / DMS10031 - orthologue cross-reactivity

Flow cytometry of cells transiently-transfected to express cynomolgous monkey or murine LRP6 was employed to assess cross-reactivity of DMS10031 / S360103E04 for LRP6 from non-human species. HEK2936E cell were transiently transfected with expression vectors encoding cynomolgous or murine full-length LRP6 (GSK) using 293fectin™ (Invitrogen) according to the manufacturer's protocol. Mock transfected cells received irrelevant DNA plasmids. Cells were cultured for 24 to 48 hours and labelled with either irrelevant negative isotype control, anti-HLA Class I (positive control) or chimeric anti-LRP6 antibodies as specified at 10 pg/ml. Primary antibody binding was detected using FITC- conjugated donkey anti-Human IgG at 1 g/ml. Propidium iodide at 2 g/ml final concentration was included in the buffer for final cell suspension to enable discrimination between live and dead cells by flow cytometry.

To determine the degree of cross-reactivity, a gate of 0.5% positivity was set for the negative isotype control histogram and used to determine relative positivity of test antibody-treated cells (Figure 5). Table 3 summarises the degree of cross-reactivity by DMS10031 to both mouse and cynomolgous LRP6 by FACS. DMS10031 showed cross-reactivity to cynomolgous LRP6 while no cross-reactivity was identified to mouse LRP6. Mock transfection did not alter background staining in all experiments in comparison to untransfected HEK2936E cells (data not shown). Table 3. Cross reactivity of DMS10031 with cynomolgous or mouse LRP6 in transiently- transfected HEK2936E cells by flow cytometry.

^*: Figures are given as percentage of positivity Example 5: Characterisation of S360103E04 / DMS10031 - LRP5 non-cross-reactivity

To confirm that the specificity of DMS10031 / S360103E04 is restricted to LRP6 and does not extend to the closely-related protein LRP5, an ELISA was used to assess binding to recombinant LRP5.

0.5 Mg/ml recombinant LRP5 protein (GSK) was coated per well in a 384-well plate and incubated overnight. Dilution ranges (1.67x10^-07 M - 1 .12x10^"13 M) of anti-LRP6 monoclonal antibodies, including a commercial anti-LRP5/LRP6 dual-cross-reactive positive control antibody (Abeam), were added to the plate for 1 hour at room temperature prior to washing with PBS + 0.05% Tween. Antibody binding was detected with a HRP-conjugated anti-mouse antibody (DAKO, 1 in 2000 dilution). HRP was detected using TMB substrate (Sigma-Aldrich) and the reaction stopped using 1 M sulphuric acid stop solution. Absorbance was read at 450nm using a Spectramax plate reader.

Treatment with the positive control antibody resulted in concentration-dependent binding, confirming the integrity of the assay. In contrast and consistent with its specificity for LRP6, S360103E04 monoclonal antibody failed to bind to LRP5 (Figure 6).

To confirm that the observed lack of binding to LRP5 was not the result of an experimental artefact related to use of a recombinant protein, the capacity of DMS10031 / S360103E04 to bind cellular LRP5 was also investigated using cells transfected to express LRP5 and flow cytometry.

HEK293 cells were transfected with an expression plasmid for human LRP5, cynomolgous LRP6 or were mock transfected using a liposome-based transfection reagent (Gemini, GEM-103, GSK) and incubated for 48 hours to allow LRP5 or LRP6 protein expression. Approximately 2.5 E+05 LRP5- or LRP6-transfected cells were incubated with l O g/ml of S360103E04 monoclonal antibody or an irrelevant mouse IgG diluted in FACS buffer (0.5% BSA + 0.1 % Sodium Azide in DPBS) for 60 minutes at 4°C. Cells were washed once in FACS buffer and incubated with a goat anti-mouse IgG conjugated to PE (BD Pharmingen, cat # 550589) for 30 minutes at 4°C. Cells were washed once in FACS buffer and resuspended in 500μΙ of FACS buffer for flow analysis using the FC500 detecting in the FL2 channel.

Analysis of cytometry data showed that S360103E04 bound to approximately 30.1 % of the cynomolgous LRP6-transfected HEK293 cells whereas negligible binding to LRP5-transfected and mock-transfected cells was seen (1.3% and 0.9%, respectively) (Figure 7). No binding was observed with the mouse isotype control in any of the transfected cell lines. These data confirm that S360103E04 does not bind to LRP5.

Example 6: Characterisation of S360103E04 / DMS10031 - epitope competition experiments Due to the unexpected observation that DMS10031 / S360103E04 can achieve antagonism of two ligand classes, we sought to investigate whether it achieves such activity by binding to a unique epitope distinct from the epitopes recognised by prototypical antigen-binding proteins that exhibit reciprocal antagonism and potentiation of alternate Wnt ligand classes. Several such mAbs have been described. For example, Arca135 (Binnerts ef a/, 2009) and YW210.09 (Gong ef a/, 2010; US Patent Application US201 1/0256127 A1 ) are anti-LRP6 antibodies with reported capacity to antagonise Wnt 1-mediated signalling and potentiate Wnt 3a signalling in a manner analogous to the activity of the LRP6-binding protein H037 described in Example 2. Conversely, YW21 1.31.57 and YW21 1.31.62 (Gong ef a/, 2010; US Patent Application US201 1/0256127 A1 ) are anti-LRP6 antibodies with reported capacity to antagonise Wnt 3a-mediated signalling and potentiate Wnt 1- mediated signalling in a manner analogous to the LRP6-binding protein K020 described in Example 2. Competition ELISAs were performed to assess binding of DMS10031 / S360103E04 in the presence or absence of Arca135, YW210.09, YW21 1.31.57, YW21 1.31.62, the Wnt 1 antagonist H037 or the Wnt 3a antagonist k020 to determine whether they bind similar or distinct/non- overlapping epitopes on LRP6. Since Arca135, YW210.09, YW21 1.31.57, YW21 1.31.62, H027 and k020 have a human lgG1 framework, S360103E04 (murine IgG framework) was selected for use in this assay to enable anti-mouse mAb detection to quantitate S360103E04 binding to LRP6 binding. 1 Mg/ml recombinant LRP6 protein was coated per well in a 384-well plate and incubated overnight. After washing, Arca135, YW210.09, YW21 1.31.57, YW21 1.31.62, H027 or k020 ("test mAbs") was added at a fixed concentration of 1.67E-07 M and incubated for 30 minutes. A dilution range of S360103E04 was prepared and added to the plate in equal volume to the test mAb to give final concentration range of 1.667E-07 M - 1.12E-13 M. Plates were incubated for 1 hour at room temperature prior to washing with PBS + 0.05% Tween. Binding of S360103E04 to LRP6 was detected using a HRP-conjugated anti-mouse antibody (DAKO) at 1 in 2000 dilution. The ELISA was developed using TMB substrate (Sigma Aldrich) with 1 M sulphuric acid stop solution. Absorbance was read at 450nm using the Spectramax. S360103E04 monoclonal antibody bound LRP6 in a dose dependent response. Pre-incubation with fixed concentration (1.67E-07 M) of any of the test mAbs failed to prevent binding by S360103E04 (Figure 8). These data are consistent with S360103E04 / DMS10031 achieving dual ligand class antagonism as a result of binding an epitope on LRP6 that is non-overlapping and distinct from those recognised by protypical LRP6 binding proteins that exhibit reciprocal Wnt ligand class antagonism / potentiation e.g. Arca135, YW210.09, YW21 1.31.57, YW21 1.31.62, H027 or k020.

Example 7: Characterisation of DMS10031 / S360103E04 - antagonism of additional Wnt ligands

The binding sites of all known Wnt ligands on LRP6 remain to be fully elucidated. LRP6 domains with binding sites for two classes of Wnts have been described. The two classes of ligand may comprise Wnts 1 , 2, 2b, 6, 8a, 9a, 9b and 10b (denoted herein as Wnt 1 Class and represented by Wnt 1 ) and Wnts 3 and 3a (denoted herein as Wnt 3 Class and represented by Wnt 3a). However, it remains to be established whether one or more of Wnts 4, 7a, 7b and 10a may also be described as members of Class 1 or if they represent a third ligand class (denoted herein as Wnt 7 Class and represented by Wnt7b) which may bind to a ligand binding site or sites on LRP6 distinct from those for ligands of classes 1 and 2. This hypothesis is based, in part, on the observation that the Wnt 3a antagonising/Wnt 1 potentiating mAb YW21 1.31.57 potentiates signalling mediated by putative class 7 ligands yet the Wnt 3a-potent.iat.ing/Wnt 1-antagonising mAb YW210.09 fails to antagonise signalling mediated by putative class 7 ligands (Gong ef a/, 2010; US Patent Application US201 1/0256127 A1 ).

To assess whether the observed antagonism of Wnt 1 Class (represented by Wnt 1 ) and Wnt 3 Class (represented by Wnt 3a) ligands of DMS10031 / S360103E04 extended to antagonism of putative Wnt 7 Class ligands, Topflash assays were employed to assess the capacity of DMS10031 / S360103E04 to modulate Wnt7b-mediated cellular signalling.

HT1080 cells (ATCC) were routinely maintained in EMEM medium (MediaTech) supplemented with 10% fetal bovine serum. Cells were lifted using 0.25% trypsin (SAFC) with 0.1 % EDTA, washed, resuspended in growth medium and seeded into white 96-well tissue culture plates to achieve 30,000 cells per well in 75 μΙ volume. Cells were incubated overnight in a humidified incubator at 37°C 5% C0₂ prior to co-transfection with the Topflash or Fopflash vectors (Millipore) and expression plasmids for, Wnt 1 , Wnt 3a and Wnt7b (GSK in-house). Per well, 0.25 μΙ Lipofectamine 2000 (Invitrogen) was added to 15 ul of OptiMem (Gibco). To this, 0.075 pg Topflash or Fopflash and 0.075 pg Wnt expression plasmids were added. The mixture was incubated for 20 minutes at room temperature prior to addition of 35 μΙ OptiMem with 0.5% fetal bovine serum. Medium was removed from the wells containing plated cells, and the resulting 50 μΙ of transfection mixture was added to cells. Plates were typically incubated for 2-4 hours in a humidified incubator at 37°C 5% C0₂. Purified monoclonal antibodies in PBS were diluted to appropriate concentrations in OptiMem with 0.5% fetal bovine serum. Transfected cells were dosed in triplicate and incubated for 24 hours. Luciferase expression was determined using the Bright-Glo reagent according to the manufacturer's instructions (Promega) and luminescence was detected using a multifunctional plate-reader (Wallac Envision).

Ratios of Topflash:Fopflash data were used to describe modulation of Wnt-mediated signalling. Consistent with data obtained using Hek293 assays, treatment of transfected HT1080 cells with DMS10031 , S360103E04 or the humanised variant DMS10064 (see Example 9) resulted in a dose- dependent antagonism of Wnt 3a-mediated signalling (Figure 9A). The antagonism of Wnt 1-mediated signalling was modest in comparison to that achieved in the Hek293 Topflash assays (Figure 9B). These data suggest that although the magnitude of antagonism of Wnt-mediated signalling by DMS10031 / S360103E04 may be cell line dependent, the unique feature of Wnt 3a-mediated signalling antagonism in the absence of reciprocal potentiation of Wnt 1-mediated signalling is retained across the cell lines tested. Importantly, Wnt signalling mediated by Wnt7b, as a representative member of the putative third ligand class, was not affected by treatment with DMS10031 or S360103E04. In contrast, the Wnt 1 antagonist, YW210.09, and the Wnt 3a antagonist, YW31 1.31.57, both caused potentiation of Wnt7b-mediated signalling (Figure 9C).

Example 8: Characterisation of DM S10031 / S360103E04 - affinity determination

The affinity of DMS10031 (chimeric HulgGI ) and DMS10064 (humanised lgG1 - See Example 9) for LRP6 was determined by surface plasmon resonance.

Hybridoma samples that were identified in the initial ELISA screen were tested by Biacore for binding to LRP6-ECD (GSK GRITS40831 ), LRP5-Fc(GSK GRITS41767) and human IgG protein (Sigma, Technical Grade) respectively.

A protein A CM5 sensorchip was prepared in the Biacore 4000 instrument. The sensorchip was docked into the instrument and run through normalising and hydrodynamic addressing protocols prior to protein immobilisation. Immobilisation of the protein A was achieved using the standard immobilisation program for amine coupling methodology in which sensorchip surface carboxyl groups were activated using (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N- hydroxysuccinimide (NHS). The protein A ligand was injected and covalently immobilised through primary amine groups to the activated carboxyl groups. Any residual activated carboxyl groups were then capped by injecting a 1 M ethanolamine pH8.5 solution. Approximately 3000RU of protein A was immobilised on spots 1 , 2, 4 and 5 in all four flowcells.

Biacore 4000 experiment methodology

Reagents were prepared as follows: HBS-EP buffer: HBS-EP run buffer (Prepared by dilution of BR- 1006-69, GE Healthcare, 0.01 M HEPES pH 7.4, 0.15 M NaCI, 3mM EDTA, 0.005% v/v Surfactant P20); human LRP6-ECD protein (GSK GRITS40831 ) at 20, 10, 5, 2.5 and Opg/ml in HBS-EP buffer; Purified anti-LRP6 antibodies including DMS10031 and DMS10064 using the following sequential protocol at 25°C: 1 ) injection of the antibody sample on to the sensorchip to capture human/chimera IgG (60s); 2) injection of the LRP6-ECD on to the captured antibody sample to observe binding (association 240s, dissociation 600s); 3) regeneration of the sensorchip using two injections of 50mM NaOH for 30s each to remove the captured antibody and LRP6-ECD. Steps 1-3 were repeated for each concentration of LRP6-ECD protein for the respective antibodies.

The Biacore 4000 evaluation software was used to analyse the sensorgram curves. The sensorgrams were "double referenced" using firstly a reference sensorspot in the respective flowcell, then subtracting away the O g/ml LRP6-ECD sensorgram curve for the respective antibody sample. This removed any instrument noise from the antibody sample sensorgrams. The curve-fit was done using the 1 :1 binding model to obtain kinetic rate constants. Representee sensorgrams are shown on Figure 10. Very similar binding kinetic data (Table 4) for the chimeric and humanized antibody variants were obtained.

Table 4 Binding kinetics for DMS10031 and DMS10064

Example 9: Humanisation of DMS10031 / S360103E04 - confirmation of activity in Topflash assay

S360103E04 and DMS10031 are murine and chimeric mAbs, respectively. It is preferable to use humanised monoclonal antibodies as therapeutic agents. The humanisation of DMS10031 was therefore undertaken.

A comparison was made between the sequences of the S360103E04 variable regions and various human immunoglobulin sequences using the BLAST program. A suitable human acceptor framework for the S360103E04 VH was identified (IGHV1-69 and the IGHJ1 human J segment sequence), in addition to a suitable human acceptor framework for the S360103E04 VL (IGKV1-39 and the IGKJ2 human J segment sequence). In CDR grafting, it is typical to require one or more framework residues from the donor antibody to be included in place of their orthologues in the acceptor frameworks in order to obtain satisfactory binding. Plasmids carrying the verified constructs were constructed using standard molecular biology techniques and were expressed in HEK293/6E cells. Antibodies were purified from the HEK cell supernatant using immobilised Protein-A columns and quantified by reading the absorbance at 280 nm.

The same signal peptide was utilised for both the heavy and light chain, the amino acid sequence of which is shown in SEQ ID NO: 15. A single variable light chain was produced according to SEQ ID NO: 14 (full length sequence is shown in SEQ ID NO: 17). A number of different variable heavy humanised sequences were produced, and the designations of the resultant antibodies are indicated in Table 5 below.

A non-potentiated Topflash assay was used to confirm that humanised variants such as DMS10064 retained the capacity to antagonise Wnt 1- and Wnt 3a-mediated signalling (Figure 11 ).

Table 5:

Example 10: Prophetic Example: in vivo characterisation of Wnt signalling antagonism - Phamacodynamics and efficacy

A variety of means is available to determine whether DMS10031 / S360103E04 or associated molecules achieve modulation of Wnt signalling in vivo, including in models of disease. Such experiments have utility in assessing whether these molecules may have therapeutic utility, for example in the treatment of cancer.

To assess whether test molecules can achieve modulation of Wnt signalling in vivo, mice may be implanted with tumour cells in which LRP6 expression and Wnt signalling have been characterised (e.g. MDA-MB-231 ) to generate a solid tumour mass. If human tumour cell lines are used, immunocompromised animals may be employed. Test molecules can be administered in an appropriate diluent (e.g. phosphate buffered saline) by an appropriate route, e.g. intraperitoneal or intravenous, and tumour tissues harvested after an appropriate interval (e.g. 24 hours). Molecular techniques including quantitative polymerase chain reaction methodologies (e.g. qRT-PCR) may be employed to analyse expression levels in the harvested samples of genes known to be responsive to Wnt-mediated signalling, e.g. Axin2 and Sp5, by use of primers specific to genes of interest. Comparison of these data to those from untreated control tumour samples would permit an assessment of the modulation of Wnt signalling achieved by the test molecules. Such studies could be extended to assess the effects of different doses of test molecules and to determine the kinetics of any observed response, for example, by harvesting samples at various intervals following dosing.

Other readouts of LRP6-specific antagonism are also available. For example, quantitative and semiquantitative determination of levels of phosphorylated LRP6 in treated versus untreated samples could be determined using e.g. ELISA or western blot methods employing an antibody or antibodies with specificity for phosphorylated amino acid residues on LRP6 that are known to be phosphorylated as part of Wnt-mediated signalling cascades. A decrease in phosphorylated LRP6 in treated versus untreated samples may be indicative of successful antagonism of Wnt-mediated signalling by test molecules.

Immunohistochemical methods could also be employed, for example, to examine the cellular localisation of beta-catenin in sections of treated tissues. Nuclear localisation of beta-catenin is indicative of active Wnt signalling, where as a relative absence of nuclear and/or predominance of cytosolic beta-catenin in treated versus untreated samples may indicate antagonism of Wnt signalling. The above studies could also be extended to include analysis of the modulation of physiological Wnt signalling in normal tissues for example in intestinal epithelium. Furthermore, such studies may be extended to include non-oncological models where Wnt signalling may be a relevant factor and/or a potential target for therapeutic modulation.

The above studies could be modified to assess the potential of test molecules to achieve efficacy in disease models which may be indicative or predictive for successful therapeutic intervention in human or animal disease. For example, mice bearing established primary tumours (syngeneic or xenografts) resulting from inoculation with tumour cell lines in which LRP6 expression and Wnt signalling have been characterised could be repeatedly treated with test molecules and the kinetics of tumour growth (e.g. determined by physical measurement) compared to that of untreated animals. Such experiments could be performed in a prophylactic setting e.g. when inoculation of tumour cells is coincident with the commencement of treatment, or in a therapeutic setting in which tumour growth is permitted to a threshold size prior to commencement of treatment. Alternative models, including the use of orthotopic primary tumour models, metastatic tumour models or transgenic animals with a predisposition of development of cancer or other diseases of interest may be employed. Where appropriate for the model used, disease progression may be monitored by means other than physical measurement, for example, bioluminescence or other imaging modalities. Alternatively, immunocompromised animals may be inoculated or engrafted with minimally-passaged human patient-derived tumour cells of pieces, which may be considered a more translationally-relevant means of predicting human disease response. Such tumour samples can be prospectively or retrospectively characterised for features including wnt signalling activity, wnt ligand expression and signalling pathway mutational status.

Example 11 : Prophetic Examples for Epitope mapping of DMS10031

The DMS10031 has been shown to bind to the LRP6 protein and to antagonise the binding of both Wnt 1 and Wnt 3a ligands. It has previously been demonstrated that these two ligands bind to spatially distinct sites on the LRP6 tertiary structure. LRP6 is comprised of 4 distinct domains, designated E1-E4, each of which is composed of a series of B-sheets in a B-propeller arrangement. It is believed that the binding sites for the Wnt ligands are located in the clefts between domains E1-E2 and E3-E4 respectively. A feature of antibody mediated LRP6 antagonism is that blocking the binding of one class of Wnt is associated with a reciprocal potentiation on binding of a Wnt of the opposite class, therefore a dual antagonist mAb may be assumed to be mediating its effect via a distinct mechanism since it is likely to be sterically impossible for a single mAb to block both Wnt binding sites simultaneously.

With the availability of a number of mAbs that are known to be competitive with ligands, for which the binding sites are known, it is possible as a minimum to state that a novel mAb is non-competitive with these other mAbs by competition binding analysis. This has been demonstrated for DMS10031 in both an ELISA and an SPR based format, highlighting that the epitope is distinct from those of competitive Wnt antagonist mAbs.

In order to ascertain which residues on the LRP6 protein form the epitope to which the DMS10031 binds, a co-crystal structure of the LRP6 extracellular domain and the Fab fragment of DMS10031 could be solved using conventional crystallography techniques. Since several structures for LRP6 have been solved (i.e. PDB 3S94 & 3S8Z: Cheng, Z. et al (201 1 ) Nat. Struct. Mol. Biol 18: 1204- 1210), 2.8 A resolution & PDB 3S0B; Bourhis et al (201 1 , unpublished), at 1.9 A resolution) and antibody Fab crystal structures are plentiful, it is conceivable that a co-crystal structure of DMS10031 in complex with LRP6 might be determined and thus the key contacts between the antibody CDRs and the LRP6 protein determined. When compared with existing information relating to the binding sites for Wnt ligands on LRP6, such information should be able to distinguish the epitope of

DMS10031 from that of other mAbs that have been shown by other methods to be ligand competitive. In the absence of crystallography data or to address the contention that such structures may be artefactual and not representative of the in vivo interaction, other functional approaches may be employed. For example, cells may be transfected with variants of LRP6 whereby amino acid residues within the putative binding sites for Wnt ligands or the mAb have been converted to alanine residues (so called 'alanine scanning'). The substitution of key amino acids for the relatively simple amino acid alanine is often used to probe structure-activity relationships; the presence of an alanine is considered generally unlikely to cause major disruption to the tertiary structure of the protein, and this can be proven by comparison with the parent molecule. However the affinity shown by binding agents such as mAbs will be impacted if a key contact residue is substituted for an alanine. Such an approach could be used to augment structural data.

As the LRP6 is a modular protein forming 4 distinct functional domains in its extracellular domains, protein engineering techniques may be used to alter the domain architecture in a more wholesale attempt to map epitopes that may be located within domain boundaries. Such profound reshuffling of domain order or deletion of domains may result in failed protein expression or misfolded protein, however if suitable positive controls are available to authenticate the material then the abolition of binding by deletion or rearrangement of a domain may provide strong evidence for that domain being implicated in the antibody epitope. Alternatively, individual domains may be expressed (either alone or in combination) and the binding of the mAb assessed in comparison with binding to the intact protein. Such approaches are also subject to assurances that isolated domains are correctly folded and this may be a more difficult feature to probe if binding sites for ligands and mAbs are not wholly located within the sub-domains being tested.

Sequence Summary (Table A)

CDRH2 for YW31 1.31.57 SEQ.I.D.NO:37 n/a

CDRH3 for YW31 1.31.57 SEQ.I.D.NO:38 n/a

CDRL1 for YW31 1.31.57 SEQ.I.D.NO:39 n/a

CDRL2 for YW31 1.31.57 SEQ.I.D.NO:40 n/a

CDRL3 for YW31 1.31.57 SEQ.I.D.NO:41 n/a

Heavy chain Variable region of SEQ.I.D.NO:42 n/a

YW31 1.31.57

Light chain Variable region of SEQ.I.D.NO:43 n/a

YW31 1.31.57

Heavy chain of YW31 1.31.57 SEQ.I.D.NO:44 SEQ.I.D.NO:45

Light chain of YW31 1.31.57 SEQ.I.D.NO:46 SEQ.I.D.NO:107

CDRH1 for YW31 1.31.62 SEQ.I.D.NO:47 n/a

CDRH2 for YW31 1.31.62 SEQ.I.D.NO:48 n/a

CDRH3 for YW31 1.31.62 SEQ.I.D.NO:49 n/a

CDRL1 for YW31 1.31.62 SEQ.I.D.NO:50 n/a

CDRL2 for YW31 1.31.62 SEQ.I.D.NO:51 n/a

CDRL3 for YW31 1.31.62 SEQ.I.D.NO:52 n/a

Heavy chain Variable region of SEQ.I.D.NO:53 n/a

YW31 1.31.62

Light chain Variable region of SEQ.I.D.NO:54 n/a

YW31 1.31.62

Heavy chain of YW31 1.31.62 SEQ.I.D.NO:55 SEQ.I.D.NO:56

Light chain of YW31 1.31.62 SEQ.I.D.NO:57 SEQ.I.D.NO:58

CDRH1 for YW210.09 SEQ.I.D.NO:59 n/a CDRH2 for YW210.09 SEQ.I.D.NO:60 n/a

CDRH3 for YW210.09 SEQ.I.D.NO:61 n/a

CDRL1 for YW210.09 SEQ.I.D.NO:62 n/a

CDRL2 for YW210.09 SEQ.I.D.NO:63 n/a

CDRL3 for YW210.09 SEQ.I.D.NO:64 n/a

Heavy chain Variable region of SEQ.I.D.NO:65 n/a

YW210.09

Light chain Variable region of SEQ.I.D.NO:66 n/a

YW210.09

Heavy chain of YW210.09 SEQ.I.D.NO:67 SEQ.I.D.NO:68

Light chain of YW210.09 SEQ.I.D.NO:69 SEQ.I.D.NO:70

CDRH1 for ARCA 135-16 and ARCA SEQ.I.D.NO:71 n/a

135-16A

CDRH2 for ARCA 135-16 and ARCA SEQ.I.D.NO:72 n/a

135-16A

CDRH3 for ARCA 135-16 and ARCA SEQ.I.D.NO:73 n/a

135-16A

CDRL1 for ARCA 135-16 and ARCA SEQ.I.D.NO:74 n/a

135-16A

CDRL2 for ARCA 135-16 and ARCA SEQ.I.D.NO:75 n/a

135-16A

CDRL3 for ARCA 135-16 and ARCA SEQ.I.D.NO:76 n/a

135-16A

Heavy chain Variable region of ARCA SEQ.I.D.NO:77 SEQ.I.D.NO:78 135-16A

Heavy chain Variable region of ARCA SEQ.I.D.NO:79 SEQ.I.D.NO:80 135-16 Light chain Variable region of ARCA SEQ.I.D.NO:81 SEQ.I.D.NO:82 135-16

Heavy chain of ARCA 135-16A SEQ.I.D.NO:83 SEQ.I.D.NO:84

Heavy chain of ARCA 135-16 SEQ.I.D.NO:85 SEQ.I.D.NO:86

Light chain of ARCA 135-16 SEQ.I.D.NO:87 SEQ.I.D.NO:88

Humanised variable heavy chain for N/A SEQ.I.D.NO:90 DMS10064

Humanised variable light chain N/A SEQ.I.D.NO:92

Humanised variable heavy chain for SEQ.I.D.NO:93 SEQ.I.D.NO:94 DMS10079

Humanised variable heavy chain for SEQ.I.D. NO:95 SEQ.I.D.NO:96 DMS10080

Humanised variable heavy chain for SEQ.I.D. NO:97 SEQ.I.D. NO:98 DMS10081

Humanised variable heavy chain for SEQ.I.D. NO:99 SEQ.I.D. NO: 100 DMS10082

Humanised variable heavy chain for SEQ.I.D.NO: 101 SEQ.I.D. NO: 102 DMS10083

Humanised variable heavy chain for SEQ.I.D.NO: 103 SEQ.I.D. NO: 104 DMS10084

Humanised variable heavy chain for SEQ.I.D.NO: 105 SEQ.I.D. NO: 106 DMS10085

NB There are no associated sequences for SEQ ID NO:89 or SEQ ID NO:91. SEQUENCE LISTING

SEQ ID 1 - 3E04 CDRH1

SYWMH

SEQ ID NO: 2 - 3E04 CDRH2 RIHPSDSDTNYNQKFKG SEQ ID NO: 3 - 3E04 CDRH3 GYYYDSSYDY SEQ ID NO: 4 - 3E04 CDRL1

RASENIYSYLA SEQ ID NO: 5 - 3E04 CDRL2 NAKTLAE

SEQ ID NO: 6 - 3E04 CDRL3 QHHYGTPLT

SEQ ID NO: 7 - Mouse monoclonal 3E04 VH

QVQLQQPGAELVKPGASVKVSCKASGYTFTSYWMHWVKQRPGQGLEWIGRIHPSDSDTNYNQKFK GKATLTVDKSSSTAFMQLSSLTSEDSAVYYCAIGYYYDSSYDYWGQGTTLTVSS

SEQ ID NO: 8 - Mouse monoclonal 3E04 VL

DIQMTQSPASLSASVGETITITCRASENIYSYLAWYQQKQGKSPQLLVYNAKTLAEGVPSRFSGSGSG TQFSLKINSLQPEDFGSYYCQHHYGTPLTFGAGTKLELK

SEQ ID NO: 9 - Murine signal sequence VH MKCSWVMFFLVATATGVHS SEQ ID NO: 10 - Murine signal sequence VL MKLPVRLVVLLWLTGARC

SEQ ID NO: 1 1 - 3E04 chimera - DMS10031 heavy chain

QVQLQQPGAELVKPGASVKVSCKASGYTFTSYWMHWVKQRPGQGLEWIGRIHPSDSDTNYNQKFK GKATLTVDKSSSTAFMQLSSLTSEDSAVYYCAIGYYYDSSYDYWGQGTTLTVSSAKTTPPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAP

lEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 12 - 3E04 chimera - DMS10031 light chain

DIQMTQSPASLSASVGETITITCRASENIYSYLAWYQQKQGKSPQLLVYNAKTLAEGVPSRFSGSGSG TQFSLKINSLQPEDFGSYYCQHHYGTPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASWCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC

SEQ ID NO: 13 - 3E04 humanised VH

QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIHPSDSDTNYNQKF KGRVTITADKSTSTAYMELSSLRSEDTAVYYCAIGYYYDSSYDYWGQGTLVTVSS

SEQ ID NO: 14 - 3E04 humanised VL

DIQMTQSPSSLSASVGDRVTITCRASENIYSYLAWYQQKPGKAPKLLIYNAKTLAEGVPSRFSGSGSG TDFTLTISSLQPEDFATYYCQHHYGTPLTFGQGTKLEIK

SEQ ID NO: 15 - Signal peptide sequence for the humanised VH and VL

MGWSCIILFLVATATGVHS SEQ ID NO: 16 - Humanised heavy chain for DMS10064

QLVQSGAEVKKPGSSVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIHPSDSDTNYNQKFKG RVTITADKSTSTAYMELSSLRSEDTAVYYCAIGYYYDSSYDYWGQGTLVTVSSASTKGPSVFPLAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 17 - Humanised light chain for DMS10064

DIQMTQSPSSLSASVGDRVTITCRASENIYSYLAWYQQKPGKAPKLLIYNAKTLAEGVPSRFSGSGSG TDFTLTISSLQPEDFATYYCQHHYGTPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC

SEQ ID NO: 18 - Humanised heavy chain for DMS10079 QVQLVQSGAEVKKPGSSVKVSCKASGGTFTSYWMHWVRQAPGQGLEWMGRIHPSDSDTNYNQKF KGRVTITADKSTSTAYMELSSLRSEDTAVYYCAIGYYYDSSYDYWGQGTLVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAP

SEQ ID NO: 19 - Humanised heavy chain for DMS10080 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSSYWMHWVRQAPGQGLEWMGRIHPSDSDTNYNQKF KGRVTITADKSTSTAYMELSSLRSEDTAVYYCAIGYYYDSSYDYWGQGTLVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAP

SEQ ID NO: 20 - Humanised heavy chain for DMS10081 QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIHPSDSDTNYNQKF KGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGYYYDSSYDYWGQGTLVTVSSASTKGPSVFPLA PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 21 - Humanised heavy chain for DMS10082

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYWMHWVRQAPGQGLEWMGRIHPSDSDTNYNQKF KGRVTITADKSTSTAYMELSSLRSEDTAVYYCAIGYYYDSSYDYWGQGTLVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAP

SEQ ID NO: 22 - Humanised heavy chain for DMS10083

QVQLVQSGAEVKKPGSSVKVSCKASGYTFSSYWMHWVRQAPGQGLEWMGRIHPSDSDTNYNQKF KGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGYYYDSSYDYWGQGTLVTVSSASTKGPSVFPLA PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 23 - Humanised heavy chain for DMS10084

QVQLVQSGAEVKKPGSSVKVSCKASGGTFTSYWMHWVRQAPGQGLEWMGRIHPSDSDTNYNQKF KGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGYYYDSSYDYWGQGTLVTVSSASTKGPSVFPLA PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 24 - Humanised heavy chain for DMS10085

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYWMHWVRQAPGQGLEWMGRIHPSDSDTNYNQKF KGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGYYYDSSYDYWGQGTLVTVSSASTKGPSVFPLA PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 25 - 3E04 chimera - DMS10031 heavy chain (polynucleotide)

CAGGTCCAACTGCAGCAGCCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAGTGAAGGTGTCC TGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATGCACTGGGTGAAGCAGAGGCCTGGCC AAGGCCTTGAGTGGATTGGAAGGATTCATCCTTCTGATAGTGATACTAACTACAATCAAAAGTTCA AGGGCAAGGCCACATTGACTGTAGACAAATCCTCCAGCACAGCCTTCATGCAGCTCAGCAGCCT GACATCTGAGGACTCTGCGGTCTATTACTGTGCAATAGGTTATTACTACGATAGTAGCTACGACT ACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCCAAAACAACACCCCCCAGCGTGTTCCC CCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGG ACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACA CCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCA GCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGT GGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCC CGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGAT CAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAA GTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCA GTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGG CAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGC AAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAGCTG ACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTG GAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGC GATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAAC GTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCC TGTCCCCTGGCAAG

SEQ ID NO: 26 - 3E04 chimera - DMS10031 light chain (polynucleotide)

GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTATCACCATCAC ATGTCGAGCAAGTGAGAATATTTACAGTTATTTAGCATGGTATCAGCAGAAACAGGGAAAATCTC CTCAGCTCCTGGTCTATAATGCAAAAACCTTAGCAGAAGGTGTGCCATCAAGGTTCAGTGGCAGT GGATCAGGCACACAGTTTTCTCTGAAGATCAACAGCCTGCAGCCTGAAGATTTTGGGAGTTATTA CTGTCAACATCATTATGGTACTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGT ACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGCAGCTGAAGAGCGGCACC GCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTG GACAATGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACTC CACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTA CGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACCGGGGCGA GTGC

SEQ ID NO: 27 - Humanised heavy chain for DMS10064 (polynucleotide)

CAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCAGCAGCGTGAAGGTGAGCTGCAA GGCCAGCGGCTACACCTTCACCAGCTACTGGATGCACTGGGTGAGGCAGGCCCCCGGCCAGG GCCTGGAGTGGATGGGCAGGATCCACCCCAGCGACAGCGACACCAACTACAACCAGAAGTTCA AGGGCAGGGTGACCATCACCGCCGACAAGAGCACCAGCACCGCCTACATGGAGCTGAGCAGCC TGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCATCGGCTACTACTACGACAGCAGCTACG ACTACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGCGCCAGCACCAAGGGCCCCAGCGTGT TCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTG AAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCAGCGGCGTG CACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTG CCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCA AGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTG CCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGA TGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGG TGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGG AGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGA ACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGAAAACCAT CAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGA GCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGC CGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGA CAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGG CAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTG AGCCTGTCCCCTGGCAAG

SEQ ID NO: 28 - Humanised light chain for DMS10064 (polynucleotide)

GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGGGTGACCATC ACCTGCAGGGCCAGCGAGAACATCTACAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCAAG GCCCCCAAGCTGCTGATCTACAACGCCAAGACCCTGGCCGAGGGCGTGCCCAGCAGGTTCAGC GGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCC ACCTACTACTGCCAGCACCACTACGGCACCCCCCTGACCTTCGGCCAGGGCACCAAGCTGGAG ATCAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGCAGCTGAAG AGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAG TGGAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAG CAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCA CAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAA CCGGGGCGAGTGC

SEQ ID NO: 29 - Humanised heavy chain for DMS10079 (polynucleotide)

CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCAGCAGCGTGAAGGTGAG CTGCAAGGCCAGCGGCGGCACCTTCACCAGCTACTGGATGCACTGGGTGAGGCAGGCCCCCG GCCAGGGCCTGGAGTGGATGGGCAGGATCCACCCCAGCGACAGCGACACCAACTACAACCAGA AGTTCAAGGGCAGGGTGACCATCACCGCCGACAAGAGCACCAGCACCGCCTACATGGAGCTGA GCAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCATCGGCTACTACTACGACAGCA GCTACGACTACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGCGCCAGCACCAAGGGCCCCA GCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGC CTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCAGC GGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTG ACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCCAGC AACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCC TGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGAC ACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGAC CCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCC AGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGAT TGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGA AAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCA GAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCG ACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTG TGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCA GCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAA GAGCCTGAGCCTGTCCCCTGGCAAG

SEQ ID NO: 30 - Humanised heavy chain for DMS10080 (polynucleotide)

CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCAGCAGCGTGAAGGTGAG CTGCAAGGCCAGCGGCTACACCTTCAGCAGCTACTGGATGCACTGGGTGAGGCAGGCCCCCGG CCAGGGCCTGGAGTGGATGGGCAGGATCCACCCCAGCGACAGCGACACCAACTACAACCAGAA GTTCAAGGGCAGGGTGACCATCACCGCCGACAAGAGCACCAGCACCGCCTACATGGAGCTGAG CAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCATCGGCTACTACTACGACAGCAG CTACGACTACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGCGCCAGCACCAAGGGCCCCAG CGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCC TGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCAGCG GCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGA CCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCCAGCA ACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCT GCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACA CCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACC CTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCA GGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATT GGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGA AAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCA GAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCG ACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTG TGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCA GCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAA GAGCCTGAGCCTGTCCCCTGGCAAG

SEQ ID NO: 31 - Humanised heavy chain for DMS10081 (polynucleotide)

CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCAGCAGCGTGAAGGTGAG CTGCAAGGCCAGCGGCTACACCTTCACCAGCTACTGGATGCACTGGGTGAGGCAGGCCCCCGG CCAGGGCCTGGAGTGGATGGGCAGGATCCACCCCAGCGACAGCGACACCAACTACAACCAGAA GTTCAAGGGCAGGGTGACCATCACCGCCGACAAGAGCACCAGCACCGCCTACATGGAGCTGAG CAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCTACTACTACGACAGCAG CTACGACTACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGCGCCAGCACCAAGGGCCCCAG CGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCC TGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCAGCG GCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGA CCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCCAGCA ACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCT GCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACA CCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACC CTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCA GGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATT GGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGA AAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCA GAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCG ACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTG TGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCA GCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAA GAGCCTGAGCCTGTCCCCTGGCAAG

SEQ ID NO: 32 - Humanised heavy chain for DMS10082 (polynucleotide) CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCAGCAGCGTGAAGGTGAG CTGCAAGGCCAGCGGCGGCACCTTCAGCAGCTACTGGATGCACTGGGTGAGGCAGGCCCCCG GCCAGGGCCTGGAGTGGATGGGCAGGATCCACCCCAGCGACAGCGACACCAACTACAACCAGA AGTTCAAGGGCAGGGTGACCATCACCGCCGACAAGAGCACCAGCACCGCCTACATGGAGCTGA GCAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCATCGGCTACTACTACGACAGCA GCTACGACTACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGCGCCAGCACCAAGGGCCCCA GCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGC CTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCAGC GGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTG ACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCCAGC AACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCC TGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGAC ACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGAC CCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCC AGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGAT TGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGA AAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCA GAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCG ACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTG TGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCA GCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAA GAGCCTGAGCCTGTCCCCTGGCAAG

SEQ ID NO: 33 - Humanised heavy chain for DMS10083 (polynucleotide)

CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCAGCAGCGTGAAGGTGAG CTGCAAGGCCAGCGGCTACACCTTCAGCAGCTACTGGATGCACTGGGTGAGGCAGGCCCCCGG CCAGGGCCTGGAGTGGATGGGCAGGATCCACCCCAGCGACAGCGACACCAACTACAACCAGAA GTTCAAGGGCAGGGTGACCATCACCGCCGACAAGAGCACCAGCACCGCCTACATGGAGCTGAG CAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCTACTACTACGACAGCAG CTACGACTACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGCGCCAGCACCAAGGGCCCCAG CGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCC TGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCAGCG GCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGA CCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCCAGCA ACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCT GCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACA CCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGACC CTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCA GGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATT GGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGA AAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCA GAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCG ACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTG TGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCA GCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAA GAGCCTGAGCCTGTCCCCTGGCAAG

SEQ ID NO: 34 - Humanised heavy chain for DMS10084 (polynucleotide)

CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCAGCAGCGTGAAGGTGAG CTGCAAGGCCAGCGGCGGCACCTTCACCAGCTACTGGATGCACTGGGTGAGGCAGGCCCCCG GCCAGGGCCTGGAGTGGATGGGCAGGATCCACCCCAGCGACAGCGACACCAACTACAACCAGA AGTTCAAGGGCAGGGTGACCATCACCGCCGACAAGAGCACCAGCACCGCCTACATGGAGCTGA GCAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCTACTACTACGACAGCA GCTACGACTACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGCGCCAGCACCAAGGGCCCCA GCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGC CTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCAGC GGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTG ACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCCAGC AACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCC TGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGAC ACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGAC CCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCC AGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGAT TGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGA AAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCA GAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCG ACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTG TGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCA GCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAA GAGCCTGAGCCTGTCCCCTGGCAAG

SEQ ID NO: 35 - Humanised heavy chain for DMS10085 (polynucleotide)

CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCAGCAGCGTGAAGGTGAG CTGCAAGGCCAGCGGCGGCACCTTCAGCAGCTACTGGATGCACTGGGTGAGGCAGGCCCCCG GCCAGGGCCTGGAGTGGATGGGCAGGATCCACCCCAGCGACAGCGACACCAACTACAACCAGA AGTTCAAGGGCAGGGTGACCATCACCGCCGACAAGAGCACCAGCACCGCCTACATGGAGCTGA GCAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCTACTACTACGACAGCA GCTACGACTACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGCGCCAGCACCAAGGGCCCCA GCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGC CTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCAGC GGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTG ACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCCAGC AACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCC TGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGAC ACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAGGAC CCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCC AGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGAT TGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAGA AAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCA GAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCG ACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTG TGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCA GCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAA GAGCCTGAGCCTGTCCCCTGGCAAG

SEQ.I.D.NO:36 - CDRH 1 for YW31 1.31.57

SYYIS

SEQ.I.D.NO:37 - CDRH2 for YW31 1.31.57 EISPYSGSTYYADSVKG

SEQ.I.D.NO:38 - CDRH3 for YW31 1.31.57 RARPPIRLYPRGSVMDY

SEQ.I.D.NO:39 - CDRL1 for YW31 1.31.57 RASQDVSTAVA SEQ.I.D.NO:40 - CDRL2 for YW31 1.31.57 SASFLYS

SEQ.I.D.NO:41 - CDRL3 for YW31 1.31.57 QQSYTLPTT

SEQ.I.D.NO:42 - Heavy chain Variable region of YW31 1.31.57 EVQLVESGGGLVQPGGSLRLSCAASGFTFTSYYISWVRQAPGKGLEWVAEISPYSGSTYYADSVKG RFTISADTSKNTAYLQMNSLRAEDTAVYYCALRARPPIRLYPRGSVMDYWGQGTLVTVSS

SEQ.I.D.NO:43 - Light chain Variable region of YW31 1.31.57 DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQSYTLPTTFGQGTKVEIK

SEQ.I.D.NO:44 - Heavy chain of YW31 1.31.57 EVQLVESGGGLVQPGGSLRLSCAASGFTFTSYYISWVRQAPGKGLEWVAEISPYSGSTYYADSVKG RFTISADTSKNTAYLQMNSLRAEDTAVYYCALRARPPIRLYPRGSVMDYWGQGTLVTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ.I.D.NO:45 - Heavy chain of YW31 1.31.57 (polynucleotide) GAGGTGCAGCTGGTGGAGAGCGGCGGAGGCCTGGTGCAGCCCGGCGGCAGCCTCAGGCTGA GCTGCGCCGCCAGCGGCTTCACCTTCACCAGCTACTACATCAGCTGGGTCAGACAGGCCCCCG GCAAGGGACTCGAGTGGGTGGCCGAGATCAGCCCCTACAGCGGCTCCACTTACTACGCCGACA GCGTGAAGGGCAGGTTCACCATCAGCGCCGACACCAGCAAGAACACCGCCTACCTGCAAATGA ACTCTCTGAGGGCCGAGGACACTGCCGTGTACTACTGCGCCCTGAGGGCTAGGCCTCCCATCA GGCTGTACCCCAGGGGCAGCGTGATGGACTACTGGGGCCAGGGCACACTAGTGACCGTGTCCA GCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGC GGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGG AACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCT GTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTG TAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGA CAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCT GTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGT GGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGT GCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGT GCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAA GGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCA GGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCT GGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGA ACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCT GACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGC CCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAG

SEQ.I.D.NO:46 - Light chain of YW31 1.31.57 DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQSYTLPTTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC SEQ.I.D.NO:47 - CDRH 1 for YW31 1.31.62

YYYIS

SEQ.I.D.NO:48 - CDRH2 for YW31 1.31.62

EISPYSGSTYYADSVKG

SEQ.I.D.NO:49 - CDRH3 for YW31 1.31.62

RARPPIRLHPRGSVMDY SEQ.I.D.NO:50 - CDRL1 for YW31 1.31.62

R A S Q D V S T A V A

SEQ.I.D.NO:51 - CDRL2 for YW31 1.31.62 S A S F L Y S SEQ.I.D.NO:52 - CDRL3 for YW31 1.31.62 Q Q S Y T T P P T

SEQ.I.D.NO:53 - Heavy chain Variable region of YW31 1.31.62 EVQLVESGGGLVQPGGSLRLSCAASGFTFGYYYISWVRQAPGKGLEWVAEISPYSGSTYYADSVKG RFTISADTSKNTAYLQMNSLRAEDTAVYYCALRARPPIRLHPRGSVMDYWGQGTLVTVSS

SEQ.I.D.NO:54 - Light chain Variable region of YW31 1.31.62

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKVEIK

SEQ.I.D.NO:55 - Heavy chain of YW31 1.31.62

EVQLVESGGGLVQPGGSLRLSCAASGFTFGYYYISWVRQAPGKGLEWVAEISPYSGSTYYADSVKG RFTISADTSKNTAYLQMNSLRAEDTAVYYCALRARPPIRLHPRGSVMDYWGQGTLVTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ.I.D.NO:56 - Heavy chain of YW31 1.31.62 (polynucleotide)

GAGGTGCAGCTGGTGGAGAGCGGCGGAGGCCTGGTGCAGCCCGGCGGCAGCCTCAGGCTGA GCTGCGCCGCAAGCGGCTTCACCTTCGGCTACTACTACATCAGCTGGGTGAGGCAGGCTCCCG GCAAGGGCCTGGAGTGGGTGGCCGAGATCAGCCCCTACAGCGGCAGCACCTACTACGCCGACT CCGTGAAGGGCCGCTTCACCATCAGCGCCGACACCAGCAAGAACACCGCCTACCTGCAAATGA ACTCTCTGAGGGCCGAGGACACCGCCGTGTACTATTGCGCCCTGAGGGCCAGGCCTCCCATCA GGCTGCACCCCAGGGGCAGCGTGATGGACTACTGGGGCCAGGGCACACTAGTGACCGTGTCCA GCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGC GGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGG AACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCT GTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTG TAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGA CAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCT GTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGT GGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGT GCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGT GCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAA GGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCA GGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCT GGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGA ACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCT GACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGC CCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAG

SEQ.I.D.NO:57 - Light chain of YW31 1.31.62

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC

SEQ.I.D.NO:58 - Light chain of YW31 1.31.62 (polynucleotide)

GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCAAGCGTCGGAGACAGGGTGACCATT ACCTGCAGGGCCAGCCAGGACGTGAGCACCGCCGTGGCCTGGTATCAGCAGAAACCCGGCAA GGCCCCCAAGCTGCTGATCTACAGCGCCAGCTTCCTGTACAGCGGCGTGCCCTCTAGGTTTAGC GGCAGCGGCAGCGGCACTGACTTCACCCTCACCATCTCCAGCCTGCAGCCCGAGGACTTCGCC ACCTACTACTGCCAGCAGAGCTACACCACCCCTCCCACCTTCGGCCAGGGCACCAAGGTGGAG ATCAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGCAGCTGAAG AGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAG TGGAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAG CAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCA CAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAA CCGGGGCGAGTGC SEQ.I.D.NO:59 - CDRH 1 for YW210.09

NSYIH

SEQ.I.D.NO:60 - CDRH2 for YW210.09

WITPYGGYTNYADSVKG SEQ.I.D.NO:61 - CDRH3 for YW210.09 GSGHVNAVKNYGYVMDY

SEQ.I.D.NO:62 - CDRL1 for YW210.09 RASQDVSTAVA SEQ.I.D.NO:63 - CDRL2 for YW210.09

SASFLYS SEQ.I.D.NO:64 - CDRL3 for YW210.09 QQSYTTPPT

SEQ.I.D.NO:65 - Heavy chain Variable region of YW210.09

EVQLVESGGGLVQPGGSLRLSCAASGFTFTNSYIHWVRQAPGKGLEWVGWITPYGGYTNYADSVK GRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGSGHVNAVKNYGYVMDYWGQGTLVTVSS

SEQ.I.D.NO:66 - Light chain Variable region of YW210.09

SEQ.I.D.NO:67 - Heavy chain of YW210.09

EVQLVESGGGLVQPGGSLRLSCAASGFTFTNSYIHWVRQAPGKGLEWVGWITPYGGYTNYADSVK G RFTI SADTSKNTAYLQ M NSLRAE DTAVYYCARGSG HVN AVKN YG YVMD YWGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ.I.D.NO:68 - Heavy chain of YW31 1.31.62 (polynucleotide)

GAGGTGCAGCTGGTGGAGAGCGGCGGAGGCCTGGTGCAGCCCGGCGGCAGCCTCAGGCTGA GCTGCGCCGCTAGCGGCTTCACCTTCACCAACAGCTACATCCACTGGGTGAGGCAGGCCCCCG GCAAAGGACTGGAGTGGGTGGGCTGGATCACCCCCTACGGCGGCTACACCAACTACGCCGACA GCGTCAAGGGGAGGTTCACCATCAGCGCCGACACCAGCAAGAACACCGCCTACCTGCAAATGA ACTCTCTGAGGGCCGAGGACACTGCCGTGTACTATTGCGCCAGGGGCTCCGGCCACGTGAACG CCGTGAAGAACTACGGCTACGTGATGGACTACTGGGGCCAGGGCACACTAGTGACCGTGTCCA GCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGC GGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGG AACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCT GTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTG TAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGA CAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCT GTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGT GGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGT GCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGT GCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAA GGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCA GGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCT GGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGA ACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCT GACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGC CCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAG SEQ.I.D.NO:69 - Light chain of YW210.09

SEQ.I.D.NO:70 - Light chain of YW31 1.31.62 (polynucleotide)

GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCAAGCGTCGGAGACAGGGTGACCATT

ACCTGCAGGGCCAGCCAGGACGTGAGCACCGCCGTGGCCTGGTATCAGCAGAAACCCGGCAA

GGCCCCCAAGCTGCTGATCTACAGCGCCAGCTTCCTGTACAGCGGCGTGCCCTCTAGGTTTAGC GGCAGCGGCAGCGGCACTGACTTCACCCTCACCATCTCCAGCCTGCAGCCCGAGGACTTCGCC ACCTACTACTGCCAGCAGAGCTACACCACCCCTCCCACCTTCGGCCAGGGCACCAAGGTGGAG ATCAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGCAGCTGAAG AGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAG TGGAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAG CAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCA CAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAA CCGGGGCGAGTGC

SEQ.I.D.NO:71 - CDRH1 for Area 135-16 and ARCA 135-16A

SYPIE

SEQ.I.D.NO:72 - CDRH2 for Area 135-16 and ARCA 135-16A

NFHPYNDNTKYNEKFKG SEQ.I.D.N073 - CDRH3 for Area 135-16 and ARCA 135-16A GYSGNYFSAMDY

SEQ.I.D.N074 - CDRL1 for Area 135-16 and ARCA 135-16A KASQNVRNDVA SEQ.I.D.N075 - CDRL2 for Area 135-16 and ARCA 135-16A LASNRHT

SEQ.I.D.N076 - CDRL3 for Area 135-16 and ARCA 135-16A

LQHWNYPYT

SEQ.I.D.N077 - Heavy chain Variable region of Area 135-16A QVQLQQSGAELVKPGASVKMSCKAFGYTFTSYPIEWMKQNHGKSLEWIGNFHPYNDNTKYNEKFKG KAKLTVEKSSSTVYLELSRSTSDDSAVYYCARGYSGNYFSAMDYWGQGTLVTVSS

SEQ.I.D.N078 - Heavy chain Variable region of Area 135-16A (polynucleotide) CAGGTGCAGCTGCAGCAGAGCGGAGCCGAACTGGTGAAGCCCGGCGCTAGCGTGAAGATGAG CTGCAAGGCCTTCGGCTACACCTTCACCAGCTACCCCATCGAGTGGATGAAGCAGAACCACGGC AAGAGCCTGGAGTGGATCGGCAATTTCCACCCCTACAACGACAACACCAAATACAACGAGAAGT TCAAGGGCAAGGCCAAGCTGACCGTGGAGAAGAGCAGCTCCACCGTCTATCTGGAGCTGAGCA GGAGCACCAGCGACGACAGCGCCGTGTACTATTGCGCCAGGGGGTACAGCGGCAACTACTTTA GCGCCATGGACTACTGGGGCCAGGGAACACTAGTGACCGTGTCCAGC

SEQ.I.D.N079 - Heavy chain Variable region of Area 135-16

QVQLQQSGAELVKPGASVKMSCKAFGYTFTSYPIEWMKQNHGKSLEWIGNFHPYNDNTKYNEKFKG KAKLTVEKSSSTVYLELSRSTSDDSAVYYCARGYSGNYFSAMDYWGQGTSVTVSS

SEQ.I.D.NO:80 - Heavy chain Variable region of Area 135-16 (polynucleotide)

CAGGTGCAGCTGCAGCAGAGCGGAGCCGAACTGGTGAAGCCCGGCGCTAGCGTGAAGATGAG CTGCAAGGCCTTCGGCTACACCTTCACCAGCTACCCCATCGAGTGGATGAAGCAGAACCACGGC AAGAGCCTGGAGTGGATCGGCAATTTCCACCCCTACAACGACAACACCAAATACAACGAGAAGT TCAAGGGCAAGGCCAAGCTGACCGTGGAGAAGAGCAGCTCCACCGTCTATCTGGAGCTGAGCA GGAGCACCAGCGACGACAGCGCCGTGTACTATTGCGCCAGGGGGTACAGCGGCAACTACTTTA GCGCCATGGACTACTGGGGCCAGGGAACATCAGTGACCGTGTCCAGC

SEQ.I.D.NO:81 - Light chain Variable region of Area 135-16A

DIVMTQSQKFMSTSVGDRVSITCKASQNVRNDVAWYQQKPGQSPKSLIYLASNRHTGVPDRFTGSG SGTDFTLTISNVQSEDLADYFCLQHWNYPYTFGGGTKLEIK

SEQ.I.D.NO:82 - Light chain Variable region of Area 135-16A (polynucleotide)

GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCAC CTGCAAGGCCAGTCAGAATGTTCGTAATGATGTAGCCTGGTATCAACAGAAACCAGGGCAGTCT CCTAAATCACTGATTTACTTGGCATCCAACCGGCACACTGGAGTCCCTGATCGCTTCACAGGCAG TGGATCTGGGACAGATTTCACTCTCACCATTAGCAATGTGCAATCTGAAGACCTGGCAGATTATT TCTGTCTGCAACATTGGAATTATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA

SEQ.I.D.NO:83 - Heavy chain of Area 135-16A

QVQLQQSGAELVKPGASVKMSCKAFGYTFTSYPIEWMKQNHGKSLEWIGNFHPYNDNTKYNEKFKG KAKLTVEKSSSTVYLELSRSTSDDSAVYYCARGYSGNYFSAMDYWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ.I.D.NO:84 - Heavy chain of Area 135-16A (polynucleotide) CAGGTGCAGCTGCAGCAGAGCGGAGCCGAACTGGTGAAGCCCGGCGCTAGCGTGAAGATGAG CTGCAAGGCCTTCGGCTACACCTTCACCAGCTACCCCATCGAGTGGATGAAGCAGAACCACGGC AAGAGCCTGGAGTGGATCGGCAATTTCCACCCCTACAACGACAACACCAAATACAACGAGAAGT TCAAGGGCAAGGCCAAGCTGACCGTGGAGAAGAGCAGCTCCACCGTCTATCTGGAGCTGAGCA GGAGCACCAGCGACGACAGCGCCGTGTACTATTGCGCCAGGGGGTACAGCGGCAACTACTTTA GCGCCATGGACTACTGGGGCCAGGGAACACTAGTGACCGTGTCCAGCGCCAGCACCAAGGGCC CCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGC TGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTGACC AGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTG GTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCC AGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCC CCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAG GACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAG GACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAG CCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAG GATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCG AGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTA GCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCA GCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCC CTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATG GCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCA GAAGAGCCTGAGCCTGTCCCCTGGCAAG

SEQ.I.D.NO:85 - Heavy chain of Area 135-16 QVQLQQSGAELVKPGASVKMSCKAFGYTFTSYPIEWMKQNHGKSLEWIGNFHPYNDNTKYNEKFKG KAKLTVEKSSSTVYLELSRSTSDDSAVYYCARGYSGNYFSAMDYWGQGTSVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ.I.D.NO:86 - Heavy chain of Area 135-16 (polynucleotide) CAGGTGCAGCTGCAGCAGAGCGGAGCCGAACTGGTGAAGCCCGGCGCTAGCGTGAAGATGAG CTGCAAGGCCTTCGGCTACACCTTCACCAGCTACCCCATCGAGTGGATGAAGCAGAACCACGGC AAGAGCCTGGAGTGGATCGGCAATTTCCACCCCTACAACGACAACACCAAATACAACGAGAAGT TCAAGGGCAAGGCCAAGCTGACCGTGGAGAAGAGCAGCTCCACCGTCTATCTGGAGCTGAGCA GGAGCACCAGCGACGACAGCGCCGTGTACTATTGCGCCAGGGGGTACAGCGGCAACTACTTTA GCGCCATGGACTACTGGGGCCAGGGAACATCAGTGACCGTGTCCAGCGCCAGCACCAAGGGCC CCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGC TGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACCGTGTCCTGGAACAGCGGAGCCCTGACC AGCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTG GTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCC AGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCC CCCTGCCCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAG GACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCCACGAG GACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAG CCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAG GATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCG AGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTA GCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCA GCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCC CTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATG GCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCA GAAGAGCCTGAGCCTGTCCCCTGGCAAG

SEQ.I.D.NO:87- Light chain of Area 135-16

DIVMTQSQKFMSTSVGDRVSITCKASQNVRNDVAWYQQKPGQSPKSLIYLASNRHTGVPDRFTGSG SGTDFTLTISNVQSEDLADYFCLQHWNYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC

SEQ.I.D.NO:88 - Light chain of Area 135-16 (polynucleotide)

GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCAC CTGCAAGGCCAGTCAGAATGTTCGTAATGATGTAGCCTGGTATCAACAGAAACCAGGGCAGTCT CCTAAATCACTGATTTACTTGGCATCCAACCGGCACACTGGAGTCCCTGATCGCTTCACAGGCAG TGGATCTGGGACAGATTTCACTCTCACCATTAGCAATGTGCAATCTGAAGACCTGGCAGATTATT TCTGTCTGCAACATTGGAATTATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACG GACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGCAGCTGAAGAGCGGCAC CGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGT GGACAATGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACT CCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGT ACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACCGGGGCG AGTGC

SEQ.I.D.NO:89 - Humanised variable heavy chain for DMS10064 QLVQSGAEVKKPGSSVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIHPSDSDTNYNQKFKG RVTITADKSTSTAYMELSSLRSEDTAVYYCAIGYYYDSSYDYWGQGTLVTVSS

SEQ.I.D.NO:90 - Humanised variable heavy chain for DMS10064 (polynucleotide)

CAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCAGCAGCGTGAAGGTGAGCTGCAA GGCCAGCGGCTACACCTTCACCAGCTACTGGATGCACTGGGTGAGGCAGGCCCCCGGCCAGG GCCTGGAGTGGATGGGCAGGATCCACCCCAGCGACAGCGACACCAACTACAACCAGAAGTTCA AGGGCAGGGTGACCATCACCGCCGACAAGAGCACCAGCACCGCCTACATGGAGCTGAGCAGCC TGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCATCGGCTACTACTACGACAGCAGCTACG ACTACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGC SEQ.I.D.NO:91 - Humanised variable light chain

DIQMTQSPSSLSASVGDRVTITCRASENIYSYLAWYQQKPGKAPKLLIYNAKTLAEGVPSRFSGSGSG TDFTLTISSLQPEDFATYYCQHHYGTPLTFGQGTKLEIK SEQ.I.D.NO:92 - Humanised variable light chain (polynucleotide)

GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGGGTGACCATC ACCTGCAGGGCCAGCGAGAACATCTACAGCTACCTGGCCTGGTACCAGCAGAAGCCCGGCAAG GCCCCCAAGCTGCTGATCTACAACGCCAAGACCCTGGCCGAGGGCGTGCCCAGCAGGTTCAGC GGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCC ACCTACTACTGCCAGCACCACTACGGCACCCCCCTGACCTTCGGCCAGGGCACCAAGCTGGAG ATCAAG

SEQ.I.D.NO:93 - Humanised variable heavy chain for DMS10079

QVQLVQSGAEVKKPGSSVKVSCKASGGTFTSYWMHWVRQAPGQGLEWMGRIHPSDSDTNYNQKF KGRVTITADKSTSTAYMELSSLRSEDTAVYYCAIGYYYDSSYDYWGQGTLVTVSS

SEQ.I.D.NO:94 - Humanised variable heavy chain for DMS10079 (polynucleotide)

CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCAGCAGCGTGAAGGTGAG CTGCAAGGCCAGCGGCGGCACCTTCACCAGCTACTGGATGCACTGGGTGAGGCAGGCCCCCG GCCAGGGCCTGGAGTGGATGGGCAGGATCCACCCCAGCGACAGCGACACCAACTACAACCAGA AGTTCAAGGGCAGGGTGACCATCACCGCCGACAAGAGCACCAGCACCGCCTACATGGAGCTGA GCAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCATCGGCTACTACTACGACAGCA GCTACGACTACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGC

SEQ.I.D.NO:95 - Humanised variable heavy chain for DMS10080

QVQLVQSGAEVKKPGSSVKVSCKASGYTFSSYWMHWVRQAPGQGLEWMGRIHPSDSDTNYNQKF KGRVTITADKSTSTAYMELSSLRSEDTAVYYCAIGYYYDSSYDYWGQGTLVTVSS

SEQ.I.D.NO:96 - Humanised variable heavy chain for DMS10080 (polynucleotide)

CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCAGCAGCGTGAAGGTGAG CTGCAAGGCCAGCGGCTACACCTTCAGCAGCTACTGGATGCACTGGGTGAGGCAGGCCCCCGG CCAGGGCCTGGAGTGGATGGGCAGGATCCACCCCAGCGACAGCGACACCAACTACAACCAGAA GTTCAAGGGCAGGGTGACCATCACCGCCGACAAGAGCACCAGCACCGCCTACATGGAGCTGAG CAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCATCGGCTACTACTACGACAGCAG CTACGACTACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGC SEQ.I.D.NO:97 - Humanised variable heavy chain for DMS10081

QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIHPSDSDTNYNQKF KGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGYYYDSSYDYWGQGTLVTVSS

SEQ.I.D.NO:98 - Humanised variable heavy chain for DMS10081 (polynucleotide)

CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCAGCAGCGTGAAGGTGAG CTGCAAGGCCAGCGGCTACACCTTCACCAGCTACTGGATGCACTGGGTGAGGCAGGCCCCCGG CCAGGGCCTGGAGTGGATGGGCAGGATCCACCCCAGCGACAGCGACACCAACTACAACCAGAA GTTCAAGGGCAGGGTGACCATCACCGCCGACAAGAGCACCAGCACCGCCTACATGGAGCTGAG CAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCTACTACTACGACAGCAG CTACGACTACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGC SEQ.I.D.NO:99 - Humanised variable heavy chain for DMS10082

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYWMHWVRQAPGQGLEWMGRIHPSDSDTNYNQKF KGRVTITADKSTSTAYMELSSLRSEDTAVYYCAIGYYYDSSYDYWGQGTLVTVSS SEQ.I.D.NO:100 - Humanised variable heavy chain for DMS10082 (polynucleotide)

CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCAGCAGCGTGAAGGTGAG CTGCAAGGCCAGCGGCGGCACCTTCAGCAGCTACTGGATGCACTGGGTGAGGCAGGCCCCCG GCCAGGGCCTGGAGTGGATGGGCAGGATCCACCCCAGCGACAGCGACACCAACTACAACCAGA AGTTCAAGGGCAGGGTGACCATCACCGCCGACAAGAGCACCAGCACCGCCTACATGGAGCTGA GCAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCATCGGCTACTACTACGACAGCA GCTACGACTACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGC

SEQ.I.D.NO:101 - Humanised variable heavy chain for DMS10083

QVQLVQSGAEVKKPGSSVKVSCKASGYTFSSYWMHWVRQAPGQGLEWMGRIHPSDSDTNYNQKF KGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGYYYDSSYDYWGQGTLVTVSS

SEQ.I.D.NO:102 - Humanised variable heavy chain for DMS10083 (polynucleotide)

CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCAGCAGCGTGAAGGTGAG CTGCAAGGCCAGCGGCTACACCTTCAGCAGCTACTGGATGCACTGGGTGAGGCAGGCCCCCGG CCAGGGCCTGGAGTGGATGGGCAGGATCCACCCCAGCGACAGCGACACCAACTACAACCAGAA GTTCAAGGGCAGGGTGACCATCACCGCCGACAAGAGCACCAGCACCGCCTACATGGAGCTGAG CAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCTACTACTACGACAGCAG CTACGACTACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGC

SEQ.I.D.NO:103 - Humanised variable heavy chain for DMS10084

QVQLVQSGAEVKKPGSSVKVSCKASGGTFTSYWMHWVRQAPGQGLEWMGRIHPSDSDTNYNQKF KGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGYYYDSSYDYWGQGTLVTVSS

SEQ.I.D.NO:104 - Humanised variable heavy chain for DMS10084 (polynucleotide)

CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCAGCAGCGTGAAGGTGAG CTGCAAGGCCAGCGGCGGCACCTTCACCAGCTACTGGATGCACTGGGTGAGGCAGGCCCCCG GCCAGGGCCTGGAGTGGATGGGCAGGATCCACCCCAGCGACAGCGACACCAACTACAACCAGA AGTTCAAGGGCAGGGTGACCATCACCGCCGACAAGAGCACCAGCACCGCCTACATGGAGCTGA GCAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCTACTACTACGACAGCA GCTACGACTACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGC

SEQ.I.D.NO:105 - Humanised variable heavy chain for DMS10085 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYWMHWVRQAPGQGLEWMGRIHPSDSDTNYNQKF KGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGYYYDSSYDYWGQGTLVTVSS

SEQ.I.D.NO:106 - Humanised variable heavy chain for DMS10085 (polynucleotide) CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCAGCAGCGTGAAGGTGAG CTGCAAGGCCAGCGGCGGCACCTTCAGCAGCTACTGGATGCACTGGGTGAGGCAGGCCCCCG GCCAGGGCCTGGAGTGGATGGGCAGGATCCACCCCAGCGACAGCGACACCAACTACAACCAGA AGTTCAAGGGCAGGGTGACCATCACCGCCGACAAGAGCACCAGCACCGCCTACATGGAGCTGA GCAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCTACTACTACGACAGCA GCTACGACTACTGGGGCCAGGGCACACTAGTGACCGTGTCCAGC

SEQ.I.D.NO:107 - Light chain of YW31 1.31.57 (polynucleotide)

GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCAAGCGTCGGAGACAGGGTGACCATT ACCTGCAGGGCCAGCCAGGACGTGAGCACCGCCGTGGCCTGGTATCAGCAGAAACCCGGCAA GGCCCCCAAGCTGCTGATCTACAGCGCCAGCTTCCTGTACAGCGGCGTGCCCTCTAGGTTTAGC GGCAGCGGCAGCGGCACTGACTTCACCCTCACCATCTCCAGCCTGCAGCCCGAGGACTTCGCA ACCTACTACTGCCAGCAGAGCTACACCCTGCCCACCACCTTTGGCCAGGGCACCAAGGTGGAG ATCAAGCGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGCAGCTGAAG AGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAG TGGAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAG CAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCA CAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAA CCGGGGCGAGTGC

Claims

An antigen binding polypeptide which binds to LRP6 and which comprises a paired VH/VL wherein the paired VH/VL antagonises or does not potentiate the signalling of one Wnt ligand and does not potentiate or activate the signalling of a second Wnt ligand

An antigen binding polypeptide according to claim 1 which antagonises or does not potentiate signalling mediated by a Wnt 1 class ligand signalling and does not activate or potentiate signalling mediated by a Wnt 3 class ligand.

An antigen binding polypeptide according to claim 1 which antagonises or does not potentiate signalling mediated by a Wnt 3 class ligand signalling and does not activate or potentiate signalling mediated by a Wnt 1 class ligand.

An antigen binding polypeptide which specifically binds to LRP6 and which comprises a paired VH/VL wherein the paired VH/VL antagonises or does not potentiate signalling of at least 2 Wnt ligands.

The antigen binding polypeptide of any one of claims 1 to 4 wherein the antigen binding polypeptide comprises a Fab, Fab', F(ab')2, Fv.ScFv, diabody, triabody, tetrabody, miniantibody or minibody.

The antigen binding polypeptide of any one of claims1-5 wherein the antigen binding polypeptide is a monoclonal antibody

The antigen binding polypeptide according to any preceding claim wherein the antigen binding polypeptide additionally binds non-human primate LRP6.

The antigen binding polypeptide according to any one of the preceding claims and wherein the antigen binding polypeptide binds LRP6 with an affinity better than 100nM.

The antigen binding polypeptide according to any one of the preceding claims and wherein the antigen binding polypeptide antagonises or does not potentiate Wnt-mediated signalling in a Wnt-responsive gene reporter assay

The antigen binding polypeptide according to any preceding claim wherein the antigen binding polypeptide further comprises additional variable binding domains.

An antigen binding polypeptide according to any preceding claim wherein the antigen binding polypeptide binds to the extracellular domain of LRP6. An antigen binding polypeptide according to any preceding claim wherein the antigen binding polypeptide does not block and/or does not compete with DKK1 and/or SOST for binding to LRP6.

The antigen binding polypeptide according to any preceding claim wherein the antigen binding fragment does not compete with an antigen binding polypeptide with variable heavy chain according to SEQ ID NO: 77 and/or light chain according to SEQ ID NO: 81 and/or an antigen binding polypeptide with variable heavy chain according to SEQ ID NO: 79 and/or light chain according to SEQ ID NO: 81 and/or an antigen binding polypeptide with variable heavy chain according to SEQ ID NO: 42 and/or light chain according to SEQ ID NO: 43, and/or an antigen binding polypeptide with variable heavy chain according to SEQ ID NO: 53 and/or light chain according to SEQ ID NO: 54, and/or an antigen binding polypeptide with variable heavy chain according to SEQ ID NO: 65 and/or light chain according to SEQ ID NO: 66.

An antigen binding polypeptide according to claim 1 obtainable by the following steps:

a) immunising with hl_RP6.

b) selecting those antigen binding polypeptides which bind hl_RP6

c) selecting those antigen binding polypeptides which do not compete with an antigen binding polypeptide with variable heavy chain according to SEQ ID NO: 77 and/or light chain according to SEQ ID NO: 81 and/or an antigen binding polypeptide with variable heavy chain according to SEQ ID NO: 79 and/or light chain according to SEQ ID NO: 81 and/or an antigen binding polypeptide with variable heavy chain according to SEQ ID NO: 42 and/or light chain according to SEQ ID NO: 43, and/or an antigen binding polypeptide with variable heavy chain according to SEQ ID NO: 53 and/or light chain according to SEQ ID NO: 54, and/or an antigen binding polypeptide with variable heavy chain according to SEQ ID NO: 65 and/or light chain according to SEQ ID NO: 66 in a binding assay.

d) selecting from the antigen binding proteins of part c) for those antigen binding polypeptides which are Wnt 1 antagonists; and further

e) selecting from the antigen binding proteins of part d) for those for those which are Wnt 3a antagonists

The antigen binding polypeptide according to any preceding claim wherein the antigen binding polypeptide comprises CDRH3 of SEQ ID NO.3 or a variant thereof.

The antigen binding polypeptide according to claim 15 wherein the antigen binding polypeptide further comprises one or more of: CDR H1 of SEQ. ID. NO: 1 , CDRH2: SEQ. ID. NO: 2 CDRL1 : SEQ. ID. NO: 4 CDRL2: SEQ. ID. NO: 5 and/or CDRL3: SEQ. ID. NO: 6 or variants thereof.

17. The antigen binding polypeptide according to claim 16 wherein the antigen binding polypeptide comprises:

i) CDRH3 as set out in SEQ ID NO. 3

ii) CDRH1 as set out in SEQ ID NO. 1 ; and

iii) CDRH2 as set out in SEQ ID NO. 2

18. The antigen binding polypeptide according to claim 16 wherein the antigen binding

polypeptide comprises:

i) CDRH3 as set out in SEQ ID NO. 3

ii) CDRH1 as set out in SEQ ID NO. 1

iii) CDRH2 as set out in SEQ ID NO. 2

iv) CDRL1 as set out in SEQ ID NO. 4

v) CDRL2 as set out in SEQ ID NO. 5; and

vi) CDRL3 as set out in SEQ ID NO. 6.

19. The antigen binding polypeptide according to any preceding claim which comprises a heavy chain variable region encoded by any one of SEQ ID NO: 13 or SEQ ID NO:93 or SEQ ID NO:95 or SEQ ID NO:97 or SEQ ID NO:99, or SEQ ID NO: 101 , or SEQ ID NO: 103, or SEQ ID NO: 105.

20. The antigen binding polypeptide according to any preceding claim which comprises a light chain variable region encoded by any one of SEQ. ID. NO: 14.

21. The antigen binding polypeptide according to any preceding claim wherein the antigen binding polypeptide comprises a heavy chain encoded by SEQ. ID. NO: 24 or SEQ. ID. NO: 21 or SEQ. ID. NO: 22 or SEQ. ID. NO: 23 or SEQ. ID. NO: 18 or SEQ. ID. NO: 19 or SEQ. ID. NO: 20 or SEQ. ID. NO: 16 and a light chain encoded by SEQ. ID. NO: 17

22. The antigen binding polypeptide according to any preceding claim wherein the antigen binding polypeptide is a humanised monoclonal antibody.

23. An antigen binding polypeptide which binds to the same epitope as an antibody which has a heavy chain sequence of SEQ. ID. NO: 24 or SEQ. ID. NO: 21 or SEQ. ID. NO: 22 or SEQ. ID. NO: 23 or SEQ. ID. NO: 18 or SEQ. ID. NO: 19 or SEQ. ID. NO: 20 or SEQ. ID. NO: 16 and a light chain sequence of SEQ ID NO: 17

24. A pharmaceutical composition comprising an antigen binding polypeptide according to any preceding claim and a pharmaceutically acceptable carrier.

25. A method of treating a human patient afflicted with an inflammatory disorder or disease or a bone disorder or disease or cancer including non small cell lung carcinoma, breast cancer, pancreatic cancer, germ cell or embryonal or teratocarcinoma tumors which method comprises the step of administering the composition of claim 24.

26. Use of the composition of claim 24 for treating a human patient afflicted with an inflammatory disorder or disease such as ulcerative colitis or inflammatory bowel disease or a bone disorder or disease or cancer including non small cell lung carcinoma, breast cancer, pancreatic cancer, germ cell or embryonal or teratocarcinoma tumors.

Use of an antigen binding polypeptide according to claims 1-23 for use in the manufacture of a medicament for the treatment or prophylaxis of an inflammatory disorder or disease such as inflammatory bowel disease or ulcerative colitis or a bone disorder or disease or cancer including non small cell lung carcinoma, breast cancer, pancreatic cancer, germ cell or embryonal or teratocarcinoma tumors.