WO2010094981A2 - Antibody therapy - Google Patents

Abstract

Described are methods of inhibiting angiogenesis, the methods comprising the simultaneous, sequential or separate administration of (i) an anti- Cathepsin S antibody molecule, and (ii) an anti-VEGF antibody molecule. Also described are anti-Cathepsin S humanised antibodies, pharmaceutical compositions and methods of treatment using such antibodies.

Description

Antibody Therapy

Field of the Invention

This application relates to methods of treatment of conditions and diseases related to angiogenesis, antibodies and compositions of antibodies for use in such methods.

Background to the Invention

Angiogenesis, the development of microvasculature, is an integral process within many normal physiological processes such as normal development and wound healing. Angiogenesis is characterised by the stimulation of endothelial cells to form primary blood vessels where a non-clarified complex interplay exists between the endothelial cells, surrounding microenvironment and a range of pro and anti-angiogenic factors. However, uncontrolled or inappropriate angiogenesis is accepted as an underlying factor to the pathology of a wide range of diseases including tumour progression and ocular disease.

Alterations in protease control frequently underlie many human pathological processes. The deregulated expression and activity of the lysosomal cysteine protease Cathepsin S has been linked to a range of conditions including neurodegenerative disorders, autoimmune diseases and certain malignancies.

PCT/GB2006/001314 (WO2006/109045) describes a monoclonal antibody with specificity for cathepsin S which potently inhibits the proteolytic activity of cathepsin S. This antibody, known as 1 E11 , was shown to inhibit tumour cell invasion and angiogenesis. Summary of the Invention

The present inventors have further investigated the properties of 1 E11 and have surprisingly shown that, in combination with an anti-VEGF antibody the anti-angiogenic effects obtainable are very much enhanced. Indeed, as described in the Examples, significant synergism has been demonstrated for the effect of combination of the cathepsin S antibody and the anti-VEGF antibody on angiogenesis, as assessed using a HUVEC tubular assay.

Accordingly, in a first aspect, the present invention provides a method of inhibiting angiogenesis in cells , a tissue or an individual, said method comprising the simultaneous, sequential or separate administration of (i) an anti-Cathepsin S antibody molecule, wherein said anti-Cathepsin S antibody inhibits the proteolytic activity of Cathepsin S; and (ii) an anti-VEGF antibody molecule to said cells, tissue or individual.

The demonstration that angiogenesis may be significantly inhibited using such joint therapy enables the use of such combinations in the treatment of diseases associated with angiogenesis. Thus in a second aspect, the invention provides a method of treating a disease associated with angiogenesis, the method comprising the simultaneous, sequential or separate administration of (i) an anti-Cathepsin S antibody molecule, wherein said anti-Cathepsin S antibody inhibits the proteolytic activity of

Cathepsin S; and

(ii) an anti-VEGF antibody molecule. In a third aspect, there is provided an anti-Cathepsin S antibody molecule, wherein said anti-Cathepsin S antibody inhibits the proteolytic activity of Cathepsin S; and an anti-VEGF antibody molecule, for the simultaneous, sequential or separate administration for the treatment of disease associated with angiogenesis.

A fourth aspect provides the use of an anti-Cathepsin S antibody molecule, wherein said anti-Cathepsin S antibody inhibits the proteolytic activity of Cathepsin S in the preparation of a medicament for combination therapy with an anti-VEGF antibody molecule for the treatment of disease associated with angiogenesis by simultaneous, sequential or separate administration of the anti-Cathepsin S antibody molecule and the anti-VEGF antibody molecule

A fifth aspect provides the use of an anti-VEGF antibody molecule in the preparation of a medicament for combination therapy with an anti- Cathepsin S antibody molecule, wherein said anti-Cathepsin S antibody inhibits the proteolytic activity of Cathepsin S, for the treatment of a disease associated with angiogenesis by simultaneous, sequential or separate administration of the anti-Cathepsin S antibody molecule and the anti-VEGF antibody molecule.

The invention may be used in the treatment of any disease associated with angiogenesis. Such diseases may include, but are not limited to, neoplastic disease such as cancer or tumours, various autoimmune disorders, hereditary disorders, and ocular disorders. In a particular embodiment of the invention, the disease is neoplastic disease. In a sixth aspect, the invention provides a pharmaceutical composition comprising an anti-Cathepsin S antibody molecule, wherein said anti- Cathepsin S antibody inhibits the proteolytic activity of Cathepsin S, and an anti-VEGF antibody molecule.

A seventh aspect of the invention provides a pharmaceutical kit comprising (i) an anti-Cathepsin S antibody molecule, wherein said anti- Cathepsin S antibody inhibits the proteolytic activity of Cathepsin S, and (ii) an anti-VEGF antibody molecule for combination therapy by simultaneous, sequential or separate administration of the Cathepsin S antibody molecule and the anti-VEGF antibody molecule.

Any suitable anti-Cathepsin S antibody may be used in the first to seventh aspects of the present invention. For example, in one embodiment, the Cathepsin S antibody may be the 1 E11 antibody as described in

WO2006/109045 or an antibody molecule based on such an antibody. Thus, in one embodiment, the antibody molecule comprises an antigen binding domain comprising at least one of the CDRs of the V_H domain of the 1 E11 antibody and/or at least one of the CDRs of the V_L domain of the 1 E11 antibody. The CDRs may be determined using any suitable system, for example using the Kabat system.

In one embodiment, the antibody molecule comprises an antigen binding domain comprising at least one of the CDRs with an amino acid sequence selected from the group consisting of Sequence ID No: 1 , Sequence ID No: 2, Sequence ID No: 3, or a variant thereof and/or at least one of the CDRs with an amino acid sequence consisting of Sequence ID No: 4, Sequence ID No: 5 and Sequence ID No: 6, or a variant thereof. The amino acid sequences corresponding to Sequence ID Nos: 1 -6 are as follows:

Sequence ID No: 1 : SYDMS

In embodiments wherein reference is made to Sequence ID No: 1 , alternative embodiments may employ a CDR having the amino acid sequence Sequence ID No: 28:

SSYDMS

Sequence ID No: 2: YITTGGVNTYYPDTVKG

Sequence ID No: 3 HSYFDY

Sequence ID No: 4: RSSQSLVHSNGNTYLH

Sequence ID No: 5: KVSNRFS

Sequence ID No: 6: SQTTHVPPT

In one embodiment of the first to seventh aspects, the anti-Cathepsin S antibody molecule comprises an antigen binding domain comprising at least one of the CDRs with an amino acid sequence selected from the group consisting of Sequence ID No: 1 , Sequence ID No: 2, Sequence ID No: 3, and/or at least one of the CDRs with an amino acid sequence consisting of Sequence ID No: 4, Sequence ID No: 5 and Sequence ID No: 6.

In a particular embodiment, the anti-Cathepsin S antibody molecule comprises an antigen binding domain comprising all three of the CDRs of the group consisting of Sequence ID No: 1 , Sequence ID No: 2, Sequence ID No: 3, or variants thereof and all three of the CDRs of the group consisting of Sequence ID No: 4, Sequence ID No: 5 and Sequence ID No: 6, or variants thereof.

In one embodiment, the anti-Cathepsin S antibody molecule comprises an antibody V_H domain or an antibody V_κ domain, or both.

In one embodiment, the anti-Cathepsin S antibody molecule V_H domain comprises at least one of the CDRs, for example two or three CDRs, with an amino acid sequence selected from the group consisting of Sequence ID No: 1 , Sequence ID No: 2, Sequence ID No: 3,and/or the antibody V_L domain comprises at least one of the CDRs, for example two or three CDRs, with an amino acid sequence consisting of Sequence ID No: 4, Sequence ID No: 5 and Sequence ID No: 6.

In one embodiment, the anti-Cathepsin S antibody V_H domain comprises CDRs with amino acid sequences Sequence ID No: 1 , Sequence ID No: 2 and Sequence ID No: 3 as CDRs 1 , 2 and 3 respectively.

In one embodiment, the anti-Cathepsin S antibody molecule V_L domain comprises CDRs with amino acid sequences Sequence ID No: 4, Sequence ID No: 5 and Sequence ID No: 6 as CDRs 1 , 2 and 3 respectively.

The amino acid sequences of the CDRs of the 1 E11 antibody were identified using the Kabat system. However, in alternative embodiments of the invention, the amino acid sequences of the CDRs of the 1 E11 antibody for use in the invention may be as identified using an alternative system, for example the IMGT system (Brochet, X. et al., Nucl. Acids Res. 36, W503-508 (2008)). Under such a system, the CDRs of the 1 E11 antibody have amino acid sequences as shown as Sequence ID NOs: 20- 25. Sequence ID NOs: 20, 21 , 22, 23, 24, and 25 are the amino acid sequences identified using the IMGT system for the CDRs corresponding to the Kabat identified CDR sequences shown as Sequence ID No: 1 /28, Sequence ID No: 2, Sequence ID No: 3, Sequence ID No: 4, Sequence ID No: 5 and Sequence ID No: 6 respectively.

Sequence ID No: 20: GFAFSSYD

Sequence ID No: 21 : ITTGGVNT

Sequence ID No: 22 ARHSYFDY

Sequence ID No: 23: QSLVHSNGNTY

Sequence ID No: 24: KVS Sequence ID No: 25: SQTTHVPPT

Thus, in one embodiment of the first to seventh aspects of the invention, the antibody molecule comprises an antigen binding domain comprising at least one of the CDRs with an amino acid sequence selected from the group consisting of Sequence ID No: 20, Sequence ID No: 21 , Sequence ID No: 22, or a variant thereof and/or at least one of the CDRs with an amino acid sequence consisting of Sequence ID No: 23, Sequence ID No: 24 and Sequence ID No: 25, or a variant thereof.

In one embodiment of the first to seventh aspects, the anti-Cathepsin S antibody molecule comprises an antigen binding domain comprising at least one of the CDRs with an amino acid sequence selected from the group consisting of Sequence ID No: 20, Sequence ID No: 21 , Sequence ID No: 22, and/or at least one of the CDRs with an amino acid sequence consisting of Sequence ID No: 23, Sequence ID No: 24 and Sequence ID No: 25.

In a particular embodiment, the anti-Cathepsin S antibody molecule comprises an antigen binding domain comprising all three of the CDRs of the group consisting of Sequence ID No: 20, Sequence ID No: 21 , Sequence ID No: 22, or variants thereof and all three of the CDRs of the group consisting of Sequence ID No: 23, Sequence ID No: 24 and Sequence ID No: 25, or variants thereof.

In one embodiment, where the anti-Cathepsin S antibody molecule comprises an antibody V_H domain or an antibody V_κ domain, or both, the anti-Cathepsin S antibody molecule V_H domain comprises at least one of the CDRs, for example two or three CDRs, with an amino acid sequence selected from the group consisting of Sequence ID No: 20, Sequence ID No: 21 , Sequence ID No: 22, and/or the antibody V_L domain comprises at least one of the CDRs, for example two or three CDRs, with an amino acid sequence consisting of Sequence ID No: 23, Sequence ID No: 24 and Sequence ID No: 25.

In one embodiment, the anti-Cathepsin S antibody V_H domain comprises CDRs with amino acid sequences Sequence ID No: 20, Sequence ID No: 21 , and Sequence ID No: 22 as CDRs 1 , 2 and 3 respectively.

In one embodiment, the anti-Cathepsin S antibody molecule V_L domain comprises CDRs with amino acid sequences Sequence ID No: 23, Sequence ID No: 24 and Sequence ID No: 25 as CDRs 1 , 2 and 3 respectively.

In a particular embodiment, the anti-Cathepsin S antibody V_H domain comprises the amino acid sequence Sequence ID No: 7 and /or the antibody V_L domain comprises the amino acid sequence Sequence ID No: 8.

Sequence ID No: 7:

VQLQESGGVLVKPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWV AYITTGGVNTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCR HSYFDYWGQGTTVTVSS

Sequence ID No: 8:

DVLMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQS PKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQTTH VPPTFGSGTKLEIKR In one embodiment, the anti-Cathepsin S antibody molecule is a chimeric antibody molecule. In one embodiment, the anti-Cathepsin S antibody molecule is a human or humanised antibody molecule.

The present inventors have developed anti-Cathepsin S humanised antibodies which, as demonstrated in the Examples, have significantly improved binding ability to Cathepsin S compared to the murine antibody.

Accordingly, in an eighth aspect, there is provided an anti-Cathepsin S antibody molecule comprising a V_H domain having the amino acid sequence shown as Sequence ID No: 9, Sequence ID No: 10, Sequence ID No: 11 , Sequence ID No: 12, or Sequence ID No: 13 and/or an antibody VL domain having the amino acid sequence shown as Sequence ID No: 14, Sequence ID No: 15, Sequence ID No: 16, Sequence ID No: 17, Sequence ID No: 18, or Sequence ID No: 19.

Sequence ID No: 9:

EVQLVESGGGLVKPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGL

EWVAYITTGGVNTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTA VYYCARHSYFDYWGQGTTVTVSS

Sequence ID No: 10:

EVQLVESGGGLVKPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGL EWVAYITTGGVNTYYPDTVKGRFTISRDNAKNTLYLQMNSLRAEDTA VYYCARHSYFDYWGQGTTVTVSS

Sequence ID No: 11 : EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGL EWVAYITTGGVNTYYPDTVKGRFTISRDNAKNSLYLQMSSLKSEDTA VYYCARHSYFDYWGQGTTVTVSS

Sequence ID No: 12:

EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGL EWVAYITTGGVNTYYPDTVKGRFTISRDNAKNSLYLQMNSLRAEDTA VYYCARHSYFDYWGQGTTVTVSS

Sequence ID No: 13:

EVQLVESGGGLVKPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGL EWVAYITTGGVNTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTA VYYCARHSYFDYWGQGTTVTVSS

Sequence ID No: 14:

DWMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTYLHWYLQKPG QSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFC

SQTTHVPPTFGQGTKLEIK

Sequence ID No: 15:

DWMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTYLHWYLQKPG

QSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC SQTTHVPPTFGQGTKLEIK

Sequence ID No: 16: DWMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTYLHWYLQRPG QSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFC SQTTHVPPTFGQGTKLEIK

Sequence ID No: 17:

DWMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTYLHWYQQRPG QSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFC SQTTHVPPTFGQGTKLEIK

Sequence ID No: 18:

DWMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTYLHWYLQRPG QSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFC SQTTHVPPTFGQGTKLEIK

Sequence ID No: 19:

DWMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTYLHWYLQKPG QSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFC

SQTTHVPPTFGQGTKLEIK

Accordingly, in one embodiment of the first to seventh aspects of the invention, the anti-Cathepsin S antibody molecule comprises an antibody VH domain having the amino acid sequence shown as Sequence ID No: 9, Sequence ID No: 10, Sequence ID No: 11 , Sequence ID No: 12, or Sequence ID No: 13.

In another embodiment, the antibody molecule comprises an antibody VL domain having the amino acid sequence shown as Sequence ID No: 14, Sequence ID No: 15, Sequence ID No: 16, Sequence ID No: 17, Sequence ID No: 18, or Sequence ID No: 19.

In the invention, any suitable anti-VEGF antibodies may be used. The antibodies may be human or humanised. Particularly good results have been obtained by the inventors employing anti VEGF-A antibodies. Accordingly, in particular embodiments of the invention, the anti-VEGF antibody molecule is an anti-VEGF-A antibody molecule. In a particular embodiment of the invention, the anti-VEGF-A antibody is bevacizumab.

The V_H and V_L sequences for bevacizumab are as follows:

VH

EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEW VGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYC AKYPHYYGSSHWYFDVWGQGTLVTVSS (Sequence ID No: 26)

VL

DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYF TSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFG QGTKVEIKR (Sequence ID No: 27)

In another embodiment, the anti-VEGF antibody is an anti VEGF-R 2 antibody. Suitable anti VEGF-R 2 antibodies may include, for example IMC-1 C11 , (ImClone Systems), IMC-1121 B (ImClone Systems), or CDP791 (UCB). For a review of anti VEGF antibodies, see Youssoufian

Clinical Cancer Research September 15, 2007 vol. 13 no. 18 5544s- 5548s. The invention also extends to the treatment of angiogenesis associated diseases with combinations including anti-Cathepsin S antibodies which do not inhibit the proteolytic effect of Cathepsin S. Thus, in a ninth aspect of the invention, there is provided a method of inhibiting angiogenesis, said method comprising the simultaneous, sequential or separate administration of (i) an anti-Cathepsin S antibody molecule, wherein said anti-Cathepsin S antibody does not inhibit the proteolytic activity of Cathepsin S; and

(ii) an anti-VEGF-A antibody molecule to said cells.

A tenth aspect provides a method of treating disease associated with angiogenesis comprising the simultaneous, sequential or separate administration of (i) an anti-Cathepsin S antibody molecule, wherein said anti-Cathepsin S antibody does not inhibit the proteolytic activity of Cathepsin S; and

(ii) an anti-VEGF-A antibody molecule to said cells.

An eleventh aspect provides an anti-Cathepsin S antibody molecule, wherein said anti-Cathepsin S antibody does not inhibit the proteolytic activity of Cathepsin S; and an anti-VEGF-A antibody molecule for the simultaneous, sequential or separate administration for the treatment of disease associated with angiogenesis.

A twelfth aspect provides the use of an anti-Cathepsin S antibody molecule, wherein said anti-Cathepsin S antibody does not inhibit the proteolytic activity of Cathepsin S in the preparation of a medicament for combination therapy with an anti-VEGF-A antibody molecule by simultaneous, sequential or separate administration of the anti-Cathepsin S antibody molecule and the anti-VEGF-A antibody molecule for the treatment of a disease associated with angiogenesis.

A thirteenth aspect provides the use of an anti-VEGF-A antibody molecule in the preparation of a medicament for combination therapy with an anti- Cathepsin S antibody molecule, wherein said anti-Cathepsin S antibody does not inhibit the proteolytic activity of Cathepsin S, by simultaneous, sequential or separate administration of the anti-VEGF-A antibody molecule and the anti-Cathepsin S antibody molecule for the treatment of a disease associated with angiogenesis.

A fourteenth aspect provides a pharmaceutical composition comprising anti-Cathepsin S antibody molecule, wherein said anti-Cathepsin S antibody does not inhibit the proteolytic activity of Cathepsin S; and an anti-VEGF-A antibody molecule.

A fifteenth aspect provides a kit comprising anti-Cathepsin S antibody molecule, wherein said anti-Cathepsin S antibody molecule does not inhibit the proteolytic activity of Cathepsin S; and an anti-VEGF-A antibody molecule, for combination therapy by simultaneous, sequential or separate administration of the Cathepsin S antibody molecule and the anti-VEGF-A antibody molecule.

In these aspects of the invention, either or both of the anti-Cathepsin S antibody molecule and the anti-VEGF-A antibody molecule may be human or humanised.

In particular embodiments of the ninth to fifteenth aspects of the invention, the anti-Cathepsin S antibody molecule is, or is a variant of, the IE4 antibody as described in WO2008/044076. In particular embodiments of the ninth to fifteenth aspects of the invention, said anti-VEGF-A antibody molecule is bevacizumab.

As well as being concerned with combinations of anti- Cathepsin S antibodies and anti-VEGF antibodies, and their use in the inhibition of angiogenesis and the treatment of diseases associated with angiogenesis, the present invention further extends to combinations of anti-Cathepsin s antibodies and some alternative VEGF inhibitors.

Accordingly, in a sixteenth aspect of the invention, there is provided a method of inhibiting angiogenesis in cells , a tissue or an individual, said method comprising the simultaneous, sequential or separate administration of (i) an anti-Cathepsin S antibody molecule, wherein said anti-Cathepsin S antibody inhibits the proteolytic activity of Cathepsin S; and

(ii) an inhibitor of VEGF to said cells, tissue or individual.

In a seventeenth aspect, the invention provides a method of treating a disease associated with angiogenesis, the method comprising the simultaneous, sequential or separate administration of (i) an anti- Cathepsin S antibody molecule, wherein said anti-Cathepsin S antibody inhibits the proteolytic activity of Cathepsin S; and (ii) an inhibitor of VEGF.

In an eighteenth aspect, there is provided an anti-Cathepsin S antibody molecule, wherein said anti-Cathepsin S antibody inhibits the proteolytic activity of Cathepsin S; and (ii) an inhibitor of VEGF, for the simultaneous, sequential or separate administration for the treatment of disease associated with angiogenesis.

A nineteenth aspect provides the use of an anti-Cathepsin S antibody molecule, wherein said anti-Cathepsin S antibody inhibits the proteolytic activity of Cathepsin S and an inhibitor of VEGF in the preparation of a medicament for combination therapy with an inhibitor of VEGF by simultaneous, sequential or separate administration of the anti-Cathepsin S antibody molecule and the inhibitor of VEGF for the treatment of disease associated with angiogenesis.

A twentieth aspect provides the use of an inhibitor of VEGF in the preparation of a medicament for combination therapy with an anti- Cathepsin S antibody molecule, wherein said anti-Cathepsin S antibody inhibits the activity of Cathepsin S, by simultaneous, sequential or separate administration of the inhibitor of VEGF and the anti-Cathepsin S antibody molecule for the treatment of disease associated with angiogenesis.

In a twenty-first aspect, the invention provides a pharmaceutical composition comprising an anti-Cathepsin S antibody molecule, wherein said anti-Cathepsin S antibody inhibits the proteolytic activity of Cathepsin S, and an inhibitor of VEGF.

A twenty-second aspect of the invention provides a pharmaceutical kit comprising an anti-Cathepsin S antibody molecule, wherein said anti- Cathepsin S antibody inhibits the proteolytic activity of Cathepsin S, and an inhibitor of VEGF for combination therapy by simultaneous, sequential or separate administration of the Cathepsin S antibody molecule and the inhibitor of VEGF.

Any suitable anti-Cathepsin S antibody may be used in the sixteenth to twenty-second aspects of the present invention. For example, the

Cathepsin S antibody molecule may be as described in relation to the first to sixth aspects of the invention supra.

Any suitable VEGF inhibitor may be used in the sixteenth to twenty- second aspects of the invention. Examples of VEGF inhibitors are described in Youssoufian Clinical Cancer Research September 15, 2007 vol. 13 no. 18 5544s-5548s. However, in a particular embodiment, the VEGF inhibitor is selected from the group consisting of Sorafenib (Bayer and Onyx), sunitinib (Pfizer), Vatalanib (PTK787) (Novartis and Schering AG), AEE788 (Novartis), Vandetanib (Zactima, ZD6474) (AstraZeneca pic), Cediranib (Recentin, AZD2171 ) (AstraZeneca pic), Pazopanib (786034), XL999 (Exelixis, Inc.), XL880 (Exelixis, Inc.), XL647 (Exelixis, Inc.), XL184 (Exelixis, Inc.), XL820 (Exelixis, Inc.), Neovastat (AE941 ) (Aeterna Zentaris), BIBF1120 (Boehringer Ingelheim), BMS-582664 (Bristol-Myers Squibb), AG013676 (Pfizer), and VEGF-Trap (Regeneron). In a particular embodiment, the VEGF inhibitor is selected from the group consisting of Sorafenib (Bayer and Onyx), sunitinib (Pfizer), Vatalanib (PTK787) (Novartis and Schering AG), Vandetanib (Zactima, ZD6474) (AstraZeneca pic), AG013676 (Pfizer), and VEGF-Trap (Regeneron).

Certain specific VEGF inhibitors may also be used in combination with anti-Cathepsin S antibodies which do not inhibit the proteolytic effect of Cathepsin S. Accordingly, in a twenty-third aspect of the present invention, there is provided a method of inhibiting angiogenesis, said method comprising the simultaneous, sequential or separate administration of (i) an anti- Cathepsin S antibody molecule, wherein said anti-Cathepsin S antibody does not inhibit the proteolytic activity of Cathepsin S; and

(ii) a VEGF inhibitor, wherein said VEGF-inhibitor is selected from the group consisting of Sorafenib (Bayer and Onyx), sunitinib (Pfizer), AEE788 (Novartis), Vandetanib (Zactima, ZD6474) (AstraZeneca pic), Cediranib (Recentin, AZD2171 ) (AstraZeneca pic), Pazopanib (786034), XL999 (Exelixis, Inc.), XL880 (Exelixis, Inc.), XL647 (Exelixis, Inc.), XL184 (Exelixis, Inc.), XL820 (Exelixis, Inc.), Neovastat (AE941 ) (Aeterna Zentaris), BIBF1120 (Boehringer Ingelheim), BMS-582664 (Bristol-Myers Squibb), and AG013676 (Pfizer).

A twenty-fourth aspect provides a method of treating disease associated with angiogenesis comprising the simultaneous, sequential or separate administration of (i) an anti-Cathepsin S antibody molecule, wherein said anti-Cathepsin S antibody does not inhibit the proteolytic activity of Cathepsin S; and

(ii) a VEGF inhibitor, wherein said VEGF-inhibitor is selected from the group consisting of Sorafenib (Bayer and Onyx), sunitinib (Pfizer), AEE788 (Novartis), Vandetanib (Zactima, ZD6474) (AstraZeneca pic), Cediranib (Recentin, AZD2171 ) (AstraZeneca pic), Pazopanib (786034), XL999 (Exelixis, Inc.), XL880 (Exelixis, Inc.), XL647 (Exelixis, Inc.), XL184 (Exelixis, Inc.), XL820 (Exelixis, Inc.), Neovastat (AE941 ) (Aeterna Zentaris), BIBF1120 (Boehringer Ingelheim), BMS-582664 (Bristol-Myers Squibb), and AG013676 (Pfizer) to said cells. A twenty-fifth aspect provides an anti-Cathepsin S antibody molecule, wherein said anti-Cathepsin S antibody does not inhibit the proteolytic activity of Cathepsin S; and a VEGF inhibitor, wherein said VEGF-inhibitor is selected from the group consisting of Sorafenib (Bayer and Onyx), sunitinib (Pfizer), AEE788 (Novartis), Vandetanib (Zactima, ZD6474) (AstraZeneca pic), Cediranib (Recentin, AZD2171 ) (AstraZeneca pic), Pazopanib (786034), XL999 (Exelixis, Inc.), XL880 (Exelixis, Inc.), XL647 (Exelixis, Inc.), XL184 (Exelixis, Inc.), XL820 (Exelixis, Inc.), Neovastat (AE941 ) (Aeterna Zentaris), BIBF1120 (Boehringer Ingelheim), BMS-582664 (Bristol-Myers Squibb), and AG013676 (Pfizer) for the simultaneous, sequential or separate administration for the treatment of disease associated with angiogenesis.

A twenty-sixth aspect provides the use of an anti-Cathepsin S antibody molecule, wherein said anti-Cathepsin S antibody does not inhibit the proteolytic activity of Cathepsin S, in the preparation of a medicament for combination therapy with a VEGF inhibitor, wherein said VEGF-inhibitor is selected from the group consisting of Sorafenib (Bayer and Onyx), sunitinib (Pfizer), AEE788 (Novartis), Vandetanib (Zactima, ZD6474) (AstraZeneca pic), Cediranib (Recentin, AZD2171 ) (AstraZeneca pic),

Pazopanib (786034), XL999 (Exelixis, Inc.), XL880 (Exelixis, Inc.), XL647 (Exelixis, Inc.), XL184 (Exelixis, Inc.), XL820 (Exelixis, Inc.), Neovastat (AE941 ) (Aeterna Zentaris), BIBF1120 (Boehringer Ingelheim), BMS- 582664 (Bristol-Myers Squibb), and AG013676 (Pfizer) by simultaneous, sequential or separate administration of the anti-Cathepsin S antibody molecule and the VEGF inhibitor for the treatment of disease associated with angiogenesis.

A twenty-seventh aspect provides the use of a VEGF inhibitor, wherein said VEGF-inhibitor is selected from the group consisting of Sorafenib (Bayer and Onyx), sunitinib (Pfizer), AEE788 (Novartis), Vandetanib (Zactima, ZD6474) (AstraZeneca pic), Cediranib (Recentin, AZD2171 ) (AstraZeneca pic), Pazopanib (786034), XL999 (Exelixis, Inc.), XL880 (Exelixis, Inc.), XL647 (Exelixis, Inc.), XL184 (Exelixis, Inc.), XL820 (Exelixis, Inc.), Neovastat (AE941 ) (Aeterna Zentaris), BIBF1120 (Boehringer Ingelheim), BMS-582664 (Bristol-Myers Squibb), and AG013676 (Pfizer) in the preparation of a medicament for combination therapy with an anti-Cathepsin S antibody molecule, wherein said anti- Cathepsin S antibody does not inhibit the activity of Cathepsin S, by simultaneous, sequential or separate administration of the VEGF inhibitor and the anti-Cathepsin S antibody molecule for the treatment of disease associated with angiogenesis.

A twenty-eighth aspect provides a pharmaceutical composition comprising anti-Cathepsin S antibody molecule, wherein said anti- Cathepsin S antibody does not inhibit the proteolytic activity of Cathepsin S; and a VEGF inhibitor, wherein said VEGF-inhibitor is selected from the group consisting of Sorafenib (Bayer and Onyx), sunitinib (Pfizer), AEE788 (Novartis), Vandetanib (Zactima, ZD6474) (AstraZeneca pic), Cediranib (Recentin, AZD2171 ) (AstraZeneca pic), Pazopanib (786034), XL999 (Exelixis, Inc.), XL880 (Exelixis, Inc.), XL647 (Exelixis, Inc.), XL184 (Exelixis, Inc.), XL820 (Exelixis, Inc.), Neovastat (AE941 ) (Aeterna Zentaris), BIBF1120 (Boehringer Ingelheim), BMS-582664 (Bristol-Myers Squibb), and AG013676 (Pfizer).

A twenty-ninth aspect provides a kit comprising anti-Cathepsin S antibody molecule, wherein said anti-Cathepsin S antibody molecule does not inhibit the proteolytic activity of Cathepsin S; and a VEGF inhibitor, wherein said VEGF-inhibitor is selected from the group consisting of Sorafenib (Bayer and Onyx), sunitinib (Pfizer), AEE788 (Novartis), Vandetanib (Zactima, ZD6474) (AstraZeneca pic), Cediranib (Recentin, AZD2171 ) (AstraZeneca pic), Pazopanib (786034), XL999 (Exelixis, Inc.), XL880 (Exelixis, Inc.), XL647 (Exelixis, Inc.), XL184 (Exelixis, Inc.), XL820 (Exelixis, Inc.), Neovastat (AE941 ) (Aeterna Zentaris), BIBF1120 (Boehringer Ingelheim), BMS-582664 (Bristol-Myers Squibb), and

AG013676 (Pfizer)., for combination therapy by simultaneous, sequential or separate administration of the Cathepsin S antibody molecule and the VEGF inhibitor.

In one embodiment of the twenty-third to twenty-ninth aspects of the invention, the VEGF inhibitor is selected from the group consisting of Sorafenib (Bayer and Onyx), Sunitinib (Pfizer), AG013676 (Pfizer), and Vandetanib (Zactima, ZD6474) (AstraZeneca pic),.

In any of the twenty third to twenty ninth aspects of the invention, the anti- Cathepsin S antibody molecule may be human or humanised and/or may be defined as for the ninth to fifteenth aspects of the invention.

In a particular embodiment of any of the sixteenth to twenty ninth aspects of the invention, the VEGF inhibitor is Sorafenib.

In a particular embodiment of one or more of the sixteenth to twenty-ninth aspects of the invention, the anti-Cathepsin S antibody molecule and the VEGF inhibitor are provided in a potentiating ratio i.e. synergistic amounts.

Preferred and alternative features of each aspect of the invention are as for each of the other aspects mutatis mutandis unless the context demands otherwise.

Detailed Description Antibody molecules

In the context of the present invention, an antibody molecule (or antibody molecule) is a molecule which has binding specificity for another molecule. The antibody molecule may be an antibody or fragment thereof.

An antibody should be understood to refer to an immunoglobulin or part thereof or any polypeptide comprising a binding domain which is, or is homologous to, an antibody binding domain. Specific antibody molecules include but are not limited to polyclonal, monoclonal, monospecific, polyspecific antibodies and fragments thereof and chimeric antibodies comprising an immunoglobulin binding domain fused to another polypeptide. Antibody mimetics are also encompassed by antibody molecules.

Intact (whole) antibodies comprise an immunoglobulin molecule consisting of heavy chains and light chains, each of which carries a variable region designated VH and VL, respectively. The variable region consists of three complementarity determining regions (CDRs, also known as hypervahable regions) and four framework regions (FR) or scaffolds. The CDR forms a complementary steric structure with the antigen molecule and determines the specificity of the antibody.

Fragments of antibodies may retain the binding ability of the intact antibody and may be used in place of the intact antibody. Accordingly, for the purposes of the present invention, unless the context demands otherwise, the term "antibody molecules" should be understood to encompass antibody fragments. Examples of antibody fragments include Fab, Fab', F (ab')2, Fd, dAb, and Fv fragments, scFvs, bispecific scFvs, diabodies, linear antibodies (see US patent 5, 641 , 870, Example 2 ; Zapata etal., Protein Eng 8 (10) : 1057-1062 [1995]) ; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

The Fab fragment consists of an entire L chain ( VL and CL), together with VH and CH1. Fab' fragments differ from Fab fragments by having additional few residues at the carboxy terminus of the CH1 domain including one or more cysteines from the antibody hinge region. The F (ab¹) 2 fragment comprises two disulfide linked Fab fragments.

Fd fragments consist of the VH and CH1 domains.

Fv fragments consist of the VL and VH domains of a single antibody.

Single-chain Fv fragments are antibody fragments that comprise the VH and VL domains connected by a linker which enables the scFv to form an antigen binding site, (see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

Diabodies are small antibody fragments prepared by constructing scFv fragments (see preceding paragraph) with short linkers (about 5-10 residues) between the VH and VL domains such that inter-chain but not intra-chain pairing of the V domains is achieved, resulting in a multivalent fragment, i.e. a fragment having two antigen-binding sites (see, for example, EP 404 097 ; WO 93/11161 ; and Hollinger et a/., Proc. Natl. Acad. Sci. USA, 90 : 6444-6448 (1993)) Further encompassed by fragments are individual CDRs.

As described above, the anti-Cathepsin S antibody molecules for use in the present invention are not limited to the specific antibodies described, such as the 1 E11 antibody, but also extend to other antibodies which maintain the ability to inhibit angiogenesis. Thus, the CDR amino acid sequences of such antibodies in which one or more amino acid residues are modified may also be used as the CDR sequence. The modified amino acid residues in the amino acid sequences of the CDR variant are preferably 30% or less, more preferably 20% or less, most preferably 10% or less, within the entire CDR. Such variants may be provided using the teaching of the present application and techniques known in the art. The CDRs may be carried in a framework structure comprising an antibody heavy or light chain sequence or part thereof. Preferably such CDRs are positioned in a location corresponding to the position of the CDR(s) of naturally occurring VH and VL domains. The positions of such CDRs may be determined using any suitable method, for example using the method as described in Kabat et al, Sequences of Proteins of Immunological Interest, US Dept of Health and Human Services, Public Health Service, Nat'l Inst, of Health, NIH Publication No. 91 -3242, 1991 and online at www.kabatdatabase.com htt{3^/jmmujτgj3jτ^^ or the IMGT system (Brochet, X. et al., Nucl. Acids Res. 36, W503-508 (2008)).

Furthermore, modifications may alternatively or additionally be made to the Framework Regions of the variable regions. Such changes in the framework regions may improve stability and reduce immunogenicity of the antibody. The antibodies of the invention herein include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain (s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U. S. Patent No. 4, 816, 567 ; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81 : 6851 -6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen- binding sequences derived from a non-human phmate(e. g. Old World Monkey, Ape etc), and human constant region sequences and humanised antibodies.

As described above, the inventors have developed some anti-Cathepsin S humanised antibodies for use in the present invention. Techniques for the production of humanized antibodies are well known in the art . Such techniques re derived from the methods originally devised by Winter et al Suitable methods for humanizing antibodies include CDR-grafting (EP 239,400; WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101 ; and 5,585,089), veneering or resurfacing (e.g. see EP 592,106; EP 519,596); and chain shuffling ( e.g. see U.S. Pat. No. 5,565,332). Further Composite Human Antibody technology such as that provided by Antitope (Cambridge, UK) may be used to produce antibodies from fully human sequences.

Modifications may be made in the VH, VL or CDRs of the antibody molecules, or indeed in the FRs using any suitable technique known in the art. For example, variable VH and/or VL domains may be produced by introducing a CDR, e.g. CDR3 into a VH or VL domain lacking such a CDR. Marks et al. (1992) Bio/ Technology, 10: 779-783 describe a shuffling technique in which a repertoire of VH variable domains lacking CDR3 is generated and is then combined with a CDR3 of a particular antibody to produce novel VH regions. Using analogous techniques, novel VH and VL domains comprising CDR derived sequences of and for use the present invention may be produced.

Modifications in framework residues in the human framework regions may be made, for example to improve binding. Such modifications may be with the corresponding residue from the CDR donor antibody. Methods for identifying and producing such framework substitutions are well known in the art; for example see, U.S. Pat. No. 5,585,089; Riechmann, et al., Nature 332:323 (1988).

Alternative techniques of producing variant antibodies for use in the invention may involve random mutagenesis of gene(s) encoding the VH or VL domain using, for example, error prone PCR (see Gram et al, 1992, P.N.A.S. 89 3576-3580. Additionally or alternatively, CDRs may be targeted for mutagenesis e.g. using the molecular evolution approaches described by Barbas et al 1991 PNAS 3809-3813 and Scier 1996 J MoI Biol 263 551 -567.

Antibody molecules of and for use in the present invention may be produced in any suitable way, either naturally or synthetically. Such methods may include, for example, traditional hybridoma techniques (Kohler and Milstein (1975) Nature, 256 :495-499), recombinant DNA techniques (see e.g. U. S. Patent No. 4,816, 567), or phage display techniques using antibody libraries (see e.g. Clackson et al. (1991 ) Nature, 352: 624-628 and Marks et al. (1992) Bio/ Technology, 10: 779- 783). Other antibody production techniques are described in Antibodies: A Laboratory Manual, eds. Harlow et al., Cold Spring Harbor Laboratory, 1988.

Traditional hybridoma techniques typically involve the immunisation of a mouse or other animal with an antigen in order to elicit production of lymphocytes capable of binding the antigen. The lymphocytes are isolated and fused with a myeloma cell line to form hybridoma cells which are then cultured in conditions which inhibit the growth of the parental myeloma cells but allow growth of the antibody producing cells. The hybridoma may be subject to genetic mutation, which may or may not alter the binding specificity of antibodies produced. Synthetic antibodies can be made using techniques known in the art (see, for example, Knappik et al, J. MoI. Biol. (2000) 296, 57-86 and Krebs et al, J. Immunol. Meth. (2001 ) 2154 67-84.

Antibodies and fragments may be tested for binding to Cat S and for the ability to inhibit the proteolytic activity of cathepsin S.

As described herein, the inventors have demonstrated that antibody molecules according to the invention have an anti-angiogenic effect. This therefore enables the use of the antibody molecules of the invention as active therapeutic agents. Accordingly in one embodiment of the invention, the antibody molecule is a "naked" antibody molecule. A "naked" antibody molecule is an antibody molecule which is not conjugated with an "active therapeutic agent".

In the context of the present application, an "active therapeutic agent" is a molecule or atom which is conjugated to an antibody moiety (including antibody fragments, CDRs etc) to produce a conjugate. Examples of such "active therapeutic agents" include drugs, toxins, radioisotopes, immunomodulators, chelators, boron compounds , dyes, nanoparticles etc.

In another embodiment of the invention, the antibody molecule is in the form of an immunoconjugate, comprising an antibody fragment conjugated to an "active therapeutic agent".

Methods of producing immunoconjugates are well known in the art; for example, see U. S. patent No. 5,057,313, Shih et al., Int. J. Cancer 41 : 832-839 (1988); Shih et al., Int. J.Cancer 46: 1101 -1106 (1990), Wong, Chemistry Of Protein Conjugation And Cross-Linking (CRC Press 1991 ); Upeslacis et al., "Modification of Antibodies by Chemical Methods, "in Monoclonal Antibodies: Principles And Applications, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc. 1995); Price, "Production and Characterization of Synthetic Peptide-Derived Antibodies," in Monoclonal Antibodies: Production, Engineering And Clinical Application, Ritter et a/.(eds.), pages 60-84 (Cambridge University Press 1995).

The antibody molecules of and for use in the invention may comprise further modifications. For example the antibodies can be glycosylated, pegylated, or linked to albumin or a nonproteinaceous polymer. The antibody molecule may be in the form of an immunoconjugate.

In one embodiment of the invention, the antibody molecules are non- fucosylated. ADCC activity of antibodies has been found to be dependent on the amount of fucose attached to the antibody. Non-fucosylated therapeutic antibodies have been shown to be more potent than corresponding fucosylated antibodies (Mori et al, Cytotechnology (2007) 55:109-114). A number of techniques exist for the production of non- fucosylated antibodies by someone skilled in genetic engineering and molecular biology. These include, but are not limited to, removal of fucose post-production via incubation of the antibody with α-1 ,6 fucosidase; the genetic engineering of a mammalian cell to have reduced or no fucosylation by mutation of the fucosyltransferase genes, FUT8 or GMD; prevention of fucose being added by transfection with the glycosyl-N- transferase gene, GnTIII; or disruption of the FUT8 and/or GMD genes using siRNA. (see, for example, Jefferis R. Trends Pharmacol Sci. 2009 Jul;30(7):356-62; Yamane-Ohnuki N, Satoh M. MAbs. 2009 May;1 (3):230- 6; Mori K, et al. Cytotechnology. 2007 Dec;55(2-3):109-14.

Antibodies of the invention may be labelled. Labels which may be used include radiolabels, enzyme labels such as horseradish peroxidase, alkaline phosphatase, or biotin.

As noted above, antibodies of and for use in the first to seventh aspects of the invention inhibit the proteolytic effect of cathepsin S with those for use in the ninth to fifteenth aspects having the property of not inhibiting the proteolytic effect of cathepsin S. The ability of an antibody molecule to inhibit the proteolytic activity of cathepsin S may be tested using any suitable method. For example the ability of an antibody molecule to inhibit the proteolytic activity of cathepsin S may be tested using a fluorimetric assay. In such an assay, any suitable fluohgenic substrate may be used, for example Cbz-Val-Val-Arg-AMC. An antibody molecule is considered to inhibit the proteolytic activity of cathepsin S if it has the ability to inhibit its activity by a significant amount. For example, in one embodiment, the antibody molecule is considered not to inhibit the proteolytic activity if it inhibits the proteolytic activity by no more than 10%, for example no more than 5%, such as no more than 2%, for example less than 1 %, such as 0% compared to an appropriate control antibody known to inhibit the proteolytic effect of cathepsin S. The ability of an antibody molecule to inhibit angiogenesis may be tested using any suitable assay known in the art. Many in vitro and in vivo assays are known in the art. These include Matrigel plug and corneal neovascularization assays, the in vivo/in vitro chick chorioallantoic membrane (CAM) assay, and the in vitro cellular (proliferation, migration, tube formation) and organotypic (aortic ring) assays, the chick aortic arch and the Matrigel sponge assays. Further details of such assays may be found, for example, in Auerbach et al, Clinical Chemistry 49: 32-40, 2003; 10.1373/49.1.32. Further details are also provided in the Examples.

The ability of an antibody molecule to inhibit tumour cell invasion may be tested using any suitable invasion assay known in the art. For example, such ability may be tested using a modified Boyden chamber as described in the Examples. The antibody molecule may be tested using any suitable tumour cell line, for example a prostate carcinoma cell line, e.g. PC3, an astrocytoma cell line e.g.U251 mg, a colorectal carcinoma cell line, e.g. HCT116, or a breast cancer cell line, e.g. MDA-MB-231 or MCF7. An antibody molecule is considered to inhibit tumour cell invasion if it has the ability to inhibit invasion by a statistically significant amount. For example, in one embodiment, the antibody molecule is able to inhibit invasion by at least 10%, for example at least 25%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% when compared to an appropriate control antibody.

Nucleic Acid

It is also within the scope of the present invention to employ gene therapy, specifically utilizing nucleic acid molecules which encode an anti- Cathepsin molecule, an anti-VEGF molecule or both molecules in the methods of the invention. Accordingly, in one embodiment of each aspect the invention, nucleic acid molecules encoding said antibody molecules may be used. Thus, in such embodiments, reference to the antibody molecule should be understood to encompass an alternative embodiment in which nucleic acid encoding such an antibody molecule is used. Thus, as an example, in one such embodiment related to the first aspect of the invention, there is provided a method of inhibiting angiogenesis in cells , a tissue or an individual, said method comprising the simultaneous, sequential or separate administration of (i) an anti-Cathepsin S antibody molecule, or a nucleic acid encoding said anti-Cathepsin S antibody molecule, wherein said anti-Cathepsin S antibody inhibits the proteolytic activity of Cathepsin S; and (ii) an anti-VEGF antibody molecule, or nucleic acid encoding said anti-VEGF antibody molecule, to said cells, tissue or individual.

Nucleic acid of and for use in the present invention may comprise DNA or RNA. It may be produced recombinantly, synthetically, or by any means available to those in the art, including cloning using standard techniques.

The nucleic acid may be inserted into any appropriate vector. A vector comprising a nucleic acid of the invention forms a further aspect of the present invention. In one embodiment the vector is an expression vector and the nucleic acid is operably linked to a control sequence which is capable of providing expression of the nucleic acid in a host cell. A variety of vectors may be used. For example, suitable vectors may include viruses (e. g. vaccinia virus, adenovirus, etc.), baculovirus); yeast vectors, phage, chromosomes, artificial chromosomes, plasmids, or cosmid DNA.

The vectors may be used to introduce the nucleic acids of the invention into a host cell. A wide variety of host cells may be used for expression of the nucleic acid of the invention. Suitable host cells for use in the invention may be prokaryotic or eukaryotic. They include bacteria, e.g. E. coli, yeast, insect cells and mammalian cells. Mammalian cell lines which may be used include Chinese hamster ovary cells, baby hamster kidney cells, NSO mouse melanoma cells, monkey and human cell lines and derivatives thereof and many others.

A host cell strain that modulates the expression of, modifies, and/or specifically processes the gene product may be used. Such processing may involve glycosylation, ubiquitination, disulfide bond formation and general post-translational modification.

Also encompassed by the invention is a method of production of an antibody molecule of the invention, the method comprising culturing a host cell comprising a nucleic acid of the invention under conditions in which expression of the nucleic specific binding members from the nucleic acid occurs and, optionally, isolating and/or purifying the antibody molecule.

For further details relating to known techniques and protocols for manipulation of nucleic acid, for example, in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, see, for example, Current Protocols in Molecular Biology, 5th ed.,Ausubel et al. eds., JohnWiley & Sons, 2005 and, Molecular Cloning: a Laboratory Manual: 3^rd edition Sambrook et al., Cold Spring Harbor Laboratory Press, 2001.

Treatment

The antibody molecules, nucleic acids compositions, and methods of the invention may be used in the treatment of a number of medical conditions. "Treatment" includes any regime that can benefit a human or non-human animal. The treatment may be in respect of an existing condition or may be prophylactic (preventative treatment). Treatment may include curative, alleviation or prophylactic effects.

The antibody molecules, nucleic acids, compositions and methods of the invention may be used in the treatment of cancers. "Treatment of cancer" includes treatment of conditions caused by cancerous growth and/or vascularisation and includes the treatment of neoplastic growths or tumours. Examples of tumours that can be treated using the invention are, for instance, sarcomas, including osteogenic and soft tissue sarcomas, carcinomas, e.g., breast-, lung-, bladder-, thyroid-, prostate-, colon-, rectum-, pancreas-, stomach-, liver-, uterine-, prostate , cervical and ovarian carcinoma, non-small cell lung cancer, hepatocellular carcinoma, lymphomas, including Hodgkin and non-Hodgkin lymphomas, neuroblastoma, melanoma, myeloma, Wilms tumor, and leukemias, including acute lymphoblastic leukaemia and acute myeloblastic leukaemia, astrocytomas, gliomas and retinoblastomas.

The invention may be particularly useful in the treatment of existing cancer and in the prevention of the recurrence of cancer after initial treatment or surgery.

The antibody molecules, nucleic acids and compositions of the invention may also be used in the treatment of other disorders mediated by or associated with angiogenesis. Such conditions include, for example, various autoimmune disorders, hereditary disorders, ocular disorders. Particular ocular disorders associated with angiogenesis which may be treated using the methods and antibody molecules of the invention include corneal graft rejection, neovascularization following injury or infection, rubeosis, diabetic retinopathy, retrolental fibroplasia and neovascular glaucoma, corneal diseases and macular degeneration.

The methods of the present invention may be used to treat other angiogenesis-mediated disorders including hemangioma, metastasis, telangiectasia, psoriasis, scleroderma, pyogenic granuloma, myocardial angiogenesis, Crohn's disease, plaque neovascularization, coronary collaterals, cerebral collaterals, arteriovenous malformations, ischemic limb angiogenesis, corneal diseases, retrolental fibroplasia, arthritis, diabetic neovascularization, peptic ulcer, Helicobacter related diseases, fractures, keloids, and vasculogenesis.

Specific disorders that can be treated, and compounds and compositions for use in the methods of the present invention, are described in more detail below.

Ocular Disorders Mediated by Angiogenesis

Various ocular disorders are mediated by angiogenesis, and may be treated using the methods described herein. One example of a disease mediated by angiogenesis is ocular neovascular disease, which is characterized by invasion of new blood vessels into the structures of the eye and is the most common cause of blindness. In age-related macular degeneration, the associated visual problems are caused by an ingrowth of choroidal capillaries through defects in Bruch's membrane with proliferation of fibrovascular tissue beneath the retinal pigment epithelium. In the most severe form of age-related macular degeneration (known as "wet" ARMD) abnormal angiogenesis occurs under the retina resulting in irreversible loss of vision. The loss of vision is due to scarring of the retina secondary to the bleeding from the new blood vessels. Current treatments for "wet" ARMD utilize laser based therapy to destroy offending blood vessels. However, this treatment is not ideal since the laser can permanently scar the overlying retina and the offending blood vessels often re-grow. An alternative treatment strategy for macular degeneration is the use of antiangiogenesis agents to inhibit the new blood vessel formation or angiogenesis which causes the most severe visual loss from macular degeneration.

Other conditions associated with or caused by angiogenic damage for which the present invention may be used include diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, neovascular glaucoma and retrolental fibroplasia, diseases associated with corneal neovascularization including, but are not limited to, epidemic keratoconjunctivitis, Vitamin A deficiency, atopic keratitis, superior limbic keratitis, pterygium keratitis sicca, and periphigoid radial keratotomy, diseases associated with retinal/choroidal neovascularization including, but are not limited to, macular degeneration, presumed myopia, optic pits, chronic retinal detachment, hyperviscosity syndromes, trauma and post-laser complications. Other diseases which may be treated using the invention include, but are not limited to, diseases associated with rubeosis (neovascularization of the angle) and diseases caused by the abnormal proliferation of fibrovascular or fibrous tissue including all forms of proliferative vitreoretinopathy, neovascular glaucoma, retinoblastoma, retrolental fibroplasia, rubeosis, uveitis, and corneal graft neovascularization other eye inflammatory diseases, ocular tumors, and diseases associated with choroidal or iris neovascularization. Pharmaceutical Compositions

Anti-Cathepsin S antibody molecules (and indeed nucleic acid molecules), VEGF inhibitors (including anti-VEGF antibodies), and chemotherapeutic agents may be administered simultaneously, separately or sequentially.

Where administered separately or sequentially, they may be administered within any suitable time period e.g. within 1 , 2, 3, 6, 12, 24, 48 or 72 hours of each other. In preferred embodiments, they are administered within 6, preferably within 2, more preferably within 1 , most preferably within 20 minutes of each other.

In preferred embodiments of the invention, the anti-Cathepsin S and anti- VEGF antibody moleculesA/EGF inhibitors are administered in a potentiating ratio. The term "potentiating ratio" in the context of the present invention is used to indicate that the anti-Cathepsin S and anti-VEGF antibody molecule/VEGF inhibitor (and optionally chemotherapeutic agent) are present in a ratio such that the cytotoxic activity of the combination is greater than that of either component alone or of the additive activity that would be predicted for the combinations based on the activities of the individual components.

Thus in a potentiating ratio, the individual components act synergistically.

Synergism may be defined using a number of methods.

For example, synergism may be defined as an Rl of greater than unity using the method of Kern et al (Cancer Res, 48: 117-121 , 1988) as modified by Romaneli et al (Cancer Chemother Pharmacol, 41: 385-390, 1998). The Rl may be calculated as the ratio of expected cell survival (Sexp) defined as the product of the survival observed with drug/antibody A alone and the survival observed with drug/antibody B alone) to the observed cell survival (Sobs) for the combination of A and B (RI=Sexp/Sobs).

Synergism may then be defined as an Rl of greater than unity. For example, in one embodiment of the invention, the anti-Cathepsin S antibody molecule and the anti-VEGF antibody molecule/VEGF inhibitor are provided in concentrations sufficient to produce an Rl of greater than 1.5, more preferably greater than 2.0, most preferably greater than 2.25.

In another method, synergism may be determined by calculating the combination index (Cl) according to the method of Chou and Talalay (Adv Enzyme Regul, 22: 27-55, 1984). Cl values of 1 , < 1 , and > 1 indicate additive, synergistic and antagonistic effects respectively. In one embodiment of the invention, the anti-Cathepsin S antibody molecule and the anti-VEGF antibody molecule/VEGF inhibitor are present in concentrations sufficient to produce a Cl of less than 1 , preferably less than 0.85.

The combined medicament thus preferably produces a synergistic effect when used to treat tumour cells.

Dose

The antibody molecules, or indeed nucleic acid molecules encoding said antibody molecules, inhibitors and chemotherapeutic agents, where appropriate are preferably administered to an individual in a "therapeutically effective amount", this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. As described herein, the concentrations are preferably sufficient to show a synergistic effect. Prescription of treatment, e.g. decisions on dosage etc, is ultimately within the responsibility and at the discretion of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners.

The optimal dose can be determined by physicians based on a number of parameters including, for example, age, sex, weight, severity of the condition being treated, and the active ingredient being administered. For example, with respect to antibodies, in general, a serum concentration of antibodies that permits saturation of receptors is desirable. A concentration in excess of approximately 0.1 nM is normally sufficient. For example, a dose of 100mg/m² of antibody provides a serum concentration of approximately 2OnM for approximately eight days.

As a rough guideline, doses of antibodies may be given in amounts of 1 ng/kg- 500mg/kg of patient weight. Equivalent doses of antibody fragments should be used at the same or more frequent intervals in order to maintain a serum level in excess of the concentration that permits saturation of the target e.g. Cathepsin S or VEGF.

Doses of the antibody molecules may be given at any suitable dose interval e.g. daily, once, twice or thrice weekly.

For example, the periods of administration of a humanised antibody could be from 1 bolus injection to weekly administration for up to one year in combination with chemotherapeutic agents. The likely dose is upwards of 1 mg/per kg/per patient.

In a preferred embodiment, they are administered as a pharmaceutical composition, which will generally comprise a suitable pharmaceutical excipient, diluent or carrier selected dependent on the intended route of administration.

Antibody molecules, nucleic acid molecules, inhibitors and chemotherapeutic agents of and for use in the present invention may be administered to a patient in need of treatment via any suitable route. The precise dose will depend upon a number of factors, including the precise nature of the member (e.g. whole antibody, fragment or diabody) and chemotherapeutic agent.

The antibody molecules, nucleic acid molecules, inhibitors and chemotherapeutic agents, as appropriate, may be administered as a pharmaceutical composition. Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention may comprise, in addition to active ingredients, a pharmaceutically acceptable excipient, a carrier, buffer stabiliser or other materials well known to those skilled in the art (see, for example, (Remington: the Science and Practice of Pharmacy, 21^st edition, Gennaro AR, et al, eds., Lippincott Williams & Wilkins, 2005.). Such materials may include buffers such as acetate, Tris, phosphate, citrate, and other organic acids ; antioxidants; preservatives; proteins, such as serum albumin, gelatin, or immunoglobulins ; hydrophilic polymers such aspolyvinylpyrrolidone ; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine ; carbohydrates; chelating agents; tonicifiers; and surfactants. The pharmaceutical compositions may also contain one or more further active compound selected as necessary for the particular indication being treated, preferably with complementary activities that do not adversely affect the activity of the antibody molecule, nucleic acid or composition of the invention. For example, in the treatment of cancer, in addition to an anti-Cathepsin S antibody molecule and an anti VEGF inhibitor, for example and anti-VEGF antibody molecule, the formulation may comprise an additional antibody, for example which may bind a different epitope on Cats, a different VEGF epitope, and/or a chemotherapeutic agent.

The active ingredients (e.g. antibody molecules and/or inhibitors and/or chemotherapeutic agents) may be administered via microspheres, microcapsules liposomes, other microparticulate delivery systems. For example, active ingredients may be entrapped within microcapsules which may be prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly- (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. For further details, see Remington: the Science and Practice of Pharmacy, 21^st edition, Gennaro AR, et al, eds., Lippincott Williams & Wilkins, 2005.

Sustained-release preparations may be used for delivery of active agents. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e. g. films, suppositories or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly (2-hydroxyethyl- methacrylate), or poly (vinylalcohol)), polylactides (U. S. Pat. No. 3, 773, 919), copolymers of L-glutamic acid and ethyl-Lglutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, and poly-D- (-)-3-hydroxybutyhc acid.

As described above nucleic acids encoding anti-cathepsin S antibodies or anti-VEGF antibodies may also be used in methods of treatment. Nucleic acid of the invention may be delivered to cells of interest using any suitable technique known in the art. Nucleic acid (optionally contained in a vector) may be delivered to a patient's cells using in vivo or ex vivo techniques. For in vivo techniques, transfection with viral vectors (such as adenovirus, Herpes simplex I virus, or adeno-associated virus) and lipid- based systems (useful lipids for lipid-mediated transfer of the gene are DOTMA, DOPE and DC-Choi, for example) may be used (see for example, Anderson et al., Science 256 : 808-813 (1992). See also WO 93/25673 ).

In ex vivo techniques, the nucleic acid is introduced into isolated cells of the patient with the modified cells being administered to the patient either directly or, for example, encapsulated within porous membranes which are implanted into the patient (see, e. g. U. S. Patent Nos. 4, 892, 538 and 5, 283, 187). Techniques available for introducing nucleic acids into viable cells may include the use of retroviral vectors, liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc.

The antibody molecule, agent, inhibitor, product or composition may be administered in a localised manner to a site of desired action, for example a tumour site or other desired site or may be delivered in a manner in which it targets tumour or other cells. Targeting therapies may be used to deliver the active agents more specifically to certain types of cell, by the use of targeting systems such as antibody or cell specific ligands. Targeting may be desirable for a variety of reasons, for example if the agent is unacceptably toxic, or if it would otherwise require too high a dosage, or if it would not otherwise be able to enter the target cells.

The invention will now be described further in the following non-limiting examples. Reference is made to the accompanying figures in which:

Figure 1A shows a bar chart illustrating the results of a HUVEC tube assay to assess the effect of a combination of anti-VEGF (250ng/ml) and 1 E11 (40OnM) on angiogenesis, in which the number of tubes was measured;

Figure 1 B shows a bar chart illustrating the results of a HUVEC tube assay to assess the effect of a combination of anti-VEGF (250ng/ml) and 1 E11 (20OnM) on angiogenesis, in which the number of tubes was measured;

Figure 2A shows a bar chart illustrating the results of a HUVEC tube assay to assess the effect of a combination of anti-VEGF (250ng/ml) and 1 E11 (20OnM) on tube disruption;

Figure 2B shows a bar chart illustrating the results of a HUVEC tube assay to assess the effect of a combination of anti-VEGF (250ng/ml) and 1 E11 (20OnM) on tube formation;

Figure 3 illustrates shows a bar chart illustrating the results of a HUVEC tube assay to assess the effect of a combination of anti-VEGF (10ng/ml) and 1 E11 (40OnM) on tube formation as assessed by the number of nodes having 1 , 2, 3 or 4 branch points; Figure 4 illustrates sequence alignments for anti-Cathepsin S humanised Vh and Vk chains;

Figure 5 illustrates the results of a Chimeric IgG competition ELISA;

Figure 6 illustrates the results of a comparison of composite humanized Antibodies Activities with a mouse Reference Antibody;

Figure 7 illustrates the results of a comparison of composite Humanized Antibodies Activities with a mouse Reference Antibody;

Figure 8 is a table summarising IC50 Values for some Anti-Cathepsin S Composite and CDR Human Antibody Sequence Variants;

Figure 9 is a schematic diagram illustrating the variable regions of the composite human antibodies 4, 15 and 23 used in the examples;

Figure 10 is a bar chart illustrating the dose-dependent reduction in tumour cell invasion obtained using humanised antibodies 4, 15 and 23;

Figure 11 a is a bar chart summarising the effect of humanised antibodies of the invention in comparison to murine antibody in 4T1 breast cancer cells;

Figure 11 b is a bar chart summarising the effect of humanised antibodies of the invention in comparison to murine antibody in H460 lung cancer cells;

Figure 12 illustrates the effect of the human antibodies in a HUVEC tube formation assay. Figure 12a is a bar chart illustrating the average branch count in the presence of various concentrations of antibodies; figure 12b is a bar chart summarising the effect of the antibodies on the number of tubes formed; figure 12c is a representative photograph showing branching of tubes in the tube formation assay; and figure 12d is a table summarising the percentage reduction in tube formation with each antibody at three concentrations;

Figure 13 is bar chart comparing the effect of the Cathepsin S humanised antibody 15 against that of herceptin on ADCC in two different colorectal cancer cell lines;

Figure 14 is a bar chart illustrating % cytotoxicity observed with various anti-cathepsin humanised antibodies, herceptin and controls in the LoVo colorectal cancer cell line with Figure 14b showing that the effect of the anti-cathepsin antibodies was neutralised by anti-CD16 antibodies;

Figure 15a is a bar chart illustrating % cytotoxicity observed with humanised antibody 4 in the colorectal cancer cell line Colo205;

Figure 15b is a bar chart illustrating % cytotoxicity observed with humanised antibody 15 in the colorectal cancer cell line Colo205;

Figure 15c is a bar chart illustrating % cytotoxicity observed with humanised antibody 23 in the colorectal cancer cell line Colo205;

Figure 16 is a bar chart illustrating % cytotoxicity observed with humanised antibodies 4, 15 and 23 in the bladder cancer cell line EJ28;

Figure 17 is a graph illustrating the effect of cathepsin S humanised antibodies on tumour growth in Colo205 xenograft model; Figure 18 is a graph illustrating the effect of cathepsin S antibodies on tumour growth in a HCT116 xenograft model; and

Figure 19 is a bar chart illustrating the effect of the cathepsin S 1 E11 antibody on a tube formation assay in the presence and absence of sorafenib.

Examples

Example 1 Effect of Anti-Cathepsin S and Anti- VEGF antibodies on angiogenesis in a HUVEC Tube Formation assay

The effect of the Anti-Cathepsin S antibody 1 E11 and the Anti- VEGF antibody at different concentrations on angiogenesis was tested using the human vascular endothelial cell tube formation assay. Briefly, human umbilical vein endothelial cells (HUVECs) suspended in MCBD 131 growth medium, containing target antibodies or isotype control were seeded onto mathgel coated tissue culture plates. After incubation at 37°C and 5% CO2 for 6-24 hrs, cells were viewed using a Nikon Eclipse TE300 microscope and images taken using a Nikon DXM1200 digital camera at magnification x20. Total tubule length and branching was counted and results were expressed as a percentage relative to the control.

The results are shown in Figures 1 A, 1 B, 2A, 2B and 3. As shown in

Figure 1 , the total number of tubes formed in the presence of either 1 E11 or Anti- VEGF antibody was reduced compared to control (VEGF only). However, results were only significant in the presence of both Anti- Cathepsin S antibody 1 E11 and the Anti- VEGF antibody. The Rl value was used to assess whether or not the effect of using the combination of Anti-Cathepsin S antibody 1 E11 and the Anti- VEGF antibody was synergistic; the result obtained (Figure 1A) ( Rl=2.6) indicates very strong synergy. The experiment was repeated with Anti-Cathepsin S antibody 1 E11 at 20OnM (Figure 1 B). The result obtained ( RI=4.0) again indicates very strong synergy The effect of the antibody combination was further investigated by measuring the mean count of branching points with the results shown in Figures 2A and 2B. The results clearly show that in the presence of the combination of both antibodies the percentage of tubules branching is significantly reduced. Figure 3 further summarises the effect of combination treatment with both antibodies and shows that combination therapy results in a reduction in the number of branch points per tubule.

Example 2 Development of Humanized cathepsin S antibodies

Having established that the combination of anti-Cathepsin S antibody molecules and anti-VEGF antibody molecules were particularly efficacious in the inhibition of angiogenesis, the inventors proceeded to develop anti- Cathepsin S humanised antibodies for use in the invention. Two methods were employed: conventional CDR grafting and Composite Human Antibody technology ™, (Antitope, Cambridge, UK). Composite Human Antibodies are entirely human in origin and comprise multiple segments of human variable region sequence from different human antibodies. Briefly, the Cathepsin S humanised antibodies were developed as follows. The antigen binding regions of the variable regions of the anti-Cathepsin S 1 E11 antibody were used as a reference to identify sequence segments from a database of unrelated human antibody variable regions; composite human variable regions with similar binding properties to the IE11 were created and screened to determine the presence of potential T cell epitopes, with only those sequence segments which do not comprise such epitopes selected for further study. These selected segments were then tested for cathepsin S binding and the segments utilised to construct the Composite Human Antibodies with properties of 1 E11.

Figure 4 shows an alignment of the humanised V_H and V_κ chains developed using the CDR and Composite Human Antibody technology with the corresponding 1 EII murine V_H and V_κ chains. For each of the heavy chain and light chain alignments, a consensus sequence is shown below. For the heavy chains, Human V_H3.7 framework was chosen for CDR grafted antibodies, with back mutations to the corresponding mouse amino acids at residues 82a, 83 and 84 (Kabat numbering) in order to maintain structure. These back mutations were omitted in Vh4. Three different V_H frameworks were chosen to construct the Composite V_H sequences: FW1 was based upon V_H3-21 , FW2 upon V_H3-7 and FW3 upon V_H3-74. FW3 of Vh1 also included a fragment of sequence (79-86) derived from human antibody heavy chain accession number AAX82494. All heavy chains included the germline J6 segment

For the light (kappa) chains, Human VK2.30 framework was chosen for CDR grafted antibodies with back mutations at residues 36, 46 and 87 (Kabat numbering) in Vk3 and Vk4 in order to maintain structure, with an additional back mutation (residue 37) in Vk3. Two different V_κ frameworks were chosen to construct the Composite V_κ sequences: FW1 was based upon V_K2-30, with FW2 and FW3 based upon V_K2-28. FW3 of Vk1 also included a fragment of sequence (76-88) derived from human antibody light chain accession number BAE94187. All light chains included the germline J2 segment. Vk5 was made additionally to investigate the contribution to antigen binding of residue K45. Vh1 and Vh2 and Vk1 and Vk2 are Composite Human Antibodies™ chains. Vh3 and Vh4 and Vk3, Vk4 and Vk5 were generated by CDR grafting. (NB Herein the nomenclature VH, Vh, and V_H are used interchangeably and VK, Vk, and V_κ are also used interchangeably.)

Humanised antibody molecules were produced using the following combinations of V_H and V_L chains:

VK1/VH1 (Humanised Antibody 4); VK1A/H2 (Humanised Antibody 53); VK2A/H1 (Humanised Antibody 15); VK2A/H2 (Humanised Antibody 23); VK3A/H3 (Humanised Antibody 43); VK3A/H4 (Humanised Antibody 56); VK4A/H3 (Humanised Antibody 36); VK4A/H4 (Humanised Antibody 28); VK5A/H3 (Humanised Antibody 13); and VK5/VH4 (Humanised Antibody 76).

Example 3 Binding activity of the Human Cathepsin S antibodies

The binding of the purified antibodies to human cathepsin S was tested via competition ELISA. The results are shown in Figures 5, 6 and 7. Briefly, for each of the chimeric and composite human antibodies tested, a dilution series of antibodies were tested against a fixed concentration of biotinylated mouse anti-CatS (0.5μg/ml) for binding to human cathepsin S (coated at 100ng/ml). Binding of biotinylated antibody decreases with increasing amounts of chimeric and control antibodies. As shown in Figure 5, which shows the result of testing chimeric IgGI , lgG4 versus control mouse antibodies, the IC50 of the chimeric IgGI (0.09μg/ml) and chimeric lgG4 antibodies (0.11 μg/ml) was considerably less than that of the murine antibody (0.17μg/ml).

Figure 6 shows the binding of each of the Vk2Vh1 , Vk2Vh2, Vk3Vh3 and Vk3Vh4 compared to the mouse antibody with Figure 7 showing the binding of each of the Vk4Vh3, Vk4Vh4, Vk5Vh3 and Vk5Vh4 antibodies compared to the mouse antibody. IC50 values were calculated and normalized against the binding of mouse. A normalized value of 1 is equal to mouse binding and a value <1 is improved binding relative to mouse antibody. The results are shown in Figure 8, which shows improved binding for each of the humanized antibody molecules.

In order to confirm that the humanised antibodies bound to the same target as the 1 E11 antibody, epitope mapping was performed. pQE30 with cloned fragments of Cats proform were sequenced and TOP10F' cells were transformed with the constructs. 3ml cultures were grown and induced using standard IPTG induction (after 3hrs of growth at 37C, 1 δOrpm for another 3hrs of growth in presence of 1 mM IPTG). After induction urea lysates were prepared from each culture and loaded onto PAGE gel to perform control Western Blot with antiHis antibody (anti- polyhistidide-peroxidase antibody (Sigma, #A7058-1VL) 1 :2000). The fragments were then probed with the 1 E11 antibody 1 :5000 with goat anti- mouse-peroxidase used as secondary antibody in 1 :10000 dilution (BioradHRP , # 172-1011 ). The gel was then probed with each of the human cathepsin S antibodies in turn (antibodies 4, 15 and 23, each ) 1 :5000 with rabbit anti-human-peroxidase used as the secondary antibody in 1 :10000 dilution, In each case, HRP was detected using DAB.

The same pattern of binding was shown for each of the cathepsin S human antibodies and the 1 E11 antibody, confirming that the antibodies bind the same epitope.

Example 4 Effect of the Human Cathepsin S antibodies on Tumour cell invasion The effect of the humanized antibodies on tumour cell invasion was assessed in-vitro using some of the developed humanised antibodies and a number of different cancer cell lines, including the breast cancer cell line MDA-MB-231 , the glioblastoma cell line U251 mg, the breast cancer cell line 4T1 , the prostate cancer cell line PC3, the bladder cancer cell lines J82 and EJ28 and the lung cancer cell line H460. Briefly, invasion assays were performed using a modified Boyden chamber with 12-μrτn pore membranes (Costar Transwell plates, Corning Costar Corp., Cambridge, MA, USA). The membranes were coated with Matrigel (100 μg/cm²) (Becton Dickinson, Oxford, UK) and allowed to dry overnight in a laminar flow hood. Cells were added to each well in 500 μl of serum-free medium in the presence of predetermined concentrations of the antibodies or control antibody. Invasion plates were incubated at 37 ⁰C and 5% CO2 for 24 hours after which cells remaining on the upper surface of the membrane were removed and invaded cells fixed in Carnoy's fixative for 15 minutes. After drying, the nuclei of the invaded cells were stained with Hoechst 33258 (50 ng/ml) in PBS for 30 minutes at room temperature. The chamber insert was washed twice in PBS, mounted in Citifluor and invaded cells were viewed with a fluorescent microscope. Digital images of representative fields from each of the triplicate membranes were taken using a digital camera at magnification of x20 and the results analysed.

In each of the cell lines tested, the humanised antibodies were shown to attenuate invasion of the tumour cell lines. Figure 10 is a bar chart illustrating the dose-dependent reduction in U251 mg tumour cell invasion obtained using antibodies 4, 15 and 23 and shows that each of these antibodies causes a dose-dependent reduction in invasion in the cell line. A similar result was obtained using the breast cancer cell line MDA-MB- 231 , in which the VK2VH2 antibody (antibody 23), VK2VH1 antibody (antibody 15) and the VK1VH1 (antibody 4) antibody demonstrated significant inhibition of tumour cell invasion compared to an isotype control.

Figure 11 summarises the effect of humanised antibodies of the invention in comparison to murine antibody in 4T1 breast cancer cells; and in H460 lung cancer cells and show a significant attenuation of invasion of the cancer cells in comparison to control isotype antibodies, with the attenuation similar to that obtained with the murine antibody 1 E11. Similar results were found with each of the other cancer cell lines tested.

Example 5 Effect of the Human Cathepsin S antibodies on angiogenesis in a HUVEC Tube Formation assay

The effect of the humanised antibodies at different concentrations on angiogenesis was tested using the human vascular endothelial cell tube formation assay. Briefly, human umbilical vein endothelial cells (HUVECs) suspended in MCBD 131 growth medium, containing target antibodies or isotype control were seeded onto matrigel coated tissue culture plates. After incubation at 37°C and 5% CO2 for 6-24 hrs, cells were viewed using a Nikon Eclipse TE300 microscope and images taken using a Nikon DXM1200 digital camera at magnification x20. Total tubule length and branching was counted and results were expressed as a percentage relative to the control. The results are shown in figure 12.

Figure 12a illustrates that, in the presence of each of the humanised antibodies, the average branch count decreased with figure 12b showing that there was a dose-dependent reduction in total tubule length in the presence of each of the humanised antibodies. The table in figure 12d summarises the reduction in total tubule length in the presence of each concentration of each antibody. Example 6 Effect of the Human Cathepsin S antibodies on ADCC

Each of humanised antibodies 4, 15 and 23 were tested for induction of antibody dependent cell mediated cytotoxicity (ADCC) using a Cytotox 96 ® non-radioactive cytotoxicity assay. The assay employs quantitative measurement of LDH (lactose dehydrogenase), a stable cytosolic enzyme that is released upon cell lysis, as an indicator of cytotoxicity. Briefly, the cancer cells (e.g. LoVo / CoLo205 / HCT116 / EJ28 / MDA361 etc.) were washed once with PBS. After removal of the PBS 3ml of trypsin was added to your target cells and the cells incubated at 37⁰C for 1 -2 minutes. 7ml of PBS was added into the trypsin and the mixture spun down at 200- 500 x g for 3-5 minutes Prior to resuspension in 10ml ADCC culture media (RPMI 1640 containing 5% FBS +15mM HEPES). The cell count was checked with Trypan Blue stain and cells adjusted to a concentration 200- 320 cells/μl. Plating 25μl of this gave 5,000-8,000 cells/well.

PBMCs from buffy pack: 50ml of Ficoll was pipetted into a 50ml tube. 40ml of RPMI 1640 diluted blood (1 :1 ratio) was layered on the Ficoll and centhfuged at 400 x g for 25 minutes. The middle layer containing the

PBMCs was aspirated into another tube and the PBMCs washed with 1 -2 volumes of RPMI 1640, prior to centrifuge at 400 x g for 5 minutes. After the wash, the supernatant was decanted and the cells pellet gently vortexed.

Red Blood Cells were lysed as follows: The cell pellet was resuspended in 20ml of 0.2% NaCI for 30 seconds to lyse RBCs (gently agitate the tube to ensure proper mixing) 20ml of 1.6% NaCI was added to neutralise the osmolarity of the cells, after which the cells were centrifuged at 400 x g for 5 minutes and the pellet resuspended in 2-5ml ADCC culture media. Cells were counted with Trypan blue at a 1 :100 dilution. Bring cells to 10,000- 16,000 cells/μl. Plating 50μl of this will give 500,000-800,000 cells/well and a effectontarget cell ratio of 100:1 in the wells.

The antibody samples (e.g. Humanized Anti-CatS / HER2 +ve control Abs) were prepared in triplicate as follows: The antibody was diluted to 200μl/ml in ADCC culture media. The antibody was serially diluted 4-fold 7 times across the plate. Plating 25μl of each antibody concentration per well yielded a range from 50μg/ml to 0.12μg/ml (final concentrations). Starting antibody concentrations were 4x since they were subsequently diluted 1 in 4 by the addition of target and effector cells. For the zero antibody control, 25μl of media was plated only (control 5)

The ADCC Reaction was performed as follows: The target cells were plated in a 96 well plate at 5,000-8,000 cells/well in 25μl of media in triplicate, with the following controls: 1 ) Effector Cell Spontaneous LDH Release: Corrects for spontaneous release of LDH from effector cells; .2) Target Cell Spontaneous LDH Release: Corrects from spontaneous release of LDH from target cells; 3) Target Cell Maximum LDH Release: Required in calculations to determine 100% release of LDH; 4) Volume Correction Control: Corrects for volume change caused by addition of Lysis Solution (10x); and 5)Culture Medium Background: Corrects for LDH activity contributed by serum in culture medium and the varying amounts of phenol red in the culture medium. 25μl of each dilution of the antibody was plated in the wells containing the target cells. The plate was incubated at 37⁰C for 15 minutes to allow opsonisation of antibody to occur. (This optional step should be included based on the target cell line.) 50μl of the effector cells were added to start the reaction. The plate was covered with a 96-well plate cover and incubated at 37⁰C for 2-4 hours. Forty-five minutes prior to harvesting supernatants, 10μl of Lysis Solution (10X) was added for every 100μl of target cells to the wells containing the Target Cell Maximum LDH Release Control. After the 2-4 hours incubation, the plate was centrifuged at 250 x g for 4 minutes.

The LDH Measurement was performed as follows: 50μl aliquots were transferred from all wells using a multichannel pipettor to a fresh 96-well flat-bottom (enzymatic assay) plate.50μl of reconstituted Substrate Mix was added to each well of the enzymatic assay plate containing samples transferred from the cytotoxicity assay plate. The plate was covered with foil to protect it from light and incubated for 30 minutes at room temperature, prior to addition of 50μl of Stop Solution to each well. The absorbance was measured at 490 or 492nm within 1 hour after the addition of Stop Solution.

The results were calculated as follows: The average of absorbance values of the Culture Medium Background was subtracted from all absorbance values of Experimental, Target Cell Spontaneous LDH Release and Effector Cell Spontaneous LDH Release. The average of the absorbance values of the Volume Correction Control was subtracted from the absorbance values obtained from the Target Cell Maximum LDH Release Control. Using the corrected values obtained in Steps 1 and 2 in the following formula the percent cytotoxicity for each effectontarget cell ratio was calculated.

%Cytotoxicity =

Experimental-Effector Spontaneous -Target Spontaneous x 100 Target maximum-Target Spontaneous

As a positive control, herceptin was used, The humanized antibody 15 was first tested for cytotoxicity against colorectal cancer cells (LoVo and MDA361 cells). The results are shown in figure 13. With each cell line, significant cytotoxicity was observed. The experiment was repeated in the presence of anti-CD16 antibodies (trastuzumab), and the presence of which no cytotoxicity was observed. This confirms that the effect seen with the humanised antibody is attributable to ADCC. The assay was repeated with humanised antibody 15 and 23 with similar results obtained (see figure 14)

Similar ADCC assays were performed with each of the humanised antibodies in other cancer cell lines. The results for CoLo205 colorectal cancer cells and EJ28 bladder cancer cells are shown in figures 15 and 16 respectively. In the examples shown, the cytotoxicity measured in CoLo205 cells for antibodies 4, 15, and 23 was approximately 27%, 19% and 15% respectively (figures 15a, 15b, and 15c) with the cytotoxicity measured in EJ28 cells for antibodies 4, 15, and 23 measured as approximately 23%, 8% and 9% respectively.

Example 7 Effect of the Human Cathepsin S antibodies on Tumour Growth

The Cathepsin S human antibodies were then tested in a Colo205 Xenograft model to determine if they had any effect on established tumours. Briefly, Colo205 cells were inoculated onto the flanks of MF1 nude mice. Tumours were allowed to grow to ~100mm³ prior to grouping for various treatments. Treatment started at day 17 with a dose of antibody 15 of 20mg / kg administered 5 times weekly i.v. The results of the study are summarised in Figure 17, which shows that the antibody caused tumour growth inhibition when compared to treatment with a human isotype control antibody. A similar study was performed on an HCT116 xenograft model, but using humanised antibody 4, with results shown in figure 18. In common with results shown in figure 17, the humanised antibody caused an attenuation of tumour growth in the model. In addition, the antibody was tested in combination with the chemotherapeutic agent CPT11 with treatment started at day 17 (4mg/kg CPT11 and 20mg / kg antibody 4 each administered three times wkly). As can be seen from the graph, the combination treatment provided enhanced attenuation of tumour growth compared to the chemotherapy alone.

Example 8 Effect of Cathepsin S antibodies in combination with VEGF inhibitors on Angiogenesis

The effect on angiogenesis of the cathepsin S antibodies in combination with the VEGF inhibitor Sorafenib at different concentrations was tested using the human vascular endothelial cell tube formation assay, as described above. The results are shown in figure 19. As can be seen from the figure the total number of nodes formed in the presence of either the 1 E11 or Sorafenib was reduced compared to the presence of the isotype control. The combination of both the 1 E11 and Sorafenib resulted in a further significant decrease in the number of nodes.

All documents referred to in this specification are herein incorporated by reference. Various modifications and variations to the described embodiments of the inventions will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the invention which are obvious to those skilled in the art are intended to be covered by the present invention.

Claims

1. A method of inhibiting angiogenesis, said method comprising the simultaneous, sequential or separate administration of (i) an anti-Cathepsin S antibody molecule, wherein said anti-Cathepsin S antibody inhibits the proteolytic activity of Cathepsin S, or a nucleic acid encoding said anti-Cathepsin S antibody molecule; and (ii) an anti-VEGF antibody molecule, or a nucleic acid encoding said anti-VEGF antibody molecule, to said cells.

2. A method of treating a disease associated with angiogenesis comprising the simultaneous, sequential or separate administration of (i) an anti-Cathepsin S antibody molecule, wherein said anti-Cathepsin S antibody inhibits the proteolytic activity of Cathepsin S, or a nucleic acid encoding said anti-Cathepsin S antibody molecule; and (ii) an anti-VEGF antibody molecule, or a nucleic acid encoding said anti-VEGF antibody molecule.

3. The method according to claim 1 or claim 2, wherein the antibody molecule comprises an antigen binding domain comprising (i) at least one of the CDRs with an amino acid sequence selected from the group consisting of Seq ID No: 1 , Seq ID No: 2, Seq ID No: 3, or a variant thereof in which 5 or less, for example 4, 3, 2, or 1 amino acid substitutions have been made in at least one CDR, and/or (ii) at least one of the CDRs with an amino acid sequence consisting of Seq ID No: 4, Seq ID No: 5 and Seq ID No: 6, or a variant thereof, in which 5 or less, for example 4, 3, 2, or 1 amino acid substitutions have been made in at least one CDR; wherein the antibody molecule retains the ability to inhibit the proteolytic activity of Cathepsin S.

4. The method according to any one of claims 3 to 5, wherein the antibody molecule comprises an antibody V_H domain which comprises CDRs with amino acid sequences Seq ID No: 1 , Seq ID No: 2 and Seq ID No: 3 as CDRs 1 , 2 and 3 respectively.

5. The method according to claim 4, wherein the antibody V_H domain comprises the amino acid sequence Seq ID No: 7.

6. The method according to any one of the preceding claims 3 to 8, wherein the antibody V_L domain comprises CDRs with amino acid sequences Seq ID No: 4, Seq ID No: 5 and Seq ID No: 6 as CDRs 1 , 2 and 3 respectively.

7. The method according to any one of claims 1 to 6, wherein the antibody V_L domain comprises the amino acid sequence Seq ID No: 8.

8. The method according to any one of claims 1 to 4 or claim 6, wherein said antibody molecule is a human or humanised antibody molecule.

9. The method according to claim 8, wherein the antibody molecule comprises an antibody V_H domain having the amino acid sequence shown as Sequence ID No: 9, Sequence ID No: 10, Sequence ID No: 11 , Sequence ID No: 12, or Sequence ID No: 13.

10. The method according to claim 8 or claim 9, wherein the antibody molecule comprises an antibody V_L domain having the amino acid sequence shown as Sequence ID No: 14, Sequence ID No: 15, Sequence ID No: 16, Sequence ID No: 17, Sequence ID No: 18, or Sequence ID No: 19.

11. The method according to any one of the preceding claims, wherein said anti-VEGF antibody molecule is an anti-VEGF-A antibody molecule.

12. The method according to claim 1 1 , wherein said anti- VEGF-A antibody molecule is a humanised or human antibody molecule.

13. The method according to claim 1 1 , wherein said antibody molecule is bevacizumab.

14. A method of inhibiting angiogenesis, said method comprising the simultaneous, sequential or separate administration of (i) an anti-Cathepsin S antibody molecule, wherein said anti-Cathepsin S antibody does not inhibit the proteolytic activity of Cathepsin S, or a nucleic acid encoding said anti-Cathepsin S antibody molecule; and (ii) an anti-VEGF-A antibody molecule, or a nucleic acid encoding said anti-VEGF-A antibody molecule, to said cells.

15. A method of treating a disease associated with angiogenesis comprising the simultaneous, sequential or separate administration of (i) an anti-Cathepsin S antibody molecule, wherein said anti-Cathepsin S antibody does not inhibit the proteolytic activity of Cathepsin S, or a nucleic acid encoding said anti- Cathepsin S antibody molecule; and (ii) an anti-VEGF-A antibody molecule, or a nucleic acid encoding said anti-VEGF-A antibody molecule, to said cells.

16. The method according to claim 15 or claim 16, wherein said anti-VEGF-A antibody molecule is a humanised or human antibody molecule.

17. The method according to claim 16, wherein said antibody molecule is bevacizumab.

18. An anti-Cathepsin S antibody molecule, wherein said anti-Cathepsin S antibody inhibits the proteolytic activity of Cathepsin S, or a nucleic acid encoding said anti- Cathepsin S antibody molecule; and (ii) an anti-VEGF antibody molecule, or a nucleic acid encoding said anti-VEGF antibody molecule, for simultaneous, sequential or separate administration for the treatment of a disease associated with angiogenesis.

19. The (i) anti-Cathepsin S antibody molecule, or nucleic acid encoding said anti-Cathepsin S antibody molecule and (ii) anti-VEGF antibody molecule, or nucleic acid encoding said anti-VEGF antibody molecule, according to claim 18, wherein the anti-Cathepsin S antibody molecule comprises an antigen binding domain comprising (i) at least one of the CDRs with an amino acid sequence selected from the group consisting of Seq ID No: 1 , Seq ID No: 2, Seq ID No: 3, or a variant thereof in which 5 or less, for example 4, 3, 2, or 1 amino acid substitutions have been made in at least one CDR, and/or (ii) at least one of the CDRs with an amino acid sequence consisting of Seq ID No: 4, Seq ID No: 5 and Seq ID No: 6, or a variant thereof, in which 5 or less, for example 4, 3, 2, or 1 amino acid substitutions have been made in at least one CDR; wherein the anti-Cathepsin S antibody molecule retains the ability to inhibit the proteolytic activity of Cathepsin S.

20. The (i) anti-Cathepsin S antibody molecule, or nucleic acid encoding said anti-Cathepsin S antibody molecule and (ii) anti-VEGF antibody molecule, or nucleic acid encoding said anti-VEGF antibody molecule, according to claim 19, wherein the anti-Cathepsin S antibody molecule comprises an antibody V_H domain which comprises CDRs with amino acid sequences Seq ID No: 1 , Seq ID No: 2 and Seq ID No: 3 as CDRs 1 , 2 and 3 respectively.

21. The (i) anti-Cathepsin S antibody molecule, or nucleic acid encoding said anti-Cathepsin S antibody molecule and (ii) anti-VEGF antibody molecule, or nucleic acid encoding said anti-VEGF antibody molecule, according to claim 20, wherein the anti-Cathepsin S antibody molecule V_H domain comprises the amino acid sequence Seq ID No: 7.

22. The (i) anti-Cathepsin S antibody molecule, or nucleic acid encoding said anti-Cathepsin S antibody molecule and (ii) anti-VEGF antibody molecule, or nucleic acid encoding said anti-VEGF antibody molecule, according to any one of claims 18 to 21 , wherein the anti- Cathepsin S antibody molecule V_L domain comprises CDRs with amino acid sequences Seq ID No: 4, Seq ID No: 5 and Seq ID No: 6 as CDRs 1 , 2 and 3 respectively.

23. The (i) anti-Cathepsin S antibody molecule, or nucleic acid encoding said anti-Cathepsin S antibody molecule and (ii) anti-VEGF antibody molecule, or nucleic acid encoding said anti-VEGF antibody molecule, according to any one of claims 18 to 22, wherein the anti- Cathepsin S antibody molecule V_L domain comprises the amino acid sequence Seq ID No: 8.

24. The (i) anti-Cathepsin S antibody molecule, or nucleic acid encoding said anti-Cathepsin S antibody molecule and (ii) anti-VEGF antibody molecule, or nucleic acid encoding said anti-VEGF antibody molecule, according to any one of claims 18 to 20 or claim 22, wherein said anti-Cathepsin S antibody molecule is a human or humanised antibody molecule.

25. The (i) anti-Cathepsin S antibody molecule, or nucleic acid encoding said anti-Cathepsin S antibody molecule and (ii) anti-VEGF antibody molecule, or nucleic acid encoding said anti-VEGF antibody molecule, according to claim 24, wherein the anti-Cathepsin S antibody molecule comprises an antibody V_H domain having the amino acid sequence shown as Sequence ID No: 9, Sequence ID No: 10, Sequence ID No: 11 , Sequence ID No: 12, or Sequence ID No: 13.

26. The (i) anti-Cathepsin S antibody molecule, or nucleic acid encoding said anti-Cathepsin S antibody molecule and (ii) anti-VEGF antibody molecule, or nucleic acid encoding said anti-VEGF antibody molecule, according to claim 24 or claim 25, wherein the anti-Cathepsin S antibody molecule comprises an antibody V_L domain having the amino acid sequence shown as Sequence ID No: 14, Sequence ID No: 15, Sequence ID No: 16, Sequence ID No: 17, Sequence ID No: 18, or Sequence ID No: 19.

27. The (i) anti-Cathepsin S antibody molecule, or nucleic acid encoding said anti-Cathepsin S antibody molecule and (ii) anti-VEGF antibody molecule, or nucleic acid encoding said anti-VEGF antibody molecule, according to any one of claims 18 to 26, wherein said anti-VEGF antibody molecule is an anti-VEGF-A antibody molecule.

28. The (i) anti-Cathepsin S antibody molecule, or nucleic acid encoding said anti-Cathepsin S antibody molecule and (ii) anti-VEGF antibody molecule, or nucleic acid encoding said anti-VEGF antibody molecule, according to claim 27, wherein said anti-VEGF-A antibody molecule is a humanised or human antibody molecule.

29. The (i) anti-Cathepsin S antibody molecule, or nucleic acid encoding said anti-Cathepsin S antibody molecule and (ii) anti-VEGF antibody molecule, or nucleic acid encoding said anti-VEGF antibody molecule, according to claim 28, wherein said anti-VEGF-A antibody molecule is bevacizumab.

30. An anti-Cathepsin S antibody molecule, wherein said anti-Cathepsin S antibody does not inhibit the proteolytic activity of Cathepsin S, or a nucleic acid encoding said anti-Cathepsin S antibody molecule, and an anti-VEGF-A antibody molecule, or a nucleic acid encoding said anti-VEGF-A antibody molecule, for simultaneous, sequential or separate administration for the treatment of a disease associated with angiogenesis.

31 . The anti-Cathepsin S antibody molecule, or nucleic acid encoding said anti-Cathepsin S antibody molecule and anti-VEGF antibody molecule, or nucleic acid encoding said anti-VEGF antibody molecule, according to claim 30, wherein said anti-VEGF-A antibody molecule is a humanised or human antibody molecule.

32. The anti-Cathepsin S antibody molecule, or nucleic acid encoding said anti-Cathepsin S antibody molecule and anti-VEGF antibody molecule, or nucleic acid encoding said anti-VEGF antibody molecule, according to claim 30 or claim 31 , wherein said anti-VEGF antibody molecule is bevacizumab.

33. The use of (i) an anti-Cathepsin S antibody molecule, wherein said anti-Cathepsin S antibody inhibits the proteolytic activity of Cathepsin S, and (ii) an anti-VEGF antibody molecule in the preparation of a medicament for combination therapy by simultaneous, sequential or separate administration of the anti-Cathepsin S antibody molecule and the anti-VEGF antibody molecule for the treatment of a disease associated with angiogenesis.

34. The use of (i) anti-Cathepsin S antibody molecule, wherein said anti-Cathepsin S antibody does not inhibit the proteolytic activity of Cathepsin S, and an anti-VEGF-A antibody molecule in the preparation of a medicament for combination therapy by simultaneous, sequential or separate administration of the anti-Cathepsin S antibody molecule and the anti-VEGF-A antibody molecule for the treatment of a disease associated with angiogenesis.

35. An anti-Cathepsin S antibody molecule comprising a VH domain having the amino acid sequence shown as Sequence ID No: 9, Sequence ID No: 10, Sequence ID No: 1 1 , Sequence ID No: 12, or Sequence ID No: 13 and/or an antibody V_L domain having the amino acid sequence shown as Sequence ID No: 14, Sequence ID No: 15, Sequence ID No: 16, Sequence ID No: 17, Sequence ID No: 18, or Sequence ID No: 19.

36. A nucleic acid encoding the anti-Cathepsin S antibody molecule according to claim 35.

37. A pharmaceutical composition comprising (i) an anti- Cathepsin S antibody molecule, wherein said anti- Cathepsin S antibody inhibits the proteolytic activity of Cathepsin S, or a nucleic acid encoding said anti- Cathepsin S antibody molecule; and (ii) an anti-VEGF antibody molecule, or a nucleic acid encoding said anti- VEGF antibody molecule.

38. A pharmaceutical kit comprising (a)(i) an anti-Cathepsin S antibody molecule, wherein said anti-Cathepsin S antibody inhibits the proteolytic activity of Cathepsin S, and (ii) an anti-VEGF antibody molecule; or (b)(i)a nucleic acid encoding an anti-Cathepsin S antibody molecule, wherein said anti-Cathepsin S antibody inhibits the proteolytic activity of Cathepsin S, and (ii) a nucleic acid encoding an anti-VEGF antibody molecule; for combination therapy by simultaneous, sequential or separate administration of the Cathepsin S antibody molecule and the anti-VEGF antibody molecule, or nucleic acids encoding said antibody molecules.

39. The pharmaceutical composition according to claim 37 or the kit according to claim 38, wherein the anti- Cathepsin S antibody molecule comprises an antigen binding domain comprising (i) at least one of the CDRs with an amino acid sequence selected from the group consisting of Seq ID No: 1 , Seq ID No: 2, Seq ID No: 3, or a variant thereof in which 5 or less, for example 4, 3, 2, or 1 amino acid substitutions have been made in at least one CDR, and/or (ii) at least one of the CDRs with an amino acid sequence consisting of Seq ID No: 4, Seq ID No: 5 and Seq ID No: 6, or a variant thereof, in which 5 or less, for example 4, 3, 2, or 1 amino acid substitutions have been made in at least one CDR; wherein the antibody molecule retains the ability to inhibit the proteolytic activity of Cathepsin S.

40. The pharmaceutical composition according to claim 37 or claim 39 or the kit according to claim 38 or claim 39, wherein the anti-Cathepsin S antibody molecule comprises an antibody V_H domain which comprises CDRs with amino acid sequences Seq ID No: 1 , Seq ID No: 2 and Seq ID No: 3 as CDRs 1 , 2 and 3 respectively.

41 . The pharmaceutical composition according to any one of claims 37 or 39-40 or the kit according to any one of claims 38-40, wherein the antibody V_H domain comprises the amino acid sequence Seq ID No: 7.

42. The pharmaceutical composition according to any one of claims 37 or 39-41 or the kit according to any one of claims 38-41 , wherein the antibody V_L domain comprises CDRs with amino acid sequences Seq ID No: 4, Seq ID No: 5 and Seq ID No: 6 as CDRs 1 , 2 and 3 respectively.

43. The pharmaceutical composition according to any one of claims 37 or 39-42 or the kit according to any one of claims 38-42, wherein the antibody V_L domain comprises the amino acid sequence Seq ID No: 8.

44. The pharmaceutical composition according to any one of claims 37, 39-40 or 42 or the kit according to any one of claims 38-40, and 42, wherein said anti-Cathepsin S antibody molecule is a human or humanised antibody molecule.

45. The pharmaceutical composition or kit according to claim 44, wherein anti-Cathepsin S antibody molecule is the antibody molecule according to claim 35.

46. The pharmaceutical composition according to any one of claims 37 or 39-45 or the kit according to any one of claims 38-45, wherein said anti-VEGF antibody molecule is an anti-VEGF-A antibody molecule.

47. The pharmaceutical composition or kit according to claim 46, wherein said anti-VEGF-A antibody molecule is a humanised or human antibody molecule.

48. The pharmaceutical composition or kit according to claim 46, wherein said antibody molecule is bevacizumab.

49. A pharmaceutical composition comprising an anti- Cathepsin S antibody molecule, wherein said anti- Cathepsin S antibody does not inhibit the proteolytic activity of Cathepsin S, or a nucleic acid encoding said anti-Cathepsin S antibody molecule; and an anti-VEGF- A antibody molecule, or a nucleic acid encoding said anti-VEGF-A antibody molecule,.

50. A kit comprising a) (i) an anti-Cathepsin S antibody molecule, wherein said anti-Cathepsin S antibody molecule does not inhibit the proteolytic activity of Cathepsin S, and (ii) an anti-VEGF-A antibody molecule or (b) (i) a nucleic acid encoding said anti-Cathepsin S antibody molecule and (ii) a nucleic acid encoding said anti-VEGF-A antibody molecule, for combination therapy by simultaneous, sequential or separate administration of the anti-Cathepsin S antibody molecule and the anti-VEGF-A antibody molecule or said nucleic acid encoding said anti-Cathepsin S antibody molecule and the anti-VEGF-A antibody molecule

51 . The pharmaceutical composition according to claim 49 or the kit according to claim 50, wherein said anti- VEGF-A antibody molecule is a humanised or human antibody molecule.

52. The pharmaceutical composition or the kit according to claim 51 , wherein said anti-VEGF-A antibody molecule is bevacizumab.