Antibodies and therapeutic uses thereof

Abstract

The present invention relates to antibodies or antigen-binding fragments thereof that specifically bind to Cartilage Oligomeric Matrix Protein (COMP) for use in therapy in a human subject.

Description

[0001] ANTIBODIES AND THERAPEUTIC USES THEREOF

[0002] Field of the invention

[0003] The present invention relates to novel antibodies and antigen-binding fragments thereof that bind to Cartilage Oligomeric Matrix Protein (COMP) and their use in therapy in a human subject.

[0004] Background of the invention

[0005] Cartilage Oligomeric Matrix Protein (COMP) is a pentameric molecule in which monomers are linked together in N-terminus with disulphide bonds. Every monomer of COMP consists of distinct functional domains: a globular C-terminus, 8 thrombospondin domains (TSP), 4 epidermal growth factor domains (EGF), and a coiled-coil structure in N-terminus. Under normal conditions, COMP is primarily expressed in specialized connective tissues, such as in cartilage and tendons. The most known function of COMP is to contribute to the supramolecular assembly and organization of the cartilage ECM and the homeostasis of that tissue. Accordingly, mutations in the COMP gene are associated with pseudoachondroplasia and multiple epiphyseal dysplasia. Accidental or pathologically induced damage of cartilage leads to significantly elevated levels of fragmented COMP in the patient’s serum, allowing these fragments to serve as a biomarker of cartilage damage and turnover [1], Serum levels of fragmented COMP are therefore used to assess the extent of cartilage destruction in patients with rheumatoid arthritis and osteoarthritis and to predict the course of such diseases.

[0006] COMP has also been found to be de novo expressed in breast cancer cells and particularly high expression levels of COMP in breast cancer lesions correlate with reduced survival and metastasis formation [2], Normal epithelial cells of the breast tissue do not express COMP. Several studies have shown that COMP has a strong oncogenic effect via several mechanisms including maintenance of cancer stem cells, enhanced cancer invasiveness of cancer cells and resistance to apoptosis [3], Currently, there is no publicly available information on the potential consequences of perturbing COMP’s extracellular functions through the use of antibodies directed against the macromolecule.

[0007] COMP overexpression relative to that of normal tissues has been observed in many types of solid human malignancies including breast, colorectal, pancreatic, ovarian, gastric, prostate, liver, thyroid, head and neck, renal, bladder, nasopharyngeal, gliomas, osteosarcomas and melanomas (Table 1 and). Due to the selective ectopic expression of COMP in solid tumours, thereby providing a highly specific tumour target and due to the paucity of resolutive treatment option for most solid tumour in their advanced stage, therapeutic targeting may provide a valuable option in the treatment of cancer patients.

[0008] Summary of the invention

[0009] The present invention relates to antibodies or antigen-binding fragments thereof that specifically bind to Cartilage Oligomeric Matrix Protein (COMP), and their use in therapy, particularly for the treatment of COMP-expressing cancers. The invention provides a panel of antibodies characterized by defined heavy and light chain variable regions and complementarity-determining regions (CDRs), including variants with at least 75% sequence identity to specified sequences.

[0010] The antibodies may be humanized and formulated as pharmaceutical compositions for administration to human subjects. In certain embodiments, the antibodies are used to treat solid tumors, including breast, colorectal, pancreatic, ovarian, gastric, prostate, liver, and thyroid cancers, wherein COMP is expressed in the tumor.

[0011] The therapeutic use of the antibodies is associated with the induction of Granzyme B-positive immune cell infiltration into the tumor microenvironment and a reduction in the proportion of cancer stem cells. In some embodiments, the antibodies are administered in combination with immune checkpoint inhibitors or are conjugated to cytotoxic agents or immune modulators to enhance therapeutic efficacy.

[0012] FIGURES

[0013] Figure 1. A) shows a graphical representation of COMP monomer and the areas of monoclonal antibodies binding; B) shows western blotting on lysates of breast cancer cells expressing deletion mutants of COMP lacking specified domains.

[0014] Figure 2. Pairwise alignment of the amino acid sequences of the variable regions of A) the heavy (VH) and B) light (VL) chains of antibody 4 performed with the corresponding sequences of the humanized antibodies.

[0015] Figure 3. ELISA dissociation curves of Antibody from COMP for Antibodies 1 , 2, 3, 4 and V7. See Example 1. X-axis: concentration of monoclonal (nM); Y-axis: Absorbance 450 nm; (•) COMP, (**square**) EDTA-COMP, (**inverted triangle**) DTT-COMP, ( **asterisk**) isotype. (A) Antibody 1 , (B) Antibody 2, (C) Antibody 3, (D) Antibody 4, (E) Antibody V7.

[0016] Figure 4. The human breast cancer cell lines MDA-MB-231 (A-D) and BT-20 (E-l) stably expressing COMP protein, and the respective control mock cells, incubated with the monoclonal antibodies. See also Example 2. The florescence signal measured with FACS analysis. X-axis: concentration of monoclonal (nM); y-axis: GMFI (AF647). (•) COMP, (**square**) Mock transfected, (**inverted triangle**) Isotype antibody (Rat-lgG2a, BD Pharmingen; Rat-lgG2b, BioLegend; Mouse- IgGl K, lgG2b, BioLegend). Panels (A) Antibody 1 , (B) Antibody 2, (C) Antibody 3, (D) Antibody 4.

[0017] Figure 5. Tumor development BALB / cJRj mice orthotopically transplanted with the 4T1-Luc2 expressing human COMP and treated with anti-COMP antibodies. In both experiments the treatment with the monoclonal antibodies reduced the volume of the primary tumor reduce tumour formation and tumor size in mice. See also Example 3. X-axis: Days post transplantation; y-axis: Tumor volume (mm3). Panel A: Experiment A: (•) Phosphate-buffered saline, PBS, (** empty square) Antibody 1 , (**empty triangle**) Antibody 2. Panel B: Experiment B: (•) Phosphate- buffered saline, PBS, (** empty square) Antibody 3, (**empty triangle**) Antibody 4.

[0018] Figure 6. CD 8-positive cells infiltration in tumour samples from antibody treated mice. See Examples 4 and 5. Graph indicates the number of positives per mm2 for PBS treated mice (darker bar) and mice treated with Antibody 4 (lighter bar). * indicates significance; (•) represents an individual sample. Figure 7. Tumorspheres formed in the presence of the monoclonal antibodies. Three tested antibodies reduced the size of the tumorspheres.

[0019] Figure 8. Comparison of the humanized monoclonal antibodies VH and VL engrafted in backbones of mouse lgG1 and lgG2a. See also Example 3.

[0020] Figure 9. Infiltration of tumor tissue with granzyme B positive immune cells in animals treated with lgG2a and lgG1 isotype of antibody V7.

[0021] Detailed description of the invention

[0022] The inventors have developed unique anti-COMP antibodies with unparalleled antigen-binding properties and an unexpected anti-tumour effect.

[0023] A first aspect of the invention relates to an antibody or antigen-binding fragment thereof that specifically binds to Cartilage Oligomeric Matrix Protein (COMP) for use in therapy in a human subject, selected from:

[0024] (i) an antibody or antigen-binding fragment thereof which comprises: a. a heavy chain variable region (VH) comprising the Complementarity Determining Regions (CDRs) i. CDR-H1 (SEQ ID NO. 2), ii. CDR-H2 (SEQ ID NO. 3), and

[0025] Hi. CDR-H3 (SEQ ID NO. 4), or a variant of said heavy chain variable region having at least 75% sequence identity to SEQ ID NO. 1 ; and b. a light chain variable region (VL) comprising the CDRs i. CDR-L1 (SEQ ID NO. 6), ii. CDR-L2 (SEQ ID NO. 7), and

[0026] Hi. CDR-L3 (SEQ ID NO. 8), or a variant said light chain variable region having at least 75% sequence identity with SEQ ID NO. 5;

[0027] (ii) an antibody or antigen-binding fragment thereof which comprises: a. a heavy chain variable region (VH) comprising the Complementarity Determining Regions (CDRs) i. CDR-H1 (SEQ ID NO. 17), ii. CDR-H2 (SEQ ID NO. 18), and

[0028] Hi. CDR-H3 (SEQ ID NO. 19), or a variant of said heavy chain variable region having at least 75% sequence identity to SEQ ID NO. 16; and b. a light chain variable region (VL) comprising the CDRs i. CDR-L1 (SEQ ID NO. 21), ii. CDR-L2 (SEQ ID NO. 22), and

[0029] Hi. CDR-L3 (SEQ ID NO. 23), or a variant of said light chain variable region having at least 75% sequence identity with SEQ ID NO. 20;

[0030] (iii) an antibody or antigen-binding fragment thereof which comprises: a. a heavy chain variable region (VH) comprising the Complementarity Determining Regions (CDRs) i. CDR-H1 (SEQ ID NO. 25), ii. CDR-H2 (SEQ ID NO. 26), and iii. CDR-H3 (SEQ ID NO. 27), or a variant of said heavy chain variable region having at least 75% sequence identity to SEQ ID NO. 24; and b. a light chain variable region (VL) comprising the CDRs i. CDR-L1 (SEQ ID NO. 29), ii. CDR-L2 (SEQ ID NO. 30), and iii. CDR-L3 (SEQ ID NO. 31), or a variant said light chain variable region having at least 75% sequence identity with SEQ ID NO. 28; and

[0031] (iv) an antibody or antigen-binding fragment thereof which comprises: a. a heavy chain variable region (VH) comprising the Complementarity Determining Regions (CDRs) i. CDR-H1 (SEQ ID NO. 33), ii. CDR-H2 (SEQ ID NO. 34), and iii. CDR-H3 (SEQ ID NO. 35), or a variant of said heavy chain variable region having at least 75% sequence identity to SEQ ID NO. 32; and b. a light chain variable region (VL) comprising the CDRs i. CDR-L1 (SEQ ID NO. 37), ii. CDR-L2 (SEQ ID NO. 38), and iii. CDR-L3 (SEQ ID NO. 39), or a variant said light chain variable region having at least 75% sequence identity with SEQ ID NO. 36.

[0032] Cartilage Oligomeric Matrix Protein (COMP) is a pentameric molecule in which monomers are linked together at the N-terminus through disulphide bonds. Every monomer of COMP consists of distinct functional domains: a globular C-terminus, 8 thrombospondin domains (TSP), 4 epidermal growth factor domains (EGF), and a coiled-coil structure at the N-terminus (see Figure 1).

[0033] The antibody or antigen-binding fragment thereof may bind within any domain of COMP. The antibody or antigen-binding fragment thereof may bind COMP in its intact or fragmented form. The antibody or antigen-binding fragment thereof may bind COMP in a monomeric or multimeric arrangement. The antibody or antigen-binding fragment thereof may bind COMP by recognizing a linear or a conformational epitope. In some embodiments the antibody or antigen-binding fragment binds the N-terminal portion of COMP.

[0034] In some embodiments the antibody or antigen-binding fragment binds preferentially the pentameric form of COMP. Most of the COMP protein circulating in the blood in pathological conditions is in its fragmented form. Therefore, preferential binding to the pentameric form subsequently is expected to give a unique recognition of the antigen.

[0035] In some embodiments, the antibody or antigen-binding fragment binds to N-terminal domain of COMP and therefore preferentially the pentameric form of COMP.

[0036] The CDR sequences described herein are defined according to IMGT system as defined in Lefranc, M.P., Unique database numbering system for immunogenetic analysis. Immunol Today, 1997. 18(11): p. 509; and Lefranc, M.P., et al., IMGT, the international ImMunoGeneTics database. Nucleic Acids Res, 1999. 27(1): p. 209-12.

[0037] In one embodiment, the antibody or antigen-binding fragment thereof for use in therapy in a human subject comprises: a heavy chain variable region (VH) comprising the Complementarity Determining Regions (CDRs) CDR-H1 (SEQ ID NO. 2), CDR-H2 (SEQ ID NO. 3), and CDR-H3 (SEQ ID NO. 4), or a variant of said heavy chain variable region having at least 75% sequence identity to SEQ ID NO. 1 ; and a light chain variable region (VL) comprising the CDRs CDR-L1 (SEQ ID NO. 6), CDR-L2 (SEQ ID NO. 7), and CDR-L3 (SEQ ID NO. 8), or a variant of said light chain variable region having at least 75% sequence identity with SEQ ID NO. 5.

[0038] In some embodiments, the VH comprises the CDRs of SEQ ID NO. 1 and the VL comprises the CDRs of SEQ ID NO. 5, i.e. the CDR sequences of SEQ ID NOs. 2-4 and 6-8, respectively.

[0039] In some embodiments, the variant of said heavy chain variable region (VH) has at least 79% sequence identity to SEQ ID NO: 1 , for example at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0040] In some embodiments, the variant of said heavy chain variable region (VH) has at least 87.7% sequence identity to SEQ ID NO: 1 , for example at least 89.5% identity.

[0041] In some embodiments, the variant of said light chain variable region (VL) has at least 79% sequence identity to SEQ ID NO: 5, for example at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0042] In some embodiments, the variant of said light chain variable region (VL) has at least 79.5% sequence identity to SEQ ID NO: 5, for example at least 81 .2%, or at least 88.4% identity.

[0043] In some embodiments, the variant of said heavy chain variable region (VH) has at least 87.7% sequence identity to SEQ ID NO: 1 and the variant of said light chain variable region (VL) has at least 79.5% sequence identity to SEQ ID NO: 5.

[0044] In some embodiments, the variant of said heavy chain variable region (VH) has at least 87.7% sequence identity to SEQ ID NO: 1 and the variant of said light chain variable region (VL) has at least 81 .2% sequence identity to SEQ ID NO: 5.

[0045] In some embodiments, the variant of said heavy chain variable region (VH) has at least 87.7% sequence identity to SEQ ID NO: 1 and the variant of said light chain variable region (VL) has at least 88.4% sequence identity to SEQ ID NO: 5. In some embodiments, the variant of said heavy chain variable region (VH) has at least 89.5% sequence identity to SEQ ID NO: 1 and the variant of said light chain variable region (VL) has at least 79.5% sequence identity to SEQ ID NO: 5.

[0046] In some embodiments, the variant of said heavy chain variable region (VH) has at least 89.5% sequence identity to SEQ ID NO: 1 and the variant of said light chain variable region (VL) has at least 81 .2% sequence identity to SEQ ID NO: 5.

[0047] In some embodiments, the variant of said heavy chain variable region (VH) has at least 89.5% sequence identity to SEQ ID NO: 1 and the variant of said light chain variable region (VL) has at least 88.4% sequence identity to SEQ ID NO: 5.

[0048] In some embodiments, the variant of said heavy chain variable region (VH) and / or the variant of said light chain variable region (VL) does not include any sequence variation in the CDR regions as compared to the specified sequences, e.g. as compared to SEQ ID NO: 1 and SEQ ID NO: 5, respectively.

[0049] In some embodiments the antibody or antigen-binding fragment thereof comprises: a. a heavy chain variable region (VH) comprising the Complementarity Determining Regions (CDRs): i. CDR-H1 (SEQ ID NO. 2), ii. CDR-H2 (SEQ ID NO. 3), and

[0050] Hi. CDR-H3 (SEQ ID NO. 4); and b. a light chain variable region (VL) comprising the CDRs: i. CDR-L1 (SEQ ID NO. 6), ii. CDR-L2 (SEQ ID NO. 7), and

[0051] Hi. CDR-L3 (SEQ ID NO. 8).

[0052] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a. a heavy chain variable region (VH) selected from the group of: i. SEQ ID NO. 9, ii. SEQ ID NO. 10,

[0053] Hi. SEQ ID NO. 11 , and iv. SEQ ID NO. 12; or a variant of said heavy chain variable region having at least 75% sequence identity to SEQ ID NO. 1 ; and b. a light chain variable region (VL) selected from the group of: i. SEQ ID NO. 13, ii. SEQ ID NO. 14, and

[0054] Hi. SEQ ID NO. 15; or a variant of said light chain variable region having at least 75% sequence identity with SEQ ID NO. 5.

[0055] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a. a heavy chain variable region (VH) selected from the group of: i. SEQ ID NO. 9, or a variant having at least 75% sequence identity thereto; ii. SEQ ID NO. 10, or a variant having at least 75% sequence identity thereto;

[0056] Hi. SEQ ID NO. 1 1 , or a variant having at least 75% sequence identity thereto; and iv. SEQ ID NO. 12; or a variant having at least 75% sequence identity thereto; and b. a light chain variable region (VL) selected from the group of: i. SEQ ID NO. 13, or a variant having at least 75% sequence identity thereto; ii. SEQ ID NO. 14, or a variant having at least 75% sequence identity thereto; and

[0057] Hi. SEQ ID NO. 15; or a variant having at least 75% sequence identity thereto.

[0058] In some embodiments, the variant of said heavy chain variable region (VH) has at least 79% sequence identity to the specified sequence, for example at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0059] In some embodiments, the variant of said light chain variable region (VL) has at least 79% sequence identity to the specified sequence, for example at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0060] In particular embodiments, the variant heavy chain variable region (VH) comprises the Complementarity Determining Regions (CDRs) CDR-H1 (SEQ ID NO. 2), CDR-H2 (SEQ ID NO. 3), and CDR-H3 (SEQ ID NO. 4); and / or the variant light chain variable region (VL) comprises the CDRs: CDR-L1 (SEQ ID NO. 6), CDR-L2 (SEQ ID NO. 7), and CDR-L3 (SEQ ID NO. 8). The sequence variation may therefore occur exclusively outside of the CDR sequences.

[0061] In some embodiments, the antibody or antigen-binding fragment thereof comprises the light chain variable region of SEQ ID NO: 14.

[0062] In some embodiments, the antibody or antigen-binding fragment according thereof comprises the heavy chain variable region of SEQ ID NO: 10.

[0063] In some embodiments, the antibody or antigen-binding fragment thereof comprises VH and VL domains selected from the following combinations: a. SEQ ID NO. 10 and SEQ ID. NO. 14; b. SEQ ID NO. 9 and SEQ ID. NO. 13; c. SEQ ID NO. 9 and SEQ ID. NO. 14; d. SEQ ID NO. 12 and SEQ ID. NO. 14; e. SEQ ID NO. 9 and SEQ ID. NO. 15; f. SEQ ID NO. 10 and SEQ ID. NO. 13; g. SEQ ID NO. 10 and SEQ ID. NO. 15; h. SEQ ID NO. 11 and SEQ ID. NO. 13; i. SEQ ID NO. 1 1 and SEQ ID. NO. 14; j. SEQ ID NO. 1 1 and SEQ ID. NO. 15; k. SEQ ID NO. 12 and SEQ ID. NO. 13; or l. SEQ ID NO. 12 and SEQ ID. NO. 15

[0064] In one embodiment the invention relates to an antibody or antigen-binding fragment thereof that specifically binds to Cartilage Oligomeric Matrix Protein (COMP) for use in therapy in a human subject, wherein the antibody or antigen-binding fragment thereof comprises: a. a heavy chain variable region (VH) comprising the Complementarity Determining Regions (CDRs) i. CDR-H1 (SEQ ID NO. 17), ii. CDR-H2 (SEQ ID NO. 18), and

[0065] Hi. CDR-H3 (SEQ ID NO. 19), or a variant of said heavy chain variable region having at least 75% sequence identity to SEQ ID NO. 16; and b. a light chain variable region (VL) comprising the CDRs i. CDR-L1 (SEQ ID NO. 21), ii. CDR-L2 (SEQ ID NO. 22), and

[0066] Hi. CDR-L3 (SEQ ID NO. 23), or a variant of said light chain variable region having at least 75% sequence identity with SEQ ID NO. 20.

[0067] In one embodiment, the antibody or antigen-binding fragment thereof for use comprises: a heavy chain variable region (VH) comprising the Complementarity Determining Regions (CDRs) CDR- H1 (SEQ ID NO. 17), CDR-H2 (SEQ ID NO. 18), and CDR-H3 (SEQ ID NO. 19), or a variant of said heavy chain variable region having at least 75% sequence identity to SEQ ID NO. 16; and a light chain variable region (VL) comprising the CDRs CDR-L1 (SEQ ID NO. 21), CDR-L2 (SEQ ID NO. 22), and CDR-L3 (SEQ ID NO. 23), or a variant of said light chain variable region having at least 75% sequence identity with SEQ ID NO. 20.

[0068] In some embodiments of the first aspect of the invention, the VH comprises the CDRs of SEQ ID NO. 16 and the VL comprises the CDRs of SEQ ID NO. 20, i.e. the CDR sequences of SEQ ID NOs. 16-19 and 21-23.

[0069] In some embodiments, the variant of said heavy chain variable region (VH) has at least 79% sequence identity to SEQ ID NO: 16, for example at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0070] In some embodiments, the variant of said light chain variable region (VL) has at least 79% sequence identity to SEQ ID NO: 20, for example at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0071] In some embodiments, the variant of said heavy chain variable region (VH) and / or the variant of said light chain variable region (VL) does not include any sequence variation in the CDR regions as compared to the specified sequences, e.g. as compared to SEQ ID NO: 16 and SEQ ID NO: 20, respectively.

[0072] In some embodiments the antibody or antigen-binding fragment thereof comprises: a. a heavy chain variable region (VH) comprising the Complementarity Determining Regions (CDRs): i. CDR-H1 (SEQ ID NO. 17), ii. CDR-H2 (SEQ ID NO. 18), and

[0073] Hi. CDR-H3 (SEQ ID NO. 19); and b. a light chain variable region (VL) comprising the CDRs: i. CDR-L1 (SEQ ID NO. 21), ii. CDR-L2 (SEQ ID NO. 22), and

[0074] III. CDR-L3 (SEQ ID NO. 23).

[0075] In one embodiment the invention relates to an antibody or antigen-binding fragment thereof that specifically binds to Cartilage Oligomeric Matrix Protein (COMP) for use in therapy in a human subject, wherein the antibody or antigen-binding fragment thereof comprises: a. a heavy chain variable region (VH) comprising the Complementarity Determining Regions (CDRs) i. CDR-H1 (SEQ ID NO. 25), ii. CDR-H2 (SEQ ID NO. 26), and

[0076] Hi. CDR-H3 (SEQ ID NO. 27), or a variant of said heavy chain variable region having at least 75% sequence identity to SEQ ID NO. 24; and b. a light chain variable region (VL) comprising the CDRs i. CDR-L1 (SEQ ID NO. 29), ii. CDR-L2 (SEQ ID NO. 30), and

[0077] Hi. CDR-L3 (SEQ ID NO. 31), or a variant of said light chain variable region having at least 75% sequence identity with SEQ ID

[0078] NO. 28.

[0079] In one embodiment, the antibody or antigen-binding fragment thereof for use comprises: a heavy chain variable region (VH) comprising the Complementarity Determining Regions (CDRs) CDR- H1 (SEQ ID NO. 25), CDR-H2 (SEQ ID NO. 26), and CDR-H3 (SEQ ID NO. 27), or a variant of said heavy chain variable region having at least 75% sequence identity to SEQ ID NO. 24; and a light chain variable region (VL) comprising the CDRs CDR-L1 (SEQ ID NO. 29), CDR-L2 (SEQ ID NO. 30), and CDR-L3 (SEQ ID NO. 31), or a variant of said light chain variable region having at least 75% sequence identity with SEQ ID NO. 28.

[0080] In some embodiments of the first aspect of the invention, the VH comprises the CDRs of SEQ ID NO. 24 and the VL comprises the CDRs of SEQ ID NO. 28, i.e. the CDR sequences of SEQ ID NOs. 25-27 and 29-31.

[0081] In some embodiments, the variant of said heavy chain variable region (VH) has at least 79% sequence identity to SEQ ID NO: 24, for example at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0082] In some embodiments, the variant of said light chain variable region (VL) has at least 79% sequence identity to SEQ ID NO: 28, for example at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0083] In some embodiments, the variant of said heavy chain variable region (VH) and / or the variant of said light chain variable region (VL) does not include any sequence variation in the CDR regions as compared to the specified sequences, e.g. as compared to SEQ ID NO: 24 and SEQ ID NO: 28, respectively. In some embodiments the antibody or antigen-binding fragment thereof comprises: a. a heavy chain variable region (VH) comprising the Complementarity Determining Regions (CDRs): i. CDR-H1 (SEQ ID NO. 25), ii. CDR-H2 (SEQ ID NO. 26), and

[0084] Hi. CDR-H3 (SEQ ID NO. 27); and b. a light chain variable region (VL) comprising the CDRs: i. CDR-L1 (SEQ ID NO. 29), ii. CDR-L2 (SEQ ID NO. 30), and

[0085] Hi. CDR-L3 (SEQ ID NO. 31).

[0086] In one embodiment the invention relates to an antibody or antigen-binding fragment thereof that specifically binds to Cartilage Oligomeric Matrix Protein (COMP) for use in therapy in a human subject, wherein the antibody or antigen-binding fragment thereof comprises: a. a heavy chain variable region (VH) comprising the Complementarity Determining Regions (CDRs) i. CDR-H1 (SEQ ID NO. 33), ii. CDR-H2 (SEQ ID NO. 34), and

[0087] Hi. CDR-H3 (SEQ ID NO. 35), or a variant of said heavy chain variable region having at least 75% sequence identity to SEQ ID NO. 32; and b. a light chain variable region (VL) comprising the CDRs i. CDR-L1 (SEQ ID NO. 37), ii. CDR-L2 (SEQ ID NO. 38), and

[0088] Hi. CDR-L3 (SEQ ID NO. 39), or a variant of said light chain variable region having at least 75% sequence identity with SEQ ID

[0089] NO. 36.

[0090] In one embodiment, the antibody or antigen-binding fragment thereof for use comprises: a heavy chain variable region (VH) comprising the Complementarity Determining Regions (CDRs) CDR- H1 (SEQ ID NO. 33), CDR-H2 (SEQ ID NO. 34), and CDR-H3 (SEQ ID NO. 35), or a variant of said heavy chain variable region having at least 75% sequence identity to SEQ ID NO. 32; and a light chain variable region (VL) comprising the CDRs CDR-L1 (SEQ ID NO. 37), CDR-L2 (SEQ ID NO. 38), and CDR-L3 (SEQ ID NO. 39), or a variant of said light chain variable region having at least 75% sequence identity with SEQ ID NO. 36.

[0091] In some embodiments of the first aspect of the invention, the VH comprises the CDRs of SEQ ID NO. 32 and the VL comprises the CDRs of SEQ ID NO. 36, i.e. the CDR sequences of SEQ ID NOs. 33-35 and 37-39.

[0092] In some embodiments, the variant of said heavy chain variable region (VH) has at least 79% sequence identity to SEQ ID NO: 32, for example at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. In some embodiments, the variant of said light chain variable region (VL) has at least 79% sequence identity to SEQ ID NO: 36, for example at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0093] In some embodiments, the variant of said heavy chain variable region (VH) and / or the variant of said light chain variable region (VL) does not include any sequence variation in the CDR regions as compared to the specified sequences, e.g. as compared to SEQ ID NO: 32 and SEQ ID NO: 36, respectively.

[0094] In some embodiments the antibody or antigen-binding fragment thereof comprises: a. a heavy chain variable region (VH) comprising the Complementarity Determining Regions (CDRs): i. CDR-H1 (SEQ ID NO. 33), ii. CDR-H2 (SEQ ID NO. 34), and

[0095] Hi. CDR-H3 (SEQ ID NO. 35); and b. a light chain variable region (VL) comprising the CDRs: i. CDR-L1 (SEQ ID NO. 37), ii. CDR-L2 (SEQ ID NO. 38), and

[0096] Hi. CDR-L3 (SEQ ID NO. 39).

[0097] By “antibody” we include substantially intact antibody molecules, as well as chimeric antibodies, humanised antibodies, human antibodies (wherein at least one amino acid is mutated relative to the naturally occurring human antibodies), single chain antibodies, biparatopic antibodies, antibody heavy chains, antibody light chains, homodimers and heterodimers of antibody heavy and / or light chains, and antigen binding fragments and derivatives of the same.

[0098] By “antigen-binding fragment” we mean any functional fragment of an antibody that is capable of binding to COMP. The antibody fragment may exclude the Fc region of a whole antibody. The antibody fragment may not include the CH2 and CH3 regions of a whole antibody.

[0099] For example, the antigen-binding fragment may include any monomeric, dimeric or trimeric immunoglobulin configurations, including bispecific arrangements, derived from genetic-molecular approaches, phage display, recombinantly and / or by immunizations procedures on any type of animal.

[0100] In some embodiments, the antigen-binding fragment is selected from the following format: scFv, Fv, Fab, F(ab)2, Fab-SH, dsFv, sdAb, di-scFvs bi-scFv, Fcabs, diabodies, scFv-Fc / minibody, triabody, tetrabody, tandAb, half antibody (Unibody), and domain antibodies.

[0101] Antibody fragments can be produced by chemical or enzymatic cleavage, but, more preferably, are produced using recombinant DNA technology. The latter allows for indefinite protein expression in prokaryotic or eukaryotic cell lines and genetic modification leading to fragments with enhanced or additional properties. Antibody fragments normally possess at least one variable (V-) domain because V-domains contain the complementarity-determining regions (CDRs) or loops for antigen binding. Alternatively, CDR-like loops can be inserted into non-variable domains (e.g. constant- heavy-3, CH3 domains) enabling these domains to bind to useful or predetermined targets. As discussed herein, it will be appreciated that the antibodies and antigen binding fragments of the invention may comprise variants of the above-defined sequences. The antibodies and antigen binding fragments thereof may have at least 60% sequence identity with any of the sequences disclosed herein. For example, they may have at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity.

[0102] The antibodies and antigen binding fragments thereof may alternatively be a variant of a specific sequence disclosed herein, wherein said variant comprises mutations at one or more positions relative to the parent sequence. By the term “mutation” we include insertions, deletions and substitutions. Accordingly, a variant may be a substitution, deletion or addition variant. A variant polypeptide may comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, up to 10, up to 20, up to 30, up to 40, up to 50, up to 75 or more amino acid mutations, in a preferred embodiment. The mutations may be either conservative or non-conservative. For example, conservative substitution refers to the substitution of an amino acid within the same general class (e.g. an acidic amino acid, a basic amino acid, a non-polar amino acid, a polar amino acid or an aromatic amino acid) by another amino acid within the same class. Thus, the meaning of a conservative amino acid substitution and non-conservative amino acid substitution is well known in the art.

[0103] “Deletion” variants may comprise the deletion of individual amino acids, deletion of small groups of amino acids such as 2, 3, 4 or 5 amino acids, or deletion of larger amino acid regions, such as the deletion of specific amino acid domains or other features. "Substitution" variants preferably involve the replacement of one or more amino acids with the same number of amino acids and making conservative amino acid substitutions. For example, an amino acid may be substituted with an alternative amino acid having similar properties, for example, another basic amino acid, another acidic amino acid, another neutral amino acid, another charged amino acid, another hydrophilic amino acid, another hydrophobic amino acid, another polar amino acid, another aromatic amino acid or another aliphatic amino acid. Some properties of the 20 main amino acids which can be used to select suitable substituents are as follows:

[0104] Amino acids herein may be referred to by full name, three letter code or single letter code. Preferred "variants" include those in which instead of the naturally occurring amino acid the amino acid which appears in the sequence is a structural analogue thereof. Amino acids used in the sequences may also be derivatised or modified, e.g. labelled, providing the function of the antibody is not significantly adversely affected.

[0105] Derivatives and variants as described above may be prepared during synthesis of the antibody or by post-production modification or by peptide synthesis or by native chemical ligation of peptides, or when the antibody is in recombinant form using the known techniques of site-directed mutagenesis, random mutagenesis, or enzymatic cleavage and / or ligation of nucleic acids.

[0106] Suitable variants may be at least 75% homologous to a sequence disclosed herein, preferably at least 79% or 80% or 90% and more preferably at least 95%, 97% or 99% homologous thereto.

[0107] Preferably variants have an amino acid sequence which has more than 75%, or more than 80%, preferably more than 85%, e.g. more than 90 or 95% amino acid identity to a sequence as shown in the sequences disclosed herein (e.g. the VH or VL region sequences, or CDR sequences therein). This level of amino acid identity may be seen across the full length of the relevant SEQ ID NO sequence or over a part of the sequence, such as across 20, 30, 50, 75, 100, 150, 200 or more amino acids, depending on the size of the full-length polypeptide.

[0108] For example, variants of the above CDR sequences may comprise one, two three, four, five, six, seven, eight or more amino acid mutations relative to the reference sequence (such as a deletion, substitution and / or insertion of an amino acid).

[0109] The percent sequence identity between two polypeptides may be determined using suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group and it will be appreciated that percent identity is calculated in relation to polypeptides whose sequences have been aligned optimally. Methods for determining sequence identity are known to those skilled in the art.

[0110] The alignment may alternatively be carried out using the Clustal W program (as described in

[0080] , which is incorporated herein by reference). The parameters used may be as follows:

[0111] Fast pairwise alignment parameters: K-tuple(word) size; 1 , window size; 5, gap penalty; 3, number of top diagonals; 5. Scoring method: x percent.

[0112] Multiple alignment parameters: gap open penalty; 10, gap extension penalty; 0.05.

[0113] Scoring matrix: BLOSUM.

[0114] Alternatively, the BESTFIT program may be used to determine local sequence alignments.

[0115] Also included within the scope of the invention are versions of antibodies and antigen-binding fragments thereof with covalently attached polyethylene glycol or other suitable polymers (see below).

[0116] Methods of generating antibodies and antibody fragments are well known in the art. For example, antibodies may be generated via any one of several methods which employ induction of in vivo production of antibody molecules, screening of immunoglobulin libraries (Orlandi et al, 1989. Proc. Natl. Acad. Sci. U.S.A. 86:3833-3837; Winter et al., 1991 , Nature 349:293-299) or generation of monoclonal antibody molecules by cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the Epstein-Barr virus (EBV)- hybridoma technique (Kohler et al., 1975. Nature 256:4950497; Kozbor et al., 1985. J. Immunol. Methods 81 :31-42; Cote et al., 1983. Proc. Natl. Acad. Sci. USA 80:2026-2030; Cole et al., 1984. Mol. Cell. Biol. 62:109-120).

[0117] Suitable monoclonal antibodies to selected antigens may be prepared by known techniques, for example those disclosed in “Monoclonal Antibodies: A manual of techniques”, H Zola (CRC Press, 1988) and in “Monoclonal Hybridoma Antibodies: Techniques and Applications”, J G R Hurrell (CRC Press, 1982).

[0118] Likewise, antibody fragments can be obtained using methods well known in the art (see, for example, Harlow & Lane, 1988, “Antibodies: A Laboratory Manual”, Cold Spring Harbor Laboratory, New York). For example, antibody fragments according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment. Alternatively, antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.

[0119] It will be appreciated by persons skilled in the art of monoclonal antibody production that independently of the sequence of the paratope and the sequence of the epitope, and / or its location on the antigen, the antibody may not perturb the function of the antigen.

[0120] It may similarly be appreciated by a skilled person that any antibody produced against COMP and that affects any of its functions may not necessarily perturb the pro-tumorigenic function of the antigen unless being produced as described for antibody of this invention.

[0121] It will be appreciated by persons skilled in the art that for human therapy, human or humanised antibodies are preferably used. Humanised forms of non-human (e.g. murine) antibodies are genetically engineered chimeric antibodies or antibody fragments having preferably minimal portions derived from non-human antibodies. Humanised antibodies include antibodies in which complementary determining regions of a human antibody (recipient antibody) are replaced by residues from a complementary determining region of a non-human species (donor antibody) such as mouse, rat of rabbit having the desired functionality. In some instances, Fv framework residues of the human antibody are replaced by corresponding non-human residues. Humanised antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported complementarity determining region or framework sequences. In general, the humanised antibody will comprise the entire portion of at least one, and typically two, variable domains, in which all or substantially all of the complementarity determining regions correspond to those of a non-human antibody and all, or substantially all, of the framework regions correspond to those of a relevant human consensus sequence. Humanised antibodies optimally also include at least a portion of an antibody constant region, such as an Fc region, typically derived from a human antibody (see, for example, Jones et al., 1986. Nature 321 :522-525; Riechmann et al., 1988, Nature 332:323-329; Presta, 1992, Curr. Op. Struct. Biol. 2:593-596).

[0122] Methods for humanising non-human antibodies are well known in the art. Generally, the humanised antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues, often referred to as imported residues, are typically taken from an imported variable domain. Humanisation can be essentially performed as described (see, for example, Jones et al., 1986, Nature 321 :522-525; Reichmann et al., 1988. Nature 332:323-327; Verhoeyen et al., 1988, Science 239:1534-15361; US 4,816,567) by substituting the rodent complementarity determining regions with the corresponding human complementarity determining regions. Accordingly, such humanised antibodies are chimaeric, partly humanized antibodies, wherein less than the intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanised antibodies may be typically human antibodies in which some complementarity determining region residues and possibly some framework residues are substituted by residues from analogous sites in rodent antibodies.

[0123] Human antibodies can also be identified using various techniques known in the art, including phage display libraries (see, for example, Hoogenboom & Winter, 1991 , J. Mol. Biol. 227:381 ; Marks et al., 1991 , J. Mol. Biol. 222:581 ; Cole et al., 1985, In: Monoclonal antibodies and Cancer Therapy, Alan R. Liss, pp. 77; Boerner et al., 1991 . J. Immunol. 147:86-95).

[0124] Once suitable antibodies are obtained, they may be tested for binding activity, for example by ELISA.

[0125] In some embodiments, the antibody has been produced using a native, tissue-extracted form of human COMP.

[0126] In some embodiments, the antibody has been produced by a readaptation of the Kohler and Milstein hybridoma procedure entailing a specific subtractive immunization protocol applied to rats.

[0127] In some embodiments, the antibody or antigen-binding fragment thereof disclosed herein comprises a heavy chain constant region, or part thereof.

[0128] In some embodiments, the heavy chain constant region is of an immunoglobulin subtype selected from the group consisting of lgG1 , lgG2, lgG3 and lgG4. In some embodiments, the heavy chain constant region is of an immunoglobulin subtype lgG1.

[0129] In some embodiments, the antibody or antigen-binding fragment thereof disclosed herein comprises a light chain constant region, or part thereof.

[0130] In some embodiments, the light chain constant region is of a kappa or lambda light chain. In some embodiments, the constant region is a human or humanised constant region.

[0131] In some embodiments, the antibody or antigen-binding fragment thereof binds COMP with high affinity, or with higher affinity than an alternative anti-COMP antibody. Affinity can be expressed by measuring the KD (dissociation constant, or equilibrium dissociation constant, expressed in M) or KA (association constant, or equilibrium association constant, expressed in M1) of a particular antibody or antigen-binding fragment when binding COMP. The KD and KA may be calculated by measuring the association rate constant (ka, expressed in M1s-1) and dissociation rate constant (kd, expressed in s1). KA is calculated by ka / kd. KD is calculated by kd / ka.

[0132] Therefore, by “higher affinity” we mean that the antibody or antigen binding fragment thereof has a higher KA and / or a lower KD than an alternative anti-COMP antibody or antigen binding fragment. In some preferred embodiments, the antibody or antigen binding fragment has a lower KD (i.e. a higher binding affinity) than an alternative anti-COMP antibody or antigen binding fragment. In some embodiments, the antibody or antigen binding fragment thereof has a higher KA and / or a lower KD than an alternative anti-COMP antibody or antigen binding fragment.

[0133] In some embodiments, the antibody or antigen-binding fragment thereof binds COMP with an affinity KD of less than 2000nM. In some embodiments, the antibody or antigen-binding fragment thereof binds COMP with an affinity KD of less than 1000mM, or less than 900nM. In some embodiments, the antibody or antigen-binding fragment thereof binds COMP with an affinity KD of less than 500nM. In some embodiments, the antibody or antigen-binding fragment thereof binds COMP with an affinity KD of less than 300nM. In some embodiments, the antibody or antigenbinding fragment thereof binds COMP with an affinity KD of less than 200nM. In some embodiments, the antibody or antigen-binding fragment thereof binds COMP with an affinity KD of less than 160nM. In some embodiments, the antibody or antigen-binding fragment thereof binds COMP with an affinity KD of less than 110nM.

[0134] In another aspect of the invention there is provided a pharmaceutical composition, comprising the antibody or antigen-binding fragment thereof as described in relation to the first aspect and a pharmaceutically acceptable buffer, carrier, excipient or diluent, for use in therapy in a human subject.

[0135] Additional compounds may also be included in the compositions, including, chelating agents such as EDTA, citrate, EGTA or glutathione.

[0136] The pharmaceutical compositions may be prepared in a manner known in the art that is sufficiently storage stable and suitable for administration to humans. For example, the pharmaceutical compositions may be lyophilised, e.g. through freeze drying, spray drying, spray cooling, orthrough use of particle formation from supercritical particle formation.

[0137] By “pharmaceutically acceptable" we mean a non-toxic material that does not decrease the effectiveness of the COMP-binding activity of the agent of the invention. Such pharmaceutically acceptable buffers, carriers or excipients are well-known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A.R Gennaro, Ed., Mack Publishing Company (1990) and handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press (2000), the disclosures of which are incorporated by reference).

[0138] The term "buffer" is intended to mean an aqueous solution containing an acid-base mixture with the purpose of stabilising pH. Examples of buffers are Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS, Tris, Hepes, HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate, borate, ACES, ADA, tartrate, AMP, AMPD, AMPSO, BES, CABS, cacodylate, CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole, imidazolelacetic acid, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO and TES.

[0139] The term "diluent" is intended to mean an aqueous or non-aqueous solution with the purpose of diluting the agent in the pharmaceutical preparation. The diluent may be one or more of saline, water, polyethylene glycol, propylene glycol, ethanol or oils (such as safflower oil, corn oil, peanut oil, cottonseed oil or sesame oil).

[0140] The term "adjuvant" is intended to mean any compound added to the formulation to increase the biological effect of the agent of the invention. The adjuvant may be one or more of zinc, copper or silver salts with different anions, for example, but not limited to fluoride, chloride, bromide, iodide, thiocyanate, sulfite, hydroxide, phosphate, carbonate, lactate, glycolate, citrate, borate, tartrate, and acetates of different acyl composition. The adjuvant may also be cationic polymers such as cationic cellulose ethers, cationic cellulose esters, deacetylated hyaluronic acid, chitosan, cationic dendrimers, cationic synthetic polymers such as poly(vinyl imidazole), and cationic polypeptides such as polyhistidine, polylysine, polyarginine, and peptides containing these amino acids.

[0141] The excipient may be one or more of carbohydrates, polymers, lipids and minerals. Examples of carbohydrates include lactose, glucose, sucrose, mannitol, and cyclodextrines, which are added to the composition, e.g. for facilitating lyophilisation. Examples of polymers are starch, cellulose ethers, cellulose carboxymethylcellulose, hydroxypropylmethyl cellulose, hydroxyethyl cellulose, ethylhydroxyethyl cellulose, alginates, carrageenans, hyaluronic acid and derivatives thereof, polyacrylic acid, polysulphonate, polyethylene glycol / polyethylene oxide, polyethylene oxide / polypropylene oxide copolymers, polyvinylalcohol / polyvinylacetate of different degree of hydrolysis, and polyvinylpyrrolidone, all of different molecular weight, which are added to the composition, e.g. for viscosity control, for achieving bioadhesion, or for protecting the lipid from chemical and proteolytic degradation. Examples of lipids are fatty acids, phospholipids, mono-, di- , and triglycerides, ceramides, sphingolipids and glycolipids, all of the different acyl chain length and saturation, egg lecithin, soy lecithin, hydrogenated egg and soy lecithin, which are added to the composition for reasons similar to those for polymers. Examples of minerals are talc, magnesium oxide, zinc oxide and titanium oxide, which are added to the composition to obtain benefits such as reduction of liquid accumulation or advantageous pigment properties.

[0142] The agents of the invention may be formulated into any type of pharmaceutical composition known in the art to be suitable for the delivery thereof.

[0143] In one embodiment, the pharmaceutical compositions of the invention may be in the form of a liposome, in which the agent is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids, which exist in aggregated forms as micelles, insoluble monolayers and liquid crystals. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Suitable lipids also include the lipids above modified by polyethylene glycol) in the polar headgroup for prolonging bloodstream circulation time.

[0144] The pharmaceutical compositions of the invention may also be in the form of biodegradable microspheres. Aliphatic polyesters, such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), copolymers of PLA and PGA (PLGA) or poly(caprolactone) (PCL), and polyanhydrides have been widely used as biodegradable polymers in the production of microspheres.

[0145] In a further embodiment, the pharmaceutical compositions of the invention are provided in the form of polymer gels, where polymers such as starch, cellulose ethers, cellulose carboxymethylcellulose, hydroxypropylmethyl cellulose, hydroxyethyl cellulose, ethylhydroxyethyl cellulose, alginates, carrageenans, hyaluronic acid and derivatives thereof, polyacrylic acid, polyvinyl imidazole, polysulphonate, polyethylene glycol / polyethylene oxide, polyethylene oxide / polypropylene oxide copolymers, polyvinylalcohol / polyvinylacetate of different degree of hydrolysis, and polyvinylpyrrolidone are used for thickening of the solution containing the agent. The polymers may also comprise gelatin or collagen.

[0146] Alternatively, the agents may simply be dissolved in saline, water, polyethylene glycol, propylene glycol, ethanol or oils (such as safflower oil, corn oil, peanut oil, cottonseed oil or sesame oil), tragacanth gum, and / or various buffers.

[0147] It will be appreciated that the pharmaceutical compositions of the invention may include ions and a defined pH for potentiation of action of the active agent. Additionally, the compositions may be subjected to conventional pharmaceutical operations such as sterilisation and / or may contain conventional adjuvants such as preservatives, stabilisers, wetting agents, emulsifiers, buffers, fillers, etc.

[0148] The pharmaceutical compositions according to the invention may be administered via any suitable route known to those skilled in the art. Thus, possible routes of administration include parenteral (intravenous, subcutaneous, and intramuscular), topical, ocular, nasal, pulmonary, buccal, oral, parenteral, vaginal and rectal. Also, administration from implants is possible.

[0149] In one preferred embodiment, the pharmaceutical compositions are administered parenterally, for example, intravenously, intracerebroventricularly, intraarticularly, intra-arterially, intraperitoneally, intrathecally, intraventricularly, intrastern ally, intracranially, intramuscularly or subcutaneously, or they may be administered by infusion techniques. They are conveniently used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.

[0150] Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

[0151] Thus, the pharmaceutical compositions of the invention are particularly suitable for parenteral, e.g. intravenous, administration. In some preferred embodiments, the pharmaceutical compositions of the invention are administered intravenously.

[0152] Alternatively, the pharmaceutical compositions may be administered intranasally or by inhalation (for example, in the form of an aerosol spray presentation from a pressurised container, pump, spray or nebuliser with the use of a suitable propellant, such as dichlorodifluoromethane, trichlorofluoro-methane, dichlorotetrafluoro-ethane, a hydrofluoroalkane such as 1 , 1 ,1 ,2- tetrafluoroethane (HFA 134A3 or 1 ,1 ,1 ,2,3,3,3-heptafluoropropane (HFA 227EA3), carbon dioxide or other suitable gas). In the case of a pressurised aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurised container, pump, spray or nebuliser may contain a solution or suspension of the active polypeptide, e.g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g. sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of a compound of the invention and a suitable powder base such as lactose or starch.

[0153] The pharmaceutical compositions will be administered to a patient in a pharmaceutically effective dose. A ‘therapeutically effective amount’, or ‘effective amount’, or ‘therapeutically effective’, as used herein, refers to that amount which provides a therapeutic effect for a given condition and administration regimen. This is a predetermined quantity of active material calculated to produce a desired therapeutic effect in association with the required additive and diluent, i.e. a carrier or administration vehicle. Further, it is intended to mean an amount sufficient to reduce and most preferably prevent, a clinically significant deficit in the activity, function and response of the host. Alternatively, a therapeutically effective amount is sufficient to cause an improvement in a clinically critical condition in a host. As is appreciated by those skilled in the art, the amount of a compound may vary depending on its specific activity. Suitable dosage amounts may contain a predetermined quantity of active composition calculated to produce the desired therapeutic effect in association with the required diluent. In the methods and use for manufacture of compositions of the invention, a therapeutically effective amount of the active component is provided. A therapeutically effective amount can be determined by the ordinary skilled medical worker based on patient characteristics, such as age, weight, sex, condition, complications, other diseases, etc., as is well known in the art. The administration of the pharmaceutically effective dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units and also by multiple administrations of subdivided doses at specific intervals. Alternatively, the does may be provided as a continuous infusion over a prolonged period.

[0154] The compositions of the invention can be formulated at various concentrations, depending on the efficacy / toxicity of the compound being used. The skilled person will be aware of suitable techniques for determining formulations and dosages to be used in practice for administration to patients.

[0155] Another aspect of the invention provides the antibody or antigen-binding fragment or pharmaceutical composition as defined herein for use for the treatment and / or prevention of a disease, such as cancer.

[0156] Another aspect of the invention provides an antibody or antigen-binding fragment or pharmaceutical composition as defined herein for use in the manufacture of a medicament for the treatment and / or prevention of a disease, such as cancer.

[0157] In another aspect of the invention there is provided a method of treatment of a human subject in need thereof, comprising administering the antibody or antigen-binding fragment thereof or the pharmaceutical composition as defined in the abovementioned aspects of the invention, to the individual. In some embodiments, the method of treatment is a method for treating cancer. COMP overexpression or de novo expression, relative to the condition of normal tissues, has been observed in many types of solid human malignancies, including the following cancer conditions: different variants of breast, gastric, colorectal, pancreatic, lung, head and neck, ovarian, renal, prostate, liver, skin, thyroid, bladder and nasopharyngeal carcinomas and soft-tissue and musculoskeletal sarcomas (Guo B, Wang Y, Liu W, Zhang S. Clin Transl Oncol. 2023 Feb;25(2):535-554. and Table 1 below).

[0158] Table 1:

[0159] Consequently, in some embodiments of the invention, the antibody or antigen-binding fragment thereof or the pharmaceutical composition comprising the antibody or antigen-binding fragment thereof is for use in treating cancer.

[0160] In some embodiments, the cancer is a cancer type characterized by significant COMP expression. By “characterized by significant COMP expression” we include the tumour type that expresses COMP at detectable levels in situ and / or the cancer condition that has induced an increase in the levels of intact or fragmented COMP in the blood of the individual affected by the cancer condition as compared to a healthy individual. Preferably, the cancer is a cancer in which COMP is expressed in the tumour, e.g. at detectable levels in situ. Expression of COMP can be measured in a biopsy from the tumour using standard immunohistochemistry, i.e. detection with antibodies, and / or COMP can be measured in the blood using ELISA.

[0161] In some embodiments the cancer comprises a solid tumour.

[0162] In some embodiments the cancer is breast, colorectal, pancreatic, ovarian, gastric, prostate, liver, and / or thyroid cancer. In some embodiments the cancer is breast cancer.

[0163] In some embodiments, upon binding to COMP, the antibody or antigen-binding fragment thereof reduces or entirely inhibits growth and expansion tumour cells in vitro and / or formation of tumour spheres. Such properties can be measured by techniques well known in the art and / or described herein.

[0164] In some embodiments, upon binding to COMP, the antibody or antigen-binding fragment reduces or entirely prevents local tumour growth and / or metastasis formation, in a syngeneic or xenogeneic murine tumour growth model based on an immunocompetent or immunodeficient recipient mouse phenotype. Such properties can be measured by techniques well known in the art and / or described herein.

[0165] In some embodiments, administration of the anti-COMP antibody or antigen-binding fragment thereof reduces or entirely prevents T-cell infiltration into a tumour lesion generated by implantation into an animal host of human cancer cells, for example corresponding to one or more of the cancers defined herein. Such properties can be measured by techniques well known in the art and / or described herein.

[0166] In some embodiments, upon binding to COMP, the antibody or antigen-binding fragment interferes with or inhibits epithelial-mesenchymal transitions (EMTs) in vitro and / or in vivo. Such properties can be measured by techniques well known in the art and / or described herein.

[0167] In some embodiments, the antibody or antigen-binding fragment induces infiltration of Granzyme B-positive immune cells into the tumor, preferably into a tumour expressing COMP, more preferably in a breast tumour.

[0168] In some embodiments, the antibody or antigen-binding fragment thereof induces a reduction in the proportion of cancer stem cells within the tumor. Cancer stem cells (CSCs) are a subpopulation of tumor cells characterized by their ability to self-renew, differentiate, and initiate tumor formation. These cells are often associated with therapy resistance, tumor recurrence, and metastasis.

[0169] The reduction in CSCs may be observed as a decrease in tumorsphere formation capacity, a reduction in the expression of sternness-associated markers (e.g., CD44Ahigh / CD24Alow, ALDH1), or diminished tumor-initiating potential in in vivo models. This effect may result from direct targeting of COMP-expressing CSCs or from enhanced immune-mediated clearance facilitated by the antibody-induced infiltration of cytotoxic immune cells, such as Granzyme B-positive T lymphocytes and NK cells.

[0170] This mechanism contributes to the therapeutic efficacy of the antibody by not only reducing tumor mass but also impairing the tumor’s capacity to regenerate and resist treatment.ln some embodiments, the antibody or antigen-binding fragment thereof is administered in combination with an immune checkpoint inhibitor. Immune checkpoint inhibitors are agents that block inhibitory pathways in T cells, thereby enhancing the immune system’s ability to recognize and destroy tumor cells. Examples include antibodies targeting programmed cell death protein 1 (PD-1), its ligand PD- L1 , and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4).

[0171] The combination of the anti-COMP antibody with an immune checkpoint inhibitor may result in a synergistic therapeutic effect. While the anti-COMP antibody facilitates immune cell infiltration into the tumor microenvironment — particularly of Granzyme B-positive cytotoxic T lymphocytes and NK cells — the checkpoint inhibitor enhances the activation and persistence of these effector cells by preventing their functional exhaustion.

[0172] In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to a cytotoxic agent or an immune modulator. Such conjugates, often referred to as antibody-drug conjugates (ADCs) or immunoconjugates, combine the specificity of the antibody for COMP-expressing tumor cells with the potent activity of a therapeutic payload.

[0173] In some embodiments, upon binding to COMP, the antibody or antigen-binding fragment reduces or inhibits Notch signalling in vitro. This can be measured by techniques well known in the art.

[0174] In another aspect, the present invention refers to an antibody or antigen-binding fragment thereof that specifically binds to COMP, particularly its N-terminal domain, for use in the treatment of COMP-expressing tumours. The antibody is capable of inducing the infiltration of granzyme B- positive immune cells into the tumor, thereby enhancing anti-tumor immunity.

[0175] The use of the terms "a" and "an“, and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising”, “comprises”, “consisting”, “having”, “including”, “at least” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method for referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0176] As used herein and in the appended claims, the singular forms “a,” “or,” and “the” include plural referents unless the context clearly dictates otherwise.

[0177] It is understood that the embodiments described herein include “consisting” and / or “consisting essentially of’ embodiments. As used herein, except where the context requires otherwise due to express language or necessary implication, the word “comprise” or variations such as “comprises” or “comprising” is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments.

[0178] The listing or discussion of an apparently prior published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0179] Preferences, options and embodiments for a given aspect, feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences, options and embodiments for all other aspects, features and parameters of the invention. Embodiments and features of the present invention are also outlined in the following items and also illustrated by the following non-limiting examples.

[0180] References

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[0183] 3. Papadakos, K.S., et al., Cartilage Oligomeric Matrix Protein initiates cancer stem cells through activation of Jagged1-Notch3 signaling. Matrix Biol, 2019. 81 : p. 107-121.

[0184] 4. Kong, L., et al., Interaction between cartilage oligomeric matrix protein and extracellular matrix protein 1 mediates endochondral bone growth. Matrix Biol, 2010. 29(4): p. 276-86.

[0185] 5. Lai, Y., et al., Enhanced COMP catabolism detected in serum of patients with arthritis and animal disease models through a novel capture ELISA. Osteoarthritis Cartilage, 2012. 20(8): p. 854-62.

[0186] 6. Hedbom, E., et al., Cartilage matrix proteins. An acidic oligomeric protein (COMP) detected only in cartilage. J Biol Chem, 1992. 267(9): p. 6132-6.

[0187] 7. DiCesare, P.E., et al., Cartilage oligomeric matrix protein and thrombospondin 1 . Purification from articular cartilage, electron microscopic structure, and chondrocyte binding. Eur J Biochem, 1994. 223(3): p. 927-37.

[0188] 8. DiCesare, P.E., et al., Cartilage oligomeric matrix protein: isolation and characterization from human articular cartilage. J Orthop Res, 1995. 13(3): p. 422-8.

[0189] 9. Lefranc, M.P., Unique database numbering system for immunogenetic analysis. Immunol Today, 1997. 18(11): p. 509.

[0190] 10. Lefranc, M.P., et al., IMGT, the international ImMunoGeneTics database. Nucleic Acids Res, 1999. 27(1): p. 209-12.

[0191] EXAMPLES

[0192] Example 1 - Production of antibodies

[0193] Table 2: Antibody amino acid sequences

[0194] Materials and Methods

[0195] Production of Monoclonal antibodies

[0196] Monoclonal antibody 2 (also referred to herein as MAB2 or Antibody 2) was raised by conventional immunizations of Balb / c female mice with a recombinant GST-flagged fragment of human COMP corresponding to the type III domain produced in insect cells (Bac-to-Bac Baculuvirus Expression System), as described in our papers [4, 5], myeloma spleen cell fusion and differential section against of hybridoma clones against the immunogen and purified human articular cartilage COMP (see below). Subcloning of positively scoring hybridoma clones was performed by conventional limiting dilution procedures.

[0197] Monoclonal antibodies 1 , 3 and 4 (also referred to herein as MAB1 , MAB3 and MAB4 or Antibody 1 , Antibody 3 and Antibody 4, respectively) were generated in Wistar female rats by immunization (three intravenous injections) with low doses (10-50 mg / rat / injection) of COMP extracted and purified from articular cartilage as originally described [6], followed by mild systemic treatment with methotrexate (1 mg / kg) and higher doses (three repeated injections - 150 mg / rat / injection) of COMP isolated from human articular cartilage as described by [7, 8], Spleen cells were fused with human myeloma cells and hybridoma clones selected by ELISA against the human COMP immunogen. Subcloning of positively scoring hybridoma clones was performed by conventional limiting dilution procedures. The isotype of the antibodies is as follow: Antibody 1 , Rat-lgG2a; Antibody 2, Mouse- IgG 1 K; Antibody 3 and 4, Rat-lgG2b.

[0198] Humanization of antibodies

[0199] Based on antibody amino acid sequence of the antibody 4, a humanization process was performed in silico. The antibody was humanized following a standard CDR grafting process. In brief, heavy and light chain human germline sequences were selected based on sequence homology to the parental mouse sequences. CDRs, as defined by the IMGT definition [9, 10], were grafted from the parental variable domain onto the human germline. As required back mutations were introduced at locations outside of the CDRs that were deemed to be potentially critical for maintenance of antibody binding. By this process, four humanized VH sequences and four humanized VL sequences were designed and then paired in all possible combinations to create a total of 16 humanized antibodies. From these 16 antibodies the 13 were deemed to be capable to bind to COMP protein. The sequence identity of the variable regions of the light and heavy chain of the humanized antibodies with the variable light chain of antibody 4 and variable heavy chain of the antibody 4 are presented in Table 3.

[0200] Table 3: Sequence Identity of Humanized antibodies to Antibody 4

[0201] Example 1 : Anti-COMP antibodies bind COMP in ELISA Method

[0202] The COMP protein (P49747, Uniprot), tagged at the C-terminus with six histidines (6His), was expressed in 293-F freestyle cells (Thermo Fisher Scientific) and subsequently purified by affinity chromatography using a nickel column (Cytiva). The purity of COMP was verified by polyacrylamide gel electrophoresis and Coomassie blue staining. Before testing with monoclonal antibodies, the recombinant purified COMP was analyzed by western blot using a polyclonal rabbit antibody to confirm the appropriate molecular weight and immunoreactivity with the antibody. The polyclonal rabbit antibody against COMP has been produced in-house and fully characterised in the past (Englund, E., et al., Cartilage oligomeric matrix protein contributes to the development and metastasis of breast cancer. Oncogene, 2016. 35: p. 5585-5596). In the ELISA method, 50pl of PBS-diluted COMP (10pg / ml) and EDTA- or DTT-treated COMP were coated in a 96-well MaxiSorp plate (Thermo Fisher Scientific) overnight. EDTA-treated COMP was produced by incubating recombinant purified COMP with 5 mM of EDTA overnight. DTT-treated COMP was produced by incubating recombinant purified COMP with 25 mM of DTT at 95°C for 10 minutes. Since COMP incorporates several calcium ions into its molecule, their removal with EDTA results in structural changes to the pentameric molecule that can lead to potential loss of binding by a monoclonal antibody. DTT-treated COMP represents a monomeric version of the molecule, as the pentamers are held together by disulfide bonds. The next day, the plate was washed with TBST buffer and blocked with 3% non-fat milk for 1 hour, and different concentrations of the monoclonal antibodies diluted in 3% non-fat milk were incubated for 1 hour at room temperature. This was followed by with TBST buffer wash, and a 1-hour incubation with the appropriate secondary HRP-conjugated antibodies (Anti-rat HRP antibody produced by Jackson ImmunoResearch and anti-mouse HRP antibody produced by Dako). Finally, 50pl of TMB substrate (Kem-En-Tech) was added to every well, the reactions were stopped with sulfuric acid, and absorbance was measured with a Cytation 5 (Bio tek). The curve was fitted to the measurements using Prism software to model specific binding with a Hill slope.

[0203] Results and conclusion

[0204] All antibodies bound well to COMP in ELISA. The dissociation constants (Kd) of the antibodies were as follows: Kd=495.2 nM for antibody 1 , Kd=31.6 nM for antibody 2, Kd=8.2 nM for antibody 3, Kd=81 .9 nM for antibody 4, and finally Kd=105.9 nM for antibody V7 (Figure 3). EDTA did not affect the binding while binding of antibody 3 and 4 were decreased when COMP was reduced with DTT. Example 2: Anti-COMP antibodies bind surface-bound COMP in human breast cancer cell lines

[0205] Method 1

[0206] Western blotting analysis of lysates of breast cancer cells BT-20 expressing wild type full length human COMP and deletion mutants of COMP have been performed. These mutans lack one of the following each: the whole polymerisation domain, all EGF domains, all TSP domains and the C- terminal globular domain. Reduced sample buffer was used for electrophoresis, and proteins were transferred to PVDF membranes after separation, blocked and incubated with antibodies. After incubation with secondary antibodies conjugated with horseradish peroxidase, binding was visualized using Immobilon ECL kit and CCD camera (Biorad).

[0207] Antibody 1 , 2, 3 and 4 as well as humanised version of 4 (V7) have been tested alongside a polyclonal rabbit antibody recognizing all domains of COMP.

[0208] Results and conclusions

[0209] The results are indicate that the main binding site for antibodies 1 , 3 and 4 (and its humanised version) are in the polymerisation domain. Antibody 2 requires the presence of EGF domains (Figure 1 A and B).

[0210] Method 2

[0211] Human breast cancer cell lines MDA-MB-231 and BT-20 (ATCC) were transfected with the pcDNA3 plasmid (Thermo Fisher Scientific) expressing the human COMP protein. Several clones stably expressing the COMP protein were isolated. In parallel, control mock cells were transfected with empty pcDNA3 and then isolated (Mock control cells).

[0212] The breast cancer cell lines were detached from the culturing flasks with Versene (Thermofisher scientific) retaining the surface bound COMP. Cells were then counted and 1x105cells added per well in a 96-well plate (Thermo Fisher Scientific) followed by incubation for 1 h with 3% BSA in FACS buffer at room temperature (10 mM HEPES, 140 mM NaCI, 5 mM KCI, 1 mM MgCh, 2 mMCaCh, 0.02% NaNs) to block the non-specific epitopes. Then cells were washed with FACS buffer and incubated for 1 h with increasing concentrations of monoclonal antibodies diluted in FACS buffer supplemented with 3% BSA at room temperature. Moreover, incubation with isotype control antibodies were used as a negative control (Rat-lgG2a, BD Pharmingen; Rat-lgG2b, BioLegend; Mouse lgGI K, lgG2b, BioLegend). Finally, cells were washed with FACS buffer and incubated with the appropriate secondary antibody conjugated with Alexa Fluor 647 at room temperature (Thermo Fisher Scientific).

[0213] Results and Conclusions

[0214] The cells were analysed with FACS using the CytoFLEX (Beckman coulter). All four antibodies were able to bind to human breast cancer cells expressing COMP. In contrast, the antibodies did not bind control mock-transfected cells. Control isotype antibodies did not bind to COMP expressing cells. The results are shown in Figure 4 for both MDA-MB-231 cells and BT-20 cells.

[0215] Example 3: Anti-COMP antibodies reduce tumor development in mouse models of breast cancer

[0216] Method

[0217] The mouse breast cancer cell line 4T1-Luc2 (CRL-2539-LUC2) was transfected with the pcDNA3 plasmid (Thermo Fisher Scientific) expressing the human COMP protein. Several clones were isolated that were stably expressing human COMP. Control mock cell clones were produced by transfection with empty pcDNA3 plasmid and then isolated.

[0218] In a syngeneic mouse model, BALB / cJRj mice (Janvier labs) were orthotopically transplanted in the 4thleft mammary fat pad with 2x106cells combining three different clones of 4T1-Luc2 cells expressing human COMP. After 7 days, at least 10 mice were included in each group (Groups: PBS, antibody 1 , antibody 2, antibody 3 and antibody 4) of the experiment, and the injection of the antibodies was initiated. Each mouse was injected intraperitoneally twice per week with 200pg of monoclonal antibody diluted in PBS, or with PBS alone, which served as the untreated control. Measurements of the primary tumour volume were performed using a caliper to measure the longer and smaller dimension of the tumour. Tumour volume was calculated using the mathematical equation: V=AxAxBx0.52 (V = tumor volume, A = tumor shortest diameter, B = tumor longest diameter).

[0219] Results and Conclusions

[0220] The results are shown in Figure 5. All mice treated with the monoclonal antibodies developed statistically significantly smaller tumors compared to the untreated PBS group. More specifically, in the first experiment (Figure 5A), in the control group, 5 mice developed tumors with an average volume of 639 mm3at the day of experiment termination. The mice treated with antibody 1 developed tumors with an average volume of 244 mm3at the day of experiment termination, with 5 mice developing tumors. The group of mice treated with antibody 2 developed tumors with an average volume of 363 mm3at the day of experiment termination, with 8 mice developing tumors. In the second experiment (Figure 5B), the control group had an average tumor volume of 578 mm3at the day of experiment termination, with 6 mice developing tumors. The group of mice treated with antibody 3 had 8 mice that developed tumors, with an average volume of 321 mm3at the day of experiment termination. Finally, the group of mice treated with antibody 4 had an average tumor volume of 160 mm3at the day of experiment termination, with 6 mice developing primary tumors. The same experiment was repeated for the comparison of the humanized antibody V7 (Figure 8). Mice were treated with antibody 4 and PBS as a positive and negative control respectively. The VH and VL (Table 3) of the humanized antibody was engrafted in lgG1 (V7-lgG1) and lgG2a (V7-lgG2a) mouse backbone. The lgG1 class of mouse antibody are equivalent to human lgG4 and the lgG2a class of mouse antibody are equivalent to human IgG 1 with the characteristic of immune system activation. At least 10 mice were injected per group with the 4T1-Luc2 cells expressing human COMP. The group of mice injected with the PBS had an average tumor volume of 558mm3 with 6 mice developing tumors. The group of mice injected with antibody 4 had an average tumor volume of 234mm3at the day of experiment termination, with 5 mice developing primary tumors. The group of mice injected with antibody V7-lgG1 had an average tumor volume of 548mm3at the day of experiment termination, with 7 mice developing primary tumors. Finally, the group of mice treated with antibody V7-lgG2a had an average tumor volume of 240mm3at the day of experiment termination, with 6 mice developing primary tumors.

[0221] Example 4: Anti-COMP antibodies inhibit formation of tumorspheres Method

[0222] The human breast cancer cell line MDA-MB-231 (ATCC) expressing COMP and their counterpart control mock cells were used to form tumorspheres. Cells (4x104) were seeded in ultra-low attachment 6-well plates (Corning) in complete MammoCult medium (Stem cell technologies). Cells expressing COMP were treated with 50pg of monoclonal antibodies or PBS as a negative control. The mock-transfected control cells that did not express COMP were used as a baseline. After seven days images of the tumorspheres were captured with the EVOS microscope (ThermoFischer Scientific). The long axis of the tumorspheres was measured with the Image J software.

[0223] Results and conclusions

[0224] Antibodies 1 and 4 treatments reduced the size of the tumorspheres in comparison with the COMP expressing untreated tumorspheres. The size of the Antibodies 1 and 4 treated tumorspheres were almost equal with the size of control mock cells that did not express COMP, see Figure 7.

[0225] The antibody 3 statistically significantly reduced the size of the tumorspheres compared with the COMP expressing untreated cells. Lastly, the antibody 2 had no effect in the size of the tumorspheres in COMP expressing cells. Reduction of the tumorspheres size is associated with the percentage of the cancer stem cells in the cell population.

[0226] Example 5: Anti-COMP antibodies promote T-cell infiltration Method

[0227] Paraffin-embedded primary tumor samples from the syngenic mouse models treated with the monoclonals were sliced and placed on microscopy slides (Dako). Epitope retrieval was performed with Envision Flex high pH kit (Dako) using a PT-link module (Dako). Tissue was stained overnight at 4°C with rabbit anti-CD8 (Abeam) and rabbit anti-granzyme B (R&D Systems).

[0228] Slides were scanned with Aperio Scanner system (Leica) at 40X and the number of positive cells calculated with Qupath software.

[0229] Results and conclusions

[0230] The animals treated with all four monoclonal antibodies had statistically significantly more tumor infiltrating CD8+T-cells as compared to Control untreated tumors, see Figure 6. This indicates a more activate immune response in the treated mice. Furthermore, treatment of mice with V7-lgG2a resulted in a significant infiltration of granzyme B-positive cells compared to V7-lgG1. Since V7- lgG2a demonstrated a therapeutic effect in the mouse model, whereas V7-lgG1 did not (Figure 8), this supports the notion that the antibody’s mode of action depends on the presence of active granzyme B-positive immune cells.

[0231] ITEMS

[0232] Certain embodiments of the invention are defined by reference to the following items:

[0233] 1. An antibody or antigen-binding fragment thereof that specifically binds to Cartilage Oligomeric Matrix Protein (COMP) for use in therapy in a human subject, selected from: (i) an antibody or antigen-binding fragment thereof which comprises: a. a heavy chain variable region (VH) comprising the Complementarity Determining Regions (CDRs) i. CDR-H1 (SEQ ID NO. 2), ii. CDR-H2 (SEQ ID NO. 3), and

[0234] Hi. CDR-H3 (SEQ ID NO. 4), or a variant of said heavy chain variable region having at least 75% sequence identity to SEQ ID NO. 1 ; and b. a light chain variable region (VL) comprising the CDRs i. CDR-L1 (SEQ ID NO. 6), ii. CDR-L2 (SEQ ID NO. 7), and iii. CDR-L3 (SEQ ID NO. 8), or a variant said light chain variable region having at least 75% sequence identity with SEQ ID NO. 5;

[0235] (ii) an antibody or antigen-binding fragment thereof which comprises: a. a heavy chain variable region (VH) comprising the Complementarity Determining Regions (CDRs) i. CDR-H1 (SEQ ID NO. 17), ii. CDR-H2 (SEQ ID NO. 18), and iii. CDR-H3 (SEQ ID NO. 19), or a variant of said heavy chain variable region having at least 75% sequence identity to SEQ ID NO. 16; and b. a light chain variable region (VL) comprising the CDRs i. CDR-L1 (SEQ ID NO. 21), ii. CDR-L2 (SEQ ID NO. 22), and iii. CDR-L3 (SEQ ID NO. 23), or a variant of said light chain variable region having at least 75% sequence identity with SEQ ID NO. 20;

[0236] (iii) an antibody or antigen-binding fragment thereof which comprises: a. a heavy chain variable region (VH) comprising the Complementarity Determining Regions (CDRs) i. CDR-H1 (SEQ ID NO. 25), ii. CDR-H2 (SEQ ID NO. 26), and iii. CDR-H3 (SEQ ID NO. 27), or a variant of said heavy chain variable region having at least 75% sequence identity to SEQ ID NO. 24; and b. a light chain variable region (VL) comprising the CDRs i. CDR-L1 (SEQ ID NO. 29), ii. CDR-L2 (SEQ ID NO. 30), and iii. CDR-L3 (SEQ ID NO. 31), or a variant said light chain variable region having at least 75% sequence identity with SEQ ID NO. 28; and

[0237] (iv) An antibody or antigen-binding fragment thereof which comprises: a. a heavy chain variable region (VH) comprising the Complementarity Determining Regions (CDRs) i. CDR-H1 (SEQ ID NO. 33), ii. CDR-H2 (SEQ ID NO. 34), and iii. CDR-H3 (SEQ ID NO. 35), or a variant of said heavy chain variable region having at least 75% sequence identity to SEQ ID NO. 32; and b. a light chain variable region (VL) comprising the CDRs i. CDR-L1 (SEQ ID NO. 37), ii. CDR-L2 (SEQ ID NO. 38), and

[0238] Hi. CDR-L3 (SEQ ID NO. 39), or a variant said light chain variable region having at least 75% sequence identity with SEQ ID NO. 36. The antibody or antigen-binding fragment thereof for use of item 1 wherein the antibody or antigen-binding fragment thereof comprises: a. a heavy chain variable region (VH) comprising the Complementarity Determining Regions (CDRs): i. CDR-H1 (SEQ ID NO. 2), ii. CDR-H2 (SEQ ID NO. 3), and

[0239] Hi. CDR-H3 (SEQ ID NO. 4); and b. a light chain variable region (VL) comprising the CDRs: i. CDR-L1 (SEQ ID NO. 6), ii. CDR-L2 (SEQ ID NO. 7), and

[0240] Hi. CDR-L3 (SEQ ID NO. 8). An antibody or antigen-binding fragment thereof according to item 1 or 2, comprising: a. a heavy chain variable region (VH) selected from the group of: i. SEQ ID NO. 9, ii. SEQ ID NO. 10,

[0241] Hi. SEQ ID NO. 11 , and iv. SEQ ID NO. 12; or a variant of said heavy chain variable region having at least 75% sequence identity to SEQ ID NO. 1 ; and b. a light chain variable region (VL) selected from the group of: i. SEQ ID NO. 13, ii. SEQ ID NO. 14, and

[0242] Hi. SEQ ID NO. 15; ora variant of said light chain variable region having at least 75% sequence identity with SEQ ID NO. 5. An antibody or antigen-binding fragment thereof according to any of the preceding items, wherein the variants have a sequence identity of at least 79% with said sequence. An antibody or antigen-binding fragment thereof according to any of the preceding items, where the light chain variable region is SEQ ID NO: 14. An antibody or antigen-binding fragment according thereof to item 5, further wherein the heavy chain variable region is SEQ ID NO: 10. An antibody or antigen-binding fragment thereof according to any of the previous items, wherein the VH and VL domains comprise: a. SEQ ID NO. 10 and SEQ ID. NO. 14; b. SEQ ID NO. 9 and SEQ ID. NO. 13; c. SEQ ID NO. 9 and SEQ ID. NO. 14; d. SEQ ID NO. 12 and SEQ ID. NO. 14; e. SEQ ID NO. 9 and SEQ ID. NO. 15; f. SEQ ID NO. 10 and SEQ ID. NO. 13; g. SEQ ID NO. 10 and SEQ ID. NO. 15; h. SEQ ID NO. 11 and SEQ ID. NO. 13; i. SEQ ID NO. 11 and SEQ ID. NO. 14; j. SEQ ID NO. 11 and SEQ ID. NO. 15; k. SEQ ID NO. 12 and SEQ ID. NO. 13; or l. SEQ ID NO. 12 and SEQ ID. NO. 15 An antibody or antigen-binding fragment thereof according to any of the previous items, wherein said antibody or antigen-binding fragment is humanized. The antibody or antigen-binding fragment thereof according to any of the previous items wherein the antigen-binding fragment is selected: scFv, Fv, Fab, F(ab)2, Fab-SH, dsFv, sdAb, di-scFvs bi-scFv, Fcabs, diabodies, scFv-Fc / minibody, triabody, tetrabody, tandAb, half antibody (Unibody), and domain antibodies. The antibody or antigen-binding fragment thereof according to any of the previous items, wherein said antibody or antigen-binding fragment binds the N-terminus of COMP. The antibody or antigen-binding fragment thereof according to any of the preceding items wherein the antigen-binding fragment binds COMP with an affinity Kd of less than 2000 nM. The antibody or antigen-binding fragment thereof according to any of the previous items, wherein said antibody or antigen-binding fragment reduces or entirely inhibits growth, expansion or formation of tumour spheres in vitro. The antibody or antigen-binding fragment thereof according to any of the previous items, wherein said antibody or antigen-binding fragment reduces or entirely prevents local tumour growth or metastasis formation, in a syngeneic or xenogeneic murine tumour growth model based on an immunocompetent or immunodeficient recipient mouse phenotype. The antibody or antigen-binding fragment thereof according to any of the previous items, wherein said antibody or antigen-binding fragment interferes or inhibits epithelial- mesenchymal transitions (EMTs), in vitro or in vivo. The antibody or antigen-binding fragment thereof according to any of the previous items, wherein said antibody or antigen-binding fragment reduces or inhibits Notch signalling in vitro. A pharmaceutical composition for use in therapy in a human subject comprising the antibody or antigen-binding fragment thereof according to any of items 1 to 15 and a pharmaceutically acceptable buffer, carrier, excipient or diluent. The antibody or antigen-binding fragment thereof for use according to any of items 1 to 15; or the pharmaceutical composition for use according to item 16, wherein said use in therapy is use in treatment of cancer. The antibody or antigen-binding fragment thereof, or the pharmaceutical composition, for use according to item 17, wherein said cancer is characterized by COMP expression, for example a cancer in which COMP is expressed in the tumour. The antibody or antigen-binding fragment thereof, or the pharmaceutical composition, for use according to item 17 or 18, wherein said cancer is a solid tumour, such as breast, colorectal, pancreatic, ovarian, gastric, prostate, liver, and / or thyroid cancer. The antibody or antigen-binding fragment thereof, or the pharmaceutical composition, for use according to any one of items 17-19 wherein the cancer is breast cancer. A method of treating a human subject in need thereof comprising administering to the subject the antibody or antigen-binding fragment thereof or pharmaceutical composition as defined in any previous item, optionally wherein the subject has cancer.

Claims

CLAIMS1. An antibody or antigen-binding fragment thereof that specifically binds to Cartilage Oligomeric Matrix Protein (COMP) for use in therapy in a human subject, selected from:(i) an antibody or antigen-binding fragment thereof which comprises: a. a heavy chain variable region (VH) comprising the Complementarity Determining Regions (CDRs) i. CDR-H1 (SEQ ID NO. 2), ii. CDR-H2 (SEQ ID NO. 3), andHi. CDR-H3 (SEQ ID NO. 4), or a variant of said heavy chain variable region having at least 75% sequence identity to SEQ ID NO. 1 ; and b. a light chain variable region (VL) comprising the CDRs i. CDR-L1 (SEQ ID NO. 6), ii. CDR-L2 (SEQ ID NO. 7), andHi. CDR-L3 (SEQ ID NO. 8), or a variant said light chain variable region having at least 75% sequence identity with SEQ ID NO. 5;(ii) an antibody or antigen-binding fragment thereof which comprises: a. a heavy chain variable region (VH) comprising the Complementarity Determining Regions (CDRs) i. CDR-H1 (SEQ ID NO. 17), ii. CDR-H2 (SEQ ID NO. 18), andHi. CDR-H3 (SEQ ID NO. 19), or a variant of said heavy chain variable region having at least 75% sequence identity to SEQ ID NO. 16; and b. a light chain variable region (VL) comprising the CDRs i. CDR-L1 (SEQ ID NO. 21), ii. CDR-L2 (SEQ ID NO. 22), andHi. CDR-L3 (SEQ ID NO. 23), or a variant of said light chain variable region having at least 75% sequence identity with SEQ ID NO. 20;(iii) an antibody or antigen-binding fragment thereof which comprises: a. a heavy chain variable region (VH) comprising the Complementarity Determining Regions (CDRs) i. CDR-H1 (SEQ ID NO. 25), ii. CDR-H2 (SEQ ID NO. 26), and iii. CDR-H3 (SEQ ID NO. 27), or a variant of said heavy chain variable region having at least 75% sequence identity to SEQ ID NO. 24; andb. a light chain variable region (VL) comprising the CDRs i. CDR-L1 (SEQ ID NO. 29), ii. CDR-L2 (SEQ ID NO. 30), andHi. CDR-L3 (SEQ ID NO. 31), or a variant said light chain variable region having at least 75% sequence identity with SEQ ID NO. 28; and(iv) An antibody or antigen-binding fragment thereof which comprises: a. a heavy chain variable region (VH) comprising the Complementarity Determining Regions (CDRs) i. CDR-H1 (SEQ ID NO. 33), ii. CDR-H2 (SEQ ID NO. 34), andHi. CDR-H3 (SEQ ID NO. 35), or a variant of said heavy chain variable region having at least 75% sequence identity to SEQ ID NO. 32; and b. a light chain variable region (VL) comprising the CDRs i. CDR-L1 (SEQ ID NO. 37), ii. CDR-L2 (SEQ ID NO. 38), andHi. CDR-L3 (SEQ ID NO. 39), or a variant said light chain variable region having at least 75% sequence identity with SEQ ID NO. 36.

2. The antibody or antigen-binding fragment thereof for use of claim 1 wherein the antibody or antigen-binding fragment thereof comprises: a. a heavy chain variable region (VH) comprising the Complementarity Determining Regions (CDRs): i. CDR-H1 (SEQ ID NO. 2), ii. CDR-H2 (SEQ ID NO. 3), andHi. CDR-H3 (SEQ ID NO. 4); and b. a light chain variable region (VL) comprising the CDRs: i. CDR-L1 (SEQ ID NO. 6), ii. CDR-L2 (SEQ ID NO. 7), andHi. CDR-L3 (SEQ ID NO. 8).

3. An antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising: a. a heavy chain variable region (VH) selected from the group of: i. SEQ ID NO. 9, ii. SEQ ID NO. 10,Hi. SEQ ID NO. 11 , and iv. SEQ ID NO. 12;or a variant of said heavy chain variable region having at least 75% sequence identity to SEQ ID NO. 1 ; and b. a light chain variable region (VL) selected from the group of: i. SEQ ID NO. 13, ii. SEQ ID NO. 14, andHi. SEQ ID NO. 15; ora variant of said light chain variable region having at least 75% sequence identity with SEQ ID NO. 5.

4. An antibody or antigen-binding fragment thereof according to any of the previous claims, wherein the VH and VL domains comprise: a. SEQ ID NO. 10 and SEQ ID. NO. 14; b. SEQ ID NO. 9 and SEQ ID. NO. 13; c. SEQ ID NO. 9 and SEQ ID. NO. 14; d. SEQ ID NO. 12 and SEQ ID. NO. 14; e. SEQ ID NO. 9 and SEQ ID. NO. 15; f. SEQ ID NO. 10 and SEQ ID. NO. 13; g. SEQ ID NO. 10 and SEQ ID. NO. 15; h. SEQ ID NO. 11 and SEQ ID. NO. 13; i. SEQ ID NO. 11 and SEQ ID. NO. 14; j. SEQ ID NO. 11 and SEQ ID. NO. 15; k. SEQ ID NO. 12 and SEQ ID. NO. 13; or l. SEQ ID NO. 12 and SEQ ID. NO.

155. An antibody or antigen-binding fragment thereof according to any of the previous claims, wherein said antibody or antigen-binding fragment is humanized.

6. A pharmaceutical composition for use in therapy in a human subject comprising the antibody or antigen-binding fragment thereof according to any of claims 1 to 5 and a pharmaceutically acceptable buffer, carrier, excipient or diluent.

7. The antibody or antigen-binding fragment thereof for use according to any of claims 1 to 5; or the pharmaceutical composition for use according to claim 6, wherein said use in therapy is use in treatment of cancer.

8. The antibody or antigen-binding fragment thereof, or the pharmaceutical composition, for use according to claim 7, wherein said cancer is a cancer in which COMP is expressed in the tumour.

9. The antibody or antigen-binding fragment thereof, or the pharmaceutical composition, for use according to any one of claims 7-8, wherein said cancer is a solid tumour.

10. The antibody or antigen-binding fragment thereof, or the pharmaceutical composition, for use according to claim 9, wherein said solid tumour is selected from the group consisting of: breast, colorectal, pancreatic, ovarian, gastric, prostate, liver, and / or thyroid cancer.

11. The antibody or antigen-binding fragment thereof, or the pharmaceutical composition, for use according to any one of claims 7-10 wherein the cancer is breast cancer.

12. The antibody or antigen-binding fragment thereof, or the pharmaceutical composition, for use according to any one of claims 7-11 wherein the treatment induces infiltration of Granzyme B-positive immune cells into the tumor.

13. The antibody or antigen-binding fragment thereof, or the pharmaceutical composition, for use according to any one of claims 7-12 wherein the antibody binds to an epitope within the N-terminal domain of COMP.

14. The antibody or antigen-binding fragment thereof, or the pharmaceutical composition, for use according to any one of claims 7-13, wherein the treatment results in a reduction in the proportion of cancer stem cells in the tumor.

15. The antibody or antigen-binding fragment thereof, or the pharmaceutical composition, for use according to any one of claims 7-14, wherein the antibody is administered in combination with an immune checkpoint inhibitor.

16. The antibody or antigen-binding fragment thereof, or the pharmaceutical composition, for use according to any one of claims 7-15, wherein the antibody is conjugated to a cytotoxic agent or immune modulator.

17. An antibody or antigen-binding fragment thereof, that specifically binds to Cartilage Oligomeric Matrix Protein (COMP) for use in the treatment of a tumor expressing COMP, wherein the antibody or antigen-binding fragment is capable of inducing infiltration of granzyme B-positive immune cells into the tumor.

18. The antibody according to claim 17, wherein the antibody or the antigen-binding fragment binds to the N-terminal domain of COMP.

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